Lymphocytic Infiltration Research Articles

A novel vaccine strategy against Brucellosis using Brucella abortus multi-epitope OMPs vaccine based on Lactococcus lactis live bacterial vectors

Brucella infections typically occur in mucosal membranes, emphasizing the need for mucosal vaccinations. This study evaluated the effectiveness of orally administering Lactococcus lactis (L. lactis) for producing the Brucella abortus multi-epitope OMPs peptide. A multi-epitope plasmid was generated through a reverse vaccinology method, and mice were administered the genetically modified L. lactis orally as a vaccine. The plasmid underwent digestion, synthesizing a 39 kDa-sized protein known as OMPs by the target group. The sera of mice that were administered the pNZ8124-OMPs-L. lactis vaccine exhibited a notable presence of IgG1 antibodies specific to outer membrane proteins (OMPs), heightened levels of interferon (IFN-λ) and tumor necrosis factor alpha (TNF-α), and enhanced transcription rates of interleukin 4 (IL-4) and interleukin 10 (IL-10). The spleen sections from the pNZ8124-OMPs-L. lactis and IRIBA group had less morphological damage associated with inflammation, infiltration of lymphocytes, and lesions to the spleen. The findings present a novel approach to utilizing the food-grade, non-pathogenic L. lactis as a protein cell factory to synthesize innovative immunological candidate OMPs. This approach offers a distinctive way to evaluate experimental medicinal items' practicality, safety, affordability, and long-term sustainability.

Abstract PO2-24-11: Hedgehog signaling influences tumor immunogenicity by the modulation of T cell populations in triple-negative breast cancer

Abstract Breast cancer is becoming more prevalent as it now makes up 30% of all new cancer cases yearly for U.S. women. Additionally, highly aggressive triple-negative breast cancer (TNBC) presents as immunologically “cold” tumors characterized by limited infiltration of anti-tumorigenic lymphocytes and the promotion of immunosuppressive cell populations. Therefore, it is essential to consider possible combinational therapies in treating primary tumors and in preventing metastases and recurrence. In the breast tumor microenvironment (TME), the presence and activity of regulatory T cells (Tregs) have been associated with poorer prognoses in breast cancer due to their ability to promote the progression of tumor growth and metastasis. Our group has extensively studied how the Hedgehog (Hh) signaling pathway can modulate the aggressiveness and the immune portfolio of the mammary TME, and we have recently reported the role of this pathway in regulating Treg activity and abundance. We have discovered that Hh inhibition can influence Treg-to-Th17 plasticity, promoting Tregs into a pro-inflammatory Th17-like phenotype (Tr17). As Tregs are highly influential to other immune populations of the TME, it is imperative to determine what phenotypic changes are induced in other T cell populations after systemic Hh inhibition in the mammary TME. We hypothesize that systemic Hh inhibition reshapes the tumor-immune microenvironment to be tumor-eradicating by influencing key immune populations of the mammary TME. Therefore, we further investigated how CD4+ and CD8+ T cells are influenced by systemic Hh blockade in a mammary carcinoma mouse model using single-cell RNA-Sequencing (scRNA-Seq) and R programming for data analyses and visualization. We sought to assess transcriptomic changes in tumoral T cells and to interpret how these elicited changes may be influencing the immunogenicity of the primary tumor. In these investigations, we found that systemic Hh inhibition modified the abundance and activation profile of CD4+ and CD8+ T cell populations within the primary tumor. As the infiltration and persistence of certain T cell subsets within the TME can influence tumor growth, dictate tumor recurrence, and contribute to immunotherapy responsiveness, it is essential to determine the mechanisms that modulates tumoral CD4+ and CD8+ T cells in the mammary TME. These studies will expand on the mechanism by which Hh signaling modulates mammary tumorigenesis systemically. This comprehensive work will also contribute to understanding the role of Hh signaling in modulating T cell subsets and inform future studies for innovative combinational therapeutic strategies to treat highly aggressive TNBC. Citation Format: Courtney Swain, Dominique Hinshaw, Heba Alsheikh, Brandon Metge, Lalita Shevde-Samant. Hedgehog signaling influences tumor immunogenicity by the modulation of T cell populations in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-11.

Abstract PS18-10: Tumor-Infiltrating Lymphocytes and Breast Cancer Mortality in Racially and Ethnically Diverse Participants of the Northern California Breast Cancer Family Registry

Abstract Purpose: Greater stromal tumor-infiltrating lymphocyte (sTIL) percentage in pre-treatment breast tumor specimens is associated with superior response to neoadjuvant chemotherapy and survival. We hypothesized that sTIL-survival associations vary by race and ethnicity and mediate survival disparities. Patients and Methods: We evaluated pre-treatment percentages of sTILs in deciles for 284 women enrolled in the Northern California Breast Cancer Family Registry and diagnosed with a first primary invasive breast cancer from 1995-2005. We assessed associations of continuous sTIL scores and lymphocyte-predominant breast cancer (LPBC, defined as ≥50% lymphocytic infiltration of tumor stroma or cell nests) with clinical and epidemiologic characteristics using regression analysis and with breast cancer-specific mortality (BCM) and overall mortality (OM) using Cox proportional hazards regression. Results: The cohort was diverse (64% from racially and ethnically minoritized populations) with near-complete (98%) germline BRCA1/2 testing and long-term follow-up (average 16.2 years, range: 1.3-26.7). In multivariable analyses, higher sTIL score was associated with reduced BCM [LPBC versus non-LPBC: HR=0.44 (95% confidence interval 0.20-0.98); and per decile increase in sTIL score: HR=0.80 (0.69-0.93)], with the strongest association in the subset of patients with hormone receptor-negative disease [HR=0.71 (0.57-0.88) and HR=0.37 (0.14-0.94), respectively]. While there was no significant difference in sTIL score between racial and ethnic groups, the continuous sTIL-survival association was statistically significant among non-Hispanic White [HR=0.73 (0.57-0.94)] and Asian American [HR=0.56 (0.35-0.89)] women but was not seen among African American and Hispanic women. Conclusion: We identified novel sTIL-BCM differences by race and ethnicity, with better survival associated with sTIL enrichment among non-Hispanic White and Asian American but not among African American or Hispanic women. Additional studies are needed to determine whether survival and particularly the response to immunotherapy are differentially mediated by immune factors between racial and ethnic groups. Multivariable Cox Proportional Hazards Regression Models of Breast Cancer-Specific and Overall Mortality. Citation Format: Julia Ransohoff, Iain Miller, Jocelym Koo, Vishal Joshi, Allison Kurian, Kimberly Allison, Esther John, Melinda Telli. Tumor-Infiltrating Lymphocytes and Breast Cancer Mortality in Racially and Ethnically Diverse Participants of the Northern California Breast Cancer Family Registry [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS18-10.

