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Purpose: The preplanned exploratory analysis of the BERIL-1 trial presented here aimed to identify biomarkers of response to the combination of buparlisib and paclitaxel.Patients and Methods: BERIL-1 was a multicenter, randomized, double-blind, placebo-controlled phase II study. Patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN) progressing on/after one previous platinum-based chemotherapy regimen in the recurrent or metastatic setting were treated with either buparlisib plus paclitaxel or placebo plus paclitaxel. Archival tumor tissue and ctDNA samples were analyzed for molecular alterations and immune infiltration using next-generation sequencing or immunohistochemistry.Results: Biomarker analyses were performed in randomized patients (n = 158) with available biomarker data. The most frequently (>5%) mutated genes were TP53, FAT1, TET2, KMT2D, PIK3CA, NOTCH1, NFE2L2, NOTCH2, CCND1, and CDKN2A. Patients with SCCHN tumors (from various primary sites) having HPV-negative status (HR = 0.51), TP53 alterations (HR = 0.55) or low mutational load (HR = 0.57) derived overall survival (OS) benefit with the combination of buparlisib and paclitaxel. OS benefit with this combination was also increased in patients with presence of intratumoral TILs ≥10% (HR = 0.51), stromal TILs ≥15% (HR = 0.53), intratumoral CD8-positive cells ≥5% (HR = 0.45), stromal CD8-positive cells ≥10% (HR = 0.47), or CD8-positive cells in invasive margins >25% (HR = 0.37). A trend for improved progression-free survival with the combination of buparlisib and paclitaxel was also observed in these patients.Conclusions: The BERIL-1 biomarker analyses showed that patients with TP53 alterations, HPV-negative status, low mutational load, or high infiltration of TILs or CD8-positive cells derived survival benefit with the combination of buparlisib and paclitaxel. Clin Cancer Res; 24(11); 2505–16. ©2018 AACR.

Purpose:Ribociclib plus endocrine therapy (ET) demonstrated a statistically significant progression-free survival and overall survival (OS) benefit in the phase III MONALEESA-7 trial of pre-/perimenopausal patients with hormone receptor (HR)-positive (HR+), HER2-negative (HER2−) advanced breast cancer (ABC). The median OS was not reached in the ribociclib arm in the protocol-specified final analysis; we hence performed an exploratory OS and additional outcomes analysis with an extended follow-up (median, 53.5 months).Patients and Methods:Patients were randomized to receive ET [goserelin plus nonsteroidal aromatase inhibitor (NSAI) or tamoxifen] with ribociclib or placebo. OS was evaluated with a stratified Cox proportional hazard model and summarized with Kaplan–Meier methods.Results:The intent-to-treat population included 672 patients. Median OS was 58.7 months with ribociclib versus 48.0 months with placebo [hazard ratio = 0.76; 95% confidence interval (CI), 0.61–0.96]. Kaplan–Meier estimated OS at 48 months was 60% and 50% with ribociclib and placebo, respectively. Subgroup analyses were generally consistent with the OS benefit, including patients who received NSAI and patients aged less than 40 years. Subsequent antineoplastic therapies following discontinuation were balanced between the ribociclib (77%) and placebo (78%) groups. Use of cyclin-dependent kinase 4/6 inhibitors after discontinuation was higher with placebo (26%) versus ribociclib (13%). Time to first chemotherapy was significantly delayed with ribociclib versus placebo. No drug–drug interactions were observed between ribociclib and either NSAI.Conclusions:Ribociclib plus ET continued to show significantly longer OS than ET alone in pre-/perimenopausal patients, including patients aged less than 40 years, with HR+/HER2− ABC with 53.5 months of median follow-up (ClinicalTrials.gov, NCT02278120).

Supplementary methods and results Supplementary Table 1: Mutational profiles of patients with HPV-positive vs HPV-negative tumors Supplementary Figure 1: Interaction between immune markers and other biomarkers

Supplementary Figure from Updated Overall Survival of Ribociclib plus Endocrine Therapy versus Endocrine Therapy Alone in Pre- and Perimenopausal Patients with HR+/HER2− Advanced Breast Cancer in MONALEESA-7: A Phase III Randomized Clinical Trial

Supplementary Table from Updated Overall Survival of Ribociclib plus Endocrine Therapy versus Endocrine Therapy Alone in Pre- and Perimenopausal Patients with HR+/HER2− Advanced Breast Cancer in MONALEESA-7: A Phase III Randomized Clinical Trial

In the context of cancer, clonal hematopoiesis of indeterminate potential (CHIP) is associated with the development of therapy-related myeloid neoplasms and shorter overall survival. Cell-free DNA (cfDNA) sequencing is becoming widely adopted for genomic screening of patients with cancer but has not been used extensively to determine CHIP status because of a requirement for matched blood and tumor sequencing. We present an accurate classification approach to determine the CH status from cfDNA sequencing alone, applying our model to 4324 oncology clinical cfDNA samples. Using this method, we determined that 30.3% of patients in this cohort have evidence of CH, and the incidence of CH varies by tumor type. Matched RNA sequencing data show evidence of increased inflammation, especially neutrophil activation, within the tumors and tumor microenvironments of patients with CH. In addition, patients with CH had evidence of neutrophil activation systemically, pointing to a potential mechanism of action for the worse outcomes associated with CH status. Neutrophil activation may be one of many mechanisms, however, because patients with estrogen receptor–positive breast cancer harboring TET2 frameshift mutations had worse outcomes but similar neutrophil frequencies to patients without CH. Together, these data show the feasibility of detecting CH through cfDNA sequencing alone and an application of this method, demonstrating increased inflammation in patients with CH both systemically and in the tumor microenvironment.

Background: In the ML program, PAM50-based intrinsic subtypes (ie, luminal A [LumA], luminal B [LumB], HER2 enriched [HER2E], and basal-like [Basal]) were found to be prognostic and predictive of ribociclib benefit in ABC (Prat et al. J Clin Oncol. 2021). While ribocliclib demonstrated benefit in all subtypes (except Basal, with a limited sample size), LumB and HER2E derived the largest benefit. However, DNA features of the intrinsic subtypes in the advanced setting remain unknown. Here, we report the results of genomic profiling of baseline circulating tumor DNA (ctDNA) by PAM50-based intrinsic subtypes across ML studies. Methods: A total of 883 of 2066 patients recruited in the ML-2, -3, and -7 phase 3 trials had both tumor intrinsic subtype and plasma ctDNA next-generation sequencing (NGS)-based data obtained at baseline (ie, before starting treatment). The NGS-based panel targeted exonic regions in approximately 550 genes sequenced on an Illumina HiSeq instrument. A total of 130 patients had the normal-like subtype and were excluded from further analyses. For genes altered in > 5% of patients, we assessed the differences in frequency across intrinsic subtypes. Genetic alterations included mutations, indels, and copy number alterations. For each gene, a Fisher exact test was used to test for differences in frequency across the subtypes. A false discovery rate (FDR) correction was used to adjust for multiple testing. For genes with FDR < 0.10, a logistic regression model was used to quantify the relationship between subtypes and alteration status. Also, we evaluated differences across subtypes for tumor mutational burden (TMB) using analysis of variance and for ctDNA fraction using a Kruskal-Wallis test. Results: Overall, gene amplifications were more frequent in the LumB, HER2E, and Basal subtypes. CCND1 (and genes FGF3/4/19 found in the same amplicon) was more frequently altered in HER2E (14.6%) and LumB (14.3%) than in the LumA (4.8%) subtype. Similarly, FGFR1 and MYC were more frequently altered in HER2E (13% and 9.8%), Basal (12.5% and 12.5%), and LumB (8.6% and 10%) than in the LumA (3.3% and 2.3%) subtype. PIK3CA alterations, including hotspot somatic mutations, were less frequent in Basal (12.5%) than in the LumB (27.6%), LumA (33.8%), and HER2E (37.4%) subtypes. In contrast, TP53 alterations were more frequent in Basal (66.7%) and HER2E (29.3%) than in the LumB (16.2%) and LumA (12.4%) subtypes. ERBB2 alterations (n = 25) were found in the LumA, LumB, and HER2E subtypes at similar frequencies (3%-4%). ESR1 did not show any significant difference across subtypes. TMB did not differ by subtype (P = .20), even when a TMB cutoff ≥ 10 was used (P = .23). Finally, ctDNA fraction differed across subtypes (P < .001), being significantly higher in the LumB (P < .001) and HER2E (P < .001) than in the LumA subtype. Conclusions: This is the first combined report of ctDNA NGS profiling and intrinsic molecular subtype in ABC. Differences in tumor DNA profiles were observed across PAM50 subtypes, with a trend for higher copy number alterations in HER2E and LumB than in the LumA subtype. LumA and Basal subtypes were found to have the most distinct genomic features. The Basal subtype is known to be similar to triple-negative BC from a clinical and biological perspective, which may explain the limited activity of ribociclib in this subgroup, as shown previously (Prat et al. J Clin Oncol. 2021). The pronounced activity of ribociclib in HER2E and LumB subtypes, which are enriched with somatic alterations associated with endocrine therapy resistance and tend to have a worse prognosis, warrants further investigation. Citation Format: Aleix Prat, Nadia Solovieff, Faye Su, Aditya Bardia, Patrick Neven, Gabriel N. Hortobagyi, Debu Tripathy, Stephen Chia, Dennis Slamon, Yen-Shen Lu, Tetiana Taran, Agnes Lteif, Carlos L. Arteaga, Fabrice André. Genomic profiling of PAM50-based intrinsic subtypes in HR+/HER2- advanced breast cancer (ABC) across the MONALEESA (ML) studies [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 PD2-05.

Background: In addition to inhibiting cell cycle progression, CDK4/6 inhibitors have been found to modulate anti-cancer immune response (Goel S, et al. Nature. 2017;548:471-475). Data from preclinical studies suggested multiple mechanisms—involving changes in both cancer cells and immune cells—by which CDK4/6 inhibition can enhance anti-cancer immunity. These mechanisms include increased antigen presentation, interferon response triggering, and regulatory T-cell suppression. Clinical correlates to preclinical observations have so far been obtained using paired biopsies from neoadjuvant breast cancer trials (Johnston S, et al. J Clin Oncol. 2019;37:178-189; Hurvitz S, et al. Clin Cancer Res. 2020; ;26:566-80). Here, we describe a gene expression analysis of paired pre- and on-treatment biopsies from CLEE011A2115C and CLEE011X2107, two phase I clinical trials evaluating ribociclib in combination with ET in the metastatic breast cancer (MBC) setting. Methods: The nCounter PanCancer IO 360 Panel (NanoString) was used to quantify expression of 770 genes in 7 pairs of tumor samples (baseline vs. cycle 1 day 15) from two phase I trials in the MBC setting (5 patients from CLEE011A2115C and 2 from CLEE011X2107). All patients were treated with ribociclib and an ET. Pairwise differential gene expression analysis of individual genes and previously published immune-related gene signatures was conducted (Bedognetti D, et al. Curr Opin Oncol. 2015;27:433-444). Results: Many genes that were markedly suppressed with treatment (eg, MKI67, MYC, CDK2, CCNB1, TYMS, and DNMT1) were related to proliferation, DNA replication, and G1/S transition of the cell cycle, as expected. Interestingly, expression of numerous genes involved in immune response was increased. These were mostly interferon-regulated genes; particularly, a gene signature indicative of a T cell-inflamed tumor microenvironment (Ayers M, et al. J Clin Invest. 2017;127:2930-2940) was upregulated. Immune-related genes tended to preferentially increase with treatment in patients who later experienced a clinical response. Since bulk RNA from tumor biopsies was analyzed, we could not distinguish whether the observed expression changes reflected increased infiltration by immune cells or an endogenous interferon response in cancer cells. The latter could be triggered by DNMT1 suppression (as previously described), which then promotes T cell-mediated immunity by increasing antigen presentation and chemokine production in cancer cells. Conclusions: This analysis was conducted with a small sample size and is considered hypothesis generating; further investigation is needed. Cell cycle and proliferation markers showed robust suppression by ribociclib-based treatment regimens, as expected. Conversely, a notable increase in immune-related genes was detected that tended to occur preferentially in patients who later experienced more favorable clinical outcomes. Both changes occurred early (cycle 1 day 15). To our knowledge, this is the first clinical biomarker report of immune activation mediated by CDK4/6 inhibition in MBC. Citation Format: Dejan Juric, Chong Ma, Ralph Tiedt, Yoon-Sim Yap, Joanne Chiu, Pamela Munster, Roohi Ismail-Khan, Laura Garcia-Estevez, Ingrid A. Mayer, Carlos Becerra, Nadia Solovieff, Agnes Lteif, Faye Su, Yen-Shen Lu. Upregulation of immune response biomarkers by ribociclib plus endocrine therapy (ET) in paired tumor samples from phase I studies [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 P5-13-18.

