PD-1 Staining Research Articles

Increased PD-1/PD-L1 Immune Checkpoint Expression Is Associated With Oral Squamous Cell Carcinoma in Never-Smokers and Never-Drinkers.

This study aimed to explore the disparities in PD-1 and PD-L1 expression among oral squamous cell carcinomas (OSCCs) in individuals categorized as never-smokers/never-drinkers versus smokers/drinkers. Immunohistochemical staining for PD-1 and PD-L1, along with PDCD1LG2/cen9 dual color probe analysis, was conducted on 130 OSCC specimens from both smoker/drinker and never-smoker/never-drinker cohorts. Associations between smoking/drinking status, clinicopathologic data, immunohistochemical antibody expression, fluorescence insitu hybridization, and survival outcomes were assessed. OSCC in never-smokers/never-drinkers exhibited significantly elevated PD-1 expression (p = 0.003), increased PD-L1-TPS expression (p = 0.044), and elevated PD-L1-CPS expression (p < 0.001). High PD-L1-ICS expression was more prevalent in never-smokers (p = 0.042). Moreover, never-smokers and never-drinkers demonstrated augmented PD-L1 gene copy numbers (p = 0.081 and p = 0.054, respectively). Increased PD-L1 gene copy number, particularly amplification, correlated with PD-L1-TPS (p = 0.039 and p < 0.001). Conversely, PD-L1 gene copy loss was associated with negative PD-L1-CPS (p = 0.023). Notably, positive PD-L1-CPS was significantly linked with improved overall survival (p = 0.023). OSCC arising in never-smokers/never-drinkers exhibit heightened PD-1/PD-L1 signaling, suggesting potential efficacy of immune checkpoint therapy in this subgroup of tumors.

Utility of PD-1, PD-L1, and IDO-1 Stains in Ocular Extranodal Marginal Zone Lymphoma (MZL) and Diffuse Large B-cell Lymphoma (DLBCL).

Extranodal marginal zone lymphoma (EMZL) is the most common subtype of ocular lymphomas. Diffuse large B-cell lymphoma (DLBCL) and EMZL with large-cell transformation present diagnostic challenges. Radiotherapy is the standard treatment for ocular lymphomas, but complications and relapse are common. Diagnostic utility in challenging cases, as well as treatment options using immune checkpoint inhibitors, are unclear in ocular lymphomas. We herein investigated the PD-1, PD-L1, and IDO1 staining patterns in 20 cases of ocular lymphomas, including EMZL (n=14), EMZL with increased large cells (n=2), and DLBCL (n=4). PD-1, PD-L1, and IDO1 staining was not detected in lymphoma cells in any cases but was observed within the tumor microenvironment in all cases. Positivity for PD-1, PD-L1, and IDO1 in inflammatory cells was seen either intratumorally or peritumorally. In all 6 cases with significantly more large B cells, the density of PD-1, PD-L1, and IDO1 expression in the tumor microenvironment was higher than that of the remaining 14 cases without large B cells ( P -value<0.0001), whereas other clinicopathologic features showed no statistical correlation. Increased expression of PD-1, PD-L1, and IDO1 in the inflammatory milieu in cases with large cells may provide diagnostic utility in small biopsies as well as therapeutic potential.

Prognostic Value of Insulin Growth Factor-Like Receptor 1 (IGFLR1) in Stage II and III Colorectal Cancer and Its Association with Immune Cell Infiltration.

IGFLR1 is a novel biomarker, and some evidences suggested that is involved in the immune microenvironment of CRC. Here, we explored the expression of IGFLR1 and its association with the prognosis as well as immune cell infiltration in CRC, with the aim to provide a basis for further studies on IGFLR1. Immunohistochemical staining for IGFLR1, TIM-3, FOXP3, CD4, CD8, and PD-1 was performed in eligible tissues to analyze the expression of IGFLR1 and its association with prognosis and immune cell infiltration. Then, we screened colon cancer samples from TCGA and grouped patients according to IGFLR1-related genes. We also evaluated the co-expression and immune-related pathways of IGFLR1 to identify the potential mechanism of it in CRC. When P < 0.05, the results were considered statistically significant. IGFLR1 and IGFLR1-related genes were associated with the prognosis and immune cell infiltration (P < 0.05). In stage II and III CRC tissue and normal tissue, we found (1) IGFLR1 was expressed in both the cell membrane and cytoplasm and which was differentially expressed between cancer tissue and normal tissue. IGFLR1 expression was associated with the expression of FOXP3, CD8, and gender but was not associated with microsatellite instability. (2) IGFLR1 was an independent prognostic factor and patients with high IGFLR1 had a better prognosis. (3) A model including IGFLR1, FOXP3, PD-1, and CD4 showed good prognostic stratification ability. (4) There was a significant interaction between IGFLR1 and GATA3, and IGFLR1 had a significant co-expression with related factors in the INFR pathway. IGFLR1 has emerged as a new molecule related to disease prognosis and immune cell infiltration in CRC patients and showed a good ability to predict the prognosis of patients.