Myeloid-derived growth factor ameliorates dextran sodium sulfate-induced colitis by regulating macrophage polarization

Inflammatory bowel disease (IBD) is characterized by inflammatory conditions in the gastrointestinal tract. According to reports, IBD prevalence is increasing globally, with heavy economic and physical burdens. Current IBD clinical treatment is limited to pharmacological methods; therefore, new strategies are needed. Myeloid-derived growth factor (MYDGF) secreted by bone marrow-derived mononuclear macrophages has beneficial effects in multiple inflammatory diseases. To this end, the present study aimed to establish an experimental IBD mouse model using dextran sulfate sodium in drinking water. MYDGF significantly alleviated DSS-induced colitis, suppressed lymphocyte infiltration, restored epithelial integrity in mice, and decreased apoptosis in the colon tissue. Moreover, the number of M1 macrophages was decreased and that of M2 macrophages was increased by the action of MYDGF. In MYDGF-treated mice, the NF-κB and MAPK pathways were partially inhibited. Our findings indicate that MYDGF could mitigate DSS-induced mice IBD by reducing inflammation and restoring epithelial integrity through regulation of intestinal macrophage polarization via NF-κB and MAPK pathway inhibition. Key messagesMYDGF alleviated DSS-induced acute colitis.MYDGF maintains colon epithelial barrier integrity and relieves inflammation.MYDGF regulates colon macrophage polarization.MYDGF partially inhibited the activation of NF-κB and MAPK pathway.

Abstract PO1-15-09: Vitamin A Metabolism Induces Ferroptosis to Enhance Immune Therapy Efficacy in HR+/HER2- Breast Cancer

Abstract Background: Immune checkpoint blockade (ICB) has substantially improved patient overall and progression-free survival in triple-negative breast cancer (TNBC), but its efficacy remains to be elucidated in the hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) breast cancer, which is characterized by generally impotent lymphocytic infiltration. In the I-SPY2 trial (NCT01042379), pembrolizumab- or durvalumab-containing neoadjuvant therapy increased the pathologic complete response of HR+/HER2- patients. However, pembrolizumab-containing therapy failed to improve the survival of metastatic HR+/HER2- breast cancer patients (NCT03051659). These inconsistent results underscored the necessity of the investigation of predictive biomarkers to pinpoint potential ICB responders in HR+/HER2- breast cancer. Method: In this study, we collected pretreatment tumor samples from 16 patients in two respective clinical trials (NCT: 04215003 and 04355858) and profiled droplet-based single-cell sequencing, bulk RNA-Seq. Patients were treated with camrelizumab (PD-1 inhibitor)-containing therapy, and five of them achieved partial relief after an 8-week course. We also leveraged the bulk RNA-Seq data from the I-SPY2 trial and Fudan University Shanghai Cancer Center (FUSCC) TNBC ICB trials (NCT04418154 and NCT04613674) for external validation. Another FUSCC transcriptome dataset consisting of 933 treatment-naïve HR+/HER2- or TNBC breast cancer patients was used to detect the abundance of cell types. In vitro and in vivo experiments were used to explore mechanisms of anti-tumor immune response. Results: Based on the single-cell sequencing results, we comprehensively delineated the microenvironmental landscape of HR+/HER2- breast cancer. We discovered responders of immunotherapy showed a higher presence of CXCL9/10+ M1-like macrophages and exhibited enrichment of tumor cells with ferroptosis. We further identified the upregulation of vitamin A metabolism and the vitamin A metabolic gene CRABP1 in tumor cells with ferroptosis. We verified the vitamin A metabolism could induce ferroptosis in HR+/HER2- breast cancers rather than TNBC by CRABP1-ERK axis, which could further recruit CXCL9/10+ M1-like macrophages and promote anti-tumor immune response in HR+/HER2- breast cancer. Combining anti-PD1 with enhancing vitamin A metabolism via retinoid acid possessed greater therapeutic efficacy than monotherapy in HR+/HER2- breast cancers. Finally, we developed an immune therapy response score and validated its reliability in predicting the immunotherapy efficacy for HR+/HER2-breast cancers through both internal and external cohorts. Conclusion: Our study represents the earliest efforts to decipher the microenvironmental landscape and the mechanisms underlying the response to ICB in HR+/HER2- breast cancer at single-cell resolution. We unraveled a HR+/HER2- breast cancer-specific Vitamin A-ferroptosis axis that mediated immunotherapy response and constructed a robust immunotherapy efficacy prediction score based on this Vitamin A-ferroptosis axis. Citation Format: Yi-Fan Zhou, Hu-Yun-Long Zhang, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. Vitamin A Metabolism Induces Ferroptosis to Enhance Immune Therapy Efficacy in HR+/HER2- Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-09.

Decellularized porcine vena cava grafts are fully repopulated after orthotopic implantation

Tissue-engineered organs, based on native extracellular matrix (ECM) scaffolds, could be a game changer in regenerative medicine applications. Decellularization technology provides such scaffolds with organ-typic ECM composition and architecture. Despite limitations such as the requirement of huge cell numbers and finding the optimal route of entry, recellularized scaffolds provide alternative grafts for transplantation. In this study we assessed whether decellularized scaffolds, when implanted, are repopulated from the adjacent tissue. Since the vasculature plays an important role in tissue functionality, our main focus was to evaluate in situ repopulation of decellularized veins in a pig model. For this, porcine inferior vena cava grafts were decellularized and orthotopically implanted in recipient pigs (n = 12). To evaluate possible immune responses to the scaffolds and to assess potential thrombus formation, cellular allogeneic vena cava grafts were transplanted in control pigs (n = 8). Within 28 days after implantation, the decellularized veins were fully recellularized with endothelial cells and smooth muscle cells. Quantitative histological analysis showed a comparable amount of smooth muscle actin in the repopulated decellularized grafts similar to the native IVC. Lymphocyte infiltrates representing signs of graft rejection were not detected in the pigs, as opposed to the control group that received the allogeneic grafts. The decellularized grafts provoked a higher incidence of thrombosis in comparison with allogeneic grafts (33.3 vs. 12.5%). With this study, we show efficient in situ repopulation of decellularized vein grafts. These findings are insightful and promising to further explore the use of decellularized tissue without the need for full pre-transplant recellularization.Graphical abstract