Background: The Phase III MONALEESA (ML)-2, -3, and -7 trials showed significant improvement in progression-free survival (PFS) and overall survival (OS) with ribociclib (RIB) + endocrine therapy (ET) over placebo (PBO) + ET in patients (pts) with HR+/HER2− advanced breast cancer (ABC); improvement in OS with cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) has been observed in some, but not all clinical trials. Gene expression analyses for each separate ML study were reported previously. Given the differences in CDK4 vs CDK6 inhibition between RIB and other CDK4/6i, we evaluated the association between cell cycle (CC)–related genes and outcomes based on pooled analysis of gene expression using tumor samples from the ML-2, -3, and -7 trials. Methods: Gene expression data were generated from pre-treatment archival tumor samples (primary, 73%; metastatic, 27%) with a customized NanoString nCounter panel (781 genes) including genes involved in CC, other signaling pathways, and breast cancer biology. Samples were pooled from 1139 pre- and postmenopausal pts with HR+/HER2− ABC across the 3 ML studies, which included pts on first- and second-line therapy. Data were categorized into training (80%) and test (20%) datasets. The training dataset was used to analyze each gene (modeled continuously) individually for an association with PFS, and genes with a gene × treatment (tx) interaction P value Results: This report focused on CC-related genes and signatures. Gene expression levels of CDKN2B and the expression ratio of CCND1/CDKN2A showed a predictive relationship with benefit from RIB in both training and test sets (Table). PFS benefit with RIB was consistent regardless of the CDK4/CDK6 expression ratio or level of expression of CCNE1, CDK2, RB1, combined CC-related genes, E2F gene signatures, RB gene signature, combined DNA-replication genes, or combined proliferation-related genes. A machine learning approach identified a clinico-genomic signature that was prognostic for PFS benefit with RIB. Selected variables included gene expression levels of FXBO5, PGR, RBBP8, and STC2 and several clinical features (tx arm, de novo disease, prior ET, and visceral disease). Pts with a low signature score had a longer mPFS vs pts with a high signature score, in the RIB (HR, 0.37; 95% CI, 0.22-0.62) and PBO (HR, 0.30; 95% CI, 0.15-0.59) arms. Conclusion: In the largest pooled analysis of the association of gene expression profile data with CDK4/6i tx response in pts with HR+/HER2− ABC, the PFS benefit with RIB + ET over ET alone was consistent irrespective of expression levels of most CC genes. Variation in magnitude of RIB benefit was observed, depending on CDKN2B expression levels, CCND1/CDKN2A expression ratio, and machine learning–derived signature scores. The clinico-genomic CDK4/6i signature requires validation in additional datasets. Table 1: Progression-Free Survival by Gene Expression Subgroup Citation Format: Aditya Bardia, Faye Su, Nadia Solovieff, Fabrice Andre, Carlos Arteaga, Patrick Neven, Yoon-Sim Yap, Yen-Shen Lu, Stephen K. Chia, Dennis Slamon, Seock-Ah Im, Arunava Chakravartty, Agnes Lteif, Tetiana Taran, Debu Tripathy. Pooled gene expression analysis and association with treatment response in patients with HR+/HER2− advanced breast cancer in the MONALEESA-2, -3, and -7 trials [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 PD17-08.

Background: The prognostic and predictive value of the 4 main intrinsic subtypes of breast cancer (ie, luminal A [LumA], luminal B [LumB], human epidermal growth factor receptor 2 enriched [HER2E], and basal-like) in hormone receptor-positive, HER2− advanced breast cancer (ABC) treated with endocrine therapy (ET) and ribociclib (RIB) is currently unknown. The MONALEESA-2, -3, and -7 trials all showed a significant benefit in progression-free survival (PFS) with RIB over placebo (PBO; Hortobagyi et al. Ann Oncol. 2018; Slamon et al. J Clin Oncol. 2018; Tripathy et al. Lancet Oncol. 2018). Here, we correlate ABC intrinsic subtypes with the PFS benefit of RIB in the MONALEESA trials. Methods: Patient samples from the MONALEESA-2, -3, and -7 trials underwent PAM50-based subtyping (blinded from clinical data), and the correlation between intrinsic subtype and PFS was analyzed. Gene expression profiling of formalin-fixed, paraffin-embedded tumor samples was performed using a customized NanoString nCounter GX 800-gene panel. The prognostic and/or predictive relationship of PAM50-based subtypes with PFS and the risk of tumor progression by subtype were evaluated using univariate and multivariable Cox proportional hazards models. Multivariable models were adjusted for known clinical prognostic factors, including age, prior chemotherapy, prior ET, ECOG performance status, visceral disease (presence of liver/lung metastases), bone-only metastases, histological grade, number of metastatic sites, and de novo metastatic disease. Results: A total of 1160 tumor samples from both the RIB (n = 672) and PBO (n = 488) treatment arms of the MONALEESA trials were profiled. Subtype distribution was generally consistent across treatment arms (Table). The associations between intrinsic subtypes and PFS were statistically significant in both treatment arms (P < .0001). Compared with patients with LumA subtype, which is the subtype that is the most prevalent and has the best prognostic outcome, patients with LumB, HER2E, and basal-like subtypes had a 1.41, 2.30, and 3.97 times higher risk of tumor progression, respectively, after adjusting for other clinical-pathologic variables and treatment arm. In terms of treatment benefit, all subtypes except for basal-like showed a significant PFS benefit with RIB treatment (Table). Patients with HER2E (hazard ratio [HR], 0.389; P < .0001), LumB (HR, 0.521; P = .0001), LumA (HR, 0.633; P = .0007), and normal-like (HR, 0.467; P = .0005) subtypes all derived benefit from RIB treatment, with HER2E demonstrating the greatest benefit. Patients with the basal-like subtype (n = 30) did not derive benefit from RIB (HR, 1.15; P = .767), although these results should be interpreted with caution due to the small sample size (RIB: 2%; PBO: 3%). Conclusions: This is the largest analysis evaluating the correlation of intrinsic ABC subtype with efficacy outcomes in patients treated with CDK4/6 inhibitors. Patients with HER2E, LumA, LumB, and normal-like subtypes all exhibited a consistent PFS benefit with RIB treatment, while patients with basal-like ABC (RIB: 2%; PBO: 3%) did not. The HER2E subtype (RIB: 14%; PBO: 11%) exhibited the greatest relative reduction in risk of progression or death (61%) with RIB plus ET. Table.SubtypeTreatment ArmDistribution, n (%)Median PFS, months, (95% CI)HRP ValueLuminal ARIB320 (48)29.60 (23.03-NA)0.63.0007PBO222 (45)19.48 (15.61-24.80)Luminal BRIB154 (23)22.21 (18.79-NA)0.52< .0001PBO124 (25)12.85 (10.98414.82)HER2-enrichedRIB95 (14)16.39 (12.71-24.6)0.39< .0001PBO52 (11)5.52 (3.12-9.17)BasalRIB16 (2)3.71 (1.91-13)1.15.77PBO14 (3)3.58 (1.87-NA)NormalRIB87 (13)22.34 (16.56-NA)0.47.0005PBO76 (16)11.10 (7.39-16.56)NA, not achieved. Citation Format: Aleix Prat, Anwesha Chaudhury, Nadia Solovieff, Laia Paré, Debora Martinez, Nuria Chic, Olga Martínez, Fara Brasó-Maristany, Karen Rodriguez-Lorenc, Tetiana Taran, Naveen Babbar, Faye Su. Correlative biomarker analysis of intrinsic subtypes and efficacy across the MONALEESA Phase III studies [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 GS1-04.

Background: Genetic alterations that contribute to resistance to therapy may be acquired during treatment (tx) for hormone receptor−positive/human epidermal growth factor receptor−negative (HR+/HER2−) advanced breast cancer (ABC). A previous pooled analysis of circulating tumor DNA (ctDNA) in MONALEESA (ML)-2, -3, and -7 identified potential predictive biomarkers for response and resistance to ribociclib (RIB) at baseline (BL). Here, we describe an analysis of paired BL and end of treatment (EOT) samples from ML-2, -3, and -7 to identify acquired mechanisms that may impact resistance to RIB + endocrine therapy (ET) vs placebo (PBO) + ET. Methods: ML-2 (NCT01958021), ML-3 (NCT02422615), and ML-7 (NCT02278120) evaluated efficacy and safety of RIB + ET vs PBO + ET in pre- and postmenopausal patients (pts) with HR+/HER2− ABC treated in first-line (1L) and second-line (2L) settings. Plasma samples were collected at cycle 1 day 1 (C1D1; prior to first therapy exposure) and at EOT (± 28 days of recorded progression). ctDNA was sequenced using a targeted next-generation sequencing panel of ≈550 genes. Genes with an alteration frequency of >5% at EOT, regardless of their frequency at BL, were included. Tumor mutational burden (TMB) was assessed by tx arm; a TMB cutoff of 10 mutations/MB was used to categorize pts as TMB high vs low. To assess differences in the presence of alterations, a McNemar test was performed on paired samples and adjusted (adj) for multiple testing using the false discovery rate (FDR). A Bayesian mixed effects model was used to account for ctDNA fraction and trial and to test for tx-specific resistance by including a tx × visit interaction term. Results: A total of 905 paired samples from ML-2, -3, and -7 were included in this analysis, 441 and 464 samples from pts treated with RIB + ET and PBO + ET, respectively. Overall, 17 genes had an alteration frequency of >5% at EOT. The ctDNA fraction was higher at EOT vs C1D1 in both the RIB (P=.037) and PBO (P=.033) arms. The frequency of alterations in RB1 (10.4% vs 2.0%), ATM (11.3% vs 8.4%), FAT1 (4.8% vs 3.0%), and FAT3 (5.0% vs 2.5%) was higher at EOT vs C1D1 in the RIB arm (FDR-adj P10) at EOT increased from 1.1% to 5.7% in the RIB arm and from 1.7% to 3% in the PBO arm. After accounting for ctDNA fraction and trial, a larger numerical increase in TMB was observed for RIB (odds ratio [OR], 9.0; 95% CI, 2.9-32.7) vs PBO (OR, 2.1; 95% CI, 0.7-6.5); however, the model did not support a differential tx effect. Conclusions: This comprehensive analysis of pooled samples from ML-2, -3, and -7 identified acquired gene alterations in pts with HR+/HER2− ABC treated with 1L or 2L RIB + ET or PBO + ET. The frequency of several genes known to contribute to resistance (ESR1, RB1, ATM, FAT1, and FAT3) was higher at EOT vs C1D1 in pts treated with RIB + ET, while ESR1 and GATA3 alterations were higher at EOT vs C1D1 in pts treated with PBO + ET. This paired dataset of BL and EOT samples from pts with HR+/HER2− ABC treated with a CDK4/6 inhibitor and ET is the largest to date and could be used to validate and confirm acquired resistance mechanisms with low alteration frequency. Citation Format: Fabrice Andre, Nadia Solovieff, Faye Su, Aditya Bardia, Patrick Neven, Yoon-Sim Yap, Debu Tripathy, Yen-Shen Lu, Dennis Slamon, Stephen K. Chia, Mukta Joshi, Arunava Chakravartty, Agnes Lteif, Tetiana Taran, Carlos Arteaga. Identification of mechanisms of acquired resistance to ribociclib plus endocrine therapy using baseline and end-of-treatment circulating tumor DNA samples in the MONALEESA-2, -3, and -7 trials [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-14.