Primary cutaneous lymphoproliferations in the gray zone between marginal zone lymphoma and CD4+ small/medium T-cell lymphoproliferative disease.

Primary cutaneous marginal zone lymphoma (PCMZL) and primary cutaneous CD4+ small/medium T-cell lymphoproliferative disease (CD4+ TLPD) are two distinct entities with excellent prognosis; however, they show profound clinical and histopathological similarities, leading to differential diagnostic uncertainty. Our aim was to review and reanalyze cases of primary cutaneous lymphoproliferations diagnosed at Semmelweis University, featuring characteristics of PCMZL and CD4+ TLPD. Cutaneous lymphoma biopsy specimens between 2018 and 2022 were collected and re-evaluated. Medical history, clinical picture, imaging, and laboratory findings were collected. Immunohistochemical staining for CD20, CD3, BCL6, CD10, PD1, CD3, CD4, CD8, and PCR tests for IGH, IGK, TCRB, and TCRG were repeated in selected cases. Among 55 cases diagnosed as PCMZL (16) or CD4+ TLPD (39), 3 patients had been diagnosed with both LPDs at different time points of their disease course. Four additional patients were identified with single lesions featuring overlapping histopathological characteristics of both LPDs and both monoclonal IGH and TCR rearrangements. All patients are currently in complete remission with local treatment. We propose that besides the overlapping histopathological, molecular, and clinical features, the subsequent appearance of PCMZL and CD4+ TLPD in a short timeframe in the same patients may suggest a common pathogenic background.

Immune checkpoint inhibitors induce acute interstitial nephritis in mice with increased urinary MCP1 and PD-1 glomerular expression

IntroductionImmune checkpoint inhibitors (ICIs) induce acute interstitial nephritis (AIN) in 2–5% of patients, with a clearly higher incidence when they are combined with platinum derivatives. Unfortunately, suitable disease models and non-invasive biomarkers are lacking. To fill this gap in our understanding, we investigated the renal effects of cisplatin and anti-PD-L1 antibodies in mice, assessing PD-1 renal expression and cytokine levels in mice with AIN, and then we compared these findings with those in AIN-diagnosed cancer patients.MethodsTwenty C57BL6J mice received 200 µg of anti-PD-L1 antibody and 5 mg/kg cisplatin intraperitoneally and were compared with those receiving cisplatin (n = 6), anti-PD-L1 (n = 7), or saline (n = 6). After 7 days, the mice were euthanized. Serum and urinary concentrations of TNFα, CXCL10, IL-6, and MCP-1 were measured by Luminex. The kidney sections were stained to determine PD-1 tissue expression. Thirty-nine cancer patients with AKI were enrolled (AIN n = 33, acute tubular necrosis (ATN) n = 6), urine MCP-1 (uMCP-1) was measured, and kidney sections were stained to assess PD-1 expression.ResultsCisplatin and anti PD-L1 treatment led to 40% AIN development (p = 0.03) in mice, accompanied by elevated serum creatinine and uMCP1. AIN-diagnosed cancer patients also had higher uMCP1 levels than ATN-diagnosed patients, confirming our previous findings. Mice with AIN exhibited interstitial PD-1 staining and stronger glomerular PD-1 expression, especially with combination treatment. Conversely, human AIN patients only showed interstitial PD-1 positivity.ConclusionsOnly mice receiving cisplatin and anti-PDL1 concomitantly developed AIN, accompanied with a more severe kidney injury. AIN induced by this drug combination was linked to elevated uMCP1, consistently with human AIN, suggesting that uMCP1 can be potentially used as an AIN biomarker.

1241 Sustained Localized Delivery of Anti-PD-1 to Tumor Draining Lymph Nodes Increases Survival and Reverses Immunosuppression in Murine Glioblastoma Models

