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Purpose: Clinical biomarkers to identify patients unlikely to benefit from CDK4/6 inhibition (CDK4/6i) in combination with endocrine therapy (ET) are lacking. We implemented a comprehensive circulating tumor DNA (ctDNA) analysis to identify genomic features for predicting and monitoring treatment resistance. Experimental Design: ctDNA was isolated from 216 plasma samples collected from 51 patients with hormone receptor–positive (HR+)/HER2-negative (HER2−) metastatic breast cancer (MBC) on a phase II trial of palbociclib combined with letrozole or fulvestrant (NCT03007979). Boosted whole-exome sequencing (WES) was performed at baseline and clinical progression to evaluate genomic alterations, mutational signatures, and blood tumor mutational burden (bTMB). Low-pass whole-genome sequencing was performed at baseline and serial timepoints to assess blood copy-number burden (bCNB). Results: High bTMB and bCNB were associated with lack of clinical benefit and significantly shorter progression-free survival (PFS) compared with patients with low bTMB or low bCNB (all P < 0.05). Dominant APOBEC signatures were detected at baseline exclusively in cases with high bTMB (5/13, 38.5%) versus low bTMB (0/37, 0%; P = 0.0006). Alterations in ESR1 were enriched in samples with high bTMB (P = 0.0005). There was a high correlation between bTMB determined by WES and bTMB determined using a 600-gene panel (R = 0.98). During serial monitoring, an increase in bCNB score preceded radiographic progression in 12 of 18 (66.7%) patients. Conclusions: Genomic complexity detected by noninvasive profiling of bTMB and bCNB predicted poor outcomes in patients treated with ET and CDK4/6i and identified early disease progression before imaging. Novel treatment strategies including immunotherapy-based combinations should be investigated in this population.

Patient demographics.

Purpose:Clinical biomarkers to identify patients unlikely to benefit from CDK4/6 inhibition (CDK4/6i) in combination with endocrine therapy (ET) are lacking. We implemented a comprehensive circulating tumor DNA (ctDNA) analysis to identify genomic features for predicting and monitoring treatment resistance.Experimental Design:ctDNA was isolated from 216 plasma samples collected from 51 patients with hormone receptor–positive (HR+)/HER2-negative (HER2−) metastatic breast cancer (MBC) on a phase II trial of palbociclib combined with letrozole or fulvestrant (NCT03007979). Boosted whole-exome sequencing (WES) was performed at baseline and clinical progression to evaluate genomic alterations, mutational signatures, and blood tumor mutational burden (bTMB). Low-pass whole-genome sequencing was performed at baseline and serial timepoints to assess blood copy-number burden (bCNB).Results:High bTMB and bCNB were associated with lack of clinical benefit and significantly shorter progression-free survival (PFS) compared with patients with low bTMB or low bCNB (all P < 0.05). Dominant APOBEC signatures were detected at baseline exclusively in cases with high bTMB (5/13, 38.5%) versus low bTMB (0/37, 0%; P = 0.0006). Alterations in ESR1 were enriched in samples with high bTMB (P = 0.0005). There was a high correlation between bTMB determined by WES and bTMB determined using a 600-gene panel (R = 0.98). During serial monitoring, an increase in bCNB score preceded radiographic progression in 12 of 18 (66.7%) patients.Conclusions:Genomic complexity detected by noninvasive profiling of bTMB and bCNB predicted poor outcomes in patients treated with ET and CDK4/6i and identified early disease progression before imaging. Novel treatment strategies including immunotherapy-based combinations should be investigated in this population.

Gene alterations associated with shorter PFS.

Supplementary Methods

PredicineATLAS gene panel.

DNA sequencing data summary.

Supplementary Figure S1. High bTMB is associated with the presence of specific mutations. Supplementary Figure S2. ROC analysis to determine optimal bTMB cutoff. Supplementary Figure S3. High correlation between bTMB determined from WES and a 152- gene targeted sequencing panel. Supplementary Figure S4. Association between high cfDNA yield and shorter PFS. Supplementary Figure S5. High baseline tumor fraction is associated with shorter PFS. Supplementary Figure S6. bTMB scores are not associated with sites of metastatic spread. Supplementary Figure S7. Specific oncogenic signaling pathways are more frequently altered in patients with high bTMB and bCNB scores. Supplementary Figure S8. Genomic landscape of all patients at baseline. Supplementary Figure S9. bTMB and bCNB at baseline vs. progression. Supplementary Figure S10. Correlation between baseline bCNB and bTMB. Supplementary Figure S11. Comparison of ctDNA dynamics as measured by ctDNA fraction and bCNB during treatment and progression. Supplementary Figure S12. Genome-wide plots of copy number changes across treatment time points. Supplementary Figure S13. Copy number changes detected for RB1 and BRCA2 genes across treatment time points. Supplementary Figure S14. Relationship between ctDNA fraction and bTMB and bCNB Supplementary Figure S15. Relationship between tumor fraction and bCNB.

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 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.

