BACKGROUND:Stromal tumour-infiltrating lymphocytes (sTILs) are prognostic in early-stage HER2-positive breast cancer, but their role in the context of dual HER2 blockade remains undefined. We evaluated manual, digital, and artificial intelligence (AI)-based sTIL quantification, together with AI-derived spatial metrics, for prognostic and treatment-benefit stratification using tumour samples from the phase 3 APHINITY trial. METHODS:In the APHINITY trial, 4805 patients were randomly assigned to receive chemotherapy plus trastuzumab with pertuzumab or chemotherapy plus trastuzumab with placebo. Median follow-up was 74·1 months (IQR 68·3-75·4). We analysed 4262 haematoxylin and eosin-stained images using manual assessment, an automated digital approach, AI-based lymphocyte quantification (AI percentage lymphocytes), and two AI-derived spatial features (AI-TIL and immune hotspot). Interobserver reproducibility was assessed in 262 randomly chosen tumour samples scored independently by five pathologists. Multivariable Cox models were used to assess associations between TIL levels and invasive disease-free survival (primary outcome in APHINITY), distant recurrence-free interval, and overall survival. The heterogeneity of pertuzumab benefit was evaluated using subgroup analyses, subpopulation treatment effect pattern plot analyses, and nested Cox models with treatment-by-biomarker interaction terms. FINDINGS:Manual scoring showed high interobserver reproducibility (intraclass correlation coefficient 0·84 [95% CI 0·79-0·88]). Concordance between manual and automated methods was modest. AI-based scoring (AI percentage lymphocytes) reclassified 120 (11·6%) of 1035 node-positive tumours from immune-low (by manual scoring) to immune-high; this subgroup of patients showed greater separation of 5-year invasive disease-free survival curves between pertuzumab and placebo groups compared with patients whose tumours were concordantly classified as immune-low by both manual and AI-based approaches. Higher levels of TILs were associated with improved invasive disease-free survival for all sTIL measurement approaches and spatial measurements (hazard ratios [HRs] 0·41-0·93). Pertuzumab was associated with improved invasive disease-free survival at higher sTIL levels across all measurement approaches (HRs 0·36-0·48), but was not associated with higher values of spatial measures. The largest 6-year absolute improvements with pertuzumab were observed in patients with node-positive disease whose tumours scored in the highest level of immune infiltration of manual sTIL scoring (≥70·0%; mean absolute improvement 12·1 percentage points [SD 2·8]). In nested prognostic and predictive models, AI-based immune hotspot scores provided the most consistent additional information when combined with any sTIL measurement (all p<0·010). INTERPRETATION:Standardised manual sTIL scoring was reproducible, and digital and AI-based methods showed consistent prognostic stratification and potential for treatment-benefit stratification despite only modest correlation between platforms. AI spatial metrics provided complementary information beyond sTIL density and could support more scalable immune assessment. Future studies are needed to validate these approaches in independent cohorts and to clarify their clinical utility for stratifying contemporary HER2-directed therapies. FUNDING:None.

Hormone receptor-positive (HR+) breast cancers (BCs) are typically "immune-cold," poorly immune-infiltrated tumors that do not respond to immune-checkpoint blockade (ICB) therapies. Using clinical data, we report that estrogen receptor α (ERα) signaling was associated with immunosuppressive pathways and a lack of response to ICB in patients with HR+ BC. In this study, we validated ER-mediated immunosuppression by engineering and modulating the ER in preclinical models in vitro, in vivo, and ex vivo. Mechanistically, we found that ERα hijacked LCOR, a nuclear receptor corepressor, thereby preventing LCOR's function in the induction of tumor immunogenicity and immune infiltration, which is normally observed in the absence of ERα, such as in ER- BC. In HR+ BC, we demonstrate that the molecular disruption of LCOR and ERα interaction using anti-ER therapies or using a mutant of the LCOR nuclear receptor-binding domain (LSKLL into LSKAA) that does not interact with ERα, restored the immunogenic functions of LCOR. Remarkably, the LCOR-ERα disruption converted HR+ BC immune-cold tumors into immune-hot tumors responsive to ICB by increased antigen presentation machinery expression, immune infiltration, T cell recognition, and T cell-mediated killing. In conclusion, ERα inhibition and the disruption of LCOR-ERα interaction represent a therapeutic strategy and an opportunity to elicit immunotherapeutic benefit in patients with HR+ BC.

