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Purpose: To conduct an updated exploratory analysis of overall survival (OS) with a longer median follow-up of 73.3 months and evaluate the prognostic value of molecular analysis by circulating tumor DNA (ctDNA). Patients and Methods: Patients with hormone receptor–positive/human epidermal growth factor receptor 2–negative (HR+/HER2−) advanced breast cancer (ABC) were randomized 2:1 to receive palbociclib (125 mg orally/day; 3/1 week schedule) and fulvestrant (500 mg intramuscularly) or placebo and fulvestrant. This OS analysis was performed when 75% of enrolled patients died (393 events in 521 randomized patients). ctDNA analysis was performed among patients who provided consent. Results: At the data cutoff (August 17, 2020), 258 and 135 deaths occurred in the palbociclib and placebo groups, respectively. The median OS [95% confidence interval (CI)] was 34.8 months (28.8–39.9) in the palbociclib group and 28.0 months (23.5–33.8) in the placebo group (stratified hazard ratio, 0.81; 95% CI, 0.65–0.99). The 6-year OS rate (95% CI) was 19.1% (14.9–23.7) and 12.9% (8.0–19.1) in the palbociclib and placebo groups, respectively. Favorable OS with palbociclib plus fulvestrant compared with placebo plus fulvestrant was observed in most subgroups, particularly in patients with endocrine-sensitive disease, no prior chemotherapy for ABC and low circulating tumor fraction and regardless of ESR1, PIK3CA, or TP53 mutation status. No new safety signals were identified. Conclusions: The clinically meaningful improvement in OS associated with palbociclib plus fulvestrant was maintained with >6 years of follow-up in patients with HR+/HER2− ABC, supporting palbociclib plus fulvestrant as a standard of care in these patients.

Supplementary figures and tables

Supplementary Data from Overall Survival with Palbociclib and Fulvestrant in Women with HR+/HER2− ABC: Updated Exploratory Analyses of PALOMA-3, a Double-blind, Phase III Randomized Study

CDK4/6 inhibition partly overcomes NF1 loss in T47D cells

Supplementary Table 1. Targets of The Breast NGS v1.0 capture panel. Supplementary Table 2. Gene list of NanoString nCounter{trade mark, serif} custom codeset. Supplementary Table 3. Gene list of Human Estrogen Receptor RT2 Profiler PCR Array (330231, PAHS-005ZA-24, Qiagen) Supplementary Table 4. Lines of treatment of patients with ESR1 and NF1 mutations. Supplementary Table 5. Disease free survival for mutated genes with incidence {greater than or equal to}5% presented by HR+/HER2-, HER2+ and TNBC subtype. Supplementary Table 6. Advanced overall survival for mutated genes with incidence {greater than or equal to}5% presented by HR+/HER2-, HER2+ and TNBC subtype

Supplementary Figure Legend

Purpose:To conduct an updated exploratory analysis of overall survival (OS) with a longer median follow-up of 73.3 months and evaluate the prognostic value of molecular analysis by circulating tumor DNA (ctDNA).Patients and Methods:Patients with hormone receptor–positive/human epidermal growth factor receptor 2–negative (HR+/HER2−) advanced breast cancer (ABC) were randomized 2:1 to receive palbociclib (125 mg orally/day; 3/1 week schedule) and fulvestrant (500 mg intramuscularly) or placebo and fulvestrant. This OS analysis was performed when 75% of enrolled patients died (393 events in 521 randomized patients). ctDNA analysis was performed among patients who provided consent.Results:At the data cutoff (August 17, 2020), 258 and 135 deaths occurred in the palbociclib and placebo groups, respectively. The median OS [95% confidence interval (CI)] was 34.8 months (28.8–39.9) in the palbociclib group and 28.0 months (23.5–33.8) in the placebo group (stratified hazard ratio, 0.81; 95% CI, 0.65–0.99). The 6-year OS rate (95% CI) was 19.1% (14.9–23.7) and 12.9% (8.0–19.1) in the palbociclib and placebo groups, respectively. Favorable OS with palbociclib plus fulvestrant compared with placebo plus fulvestrant was observed in most subgroups, particularly in patients with endocrine-sensitive disease, no prior chemotherapy for ABC and low circulating tumor fraction and regardless of ESR1, PIK3CA, or TP53 mutation status. No new safety signals were identified.Conclusions:The clinically meaningful improvement in OS associated with palbociclib plus fulvestrant was maintained with >6 years of follow-up in patients with HR+/HER2− ABC, supporting palbociclib plus fulvestrant as a standard of care in these patients.

Genetic profile of advanced breast cancer subtypes

NCOR expression following NF1 loss

Purpose:Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC.Experimental Design:Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients.Results:We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant.Conclusions:Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.

