Background KN026 is a novel bispecific HER2-targeting antibody for HER2-positive recurrent or metastatic breast cancer that showed prolonged median PFS and lessened distinction of PFS regarding the HR subgroup in our phase II clinical trial, compared with PUFFIN study of first-line trastuzumab combined with pertuzumab therapy. A more detailed discovery of its peculiarity is needed for optimal application of KN026 treatment. Methods We performed whole-transcriptome sequencing of digital spatial profiling (DSP) on 8 pre/post-treatment tumor samples. Mechanistic explorations were conducted by plasmid transfection, co-culture, CCK8 proliferation assay and flow cytometry. Results Compared to the tumor regions with non-objective response (OR), those with OR had high expression of CALML5, TFAP2B, and ERBB2, and relatively low expression of ESR1 in tumor cells at baseline. The expression of ESR1 had a tentative association with PI3K/AKT and NOTCH signaling pathways which were downstream or interactive pathways of HER2 target and also acted as interactive pathways of ER-mediated signaling. The co-expression of ERBB2 and CDK12 emerged as a distinctive signature of OR. KN026 treatment also reshaped intratumoral activated T- and B-cell subtypes in hot-tumor regions, regardless of myeloid-derived cells. Conclusions Both HER2 and ESR1 are determinant of KN026 efficacy in advanced HER2-positive breast cancer, implying the potential of KN026 combined with endocrine therapy in HER2- and ER-positive breast cancer.

Background: Hepatitis B virus (HBV) infection (surface antigen positive, HBsAg+) has been related to the increased risk in follicular lymphoma (FL). The further understanding of features in HBV-associated FL remains lacking. Methods: We explored clinical risk factors in HBsAg-positive patients from multicentric clinical investigation retrospectively (n = 276) and integrated HBV-related factors into Follicular Lymphoma International Prognostic Index (FLIPI) scoring system for risk prediction. The methylation profiles in pre- and paired HBsAg+FL occurring progression of disease within 2 years (POD24) were determined using the Human Methylation 850K BeadChip platform. Bulk RNA sequencing was performed for gene expression in samples from the same patient and confirmed using Myc^Cd19Cre C57BL/6J chimera mice. Results: We found that HBsAg+ FL with a higher incidence of POD24. The high HBV-DNA load (> 10⁵ copies/mL) was identified as a pivotal risk factor. HBsAg+ FL with the rapidly decreasing viral load showed lower incidence of POD24 than those without viral control (P = 0.026). Integrated risk stratification incorporating HBV-related clinical parameters based on FLIPI scoring systems had potential predictive value for high-risk patients (AUC = 0.616, P = 0.002). The methylation profiles in pre-POD24-HBsAg+FL and paired POD24-HBsAg+FL showed distinguished signatures of methylated KMT2A, EP300-AS1, ARID1B, MHC I class molecular genes related to tumor cells, and TNFRSF1A, LTA, IQCE genes related to immune cells. Of note, we confirmed that the crucial CXCR5 mRNA expression with specific methylated regions was inversely correlated to featured MYC mRNA expression as “trans” regulation in both POD24-HBsAg+FL and Myc^Cd19Cre lymphoma model. Conclusion: Integrated clinicopathological features into prediction system may provide precise risk stratification for HBV-positive FL. Modifiable DNA methylation acts as the potential targets for the combined treatment strategy to delay POD24 occurrence. Plain Language Summary: Based on the retrospective clinical data from multiple clinical centers of China, we first present the clinical characteristics associated with chronic hepatitis B virus (HBV) infection and illustrated their prognostic roles in follicular lymphoma. We novelly integrated HBV-related risk factors into the canonical Follicular Lymphoma International Prognostic Index scoring. We revealed that modifiable genes methylation,especially in genes which regulated immune response, was associated with the early relapse of disease. The myelocytomatosis oncogene (MYC) is the driver for the aggressive progression of B-cell lymphoma in multiple clinical guidelines. Of note, we further confirmed that Chemokine C-X-C-Motif Receptor 5 (CXCR5) mRNA expression with specific methylated regions was inversely correlated to MYC mRNA expression during the disease progression, showing the potential promotion of immune activities on tumor progression. Our study provided the potential targets to delay the disease progression within 24 months (POD24) through reversing methylation modification in the further epigenetic molecular treatment.