Purpose: Ribociclib plus endocrine therapy (ET) demonstrated a statistically significant progression-free survival and overall survival (OS) benefit in the phase III MONALEESA-7 trial of pre-/perimenopausal patients with hormone receptor (HR)-positive (HR+), HER2-negative (HER2−) advanced breast cancer (ABC). The median OS was not reached in the ribociclib arm in the protocol-specified final analysis; we hence performed an exploratory OS and additional outcomes analysis with an extended follow-up (median, 53.5 months). Patients and Methods: Patients were randomized to receive ET [goserelin plus nonsteroidal aromatase inhibitor (NSAI) or tamoxifen] with ribociclib or placebo. OS was evaluated with a stratified Cox proportional hazard model and summarized with Kaplan–Meier methods. Results: The intent-to-treat population included 672 patients. Median OS was 58.7 months with ribociclib versus 48.0 months with placebo [hazard ratio = 0.76; 95% confidence interval (CI), 0.61–0.96]. Kaplan–Meier estimated OS at 48 months was 60% and 50% with ribociclib and placebo, respectively. Subgroup analyses were generally consistent with the OS benefit, including patients who received NSAI and patients aged less than 40 years. Subsequent antineoplastic therapies following discontinuation were balanced between the ribociclib (77%) and placebo (78%) groups. Use of cyclin-dependent kinase 4/6 inhibitors after discontinuation was higher with placebo (26%) versus ribociclib (13%). Time to first chemotherapy was significantly delayed with ribociclib versus placebo. No drug–drug interactions were observed between ribociclib and either NSAI. Conclusions: Ribociclib plus ET continued to show significantly longer OS than ET alone in pre-/perimenopausal patients, including patients aged less than 40 years, with HR+/HER2− ABC with 53.5 months of median follow-up (ClinicalTrials.gov, NCT02278120).

In the context of cancer, clonal hematopoiesis of indeterminate potential (CHIP) is associated with the development of therapy-related myeloid neoplasms and shorter overall survival. Cell-free DNA (cfDNA) sequencing is becoming widely adopted for genomic screening of cancer patients but has not been used extensively to determine CHIP status due to a requirement for matched blood and tumor sequencing. Here we present an accurate machine learning approach to determine clonal hematopoiesis (CH) status from cfDNA sequencing alone and apply our model to 4,096 oncology clinical cfDNA samples. Using this method, we determine that 26% of patients in this cohort have evidence of CH and CH is most common in lung cancer patients. Matched RNAseq data shows signals of increased inflammation, especially neutrophil activation, within the tumor microenvironment of CH-positive patients. Additionally, CH patients showed evidence of neutrophil activation systemically, pointing to a potential mechanism of action for the worse outcomes associated with CH status. Neutrophil activation may be one of many mechanisms however, as estrogen positive breast cancer patients harboring TET2 frameshift mutations had worse outcomes but similar neutrophil levels to CH-negative patients.One Sentence SummaryWe train an accurate machine learning model to detect clonal hematopoiesis in cancer and characterize associated changes in the tumor microenvironment.

Using an ORF kinome screen in MCF-7 cells treated with the CDK4/6 inhibitor ribociclib plus fulvestrant, we identified FGFR1 as a mechanism of drug resistance. FGFR1-amplified/ER+ breast cancer cells and MCF-7 cells transduced with FGFR1 were resistant to fulvestrant ± ribociclib or palbociclib. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Addition of the FGFR TKI erdafitinib to palbociclib/fulvestrant induced complete responses of FGFR1-amplified/ER+ patient-derived-xenografts. Next generation sequencing of circulating tumor DNA (ctDNA) in 34 patients after progression on CDK4/6 inhibitors identified FGFR1/2 amplification or activating mutations in 14/34 (41%) post-progression specimens. Finally, ctDNA from patients enrolled in MONALEESA-2, the registration trial of ribociclib, showed that patients with FGFR1 amplification exhibited a shorter progression-free survival compared to patients with wild type FGFR1. Thus, we propose breast cancers with FGFR pathway alterations should be considered for trials using combinations of ER, CDK4/6 and FGFR antagonists., Era+ breast cancer patients often develop resistance to endocrine therapy. Here, the authors show that FGFR1 amplification is a resistance mechanism to CDK4/6 inhibitor and endocrine therapy and that combined treatment with FGFR, CDK4/6, and anti-estrogens is a potential therapeutic strategy in Era+ breast cancer tumors.

Background: In the Phase III MONALEESA-3 (NCT02422615) study, ribociclib (RIB; cyclin-dependent kinase 4/6 inhibitor) + fulvestrant (FUL) significantly prolonged progression-free survival (PFS) vs placebo (PBO) + FUL in postmenopausal women with hormone receptor-positive (HR+), HER2-negative (HER2–) advanced breast cancer (ABC). Here we present MONALEESA-3 efficacy data by molecular alterations detected in circulating tumor DNA (ctDNA) at baseline. Methods: Postmenopausal women (N=726) with HR+, HER2– ABC (treatment naïve or received ≤1 line of prior endocrine therapy for ABC) were randomized 2:1 to RIB or PBO (600 mg/day; 3-weeks-on/1-week-off) + FUL (500 mg per label). Biomarker analysis of baseline ctDNA was an exploratory endpoint. Baseline plasma samples were collected from 692 patients (pts), and plasma ctDNA was analyzed using next-generation sequencing with a targeted panel of ˜550 genes. The subgroup analysis was performed in 600 pts (RIB + FUL n=400; PBO + FUL n=200) with evaluable ctDNA data. Results were not adjusted for multiple testing. Results: Alterations (frequency) were observed in the following genes: PIK3CA (35%), ESR1 (14%), TP53 (18%), CDH1 (12%), FGFR1 (5%), FGFR1/ZNF703/WHSC1L1 (11%), cell cycle-related (CCC) genes (16%), and genes involved in receptor tyrosine kinase (RTK) signaling (20%), and the mitogen-activated protein kinase (MAPK) pathway (10%). PFS hazard ratios favored RIB vs PBO regardless of baseline ctDNA gene alteration status (Table). Events, n/NMedian PFS, months Gene(s)RIB + FULPBO + FULRIB + FULPBO + FULHazard ratio; 95% confidence intervalPIK3CAWT108/26570/12422.3416.490.67; 0.49–0.91Altered75/13549/7616.3611.100.75; 0.52–1.08ESR1WT141/33899/17622.3414.880.66; 0.51–0.85Altered42/6220/249.236.110.71; 0.41–1.23TP53WT132/32598/17022.3414.880.62; 0.47–0.80Altered51/7521/309.177.390.75; 0.44–1.27CDH1WT156/35697/17320.6316.490.70; 0.54–0.90Altered27/4422/2712.095.130.46; 0.24–0.86FGFR1WT165/378113/19221.3214.550.65; 0.51–0.83Altered18/226/88.346.110.64; 0.23–1.75FGFR1/ZNF703/WHSC1L1WT155/356105/18121.3214.650.67; 0.52–0.85Altered28/4414/1910.976.670.73; 0.37–1.43CCCWT138/331103/17422.0814.650.61; 0.47–0.79Altered45/6916/2612.659.130.92; 0.51–1.65RTKWT126/31595/16622.3414.820.61; 0.47–0.80Altered57/8524/349.175.780.82; 0.50–1.35MAPKWT158/357105/18320.0414.820.69; 0.54–0.89Altered25/4314/1714.723.610.47; 0.22–1.00 Numerically shorter PFS was observed in pts with altered vs wildtype (WT) genes, irrespective of treatment. A slight trend towards limited RIB benefit vs PBO was observed in pts with altered CCC and RTK genes. RIB benefit was more pronounced vs PBO in pts with altered CDH1 and MAPK genes. PIK3CA and ESR1 analyses for pts receiving treatment in the first- or second-line setting will be presented at the meeting. Conclusions: Generally consistent PFS benefit for RIB + FUL vs PBO + FUL was observed irrespective of baseline ctDNA alterations; altered status trended to correlate with a shorter PFS. Results are hypothesis generating and should be interpreted with caution for some subgroups due to small sample sizes. Citation Format: Neven P, Petrakova K, Val Bianchi G, De la Cruz-Merino L, Jerusalem G, Sonke GS, Nusch A, Beck JT, Chia S, Solovieff N, Rodriguez Lorenc K, Miller M, Su F, Lm S-A. Biomarker analysis by baseline circulating tumor DNA alterations in the MONALEESA-3 study [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 PD2-05.

PURPOSE The prognostic and predictive value of intrinsic subtypes in hormone receptor–positive, human epidermal growth factor receptor 2–negative advanced breast cancer treated with endocrine therapy and ribociclib (RIB) is currently unknown. We evaluated the association of intrinsic subtypes with progression-free survival (PFS) in the MONALEESA trials. METHODS A retrospective and exploratory PAM50-based analysis of tumor samples from the phase III MONALEESA-2, MONALEESA-3, and MONALEESA-7 trials was undertaken. The prognostic relationship of PAM50-based subtypes with PFS and risk of disease progression by subtype and treatment were evaluated using a multivariable Cox proportional hazards model, adjusting for age, prior chemotherapy, performance status, visceral disease, bone-only metastases, histological grade, number of metastatic sites, prior endocrine therapy, and de novo metastatic disease. RESULTS Overall, 1,153 tumors from the RIB (n = 667) and placebo (n = 486) cohorts were robustly profiled. Subtype distribution was luminal A (LumA), 46.8%; luminal B (LumB), 24.0%; normal-like, 14.1%; HER2-enriched (HER2E), 12.6%; and basal-like, 2.4% and was generally consistent across treatment arms and trials. The associations between subtypes and PFS were statistically significant in both arms ( P < .001). The risks of disease progression for LumB, HER2E, and basal-like subtypes were 1.44, 2.32, and 3.87 times higher compared with those for LumA, respectively. All subtypes except basal-like demonstrated significant PFS benefit with RIB. HER2E (hazard ratio [HR], 0.40; P < .0001), LumB (HR, 0.52; P < .0001), LumA (HR, 0.63; P = .0007), and normal-like (HR, 0.47; P = .0005) subtypes derived benefit from RIB. Patients with basal-like subtype (n = 28) did not derive benefit from RIB (HR, 1.14; P = .78). CONCLUSION In this retrospective exploratory analysis of hormone receptor–positive and human epidermal growth factor receptor 2–negative advanced breast cancer, each intrinsic subtype exhibited a consistent PFS benefit with RIB, except for basal-like.