NCOR expression in NF1 mutated cancers

Genetic profile of ER positive primary breast cancers that 'switch' to triple negative recurrent advanced breast cancer

The study of cancer evolution continues to deliver novel insights into the biology driving cancer. International consortia working on large genome sequencing initiatives have now provided an outline of the genomic landscape for many cancers. This vast resource of genomic data has also allowed the development of advanced computational tools revealing biology shaping genomic changes. Head and neck squamous cell carcinoma (HNSCC) is represented within the international consortia projects, though there remains only modest whole genome data and data from human papillomavirus-related cancers. Data for recurrent HNSCC and longitudinal data from patients treated with therapy are lacking and should be a priority for the community. This review will discuss the available resources and approaches for understanding HNSCC evolution, and consider how this might be applied to the clinical paradigm and used to develop the next generation of clinical trials.

Blastobotrys aristata is a member of the Trichomonascaceae family in the order Saccharomycetales. Here, we present the genome sequence of B. aristata UCD613, which was isolated from soil in Dublin, Ireland. This genome is 13.3 Mb and was assembled into 4 chromosome-size scaffolds of >2.2 Mb in size plus a mitochondrial genome scaffold.

The United Kingdom has been severely affected by the coronavirus disease 2019 (COVID-19) pandemic. As the National Health Service (NHS) has urgently prioritized management of this outbreak, the UK clinical oncology community has had to adapt rapidly to maintain cancer services and training. These unprecedented times have altered countless aspects of cancer care, education, and research, providing a legacy that will extend well beyond the pandemic that catalyzed them. This editorial focuses on 3 key themes that distinguish the United Kingdom from many other countries.

Purpose: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC. Experimental Design: Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients. Results: We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant. Conclusions: Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.

Circulating tumor DNA (ctDNA) is at the forefront of liquid biopsy technology. A key advantage of ctDNA is being able to achieve genomic profiling from a blood test rather than from a more invasive tissue biopsy, reducing risk and discomfort for patients as well as costs and logistical complexity. Increasing evidence supports ctDNA as a useful clinical tool in specific indications, for example in the detection of PIK3CA mutations to identify patients suitable for alpelisib plus fulvestrant.The expectation is that indications for routine ctDNA testing will expand as more data become available and ctDNA is embedded into clinical trials and paired with novel therapies. Biological and technical factors both contribute to the feasibility of ctDNA expansion into the clinic. Representation of heterogeneity might be a key strength of ctDNA analysis over tumour biopsy analysis, as sampling the circulation compartment can in principle allow representation of a wider array of disease sites than a tumor sample acquired with a single needle. However, the factors influencing how different metastases might contribute to the circulating compartment are poorly understood, presenting a challenge to clinical interpretation. Moreover, the limit of detection of ctDNA assays, the timing of treatment and stochastic effects can contribute to uncertainty, particularly in the interpretation of negative results. Recent work analyzing tumor mutational burden using ctDNA has highlighted these challenges as applied to biomarker development. Circulating tumour DNA is likely to complement tissue biopsy in the future, and may substitute tumor biopsy in specific indications supported by the data. Ultimately, clinical utility for different ctDNA indications, as opposed to tissue biopsy, will need ongoing confirmation within clinical trials. Citation Format: Ben O’Leary. Can ctDNA substitute tissue testing [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 ED9-2A.

BACKGROUND Circulating tumor DNA (ctDNA) assays are increasingly used for clinical decision-making, but it is unknown how well different assays agree. We aimed to assess the agreement in ctDNA mutation calling between BEAMing (beads, emulsion, amplification, and magnetics) and droplet digital PCR (ddPCR), 2 of the most commonly used digital PCR techniques for detecting mutations in ctDNA. METHODS Baseline plasma samples from patients with advanced breast cancer enrolled in the phase 3 PALOMA-3 trial were assessed for ESR1 and PIK3CA mutations in ctDNA with both BEAMing and ddPCR. Concordance between the 2 approaches was assessed, with exploratory analyses to estimate the importance of sampling effects. RESULTS Of the 521 patients enrolled, 363 had paired baseline ctDNA analysis. ESR1 mutation detection was 24.2% (88/363) for BEAMing and 25.3% (92/363) for ddPCR, with good agreement between the 2 techniques (κ = 0.9l; 95% CI, 0.85–0.95). PIK3CA mutation detection rates were 26.2% (95/363) for BEAMing and 22.9% (83/363) for ddPCR, with good agreement (κ = 0.87; 95% CI, 0.81–0.93). Discordancy was observed for 3.9% patients with ESR1 mutations and 5.0% with PIK3CA mutations. Assessment of individual mutations suggested higher rates of discordancy for less common mutations (P = 0.019). The majority of discordant calls occurred at allele frequency CONCLUSIONS This large, clinically relevant comparison showed good agreement between BEAMing and ddPCR, suggesting sufficient reproducibility for clinical use. Much of the observed discordancy may be related to sampling effects, potentially explaining many of the differences in the currently available ctDNA literature.