Abstract PO3-25-07: Characterization of tertiary lymphoid structures and its association with response to immune checkpoint inhibitors in triple negative breast cancer using spatial transcriptomics

Abstract Background Tertiary lymphoid structures (TLS) are ectopic lymphoid organs playing a role in adaptive antitumor immune response. They were shown to be associated with favorable outcome and appears to be a promising biomarker for response to immune checkpoint inhibitors (ICI). Yet, there is no consensus for their detection and quantification. Here we aimed to derive a specific TLS signature using high resolution spatial transcriptomics data from a large series of triple negative breast cancer (TNBC) samples and to assess its clinical relevance including response to ICI. Methods Spatial transcriptomics (ST) was performed on a series of 94 early-stage TNBC surgical samples with detailed clinicopathological and outcome data. TLS and tumor infiltrating lymphocytes (TILs) were scored using hematoxylin and eosin and double immunohistochemistry (CD3/CD20) stained slides by a dedicated breast pathologist. Regression based on morphological annotations and deconvolution methods estimated the fraction of gene expression related to TLS in each ST spot allowing to derive specific TLS signature. Its prognostic and predictive value of response to ICI was assessed using publicly available breast cancer (METABRIC, SCAN-B, and I-SPY2) and other tumor type datasets treated with ICI (Bareche et al. Ann Oncol, 2022) from bulk gene expression data. Results Deconvolution of the TLS compartments using the ST gene expression data showed that TLS are enriched in all B cell subsets, CD4+ (central) memory and naïve T cells, as well as mast cells. A functional analysis showed an enrichment of ‘vascular endothelial cell proliferation’, ‘mitotic G2/M transition checkpoint’, ‘V/D/J recombination’ and ‘regulation of cell chemotaxis to fibroblast growth factor’ suggesting that TLS aggregates are associated with angiogenesis, immune cell proliferation, adaptative humoral response and tissue remodeling. A comparison of gene expression data between TLS and non-aggregated TILs led to the development of a specific TLS gene signature comprising B and T cell-specific genes, immunoglobulin genes as well as genes associated with TLS initiation. Its projection on tumor slides overlapped with the regions annotated as TLS by the pathologist, demonstrating its specificity for TLS detection. As expected, the highest levels of the TLS signature were observed in the immunomodulatory TNBC molecular subtype and fully inflamed tumors, whereas the lowest levels were observed in the mesenchymal molecular subtype and immune desert tumors (p< 0.05). Of note, high levels of the TLS signature were associated with better prognosis in TNBC patients from METABRIC (p=6.5 10−5) and SCAN-B datasets (p=1.6 10−3) as well as with higher pathological complete response rates in all breast cancer patients treated with neoadjuvant pembrolizumab in the I-SPY2 study (p=0.026). Similar results were found in other tumor types treated with immunotherapy in the metastatic setting including metastatic melanoma, pancreatic and bladder cancers (PFS, p=6 10−6). Of interest, the TLS signature outperformed the predictive value of other immune-related signatures including TILs suggesting a key role of TLS in obtaining a sustainable, adaptive antitumor immune response in the vicinity of the tumor area. Conclusion By leveraging the potential of spatial transcriptomics, we have successfully developed a unique TLS gene signature that demonstrates a strong correlation with the response to ICI in breast cancer and various other tumor types. This TLS gene signature outperforms other immune biomarkers, underscoring the pivotal role of TLS in eliciting a robust antitumor immune response. The universality of this TLS signature makes it a potent biomarker capable of identifying patients who would benefit from immunotherapy, pending further validation. Citation Format: Xiaoxiao Wang, David Venet, Frédéric Lifrange, Denis Larsimont, Mattia Rediti, Linnea Stenbeck, Floriane Dupont, Ghizlane Rouas, Andrea Joaquin Garcia, David Gacquer, Ligia Craciun, Laurence Buisseret, Nayanika Bhalla, Yuvarani Masarapu, Kim Thrane, Eva Gracia Villacampa, Lovisa Franzén, Sami Saarenpää, Linda Kvastad, Joakim Lundeberg, Françoise Rothé, Christos Sotiriou. Characterization of tertiary lymphoid structures and its association with response to immune checkpoint inhibitors in triple negative breast cancer using spatial transcriptomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-07.

Abstract PS16-07: Tumor infiltrating lymphocyte stratification refines AJCC TNM staging based outcomes of early-stage triple negative breast cancer treated with neoadjuvant anthracycline-free, docetaxel and carboplatin chemotherapy

