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Background: The variability in responses to neoadjuvant treatment with anti-HER2 antibodies prompts to personalized clinical management and the development of innovative treatment strategies. Tumor-infiltrating Natural Killer (TI-NK) cells can predict the efficacy of HER2-targeted antibodies independently from clinicopathological factors in primary HER2-positive breast cancer patients. Understanding the mechanism/s underlying this association would contribute to optimizing patient stratification and provide the rationale for combinatorial approaches with immunotherapy., Methods: We sought to uncover processes enriched in NK cell-infiltrated tumors as compared to NK cell-desert tumors by microarray analysis. Findings were validated in clinical trial-derived transcriptomic data. In vitro and in vivo preclinical models were used for mechanistic studies. Findings were analysed in clinical samples (tumor and serum) from breast cancer patients., Results: NK cell-infiltrated tumors were enriched in CCL5/IFNG-CXCL9/10 transcripts. In multivariate logistic regression analysis, IFNG levels underlie the association between TI-NK cells and pathological complete response to neoadjuvant treatment with trastuzumab. Mechanistically, the production of IFN-ɣ by CD16 +  NK cells triggered the secretion of CXCL9/10 from cancer cells. This effect was associated to tumor growth control and the conversion of CD16 into CD16 - CD103 +  NK cells in humanized in vivo models. In human breast tumors, the CD16 and CD103 markers identified lineage-related NK cell subpopulations capable of producing CCL5 and IFN-ɣ, which correlated with tissue-resident CD8 +  T cells. Finally, an early increase in serum CCL5/CXCL9 levels identified patients with NK cell-rich tumors showing good responses to anti-HER2 antibody-based neoadjuvant treatment., Conclusions: This study identifies specialized NK cell subsets as the source of IFN-ɣ influencing the clinical efficacy of anti-HER2 antibodies. It also reveals the potential of serum CCL5/CXCL9 as biomarkers for identifying patients with NK cell-rich tumors and favorable responses to anti-HER2 antibody-based neoadjuvant treatment., (© 2024. The Author(s).)