Background Conventional biomarkers in thyroid cancer are not disease specific and fluctuate in advanced disease, making interpretation difficult. Circulating tumour DNA (ctDNA) has been shown to be a useful biomarker in other solid tumours. This is a multimutational study of ctDNA over multiple timepoints, designed to test the hypothesis that ctDNA is a potential biomarker in patients with advanced thyroid cancer. Methods Mutational analysis of archival tumour tissue was performed using NGS with a targeted gene panel. Custom TaqMan assays were designed for plasma ctDNA testing using digital droplet polymerase chain reaction. Concentrations of detected ctDNA were correlated with the conventional biomarker concentration and axial imaging status defined by the Response Evaluation Criteria in Solid Tumours criteria. Results Tumour tissue from 51 patients was obtained, with the following histologies: 32 differentiated (differentiated thyroid cancer [DTC]), 15 medullary (medullary thyroid cancer [MTC]), three poorly differentiated and one anaplastic. NGS analysis detected variants in 42 (82%) of cases. Plasma was assayed for these patients in 190 samples, and ctDNA was detected in 67% of patients. Earlier detection of disease progression was noted in three patients with MTC. In two cases (PTC and ATC), where conventional biomarkers were not detectable, ctDNA was detected before disease progression. Changes in ctDNA concentration occurred earlier than conventional markers in response to disease progression in multiple patients with DTC receiving targeted therapies. Conclusion The majority of patients with advanced thyroid cancer had detectable ctDNA. ctDNA measurement may offer superiority over conventional markers in several scenarios: earlier detection of progression in MTC; as an alternative biomarker when conventional markers are not available; more rapid assessment of the disease status in response to targeted therapies, thereby potentially allowing prompter discontinuation of futile therapies. These early results support the hypothesis that ctDNA may be a clinically useful biomarker in thyroid cancer.

Background Selection of resistance mutations may play a major role in the development of endocrine resistance. ESR1 mutations are rare in primary breast cancer but have high prevalence in patients treated with aromatase inhibitors (AI) for advanced breast cancer. We investigated the evolution of genetic resistance to the first-line AI therapy using sequential ctDNA sampling in patients with advanced breast cancer. Patients and methods Eighty-three patients on the first-line AI therapy for metastatic breast cancer were enrolled in a prospective study. Plasma samples were collected every 3 months to disease progression and ctDNA analysed by digital droplet PCR and enhanced tagged-amplicon sequencing (eTAm-Seq). Mutations identified in progression samples by sequencing were tracked back through samples before progression to study the evolution of mutations on therapy. The frequency of novel mutations was validated in an independent cohort of available baseline plasma samples in the Study of Faslodex versus Exemestane with or without Arimidex (SoFEA) trial, which enrolled patients with prior sensitivity to AI. Results Of the 39 patients who progressed on the first-line AI, 56.4% (22/39) had ESR1 mutations detectable at progression, which were polyclonal in 40.9% (9/22) patients. In serial tracking, ESR1 mutations were detectable median 6.7 months (95% confidence interval 3.7–NA) before clinical progression. Utilising eTAm-Seq ctDNA sequencing of progression plasma, ESR1 mutations were demonstrated to be sub-clonal in 72.2% (13/18) patients. Mutations in RAS genes were identified in 15.4% (6/39) of progressing patients (4 KRAS, 1 HRAS, 1 NRAS). In SoFEA, KRAS mutations were detected in 21.2% (24/113) patients although there was no evidence that KRAS mutation status was prognostic for progression free or overall survival. Conclusions Cancers progressing on the first-line AI show high levels of genetic heterogeneity, with frequent sub-clonal mutations. Sub-clonal KRAS mutations are found at high frequency. The genetic diversity of AI resistant cancers may limit subsequent targeted therapy approaches.

BackgroundThere are no established molecular biomarkers for patients with breast cancer receiving combination endocrine and CDK4/6 inhibitor (CDK4/6i). We aimed to determine whether genomic markers in circulating tumor DNA (ctDNA) can identify patients at higher risk of early progression on fulvestrant therapy with or without palbociclib, a CDK4/6i.MethodsPALOMA-3 was a phase III, multicenter, double-blind randomized controlled trial of palbociclib plus fulvestrant (n = 347) vs placebo plus fulvestrant (n = 174) in patients with endocrine-pretreated estrogen receptor–positive (ER+) breast cancer. Pretreatment plasma samples from 459 patients were analyzed for mutations in 17 genes, copy number in 14 genes, and circulating tumor fraction. Progression-free survival (PFS) was compared in patients with circulating tumor fraction above or below a prespecified cutoff of 10% and with or without a specific genomic alteration. All statistical tests were 2-sided.ResultsPatients with high ctDNA fraction had worse PFS on both palbociclib plus fulvestrant (hazard ratio [HR] = 1.62, 95% confidence interval [CI] = 1.17 to 2.24; P = .004) and placebo plus fulvestrant (HR = 1.77, 95% CI = 1.21 to 2.59; P = .004). In multivariable analysis, high-circulating tumor fraction was associated with worse PFS (HR = 1.20 per 10% increase in tumor fraction, 95% CI = 1.09 to 1.32; P < .001), as was TP53 mutation (HR = 1.84, 95% CI = 1.27 to 2.65; P = .001) and FGFR1 amplification (HR = 2.91, 95% CI = 1.61 to 5.25; P < .001). No interaction with treatment randomization was observed.ConclusionsPretreatment ctDNA identified a group of high-risk patients with poor clinical outcome despite the addition of CDK4/6 inhibition. These patients might benefit from inclusion in future trials of escalating treatment, with therapies that may be active in these genomic contexts.

