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Background: Cyclin-dependent kinase 4/6 inhibitors (CKD4/6i) have demonstrated clinical utility extending progression-free survival (PFS) and overall survival (OS) for advanced hormone receptor positive and HER2 negative (HR+/HER2-) breast cancer patients. The efficacy in early-stage breast cancer (eBC) is unclear, with conflicting results from adjuvant CDK4/6i trials on invasive disease-free survival. Thus, there is a critical need to identify biomarkers of response (BoR) to determine which, if any, eBC patients could benefit from this treatment. This BoR could also stratify advanced BC patients for likelihood to respond to CDK4/6i. Metabolism is influenced by both genome and environment, and changes in the metabolome can be correlated with drug responsiveness. Thus, metabolite BoRs may serve to identify eBC patients for which CDK4/6i would offer a therapeutic benefit.Methods: Plasma and serum samples from 50 early-stage ER+/HER2- breast cancer patients, treated with neoadjuvant CDK4/6 inhibitor palbociclib (palbo) and aromatase inhibitor (AI) anastrozole on NeoPalAna trial (ClinicalTrials.gov identifier NCT01723774), were collected from treatment-naïve patients (BL) and 3 consecutive time points: anastrozole,1 mg daily for 4 weeks (C1D1), anastrozole plus palbo,125 mg daily, for 15 days (C1D15), and for 4-5 months before surgery (SURG). Metabolites were extracted from all samples via methanol and chloroform precipitation and quantified using an unbiased, non-destructive, nuclear magnetic resonance (NMR)-based profiling platform (Olaris®, Inc., Waltham, MA). Statistical analysis and machine learning was used to identify differential metabolites and generate predictive models. A separate validation set of samples was collected from a subset of patients (N=6) who received an additional cycle of palbo treatment prior to surgery to assess model accuracy. Results: Non-parametric differential expression analysis of BL/C1D1, BL/C1D15, and C1D1/C1D15 identified 53 ,97, and 90 differential NMR resonances in plasma (p2.7%). Analysis of the responder (R) and non-responder (NR) groups identified that 13 plasma and 14 serum resonances (21 unique resonances and 6 overlapping) were differentially expressed (p Citation Format: Chen Dong, Shana Thomas, Chandrashekhar Honrao, Leonardo O. Rodrigues, Nathalie Tessier, Bo Zhang, Souzan Sanati, Kiran Vij, Brenda J. Ernst, Karen S. Anderson, Mateusz Opyrchal, Foluso Ademuyiwa, Lindsay L. Peterson, Matthew P. Goetz, Donald Northfelt, Elizabeth O'Day, Cynthia Ma. Identifying a metabolite signature that correlates with tumor proliferation in early-stage breast cancer patients treated with CDK4/6 inhibitors from matched plasma and serum samples [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-20.

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: Cyclin-dependent kinase (CDK) 4/6 drives cell proliferation in estrogen receptor–positive (ER+) breast cancer. This single-arm phase II neoadjuvant trial (NeoPalAna) assessed the antiproliferative activity of the CDK4/6 inhibitor palbociclib in primary breast cancer as a prelude to adjuvant studies.Experimental Design: Eligible patients with clinical stage II/III ER+/HER2− breast cancer received anastrozole 1 mg daily for 4 weeks (cycle 0; with goserelin if premenopausal), followed by adding palbociclib (125 mg daily on days 1–21) on cycle 1 day 1 (C1D1) for four 28-day cycles unless C1D15 Ki67 > 10%, in which case patients went off study due to inadequate response. Anastrozole was continued until surgery, which occurred 3 to 5 weeks after palbociclib exposure. Later patients received additional 10 to 12 days of palbociclib (Cycle 5) immediately before surgery. Serial biopsies at baseline, C1D1, C1D15, and surgery were analyzed for Ki67, gene expression, and mutation profiles. The primary endpoint was complete cell cycle arrest (CCCA: central Ki67 ≤ 2.7%).Results: Fifty patients enrolled. The CCCA rate was significantly higher after adding palbociclib to anastrozole (C1D15 87% vs. C1D1 26%, P < 0.001). Palbociclib enhanced cell-cycle control over anastrozole monotherapy regardless of luminal subtype (A vs. B) and PIK3CA status with activity observed across a broad range of clinicopathologic and mutation profiles. Ki67 recovery at surgery following palbociclib washout was suppressed by cycle 5 palbociclib. Resistance was associated with nonluminal subtypes and persistent E2F-target gene expression.Conclusions: Palbociclib is an active antiproliferative agent for early-stage breast cancer resistant to anastrozole; however, prolonged administration may be necessary to maintain its effect. Clin Cancer Res; 23(15); 4055–65. ©2017 AACR.