PURPOSE: Patients with triple negative breast cancer (TNBC) who do not achieve pathological complete response (pCR) following neoadjuvant chemotherapy have a high risk of recurrence and death. Molecular characterization may identify patients unlikely to achieve pCR. This neoadjuvant trial was conducted to determine the pCR rate with docetaxel and carboplatin, and to identify molecular alterations and/or immune gene signatures predicting pCR. EXPERIMENTAL DESIGN: Patients with clinical stages II/III TNBC received 6 cycles of docetaxel and carboplatin. The primary objective was to determine if neoadjuvant docetaxel and carboplatin would increase the pCR rate in TNBC compared to historical expectations. We performed whole exome sequencing (WES) and immune profiling on pre-treatment tumor samples to identify alterations that may predict pCR. Thirteen matching on-treatment samples were also analyzed to assess changes in molecular profiles. RESULTS: Fifty-eight of 127 (45.7%) patients achieved pCR. There was a non-significant trend towards higher mutation burden for patients with residual cancer burden (RCB) 0/I versus RCB II/III (median 80, versus 68 variants, p 0.88). TP53 was the most frequently mutated gene, observed in 85.7% of tumors. EGFR, RB1, RAD51AP2, SDK2, L1CAM, KPRP, PCDHA1, CACNA1S, CFAP58, COL22A1, and COL4A5 mutations were observed almost exclusively in pre-treatment samples from patients who achieved pCR. Seven mutations in PCDHA1 were observed in pre-treatment samples from patients who did not achieve pCR. Several immune gene signatures including IDO1, PD-L1, interferon gamma signaling, CTLA4, cytotoxicity, tumor inflammation signature, inflammatory chemokines, cytotoxic cells, lymphoid, PD-L2, exhausted CD8, Tregs, and immunoproteosome were upregulated in pre-treatment samples from patients who achieved pCR. CONCLUSIONS: Neoadjuvant docetaxel and carboplatin resulted in a pCR of 45.7%. WES and immune profiling differentiated patients with and without pCR. TRIAL REGISTRATION: Clinical trial information: NCT02124902, Registered 24 April 2014, & NCT02547987, Registered 10 September 2015.

Background: Cyclin-dependent kinases 4 and 6 inhibitors (CDK4/6i) are now standard of care (SOC) for pts with HR+/HER2- MBC. However, aside from HR positivity, there are no biomarkers for pt selection. Although mutations (muts) in genes such as RB1, FAT1, or FGFR, are associated with CDK4/6i resistance, they are rare events in HR+/HER2- MBC. The goal of this study is to examine the ctDNA mut profile, and the impact of commonly mutated genes (ESR1, TP53 and PIK3CA) on progression free survival (PFS) in pts received pal as SOC in a real world experience. Methods: Chart review was performed for pts with HR+/HER2- MBC who received pal and ctDNA Guardant360® (G360) testing between 01/2015 and 06/2020 at Siteman Cancer Center. The Kaplan-Meier method was used to generate time-to-event curves with calculated mPFS. A stratified log-rank test was used for all comparisons with P-values reported. Hazard ratios (HRs) and associated 95% confidence intervals (CI) were calculated with a stratified Cox proportional-hazards model. Results: Among the 73 pts identified, median age was 61.5 years (range 33.7-83.3). 15 (20.5%) pts received pal as 1st-line, 22 (30.1%) pts as 2nd-line, and 36 (49.3%) pts as 3rd-line and beyond. All 73 pts had muts by G360. Top 3 mutated genes are PIK3CA (n=25, 34.2%), TP53 (n=20, 27.4%) and ESR1 (n=17, 23.3%), followed by GATA3 (n=12, 16.4%) and MYC (n=6, 8.2%). For association analysis with PFS, 17 pts were excluded from further analysis due to having had multiple interim therapies between G360 testing and pal therapy, leaving 56 pts who had G360 immediately prior to pal (n=11), during pal (n=13), at progressive disease (PD) on pal (n=20), or had 1 additional therapy after PD on pal (n=12). Among the 56 pts, the median age on pal was 62.8 years (36.6-78.4). Median duration on pal was 7.7 months (mo). Pal was received as 1st-line in 14 (25.0%), 2nd-line in 17 (30.4%) and 3rd-line plus in 25 (44.6%) pts. Muts in PIK3CA, TP53 and ESR1, were identified in 18 (32.1%), 16 (28.6%), and 13 (23.2%) pts, respectively, with rates similar to the entire 73-pt cohort. Among 13 ESR1 mutant pts, 7 had letrozole and 6 had fulvestrant as pal’s endocrine partner. Concurrent muts in 2 of 3 genes were observed in 11 (19.6%) pts (6 with PIK3CA and TP53, and 5 with ESR1 and TP53). The mPFS was 7.4 and 12.6 mo (p=0.03) in pts with or without PIK3CA mut, 7.9 and 12.8 mo (p=0.85) with or without ESR1 mut, and 16.0 and 13.3 mo (p=0.46), with or without TP53 mut, respectively. mPFS for pts with concurrent muts in 2 of the 3 genes was 7.4 mo, compared to 11.7 mo and 13.3 mo in pts with 1 or no mut in these 3 genes (p=0.46). In multivariate analysis that included age, visceral mets and line of therapy, the HRs for PFS were 10 muts, respectively. Conclusions: In this retrospective real world experience, ctDNA muts in PIK3CA and ESR1 as well as higher number of muts in ctDNA were associated with worse outcome in pts received pal. Although several studies have shown CDK4/6i benefit regardless of PIK3CA or ESR1 mut status, our data indicates that muts in these genes render worse prognosis on CDK4/6i and supports PIK3CA and ESR1 muts as driver events in HR+ MBC. PI3Ki or novel ER targeted agents are combined with CDK4/6i in clinical trials and results are eagerly awaited. In addition, further study for pts with higher ctDNA mut number is warranted. Multivariate Cox Proportional Hazards Regression Model on PFSVariablesPFSHR (95% CI)P-valueESR1 (non-mut vs. mut)0.64 (0.26-1.56)0.32TP53 (non-mut vs. mut)1.70 (0.80-3.60)0.16PIK3CA (non-mut vs. mut)0.42 (0.17-1.02)0.06Age (=65 yo)0.92 (0.45-1.87)0.81No. Lines of pal (1st- vs. 3rd-line)0.61 (0.25-1.48)0.28No. Lines of pal (2nd- vs. 3rd-line)0.46 (0.20-1.04)0.06No. of muts (1-5 vs. >10)0.74 (0.24-2.33)0.61No. of muts (6-10 vs. >10)0.64 (0.20-2.07)0.46Visceral mets (no vs. yes)1.25 (0.52-2.98)0.61 Citation Format: Jing Xi, Nusayba Bagegni, Foluso Ademuyiwa, Ashley Frith, Rama Suresh, Anna Roshal, Caron Rigden, Leonel Hernandez-Aya, Lindsay Peterson, Mateusz Opyrchal, Katherine Clifton, Katherine Weilbaecher, Ron Bose, Cynthia Ma. Circulating tumor DNA (ctDNA) mutation (mut) profile in relation to paboclicib (pal) efficacy in hormone receptor positive (HR+) and HER2 negative (HER2-) metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-18.

