Hirschsprung disease (HSCR) is a common cause of intestinal obstruction in the newborn. Hirschsprung‐associated enterocolitis (HAEC) is a significant and life‐threatening complication of HSCR, affecting up to 60% of patients. Animal models of endothelin receptor B (EdnrB) mutation reliably model human HSCR and HAEC. We previously demonstrated intestinal dysbiosis and a gut‐specific deficiency of B‐lymphocyte–produced secretory IgA (sIgA), the primary effector molecule of mucosal immunity, in mice with homozygous neural crest cell–conditional deletion of EdnrB (EdnrBNCC−/−). To determine mechanisms for sIgA deficiency, we examined intrinsic and extrinsic aspects of B‐lymphocyte development and function. Expression of the endothelin axis components [endothelin‐1 (ET‐1), endothelin‐3 (ET‐3), endothelin receptor A (EdnrA), EdnrB] were determined over a developmental time course. B‐lymphocyte survival and Ig production were assayed in vitro. Polymeric Ig receptor (pIgR)–mediated IgA transport into the intestinal lumen was interrogated. We found endothelin axis component (EdnrA, EdnrB, ET‐1, ET‐3) expression in developing extramedullary hematopoietic organs and that some splenic B lymphocytes express EdnrB. Splenic B lymphocytes from EdnrBNCC−/− mice showed no intrinsic defect in survival vs. wild‐type (WT) B lymphocytes. In vitro stimulation of splenic B lymphocytes demonstrated decreased IgA, IgG, and IgM production in EdnrBNCC−/− vs. WT mice. Additionally, small intestinal pIgR was decreased ∼50% in EdnrBNCC−/− mice. These results suggest an intrinsic B‐lymphocyte defect in antibody production as well as an extrinsic defect in IgA transport in the EdnrBNCC−/− model of HAEC. Our results are consistent with human HAEC observations of decreased luminal sIgA and mouse models of other inflammatory bowel diseases, in which decreased pIgR is seen in concert with a dysregulated microbiota. Finally, our results suggest targeting the dysbiotic microbiome and pIgR‐mediated sIgA transport as potential therapeutic approaches in prevention and treatment of HAEC.—Medrano, G., Cailleux, F., Guan, P., Kuruvilla, K., Barlow‐Anacker, A. J., Gosain, A. B‐lymphocyte–intrinsic and –extrinsic defects in secretory immunoglobulinA production in the neural crest–conditional deletion of endothelin receptor B model of Hirschsprung‐associated enterocolitis. FASEB J. 33, 7615–7624 (2019). http://www.fasebj.org

601 Background: Personalized circulating tumor DNA (ctDNA) assays are being evaluated in early breast cancer (BC). We investigated the value of ctDNA monitoring in the NeoRHEA study. Methods: NeoRhea (NCT03065621) is a single arm phase 2 study in which patients with estrogen receptor (ER)+/HER2- early breast cancer were treated with neoadjuvant palbociclib + endocrine therapy (ET) for 4 months. Plasma samples were collected at four timepoints: pre-treatment (BL), after the first treatment cycle (C1D28), before surgery (Surgery) and one month post Surgery (End of Study). ctDNA detection was evaluated using the personalized RaDaR assay. Whole-exome sequencing (WES) was performed on BL tumor biopsies followed by a personalized assay development using tumor, plasma and normal DNA in order to track up to 48-patient specific somatic variants in plasma cell-free DNA (cfDNA) using next generation sequencing. Associations between ctDNA detection and BL clinical-pathological characteristics, cell cycle arrest (CCCA) defined as Ki67 ≤2.7% at surgery, residual cancer burden (RCB) rate and breast cancer free survival (BCFS) were investigated. Results: Of 100 patients enrolled, 78 patients and 302 plasma samples were successfully profiled by the RaDaR assay. The number of variants targeted by the assay ranged between 7-48 (median = 25). The median estimated variant allele fraction for ctDNA positive samples was 0.02% (range 0.0009%-0.91%). A total of 42/76 patients were ctDNA positive at BL, 4/76 at C1D28, 4/75 at Surgery and 0/75 at End of Study. Out of 78 patients, 68% were postmenopausal, 78 % had cT2 and 69% cN0 tumors, 20% had multifocal/multicentric tumors, 18% had histological grade 3 tumors, 76% had CCCA (Ki67 not available in 20 patients) and 34% RCB 3 at surgery. ctDNA detection at BL was higher in histological grade 3 tumors (p = 0.03), lower in multifocal/multicentric tumors (p = 0.01), higher in RCB III tumors, (p = 0.01) but was not associated with CCCA. With a median follow-up of 3.8 years (range 1-5 years), 4 patients developed distant and one patient locoregional recurrences. ctDNA detection after one month of treatment (logrank p = 0.02), but not at baseline (p = 0.59) nor at surgery (p = 0.67) was associated with worse BCFS. Conclusions: Detection of ctDNA in early-stage breast cancer is challenging. Our data suggests association of ctDNA detection with some pathological and clinical variables. Independent validation is needed. Clinical trial information: NCT03065621 .