Follicular lymphoma (FL) is one of the most common types of indolent non-Hodgkin lymphoma that can undergo rapid progression, which is associated with poor patient outcomes. The homing of B cells into aberrant immunological niches within the germinal center (GC) is a prerequisite for disease development. The C-C chemokine receptor type 7 ( CCR7 ), expressed by circulating B cells, mediates their homing and dissemination to draining lymph nodes (drLNs). Here, we identified activated B cells emerging from adjacent GCs in drLNs as the origin of circulating memory B cells in distant LNs. Transcriptional upregulation of Ccr7 in these memory B cells was found to guide their migration. To model lymphomagenesis, we generated a mouse model with B-cell-specific Myc overexpression, achieved by CRISPR/Cas9-mediated homologous recombination to insert a Myc expression cassette into the Hipp11 locus. Histological signs of aggressive lymphoma were evident in Ccr7 wild-type mice, whereas Ccr7 knockout mice exhibited pre-malignant atypia phenotypes. We in-depth revealed that Ccr7 knockout impeded the differentiation of pre-malignant Rnaseh2a+ memory B cells toward malignant Pax5+ GC B cells. Furthermore, Ccr7 deficiency attenuated the dependency of malignant memory or GC B cells on extensive follicular dendritic cell (FDC) networks and T-cell help. Conversely, IL-1β-induced FDC re-expansion preferentially fostered the proliferation of malignant GC B cells even in the context of Ccr7 knockout. Our findings reveal an unforeseen role for CCR7 in driving the malignant evolution of memory B cells by orchestrating their migratory dynamics and reprogramming the supportive GC niche.

Triple-negative breast cancer (TNBC) is highly invasive and has an inferior prognosis, with the majority of patients deriving limited benefits from immunotherapy. Enhancing the efficacy of immune checkpoint inhibitors (ICIs) remains a formidable challenge for TNBC. Structural maintenance of chromosome 4 (SMC4), a regulator of genomic stability, has been implicated in tumor progression. However, its role in TNBC immune evasion remains unknown. SMC4 expression and spatial distribution in TNBC were analyzed using bioinformatics, immunohistochemistry, rt-qPCR and western blot. Immunohistochemistry was used to evaluated the association of SMC4 expression with immunotherapy response in advanced TNBC. Stable SMC4-knockdown and overexpression TNBC cell lines were established to assess tumor biology in vitro and in vivo, particularly its regulation of CD8⁺ T cell function in the tumor microenvironment (TME). Flow cytometry was used to characterize CD8⁺ T cell phenotypes. Western blot, RT-qPCR, ELISA, molecular docking and immunoprecipitation were used to evaluate the mechanism of SMC4 regulation of immune escape. We demonstrated that SMC4 is aberrantly overexpressed in TNBC and correlates with diminished immunotherapeutic response and unfavorable prognosis. Intriguingly, SMC4 drives tumor progression in a CD8⁺T cell-dependent manner, unveiling its non-canonical role in immune modulation. Mechanistically, SMC4 executes dual immunosuppressive functions hand, SMC4 maintains genomic stability to suppress cGAS-STING-mediated type I interferon production and CD8⁺ T cells recruitment. In contrast, SMC4 directly impaired CD8⁺T cell cytotoxicity through STING-independent PD-L1 upregulation. Knockdown SMC4 not only restored tumor immunogenicity, but also potentiated the efficacy of anti-PD-1 therapy in immunocompetent 4T1 murine models. Our findings establish SMC4 as a dual regulator of immune evasion in TNBC and propose targeting SMC4 as a promising combinatorial strategy to overcome the current limitations in immunotherapy.

Hepatitis B virus (HBV) infections are associated with an increased risk of B-cell lymphomas, including follicular lymphoma (FL). Chronic HBV infection and shifts in infection status following treatment can influence lymphomagenesis and immune reprogramming, potentially affecting clinical outcomes. Using the CosMx Spatial Molecular Imaging, after integrated with dynamic HBV infection states and duration of overt infection, this refines four recurrent patterns, especially memory B cell-like malignant cells (MBLM) subtype (PTPRCAP + ) that is traced with atypical features and latent indolence, and virus protein transport-related follicular dendritic cell (FDC) which shows interplay with MBLM. Except for virus-induced immune exhaustion, distinct contributions to POD24 occurrence from malignant cells and immunosuppressive cell communities under distinct HBV infection states are discerned such as through CCL21/CCR7 signaling pathway. Taken together, our spatial multicellular dynamics reveal an increased prevalence of MBLM and variable FDC phenotypes which is associated with POD24 occurrence in HBV-related FL tumors.