In metastasis, the dynamics of tumor-immune interactions during micrometastasis remain unclear. Identifying the vulnerabilities of micrometastases before outbreaking into macrometastases can reveal therapeutic opportunities for metastasis. Here, we report a function of T cell immunoglobulin and mucin domain 3 (TIM3) in tumor cells during micrometastasis using breast cancer (BC) metastasis mouse models. TIM3 is highly upregulated in micrometastases, promoting survival, stemness, and immune escape. TIM3+ tumor cells are specifically selected during early seeding of micrometastasis. Mechanistically, TIM3 increases β-catenin/interleukin-1β (IL-1β) signaling, leading to stemness and immune-evasion by inducing immunosuppressive γδ T cells and reducing CD8 T cells during micrometastasis. Clinical data confirm increased TIM3+ tumor cells in BC metastasis and TIM3+ tumor cells as a biomarker of poor outcome in BC patients. (Neo)adjuvant TIM3 blockade reduces the metastatic seeding and incidence in preclinical models. These findings unveil a specific mechanism of micrometastasis immune-evasion and the potential use of TIM3 blockade for subclinical metastasis.

Supplementary Table S1. Selection Criteria; Supplementary Table S2. Recruiting Sites, Principal Investigators, and Patient Numbers; Supplementary Table S3. Study Members; Supplementary Table S4. List of antibodies used for immunohistochemistry analysis; Supplementary Table S5. Cutoff values for the quantitative assessment of biomarkers by immunohistochemistry; Supplementary Table S6. The 77-gene panel by the AVENIO ctDNA Expanded Kit used for targeted NGS on plasma samples; Supplementary Table S7. Representativeness of study participants; Supplementary Table S8. Association of baseline clinical and pathologic characteristics of trial participants included in the efficacy analysis with clinical benefit; Supplementary Table S9. Relative dose intensity and drug discontinuation; Supplementary Table S10. All causality AEs according to NCI-CTCAE version 4.0.3 occurring in at least 5% of the patients; Supplementary Table S11. Adverse events of special interest according to NCI-CTCAE version 4.0.3; Supplementary Table S12. Baseline clinical and pathologic characteristics of trial participants included in the retinoblastoma and signature subsets; Supplementary Table S13. Tumor best response according to RECIST version 1.1 of trial participants included in the retinoblastoma and signature subsets; Supplementary Table S14. Association of biomarkers and composite signatures with clinical benefit.

Summary of all adverse events according to NCI-CTCAE version 4.0.3.

Kaplan-Meier analyses of investigator-assessed progression-free survival and overall survival in the efficacy population.

The level of mutant and wild-type circulating DNA in baseline plasma samples by patient.

(A) Longitudinal ctDNA quantification during study treatment. (B) Variations of ctDNA during study treatment. (C, D) Kaplan-Meier analyses of investigator-assessed progression-free survival according to the ctDNA detection after 2 weeks and 12 weeks of study treatment.

Immunotherapies are beneficial for a considerable proportion of cancer patients, but ineffective in others. In vitro modelling of the complex interactions between cancer cells and their microenvironment could provide a path to understanding immune therapy sensitivity and resistance. Here we develop MIRO, a fully humanised in vitro platform to model the spatial organisation of the tumour/stroma interface and its interaction with immune cells. We find that stromal barriers are associated with immune exclusion and protect cancer cells from antibody-dependent cellular cytotoxicity, elicited by targeted therapy. We demonstrate that IL2-driven immunomodulation increases immune cell velocity and spreading to overcome stromal immunosuppression and restores anti-cancer response in refractory tumours. Collectively, our study underscores the translational value of MIRO as a powerful tool for exploring how the spatial organisation of the tumour microenvironment shapes the immune landscape and influences the responses to immunomodulating therapies. Modelling the tumour immune microenvironment in vitro is a valuable tool to test immunotherapy efficiency but capturing its complexity is challenging. Here authors present a fully humanised in vitro platform representing tumour/stroma interface and demonstrate how modulation by IL2 may allow immune cells to overcome stromal barriers.