Background: With advances in next generation sequencing (NGS) and now approved targeted therapy in breast cancer, genomic testing to identify potentially actionable mutations has become a common practice in patients (pts) with advanced breast cancer using both ctDNA and traditional tissue-based assays. Less is known regarding physician practice patterns in obtaining NGS testing and the practical implications of testing in older adults with breast cancer.Methods: Pts with advanced breast cancer underwent molecular profiling using a plasma-based ctDNA NGS assay (Guardant360® or Tempus®) between 5/2015 and 5/2020 at Siteman Cancer Center. Pts with advanced breast cancer who underwent genomic profiling using a tissue-based NGS assay (Tempus®) between 12/2017 and 5/2020 at this institution were also included. Clinicopathological histories were obtained from the medical record. Correlations were examined using a Fisher’s exact test.Results: During 5/15-5/20, 244 pts underwent ctDNA testing and 147 pts had a tissue-based NGS assay performed. There was no significant difference between the number of pts ≥ 65 years-old who underwent ctDNA testing (n=78, 32.0%) and tissue testing (n=37, 25.2%). There was no statistically significant difference between date of metastatic diagnosis and date of NGS testing between the older and younger cohorts. In pts who underwent tissue-based NGS testing, there was no significant difference between site of tissue tested (distant recurrence vs local) in the older and younger cohorts. The most common clinical managements following both ctDNA and tissue-based testing are presented in Table 1. Out of the 391 pts who underwent testing, 27 pts had both ctDNA and tissue-based NGS performed. Pts ≥ 65 were less likely to have both assays performed (n=3, 11.1%; p Conclusion: Older adults, who are typically less likely to be included in clinical trials, may still benefit from NGS to reveal potentially targetable mutations. It is reassuring in our cohort that older adults had ctDNA and tissue-based NGS performed at similar rates as part of standard of care treatment. The clinical management following NGS testing was also not significantly different in the older adult cohort. Older adults were less likely to have both tissue and ctDNA testing performed however, given the high concordance rates between tests, this may be less clinically relevant. Table 1clinical management following NGS testing≥65 years-old Citation Format: Katherine K Clifton, Jingqin Luo, Yu Tao, Jennifer Saam, Thereasa Rich, Timothy Rearden, Anna Roshal, Ashley Frith, Caron Rigden, Foluso Ademuyiwa, Katherine Weilbaecher, Leonel Hernandez Aya, Lindsay Peterson, Nusayba Bagegni, Rama Suresh, Ron Bose, Tanya Wildes, Mateusz Opyrchal, Cynthia Ma. Next generation sequencing (NGS) in older adults with breast cancer using tissue-based and circulating tumor DNA (ctDNA) assays [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-09.

1089 Background: Although the noninvasive nature of ctDNA testing is attractive in an older adult population, less is known regarding the mutation profiles of ctDNA in the older adult breast cancer population as this population is often excluded from studies. Previous tissue testing has shown differences in mutation profiles between older and younger adults with breast cancer. The objective of this study is to assess differences in mutation profiles in the older and younger adult breast cancer population using a ctDNA assay. Methods: Patients (pts) with advanced breast cancer underwent molecular profiling using a plasma-based ctDNA NGS assay (Guardant360) between 5/2015-10/2019 at Siteman Cancer Center. Clinicopathological histories were obtained from the medical record. The results of a multicenter database of pts with advanced breast cancer who had undergone molecular profiling using Guardant360 were obtained. Associations between mutations and age were measured using a Fisher’s exact test. Results: In the single institution cohort, of the 214 patients who underwent testing, 148 (69.16%) were < 65 and 66 (30.84%) ≥ 65 years-old. The most frequently mutated genes in age < 65 pts were TP53 (48.65%), PIK3CA (35.81%), and ESR1 (30.41%) while the most frequently mutated genes in age≥65 pts were PIK3CA (56.06%), TP53 (51.52%), ESR1 (25.76%), and ATM (21.21%). ATM, BRAF and PIK3CA mutations were found more frequently in age≥ 65 pts with ER+ HER2- breast tumors (p < 0.01). MYC and ESR1 mutations were not significantly associated with age, overall or within subtype. Overall ctDNA resulted in change in management in 19.8% pts (40/202). In the larger multicenter cohort, of the 8803 pts who underwent testing, 5367 (61.0%) were < 65 and 3417 (38.8%) ≥ 65 years-old. ATM, ESR1 and PIK3CA mutations were more common in age≥65 pts (p < 0.0001) and MYC mutations were less common in age≥65 pts (p < 0.0001). Conclusions: This study found that ctDNA is a feasible, attractive alternative to traditional biopsies and may identify actionable mutations in older adults with breast cancer. When controlling for subtype, results from a single institution were similar to the larger multicenter cohort showing ATM and PIK3CA were more common in the older adult population. This data suggests there may be additional molecular differences between breast cancer in older compared to younger adults that warrants further investigation.