Invasive lobular breast cancer (ILC) represents the second most common histology of breast cancer after invasive ductal breast cancer (IDC), accounts for up to 15% of all invasive cases and generally express the estrogen receptor (ER, coded by the ESR1 gene). ESR1 mutations have been associated with resistance to endocrine therapy, however these have not been specifically evaluated in ILC. We assessed the frequency of ESR1 mutations by droplet digital PCR in a retrospective multi-centric series of matched primary tumor and recurrence samples (n = 279) from 80 metastatic ER-positive ILC patients. We further compared ESR1 mutations between IDC and ILC patients in metastatic samples from MSKCC-IMPACT (n = 595 IDC and 116 ILC) and in ctDNA from the SoFEA and PALOMA-3 trials (n = 416 IDC and 76 ILC). In the retrospective series, the metastases from seven patients (9%) harbored ESR1 mutations, which were absent from the interrogated primary samples. Five patients (6%) had a mutation in the primary tumor or axillary metastasis, which could not be detected in the matched distant metastasis. In the MSKCC-IMPACT cohort, as well as in the SoFEA and PALOMA-3 trials, there were no differences in prevalence and distribution of the mutations between IDC and ILC, with D538G being the most frequent mutation in both histological subtypes. To conclude, no patient had an identical ESR1 mutation in the early and metastatic disease in the retrospective ILC series. In the external series, there was no difference in terms of prevalence and type of ESR1 mutations between ILC and IDC., info:eu-repo/semantics/published

1000 Background: In PALOMA-3, a randomized, double-blind, placebo-controlled, phase 3 study, PAL+FUL significantly prolonged progression-free survival (PFS) compared with placebo (PBO) + FUL (1-sided P6 years of median follow-up in pts with HR+/HER2– ABC who had progressed on prior endocrine treatment. Pfizer (NCT01942135) Clinical trial information: NCT01942135 .[Table: see text]

Purpose ESR1 mutations are selected by prior aromatase inhibitor (AI) therapy in advanced breast cancer. We assessed the impact of ESR1 mutations on sensitivity to standard therapies in two phase III randomized trials that represent the development of the current standard therapy for estrogen receptor–positive advanced breast cancer. Materials and Methods In a prospective-retrospective analysis, we assessed ESR1 mutations in available archived baseline plasma from the SoFEA (Study of Faslodex Versus Exemestane With or Without Arimidex) trial, which compared exemestane with fulvestrant-containing regimens in patients with prior sensitivity to nonsteroidal AI and in baseline plasma from the PALOMA3 (Palbociclib Combined With Fulvestrant in Hormone Receptor–Positive HER2-Negative Metastatic Breast Cancer After Endocrine Failure) trial, which compared fulvestrant plus placebo with fulvestrant plus palbociclib in patients with progression after receiving prior endocrine therapy. ESR1 mutations were analyzed by multiplex digital polymerase chain reaction. Results In SoFEA, ESR1 mutations were found in 39.1% of patients (63 of 161), of whom 49.1% (27 of 55) were polyclonal, with rates of mutation detection unaffected by delays in processing of archival plasma. Patients with ESR1 mutations had improved progression-free survival (PFS) after taking fulvestrant (n = 45) compared with exemestane (n = 18; hazard ratio [HR], 0.52; 95% CI, 0.30 to 0.92; P = .02), whereas patients with wild-type ESR1 had similar PFS after receiving either treatment (HR, 1.07; 95% CI, 0.68 to 1.67; P = .77). In PALOMA3, ESR1 mutations were found in the plasma of 25.3% of patients (91 of 360), of whom 28.6% (26 of 91) were polyclonal, with mutations associated with acquired resistance to prior AI. Fulvestrant plus palbociclib improved PFS compared with fulvestrant plus placebo in both ESR1 mutant (HR, 0.43; 95% CI, 0.25 to 0.74; P = .002) and ESR1 wild-type patients (HR, 0.49; 95% CI, 0.35 to 0.70; P < .001). Conclusion ESR1 mutation analysis in plasma after progression after prior AI therapy may help direct choice of further endocrine-based therapy. Additional confirmatory studies are required.