Nipple discharge affects over 80% of women at some point in their lives, with malignancy detected in up to 23% of cases. This review highlights the shift from traditional surgical approaches to advanced imaging techniques, which enhance diagnostic accuracy and reduce unnecessary procedures. Diagnosis begins with a thorough medical history and physical examination to assess the need for imaging. Physiological nipple discharge, which is bilateral, multiductal, and non-spontaneous, typically requires no imaging. Conversely, pathological nipple discharge (PND), characteristically unilateral, uniductal, and spontaneous, requires imaging to rule out malignancy. Bloody PND is frequently associated with breast cancer, and up to 12% of non-bloody PND cases also involve malignancy. For women over 40 years, the first-line imaging modality is full-field digital mammography (FFDM) or digital breast tomosynthesis (DBT), usually combined with ultrasound (US). Men with PND undergo FFDM/DBT starting at age 25 years due to their higher risk of breast cancer. For women aged 30-39 years, US is the first assessment tool, with FFDM/DBT added, if necessary, while US is preferred for younger women and men. When initial imaging is negative or inconclusive, magnetic resonance imaging (MRI) is useful, often replacing galactography. With its high sensitivity and negative predictive value of almost 100%, a negative MRI can often obviate the need for surgery. Contrast-enhanced mammography (CEM) offers a viable alternative when MRI is not feasible. Although invasive, ductoscopy helps identify patients who may not require duct excision. This review consolidates the latest evidence and proposes an updated diagnostic algorithm for managing PND effectively. CRITICAL RELEVANCE STATEMENT: Effective management of nipple discharge requires recognising when imaging tests are needed and selecting the most appropriate diagnostic technique to rule out malignancy and avoid unnecessary interventions. KEY POINTS: First-line imaging for pathological nipple discharge (PND) assessment includes ultrasound and mammography. MRI is recommended for patients with PND and negative conventional imaging. A negative MRI is sufficient to justify surveillance rather than surgery. Contrast-enhanced mammography (CEM) is an alternative when MRI is unavailable or contraindicated.

The human gut includes plasma cells (PCs) expressing immunoglobulin A1 (IgA1) or IgA2, two structurally distinct IgA subclasses with elusive regulation, function, and reactivity. We show here that intestinal IgA1+ and IgA2+ PCs co-emerged early in life, comparably accumulated somatic mutations, and were enriched within short-lived CD19+ and long-lived CD19- PC subsets, respectively. IgA2+ PCs were extensively clonally related to IgA1+ PCs and a subset of them presumably emerged from IgA1+ precursors. Of note, secretory IgA1 (SIgA1) and SIgA2 dually coated a large fraction of mucus-embedded bacteria, including Akkermansia muciniphila. Disruption of homeostasis by inflammatory bowel disease (IBD) was associated with an increase in actively proliferating IgA1+ plasmablasts, a depletion in long-lived IgA2+ PCs, and increased SIgA1+SIgA2+ gut microbiota. Such increase featured enhanced IgA1 reactivity to pathobionts, including Escherichia coli, combined with depletion of beneficial A. muciniphila. Thus, gut IgA1 and IgA2 emerge from clonally related PCs and show unique changes in both frequency and reactivity in IBD.

Purpose: The aim of this study was to determine whether low HLA-I expression and NK cells infiltration are related to prognostic features in breast cancer, as observed in cancers in other locations and non-hormone dependent breast cancers. Particularly, we explored their relation to infiltrated axillary lymph nodes (ALNs), with the aim of finding new predictors helping to decide the extent of axillary surgery. Patients and Methods: We conducted a retrospective correlational analysis of 35 breast cancers from 35 breast cancer patients showing axillary infiltration at diagnosis and with upfront surgery. HLA-I H-score and the number of NK cells x 50 high power fields (HPF) in the biopsy specimen were correlated with pathological variables of the surgical specimen: number of infiltrated ALNs, tumor size, histological type, the presence of ductal carcinoma in situ, focality, histological grade, necrosis, lymphovascular and perineural invasion, Her2Neu status, and the percentages of tumor-infiltrating lymphocytes (TILs), estrogen receptor, progesterone receptor, ki67, and p53. Results: All tumors showed hormone receptor expression and three of them Her2Neu positivity. A positive correlation (p=0.001**) was found between HLA-I H-score and TILs and Ki67 expression. HLA H-score increased with histological grade and was higher in unifocal than in multifocal disease (p=0.044 and p=0.011, respectively). No other correlations were found. Conclusion: High HLA-I H-score values correlated with features of poor prognosis in this cohort of luminal breast tumors, but not with infiltrated ALNs. This finding highlights the differences between luminal breast cancer, and cancers in other locations and non-hormone dependent breast cancers, in which low HLA-I expression tends to be associated with poor prognostic features.

Background: The purpose of this phase of the MUltimodal Targeted Axillary Surgery trial (ClinicalGov NCT04039893, July 31 2019) was to determine whether HLA-I expression and NK cell tumor infiltration in breast cancer are related to axillary tumor burden and other prognostic features.

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.

Correlation between gene expression (y-axis) and percentages of stromal TILs (x-axis)

Expression of the 23 genes and signatures across the 3 cancer groups in the nCounter-based dataset

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 earlystage NSCLC patients that underwent up-front surgery. Methods: A total of 185 treatment-naive 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 & GE;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.

Video showing migration of activated CAFs toward the Gln-rich compartment when cultured in a Gln-concentration gradient (0.2-2 mM)

Figures S1, S2, S3, S4, S5, S6

Kaplan-Meier survival analysis based on selected genes or gene signatures.