Introduction: Palbo is approved in combination with an aromatase inhibitor or fulvestrant (FUL) for the treatment of HR+ HER2- MBC. The incidence of grade (G) 3/4 neutropenia (ANC) approaching 66% has been observed in phase 3 trials of palbo. We hypothesize that an alternative schedule of palbo, 5 days on/2 days off every 7 days, reduces the severity of neutropenia, therefore allowing continued weekly dosing and less dose reduction and discontinuation. Methods: A single arm phase II study (Alt Dose Palbo) was conducted in patients (pts) with HR+ HER2- MBC who had ≤1 prior systemic therapy in the metastatic setting (NCT03007979). Pts were treated with palbo 125 mg daily on a 5 days on/2 days off every 7-day schedule, along with letrozole (LET) or FUL per treating physician. Goserelin was administered if premenopausal. The primary objective was to determine the rate of G3/4 ANC within the first 29 days of treatment. Secondary objectives included determining the rate of G3/4 ANC during all cycles, rate of palbo dose reduction/interruption/discontinuation, adverse event (AE) profile per CTCAE v5, progression free survival (PFS), objective response rate (ORR) and clinical benefit rate (CBR) by RECIST 1.1. The sample size of 47 was calculated to provide 90% power based on one-sample binomial exact test at a 5% alpha level to test the one-sided null hypothesis of G3/4 ANC rate >62% versus the alternative of Table 1C1 D1-29 AEG1G2G3TotalWBC decreased26%50%17%93%ANC decreased10%43%21%74%Anemia38%12%0%50%Fatigue29%0%0%29%Platelets decreased14%0%2%17%Nausea12%2%0%14%All cycle AEG1G2G3TotalWBC decreased19%50%26%95%ANC decreased5%40%36%81%Anemia50%21%2%74%Fatigue36%5%0%40%Nausea26%2%0%29%Platelets decreased24%0%2%26%Alopecia21%0%0%21%Mucositis19%0%0%19%Hot flashes17%2%0%19%Constipation14%0%0%14%Arthralgia12%2%0%14%AST elevated10%0%2%12%Anorexia12%0%0%12%ALT elevated7%0%5%12% Citation Format: Jairam Krishnamurthy, Jingqin Luo, Foluso Ademuyiwa, Rama Suresh, Caron Rigden, Timothy Reardon, Katherine Weilbaecher, Ashley Frith, Anna Roshal, Pavan Tandra, Mathew Cherian, Tracy Summa, Shana Thomas, Leonel Hernandez-Aya, Lindsay Peterson, Cynthia Ma. A phase II trial assessing the safety of an alternative dosing schedule of palbociclib (palbo) in hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC): Alt Dose Palbo [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-13.

Half of hormone receptor-positive (HR+) breast cancer patients will develop joint pain, termed aromatase inhibitor-induced arthralgia (AIA), while taking aromatase inhibitor therapy. Though there is no universally accepted effective treatment for AIA, there has been some evidence to support high-dose vitamin D as a treatment. We randomized post-menopausal women who were beginning adjuvant AI therapy to receive standard-dose vitamin D3 (800 IU daily for 52 weeks), or high-dose vitamin D3 (50,000 IU weekly for 12 weeks, followed by 2000 IU daily for 40 weeks). The primary end point was development of AIA. The trial was designed to enroll 184 patients. This futility analysis was performed after 93 patients were enrolled. The high-dose vitamin D regimen was effective in raising serum vitamin D levels, but there was no significant difference in development of AIA between the two arms. In the high-dose arm, 25 patients (54%) developed AIA, compared to 27 patients (57%) in the standard-dose arm. The planned futility analysis was positive; thus, the study was terminated. Neither baseline vitamin D nor 12-week vitamin D level was predictive of AIA development. Although vitamin D levels were increased in the high-dose arm, there was no significant signal for benefit of high-dose vitamin D supplementation for AIA prevention in this unblinded trial. This study, along with several others, implies that vitamin D likely does not play a significant role in AIA for the majority of patients.

Palbociclib 3-weeks-on/1-week-off, combined with hormonal therapy, is approved for hormone receptor positive (HR+)/HER2-negative (HER2−) advanced/metastatic breast cancer (MBC). Neutropenia is the most frequent adverse event (AE). We aim to determine whether an alternative 5-days-on/2-days-off weekly schedule reduces grade 3 and above neutropenia (G3 + ANC) incidence. In this single-arm phase II trial, patients with HR+/HER2− MBC received palbociclib 125 mg, 5-days-on/2-days-off, plus letrozole or fulvestrant per physician, on a 28-day cycle (C), as their first- or second-line treatment. The primary endpoint was G3 + ANC in the first 29 days (C1). Secondary endpoints included AEs, efficacy, and serum thymidine kinase 1 (sTK1) activity. At data-cutoff, fifty-four patients received a median of 13 cycles (range 2.6–43.5). The rate of G3 + ANC was 21.3% (95% CI: 11.2–36.1%) without G4 in C1, and 40.7% (95% CI: 27.9–54.9%), including 38.9% G3 and 1.8% G4, in all cycles. The clinical benefit rate was 80.4% (95% CI: 66.5–89.7%). The median progression-free survival (mPFS) (95% CI) was 19.75 (12.11–34.89), 33.5 (17.25–not reached [NR]), and 11.96 (10.43–NR) months, in the overall, endocrine sensitive or resistant population, respectively. High sTK1 at baseline, C1 day 15 (C1D15), and C2D1 were independently prognostic for shorter PFS (p = 9.91 × 10−4, 0.001, 0.007, respectively). sTK1 decreased on C1D15 (p = 4.03 × 10−7), indicating target inhibition. Rise in sTK1 predicted progression, with the median lead time of 59.5 (inter-quartile range: −206.25–0) days. Palbociclib, 5-days-on/2-days-off weekly, met its primary endpoint with reduced G3 + ANC, without compromising efficacy. sTK1 is prognostic and shows promise in monitoring the palbociclib response. ClinicalTrials.gov#: NCT3007979.