Constrained supersymmetric models like the CMSSM might look less attractive nowadays because of fine tuning arguments. They also might look less probable in terms of Bayesian statistics. The question how well the model under study describes the data, however, is answered by frequentist p-values. Thus, for the first time, we calculate a p-value for a supersymmetric model by performing dedicated global toy fits. We combine constraints from low-energy and astrophysical observables, Higgs boson mass and rate measurements as well as the non-observation of new physics in searches for supersymmetry at the LHC. Using the framework Fittino, we perform global fits of the CMSSM to the toy data and find that this model is excluded at more than 95% confidence level., 6 pages, 7 figures; To appear in the Proceedings of the 37th International Conference on High Energy Physics (ICHEP 2014), 2-9 July 2014, Valencia, Spain

Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC.Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients.We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations inOur research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of

CDK4/6 inhibition with endocrine therapy is now a standard of care for advanced estrogen receptor–positive breast cancer. Mechanisms of CDK4/6 inhibitor resistance have been described preclinically, with limited evidence from clinical samples. We conducted paired baseline and end-of-treatment circulating tumor DNA sequencing from 195 patients in the PALOMA-3 randomized phase III trial of palbociclib plus fulvestrant versus placebo plus fulvestrant. We show that clonal evolution occurs frequently during treatment, reflecting substantial subclonal complexity in breast cancer that has progressed after prior endocrine therapy. RB1 mutations emerged only in the palbociclib plus fulvestrant arm and in a minority of patients (6/127, 4.7%, P = 0.041). New driver mutations emerged in PIK3CA (P = 0.00069) and ESR1 after treatment in both arms, in particular ESR1 Y537S (P = 0.0037). Evolution of driver gene mutations was uncommon in patients progressing early on palbociclib plus fulvestrant but common in patients progressing later on treatment. These findings inform future treatment strategies to address resistance to palbociclib plus fulvestrant. Significance: Acquired mutations from fulvestrant are a major driver of resistance to fulvestrant and palbociclib combination therapy. ESR1 Y537S mutation promotes resistance to fulvestrant. Clonal evolution results in frequent acquisition of driver mutations in patients progressing late on therapy, which suggests that early and late progression have distinct mechanisms of resistance. Cancer Discov; 8(11); 1390–403. ©2018 AACR. See related commentary by Schiff and Jeselsohn, p. 1352. This article is highlighted in the In This Issue feature, p. 1333

Novel approaches for classification, including molecular features, are needed to direct therapy for men with low-grade prostate cancer (PCa), especially men on active surveillance. Risk alleles identified from genome-wide association studies (GWAS) could improve prognostication. Those risk alleles that coincided with genes and somatic copy number aberrations associated with progression of PCa were selected as the most relevant for prognostication. In a systematic literature review, a total of 698 studies were collated. Fifty-three unique SNPs residing in 29 genomic regions, including 8q24, 10q11 and 19q13, were associated with PCa progression. Functional studies implicated 21 of these single nucleotide polymorphisms (SNPs) as modulating the expression of genes in the androgen receptor pathway and several other oncogenes. In particular, 8q24, encompassing MYC, harbours a high density of SNPs conferring unfavourable pathological characteristics in low-grade PCa, while a copy number gain of MYC in low-grade PCa was associated with prostate-specific antigen recurrence after radical prostatectomy. By combining GWAS data with gene expression and structural rearrangements, risk alleles were identified that could provide a new basis for developing a prognostication tool to guide therapy for men with early prostate cancer.

Minimally invasive circulating free DNA (cfDNA) analysis can portray cancer genome landscapes but highly sensitive and specific genetic approaches are necessary to accurately detect mutations with often low variant frequencies. We developed a targeted cfDNA sequencing technology using novel off-the-shelf molecular barcodes for error correction, in combination with custom solution hybrid capture enrichment. Modelling based on cfDNA yields from 58 patients shows that our assay, which requires 25ng of cfDNA input, should be applicable to >95% of patients with metastatic colorectal cancer. Sequencing of a 163.3 kb target region including 32 genes detected 100% of single nucleotide variants with 0.15% variant frequency in cfDNA spike-in experiments. Molecular barcode error correction reduced false positive mutation calls by 98.6%. In a series of 28 patients with metastatic colorectal cancers, 80 out of 91 (88%) mutations previously detected by tumour tissue sequencing were called in the cfDNA. Call rates were similar for single nucleotide variants and small insertions/deletions. Mutations only called in cfDNA but not detectable in matched tumour tissue included, among others, a subclonal resistance driver mutation to anti-EGFR antibodies in theKRASgene, multiple activatingPIK3CAmutations in each of two patients (indicative of parallel evolution), andTP53mutations originating from clonal haematopoiesis. Furthermore, we demonstrate that cfDNA off-target read analysis allows the reconstruction of genome wide copy number aberration profiles from 71% of these 28 cases. This error-corrected ultra-deep cfDNA sequencing assay with a target region that can be readily customized enables broad insights into cancer genomes and evolution.

CDK4/6 inhibition substantially improves progression-free survival (PFS) for women with advanced estrogen receptor-positive breast cancer, although there are no predictive biomarkers. Early changes in circulating tumor DNA (ctDNA) level may provide early response prediction, but the impact of tumor heterogeneity is unknown. Here we use plasma samples from patients in the randomized phase III PALOMA-3 study of CDK4/6 inhibitor palbociclib and fulvestrant for women with advanced breast cancer and show that relative change in PIK3CA ctDNA level after 15 days treatment strongly predicts PFS on palbociclib and fulvestrant (hazard ratio 3.94, log-rank p = 0.0013). ESR1 mutations selected by prior hormone therapy are shown to be frequently sub clonal, with ESR1 ctDNA dynamics offering limited prediction of clinical outcome. These results suggest that early ctDNA dynamics may provide a robust biomarker for CDK4/6 inhibitors, with early ctDNA dynamics demonstrating divergent response of tumor sub clones to treatment.