522 Background: CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) are standard of care in the treatment of estrogen receptor (ER) positive, HER2-negative (ER+/HER2-) advanced breast cancer (BC). We tested palbociclib + ET as neoadjuvant therapy to identify baseline biomarkers of no response. Methods: NeoRHEA (NCT03065621) was a phase II, single arm trial evaluating 4 months of neoadjuvant palbociclib and ET in pre- or post-menopausal women with ER+/HER2- early BC. The primary objective was to identify biomarkers of no response by locally-assessed ultrasound (US) using RNA-seq on baseline (pretreatment) tumor biopsies. No response to treatment was defined as stable or progressive disease (SD or PD) at post-treatment US based on WHO criteria. Secondary endpoints included residual cancer burden (RCB) of 3 and absence of complete cell cycle arrest (CCCA) defined as Ki67 by immunohistochemistry (IHC) > 2.7% at surgery. We evaluated baseline clinicopathological characteristics, RNA-seq derived PAM50 subtypes and mRNA expression of ESR1, Ki67, CCNE1, RB1 and CDK6 single genes according to US response and CCCA. We also performed gene set enrichment analysis (GSEA) for 50 gene sets related to cancer hallmarks according to US response and CCCA. Results: 73 of 100 patients enrolled had baseline frozen tumors with enough cellularity and successful RNA-sequencing performed. Among the 73 patients, 70% were post-menopausal, 84% had cT2 and 70% cN0 tumors, 75% had invasive ductal carcinoma and 66% had histological grade 2 tumors. RNA-seq derived PAM50 subtypes were Luminal A, Luminal B, and non-Luminal in 56%, 36% and 8% of patients, respectively. Among the 73 patients, 31 (42%) had absence of US response (28 and 3 patients had stable and progressive disease by US, respectively) and 23 (31%) had RCB 3 tumors. Fifty-three of 73 patients had Ki67 by IHC available at surgery out of whom, 14 (26%) had absence of CCCA. Neither baseline clinicopathological characteristics nor PAM50 subtypes nor expression of ESR1, Ki67, CCNE1 and RB1 were associated with absence of US response or absence of CCCA. Interestingly, we observed higher baseline mRNA expression of CDK6 in patients with absence of US response (p = 0.039) or those with no CCCA (p = 0.004). We observed an enrichment in inflammatory / IFN-γ response and proliferation-related, G2M checkpoint gene sets in patients with US response (NES: 2.06, 2.30, 1.47; FDR: 7.04e-9, 5.48e-17, 0.0193 respectively) and an enrichment in early estrogen response gene set in patients with absence of US response (NES: -1.78, FDR: 0.0006). No signaling pathway was enriched in patients without CCCA. Conclusions: High pre-treatment mRNA expression of the CDK6 gene was associated with no US response or absence of CCCA in patients treated with neoadjuvant palbociclib and ET. Independent validation is needed. Clinical trial information: NCT03065621 .