PURPOSE This analysis evaluated the genomic landscape of premenopausal patients with hormone receptor–positive and human epidermal growth factor receptor 2–negative advanced breast cancer and the association of genetic alterations with response to ribociclib in the phase III MONALEESA-7 trial. METHODS Premenopausal patients were randomly assigned 1:1 to receive endocrine therapy plus ribociclib or placebo. Plasma collected at baseline was sequenced using targeted next-generation sequencing for approximately 600 relevant cancer genes. The association of circulating tumor DNA alterations with progression-free survival (PFS) was evaluated to identify biomarkers of response and resistance to ribociclib. RESULTS Baseline circulating tumor DNA was sequenced in 565 patients; 477 evaluable patients had evidence of ≥ 1 alteration. The most frequent alterations included PIK3CA (29%), TP53 (19%), CCND1 (10%), MYC (8%), GATA3 (8%), receptor tyrosine kinases (18%), and the Chr8p11.23 locus (13%). A treatment benefit of ribociclib was seen with wild-type (hazard ratio [HR] 0.44 [95% CI, 0.32 to 0.61]) and altered (HR 0.56 [95% CI, 0.36 to 0.89]) PIK3CA. Overall, patients with altered CCND1 had shorter PFS regardless of treatment, suggesting CCND1 as a potential prognostic biomarker. Benefit with ribociclib was seen in patients with altered (HR 0.22 [95% CI, 0.08 to 0.55]) or wild-type (HR 0.50 [95% CI, 0.38 to 0.67]) CCND1, but greater benefit was observed with altered, suggesting predictive potential of CCND1. Alterations in TP53, MYC, Chr8p11.23 locus, and receptor tyrosine kinases were associated with worse PFS, but ribociclib benefit was independent of alteration status. CONCLUSION In this study—to our knowledge, the first large study of premenopausal patients with hormone receptor–positive and human epidermal growth factor receptor 2–negative advanced breast cancer—multiple genomic alterations were associated with poor outcome. A PFS benefit of ribociclib was observed regardless of gene alteration status, although in this exploratory analysis, a magnitude of benefits varied by alteration.

Background: The MONALEESA (ML)-2, -3, and -7 trials have shown a significant benefit in overall survival (OS) with ribociclib (RIB) + endocrine therapy (ET) over placebo (PBO) + ET in HR+/HER2− advanced breast cancer. HR+ breast cancer is a clinically and biologically heterogeneous disease, with identified intrinsic subtypes that vary in incidence, survival rate, and response to treatment. In a pooled analysis of the ML studies, patients with both luminal and HER2-enriched (HER2E) subtypes exhibited a consistent progression-free survival benefit with RIB + ET. The HER2E subtype (RIB, 14%; PBO, 11%), which is associated with ET resistance and poor outcomes, exhibited the greatest relative reduction in risk of progression or death (61%) with RIB + ET. Here, we report a pooled analysis of the ML-2, -3, and -7 trials, presenting OS by intrinsic subtype.. Methods: Tumor samples from patients enrolled in the ML-2, -3, and -7 trials underwent PAM50-based subtyping (blinded from clinical data), and the correlation between intrinsic subtype and OS was analyzed. Gene expression profiling of formalin-fixed, paraffin-embedded tumor samples was performed using a customized NanoString nCounter GX 800-gene panel. The prognostic and/or predictive relationship between PAM50-based subtypes and OS was evaluated using univariable and multivariable Cox proportional hazards models. Multivariable models were adjusted for known clinical prognostic factors, including age, prior chemotherapy, prior ET, ECOG performance status, visceral disease (presence of liver/lung metastases), bone-only metastases, histological grade, number of metastatic sites, tumor type, and de novo metastatic disease.. Results: From the pooled patient population (N = 2066), 997 tumor (71% primary) samples from the RIB (n = 585) and PBO (n = 412) arms of the ML trials (ML-2, n = 318; ML-3, n = 414; ML-7, n = 265) were profiled. Subtype distribution was consistent across treatment arms (Table). A similar benefit with RIB vs PBO was observed in the ITT (hazard ratio [HR], 0.76; 95% CI, 0.67-0.86) and biomarker (HR, 0.75; 95% CI, 0.63-0.89) populations. In both univariable and multivariable analyses, intrinsic subtype was prognostic for OS in both the RIB and PBO arms (P < .0001 for both arms); patients with luminal A subtype had the best OS outcomes in both arms, whereas patients with basal-like subtype had the worst OS outcomes. Intrinsic subtype was also predictive of OS (subtype-treatment interaction: P = .016 [univariable], P = .007 [multivariable]) with a consistent OS benefit with RIB treatment in all subtypes except for basal-like (Table). Patients with HER2E (HR, 0.60; P = .018), luminal B (HR, 0.69; P = .023), and luminal A (HR, 0.75; P = .021) subtypes all derived benefit from RIB. In patients with the basal-like subtype (n = 30), the HR was 1.89 (P = .148); these results should be interpreted with caution due to the small sample size (3% in each arm) and exploratory nature of this analysis.Conclusions: This pooled analysis of the ML trials confirmed the prognostic and predictive value of intrinsic subtype (PAM50 based) for OS. The addition of RIB to ET resulted in consistent OS benefit across all subtypes except for basal-like. The. consistent survival benefit in the HER2E subtype, which is associated with endocrine resistance and a very poor prognosis compared with luminal disease, warrants further investigation. SubtypeTreatment ArmDistribution, n (%)OS, median (95% CI), monthsHR (95% CI)P ValueaLuminal ARIB320 (55)68.0 (61.5-NR)0.75 (0.58-0.96).021PBO222 (54)54.6 (48.3-66.2)Luminal BRIB154 (26)58.8 (48.3-79.2)0.69 (0.50-0.95).023PBO124 (30)44.9 (35.5-52.6)HER2-enrichedRIB95 (16)40.3 (33.4-49.0)0.60 (0.40-0.92).018PBO52 (13)29.4 (23.9-42.0)Basal-likeRIB16 (3)19.4 (10.7-33.2)1.89 (0.80-4.47).148PBO14 (3)21.2 (12.8-NR)aP values are all two-sided. NR, not reached. Citation Format: Lisa Carey, Nadia Solovieff, Fabrice André, Joyce O'Shaughnessy, David A Cameron, Wolfgang Janni, Gabe S Sonke, Yoon-Sim Yap, Denise A Yardley, Juan Pablo Zarate, Tetiana Taran, Faye Su, Agnes Lteif, Aleix Prat. Correlative analysis of overall survival by intrinsic subtype across the MONALEESA-2, -3, and -7 studies of ribociclib + endocrine therapy in patients with HR+/HER2− advanced breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS2-00.

Background: The addition of first-line ribociclib (RIB; cyclin-dependent kinase 4/6 inhibitor) to letrozole (LET) significantly improved progression-free survival (PFS) compared with placebo (PBO) + LET in patients (pts) with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–) advanced breast cancer (ABC) in the Phase III MONALEESA-2 study. Identifying biomarkers that predict response to treatment remains a key challenge in pts with HR+ ABC. Here we analyze results from MONALEESA-2 by molecular alterations detected in circulating tumor DNA (ctDNA) at baseline, including PIK3CA mutations and other alterations considered to be important in HR+ ABC. Methods: Postmenopausal women (N=668) with HR+, HER2– ABC who had not received any prior therapy for ABC were randomized 1:1 to RIB (600 mg/day; 3-weeks-on/1-week-off) + LET (2.5 mg/day; continuous) or PBO + LET. The primary endpoint was PFS. Biomarker analysis of the ctDNA mutation profile was an exploratory endpoint. Plasma samples for ctDNA analysis were collected at baseline and end of treatment. ctDNA was analyzed using next-generation sequencing with a targeted panel of ˜550 genes. Results: Baseline ctDNA was successfully sequenced in 494 pts (RIB + LET: n=212; PBO + LET: n=215); 67 (14%) of 494 pts were removed from the analysis due to limited tumor DNA in circulation. 427 (86%) pts had ≥1 alteration, including 1,573 mutations, 513 short insertions/deletions, 166 amplifications, and 8 translocations. Alterations (frequency) were commonly observed in the following genes: PIK3CA (33%), TP53 (12%), ZNF703/FGFR1 (5%), and ESR1 (4%), and in genes involved in receptor tyrosine kinase (RTK) signaling (12%). RIB + LET treatment benefit was consistent in pts with wild-type (WT) and altered PIK3CA, and in pts with WT and altered TP53 (Table). RIB + LET improved PFS regardless of RTK or ZNF703/FGFR1 alterations. However, there was a weak trend for increased benefit in pts with WT vs altered RTK genes and in pts with WT vs altered ZNF703/FGFR1 genes. These results should be interpreted with caution due to the small number of pts with these alterations. There were too few ESR1 alterations for firm conclusions to be drawn. Events, n/NMedian PFS, months Gene(s)RIB + LETPBO + LETRIB + LETPBO + LETHazard ratio (95% confidence interval)PIK3CAWT54/14393/14229.614.70.44 (0.31–0.62)Altered40/6955/7319.212.70.53 (0.35–0.81)TP53WT72/180129/19427.614.70.44 (0.33–0.59)Altered22/3219/2110.25.50.43 (0.23–0.83)ZNF703/FGFR1WT88/202139/20524.814.60.47 (0.36–0.62)Altered6/109/1010.611.40.73 (0.23–2.29)RTKWT81/189128/18724.814.40.46 (0.35–0.61)Altered13/2320/2821.311.40.72 (0.34–1.53) Conclusions: Consistent RIB + LET treatment benefit was observed compared with PBO + LET, irrespective of the status of baseline ctDNA biomarkers. Citation Format: Hortobagyi GN, Stemmer S, Campone M, Sonke GS, Arteaga CL, Paluch-Shimon S, Petrakova K, Villanueva C, Nusch A, Grischke E-M, Chan A, Jakobsen E, Marschner N, Hart LL, Alba E, Ohnstand HO, Blau S, Yardley DA, Solovieff N, Su F, Germa C, Yap Y-S. First-line ribociclib + letrozole in hormone receptor-positive, HER2-negative advanced breast cancer: Efficacy by baseline circulating tumor DNA alterations in MONALEESA-2 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-06.

Childhood maltreatment is highly prevalent and serves as a risk factor for mental and physical disorders. Self-reported childhood maltreatment appears heritable, but the specific genetic influences on this phenotype are largely unknown. The aims of this study were to 1) identify genetic variation associated with reported childhood maltreatment, 2) calculate the relevant SNP-based heritability estimates, and 3) quantify the genetic overlap of reported childhood maltreatment with mental and physical health-related phenotypes. Genome-wide association analysis for childhood maltreatment was undertaken, using a discovery sample from the UK Biobank (UKBB) (n=124,000) and a replication sample from the Psychiatric Genomics Consortium–posttraumatic stress disorder working group (PGC-PTSD) (n=26,290). Heritability estimations for childhood maltreatment and genetic correlations with mental/physical health traits were calculated using linkage disequilibrium score regression (LDSR). Two genome-wide significant loci associated with childhood maltreatment, located on chromosomes 3p13 (rs142346759, beta=0.015, p=4.35×10−8,FOXP1) and 7q31.1 (rs10262462, beta=-0.016, p=3.24×10−8,FOXP2), were identified in the discovery dataset but were not replicated in the PGC-PTSD sample. SNP-based heritability for childhood maltreatment was estimated to be ∼6%. Childhood maltreatment was most significantly genetically correlated with depressive symptoms (rg=0.70, p=4.65×10−40). This is the first large-scale genetic study to identify specific variants associated with self-reported childhood maltreatment.FOXPgenes could influence traits such as depression and thereby be relevant to childhood maltreatment. Alternatively, these variants may be associated with a greater likelihood of reporting maltreatment. A clearer understanding of the genetic relationships of childhood maltreatment, including particular abuse subtypes, with various psychiatric disorders, may ultimately be useful in in developing targeted treatment and prevention strategies.