1010 Background: Markers of early progression on endocrine therapy in combination with CDK4/6 inhibitors remain limited despite the key role of these combinations in treating of ER+ advanced breast cancer (ABC). We investigated genomic aberrations in patients treated with fulvestrant, with and without palbociclib, with a circulating tumor DNA analysis of baseline plasma in the PALOMA-3 trial. Methods: PALOMA-3 was a phase III trial that randomized 521 patients with ER+/HER2- ABC 2:1 to palbociclib plus fulvestrant (P+F) or placebo plus fulvestrant (F). Using baseline plasma samples, somatic mutations were assessed with a 17 gene panel. Copy number aberrations (CNA) were characterized with a 14 gene panel including ~800 SNPs in 8 commonly altered regions for estimation of tumor purity, the percentage of plasma DNA that was derived from tumor cells. Results for mutations and CNAs were available in 310 pts (203 P+F, 107 F) and were associated with clinical characteristics and progression free survival (PFS) using univariable and multivariable Cox proportional hazards models, including tumor purity and treatment as variables. Results: In the multivariable analysis of the whole cohort, higher baseline tumor purity in plasma was associated with worse PFS (HR 1.20, 95% CI 1.09 – 1.32, p = 0.0001, HR per 10% increase in purity). Baseline TP53 mutation was also associated with shorter PFS (HR 1.84, 95%CI 1.27 – 2.65, p = 0.0011), as was baseline FGFR1 amplification (HR 2.91, 95%CI 1.61 – 5.25, p = 0.0004). PIK3CA and ESR1 mutations had no significant association with PFS in the multivariable model. Palbociclib treatment effect was comparable with the overall trial result (HR 0.43, 95%CI 0.32 - 0.57, p < 0.0001). TP53 mutations were significantly associated with visceral (q = 0.046) and soft tissue/LN metastases (q = 0.042) and the number of disease sites (q = 0.0086) after correction for multiple testing. Conclusions: TP53 mutation, FGFR1 amplification , and tumor purity in plasma identified patients at risk of early progression In PALOMA-3. If validated these results could inform future clinical trials of CDK4/6 inhibitors combinations.

Background: CDK4/6 inhibition combined with endocrine therapy (ET) is now the standard of care for advanced hormone receptor (HR) positive breast cancer patients (pts). Previous ctDNA analysis of paired pre-treatment (pre-T) and end of treatment (EoT) plasma samples from the PALOMA-3 trial of the CDK4/6 inhibitor P with/without F demonstrated that acquired PIK3CA, ESR1, RB1 and rare growth factor receptor mutations (mut) likely contribute to resistance (Turner et al, ASCO 2018, Abstract 1001). We now report results from an extended ctDNA analysis of paired PALOMA-3 plasma samples using hybridization capture-based, next-generation sequencing (NGS) assay covering 87 breast cancer, HR signaling, and cell cycle-related genes. Methods: Pre-/post-menopausal pts whose disease progressed after prior ET (N=521) were randomized 2:1 to receive F 500 mg + P (125 mg/d, 3 wk on/1 wk off) or placebo. Cell-free DNA extracted from paired plasma samples (n=194) was analyzed with a custom 87-gene NGS assay, to identify single nucleotide variants and copy number amplification (CNA), with molecular barcoding, high NGS coverage (10,000-20,000 reads per nucleotide position), and background error correction. Differences in ctDNA mut frequencies detected in P+F compared to F alone were assessed and their association to clinical outcome estimated. Results: Blinded assay qualification and droplet digital PCR validation suggested a mut detection frequency cutoff at 0.3%. A high coefficient of correlation with previously presented data was observed for ESR1 and PIK3CA variants (r=0.94 and 0.97, respectively), with acquisition of PIK3CA and ESR1 mutations at EoT in P+F and F groups. Gene level mut analysis of EoT plasma revealed no significant difference between P+F vs F alone (Table), although there was an increase in RB1 mutations in P+F consistent with previous data. Other acquired muts at EoT (SMAD4, NOTCH2, and CDKN1B) were observed at low frequencies. Muts in CDK4 and CDK6 were rarely observed (< 1% of pts), regardless of treatment arm. Detected Variants, Frequency, n (%) P+FF GenePre-TEoTPre-TEoTPre-T pvalEoT pvalPIK3CA47(37)51(40)19(28)22(33)0.2660.352ESR136(28)45(35)19(28)24(36)11TP5330(24)33(26)23(34)25(37)0.1280.137RB12(2)9(7)2(3)2(3)NA0.336PTEN5(4)7(6)3(4)3(4)11AKT17(6)7(6)2(3)2(3)0.7210.721 Conclusions: Our results corroborate the previously reported results demonstrating that genomic profiles of treatment resistant cancer are similar between P + F and F therapy. Expanded analysis of cell-cycle genes identified no new recurrently mutated genes, and confirmed that RB1 mutations are selected at low frequency after P+F treatment. Alterations in cell cycle genes may not be a common mechanism of resistance to CDK4/6 inhibitors in HR+ HER2- advanced breast cancer. Sponsor: Pfizer Citation Format: O'Leary B, Lira ME, Huang S, Deng S, Xie T, Kinong JK, Hardwick J, Rejto P, Liu Y, Huang X, Huang Bartlett C, André F, Loibl S, DeMichele A, Cristofanilli M, Turner NC. Longitudinal ctDNA sequencing using an expanded genomic panel in the PALOMA3 trial of palbociclib plus fulvestrant [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-02.