Stromal tumour infiltrating lymphocytes (sTIL) in haematoxylin and eosin (H&E) stained sections has been linked to better outcomes and better responses to neoadjuvant therapy in triple-negative and HER2-positive breast cancer (TNBC and HER2 +). However, the infiltrate includes different cell populations that have specific roles in the tumour immune microenvironment. Various studies have found high concordance between sTIL visual quantification and computational assessment, but specific data on the individual prognostic impact of plasma cells or lymphocytes within sTIL on patient prognosis is still unknown. In this study, we validated a deep-learning breast cancer sTIL scoring model (smsTIL) based on the segmentation of tumour cells, benign ductal cells, lymphocytes, plasma cells, necrosis, and 'other' cells in whole slide images (WSI). Focusing on HER2 + and TNBC patient samples, we assessed the concordance between sTIL visual scoring and the smsTIL in 130 WSI. Furthermore, we analysed 175 WSI to correlate smsTIL with clinical data and patient outcomes. We found a high correlation between sTIL values scored visually and semi-automatically (R = 0.76; P = 2.2e-16). Patients with higher smsTIL had better overall survival (OS) in TNBC (P = 0.0021). In the TNBC cohort, smsTIL was as an independent prognostic factor for OS. As part of this work, we introduce a new segmentation dataset of H&E-stained WSI., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Stromal tumor-infiltrating lymphocytes (sTILs) are a robust prognostic and predictive biomarker in triple-negative breast carcinoma. However, the sTIL compartment comprises different cell populations. The aim of the study is to characterize the distribution of T cells (CD3+ and CD8+), B cells, and plasma cells and explore their association with outcome in the surgical specimen of 62 patients. Furthermore, programmed death ligand 1 expression and the presence of tertiary lymphoid structures (TLSs) are explored. Patients with higher sTILs achieve better progression-free survival (PFS) (P = .0013), and tumors have more plasma cells in the infiltrate. Specifically, higher counts of T cells (both CD3+ and CD8+) have better PFS (P = .002 and P = .0086, respectively) as it is observed in tumors with higher infiltration of CD8+ T cells in the tumor core (P = .035). Higher infiltration by B cells and plasma cells shows a positive tendency toward increased PFS (P = .06 and P = .058). Programmed death ligand 1 (SP142) is positive in 56% of tumors. Tumors with at least 1 TLS (42%) show higher CD8+ T cell infiltration in the tumor core and the sTIL value doubles compared to tumors devoid of TLSs [sTIL mean: 36 ± 11% and 18 ± 5% (CI [Confidence Interval]: 95%), respectively]. Our study demonstrates that the characterization of the immune cell infiltration is as relevant as its distribution. Moreover, the importance of considering different immune cell types for classification is emphasized. Therefore, a new classification of triple-negative breast carcinoma immune infiltration with CD8+ T cell and plasma cell densities in the tumor core and infiltrative margin is proposed., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Introduction: Neoadjuvant and adjuvant immune checkpoint blockade (ICB) have recently become standard of care in resectable non-small cell lung cancer (NSCLC). Yet, biomarkers that inform patients who benefit from this approach remain largely unknown. Here, we interrogated the tumor immune microenvironment (TIME) in early-stage NSCLC patients that underwent up-front surgery., Methods: A total of 185 treatment-naïve patients with early-stage NSCLC, that underwent up-front surgical treatment between 2006 and 2018 at Hospital del Mar were included. 124 lung adenocarcinomas (LUADs), and 61 squamous cell carcinoma (LUSCs) were included in a tissue microarray. Immunohistochemistry for CD3, CD4, CD8, CD68, CD80, CD103, FOXP3, PD-1, PD-L1, PD-L2 and HLA class II were evaluated by digital image analysis (QuPath software). TIME was categorized into four groups using PD-L1 expression in tumor cells (<1 % or ≥1 %) and tumor resident memory (CD103 + ) immune cells (using the median as cut-off). We explored the association between different TIME dimensions and patient's clinicopathological features and outcomes., Results: We found increased levels of T cell markers (CD3 + , CD4 + , CD8 + cells), functional immune markers (FOXP3 + cells) as well as, higher HLA-II tumor membrane expression in LUADs compared to LUSCs (p < 0.05 for all). In contrast, LUSCs displayed higher percentage of intratumor macrophages (CD68 + cells) as well as, higher PD-L1 and PD-L2 tumor membrane expression (p < 0.05 for all). Unsupervised analysis revealed three different tumor subsets characterized by membrane tumor expression of PD-L1, PD-L2 and HLA-class II. Enrichment of T cells (CD3 + , CD8 + cells), regulatory T cells (FOXP3 + cells) and macrophages (CD68 + cells) was observed in the CD103 + /PD-L1 + group (p < 0.05 for all). Multivariate analysis showed that infiltration by CD103 + immune cells was associated with improved OS (p = 0.009)., Conclusions: TIME analysis in resected NSCLC highlighted differences by histology, PD-L1 expression and molecular subgroups. Biomarker studies using IHC might aid to individually tailor adjuvant treatment in early-stage NSCLC., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:P. Rocha reports travel support from AstraZeneca. A. Taus reports personal fees and non-financial support from Takeda, Sanofi, Roche, BMS, MSD, GSK, Astrazeneca and Pfizer, outside the submitted work. R. Chalela reports travel and personal fees from Chiesi, GSK and AstraZeneca, outside the submitted work. B. Bellosillo reports grants, personal fees and non-financial support from Roche, grants, personal fees and non-financial support from ThermoFisher, grants, personal fees and non-financial support from Astra-Zeneca, personal fees and non-financial support from Merck-Serono, personal fees and non-financial support from Novartis, personal fees and non-financial support from Qiagen, personal fees and non-financial support from Pfizer, personal fees and non-financial support from BMS. L. Comerma reports personal fees and non-financial support from Roche, personal fees and non-financial support from MSD and personal fees from Diaceutics, outside the submitted work. E. Arriola reports grants, personal fees and non-financial support from Roche, personal fees, and non-financial support from BMS, personal fees and non-financial support from MSD, personal fees from Astra Zeneca, grants and personal fees from Pfizer, personal fees and non-financial support from Boehringer Ingelheim, personal fees and non-financial support from Lilly, personal fees from Takeda, outside the submitted work. Remain authors have no competing interests to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Some studies suggested that the patients included in the Z0011 trial may represent patients with ultrasound-negative axillary nodes and axillary invasion diagnosed by sentinel node (SN) biopsy. Nevertheless, the National Comprehensive Cancer Network (NCCN) guidelines recommend SN mapping if 1 or 2 suspicious lymph nodes are identified on axillary ultrasound (AU). The aim of this preliminary phase of the Multimodal Targeted Axillary Surgery (MUTAS) trial was to establish the accuracy of SN mapping in patients with axillary involvement undergoing upfront surgery., Methods: Between September 2019 and March 2022, we recruited patients with biopsy-proven metastatic axillary nodes and upfront surgery from a single center. We performed SN mapping in these patients before the surgical intervention, which included axillary lymph node dissection. The biopsy-proven metastatic node, SNs and the remaining axillary nodes were excised separately. SN status was considered representative of the status of the remaining axillary nodes. We calculated the sensitivity, specificity, negative predictive value and positive predictive value of the SN, overall and in patients with palpable nodes, in those with non-palpable nodes and an AU leading to diagnosis of axillary involvement, in those with 1 or 2 suspicious nodes on AU, and in patients with a single suspicious node on AU. We evaluated clinical, imaging and pathology features as predictors of the status of the remaining axillary nodes, false-negatives, and false-positives., Results: We included 25 patients in this phase. The false-negative rate of SN mapping was 28% overall, 21.42% for patients with palpable nodes, 36.36% for patients with non-palpable nodes and an AU diagnosis of axillary involvement, 28.75% for those with 1 or 2 suspicious nodes on AU, and 15.38% in patients with a single suspicious node on AU. The negative predictive value was highest in patients with a single suspicious node on AU (75%). The only significant predictive factor was that FN showed a higher Ki67 index score., Conclusions: In this study, SN mapping was not reliable in patients with biopsy-proven metastatic axillary nodes and upfront surgery for any of the subgroups studied. Further research should elucidate the best staging pathways in these patients to avoid premature de-escalation., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://gs.amegroups.com/article/view/10.21037/gs-22-480/coif). The authors have no conflicts of interest to declare., (2023 Gland Surgery. All rights reserved.)