Post-traumatic stress disorder (PTSD) is a common and debilitating disorder. The risk of PTSD following trauma is heritable, but robust common variants have yet to be identified by genome-wide association studies (GWAS). We have collected a multi-ethnic cohort including over 30,000 PTSD cases and 170,000 controls. We first demonstrate significant genetic correlations across 60 PTSD cohorts to evaluate the comparability of these phenotypically heterogeneous studies. In this largest GWAS meta-analysis of PTSD to date we identify a total of 6 genome-wide significant loci, 4 in European and 2 in African-ancestry analyses. Follow-up analyses incorporated local ancestry and sex-specific effects, and functional studies. Along with other novel genes, a non-coding RNA (ncRNA) and a Parkinson’s Disease gene,PARK2, were associated with PTSD. Consistent with previous reports, SNP-based heritability estimates for PTSD range between 10-20%. Despite a significant shared liability between PTSD and major depressive disorder, we show evidence that some of our loci may be specific to PTSD. These results demonstrate the role of genetic variation contributing to the biology of differential risk for PTSD and the necessity of expanding GWAS beyond European ancestry.

1009 Background: Biomarker analyses have been presented separately for each Phase III ML trial, which tested efficacy and safety of ribociclib (RIB) with different endocrine therapy (ET) combination partners as first- or second-line treatment for hormone receptor–positive, HER2-negative (HR+/HER2−) ABC. Here, using the largest pooled biomarker dataset of a CDK4/6 inhibitor in ABC to date, we identify potential biomarkers of response or resistance to RIB across ML trials. Methods: Baseline ctDNA from 1503 patients (pts) enrolled in ML-2, 3, and 7 was assessed using next-generation sequencing with a targeted panel of 557 genes. Genes with an alteration frequency ≥2% and in ≥15 pts per treatment arm were included (83 genes). Genetic alteration was defined as presence of a mutation, short insertion/deletion, or copy number alteration. Cox proportional hazard model of progression-free survival (PFS) was fit with gene-by-treatment interaction. Genes with interaction P< 0.10 and genes of interest were investigated. Results: Pts with alterations in FRS2 and PRKCA (treatment interaction P< 0.05) as well as MDM2, ERBB2, AKT1, and BRCA1/2 ( P> 0.05 but considered actionable) had a trend for increased PFS benefit of RIB vs PBO (Table). Pts with alterations in CHD4, BCL11B, ATM, or CDKN2A/2B/2C derived little to no added PFS benefit with RIB vs PBO ( P interaction < 0.10; hazard ratio [HR] > 0.80). Data on genes implicated in the literature as potential mechanisms of resistance to ET and/or CDK4/6 inhibition ( ESR1, PTEN, FAT1, RB1, and NF1) will be presented. Conclusions: Results of this pooled analysis of the ML-2, 3, and 7 trials, the largest biomarker analysis of any CDK4/6 inhibitor in ABC, revealed several potential biomarkers of response ( FRS2, MDM2, PRKCA, ERBB2, AKT1, and BRCA1/2) or resistance ( CHD4, BCL11B, ATM, or CDKN2A/2B/2C) to RIB. Clinical trial information: NCT01958021; NCT02422615; NCT02278120 . [Table: see text]

Background: The sensitivity of plasma-based oncological assays is influenced by the amount of tumor DNA shed into the circulation (ctDNA fraction). Higher ctDNA fraction has been shown to correlate with later-stage cancers and poorer overall survival (Bettegowda C, et al. Sci Transl Med. 2014; Dawson SJ, et al. N Engl J Med. 2014). Other factors affecting ctDNA fraction are poorly understood. We present results from three large Phase III breast cancer studies (MONALEESA-2, -3, and -7) to evaluate correlations between ctDNA fraction and clinical characteristics and impact on progression-free survival (PFS) and best overall tumor response. Methods: Baseline plasma samples were collected from patients (pts) in the MONALEESA-2, -3, and -7 studies, which are Phase III registration trials testing ribociclib, a cyclin-dependent kinase 4/6 inhibitor, in hormone receptor–positive, human epidermal growth factor receptor 2–negative advanced breast cancer. For each trial, cell-free DNA was extracted from plasma and sequenced using a validated next-generation sequencing assay targeting approximately 550 genes. ctDNA fraction was estimated by analyzing short-read sequences using PureCN (Riester M, et al. Source Code Biol Med. 2016) and tested for correlations with various tumor characteristics (size at baseline, Ki67 expression, number and type of metastatic sites, hormone receptor status) and pt characteristics (type of prior therapy, line of therapy, ECOG performance status, age, body mass index, race, histological grade, menopausal status). We also evaluated the prognostic value of ctDNA fraction on PFS and best overall response. To evaluate the prognostic effect on PFS, ctDNA fraction was binned into 3 categories (not detectable; low, 0.5%-10%; high, > 10%). Results: Higher ctDNA fractions were observed in pts with more metastatic sites (P < 0.0001), larger tumor diameter (P < 0.0001), higher Ki67 expression (P = 0.0001), higher lactate dehydrogenase levels (P < 0.0001), higher-grade tumors (P = 0.0004), worse ECOG performance status (P < 0.0001), progesterone receptor–negative tumors (P = 0.01), presence of liver metastases (P < 0.0001), and shorter disease-free intervals (P < 0.0001). ctDNA fractions were also higher in younger pts (continuous variable; P < 0.0001) and pts with lower BMI (P = 0.0003), prior chemotherapy (P = 0.002), and prior endocrine therapy (P = 0.01). Lower ctDNA fractions were observed in pts with better overall response (P < 0.0001), with median ctDNA fractions lowest in pts with complete responses followed by partial response, stable disease, and progressive disease. Pts with low (0.5%-10%) ctDNA fractions had longer PFS than pts with high (> 10%) ctDNA fractions (HR [95% CI], 0.63 [0.48-0.82], 0.64 [0.51-0.80], and 0.67 [0.52-0.86] in the MONALEESA-2, -3, and -7 trials, respectively). A more pronounced effect on PFS was observed in pts with undetectable levels of ctDNA vs pts with higher ctDNA fractions (HR [95% CI], 0.35 [0.23-0.52], 0.34 [0.21-0.55], and 0.31 [0.21-0.47] in the MONALEESA-2, -3, and -7 trials, respectively). Conclusions: Generally, higher ctDNA fraction was associated with more aggressive tumor characteristics and prior exposure to therapy. Lower ctDNA fraction was associated with longer PFS and better overall response. Together these data suggest that ctDNA fraction is a robust prognostic factor. Citation Format: Nadia Solovieff, Faye Su, Rebecca Leary, Alejandro Balbin, Arunava Chakravartty, Karen Rodriguez Lorenc, Tetiana Taran, Naveen Babbar. Association of tumor DNA in circulation with clinical characteristics and treatment response in HR+/HER2− advanced breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A031. doi:10.1158/1535-7163.TARG-19-A031

Purpose: The preplanned exploratory analysis of the BERIL-1 trial presented here aimed to identify biomarkers of response to the combination of buparlisib and paclitaxel. Patients and Methods: BERIL-1 was a multicenter, randomized, double-blind, placebo-controlled phase II study. Patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN) progressing on/after one previous platinum-based chemotherapy regimen in the recurrent or metastatic setting were treated with either buparlisib plus paclitaxel or placebo plus paclitaxel. Archival tumor tissue and ctDNA samples were analyzed for molecular alterations and immune infiltration using next-generation sequencing or immunohistochemistry. Results: Biomarker analyses were performed in randomized patients (n = 158) with available biomarker data. The most frequently (>5%) mutated genes were TP53, FAT1, TET2, KMT2D, PIK3CA, NOTCH1, NFE2L2, NOTCH2, CCND1, and CDKN2A. Patients with SCCHN tumors (from various primary sites) having HPV-negative status (HR = 0.51), TP53 alterations (HR = 0.55) or low mutational load (HR = 0.57) derived overall survival (OS) benefit with the combination of buparlisib and paclitaxel. OS benefit with this combination was also increased in patients with presence of intratumoral TILs ≥10% (HR = 0.51), stromal TILs ≥15% (HR = 0.53), intratumoral CD8-positive cells ≥5% (HR = 0.45), stromal CD8-positive cells ≥10% (HR = 0.47), or CD8-positive cells in invasive margins >25% (HR = 0.37). A trend for improved progression-free survival with the combination of buparlisib and paclitaxel was also observed in these patients. Conclusions: The BERIL-1 biomarker analyses showed that patients with TP53 alterations, HPV-negative status, low mutational load, or high infiltration of TILs or CD8-positive cells derived survival benefit with the combination of buparlisib and paclitaxel. Clin Cancer Res; 24(11); 2505–16. ©2018 AACR.

The genetic architecture of post-traumatic stress disorder (PTSD) remains poorly understood with the vast majority of genetic association studies reporting on single candidate genes. We conducted a large genetic study in trauma-exposed European-American women (N=2538; 845 PTSD cases, 1693 controls) by testing 3742 SNPs across more than 300 genes and conducting polygenic analyses using results from the Psychiatric Genome-Wide Association Studies Consortium (PGC). We tested the association between each SNP and two measures of PTSD, a severity score and diagnosis. We found a significant association between PTSD (diagnosis) and SNPs (top SNP: rs363276, odds ratio (OR)=1.4, p=2.1E-05) in SLC18A2 (vesicular monoamine transporter 2). A haplotype analysis of 9 SNPs in SLC18A2, including rs363276, identified a risk haplotype (CGGCGGAAG, p=0.0046), and the same risk haplotype was associated with PTSD in an independent cohort of trauma-exposed African-Americans (p=0.049; N=748, men and women). SLC18A2 is involved in transporting monoamines to synaptic vesicles and has been implicated in a number of neuropsychiatric disorders including major depression. Eight genes previously associated with PTSD had SNPs with nominally significant associations (p

Background: MONALEESASIA (NCT02333370) is an ongoing Phase Ib study in which Asian patients (pts) with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) ABC are treated with ribociclib (a cyclin-dependent kinase 4/6 inhibitor) and either letrozole (all pts), fulvestrant, or tamoxifen (Japanese pts). Tumor biomarker analysis in MONALEESASIA using NanoString technology for gene expression was presented previously (Yap YS, et al. ESMO Asia 2018 [Abstract 400]). Here we report additional biomarker analysis of baseline ctDNA. Methods: Plasma samples were collected at baseline and end of treatment; ctDNA was extracted and used to generate next-generation sequencing libraries. The DNA libraries were then enriched for a specific 2.9 Mb of the human genome containing ≈550 oncogenes and tumor suppressor genes. Captured libraries were then combined in equimolar pools and sequenced, targeting ≥70 million sequencing reads per sample to ensure that unique coverage of the genes was >1000× or approached the maximal complexity of the library. Results: ctDNA was analyzed in baseline plasma samples collected from 77 pts (49 Japanese and 28 non-Japanese). Similar gene alteration frequencies were observed in Japanese and non-Japanese pts except for ESR1 alterations, which were more common in Japanese pts (Table). TP53 alterations were more common in pts with progressive disease, and the frequencies of TP53, ESR1, and FAT1 were slightly lower in pre- vs postmenopausal pts; however, these differences were not statistically significant. Response rates by genetic alteration are shown in the table. Conclusion: There appeared to be a trend toward a higher likelihood of alterations in TP53 and PIK3CA in pts with poorer responses, although the sample size was small. These data are hypothesis generating, and additional analysis is ongoing. Gene AlterationsPIK3CATP53CCND1FAT1ESR1FGFR1CDH1Frequency, n (%)Japanesen=4917 (35)11 (22)5 (10)3 (6)7 (14)5 (10)4 (8)Non-Japanesen=288 (29)7 (25)3 (11)5 (18)02 (7)0Best Overall Response, n of Patients with Alteration (% Response Type)PRn=359 (26)6 (17)5 (14)3 (9)2 (6)4 (11)0SDn=299 (31)7 (24)1 (3)3 (10)3 (10)03 (10)NCRNPDn=63 (50)1 (17)002 (33)1 (17)1 (17)PDn=74 (57)4 (57)2 (29)2 (29)02 (29)0CCND1, cyclin D1; CDH1, cadherin 1; ESR1, estrogen receptor 1; FAT1, FAT atypical cadherin 1; FGFR1, fibroblast growth factor receptor 1; NCRNPD, not complete response nor progressive disease; PD, progressive disease; PIK3CA, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit α; PR, partial response; SD, stable disease; TP53, tumor protein p53. Citation Format: Yoon-Sim Yap, Faye Su, Nadia Solovieff, Yoshinori Ito, Norikaza Masuda, Takashi Ishikawa, Tomoyuki Aruga, Seung Jin Kim, Wei He, Mihaela Gazdoiu, Joanne Chiu. Baseline circulating tumor DNA (ctDNA) analysis in Asian women with HR+/HER2- advanced breast cancer (ABC) receiving ribociclib + endocrine therapy in the Phase Ib MONALEESASIA trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT143.