Treatment for colorectal cancer is guided principally by stage in a classification that has changed little over 70 years. Patients presenting with stage II and III resected colorectal cancer have no detectable metastatic disease, but a proportion of these patients will have undeclared micrometastatic disease, which it may be possible to eradicate with adjuvant treatment. Adjuvant chemotherapy with 5-fluorouracil (5-FU)based regimens was the standard of care for the treatment of stage III patients [1e3] and amodest benefit of oxaliplatin has been shown [4,5]. These studies also included high-risk stage II patients, but with a lower risk of relapse, even similar hazard ratios translate into small clinical benefit and the role for adjuvant chemotherapy remains debatable [6e8]. The largest study (QUASAR) suggested possible benefits of adjuvant chemotherapy of the order of 3e4% in overall survival for stage II patients [9], although of only marginal statistical significance. CALGB9581 (albeit investigating immunotherapy in this setting) confirmed the good prognosis of many patients with low-risk, stage II colorectal cancer [10]. Despite numerous emerging biomarkers, decisions on the potential benefits of adjuvant chemotherapy in stage II disease have used subjective analyses of adverse histopathological features (derived from the initial adjuvant 5-FU studies without subsequent validation). The hope is that biomarkers will allow better resolution of the heterogeneity thought to be present in colorectal cancer and, subsequently, more effectively targeted treatment. We believe there is now a strong case for routinely assessing mismatch repair (MMR) status in stage II colorectal cancer.

We present an extensive study of the MSSM parameter space allowing for general generation mixing in the squark sector. Employing an MCMC algorithm, we establish the parameter ranges which are allowed with respect to various experimental and theoretical constraints. Based on this analysis, we propose benchmark scenarios for future studies. Moreover, we discuss aspects of signatures at the LHC., 4 pages, 3 figures. Contribution to the proceedings of the 38th International Conference of High Energy Physics (ICHEP), Chicago, Aug. 3-10, 2016

Uncontrolled cellular proliferation, mediated by dysregulation of the cell-cycle machinery and activation of cyclin-dependent kinases (CDKs) to promote cell-cycle progression, lies at the heart of cancer as a pathological process. Clinical implementation of first-generation, nonselective CDK inhibitors, designed to inhibit this proliferation, was originally hampered by the high risk of toxicity and lack of efficacy noted with these agents. The emergence of a new generation of selective CDK4/6 inhibitors, including ribociclib, abemaciclib and palbociclib, has enabled tumour types in which CDK4/6 has a pivotal role in the G1-to-S-phase cell-cycle transition to be targeted with improved effectiveness, and fewer adverse effects. Results of pivotal phase III trials investigating palbociclib in patients with advanced-stage oestrogen receptor (ER)-positive breast cancer have demonstrated a substantial improvement in progression-free survival, with a well-tolerated toxicity profile. Mechanisms of acquired resistance to CDK4/6 inhibitors are beginning to emerge that, although unwelcome, might enable rational post-CDK4/6 inhibitor therapeutic strategies to be identified. Extending the use of CDK4/6 inhibitors beyond ER-positive breast cancer is challenging, and will likely require biomarkers that are predictive of a response, and the use of combination therapies in order to optimize CDK4/6 targeting.

We present an extensive study of non-minimally flavour violating (NMFV) terms in the Lagrangian of the Minimal Supersymmetric Standard Model (MSSM). We impose a variety of theoretical and experimental constraints and perform a detailed scan of the parameter space by means of a Markov Chain Monte-Carlo (MCMC) setup. This represents the first study of several non-zero flavour-violating elements within the MSSM. We present the results of the MCMC scan with a special focus on the flavour-violating parameters. Based on these results, we define benchmark scenarios for future studies of NMFV effects at the LHC.