Purpose: To assess the efficacy and exploratory biomarkers of continuing palbociclib plus endocrine therapy (ET) beyond progression on prior palbociclib-based regimen in patients with hormone receptor-positive/HER2-negative (HR+/HER2-) advanced breast cancer (ABC)., Patients and Methods: The multicenter, open-label, phase II BioPER trial included women who had experienced a progressive disease (PD) after having achieved clinical benefit on the immediately prior palbociclib plus ET regimen. Palbociclib (125 mg, 100 mg, or 75 mg daily orally for 3 weeks and 1 week off as per prior palbociclib-based regimen) plus ET of physician's choice were administered in 4-week cycles until PD or unacceptable toxicity. Coprimary endpoints were clinical benefit rate (CBR) and percentage of tumors with baseline loss of retinoblastoma (Rb) protein expression. Additional endpoints included safety and biomarker analysis., Results: Among 33 patients enrolled, CBR was 34.4% [95% confidence interval (CI), 18.6-53.2; P < 0.001] and 13.0% of tumors (95% CI, 5.2-27.5) showed loss of Rb protein expression, meeting both coprimary endpoints. Median progression-free survival was 2.6 months (95% CI, 1.8-6.7). No new safety signals were reported. A signature that included baseline mediators of therapeutic resistance to palbociclib and ET (low Rb score, high cyclin E1 score, ESR1 mutation) was independently associated with shorter median progression-free survival (HR, 22.0; 95% CI, 1.71-282.9; P = 0.018)., Conclusions: Maintaining palbociclib after progression on prior palbociclib-based regimen seems to be a reasonable, investigational approach for selected patients. A composite biomarker signature predicts a subset of patients who may not derive a greater benefit from palbociclib rechallenge, warranting further validation in larger randomized controlled trials., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: The p53 mutation in breast cancer confers a worse prognosis and is usually associated with p53 overexpression (p53+) on immunohistochemistry. Previous studies have shown that p53+ tumors could be associated with low axillary tumor burden (ATB)., Objective: We aimed to evaluate the association between p53+ and ATB in a large series of breast cancers as an aid to personalizing axillary surgical treatment., Methods: We retrieved 1762 infiltrating breast carcinomas from our database that were treated with upfront surgery in Hospital del Mar from 2004 to 2018. We compared p53+ and p53-negative (p53-) tumors in terms of the percentage of cases with high ATB and overall survival. This comparison was made overall and for each immunophenotype., Results: Overall, 18.7% of breast tumors were p53+. High ATB was less common in p53+ tumors than in p53- tumors in the luminal B-Her2-negative immunophenotype (6.2% versus 16.9%, respectively, P = 0.025), but not in the other immunophenotypes or overall. Overall survival was worse in patients with p53+ breast cancer (P = 0.002)., Conclusion: p53+ breast cancers were associated with worse overall survival. However, low ATB was more common in these tumors than in p53- tumors in the luminal B-Her2-negative subtype. Information on p53 expression could be of use to predict ATB in some breast cancer tumors.

Ligand-dependent corepressor (LCOR) mediates normal and malignant breast stem cell differentiation. Cancer stem cells (CSCs) generate phenotypic heterogeneity and drive therapy resistance, yet their role in immunotherapy is poorly understood. Here we show that immune-checkpoint blockade (ICB) therapy selects for LCOR low CSCs with reduced antigen processing/presentation machinery (APM) driving immune escape and ICB resistance in triple-negative breast cancer (TNBC). We unveil an unexpected function of LCOR as a master transcriptional activator of APM genes binding to IFN-stimulated response elements (ISREs) in an IFN signaling-independent manner. Through genetic modification of LCOR expression, we demonstrate its central role in modulation of tumor immunogenicity and ICB responsiveness. In TNBC, LCOR associates with ICB clinical response. Importantly, extracellular vesicle (EV) Lcor-messenger RNA therapy in combination with anti-PD-L1 overcame resistance and eradicated breast cancer metastasis in preclinical models. Collectively, these data support LCOR as a promising target for enhancement of ICB efficacy in TNBC, by boosting of tumor APM independently of IFN., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objectives: To evaluate the mammographic features in women with benign breast disease (BBD) and the risk of subsequent breast cancer according to their mammographic findings., Methods: We analyzed data from a Spanish cohort of women screened from 1995 to 2015 and followed up until December 2017 (median follow-up, 5.9 years). We included 10,650 women who had both histologically confirmed BBD and mammographic findings. We evaluated proliferative and nonproliferative BBD subtypes, and their mammographic features: architectural distortion, asymmetries, calcifications, masses, and multiple findings. The adjusted hazard ratios (aHR) and 95% confidence intervals (95% CI) for breast cancer were estimated using a Cox proportional hazards model. We plotted the adjusted cumulative incidence curves., Results: Calcifications were more frequent in proliferative disease with atypia (43.9%) than without atypia (36.8%) or nonproliferative disease (22.2%; p value < 0.05). Masses were more frequent in nonproliferative lesions (59.1%) than in proliferative lesions without atypia (35.1%) or with atypia (30.0%; p value < 0.05). Multiple findings and architectural distortion were more likely in proliferative disease (16.1% and 4.7%) than in nonproliferative disease (12.8% and 1.9%). Subsequent breast cancer occurred in 268 (2.5%) women. Compared with women who had masses, the highest risk of subsequent breast cancer was found in those with architectural distortions (aHR, 2.21; 95% CI, 1.16-4.22), followed by those with multiple findings (aHR, 1.89; 95% CI, 1.34-2.66), asymmetries (aHR, 1.66; 95% CI, 0.84-3.28), and calcifications (aHR, 1.60; 95% CI, 1.21-2.12)., Conclusion: BBD subtypes showed distinct mammographic findings. The risk of subsequent breast cancer was high in those who have shown architectural distortion, multiple findings, asymmetries, and calcifications than in women with masses., Key Points: • The presence of mammographic findings in women attending breast cancer screening helps clinicians to assess women with benign breast disease (BBD). • Calcifications were frequent in BBDs with atypia, which are the ones with a high breast cancer risk, while masses were common in low-risk BBDs. • The excess risk of subsequent breast cancer in women with BBD was higher in those who showed architectural distortion compared to those with masses., (© 2021. European Society of Radiology.)

Enhancing natural killer (NK) cell-based cancer immunotherapy by overcoming immunosuppression is an area of intensive research. Here, we have demonstrated that the anti-CD137 agonist urelumab can overcome TGFβ-mediated inhibition of human NK-cell proliferation and antitumor function. Transcriptomic, immunophenotypic, and functional analyses showed that CD137 costimulation modified the transcriptional program induced by TGFβ on human NK cells by rescuing their proliferation in response to IL2, preserving their expression of activating receptors (NKG2D) and effector molecules (granzyme B, IFNγ) while allowing the acquisition of tumor-homing/retention features (CXCR3, CD103). Activated NK cells cultured in the presence of TGFβ1 and CD137 agonist recovered CCL5 and IFNγ secretion and showed enhanced direct and antibody-dependent cytotoxicity upon restimulation with cancer cells. Trastuzumab treatment of fresh breast carcinoma-derived multicellular cultures induced CD137 expression on tumor-infiltrating CD16 + NK cells, enabling the action of urelumab, which fostered tumor-infiltrating NK cells and recapitulated the enhancement of CCL5 and IFNγ production. Bioinformatic analysis pointed to IFNG as the driver of the association between NK cells and clinical response to trastuzumab in patients with HER2-positive primary breast cancer, highlighting the translational relevance of the CD137 costimulatory axis for enhancing IFNγ production. Our data reveals CD137 as a targetable checkpoint for overturning TGFβ constraints on NK-cell antitumor responses., (©2021 American Association for Cancer Research.)