Background: The genetic landscape of premenopausal hormone receptor-positive (HR+) ABC is not well understood. The Phase III MONALEESA-7 study (NCT02278120), the first trial of endocrine therapy ± a cyclin-dependent kinase 4/6 inhibitor for premenopausal patients (pts) with HR+/human epidermal growth factor receptor 2-negative (HER2-) ABC, demonstrated that the addition of ribociclib (RIB) to a nonsteroidal aromatase inhibitor (NSAI) or tamoxifen (TAM) + goserelin (GOS) significantly extended progression-free survival (PFS; Tripathy D, et al. Lancet Oncol. 2018). We conducted a comprehensive ctDNA genomic analysis from MONALEESA-7. Methods: Premenopausal pts with HR+/HER2- ABC were randomized 1:1 to RIB or placebo (PBO) + NSAI (letrozole [LET] or anastrozole) or TAM + GOS. Plasma samples for ctDNA analysis were collected at baseline and end of treatment. ctDNA was analyzed using next-generation sequencing (targeted panel of 550 genes). Results: Among the 489 pts with ctDNA analyzed at baseline, the most common alterations were in PIK3CA (28%), TP53 (19%), CCND1 (11%), MYC (8%), and GATA3 (8%). Poorer prognosis in both treatment groups was most evident in patients with TP53 and MYC alterations. A PFS treatment effect in favor of RIB was noted in all subsets, independent of biomarker status (Table). However, based on HR, a trend for more pronounced benefit with RIB + NSAI/TAM + GOS was observed in pts with altered CCND1, GATA3, and genes involved in receptor tyrosine kinase signaling. Conclusions: RIB + NSAI/TAM + GOS provided PFS benefit irrespective of baseline biomarker alteration status and represents recommended first-line therapy for pts with premenopausal HR+/HER2- ABC. The genetic landscape of premenopausal ABC might modulate the magnitude of therapeutic benefit; these novel findings require confirmation in additional biomarker studies. RIB + NSAI/TAM + GOSPBO + NSAI/TAM + GOSEvents, n/NPFS, median monthsEvents, n/NPFS, median, monthsHRa (95% CI)PIK3CAWT68/18024.6798/17012.190.45 (0.33-0.62)Alt38/6914.7546/7012.850.57(0.36-0.9)TP53WT78/20324.67109/19412.980.48(0.36-0.65)Alt28/469.2335/467.160.47(0.27-0.82)CCND1WT91/22122.11126/21712.880.52(0.39-0.68)Alt15/2811.2718/235.520.21(0.08-0.54)MYCWT90/22924.67125/22112.880.49(0.37-0.65)Alt16/207.3419/197.160.57(0.25-1.31)GATA3WT96/22622.11131/22212.850.52(0.39-0.68)Alt10/23NA13/185.520.18(0.05-0.62)Receptor tyrosine kinasesbWT76/19827.53114/20614.520.5(0.37-0.67)Alt30/5114.5530/345.650.26(0.14-0.47)8p11.23cWT84/21523.03124/21412.780.47(0.36-0.63)Alt22/3412.5220/269.130.51(0.26-1)8p11.23, chromosome 8, short arm, region 11.23; alt, alteration; CCND1, cyclin D1; CI, confidence interval; GATA3, GATA binding protein 3; GOS, goserelin; NA, not applicable; NSAI, nonsteroidal aromatase inhibitor; MYC, MYC proto-oncogene, bHLH transcription factor; PBO, placebo; PFS, progression-free survival; PIK3CA, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit α; RIB, ribociclib; TAM, tamoxifen; TP53, tumor protein p53; WT, wild type.a HR for PFS of RIB vs PBO. b Receptor tyrosine kinase genes include EGFR, ERBB2, ERBB3, ERBB4, FGFR1, IGF1, IGF1R, KDR, KIT, PDGFRA, PDGFRB, and VEGFA. c Includes FGFR1, WHSC1L1, and ZNF703. Citation Format: Aditya Bardia, Faye Su, Nadia Solovieff, Seock-Ah Im, Joohyuk Sohn, Keun Seok Lee, Saul Campos-Gomez, Kyung Hae Jung, Rafael Villanueva Vazquez, Yen-Shen Lu, Fabio Franke, Sara Hurvitz, Nadia Harbeck, Louis Chow, Karen Rodriguez Lorenc, Tetiana Taran, Naveen Babbar, Debu Tripathy. Genetic landscape of premenopausal HR+/HER2- advanced breast cancer (ABC) based on comprehensive circulating tumor DNA analysis and association with clinical outcomes in the Phase III MONALEESA-7 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT141.

Posttraumatic stress disorder (PTSD) is a common and debilitating mental disorder with a particularly high burden for women. Emerging evidence suggests PTSD may be more heritable among women and evidence from animal models and human correlational studies suggest connections between sex-linked biology and PTSD vulnerability, which may extend to the disorder's genetic architecture. We conducted a genome-wide association study (GWAS) of PTSD in a primarily African American sample of women from the Detroit Neighborhood Health Study (DNHS) and tested for replication in an independent cohort of primarily European American women from the Nurses Health Study II (NHSII). We genotyped 413 DNHS women - 94 PTSD cases and 319 controls exposed to at least one traumatic event - on the Illumina HumanOmniExpress BeadChip for >700,000 markers and tested 578 PTSD cases and 1963 controls from NHSII for replication. We performed a network-based analysis integrating data from GWAS-derived independent regions of association and the Reactome database of functional interactions. We found genome-wide significant association for one marker mapping to a novel RNA gene, lincRNA AC068718.1, for which we found suggestive evidence of replication in NHSII. Our network-based analysis indicates that our top GWAS results were enriched for pathways related to telomere maintenance and immune function. Our findings implicate a novel RNA gene, lincRNA AC068718.1, as risk factor for PTSD in women and add to emerging evidence that non-coding RNA genes may play a crucial role in shaping the landscape of gene regulation with putative pathological effects that lead to phenotypic differences.

Summary Background The ankyrin 3 gene ( ANK3 ) produces the ankyrin G protein that plays an integral role in regulating neuronal activity. Previous studies have linked ANK3 to bipolar disorder and schizophrenia. A recent mouse study suggests that ANK3 may regulate behavioral disinhibition and stress reactivity. This led us to hypothesize that ANK3 might also be associated with stress-related psychopathology such as posttraumatic stress disorder (PTSD), as well as disorders of the externalizing spectrum such as antisocial personality disorder and substance-related disorders that are etiologically linked to impulsivity and temperamental disinhibition. Methods We examined the possibility of association between ANK3 SNPs and both PTSD and externalizing (defined by a factor score representing a composite of adult antisociality and substance abuse) in a cohort of white non-Hispanic combat veterans and their intimate partners ( n = 554). Initially, we focused on rs9804190—a SNP previously reported to be associated with bipolar disorder, schizophrenia, and ankyrin G expression in brain. Then we examined 358 additional ANK3 SNPs utilizing a multiple-testing correction. Results rs9804190 was associated with both externalizing and PTSD ( p = 0.028 and p = 0.042 respectively). Analysis of other ANK3 SNPs identified several that were more strongly associated with either trait. The most significant association with externalizing was observed at rs1049862 ( p = 0.00040, p corrected = 0.60). The most significant association with PTSD ( p = 0.00060, p corrected = 0.045) was found with three SNPs in complete linkage disequilibrium (LD)—rs28932171, rs11599164, and rs17208576. Conclusions These findings support a role of ANK3 in risk of stress-related and externalizing disorders, beyond its previous associations with bipolar disorder and schizophrenia.

Sickle cell anemia is common in the Middle East and India where the HbS gene is sometimes associated with the Arab-Indian (AI) β-globin gene (HBB) cluster haplotype. In this haplotype of sickle cell anemia, fetal hemoglobin (HbF) levels are 3-4 fold higher than those found in patients with HbS haplotypes of African origin. Little is known about the genetic elements that modulate HbF in AI haplotype patients. We therefore studied Saudi HbS homozygotes with the AI haplotype (mean HbF 19.2±7.0%, range 3.6 to 39.6%) and employed targeted genotyping of polymorphic sites to explore cis- and trans- acting elements associated with high HbF expression. We also described sequences which appear to be unique to the AI haplotype for which future functional studies are needed to further define their role in HbF modulation. All cases, regardless of HbF concentration, were homozygous for AI haplotype-specific elements cis to HBB. SNPs in BCL11A and HBS1L-MYB that were associated with HbF in other populations explained only 8.8% of the variation in HbF. KLF1 polymorphisms associated previously with high HbF were not present in the 44 patients tested. More than 90% of the HbF variance in sickle cell patients with the AI haplotype remains unexplained by the genetic loci that we studied. The dispersion of HbF levels among AI haplotype patients suggests that other genetic elements modulate the effects of the known cis- and trans-acting regulators. These regulatory elements, which remain to be discovered, might be specific in the Saudi and some other populations where HbF levels are especially high.

Sickle cell anemia (SCA) is an autosomal-recessive hemoglobinopathy with a highly variable phenotype. Multiple clinical complications are characteristic of SCA including inflammatory and oxidant damage to both small and large blood vessels, hemolysis, vasoocclusion, and premature mortality. The overall severity of SCA is affected by multiple genetic modifier loci, including ARFGEF2, a gene known to modify TNF-α receptor release from human endothelial cells. In this report, we examine the effect of siRNA mediated knockdown of ARFGEF2 inhuman pulmonary artery endothelial cells and report that TNF-α induced expression of ICAM1 and VCAM1, both important mediators of endo-thelial-leukocyte adhesion, is significantly enhanced after ARFGEF2 knockdown. Levels of ICAM-1 protein are also increased in TNF-α treated endothelial cells after ARFGEF2 knockdown; the increased ICAM-1 appears to be localized in the cytoplasm. IL-1β stimulation of endothelial cells without ARFGEF2 produced enhanced ICAM1 expression only. Additionally, ARFGEF2 knockdown distinctly altered endothelial cell morphology. Large-vessel pathology in SCA is believed to begin with endothelial activation by inflammatory cytokines and adhesion of sickle erythrocytes and leukocytes, leading to a progressive vasculopathy characterized by smooth muscle cell migration and proliferation. Understanding how variability in the function of ARFGEF2 alters the response of pulmonary vasculature to TNF-α might suggest new targets for SCA treatment.