Abstract Background: Stromal tumor-infiltrating lymphocytes (sTILs) quantification is associated with pathological response to neoadjuvant chemotherapy (NAC) and long term outcomes in setting of adjuvant anthracycline based chemotherapy. A previous study in TNBC patients treated with adjuvant anthracycline chemotherapy shows that at a cut point of 30%, sTILs can up and downstage traditional AJCC stage groups. Additive impact of sTILs on refining outcomes beyond pathological response and TNM stage for patients treated with anthracycline-free chemotherapy is not known. This study aimed to investigate impact of sTILs on outcomes in a large cohort of TNBC patients treated with Docetaxel plus carboplatin NAC. Methods: Patients with stage I (T size > 1cm) to III TNBC scheduled to receive 6 cycles of NAC docetaxel (75 mg/m2) plus carboplatin (AUC 6) (TCb) every 3 weeks from two studies (NCT01560663 and NCT02302742) were combined for this analysis. sTILs were evaluated on pre-treatment H&E slide using standard criteria centrally by one of the investigators (RS). Pathological complete response (pCR) was defined as ypT0/is ypN0. Logistic regression analysis was used to examine the effect of multiple variables on Event free survival (EFS) and overall survival (OS). Results: For 474 patients included in this analysis, median age was 52 years, 8% were Black, 13% had germline BRCA1/2 mutation, 44% had clinical Lymph node (LN) positive disease and 13%, 62% and 25% respectively had TNM stage I, II and III disease. Median sTILs were 5% (range 1-95%) and 25% had >30% sTILs. pCR and RCB 0+1 rates were 50.2% and 60.4% respectively. On multivariable analysis, T stage (OR=0.51, p=0.030), nodal status (OR=0.56, p=0.028), Ki67 (OR=2.74, p< 0.001) and sTILs (OR=2.01, p=0.014) were associated with pCR. pCR rate in those with sTILs < 30 vs sTILs >30 was 45.9% and 63.9% respectively (p=0.0014). At median follow-up of 58 months, 5 years EFS was 81.05% in all patients, 93.94% in those with pCR and 68.67% in those without pCR, 5 year OS was 84.83% in all patients, 96.53% in those with pCR and 74.13% in those with residual disease. On multivariate analysis lower T stage, negative LN status and increasing sTILS were associated with better EFS (T stage: HR =1.98, p=0.018; LN status: HR=2.92, p< 0.001; sTILs: HR:0.46, p=0.04) and OS (T stage: HR=1.89, p=0.040; LN status: HR=3.13, p=0.001; sTILs: HR=0.30, p=0.008). At a cut-point of 30%, sTILs up and downstaged anatomic AJCC TNM stage groups (table 1). Conclusions and Relevance: In patients treated with anthracycline-free TCb chemotherapy, sTILs were independent predictors of EFS and OS beyond clinicopathological features. Notably, 30% sTILs cut-point stratified outcomes beyond anatomical TNM staging. These, findings can aid in patient stratification for chemotherapy backbone de-escalation in future trials and have the potential to inform patient selection for adjuvant treatment escalation and de-escalation trials. Tumor infiltrating lymphocyte stratification refines AJCC TNM staging based outcomes of early-stage triple negative breast cancer treated with neoadjuvant anthracycline-free, docetaxel and carboplatin chemotherapy. Table. Citation Format: Miguel Martín, Rachel Yoder, Roberto Salgado, María del Monte-Millán, Enrique Álvarez, Isabel Echavarria, Joshua Staley, Coralia Bueno-Muiño, Yolanda Jerez Gilarranz, Andrew Godwin, María Cebollero, Oscar Bueno, José Ángel García-Sáenz, Fernando Moreno Antón, Uriel Bohn, Henry L Gómez, Tatiana Massarrah, Sara López-Tarruella, Priyanka Sharma. Tumor infiltrating lymphocyte stratification refines AJCC TNM staging based outcomes of early-stage triple negative breast cancer treated with neoadjuvant anthracycline-free, docetaxel and carboplatin chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS16-07.

Abstract PO1-06-02: Solti-1716 TATEN phase II trial: Targeting non-Luminal disease by PAM50 with pembrolizumab and paclitaxel in Hormone Receptor-positive/HER2-negative advanced breast cancer (ABC)

Abstract Background Within HR+/HER2-negative breast cancer, PAM50 non-luminal tumors (HER2-enriched [HER2-E] and Basal-like) have higher expression of proliferation and immune-related genes and tumor infiltrating lymphocytes and might benefit from immunotherapy. Here, we report the efficacy, safety, and correlative analysis data of the TATEN trial, the first study designed to evaluate pembrolizumab and paclitaxel in HR+/HER2-negative, PAM50 non-luminal, ABC. Methods TATEN trial (NCT04251169) is a single-arm, multicenter, phase II study evaluating pembrolizumab in combination with paclitaxel in patients with HR+/HER2-, PAM50 non-luminal ABC. Key inclusion criteria include progression to prior CDK4/6 inhibitors (CDK4/6i), presence of measurable disease, no prior chemotherapy for ABC, ECOG 0-1, and non-luminal metastatic disease by PAM50. Included patients received pembrolizumab at 200 mg every 3 weeks (beginning at cycle 1) in combination with weekly paclitaxel at 80 mg/m2 beginning at cycle 2. The primary endpoint was overall response rate (ORR) according to RECIST V1.1. in patients who received at least one dose of combination treatment and had a first, post-baseline tumor assessment (evaluable population). Secondary endpoints included progression-free survival (PFS), clinical benefit rate (CBR), safety, and predictive biomarkers. The study was based on a Simon two-stage design. Stage I of the trial would be considered successful if at least 6 of 15 patients achieved a partial response and/or complete response. In that case, the trial would recruit up to 46 evaluable patients for a target ORR ≥ 41%. Metastatic biopsies from patients enrolled in pre-screening were also evaluated for tumor infiltrating lymphocytes (TILs) and were further analyzed with an expression panel of 192 genes, including PDL1 and PD1. Results From July 2020 to December 2021, 132 tumors were screened, and 27 PAM50 non-luminal tumors were identified (20%). Non-luminal tumors trended to have higher PDL1 expression (p=0.090) and TILs (p=0.084) compared to luminal tumors, while no difference was observed for PD1 (p=0.850). Of 20 recruited patients in the study (stage I+II), 18 were evaluable for the primary endpoint. Baseline characteristics were as follows: median age 55 years, ECOG 0 55%, de novo MBC 22%, and visceral disease 72.2%. Eleven patients had received paclitaxel treatment in the adjuvant setting. Regarding PAM50 subtype, 2 patients had basal-like and 16 HER2-E tumors. At the time of data cut-off (June 2023), 13 patients (72.2%) had stopped their treatment because of progressive disease and 3 (16.6%) due to toxicity. Two patients (11.1%) were still on treatment. The ORR was 61.1 % (11 of 18, 95% CI 35.7-82.7). CBR was 88.9% (16 of 18, 95% CI 65.3-98.6), and median PFS was 8.3 months (95% CI 7.3 – 14.1). Treatment-related adverse events (TRAEs) of any grade (G) occurred in 19 patients (95%), while 45% of patients experienced G3 TRAEs. No G4 or G5 TRAEs were reported in the evaluable population. Gene expression analysis was successful for all recruited patients (n=20). High expression of the PAM50 luminal A signature (p=0.049), and the luminal genes PGR (p=0.028) and RRAGA (p=0.038) were associated with worse PFS (univariate analyses). The pan-leucocyte receptor CD84 (p=0.028) was associated with better PFS (univariate analysis). Conclusions Pembrolizumab in combination with paclitaxel is safe and exhibits promising efficacy outcomes in patients with CDK4/6i resistant HR+/HER2- ABC with a PAM50 non-luminal subtype. Although meeting the criteria for stage II, the enrollment was stopped prematurely due to the lack of funding and pembrolizumab supply. More correlative analyses will be presented at the conference. This study was funded in part by MSD. Citation Format: Aleix Prat, Benedetta Conte, Fara Brasó-Maristany, Nuria Chic, Montserrat Muñoz, Cristina Hernando, Manuel Alva, Silvia Vazquez, Salvador Blanch, Mafalda Oliveira, Esther Fernández, Oleguer Castillo, Patricia Galván, Ángela Aguirre, Esther Sanfeliu, Lorea Villanueva, Tomás Pascual, Eva Ciruelos. Solti-1716 TATEN phase II trial: Targeting non-Luminal disease by PAM50 with pembrolizumab and paclitaxel in Hormone Receptor-positive/HER2-negative advanced breast cancer (ABC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-02.