Snail1 is a transcriptional factor required for epithelial to mesenchymal transition and activation of cancer-associated fibroblasts (CAF). Apart from that, tumor endothelial cells also express Snail1. Here, we have unraveled the role of Snail1 in this tissue in a tumorigenic context. Methods : We generated transgenic mice with an endothelial-specific and inducible Snail1 depletion. This murine line was crossed with MMTV-PyMT mice that develop mammary gland tumors and the consequence of Snail1 depletion in the endothelium were investigated. We also interfere Snail1 expression in cultured endothelial cells. Results : Specific Snail1 depletion in the endothelium of adult mice does not promote an overt phenotype; however, it delays the formation of mammary gland tumors in MMTV-PyMT mice. These effects are associated to the inability of Snail1-deficient endothelial cells to undergo angiogenesis and to enhance CAF activation in a paracrine manner. Moreover, tumors generated in mice with endothelium-specific Snail1 depletion are less advanced and show a papillary phenotype. Similar changes on onset and tumor morphology are observed by pretreatment of MMTV-PyMT mice with the angiogenic inhibitor Bevacizumab. Human breast papillary carcinomas exhibit a lower angiogenesis and present lower staining of Snail1, both in endothelial and stromal cells, compared with other breast neoplasms. Furthermore, human breast tumors datasets show a strong correlation between Snail1 expression and high angiogenesis. Conclusion : These findings show a novel role for Snail1 in endothelial cell activation and demonstrate that these cells impact not only on angiogenesis, but also on tumor onset and phenotype., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Triple-negative breast cancer (TNBC) is more aggressive than other breast cancer subtypes. TNBC is characterized by increased expression of Programmed Death-ligand 1 (PD-L1), a signal used by many tumors to escape the immune response. Expression of PD-L1 is a positive predictor of response to immunotherapy; therefore, it should be investigated in TNBC in order to select patients who may benefit from anti-PD-L1 therapies. While many PD-L1 assays are available, only the VENTANA platform with the anti-PD-L1 (SP142) antibody is licensed as a companion diagnostic device for selecting patients with metastatic/advanced TNBC who are candidates for treatment with atezolizumab. In this article, we provide a summary of an expert round-table discussion about PD-L1 testing, using the SP142 antibody in metastatic TNBC.

Tumors are complex tissues composed of transformed epithelial cells as well as cancer-activated fibroblasts (CAF) that facilitate epithelial tumor cell invasion. We show here that CAFs and other mesenchymal cells rely much more on glutamine than epithelial tumor cells; consequently, they are more sensitive to inhibition of glutaminase. Glutamine dependence drove CAF migration toward this amino acid when cultured in low glutamine conditions. CAFs also invaded a Matrigel matrix following a glutamine concentration gradient and enhanced the invasion of tumor cells when both cells were cocultured. Accordingly, glutamine directed invasion of xenografted tumors in immunocompromised mice. Stimulation of glutamine-driven epithelial tumor invasion by fibroblasts required previous CAF activation, which involved the TGFβ/Snail1 signaling axis. CAFs moving toward Gln presented a polarized Akt2 distribution that was modulated by the Gln-dependent activity of TRAF6 and p62 in the migrating front, and depletion of these proteins prevented Akt2 polarization and Gln-driven CAF invasion. Our results demonstrate that glutamine deprivation promotes CAF migration and invasion, which in turn facilitates the movement of tumor epithelial cells toward nutrient-rich territories. These results provide a novel molecular mechanism for how metabolic stress enhances invasion and metastasis. SIGNIFICANCE: Cancer-associated fibroblasts migrate and invade toward free glutamine and facilitate invasion of tumor epithelial cells, accounting for their movement away from the hostile conditions of the tumor towards nutrient-rich adjacent tissues. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/2/438/F1.large.jpg., (©2020 American Association for Cancer Research.)