Objectives Cyclin D–cyclin-dependent kinase (CDK) 4/6–inhibitor of CDK4/6–retinoblastoma (Rb) pathway hyperactivation is associated with hormone receptor-positive (HR+) breast cancer (BC). This study assessed the biological activity of ribociclib (LEE011; CDK4/6 inhibitor) plus letrozole compared with single-agent letrozole in the presurgical setting. Materials and methods Postmenopausal women ( N = 14) with resectable, HR+, human epidermal growth factor receptor 2-negative (HER2–) early BC were randomized 1:1:1 to receive 2.5 mg/day letrozole alone (Arm 1), or with 400 or 600 mg/day ribociclib (Arm 2 or 3). Circulating tumor DNA and tumor biopsies were collected at baseline and, following 14 days of treatment, prior to or during surgery. The primary objective was to assess antiproliferative response per Ki67 levels in Arms 2 and 3 compared with Arm 1. Additional assessments included safety, pharmacokinetics, and genetic profiling. Results Mean decreases in the Ki67-positive cell fraction from baseline were: Arm 1 69% (range 38–100%; n = 2), Arm 2 96% (range 78–100%; n = 6), Arm 3 92% (range 75–100%; n = 3). Decreased phosphorylated Rb levels and CDK4 , CDK6 , CCND2 , CCND3 , and CCNE1 gene expression were observed following ribociclib treatment. Ribociclib and letrozole pharmacokinetic parameters were consistent with single-agent data. The ribociclib plus letrozole combination was well tolerated, with no Grade 3/4 adverse events over the treatment. Conclusion The results suggest absence of a drug–drug interaction between ribociclib and letrozole and indicate ribociclib plus letrozole may reduce Ki67 expression in HR+, HER2– BC (NCT01919229).

Importance Focal cortical dysplasia (FCD), hemimegalencephaly, and megalencephaly constitute a spectrum of malformations of cortical development with shared neuropathologic features. These disorders are associated with significant childhood morbidity and mortality. Objective To identify the underlying molecular cause of FCD, hemimegalencephaly, and diffuse megalencephaly. Design, Setting, and Participants Patients with FCD, hemimegalencephaly, or megalencephaly (mean age, 11.7 years; range, 2-32 years) were recruited from Pediatric Hospital A. Meyer, the University of Hong Kong, and Seattle Children’s Research Institute from June 2012 to June 2014. Whole-exome sequencing (WES) was performed on 8 children with FCD or hemimegalencephaly using standard-depth (50-60X) sequencing in peripheral samples (blood, saliva, or skin) from the affected child and their parents and deep (150-180X) sequencing in affected brain tissue. Targeted sequencing and WES were used to screen 93 children with molecularly unexplained diffuse or focal brain overgrowth. Histopathologic and functional assays of phosphatidylinositol 3-kinase–AKT (serine/threonine kinase)–mammalian target of rapamycin (mTOR) pathway activity in resected brain tissue and cultured neurons were performed to validate mutations. Main Outcomes and Measures Whole-exome sequencing and targeted sequencing identified variants associated with this spectrum of developmental brain disorders. Results Low-level mosaic mutations ofMTORwere identified in brain tissue in 4 children with FCD type 2a with alternative allele fractions ranging from 0.012 to 0.086. Intermediate-level mosaic mutation ofMTOR(p.Thr1977Ile) was also identified in 3 unrelated children with diffuse megalencephaly and pigmentary mosaicism in skin. Finally, a constitutional de novo mutation ofMTOR(p.Glu1799Lys) was identified in 3 unrelated children with diffuse megalencephaly and intellectual disability. Molecular and functional analysis in 2 children with FCD2a from whom multiple affected brain tissue samples were available revealed a mutation gradient with an epicenter in the most epileptogenic area. When expressed in cultured neurons, allMTORmutations identified here drive constitutive activation of mTOR complex 1 and enlarged neuronal size. Conclusions and Relevance In this study, mutations ofMTORwere associated with a spectrum of brain overgrowth phenotypes extending from FCD type 2a to diffuse megalencephaly, distinguished by different mutations and levels of mosaicism. These mutations may be sufficient to cause cellular hypertrophy in cultured neurons and may provide a demonstration of the pattern of mosaicism in brain and substantiate the link between mosaic mutations ofMTORand pigmentary mosaicism in skin.

Summary A mutation in the LMNA gene is responsible for the most dramatic form of premature aging, Hutchinson–Gilford progeria syndrome (HGPS). Several recent studies have suggested that protein products of this gene might have a role in normal physiological cellular senescence. To explore further LMNA’s possible role in normal aging, we genotyped 16 SNPs over a span of 75.4 kb of the LMNA gene on a sample of long-lived individuals (LLI) (US Caucasians with age ‡ 95 years, N = 873) and genetically matched younger controls (N = 443). We tested all common nonredundant haplotypes (frequency ‡ 0.05) based on subgroups of these 16 SNPs for association with longevity. The most significant haplotype, based on four SNPs, remained significant after adjustment for multiple testing (OR = 1.56, P = 2.5 · 10 )5 , multiple-testing-adjusted P = 0.0045). To attempt to replicate these results, we genotyped 3448 subjects from four independent samples of LLI and control subjects from (i) the New England Centenarian Study (NECS) (N = 738), (ii) the Southern Italian Centenarian Study (SICS) (N = 905), (iii) France (N = 1103), and (iv) the Einstein Ashkenazi Longevity Study (N = 702). We replicated the association with the most significant haplotype from our initial analysis in the NECS sample (OR = 1.60, P = 0.0023), but not in the other three samples (P > 0.15). In a meta-analysis combining all five samples, the best haplotype remained significantly associated with longevity after adjustment for multiple testing in the initial and follow-up samples (OR = 1.18, P = 7.5 · 10 )4 , multiple-testing-adjusted P = 0.037). These results

Sickle cell anemia (SCA, HBB glu6val) is characterized by multiple complications and a high degree of phenotypic variability: some subjects have only sporadic pain crises and few acute hospitalizations, while others experience multiple serious complications, high levels of morbidity, and accelerated mortality [1]. The tumor necrosis factor-α (TNF-α) signaling pathway plays important roles in inflammation and the immune response; variation in this pathway might be expected to modify the overall severity of SCA through the pathway's effects on the vascular endothelium [2,3]. We examined plasma biomarkers of TNF-α activity and endothelial cell activation for associations with SCA severity in 24 adults (12 mild, 12 severe). Two biomarkers, tumor necrosis factor-α receptor-1 (TNF-R1) and vascular cell adhesion molecule-1 (VCAM-1) were significantly higher in subjects with severe SCA. Along with these biomarker differences, we also examined data from a genome-wide association study (GWAS) using SCA severity as a disease phenotype, and found evidence of genetic association between disease severity and a single nucleotide polymorphism (SNP) in VCAM1, which codes for VCAM-1, and several SNPs in ARFGEF2, a gene involved in TNF-R1 release [4].

In a genome-wide association study of 848 blacks with sickle cell anemia, we identified single nucleotide polymorphisms (SNPs) associated with fetal hemoglobin concentration. The most significant SNPs in a discovery sample were tested in a replication set of 305 blacks with sickle cell anemia and in subjects with hemoglobin E or β thalassemia trait from Thailand and Hong Kong. A novel region on chromosome 11 containing olfactory receptor genes OR51B5 and OR51B6 was identified by 6 SNPs (lowest P = 4.7E−08) and validated in the replication set. An additional olfactory receptor gene, OR51B2, was identified by a novel SNP set enrichment analysis. Genome-wide association studies also validated a previously identified SNP (rs766432) in BCL11A, a gene known to affect fetal hemoglobin levels (P = 2.6E−21) and in Thailand and Hong Kong subjects. Elements within the olfactory receptor gene cluster might play a regulatory role in γ-globin gene expression.

Background: Endocrine therapy resistance in hormone receptor-positive breast cancer often leads to activation of phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin pathway. In the BELLE-3 study the progression-free survival (PFS) improvement in buparlisib (BUP) vs placebo (PBO) arm was greater in patients (pts) with PIK3CA-mutant than PIK3CA-wildtype (Wt) tumors, based on circulating tumor DNA. In the current study we report the additional exploratory biomarker results from BELLE-3. Methods: The relationship between PFS and frequently mutated genes (PIK3CA, CCND1, ESR1 and 8q11.23 region) and pathways (PI3K and CDK pathways) was explored using NGS in tumor and pts were classified as altered (Alt) vs Wt. Genes and pathways with at least 10% frequency in both arms were carried forward to correlative analyses with PFS. In addition, ESR1 mutations were detected by BEAMing technology in baseline cell free DNA (cfDNA) for 167 pts. Results: NGS data were available for 185 pts and the NGS panel covered 500+ gene mutations, short insertions/deletion, copy number alterations, and translocations. PIK3CA was altered in 41% of pts and PI3K pathway was altered in 64% of pts. Benefit of BUP treatment was observed in PIK3CA altered pts, but did not extend to other alterations in the PI3K pathway. CCND1 was altered in 19% of pts and the CDK pathway was altered in 34% of pts and improved benefit of BUP was observed in altered pts in both the groups (HR = 0.31 and 0.38 respectively). Alterations in the 8q11.23 region (ZNF703, WHSC1L1 and FGFR1) were observed in 21% of pts with the benefit of BUP observed only among wildtype. In cfDNA, a higher treatment effect was observed in the ESR1 mutant subgroup (HR = 0.76 [Wt] and 0.27 [Alt]). Subgroup analyses of ESR1 mutations revealed a greater improvement in median PFS among pts with D538G mutation (BKM vs PBO = 8.25 vs 1.51; HR = 0.18) than among pts with Y537N/S/C mutation (BKM vs PBO = 4.17 vs 2.76; HR = 0.45). The PFS of different groups analyzed by NGS is mentioned in the table. Conclusions: A trend for stronger treatment effect was observed among pts with alterations in PIK3CA, CDK pathway, CCND1 (tumor), and ESR1 (ctDNA). The possibility of correlation among identified biomarkers is currently being explored. Table: Median PFSGroupTreatmentNEventsMedian PFS, moHR (95% CI)PIK3CAWtPBO34271.480.73 (0.46 - 1.16)BUP75602.83AltPBO23191.40.44 (0.25 - 0.79)BUP53344.11PI3K pathway (excluding PIK3CA Alt pts)WtPBO19171.450.51 (0.28 - 0.93)BUP47402.83Alt*PBO15104.671.1 (0.51 - 2.39)BUP28202.86CCND1WtPBO47371.480.61 (0.41 - 0.92)BUP103733.35AltPBO1091.430.31 (0.13 - 0.76)BUP25212.1CDK pathwayWtPBO40321.480.63 (0.4 - 0.98)BUP83583.42AltPBO17141.430.38 (0.19 - 0.75)BUP45362.68q11.23 regionWtPBO48391.450.49 (0.33 - 0.73)BUP98704.11AltPBO971.410.9 (0.38 - 2.13)BUP30241.48ESR1 mutation statusWtPBO29202.70.76 (0.43 - 1.31)BUP56372.8AltPBO23202.60.27 (0.14 - 0.51)BUP59246.0 * To assess the impact of genes in the PI3K pathway other than PIK3CA, the altered group includes patients who are PIK3CA wildtype but have an alteration in a different gene in the PI3K pathway. Citation Format: Nadia Solovieff, Ying A. Wang, Banu Sankaran, Nicolas Scheuer, Mona El-Hashimy, Denis Weber, Dalila Sellami, Angelo Di Leo. Biomarker analysis in HR+, HER2-, locally advanced, or metastatic breast cancer patients treated with buparlisib: results from BELLE-3 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A029.