Abstract PO2-25-03: Deciphering the immune molecular signature associated with pembrolizumab efficacy in the neoadjuvant setting of Triple-Negative Breast Cancer patients: a single-centre experience

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancers and is characterized by a high rate of recurrence, despite the improvement of the therapeutic approaches in recent years. Neoadjuvant chemotherapy (NACT) is the standard treatment for early-stage TNBC. Achieving a pathological complete response (pCR) is considered an essential prognostic factor for long-term outcomes. However, the percentage of pCR patients after NACT is limited to 30-40%, thus requiring the implementation of novel therapeutic strategies. Recently, by applying a morphology-guided spatial transcriptomic approach on Tru-Cut biopsies, we showed that intratumoral infiltrating lymphocytes (iTILs) and stromal Natural Killer (NK) cells are major determinants of chemotherapy efficacy in TNBC. These data highlight a complex three-dimensional organization of the tumor landscape and underline how spatiality and molecular activation of immune cells are essential to enhance the efficacy of neoadjuvant NACT in TNBC. The fact that immune cells are important players in this setting is corroborated by the clinical success shown by the KEYNOTE-522 study. This trial paved the way for the use of immunotherapy in neoadjuvant settings, obtaining a significant improvement in the patient’s overall response compared to standard NACT. Still, the implementation of chemotherapy with immunotherapy in the TNBC neoadjuvant treatment implicates several compelling questions, including how to further increase the percentage of patients with a complete response, overcoming resistance mechanisms, and how to improve the personalization of treatment, evaluating which patients can benefit from a therapy de-scalation or another combined therapeutic strategy. In this prospective cohort study, we decided to define the relevance of different immune populations in affecting pembrolizumab efficacy in the neoadjuvant treatment of TNBC patients. We treated 40 patients with an early-TNBC diagnosis with the KEYNOTE-522 regimen that consists of four cycles of an intravenous infusion of pembrolizumab plus paclitaxel and carboplatin, followed by four cycles of pembrolizumab plus epirubicin and cyclophosphamide. Only patients with evident side effects to immunotherapy administration were a priori excluded. Currently, 18 patients ended the neoadjuvant schedule and underwent surgical excision. Our data indicate that 15 patients displayed a pCR at the end of the therapy, whereas 3 patients displayed only partial response. To further clarify the immune dynamics that lead to a complete response to pembrolizumab in these patients, we collected peripheral blood before and after neoadjuvant treatment to isolate peripheral blood mononucleated cells (PBMCs). We applied a single-cell transcriptome analysis to shape any variations in circulating immune cells occurring during neoadjuvant treatment and associated with patient response. In addition, we collected residual tumors at surgery from partial responders and derived three-dimensional organoids that recapitulate the characteristics of the original patient tumor. These ex vivo organoid-based platforms are applied to clarify the molecular mechanisms driving neoadjuvant resistance. To this aim, we are performing co-cultures with autologous immune cells to identify novel combined therapeutic strategies to rescue pembrolizumab efficacy in these models. Overall, our study is defining the immune-related molecular mechanisms driving pembrolizumab and chemotherapy response in the neoadjuvant treatment of TNBC patients. Our findings can improve our understanding of which immune populations are determinant players in anti-TNBC immune response and can be proposed as novel biomarkers of pembrolizumab efficacy during the neoadjuvant treatment. Citation Format: Elisa Gasparini, Gloria Manzotti, Moira Ragazzi, Benedetta Donati, Federica Torricelli, Elisa Salviato, Alessandra Bisagni, Eleonora Zanetti, Alessandra Bologna, Saverio Coiro, Rita Vacondio, Guglielmo Ferrari, Giancarlo Bisagni, Carmine Pinto, Alessia Ciarrocchi, Francesca Reggiani. Deciphering the immune molecular signature associated with pembrolizumab efficacy in the neoadjuvant setting of Triple-Negative Breast Cancer patients: a single-centre experience [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-25-03.

Abstract PO4-25-09: HER2-Low and Ultra-Low expression in young women with luminal breast cancer in México

Abstract Background and objective HER2-Low in breast cancer is a biomarker with new approaches for prognosis and medical treatment decisions. However, HER2-Ultra-Low is an area of novel research. Our objective was to describe the clinicopathologic characteristics and HER2 expression status of a group of Mexican young patients with luminal breast cancer. Methods One hundred and fifty-seven young patients (< 40 years) with luminal breast cancer diagnosed from 2014 to 2020 were included. Clinicopathologic features, hormone receptors, HER2 expression, and tumor infiltrating lymphocytes (TILs) were assessed. We categorized patients in three groups: HER2 negative (n=95, 60.5%), HER2-Low (n=33, 21.0%), and HER2-Ultra-Low (n=29, 18.5%). Descriptive statistics were used to report demographic, clinical and histopathological characteristics. Kaplan-Meier curves and log-rank or Mantel-Cox tests were used to determine survival. Statistical significance was established using a P value < 0.05. This analysis was performed using R (version 4.0.4) through R Studio interface (version 1.4.1106). Results The median of age at diagnosis was 36.1 years [range 32.8-38.8], with a median of follow-up of 5.08 years [range 3.58-6.52]. The most frequent histologic type was ductal infiltrating carcinoma (81%), and 49.4% presented grade two of differentiation. T2, N0 and clinical stage II were the most common (45.2%, 44.5, and 48.1%, respectively), and fourteen patients (9.0%) presented clinical stage IV. We found statistically significant differences in Ki67 and total TILs quantification between HER2 negative and HER2-Low. Progesterone receptor levels were higher for HER2 negative and Ultra-Low in comparison to HER2-Low. The total population had a 5-year overall survival of 84.9%. We found statistically significant differences in the median of survival between HER2 groups, with a shorter survival for HER2-Low. Conclusion To date, there are no studies that describe the clinical profile and OS of HER2-Low and Ultra-Low in young patients with luminal breast cancer in México. These preliminary findings could indicate the need for further investigation in prognosis, outcomes, and specific treatment in these patients. This study contributes to better understand the clinicopathologic profile of the HER2-Ultra-Low group and its differences with the HER2-Low group. Citation Format: Jesus Edgardo Hernandez Hernandez, César Octavio Lara Torres, Bryan Moreno Moran, Cynthia Villareal Garza, Fany Iris Porras Reyes, Víctor Manuel Pérez Sánchez, Alejandro Mohar. HER2-Low and Ultra-Low expression in young women with luminal breast cancer in México [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-09.