Introduction: We aimed to assess differences in breast cancer risk across benign breast disease diagnosed at prevalent or incident screens., Materials and Methods: We conducted a retrospective cohort study with data from 629,087 women participating in a long-standing population-based breast cancer screening program in Spain. Each benign breast disease was classified as non-proliferative, proliferative without atypia, or proliferative with atypia, and whether it was diagnosed in a prevalent or incident screen. We used partly conditional Cox hazard regression to estimate the adjusted hazard ratios of the risk of breast cancer., Results: Compared with women without benign breast disease, the risk of breast cancer was significantly higher (p-value = 0.005) in women with benign breast disease diagnosed in an incident screen (aHR, 2.67; 95%CI: 2.24-3.19) than in those with benign breast disease diagnosed in a prevalent screen (aHR, 1.87; 95%CI: 1.57-2.24). The highest risk was found in women with a proliferative benign breast disease with atypia (aHR, 4.35; 95%CI: 2.09-9.08, and 3.35; 95%CI: 1.51-7.40 for those diagnosed at incident and prevalent screens, respectively), while the lowest was found in women with non-proliferative benign breast disease (aHR, 2.39; 95%CI: 1.95-2.93, and 1.63; 95%CI: 1.32-2.02 for those diagnosed at incident and prevalent screens, respectively)., Conclusion: Our study showed that the risk of breast cancer conferred by a benign breast disease differed according to type of screen (prevalent or incident). To our knowledge, this is the first study to analyse the impact of the screening type on benign breast disease prognosis., Competing Interests: Declaration of competing interest None declared., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls., Competing Interests: Competing interestsA.E. is on the Roche advisory board and has reported honoraria from Amgen, Novartis and Roche. A.J.L. is a consultant for BMS, Merck, AZ/Medimmune, and Genentech. R.S. reports research funding from Roche, Puma, Merck; advisory board and consultancy for BMS; travel funding from Roche, Merck, and Astra Zeneca. S.G. reports Lab research funding from Lilly, Clinical research funding from Eli Lilly and Novartis and is a Paid advisor to Eli Lilly, Novartis, and G1 Therapeutics. J.v.d.L. is member of the scientific advisory boards of Philips, the Netherlands and ContextVision, Sweden and receives research funding from Philips, the Netherlands and Sectra, Sweden. S.A. reports Research funding to institution from Merck, Genentech, BMS, Novartis, Celgene and Amgen and is an uncompensated consultant /steering committee member for Merck, Genentech and BMS. T.O.N. has consulted for Nanostring and received compensation and has intellectual property rights/ownership interests from Bioclassifier LLC [not related to the subject material under consideration]. S.L. receives research funding to institution from Novartis, Bristol Meyers Squibb, Merck, Roche-Genentech, Puma Biotechnology, Pfizer and Eli Lilly, has acted as consultant (not compensated) to Seattle Genetics, Pfizer, Novartis, BMS, Merck, AstraZeneca and Roche-Genentech and acted as consultant (paid to her institution) to Aduro Biotech. S.R.L. has received travel and educational funding from Roche/Ventana. A.M. is an equity holder in Elucid Bioimaging and in Inspirata Inc., a scientific advisory consultant for Inspirata Inc, has served as a scientific advisory board member for Inspirata Inc, Astrazeneca, Bristol Meyers-Squibb and Merck, has sponsored research agreements with Philips and Inspirata Inc, is involved in a NIH U24 grant with PathCore Inc, and 3 different R01 grants with Inspirata Inc. and his technology has been licensed to Elucid Bioimaging and Inspirata Inc. G.C. is on the advisory boards of Roche, BMS, Pfizer, Seattle Genetics and Ellipsis, and reports personal fees from Roche, BMS, Pfizer, Seattle Genetics, and Ellipsis, outside of the submitted work. J.H. is the director and owner of Vivactiv Ltd. J.H. is the director and owner of Slide Score B.V. F.P.L. reports funding from Astrazeneca, BMS, Roche, MSD, Pfizer, Novartis, Sanofi, Eli Lilly. J.B. reports consultancies from Insight Genetics, BioNTech AG, Biotheranostics, Pfizer, RNA Diagnostics and OncoXchange, research funding from Thermo Fisher Scientific, Genoptix, Agendia, NanoString Technologies, Stratifyer GmbH and Biotheranostics, applied for patents, including Jan 2017: Methods and Devices for Predicting Anthracycline Treatment Efficacy, US utility—15/325,472; EPO—15822898.1; Canada—not yet assigned; Jan 2017: Systems, Devices and Methods for Constructing and Using a Biomarker, US utility—15/328,108; EPO—15824751.0; Canada—not yet assigned; Oct 2016: Histone gene module predicts anthracycline benefit, PCT/CA2016/000247; Dec 2016: 95‐Gene Signature of Residual Risk Following Endocrine Treatment, PCT/CA2016/000304; Dec 2016: Immune Gene Signature Predicts Anthracycline Benefit, PCT/CA2016/000305. M.A.S. reports consulting work for Achilles Therapeutics. C.S. reports receipt of grants/research support from Pfizer, AstraZeneca, BMS and Ventana; receipt of honoraria, consultancy, or SAB Member fees from Pfizer, Novartis, GlaxoSmithKline, MSD, BMS, Celgene, AstraZeneca, Illumina, Sarah Canon Research Institute, Genentech, Roche-Ventana, GRAIL, Medicxi; Advisor for Dynamo Therapeutics; Stock shareholder in Apogen Biotechnologies, Epic Bioscience, GRAIL; Co-Founder & stock options in Achilles Therapeutics. A.H.B. is the co-founder and CEO of PathAI. J.K. is an employee of PathAI. D.D. is on the advisory board for Oncology Analytics, Inc, and a consultant for Novartis. D.L.R. is on the advisory board of Amgen, Astra Zeneca, Cell Signaling Technology, Cepheid, Daiichi Sankyo, GSK, Konica/Minolta, Merck, NanoString, Perkin Elmer, Ventana, Ultivue; receives research support from Astra Zeneca, Cepheid, Navigate BioPharma, NextCure, Lilly, Ultivue; instrument support from Ventana, Akoya/Perkin Elmer, NanoString; paid consultant for Biocept; received travel honoraria from BMS, founder and equity holder for PixelGear and received royalty from Rarecyte. A.T. reports benefits from ICR’s Inventors Scheme associated with patents for one of PARP inhibitors in BRCA1/2 associated cancers, as well as honoraria from Pfizer, Vertex, Prime Oncology, Artios, honoraria and stock in InBioMotion, honoraria and financial support for research from AstraZeneca, Medivation, Myriad Genetics and Merck Serono. This work includes contributions from, and was reviewed by, individuals at the FDA. This work has been approved for publication by the agency, but it does not necessarily reflect official agency policy. Certain commercial materials and equipment are identified in order to adequately specify experimental procedures. In no case does such identification imply recommendation or endorsement by the FDA, nor does it imply that the items identified are necessarily the best available for the purpose. This work includes contributions from, and was reviewed by, individuals who received funding from the National Institutes of Health, the U.S. Department of Veterans Affairs and the Department of Defense. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the U.S. Department of Veterans Affairs, the Department of Defense, or the United States Government., (© The Author(s) 2020.)

Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring., Competing Interests: Competing interestsJ.T. is funded by Visiopharm A/S, Denmark. A.M. is an equity holder in Elucid Bioimaging and in Inspirata Inc. He is also a scientific advisory consultant for Inspirata Inc. In addition he has served as a scientific advisory board member for Inspirata Inc, Astrazeneca, Bristol Meyers-Squibb and Merck. He also has sponsored research agreements with Philips and Inspirata Inc. His technology has been licensed to Elucid Bioimaging and Inspirata Inc. He is also involved in an NIH U24 grant with PathCore Inc, and three different R01 grants with Inspirata Inc. S.R.L. received travel and educational funding from Roche/Ventana. A.J.L. serves as a consultant for BMS, Merck, AZ/Medimmune, and Genentech. He is also provides consulting and advisory work for many other companies not relevant to this work. FPL does consulting for Astrazeneca, BMS, Roche, MSD Pfizer, Novartis, Sanofi, and Lilly. S.Ld.H., A.K., M.K., U.K., and M.B. are employees of Roche. J.M.S.B. is consultant for Insight Genetics, BioNTech AG, Biothernostics, Pfizer, RNA Diagnostics, and OncoXchange. He received funding from Thermo Fisher Scientific, Genoptix, Agendia, NanoString technologies, Stratifyer GmBH, and Biotheranostics. L.F.S.K. is a consultant for Roche and Novartis. J.K.K. and A.H.B. are employees of PathAI. D.L.R. is on the advisory board for Amgen, Astra Xeneca, Cell Signaling Technology, Cepheid, Daiichi Sankyo, GSK, Konica/Minolta, Merck, Nanostring, Perking Elmer, Roche/Ventana, and Ultivue. He has received research support from Astrazeneca, Cepheid, Navigate BioPharma, NextCure, Lilly, Ultivue, Roche/Ventana, Akoya/Perkin Elmer, and Nanostring. He also has financial conflicts of interest with BMS, Biocept, PixelGear, and Rarecyte. S.G. is a consultant for and/or receives funding from Eli Lilly, Novartis, and G1 Therapeutics. J.A.W.M.vdL. is a member of the scientific advisory boards of Philips, the Netherlands and ContextVision, Sweden, and receives research funding from Philips, the Netherlands and Sectra, Sweden. S.A. is a consultant for Merck, Genentech, and BMS, and receives funding from Merck, Genentech, BMS, Novartis, Celgene, and Amgen. T.O.N. has consulted for Nanostring, and has intellectual property rights and ownership interests from Bioclassifier LLC. S.L. receives research funding to her institution from Novartis, Bristol Meyers-Squibb, Merck, Roche-Genentech, Puma Biotechnology, Pfizer and Eli Lilly. She has acted as consultant (not compensated) to Seattle Genetics, Pfizer, Novartis, BMS, Merck, AstraZeneca and Roche-Genentech. She has acted as consultant (paid to her institution) to Aduro Biotech. J.H. is director and owner of Slide Score BV. M.M.S. is a medical advisory board member of OptraScan. R.S. has received research support from Merck, Roche, Puma; and travel/congress support from AstraZeneca, Roche and Merck; and he has served as an advisory board member of BMS and Roche and consults for BMS., (© The Author(s) 2020.)

Poly(ADP-ribose)-polymerase (PARP)-1 and PARP-2 play an essential role in the DNA damage response. Based on this effect of PARP in the tumor cell itself, PARP inhibitors have emerged as new therapeutic tools both approved and in clinical trials. However, the interactome of multiple other cell types, particularly T cells, within the tumor microenvironment are known to either favor or limit tumorigenesis. Here, we bypassed the embryonic lethality of dually PARP-1/PARP-2-deficient mice by using a PARP-1-deficient mouse with a Cd4-promoter-driven deletion of PARP-2 in T cells to investigate the understudied role of these PARPs in the modulation of T cell responses against AT-3-induced breast tumors. We found that dual PARP-1/PARP-2-deficiency in T cells promotes tumor growth while single deficiency of each protein limited tumor progression. Analysis of tumor-infiltrating cells in dual PARP-1/PARP-2-deficiency host-mice revealed a global change in immunological profile and impaired recruitment and activation of T cells. Conversely, single PARP-1 and PARP-2-deficiency tends to produce an environment with an active and partially upregulated immune response. Our findings pinpoint opposite effects of single and dual PARP-1 and PARP-2-deficiency in modulating the antitumor response with an impact on tumor progression, and will have implications for the development of more selective PARP-centered therapies.

Natural killer (NK) cells can orchestrate effective antitumor immunity. The presence of tumor-infiltrating NK cells in diagnostic biopsies predicts pathologic complete response (pCR) to HER2-specific therapeutic antibodies in patients with primary breast cancer. Here, we analyzed whether diversity in circulating NK cells might influence tumor infiltration and HER2-specific therapeutic antibody efficacy. We found that numbers of circulating CD57 + NK cells inversely correlated with pCR to HER2-specific antibody treatment in patients with primary breast cancer independently of age, traditional clinicopathologic factors, and CD16A 158F/V genotype. This association was uncoupled from the expression of other NK-cell receptors, the presence of adaptive NK cells, or changes in major T-cell subsets, reminiscent of cytomegalovirus-induced immunomodulation. NK-cell activation against trastuzumab-coated HER2 + breast cancer cells was comparable in patients with high and low proportions of CD57 + NK cells. However, circulating CD57 + NK cells displayed decreased CXCR3 expression and CD16A-induced IL2-dependent proliferation in vitro Presence of CD57 + NK cells was reduced in breast tumor-associated infiltrates as compared with paired peripheral blood samples, suggesting deficient homing, proliferation, and/or survival of NK cells in the tumor niche. Indeed, numbers of circulating CD57 + were inversely related to tumor-infiltrating NK-cell numbers. Our data reveal that NK-cell differentiation influences their antitumor potential and that CD57 + NK cells may be a biomarker useful for tailoring HER2 antibody-based therapeutic strategies in breast cancer., (©2019 American Association for Cancer Research.)