Supplementary Table S1 describes the AE profile, Supplementary S2 shows the results of clinical and radiologic responses.

Supplementary Figure Legends S1-S5

Supplementary Table S3 provides the result of the 83-gene panel next generation sequencing

Supplementary Fig. S2. Mutations in selected genes observed in this trial in contrast to that in COSMIC data

Purpose: Hyperactivation of AKT is common and associated with endocrine resistance in estrogen receptor–positive (ER+) breast cancer. The allosteric pan-AKT inhibitor MK-2206 induced apoptosis in PIK3CA-mutant ER+ breast cancer under estrogen-deprived condition in preclinical studies. This neoadjuvant phase II trial was therefore conducted to test the hypothesis that adding MK-2206 to anastrozole induces pathologic complete response (pCR) in PIK3CA mutant ER+ breast cancer.Experimental Design: Potential eligible patients with clinical stage II/III ER+/HER2− breast cancer were preregistered and received anastrozole (goserelin if premenopausal) for 28 days in cycle 0 pending tumor PIK3CA sequencing. Patients positive for PIK3CA mutation in the tumor were eligible to start MK-2206 (150 mg orally weekly, with prophylactic prednisone) on cycle 1 day 2 (C1D2) and to receive a maximum of four 28-day cycles of combination therapy before surgery. Serial biopsies were collected at preregistration, C1D1 and C1D17.Results: Fifty-one patients preregistered and 16 of 22 with PIK3CA-mutant tumors received study drug. Three patients went off study due to C1D17 Ki67 >10% (n = 2) and toxicity (n = 1). Thirteen patients completed neoadjuvant therapy followed by surgery. No pCRs were observed. Rash was common. MK-2206 did not further suppress cell proliferation and did not induce apoptosis on C1D17 biopsies. Although AKT phosphorylation was reduced, PRAS40 phosphorylation at C1D17 after MK-2206 persisted. One patient acquired an ESR1 mutation at surgery.Conclusions: MK-2206 is unlikely to add to the efficacy of anastrozole alone in PIK3CA-mutant ER+ breast cancer and should not be studied further in the target patient population. Clin Cancer Res; 23(22); 6823–32. ©2017 AACR.

Supplementary Table S3. All observed coding mutations with detailed annotations

Purpose: This trial was conducted to determine the maximum tolerated dose (MTD) and preliminary efficacy of buparlisib, an oral pan-class I PI3K inhibitor, plus fulvestrant in postmenopausal women with metastatic estrogen receptor positive (ER+) breast cancer.Experimental Design: Phase IA employed a 3+3 design to determine the MTD of buparlisib daily plus fulvestrant. Subsequent cohorts (phase IB and cohort C) evaluated intermittent (5/7-day) and continuous dosing of buparlisib (100 mg daily). No more than 3 prior systemic treatments in the metastatic setting were allowed in these subsequent cohorts.Results: Thirty-one patients were enrolled. MTD was defined as buparlisib 100 mg daily plus fulvestrant. Common adverse events (AE) included fatigue (38.7%), transaminases elevation (35.5%), rash (29%), and diarrhea (19.4%). C-peptide was significantly increased during treatment, consistent with on-target effect of buparlisib. Compared with intermittent dosing, daily buparlisib was associated with more frequent early onset AEs and higher buparlisib plasma concentrations. Among the 29 evaluable patients, the clinical benefit rate was 58.6% (95% CI, 40.7%–74.5%). Response was not associated with PIK3CA mutation or treatment cohort; however, loss of PTEN, progesterone receptor (PgR) expression, or mutation in TP53 was most common in resistant cases, and mutations in AKT1 and ESR1 did not exclude treatment response.Conclusions: Buparlisib plus fulvestrant is clinically active with manageable AEs in patients with metastatic ER+ breast cancer. Weekend breaks in buparlisib dosing reduced toxicity. Patients with PgR negative and TP53 mutation did poorly, suggesting buparlisib plus fulvestrant may not be adequately effective against tumors with these poor prognostic molecular features. Clin Cancer Res; 22(7); 1583–91. ©2015 AACR.