IMPORTANCE: Standard first-line regimens for patients with metastatic gastroesophageal adenocarcinomas have an approximate 40% objective response rate (ORR). The combination of leucovorin, fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX) has been efficacious as first-line therapy for other gastrointestinal cancers, such as pancreatic and colon cancers. OBJECTIVE: To evaluate the clinical activity and safety of FOLFIRINOX as first-line treatment for patients with advanced gastroesophageal adenocarcinoma. DESIGN, SETTING, AND PARTICIPANTS: This is an open-label, single-arm phase 2 study of first-line FOLFIRINOX in patients with advanced gastroesophageal adenocarcinoma. Estimated sample size included 41 patients with ERBB2-negative disease with 90% power to detect an ORR of 60% or greater with α of .10. No enrollment goal was planned for ERBB2-positive patients, but they were allowed to receive trastuzumab in combination with FOLFIRINOX. INTERVENTIONS: Starting doses were fluorouracil, 400 mg/m(2) bolus, followed by 2400 mg/m(2) over 46 hours; leucovorin, 400 mg/m(2); irinotecan, 180 mg/m(2); and oxaliplatin, 85 mg/m(2). Trastuzumab was administered as a 6 mg/kg loading dose, followed by 4 mg/kg every 14 days in patients with ERBB2-positive disease. MAIN OUTCOMES AND MEASURES: The primary end point was ORR by the Response Evaluation Criteria in Solid Tumors, version 1.1. Secondary end points included safety profile, progression-free survival (PFS), overall survival (OS), and duration of response. RESULTS: From November 2013 to May 2018, 67 patients were enrolled (median [range] age, 59.0 [34-78] years; including 56 [84%] men), and 26 of 67 (39%) had ERBB2-positive disease. Median follow-up was 17.4 months. The ORR was 61%(95% CI, 44.5%-75.8%) (25 of 41) in the ERBB2-negative group and 85% (95% CI, 65.1%-95.6%) (22 of 26) in the ERBB2-positive group, including 1 patient with complete response. For ERBB2-negative patients, median PFS was 8.4 months and median OS was 15.5 months; for ERBB2-positive patients, median PFS was 13.8 months and median OS was 19.6 months. Fifty-six patients (84%) had dose modifications or treatment delays. The most common toxic effects were neutropenia (91%, n = 61), diarrhea (63%, n = 42), peripheral sensory neuropathy (61%, n = 41), and nausea (48%, n = 32), with no unexpected toxic effects. CONCLUSIONS AND RELEVANCE: The FOLFIRINOX regimen with or without trastuzumab was associated with improved ORR and PFS in patients with advanced gastroesophageal adenocarcinoma in the first-line setting. This regimen may be a reasonable therapeutic option for patients with preserved performance status. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01928290

Half of hormone receptor-positive (HR+) breast cancer patients will develop joint pain, termed aromatase inhibitor-induced arthralgia (AIA), while taking aromatase inhibitor therapy. Though there is no universally accepted effective treatment for AIA, there has been some evidence to support high-dose vitamin D as a treatment.We randomized post-menopausal women who were beginning adjuvant AI therapy to receive standard-dose vitamin D3 (800 IU daily for 52 weeks), or high-dose vitamin D3 (50,000 IU weekly for 12 weeks, followed by 2000 IU daily for 40 weeks). The primary end point was development of AIA. The trial was designed to enroll 184 patients. This futility analysis was performed after 93 patients were enrolled.The high-dose vitamin D regimen was effective in raising serum vitamin D levels, but there was no significant difference in development of AIA between the two arms. In the high-dose arm, 25 patients (54%) developed AIA, compared to 27 patients (57%) in the standard-dose arm. The planned futility analysis was positive; thus, the study was terminated. Neither baseline vitamin D nor 12-week vitamin D level was predictive of AIA development.Although vitamin D levels were increased in the high-dose arm, there was no significant signal for benefit of high-dose vitamin D supplementation for AIA prevention in this unblinded trial. This study, along with several others, implies that vitamin D likely does not play a significant role in AIA for the majority of patients.

Objectives Macropinocytosis promotes internalization of albumin into cells to serve as a nutrient supply and is constitutively driven by signaling pathways frequently hyperactivated in head and neck squamous-cell carcinoma (HNSCC). In this way, drugs bound to albumin may selectively target HNSCC. nab-paclitaxel is a nanoparticle albumin-bound formulation of paclitaxel that improves drug delivery into tumor compared to paclitaxel. The primary aim of this single-arm, multicenter, phase 2 trial was to determine if nab-paclitaxel, cetuximab, and carboplatin (CACTUX regimen) would result in longer progression-free survival (PFS) than the historical regimen (EXTREME: 5-fluorouracil, cetuximab, and a platinum). Materials and methods Patients with untreated recurrent or metastatic HNSCC received six, three-week cycles of nab-paclitaxel, cetuximab, and carboplatin, followed by maintenance nab-paclitaxel and cetuximab until progression. We hypothesized the median PFS with CACTUX would be 35% longer than with EXTREME (corresponding to 7.6 vs 5.6 months; power 0.80, α = 0.05, one-sided test, n = 70). Secondary outcomes included objective response rate (ORR) and overall survival (OS). Results Seventy-four patients enrolled into the trial; seventy were evaluable. The median PFS was 6.1 months (95% CI, 4.1–7.4). The ORR was 60%. Median follow-up was 18 months (IQR: 4.7–23). The median OS was 17.8 months (95% CI, 8.5–21.7) for all patients, and 19.8 months (95% CI, 10.9–22.0) for human papillomavirus (HPV)-related oropharynx SCC and 14.0 months (95% CI, 4.6–23.3) for HPV-unrelated HNSCC. Conclusion Among patients with recurrent or metastatic HNSCC, CACTUX did not result in a longer PFS than historical EXTREME. However, CACTUX did result in a more favorable ORR and OS.