With the identification of therapeutic targets for lung adenocarcinoma, it has become mandatory to distinguish it from other entities. Some cases remain classified as non-small cell lung carcinoma, not otherwise specified (NSCLC-NOS) with immunohistochemistry. Electron microscopy (EM) can be useful, allowing the identification of glandular differentiation. The aim of this study was to determine the complementary value of immunohistochemistry and EM.Forty-eight NSCLC-NOS cases were selected (PSMAR-Biobank, Barcelona, Spain). Immunohistochemistry (TTF-1, p40) was performed. Tissue was retrieved from paraffin blocks. Results were compared to the final diagnosis, derived from combination of light microscopy, immunohistochemistry, EM, molecular studies and resection specimen.Immunohistochemistry concurred with final diagnosis in 36 cases (75%, Kappa = 0.517). EM agreed with final diagnosis in 35 (72.9%, Kappa = 0.471). Immunohistochemistry had a sensitivity = 73%, specificity = 100%, positive predictive value (PPV) = 100% and negative predictive value (NPV) = 52.4% for adenocarcinoma. All adenocarcinoma cases not solved by immunohistochemistry (n = 10) were classified by EM, and vice versa. Data from EM were identical to those of immunohistochemistry: sensitivity = 73%, specificity = 100%, PPV = 100% and NPV = 52.4%. Combining both techniques, 47 cases were coincident with final diagnosis (97.9%, Kappa = 0.943).EM can provide valuable information in subtyping NSCLC-NOS, being particularly useful when immunohistochemistry is inconclusive. EM could be considered as a complementary tool for decision-making in NSCLC-NOS.

B cells thwart antigenic aggressions by releasing immunoglobulin M (IgM), IgG, IgA, and IgE, which deploy well-understood effector functions. In contrast, the role of secreted IgD remains mysterious. We found that some B cells generated IgD-secreting plasma cells following early exposure to external soluble antigens such as food proteins. Secreted IgD targeted basophils by interacting with the CD44-binding protein galectin-9. When engaged by antigen, basophil-bound IgD increased basophil secretion of interleukin-4 (IL-4), IL-5, and IL-13, which facilitated the generation of T follicular helper type 2 cells expressing IL-4. These germinal center T cells enhanced IgG1 and IgE but not IgG2a and IgG2b responses to the antigen initially recognized by basophil-bound IgD. In addition, IgD ligation by antigen attenuated allergic basophil degranulation induced by IgE co-ligation. Thus, IgD may link B cells with basophils to optimize humoral T helper type 2-mediated immunity against common environmental soluble antigens., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mechanistic target of rapamycin (mTOR) enhances immunity in addition to orchestrating metabolism. Here we show that mTOR coordinates immunometabolic reconfiguration of marginal zone (MZ) B cells, a pre-activated lymphocyte subset that mounts antibody responses to T-cell-independent antigens through a Toll-like receptor (TLR)-amplified pathway involving transmembrane activator and CAML interactor (TACI). This receptor interacts with mTOR via the TLR adapter MyD88. The resulting mTOR activation instigates MZ B-cell proliferation, immunoglobulin G (IgG) class switching, and plasmablast differentiation through a rapamycin-sensitive pathway that integrates metabolic and antibody-inducing transcription programs, including NF-κB. Disruption of TACI-mTOR interaction by rapamycin, truncation of the MyD88-binding domain of TACI, or B-cell-conditional mTOR deficiency interrupts TACI signaling via NF-κB and cooperation with TLRs, thereby hampering IgG production to T-cell-independent antigens but not B-cell survival. Thus, mTOR drives innate-like antibody responses by linking proximal TACI signaling events with distal immunometabolic transcription programs.

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

Secretory immunoglobulin A (SIgA) enhances host-microbiota symbiosis, whereas SIgM remains poorly understood. We found that gut IgM + plasma cells (PCs) were more abundant in humans than mice and clonally related to a large repertoire of memory IgM + B cells disseminated throughout the intestine but rare in systemic lymphoid organs. In addition to sharing a gut-specific gene signature with memory IgA + B cells, memory IgM + B cells were related to some IgA + clonotypes and switched to IgA in response to T cell-independent or T cell-dependent signals. These signals induced abundant IgM which, together with SIgM from clonally affiliated PCs, recognized mucus-embedded commensals. Bacteria recognized by human SIgM were dually coated by SIgA and showed increased richness and diversity compared to IgA-only-coated or uncoated bacteria. Thus, SIgM may emerge from pre-existing memory rather than newly activated naive IgM + B cells and could help SIgA to anchor highly diverse commensal communities to mucus., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

MYC translocation is a defining feature of Burkitt lymphoma (BL), and the new category of high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 translocations, and occurs in 6% to 15% of diffuse large B-cell lymphomas (DLBCLs). The low incidence of MYC translocations in DLBCL makes the genetic study of all these lymphomas cumbersome. Strategies based on an initial immunophenotypic screening to select cases with a high probability of carrying the translocation may be useful. LMO2 is a germinal center marker expressed in most lymphomas originated in these cells. Mining gene expression profiling studies, we observed LMO2 downregulation in BL and large B-cell lymphoma (LBCL) with MYC translocations, and postulated that LMO2 protein expression could assist to identify such cases. We analyzed LMO2 protein expression in 46 BLs and 284 LBCL. LMO2 was expressed in 1/46 (2%) BL cases, 146/268 (54.5%) DLBCL cases, and 2/16 (12.5%) high-grade B-cell lymphoma cases with MYC and BCL2 and/or BCL6 translocations. All BLs carried MYC translocation (P<0.001), whereas LMO2 was only positive in 6/42 (14%) LBCL with MYC translocation (P<0.001). The relationship between LMO2 negativity and MYC translocation was further analyzed in different subsets of tumors according to CD10 expression and cell of origin. Lack of LMO2 expression was associated with the detection of MYC translocations with high sensitivity (87%), specificity (87%), positive predictive value and negative predictive value (74% and 94%, respectively), and accuracy (87%) in CD10 LBCL. Comparing LMO2 and MYC protein expression, all statistic measures of performance of LMO2 surpassed MYC in CD10 LBCL. These findings suggest that LMO2 loss may be a good predictor for the presence of MYC translocation in CD10 LBCL.