We investigate the constrained Minimal Supersymmetric Standard Model (cMSSM) in the light of constraining experimental and observational data from precision measurements, astrophysics, direct supersymmetry searches at the LHC and measurements of the properties of the Higgs boson, by means of a global fit using the program Fittino. As in previous studies, we find rather poor agreement of the best fit point with the global data. We also investigate the stability of the electro-weak vacuum in the preferred region of parameter space around the best fit point. We find that the vacuum is metastable, with a lifetime significantly longer than the age of the Universe. For the first time in a global fit of supersymmetry, we employ a consistent methodology to evaluate the goodness-of-fit of the cMSSM in a frequentist approach by deriving p-values from large sets of toy experiments. We analyse analytically and quantitatively the impact of the choice of the observable set on the p-value, and in particular its dilution when confronting the model with a large number of barely constraining measurements. Finally, for the preferred sets of observables, we obtain p-values for the cMSSM below 10%, i.e. we exclude the cMSSM as a model at the 90% confidence level., 22 pages, 16 figures, to be submitted to EPJ C

1001Background: CDK4/6 inhibition combined with endocrine therapy is now a standard of care for advanced estrogen receptor (ER) positive breast cancer. Mechanisms of resistance to CDK4/6 inhibitors...

It has been proposed that the observed diphoton excess at 750 GeV could be explained within the constrained minimal supersymmetric standard model via resonantly produced stop bound states. We reanalyze this scenario critically and extend previous work to include the constraints from the stability of the electroweak vacuum and from the decays of the stoponium into a pair of Higgs bosons. It is shown that the interesting regions of parameter space with a light stop and Higgs of the desired mass are ruled out by these constraints. This conclusion is not affected by the presence of the bound states because the binding energy is usually very small in the regions of parameter space which can explain the Higgs mass. Thus, this also leads to strong constraints on the diphoton production cross section which is in general too small., Comment: 8 pages, 5 figures; v2: added Fig. 5, matches published version

1018 Background: Palbociclib improves progression free survival (PFS) in patients with advanced, hormone receptor positive, HER2-negative breast cancer. There are currently no biomarkers to predict sensitivity to palbociclib. We investigated if early circulating tumour DNA (ctDNA) dynamics could predict clinical outcome on palbociclib (Palbo) and fulvestrant (F) in the PALOMA-3 trial. Methods: Plasma samples were prospectively collected in PALOMA-3 for ctDNA analysis at baseline, cycle 1 day 15 (D15) and end of treatment (EOT), and were screened for PIK3CA and ESR1 mutations using digital PCR. The primary objective was to assess whether early dynamic changes in PIK3CA mutations would predict PFS in patients treated with Palbo + F. Suppression of PIK3CA ctDNA levels below an optimised cut-off at cycle 1 D15, relative to baseline levels, was termed a ‘circulating DNA ratio (CDR) response. Results: PIK3CA mutations were identified in 22.0% (100/455) of screened baseline samples with 52 of these randomised to Palbo + F and having matched D15 samples. Patients on Palbo + F with a CDR response had a median PFS of 11.2 months (95%CI 11.1 – undefined) whereas patients on Palbo + F without a CDR response had a median PFS of 4.1 months (95%CI 3.6 – 5.5) (HR for no CDR response 4.92, 95% CI 1.98 – 12.26, p = 0.0002). Overall, Palbo + F suppressed D15 PIK3CA ctDNA levels to a greater extent than placebo + F (p

We re-evaluate the constraints on the parameter space of the minimal supersymmetric standard model from tunneling to charge- and/or color-breaking minima, taking into account thermal corrections. We pay particular attention to the region known as the Natural MSSM, where the masses of the scalar partners of the top quarks are within an order of magnitude or so of the electroweak scale. These constraints arise from the interaction between these scalar tops and the Higgs fields, which allows the possibility of parameter points having deep charge- and color-breaking true vacua. In addition to requiring that our electro-weak-symmetry-breaking, yet QCD- and electromagnetism-preserving vacuum has a sufficiently long lifetime at zero temperature, also demanding stability against thermal tunneling further restricts the allowed parameter space., Comment: 7 pages, 2 figures, software available from http://vevacious.hepforge.org/ - version 2 matches that accepted for publication in Phys. Lett. B

The recent discovery of a Higgs boson by the LHC experiments has profound implications for supersymmetric models. In particular, in the context of restricted models, such as the supergravity-inspired constrained minimal supersymmetric standard model, one finds that preferred regions in parameter space have large soft supersymmetry-breaking trilinear couplings. This potentially gives rise to charge- and/or color-breaking minima besides those with the correct breaking of $SU(2)_L \times U(1)_Y$. We investigate the stability of parameter points in this model against tunneling to possible deeper color- and/or charge-breaking minima of the one-loop effective potential. We find that allowed regions of the parameter space with light staus or with light stops are seriously constrained by the requirement that the tunneling time out of the normal electroweak-symmetry-breaking vacuum is more than a fifth of the age of the known Universe. We also find that "thumb rule" conditions on Lagrangian parameters based on specific directions in the tree-level potential are of limited use., 30 pages, 14 figures. V2: as published in JHEP (+1 extra ref.); while awaiting review, code error was found and tunneling times had to be re-done. Main results unchanged: quick tunneling to CCB minima restricts relevant parameter space; no analytic formula finds points with CCB global minima well. However, parameter space allowed by long tunneling times is larger: stau co-annihilation seems safe