Background: The approved 3 weeks (wks) on/1 wk off schedule of PAL results in grade (G)3+ neutropenia (ANC) up to 66%. We hypothesized that an alternative schedule (Alt Dose Pal), 5 on/2 off every 7 days, reduces the rate of G3+ ANC, allowing continued weekly dosing. In addition, based on our previous study supporting sTK1, an E2F-dependent enzyme critical for DNA synthesis, as a pharmacodynamic indicator of CDK4/6 inhibition, we hypothesized that Alt Dose Pal inhibits sTK1 and sTK1 dynamics predicts PAL response. Methods: A single arm phase II trial was conducted in HR+ HER2- MBC, ≤1 prior endocrine therapy (ET) for MBC (NCT03007979). Pts received PAL 125 mg daily, 5 on/2 off weekly, plus letrozole (LET) or fulvestrant (FUL) per physician choice, on a 28-day cycle (C). Goserelin was added if premenopausal. Complete blood count and chemistry panel were done at baseline (BL), C1&2D15, and D1 of C2+. The primary objective was to determine the rate of G3+ ANC in C1 D1-D29. Secondary objectives were to assess the rate of all cycle G3+ ANC, PAL dose reduction/discontinuation, adverse events (AE) per CTCAE v5, progression free survival (PFS), objective response rate (ORR: CR+PR (complete and partial responses)) and clinical benefit rate (CBR: no progression (PD) in 24 wks) by RECIST 1.1. The sample size of 47 provides 90% power, 1-sample binomial exact test, 5% alpha, to test the 1-sided null hypothesis of G3+ ANC rate >62% vs the alternative of 10% or G3+. The ORR was 50% (2 CR, 13 PR, 95% CI: 33.15%-66.85%) in 30 pts with measurable disease (n=29) or non-measurable but CR (n=1). The CBR was 81.63% (95% CI: 67.5%-90.76%) in 40 (2CR, 13 PR, 25 SD ≥24 wks) of 49 evaluable pts. The median PFS (mPFS) was 24.3 mo (95% CI 15~not reached (NR)) overall. The mPFS was 33.5 mo (95% CI 17.3~NR) and 12 mo (95% CI: 10.4~NR), in ET sensitive and resistant population, respectively. sTK1 was significantly reduced, 80% down to undetectable, as early as C1D15 (p=7.77E-07). BL sTK1 levels correlated with PD vs non-PD (p=0.003) as best response and negatively correlated with PFS (p=0.002). sTK1 rose significantly at PD (p=0.0003). A median lead time of 80.6 (IQ range 6.8-189.2) days was observed for rising TK before RECIST PD. Conclusion: The Alt Dose Pal trial met its primary endpoint with reduced G3+ ANC. The efficacy data is comparable to prior reports. sTK1 shows promise for PAL response prediction and monitoring. Table 1 AEAEG1G2G3G4TotalC1 D1-29 (n=47)Leukopenia13 (27.6%)22 (46.8%)7 (14.9%)042 (89%)Neutropenia6 (12.8%)18 (38.3%)10 (21.3%)034 (72%)Anemia18 (38.3%)5 (10.6%)0023 (49%)Lymphopenia6 (12.8%)8 (17%)2 (4.3%)016 (34%)Fatigue13 (27.7%)00013 (28%)Hot flashes7 (14.9%)0007 (15%)Nausea6 (12.8%)1 (2.1%)007 (15%)Thrombocytopenia5 (10.6%)1 (2.1%)006 (13%)Alopecia5 (10.6%)0005 (11%)Dizziness1 (2.1%)01 (2.1%)02 (4%)All cycles (n=54)Leukopenia10 (18.5%)22 (40.7%)21 (38.9%)053 (98%)Neutropenia4 (7.4%)21 (38.9%)21 (38.9%)1 (1.9%)47 (87%)Anemia29 (53.7%)12 (22.2%)3 (5.6%)044 (82%)Lymphopenia8 (14.8%)16 (29.6%)10 (18.5%)034 (63%)Fatigue19 (35.2%)4 (7.4%)0023 (43%)Nausea19 (35.2%)3 (5.6%)0022 (41%)Alopecia18 (33.3%)00018 (33%)Thrombocytopenia15 (27.8%)01 (1.9%) *016 (30%)ALT elevated3 (5.6%)02 (3.7%)05 (9%)AST elevated4 (7.4%)01 (1.9%)05 (9%)*pt died from subdural hematoma (G5) Citation Format: Jairam Krishnamurthy, Jingqin Luo, Rama Suresh, Foluso Ademuyiwa, Caron Rigden, Timothy Reardon, Katherine Clifton, Katherine Weilbaecher, Ashley Frith, Anna Roshal, Pavan Tandra, Mathew Cherian, Tracy Summa, Shana Thomas, Leonel Hernandez-Aya, Mattias Bergqvist, Lindsay Peterson, Cynthia X Ma. Safety and efficacy of an alternative schedule of palbociclib (PAL) in hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) and the utility of serum thymidine kinase 1 (sTK1) activity in predicting PAL response [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS12-02.