Supplementary Table S1. Adverse events that led to dose interruption or reduction Supplementary Table S2. Treatment-related Psychiatric Adverse Events (AEs) Supplementary Table S4. PgR Status of the Primary versus Metastatic (Recurrent) Disease of the 29 Evaluable Patients

Supplementary Fig. S1 is the consort diagram; Supplementary Fig. S2 describes the clinicopathologic and molecular features of the 6 palbociclib resistant tumors; Supplementary Fig S3 shows the proliferation score and correlation with Ki67 at each time point; Supplementary Fig S4 shows the heatmap of the proliferation score genes and ER regulated genes

The caption provides the title and description for the Supplementary Tables S3 adn S4.

Fig S1 Study Flowchart Fig S2 PIK3CA somatic mutation pattern Fig S3 Lack of apoptotic effect by anastrozole monotherapy (C1D1) and in combination with MK-2206 (C1D17 and surgery) Fig S4 CDH1 somatic mutation pattern Fig S5 ESR1 somatic mutation pattern

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Background & Rationale Identifying non-responders to chemotherapy with reliable predictive tests is crucial to response-guided neoadjuvant therapy. BREVITY aims to support RDA as a response assessment tool in breast cancer.RDA is based on RNA disruption, a qualitative and quantitative alteration of ribosomal RNA that correlates with chemotherapy response. RDA stratifies patients into 3 zones: non-response zone 1, partial-response zone 2 and response zone 3. Survival data in MA.22 trial showed significantly superior 5-year DFS KM-curves for zone 3 patients compared to zones 1 & 2. MA.22 and other studies indicated a potentially valuable clinical utility to be pursued. Trial Design. Study type: Prospective 2-phase interventional study. Samples: 2 core needle biopsy specimens taken at 2 time points during chemotherapy. 1 Follow-up: 60 months from date of study enrolment. Incl. Criteria. •Women 18+ years old and able to provide informed consent. •Newly diagnosed clinical stage I, II or III breast cancer with complete surgical excision after neoadjuvant therapy as treatment goal. •Confirmation of invasive breast cancer of any subtype or grade, tumor size > 1cm. •Scheduled for neoadjuvant chemotherapy +/- antibodies and +/- other drugs according to SoC. •Willing to have research core needle biopsies at 2 timepoints during neoadjuvant treatment. Excl. Criteria. •Prior local or systemic therapy for current breast cancer. •Participation in another interventional clinical trial with experimental drugs during neoadjuvant therapy. •Stage IV breast cancer. •Bilateral, multifocal or multicentric breast cancer. •Prior malignant disease except curatively treated basalioma of the skin or pTis of the cervix uteri. •Pregnancy or breast feeding. Trial Objectives & Endpoints Statistical Methods. Phase 1: training set will be used to estimate and visualize operating characteristics as functions of RDI. These include NPV, PPV, sensitivity, specificity, and related concepts. Suitable cut-offs will be determined that optimize NPV of zone 1 and PPV of zone 3. Phase 1 powered to estimate 25th RDI percentile as candidate for cut-off c1 with 9.5% precision E. Phase 2: analysis by interval-estimation of performance characteristics on an independent validation set, given cut-offs selected in phase 1. Primary target of validation is NPV of zone 1 that corresponds to RDA’s main clinical utility. PPV of zone 3 to be established for selected subgroups. Sample size for validation chosen to yield 95% CI estimate for NPV with 5.25% precision. Present and Target Accrual. Present AccrualTarget Accrual(must be fully evaluable). Phase 1 (Training set):71 (target 80). Phase 2 (Validation set):454 Other Contacts. Sponsor: Rna Diagnostics Inc Biopsy Collection Time Points for RDABiopsy(2 specimens)1st core biopsy2nd core biopsyTiming35 +/- 4 daysIf no drug change:55 +/- 5 days. If drug change:2-3 weeks after start of new drugs:•3-weekly: at 16 +/- 2 days•Bi-weekly: at day of 2nd dose•Weekly: at day of 4th dose Primary ObjectivesDescriptionEndpointsi.1.Phase 1: Determine 2 RDI cut-offs to have a diagnostic test optimized in terms of predictive values2.Phase 2: Establish performance characteristics for first cut-off in terms of NPVNPVPPVSecondary Objectivesi.Assess NPV in subgroups by subtypeNPVii.Assess PPV in HER2+PPViii.Compare pCR prevalence and DFS in zones 1-3 for all patients and by subgrouppCRDFSv.Test for association btw. cancer subgroups and test resultTest result (zones 1-3)vi.Compare RDA and other biomarker results (Ki67, tumor grade) Citation Format: Maureen Trudeau, Joke Tio, Foluso Ademuyiwa, Thierry Petit, Bryan Hennessy, Marina Cazzaniga, Daniele Generali. RNA disruption assay (RDA) - Breast cancer response evaluation for individualized therapy (brevity/brevity-02) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-03-01.