Antibody targeting of the immune checkpoint receptor PD1 produces therapeutic activity in a variety of solid tumors, but most patients exhibit partial or complete resistance to treatment for reasons that are unclear. In this study, we evaluated tumor specimens from 65 patients with melanoma, lung nonsquamous, squamous cell lung or head and neck cancers who were treated with the approved PD1-targeting antibodies pembrolizumab or nivolumab. Tumor RNA before anti-PD1 therapy was analyzed on the nCounter system using the PanCancer 730-Immune Panel, and we identified 23 immune-related genes or signatures linked to response and progression-free survival (PFS). In addition, we evaluated intra- and interbiopsy variability of PD1, PD-L1, CD8A, and CD4 mRNAs and their relationship with tumor-infiltrating lymphocytes (TIL) and PD-L1 IHC expression. Among the biomarkers examined, PD1 gene expression along with 12 signatures tracking CD8 and CD4 T-cell activation, natural killer cells, and IFN activation associated significantly with nonprogressive disease and PFS. These associations were independent of sample timing, drug used, or cancer type. TIL correlated moderately (∼0.50) with PD1 and CD8A mRNA levels and weakly (∼0.35) with CD4 and PD-L1. IHC expression of PD-L1 correlated strongly with PD-L1 (0.90), moderately with CD4 and CD8A, and weakly with PD1. Reproducibility of gene expression in intra- and interbiopsy specimens was very high (total SD <3%). Overall, our results support the hypothesis that identification of a preexisting and stable adaptive immune response as defined by mRNA expression pattern is reproducible and sufficient to predict clinical outcome, regardless of the type of cancer or the PD1 therapeutic antibody administered to patients. Cancer Res; 77(13); 3540-50. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Innate lymphoid cells (ILCs) regulate stromal cells, epithelial cells and cells of the immune system, but their effect on B cells remains unclear. Here we identified RORγt(+) ILCs near the marginal zone (MZ), a splenic compartment that contains innate-like B cells highly responsive to circulating T cell-independent (TI) antigens. Splenic ILCs established bidirectional crosstalk with MAdCAM-1(+) marginal reticular cells by providing tumor-necrosis factor (TNF) and lymphotoxin, and they stimulated MZ B cells via B cell-activation factor (BAFF), the ligand of the costimulatory receptor CD40 (CD40L) and the Notch ligand Delta-like 1 (DLL1). Splenic ILCs further helped MZ B cells and their plasma-cell progeny by coopting neutrophils through release of the cytokine GM-CSF. Consequently, depletion of ILCs impaired both pre- and post-immune TI antibody responses. Thus, ILCs integrate stromal and myeloid signals to orchestrate innate-like antibody production at the interface between the immune system and circulatory system.

A dense mucus layer in the large intestine prevents inflammation by shielding the underlying epithelium from luminal bacteria and food antigens. This mucus barrier is organized around the hyperglycosylated mucin MUC2. Here we show that the small intestine has a porous mucus layer, which permitted the uptake of MUC2 by antigen-sampling dendritic cells (DCs). Glycans associated with MUC2 imprinted DCs with anti-inflammatory properties by assembling a galectin-3-Dectin-1-FcγRIIB receptor complex that activated β-catenin. This transcription factor interfered with DC expression of inflammatory but not tolerogenic cytokines by inhibiting gene transcription through nuclear factor κB. MUC2 induced additional conditioning signals in intestinal epithelial cells. Thus, mucus does not merely form a nonspecific physical barrier, but also constrains the immunogenicity of gut antigens by delivering tolerogenic signals.

Background: A human model of gastroesophageal reflux disease was used to examine the contribution of a non-specialized columnar type of metaplasia (NSCM) and key molecular events (BMP4 and CDX2) in the development of Barrett's esophagus., Methods: Biopsies of the remnant esophagus from 18 patients undergoing esophagectomy with gastric preservation were taken at 6-36-month intervals postoperatively and examined for activation of the BMP pathway (BMP4/P-Smad 1/5/8) and CDX2 and CDX1 expression by imunohistochemistry, quantitative real-time PCR, Western blot, and in situ hybridization., Results: A short segment (mean 15.6 mm) of NSCM was detected in 10 (56%) patients, with an increasing prevalence from 17% at 6 months to 62% at 36 months. Nuclear expression of P-Smad 1/5/8 in the squamous epithelium close to the anastomosis with strong expression in all epithelial cells of NSCM areas was found. Forty-eight (63%) biopsies with NSCM showed scattered nuclear expression of CDX2. Two cases showed isolated glands at 18, 24, and 36 months that fully expressed CDX2 and co-expressed CDX1. BMP4 mRNA and CDX2 mRNA levels were significantly greater in NSCM than in squamous epithelium., Conclusions: BMP4 activation in NSCM and early expression of CDX2 are involved in the columnar epithelial differentiation of Barrett's esophagus.

Neutrophils use immunoglobulins to clear antigen, but their role in immunoglobulin production is unknown. Here we identified neutrophils around the marginal zone (MZ) of the spleen, a B cell area specialized in T cell-independent immunoglobulin responses to circulating antigen. Neutrophils colonized peri-MZ areas after postnatal mucosal colonization by microbes and enhanced their B cell-helper function after receiving reprogramming signals, including interleukin 10 (IL-10), from splenic sinusoidal endothelial cells. Splenic neutrophils induced immunoglobulin class switching, somatic hypermutation and antibody production by activating MZ B cells through a mechanism that involved the cytokines BAFF, APRIL and IL-21. Neutropenic patients had fewer and hypomutated MZ B cells and a lower abundance of preimmune immunoglobulins to T cell-independent antigens, which indicates that neutrophils generate an innate layer of antimicrobial immunoglobulin defense by interacting with MZ B cells.