4532 Background: Standard first-line regimens for patients with metastatic gastroesophageal adenocarcinomas have moderate clinical benefit with objective response rates (ORR) of approximately 40-50%. FOLFIRINOX has been shown to be an effective and well-tolerated first line therapy in other GI cancers. In this open-label, single-arm phase II study of patients with advanced gastroesophageal adenocarcinomas, we sought to evaluate the safety and clinical activity of FOLFIRINOX. Methods: The primary endpoint was ORR, and secondary endpoints included safety profile, progression free survival (PFS), overall survival (OS), time to progression (TTP), clinical benefit rate (CBR), and duration of response. Estimated sample size included 41 patients with HER2 negative disease with 90% power to detect an ORR≥60% with alpha of 0.10. No enrollment goal was planned for HER2 positive patients, but they were allowed participation to receive study treatment in combination with trastuzumab. Treatment consisted of 400mg/m2 5-FU bolus, 400 mg/m2 leucovorin, 2400 mg/m2 5-FU infusion over 46 hours, 180 mg/m2 irinotecan, and 85 mg/m2 oxaliplatin. Trastuzumab was administered intravenously as a 6 mg/kg loading dose then given 4 mg/kg every 14 days for HER2 positive patients. This trial is registered with ClinicalTrials.gov, NCT01928290. Results: From November 2013 to May 2019, 67 patients were enrolled, of which 26 (39%) had HER2 positive disease. Median follow-up was 16.1 months. ORR was 61% (25/41) for HER2 negative and 85% (22/26) for HER2 positive groups. Overall, one patient (2%) had a complete response, 36 patients (69%) had partial responses, and 13 patients (19%) had stable disease for >6 months; therefore, CBR was 96%. Median PFS was 11.9 months, median OS was 17.4 months. 41 patients (83.7%) had dose modification or treatment delay with the most common toxicities being neutropenia, diarrhea, peripheral sensory neuropathy, and nausea with no unexpected toxicities. Conclusions: FOLFIRINOX is a highly effective three-drug regimen for first-line treatment of advanced gastroesophageal cancer with expected, tolerable toxicities. Clinical trial information: NCT01928290 .

Background: Pemetrexed is an antifolate chemotherapeutic agent approved for use in non-small cell lung cancer (NSCLC). The mammalian target of rapamycin (mTOR) pathway is implicated in lung cancer development and inhibited by temsirolimus. Methods: We performed a phase I study evaluating the combination of pemetrexed and temsirolimus in advanced non-squamous NSCLC. Results: Eight patients were enrolled in this study. The dose-limiting toxicities included grade 4 thrombocytopenia, grade 3 leukopenia and grade 3 neutropenia. The maximum tolerated dose was determined to be pemetrexed 375 mg/m2 intravenously on day 1 and temsirolimus 25 mg intravenously on days 1, 8 and 15. No objective responses were noted and 3 patients had stable disease as the best response. Conclusion: The combination of pemetrexed and temsirolimus is feasible and well tolerated. This combination may be further evaluated in patients with mTOR pathway activation, particularly in those with TSC1 or STK11 mutations.

One of the principle unresolved questions in adjuvant endocrine therapy for breast cancer is whether it is more beneficial for women to receive aromatase inhibitor (AI) monotherapy or start with tamoxifen and then switch to AI therapy. This review will compare the current available efficacy, safety, and cost-effectiveness data for AIs in the initial adjuvant and switch adjuvant settings.A search of the Medline database from 1976 through 2006 was performed for the following terms: breast cancer, adjuvant, aromatase inhibitors, anastrozole, letrozole, exemestane, tamoxifen, sequential, switching. A search for relevant abstracts from the EBCC, ECCO, ASCO, and SABCS conferences was also performed.In the upfront adjuvant setting, anastrozole and letrozole have both demonstrated a significant disease-free survival (DFS) benefit over tamoxifen. Upfront therapy with a nonsteroidal AI appears to be most critical for patients at risk of an early relapse, illustrated by the finding that upfront letrozole provided a significant early DFS advantage over tamoxifen only in patients with node-positive disease (hazard ratio = 0.71, p0.001). With respect to safety, both strategies have similar adverse event profiles. From an economic perspective, AIs, whether used upfront or sequentially, are considered cost-effective compared with tamoxifen due to the cost savings associated with a reduction in the breast cancer event rate. From the efficacy standpoint, modeling studies have produced inconsistent results and do not produce definitive data.Differences in patient populations, definitions of end points, and prior tamoxifen usage between the trials discussed necessitates a careful interpretation but may provide insights in the treatment decision-making process. The BIG 1-98 trial was designed to compare letrozole monotherapy versus a letrozole-to-tamoxifen or reverse-sequence approach and should provide insights to the question of optimal therapy. Until results are available, for higher-risk patients (i.e., those with positive lymph nodes), initiation of treatment with a non-steroidal AI may be beneficial to avoid tamoxifen-associated early relapses that occur in the first 2 years after diagnosis.