Abstract PO5-05-11: The effect of eribulin treatment for patients with HER2-low breast cancer

Abstract Background: Eribulin, a nontaxane, synthetic microtuble dynamics inhibitor, induces longer overall survival (OS) in patients with advanced or metastatic breast cancer (MBC). Interestingly, it also improves the tumor immune microenvironment via vascular remodeling. DESTINY-Breast04 trial showed that trastuzumab deruxtecan resulted in significantly longer OS of human epithelial growth receptor 2 (HER2)-low breast cancer patients, and this mechanism may also be related to tumor microenvironment. In this study, we investigated the effect of eribulin for patients with HER2-low breast cancer. Materials and Methods: This retrospective study comprised 97 MBC treated with eribulin from August 2011 to April 2019 at our institute. HER2 levels were determined through tumor biopsy specimen at first medical examination. We evaluated the correlations between HER2 levels and clinicopathological features, including the prognosis and immune related markers in each breast cancer subtype. We also classified the progressive disease (PD) into progression due to pre-existing lesion (PPL) and progression due to new metastasis (PNM) based on systemic computed tomography findings, and investigated the relationships between HER2 expression and PD types. Results: HER2-high group had significantly better OS than other groups (p=0.021, log-rank). Hormone receptor (HR)-negative/HER2-low patients had significantly better progression-free survival (PFS) than HR-negative/HER2-negative (p=0.024, log-rank). Though we also examined HER2 levels of recurrent tumor specimen, it was not a significantly useful prognostic marker in patients with HR-negative breast cancer (p=0.085, log-rank). There was no significant correlation between HER2 expression and any tested clinicopathological parameter, including absolute lymphocyte count, neutrophil-to-lymphocyte ratio and tumor infiltrating lymphocytes. PD were observed in 64 patients (PPL: n=47, PNM: n=17). PD type of all HR-positive/HER2-high patients was PNM, whereas all HR-negative/HER2-high patients was PPL (p=0.035). HER2-low group tended to have a higher PPL rare than negative group (83.3% vs 69.2%). Conclusions: In HR-negative/HER2-low group, PFS was improved by eribulin treatment, which might be due to unique anticancer effect of eribulin, suppression of epithelial-mesenchymal transition. Further study of this mechanism through basic research is important. Also, assessment of HER2 from primary tumor may be more useful than recurrent specimen. Citation Format: Wataru Goto, Saeko Henmi, Mariko Nishikawa, Asuka Kouchi, Rika Kouhashi, Akimichi Yabumoto, Koji Takada, Yuka Asano, Yukie Tauchi, Kana Ogisawa, Tamami Morisaki, Shinichiro Kashiwagi. The effect of eribulin treatment for patients with HER2-low breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-05-11.

Abstract PO1-28-04: PILHLE-001: neoadjuvant pyrotinib combined with chemotherapy in HR-positive, HER2-low (IHC 2+/FISH-negative) early breast cancer

