Circulating Tumor DNA After Neoadjuvant Chemotherapy in Breast Cancer Is Associated With Disease Relapse.

PURPOSE: Detection of circulating tumor DNA (ctDNA) after neoadjuvant chemotherapy in patients with early-stage breast cancer may allow for early detection of relapse. In this study, we analyzed ctDNA using a personalized, tumor-informed multiplex polymerase chain reaction-based next-generation sequencing assay. METHODS: Plasma samples (n = 157) from 44 patients were collected before neoadjuvant therapy (baseline), after neoadjuvant therapy and before surgery (presurgery), and serially postsurgery including a last follow-up sample. The primary end point was event-free survival (EFS) analyzed using Cox regression models. RESULTS: Thirty-eight (86%), 41 (93%), and 38 (86%) patients had baseline, presurgical, and last follow-up samples, respectively. Twenty patients had hormone receptor-positive/human epidermal growth factor receptor 2-negative, 13 had triple-negative breast cancer, and 11 had human epidermal growth factor receptor 2-positive disease. Baseline ctDNA detection was observed in 22/38 (58%) patients and was significantly associated with Ki67 > 20% (P = .036) and MYC copy-number gain (P = .0025, false discovery rate = 0.036). ctDNA detection at presurgery and at last follow-up was observed in 2/41 (5%) and 2/38 (5%) patients, respectively. Eight relapses (seven distant and one local) were noted (median follow-up 3.03 years [range, 0.39-5.85 years]). After adjusting for pathologic complete response (pCR), ctDNA detection at presurgery and at last follow-up was associated with shorter EFS (hazard ratio [HR], 53; 95% CI, 4.5 to 624; P < .01, and HR, 31; 95% CI, 2.7 to 352; P < .01, respectively). Association between baseline detection and EFS was not observed (HR, 1.4; 95% CI, 0.3 to 5.9; P = .67). CONCLUSION: The presence of ctDNA after neoadjuvant chemotherapy is associated with relapse in early-stage breast cancer, supporting interventional trials for testing the clinical utility of ctDNA monitoring in this setting.

B-lymphocyte-intrinsic and -extrinsic defects in secretory immunoglobulin A production in the neural crest-conditional deletion of endothelin receptor B model of Hirschsprung-associated enterocolitis.

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.

Fecal microbiota manipulation prevents dysbiosis and alcohol-induced liver injury in mice.

BACKGROUND & AIMS: Alcoholic liver disease (ALD) is a leading cause of liver failure and mortality. In humans, severe alcoholic hepatitis is associated with key changes to intestinal microbiota (IM), which influences individual sensitivity to develop advanced ALD. We used the different susceptibility to ALD observed in two distinct animal facilities to test the efficiency of two complementary strategies (fecal microbiota transplantation and prebiotic treatment) to reverse dysbiosis and prevent ALD. METHODS: Mice were fed alcohol in two distinct animal facilities with a Lieber DeCarli diet. Fecal microbiota transplantation was performed with fresh feces from alcohol-resistant donor mice to alcohol-sensitive receiver mice three times a week. Another group of mice received pectin during the entire alcohol consumption period. RESULTS: Ethanol induced steatosis and liver inflammation, which were associated with disruption of gut homeostasis, in alcohol-sensitive, but not alcohol resistant mice. IM analysis showed that the proportion of Bacteroides was specifically lower in alcohol-sensitive mice (p<0.05). Principal coordinate analysis showed that the IM of sensitive and resistant mice clustered differently. We targeted IM using two different strategies to prevent alcohol-induced liver lesions: (1) pectin treatment which induced major modifications of the IM, (2) fecal microbiota transplantation which resulted in an IM very close to that of resistant donor mice in the sensitive recipient mice. Both methods prevented steatosis, liver inflammation, and restored gut homeostasis. CONCLUSIONS: Manipulation of IM can prevent alcohol-induced liver injury. The IM should be considered as a new therapeutic target in ALD. LAY SUMMARY: Sensitivity to alcoholic liver disease (ALD) is driven by intestinal microbiota in alcohol fed mice. Treatment of mice with alcohol-induced liver lesions by fecal transplant from alcohol fed mice resistant to ALD or with prebiotic (pectin) prevents ALD. These findings open new possibilities for treatment of human ALD through intestinal microbiota manipulation.

Toxic lipids stored by Kupffer cells correlates with their pro-inflammatory phenotype at an early stage of steatohepatitis.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is characterized by steatosis associated with liver inflammation. Steatosis causes recruitment of lymphocytes into the liver and this is worsened by lipopolysaccharides (LPS). As macrophages may be involved in the lymphocyte homing, we studied the role of lipids in determining the phenotype of Kupffer cells (KCs) at the stage of steatosis. METHODS: Steatosis was induced in mice by a high fat diet. The turnover and the recruitment of KCs were analyzed in vivo by flow cytometry. KCs phenotype was assessed by optical and electron microscopy, cell culture and lymphocyte recruitment by in vitro chemotaxis. Lipidomic analysis was carried out by mass-spectrometry and gene expression analysis by TaqMan low density array. RESULTS: Although the number of KCs was not modified in steatotic livers compared to normal livers, their phenotypes were different. Electron microscopy demonstrated that the KCs from fatty livers were enlarged and loaded with lipid droplets. Lipid synthesis and trafficking were dysregulated in fat-laden KCs and toxic lipids accumulated. Fat-laden KCs recruited more CD4+ T and B lymphocytes in response to LPS stimulation than did control KCs and produced high levels of pro-inflammatory cytokines/chemokines, which could be reversed by inhibition of lipogenesis. CONCLUSIONS: Lipid accumulation in fat-laden KCs is due to a dysregulation of lipid metabolism and trafficking. Fat-laden KCs are "primed" to recruit lymphocytes and exhibit a pro-inflammatory phenotype, which is reversible with inhibition of lipogenesis.