89 Background: Standard first-line regimens for patients with metastatic gastroesophageal adenocarcinomas have an approximate 40% objective response rate (ORR). FOLFIRINOX has been used in first line therapy in other GI cancers (i.e pancreatic and CRC) with impressive efficacy signals. Methods: This is a phase II study of first line combination chemotherapy with fluorouracil (5-FU), irinotecan, and oxaliplatin in patients with advanced gastric, esophageal, or gastroesophageal junction adenocarcinoma (NCT01928290). Starting doses were 5-FU 400mg/m2 bolus followed by 2400 mg/m2 over 46 hours with leucovorin 400 mg/m2, irinotecan 180 mg/m2, and oxaliplatin 85 mg/m2. Trastuzumab was administered as 6 mg/kg loading dose then 4 mg/kg every 14 days if patients had HER2+ cancer. The primary endpoint was overall response rate (ORR). Secondary endpoints included progression free survival (PFS), overall survival (OS), time to progression (TTP), clinical benefit rate (CBR), and duration of response. Enrollment of 41 patients with HER2- disease was planned to reach one-sided = 0.10 and power 0.90 with goal of detecting true ORR60%. No enrollment goal was planned for HER2+. Results: From Nov 2013 to July 2017, 58 patients were enrolled, 25 out of 58 (43%) had HER2+ disease. Forty-nine patients were evaluable for response as they completed at least one restaging scan. ORR was 78% (38/49) in all patients, 67% (18/27) in HER2-, 91% (20/22) in HER2+. One patient (2%) had complete response, 37 (76%) had partial response, 7 (14%) had stable disease > 6 months; therefore, CBR was 92%. Median PFS is 11.9 months, median OS is 17.4 months and median follow up time 16.1 months. 41 (83.7%) had dose modification or delay during treatment. There were no unexpected toxicities. Conclusions: FOLFIRINOX with or without trastuzumab showed remarkable ORR and PFS in patients with advanced gastroesophageal adenocarcinoma in the first-line setting. This regimen may be a reasonable therapeutic option for patients with preserved performance status. Further investigation in larger population is warranted. Clinical trial information: NCT01928290.

Preoperative radiation therapy with 5-fluorouracil chemotherapy is a standard of care for cT3-4 rectal cancer. Studies incorporating additional cytotoxic agents demonstrate increased morbidity with little benefit. We evaluate a template that: (1) includes the benefits of preoperative radiation therapy on local response/control; (2) provides preoperative multidrug chemotherapy; and (3) avoids the morbidity of concurrent radiation therapy and multidrug chemotherapy.Patients with cT3-4, any N, any M rectal cancer were eligible. Patients were confirmed to be candidates for pelvic surgery, provided response was sufficient. Preoperative treatment was 5 fractions radiation therapy (25 Gy to involved mesorectum, 20 Gy to elective nodes), followed by 4 cycles of FOLFOX [5-fluorouracil, oxaliplatin, leucovorin]. Extirpative surgery was performed 4 to 9 weeks after preoperative chemotherapy. Postoperative chemotherapy was at the discretion of the medical oncologist. The principal objectives were to achieve T stage downstaging (ypTcT) and preoperative grade 3+ gastrointestinal morbidity equal to or better than that of historical controls.76 evaluable cases included 7 cT4 and 69 cT3; 59 (78%) cN+, and 7 cM1. Grade 3 preoperative GI morbidity occurred in 7 cases (9%) (no grade 4 or 5). Sphincter-preserving surgery was performed on 57 (75%) patients. At surgery, 53 patients (70%) had ypT0-2 residual disease, including 21 (28%) ypT0 and 19 (25%) ypT0N0 (complete response); 24 (32%) were ypN+. At 30 months, local control for all evaluable cases and freedom from disease for M0 evaluable cases were, respectively, 95% (95% confidence interval [CI]: 89%-100%) and 87% (95% CI: 76%-98%). Cases were subanalyzed by whether disease met requirements for the recently activated PROSPECT trial for intermediate-risk rectal cancer. Thirty-eight patients met PROSPECT eligibility and achieved 16 ypT0 (42%), 15 ypT0N0 (39%), and 33 ypT0-2 (87%).This regimen achieved response and morbidity rates that compare favorably with those of conventionally fractionated radiation therapy and concurrent chemotherapy.

553 Background: Preoperative radiotherapy (RT) with 5FU chemotherapy (CT) is a standard of care for cT3-4 rectal cancer. Studies incorporating additional cytotoxic agents have resulted in increased morbidity with little benefit. We evaluate a template that seeks to (1) include the known benefits of preoperative RT on local response/control, (2) provide for preoperative multi-drug CT, (3) avoid the morbidity of concurrent RT and multi-drug CT. Methods: Patients with cT3-4, any N, any M rectal cancer were eligible. Patients were confirmed to be candidates for surgery, provided the response was sufficient. Preoperative treatment was 5 fractions RT (25 Gy to involved mesorectum, 20 Gy to elective nodes), followed by 4 cycles of mFOLFOX6. Postoperative CT was at the discretion of the medical oncologist. The principal objectives are to demonstrate that this regimen can achieve T stage down staging (ypT < cT) and acute grade 3+ gastrointestinal (GI) morbidity equal to or better than historical controls. Results: Accrual opened late 2009, with 60 patients enrolled through 8/2011. Forty-six have had sufficient time to proceed to surgery with 4 having grade 3 preoperative GI morbidity. Two cases are inevaluable for response: one withdrew consent prior to CT and one received no surgery due to progression of cM1 disease (with local response). The 44 evaluable cases included 4 cT4 and 40 cT3; 32 (73%) cN+, 4 cM1. At surgery 33 (75%) had ypT0-2 residual disease including 13 (30%) ypT0, 14 (32%) were ypN+. Cases were sub-analyzed by whether disease was too advanced for the upcoming ACOSOG preoperative FOLFOX vs. 5FU-RT trial. By ACOSOG eligibility, response rates were (eligible first, ineligible second) ypT0: 10/22 (45%) vs. 3/22 (14%) (p = 0.05), ypT0-2: 19/22 (86%) vs. 14/22 (64%) (p = NS). Conclusions: This regimen achieves high response rates with acceptable morbidity. The response for ACOSOG eligible cases meets pre-determined stopping criteria for proceeding to a randomized trial. Our successor study will randomize to this regimen vs. FOLFOX alone for ACOSOG eligible cases, while initially continuing as a single arm trial for ACOSOG ineligible cases.