Abstract Background: Hormone receptor (HR)-positive/HER2-low (IHC 2+/FISH negative or IHC 1+) breast cancers (BCs) represent the largest proportion of breast carcinomas. However, its response to neoadjuvant chemotherapy or endocrine therapy is the lowest, with a residual cancer burden (RCB) 0/I rate around 15%. Previous studies have demonstrated that pyrotinib (P), a pan-HER tyrosine kinase inhibitor, exhibits excellent response in HER2-positive early or advanced BC (E/ABC). In-vitro experiments have shown that pyrotinib can effectively inhibit colony formation in HER2-low (IHC 2+/FISH negative) BC cells. This PILHLE-001 study (NCT05165225) aimed to evaluate the efficacy and safety of neoadjuvant pyrotinib plus chemotherapy in HR-positive/HER2-low (IHC 2+/FISH negative) EBC patients with potential biomarkers. Methods: PILHLE-001, as a single-arm, single-center phase Ⅱ trial, enrolled patients with previously untreated HR-positive (ER or PR > 1%) and HER2-low (IHC 2+/FISH negative) invasive EBC (TNM stage II-III) to receive pyrotinib 320mg QD and four Q3W of epirubicin-cyclophosphamide followed by four Q3W of docetaxel. MammaPrint/BluePrint at baseline (t0) was assessed. Breast magnetic resonance imaging (MRI) was conducted at t0, the end of cycle 2 neoadjuvant therapy (t1), and the end of all neoadjuvant therapy. Immune cell subpopulations assay and gene sequencing of tumor tissues with whole blood control samples were performed at t0 and surgery (t2). Additional core biopsy was administered to reassess Ki67 and tumor infiltrating lymphocytes (TILs) at t1. The primary outcome was RCB 0/I rate. The short-term secondary outcomes included pathological complete response (pCR, ypT0/is ypN0) rate, objective response rate (ORR), breast conservation surgery (BCS) rate, safety, and exploratory biomarkers analysis. Results: From July 2021 to March 2023, 48 patients were enrolled and treated. The median age of the patients was 48 years (range 28-66), 43 (90%) had cT1-2 tumors, 30 (63%) showed no node involvement, 37 (77%) had tumours with TNM stage II, and 47 (98%) had ER expression ≥ 50%. Twenty-eight (61%) of 46 patients had MammaPrint high risk. Out of the 48 patients, 26 (54.2%) achieved RCB 0/I, with a 95% confidence interval of 39.2% to 68.6%. The RCB 0/I rate was particularly higher in tumors with no lymph node involvement (73.3%), TNM stage II (64.9%), or abundant tumor-infiltrating lymphocytes (66.7%). Three (6.3%) patients obtained a pCR. The rate of BCS was 60.4% (29/48). The ORR was 45.8% (22/48) at t1 and 81.3% (39/48) at the end of all neoadjuvant therapy, respectively. No patients had progressive disease. All treatment-related adverse events (AEs) were mostly of grade 1 or 2 severity. Grade ≥ 3 AEs occurred in 21 (44%) patients, with the most prevalent being diarrhea [10 (21%)]. All AEs were reversible with symptomatic treatment and no treatment-related deaths occurred. Patients with MammaPrint risk-low BC had a lower RCB 0/I rate than those with risk-high BC (60.7% vs. 44.4%), although the difference was not statistically significant. Baseline specific immune cell subpopulations (lower PD-1, higher CD3, etc.) or the presence of non-altered PIK3CA were significantly associated with a higher likelihood of achieving RCB 0/I. Similarly, objective response, greater declines in specific MRI quantitative parameters (Ktrans and Kep), greater declines in Ki67, or more increased TILs at t1 were also significantly associated with a higher rate of RCB 0/I. Conclusions: The PILHLE-001 trial first revealed that neoadjuvant pyrotinib plus chemotherapy has encouraging efficacy and acceptable toxicity in patients with HR-positive/HER2-low (IHC 2+/FISH negative) EBC, and this regimen warrants further investigation in phase III randomized controlled trials. Citation Format: Chang Gong, Yuan Xia, Yingying Zhu, Yaping Yang, Wenqian Yang, Louis W.C. Chow, Li Ling, Yunjie Zeng, Jiajie Zhong, Ziliang Cheng, Jun Shen, Qun Lin, Yinduo Zeng, Qiang Liu, Erwei Song. PILHLE-001: neoadjuvant pyrotinib combined with chemotherapy in HR-positive, HER2-low (IHC 2+/FISH-negative) early breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-28-04.

Antitumor immunity and prognosis value elicited by FAT3 and LRP1B co-mutation in endometrial cancer

Objective: FAT3 and LRP1B are two tumor suppressor genes with high mutation frequency in multiple cancer types, we sought to investigate the prognostic and immunological significance of these two genes in EC.Methods: Based on a cohort of 502 EC samples, we conducted a comprehensive analysis of its multidimensional data types including genomic, transcriptomic, and clinical information, the potential impact of FAT3 and LRP1B co-mutation on antitumor immune response and prognosis were systematically discussed.Results: We observed that FAT3 and LRP1B co-mutation was not only defined a dataset with prominently increased TMB, decreased tumor aneuploidy, and specially enriched in MSI-H subtype, but also manifested increased expression of immune-related markers, especially exclusive upregulation of PD-L1 levels and higher PD-L1+/CD8A+ proportion. Further analysis focused on lymphocyte infiltration and pathway enrichment explored the immune cell composition of the microenvironment and underlying molecular mechanisms affecting tumor development. Furthermore, EC patients with FAT3 and LRP1B co-mutation possessed significantly prolonged PFS and OS, and the co-mutation status was proved to be an independent prognostic factor. And a nomogram with high predictive performance was constructed by incorporating co-mutation with clinical features. More strikingly, the prognosis of MSI-H patients in EC with co-mutation was significantly improved, and their survival reached a level consistent with the POLE subtype.Conclusions: In endometrial cancer, co-mutation of FAT3 and LRP1B not only leads to activation of the immune state, but also represents a subgroup with an improved prognosis, particularly in the MSI-H subtype.

High immune cell infiltration predicts improved survival in cholangiocarcinoma.

Antitumoral immune response has a crucial role in constraining cancer. However, previous studies on cholangiocarcinoma (CCA), a rare and aggressive cancer, have reported contradictory findings on the prognostic impact of tumor-infiltrating T-lymphocytes. We aimed to clarify the effect of tumor-infiltrating CD3+ and CD8+ lymphocytes and PD-1/PD-L1 expression on CCA prognosis. CD3+, CD8+, and PD-1+ lymphocyte densities, as well as PD-L1 expression rate were analyzed from stained tissue microarray samples from the tumor center and invasive margin of 47 cholangiocarcinomas. The association of CD3+ and CD8+ based Immune cell score (ICS) and its components with overall survival was evaluated, adjusting for age, sex, TNM stage, radicality of surgery, tumor location, and PD-L1 expression on immune cells. Low ICS was a strong independent prognostic factor for worse overall survival (Hazard ratio 9.27, 95% confidence interval 2.72-31.64, P<0.001). Among the ICS components, high CD8+ lymphocyte infiltration at the tumor center had the most evident impact on patient outcome. PD-1 and PD-L1 expression on immune cells did not have a significant impact on overall survival alone; however, PD-L1 positivity seemed to impair survival for ICSlow subgroup. Identifying patient subgroups that could benefit from immunotherapy with PD-1/PD-L1 pathway blockade may help improve treatment strategies for this aggressive cancer. Our findings highlight the importance of evaluating the immune contexture in cholangiocarcinoma, as ICS serves as a strong independent prognostic and selective factor for patients who might benefit from immunotherapy.

Determination of the effects of sunset yellow on mouse liver and pancreas using histological methods.

Sunset Yellow (SY) is an azo synthetic food dye. Although the amount of SY consumed varies in different periods of life, it increases especially in children and adolescents. It may cause pathologic effects in organs at early ages. The aim of this study was to determine the effects of SY on the liver and pancreas of mice of different age groups using histological methods. The study included Swiss albino mice that were divided into three treatment groups and three control groups based on age (4, 8, and 10weeks old), with six mice in each group (n=6/group). SY was administered at 30mg/kg/bw/week orally for 28days to treatment groups. The liver and pancreas tissues were kept in 10% formaldehyde, then passed through alcohol and xylene series and stained with Hematoxylin-Eosin. They were evaluated using light and electron microscopy. In SY groups, the mean body weight (p: 0.026) and the mean liver weight (p: 0.013) of the mice increased, and their mean pancreas weight decreased (p: 0.045).The numbers of degenerative cells in the liver tissues of the mice in the SY groups were high. Severe dilation in the sinusoids and haemorrhages focused around the Vena Cava were detected. In the pancreatic tissues of the SY groups, increases in fibroblasts and lymphocytic infiltration were observed. Pathologies interpreted as chronic pancreatitis were more intense in the weaning group (4weeks old). SY may be more harmful at an early age, and it may be beneficial to limit its use during this period.