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.

Introduction: Cytotoxic chemotherapy for sporadic Triple Negative Breast Cancer (TNBC) remains the standard of care and the recent approval for adjuvant PD1 therapy is not biomarker guided. Pathological complete response (pCR) is often not achieved and portends poor survival. Predictive markers for individual drugs have proven elusive. Approach: Microscaled proteogenomics (MPG) was applied to snap-frozen TNBC clinical trial core needle biopsies obtained before treatment with carboplatin and docetaxel (WashU: NCT201404107 and BCM: NCT02544987). Clinical endpoints for discovery analysis were pathological complete response (pCR) and residual cancer burden (RCB). Standard non-parametric statistical tests were employed to identify proteogenomic features associated with these endpoints. Results: Copy number aberrations (CNA) are a recurrent feature of TNBC and a potential driver of chemotherapy sensitivity. We therefore sought CNA with concordant changes at the mRNA and protein levels that also associate with pCR status. Genes located within a recurrent interstitial deletion at chromosomal location 19q13.3 were the most significantly down-regulated at mRNA and protein level in chemotherapy resistant cases. 19q13.3 encodes multiple DNA damage response (DDR) genes; however, only LIG1, a DNA ligase required for lagging strand synthesis and DNA repair, showed concordant changes at both the mRNA and protein level. In multiple independent TNBC data sets, LIG1 deletion was associated lack of pCR and poor metastasis-free survival. Additionally in the BrighTNess TNBC trial lower LIG1 mRNA levels were associated with increased chemotherapy resistance in the carboplatin containing arms (no pCR and residual cancer burden I-III; p=0.0008 and 0.003 respectively). In PDX-derived short-term cultures and PDXs treated with docetaxel or carboplatin, a specific association of carboplatin resistance with LIG1 deletion was observed. LIG1 depleted-tumors did not harbor elevated scores for homologous recombination defect signature, suggesting LIG1 loss is an orthogonal pathway for TNBC pathogenesis The high chromosomal instability index in LIG1 deletion tumors in our TNBC study was robustly reproduced in multiple datasets (including TCGA-BRCA ; Metastatic breast cancer project). LIG1 copy number deletion was also associated with poor progression free survival, and high chromosomal instability in multiple other cancers (including TCGA-​UCEC HR=2.23, TCGA-HNSC HR=1.46, TCGA-PRAD HR=2.07, TCGA- COAD HR=1.75 and TCGA-KIRP HR=4.00). Conclusion: Deletion of LIG1 is associated with chromosomal instability in TNBC and occurs in tumors without genomic evidence for defects in homologous recombination. Other clinical features of LIG1 deleted TNBC and how LIG1 loss may cause chromosomal instability and tumorigenesis will be discussed. Citation Format: Meenakshi Anurag, Eric Jaehnig, Jonathan Lei, Beom-Jun Kim, Anh Minh Tran Huynh, Yongchao Dou, Tanmayi Vashist, Erik Bergstrom, Xuxu Gou, Viktoriya Korchina, Donna Marie Muzny, Kristen Otte, Harshavardhan Doddapaneni, Lacey Dobrolecki, Gloria Vittone Echeverria, Bora Lim, Mothaffar Rimawi, Karsten Krug, Ian Hageman, Henry Rodriguez, Ana I. Robles, Tara Hiltke, Kent Osborne, Michael Gillette, George Miles, Steven Carr, Michael T Lewis, Bing Zhang, Foluso Ademuyiwa, Shankha Satpathy, Matthew J. Ellis. LIG1 deletion predicts chemotherapy resistance, chromosomal instability, and poor prognosis in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1010.

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.

Additional file 1: Table S1. The race-associated hazards ratios of subsequently developing invasive breast cancer in women with LCIS during three time intervals.

Additional file 2: Table S2. The race-associated hazard ratios of subsequent invasive breast cancer in women with LCIS by age at the diagnosis of LCIS.

Additional file 1: Table S1. The race-associated hazards ratios of subsequently developing invasive breast cancer in women with LCIS during three time intervals.

Additional file 2: Table S2. The race-associated hazard ratios of subsequent invasive breast cancer in women with LCIS by age at the diagnosis of LCIS.