HbF inhibits deoxy-HbS polymerization. Patients with elevated HbF have fewer vasoocclusive complications and prolonged survival [1]. Three major HbF QTL are known. The C>T polymorphism (rs7482144) at nucleotide –158 upstream of HBG2 is associated with increased HbF in some SCA patients [2]. Polymorphisms in intron 2 of BCL11A represented by rs766432 was associated with HbF in healthy Northern Europeans [3], African Americans with SCA [4, 5], Chinese with β-thalassemia trait and Thai’s with HbE-β thalassemia [5]. BCL11A polymorphisms correlate highly with HbF levels in SCA, accounting for 7–12% of the HbF variance [6]. The HMIP polymorphisms are distributed in three LD blocks [7]. HMIP block 2 represented by rs9399137 is most significantly associated with HbF expression and might function as a distal regulatory element [8,9]. We studied a selected group of 20 African American SCA patients with exceptionally high HbF (mean 17.2%) which differed by more than 4 times the standard deviation of 30 other patients with low HbF (mean 5.0%; Table IA). All study subjects’ HBB underwent nucleotide sequencing to ascertain that they were HbS homozygotes. Multiplex ligation-dependent probe amplification (MLPA) was carried out to ensure that they did not harbor hereditary persistence of fetal hemoglobin (HPFH) 1, HPFH 2, Black (δβ)0- and Black (Aγδβ)0-thalassemia deletions [10]. Furthermore, their HBG2 and HBG1 promoters were also sequenced to be certain that they did not have promoter HPFH single nucleotide mutations [11,12]. They were unlikely to be on hydroxyurea based on their MCV being less than 100 fL. Table IA Hematologic results of high and low HbF study groups. In addition we conducted a subset analyses in 56 patients with unusually high HbF (mean 20.7%) which differed by more than 11 times the standard deviation of 489 patients with low HbF (mean 3.1%; table IB). These patients were selected from 1,086 subjects from the Cooperative Study of Sickle Cell Disease (CSSCD) who were previously investigated in a genome-wide association study (GWAS) of HbF [13]. Table IB Hematologic results of high and low HbF CSCCD groups. The MAF of rs7482144, also known as the Xmn I site, on chromosome 11p15 in the unusually high HbF group (10%) is not significantly different from that in the low HbF group (8%) as shown in Table II. The SNP rs5006884, a missense mutation (CTC>TTC or Leu172Phe) in OR51B6 (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=5006884) on chromosome 11p15 was reported to be associated with HbF in SCA in a GWAS [13]. In the present study, the MAF of rs5006884 in the unusually high HbF group (2%) is actually less than that in the low HbF group (10%), even though the difference is not statistically significant (Table II). It should be recognized that the small sample size in the current study does not afford sufficient power to discern possible MAF differences if present at these 2 SNPs between these 2 groups of patients. Table II HbF QTL minor allele frequencies in high and low HbF groups The SNP rs7482144 did not reach genome-wide significance in previous GWAS of HbF in the full CSCCD cohort [13]. In the present study with subsets from the CSCCD cohort, rs7482144 has a significantly higher MAF in the unusually high HbF group (30%) compared to the low HbF group (10%), P = 0.002. The MAF of rs5006884 in OR51B6 in the high HbF group (22%) is significantly higher than that in the low HbF group (10%), P = 0.00055. This SNP reached genome-wide significance in the GWAS of the full CSSCD cohort, however the mean HbF levels in homozygotes for this variant was only 10.6%. The present subset analysis shows that for SCA patients with unusually high HbF the frequency of this mutation is comparable to the general population (Table II). To examine the BCL11A QTL, three SNPs, rs766432, rs4671393 and rs11886868 were chosen for genotyping [6]. These 3 SNPs are in strong LD. Only data on rs766432 that is most highly correlated with HbF in SCA patients are presented. The MAF in the unusually high HbF group (45%) is significantly higher than that in the low HbF group (25%), P = 0.05 (Table II). The MAF in the subgroups from CSCCD were similar: 47% in the high and 20% in the low HbF groups, P = 6.399×10−10. The allele C of this SNP was associated with increased levels of HbF [13] and the mean HbF levels in subjects homozygous for the C allele was 8.26%. The present subset analysis in the CSSCD subgroups shows that in patients with unusually high HbF the frequency of this mutation is almost twice that of the full cohort. The QTL in HMIP is best represented by rs9399137 [9]. The MAF of rs9399137 among SCA patients of African descent without European admixture was reported to be 1–2% [14,15]. A GWAS on over 800 African American SCA patients did not detect genome-wide significance of association of this QTL with HbF [13]. The functional motif for this QTL is most likely a 3-bp (TAC) deletion which is in complete LD with the minor allele of rs9399137 [9]. The frequency of this 3-bp deletion is 23% in non-African HapMap populations, but only 5% in Africans [9]. In the current study, the MAF of rs9399137 in the African American SCA patients with unusually high HbF is 18%, significantly higher than that with low HbF (3%), P = 0.02 (Table II). Furthermore, the 3-bp deletion as reported by Farrell et al [9] was found for the first time in these African American SCA patients and it is in complete LD with the minor allele of rs9399137. Among the subset of CSSCD patients, the MAF of rs9399137 in the unusually high HbF group (9%) is also significantly higher than that in the low HbF group (3%), P = 0.006 (Table II). These results raise the possibility that some African American SCA patients with markedly elevated HbF might have inherited the minor allele of chromosome 6q23 QTL due to European genetic admixture. The minor T allele of rs7775698 tags either an ancestral T nucleotide found mostly in African populations, or a 3-bp deletion often found in European and Chinese populations [9]. In the present study, the MAF of rs7775698 in the high HbF group (40%) is higher than that in the low HbF group (21%). But the difference is not statistically significant (P = 0.07). Among the CSCCD patients, the MAF of rs7775698 in the high HbF group is 16% compared to 19% in the low HbF group (Table II). We found a 2-bp (CC) deletion plus an (A) insertion 19 bp downstream of rs9399137. This deletion/insertion was present in 33% of chromosome 6 in both high and low HbF groups. It is unlikely that it plays a significant functional role in modulating HbF expression. Its relatively high frequency in both groups makes it a probable haplotype marker in African American SCA patients. Two of the 3 known HbF major QTLs are present in the unusually high HbF patient study group at a frequency significantly higher than those with low HbF. Their cumulative effect was estimated by assigning to each minor allele at each locus a score of 1, and adding the scores to generate the Number of Minor Allele Present (NOMAP) [16]. There was an average of 2 minor alleles in the unusually high HbF group compared with 1 in the low HbF group (P = 0.001) as shown in Figure 1. Figure 1 To explore other possible genetic loci that may modulate HbF expression, we undertook nucleotide sequencing of a 14.1 kb DNA fragment between HBG1 and HBD in 15 high and 15 low HbF patients. This DNA fragment was chosen because BCL11A binds to this intergenic region and it also encompasses the Corfu deletion that in homozygotes is characterized by markedly elevated HbF [17, 18]. Thirty eight SNPs were found in both the high and low HbF groups (Supplementary Table I). SNP rs10128558 as described by Galarneau et al [19] was present in our study groups and found to be in complete LD with Xmn I polymorphism. Its MAF in the high HbF group (10%) is not different from the low HbF group (8%). Based on in silico analysis by TFSEARCH (threshold score 85), 22 of the 38 SNPs are associated with alterations in transcription factor binding including erythroid specific transcription factors. In addition, 4 SNPs have significantly higher major allele frequency in the high compared to the low HbF groups (P < 0.05). Three of these 4 SNPs result in alteration in transcription factor binding sites (Supplementary Table I). This study based on a small cohort of carefully selected African American SCA patients with unusually high HbF revealed that the MAF for rs766432 (BCL11A), rs9399137 and 3-bp deletion both within HMIP are much higher than that found in patients with low HbF. These findings also raise the possibility that some African American patients with markedly elevated HbF might have inherited the minor allele of chromosome 6q23 QTL due to European genetic admixture. Validation of these findings in a larger patient cohort and further functional investigations into these and other polymorphisms are warranted.

Schizophrenia is a common disease with a complex aetiology, probably involving multiple and heterogeneous genetic factors. Here, by analysing the exome sequences of 2,536 schizophrenia cases and 2,543 controls, we demonstrate a polygenic burden primarily arising from rare (less than 1 in 10,000), disruptive mutations distributed across many genes. Particularly enriched gene sets include the voltage-gated calcium ion channel and the signalling complex formed by the activity-regulated cytoskeleton-associated scaffold protein (ARC) of the postsynaptic density, sets previously implicated by genome-wide association and copy-number variation studies. Similar to reports in autism, targets of the fragile X mental retardation protein (FMRP, product of FMR1) are enriched for case mutations. No individual gene-based test achieves significance after correction for multiple testing and we do not detect any alleles of moderately low frequency (approximately 0.5 to 1 per cent) and moderately large effect. Taken together, these data suggest that population-based exome sequencing can discover risk alleles and complements established gene-mapping paradigms in neuropsychiatric disease., National Human Genome Research Institute (U.S.) (Grant U54HG003067)

BACKGROUND: There is considerable variation in psychological reactions to natural disasters, with responses ranging from relatively mild and transitory symptoms to severe and persistent posttraumatic stress (PTS). Some survivors also report post-traumatic growth (PTG), or positive psychological changes due to the experience and processing of the disaster and its aftermath. Gene-environment interaction (GxE) studies could offer new insight into the factors underlying variability in post-disaster psychological responses. However, few studies have explored GxE in a disaster context. METHODS: We examined whether ten common variants in seven genes (BDNF, CACNA1C, CRHR1, FKBP5, OXTR, RGS2, SLC6A4) modified associations between Hurricane Katrina exposure and PTS and PTG. Data were from a prospective study of 205 low-income non-Hispanic Black parents residing in New Orleans prior to and following Hurricane Katrina. RESULTS: We found a significant association (after correction) between RGS2 (rs4606; p=0.0044) and PTG, which was mainly driven by a cross-over GxE (p=0.006), rather than a main genetic effect (p=0.071). The G (minor allele) was associated with lower PTG scores for low levels of Hurricane exposure and higher PTG scores for moderate and high levels of exposure. We also found a nominally significant association between variation in FKBP5 (rs1306780, p=0.0113) and PTG, though this result did not survive correction for multiple testing. LIMITATIONS: Although the inclusion of low-income non-Hispanic Black parents allowed us to examine GxE among a highly vulnerable group, our findings may not generalize to other populations or groups experiencing other natural disasters. Moreover, not all participants invited to participate in the genetic study provided saliva. CONCLUSIONS: To our knowledge, this is the first study to identify GxE in the context of post-traumatic growth. Future studies are needed to clarify the role of GxE in PTS and PTG and post-disaster psychological responses, especially among vulnerable populations. Language: en

Genome-wide association studies have identified many variants that each affects multiple traits, particularly across autoimmune diseases, cancers and neuropsychiatric disorders, suggesting that pleiotropic effects on human complex traits may be widespread. However, systematic detection of such effects is challenging and requires new methodologies and frameworks for interpreting cross-phenotype results. In this Review, we discuss the evidence for pleiotropy in contemporary genetic mapping studies, new and established analytical approaches to identifying pleiotropic effects, sources of spurious cross-phenotype effects and study design considerations. We also outline the molecular and clinical implications of such findings and discuss future directions of research.

6045Background: A phase II, placebo-controlled study of buparlisib (BUP) and paclitaxel (PAC) in patients (pts) with platinum-pretreated recurrent/metastatic HNSCC (NCT01852292) met its primary end...