The Etiologic Landscape of Lymphoproliferation in Childhood: Proposal for a Diagnostic Approach Exploring from Infections to Inborn Errors of Immunity and Metabolic Diseases.

Lymphoproliferation is defined by lymphadenopathy, splenomegaly, hepatomegaly, or lymphocytic organ and tissue infiltration. The most common etiologies of lymphoproliferation are represented by infectious diseases and lymphoid malignancies. However, it is increasingly recognized that lymphoproliferative features can be the presenting sign of rare conditions, including inborn errors of immunity (IEI) and inborn errors of metabolism (IEM). Among IEI, lymphoproliferation is frequently observed in autoimmune lymphoproliferative syndrome (ALPS) and related disorders, common variable immunodeficiency (CVID), activated phosphoinositide 3-kinase δ syndrome, and Epstein-Barr virus (EBV)-related disorders. Gaucher disease and Niemann-Pick disease are the most common IEMs that can present with isolated lymphoproliferative features. Notably, other rare conditions, such as sarcoidosis, Castleman disease, systemic autoimmune diseases, and autoinflammatory disorders, should be considered in the differential diagnosis of patients with persistent lymphoproliferation when infectious and malignant diseases have been reasonably ruled out. The clinical features of lymphoproliferative diseases, as well as the associated clinical findings and data deriving from imaging and first-level laboratory investigations, could significantly help in providing the correct diagnostic suspicion for the underlying etiology. This paper reviews the most relevant diseases associated with lymphoproliferation, including infectious diseases, hematological malignancies, IEI, and IEM. Moreover, some practical indications to orient the initial diagnostic process are provided, and two diagnostic algorithms are proposed for the first-level assessment and the approach to persistent lymphoproliferation, respectively.

Pathological, morphological, and molecular characterization of the infection by Brachycladium goliath in humpback whale Megaptera novaeangliae.

The humpback whale (Megaptera novaeangliae) is a cosmopolitan migratory, seasonal mysticete that frequents the Brazilian coast. Strands of specimens may occur during the migratory stay in the country. In 2021 and 2022, three live humpback whales stranded on the coast of Rio Grande do Sul and Santa Catarina states in southern Brazil. After euthanasia, specimens were necropsied, and organs were thoroughly examined for lesions. Grossly, in all three cases, the liver exhibited multifocal, irregular, firm, white areas on the hepatic capsule, which extended into the parenchyma. On the cut surface, the livers were yellow to pale brown with orangish to greenish areas, the bile ducts were prominent, thickened, and severely dilated, and leaf-shaped flukes were found inside of them. Additionally, one case showed moderate atrophy of the right hepatic lobe. The histological findings included dilation of bile ducts, hyperplasia of the bile duct epithelium, marked inflammatory infiltration of lymphocytes, plasma cells, and eosinophils, and portal fibrosis. The parasite Brachycladium goliath was both morphologically and molecularly identified based on diagnostic key for trematodes and the original description of the species, and the amplification and sequencing of the ITS-2 region, respectively. Even though hepatic injury was not the primary cause of stranding, it may have contributed to the debilitation of the whales. To the authors' knowledge, this is the first study that reports M. novaeangliae as a definitive host of B. goliath and that describes the lesions caused by the parasite in cetaceans.

1317: Identifying Lymphocyte Infiltration for Rectal Cancer Outcome Prediction Using Phase III Trial Data

1317: Identifying Lymphocyte Infiltration for Rectal Cancer Outcome Prediction Using Phase III Trial Data

Comparative study of virus and lymphocyte distribution with clinical data suggests early high dose immunosuppression as potential key factor for the therapy of patients with BoDV-1 infection

ABSTRACT Borna disease virus 1 (BoDV-1) was just recently shown to cause predominantly fatal encephalitis in humans. Despite its rarity, bornavirus encephalitis (BVE) can be considered a model disease for encephalitic infections caused by neurotropic viruses and understanding its pathomechanism is of utmost relevance. Aim of this study was to compare the extent and distribution pattern of cerebral inflammation with the clinical course of disease, and individual therapeutic procedures. For this, autoptic brain material from seven patients with fatal BVE was included in this study. Tissue was stained immunohistochemically for pan-lymphocytic marker CD45, the nucleoprotein of BoDV-1, as well as glial marker GFAP and microglial marker Iba1. Sections were digitalized and counted for CD45-positive and BoDV-1-positive cells. For GFAP and Iba1, a semiquantitative score was determined. Furthermore, detailed information about the individual clinical course and therapy were retrieved and summarized in a standardized way. Analysis of the distribution of lymphocytes shows interindividual patterns. In contrast, when looking at the BoDV-1-positive glial cells and neurons, a massive viral involvement in the brain stem was noticeable. Three of the seven patients received early high-dose steroids, which led to a significantly lower lymphocytic infiltration of the central nervous tissue and a longer survival compared to the patients who were treated with steroids later in the course of disease. This study highlights the potential importance of early high-dose immunosuppressive therapy in BVE. Our findings hint at a promising treatment option which should be corroborated in future observational or prospective therapy studies. ABBREVIATIONS: BoDV-1: Borna disease virus 1; BVE: bornavirus encephalitis; Cb: cerebellum; CNS: central nervous system; FL: frontal lobe; GFAP: glial fibrillary acid protein; Hc: hippocampus; Iba1: ionized calcium-binding adapter molecule 1; Iba1act: general activation of microglial cells; Iba1nod: formation of microglial nodules; IL: insula; Me: mesencephalon; Mo: medulla oblongata; OL: occipital lobe; pASS: per average of 10 screenshots; patearly: patients treated with early high dose steroid shot; patlate: patients treated with late or none high dose steroid shot; Po: pons; So: stria olfactoria; Str: striatum