INTRODUCTION: Despite the advent of immunotherapy as a promising therapeutic, glioblastoma (GBM) remains resistant to using checkpoint blockade against programmed cell death protein 1 (PD-1) on T cells. The highly immunosuppressive tumor milieu of GBM prevents rescue of inactivated T cells due to decreased numbers of infiltrating T cells at the tumor site as well as increased recruitment of myeloid cells. Moreover, the current strategy of using antibodies against PD-1 (anti-PD-1) requires multiple intravenous infusions every two or three weeks with debilitating systemic effects. METHODS: Mice orthotopically implanted with GL261 glioma cells were injected with PCL:PEG:PCL hydrogel polymers loaded with anti-PD-1 in one of the following locations: cervical lymph nodes, inguinal lymph nodes, and the tumor site. Mice treated systemically with anti-PD-1 were used as comparative controls. Kaplan-Meier curves were generated for all arms, with subsequent ex vivo flow cytometric staining for CD4, CD8, IFN-y, TNF-a, Foxp3, and PD-1. RESULTS: Mice implanted with PCL:PEG:PCL hydrogels carrying anti-PD-1 at the site of their lymph nodes showed significantly improved survival outcomes compared to mice systemically treated with three doses of anti-PD-1 (p &lt; 0.001). Flow cytometric analysis of lymph nodes and brain tissue in mice injected with this gel demonstrated increased levels of IFN-y, indicating greater reversal of immunosuppression compared to standard treatment. CONCLUSIONS: Our data demonstrates proof of principle of the advantages of using localized therapy that targets lymph nodes for GBM. We propose a paradigm shift for developing new sustained local treatments with immunotherapy that are able to eliminate the need for multiple systemic infusions and their off-target effects.

Cytotoxic response of tumor-infiltrating lymphocytes of head and neck cancer slice cultures under mitochondrial dysfunction.

Head and neck squamous cell carcinomas (HNSCC) are highly heterogeneous tumors. In the harsh tumor microenvironment (TME), metabolic reprogramming and mitochondrial dysfunction may lead to immunosuppressive phenotypes. Aerobic glycolysis is needed for the activation of cytotoxic T-cells and the absence of glucose may hamper the full effector functions of cytotoxic T-cells. To test the effect of mitochondrial dysfunction on cytotoxic T cell function, slice cultures (SC) of HNSCC cancer were cultivated under different metabolic conditions. Tumor samples from 21 patients with HNSCC were collected, from which, SC were established and cultivated under six different conditions. These conditions included high glucose, T cell stimulation, and temporarily induced mitochondrial dysfunction (MitoDys) using FCCP and oligomycin A with or without additional T cell stimulation, high glucose and finally, a control medium. Over three days of cultivation, sequential T cell stimulation and MitoDys treatments were performed. Supernatant was collected, and SC were fixed and embedded. Granzyme B was measured in the supernatant and in the SC via immunohistochemistry (IHC). Staining of PD1, CD8/Ki67, and cleaved-caspase-3 (CC3) were performed in SC. Hematoxylin eosin stains showed that overall SC quality remained stable over 3 days of cultivation. T cell stimulation, both alone and combined with MitoDys, led to significantly increased granzyme levels in SC and in supernatant. Apoptosis following T cell stimulation was observed in tumor and stroma. Mitochondrial dysfunction alone increased apoptosis in tumor cell aggregates. High glucose concentration alone had no impact on T cell activity and apoptosis. Apoptosis rates were significantly lower under conditions with high glucose and MitoDys (p=0.03). Stimulation of tumor-infiltrating lymphocytes in SC was feasible, which led to increased apoptosis in tumor cells. Induced mitochondrial dysfunction did not play a significant role in the activation and function of TILs in SC of HNSCC. Moreover, high glucose concentration did not promote cytotoxic T cell activity in HNSCC SC.

High-risk histopathologic features in local advanced conjunctival melanoma.

To identify high-risk histopathologic and molecular features of local recurrence, nodal metastasis, distant metastasis (DM) and disease-specific death (DSD) in conjunctival melanoma (CoM). Ninety patients with pathologically diagnosed CoM between 2008 and 2023 were enrolled. Immunohistochemistry staining of BRAFV600E, NRASQ61R, CD117, PD-1 and PD-L1 was performed in 65 and 45 patients, respectively. Cox regression and Kaplan-Meier survival analysis were conducted to identify risk factors for local recurrence, nodal metastasis, DM and DSD. Pathologically, ulceration (hazard ratio [HR]: 3.170; 95% CI: 1.312-7.659; p = 0.01) and regression (HR: 3.196; 95% CI: 1.094-9.335; p = 0.034) were risk factors for DM. Tumour thickness ≥ 4 mm (HR: 4.889; 95% CI: 1.846-12.946; p = 0.001) and regression (HR: 4.011; 95% CI: 1.464-10.991; p = 0.007) were risk factors for DSD. For patients with tumour thickness < 4 mm, the presence of ulceration indicated a higher risk of nodal metastasis (log-rank p = 0.0011), DM (log-rank p = 0.00051) and DSD (log-rank p = 0.02). Patients with regression (+)/tumour-infiltrating lymphocytes (TILs) (+) had a higher risk for DM (log-rank p = 0.011) and DSD (log-rank p = 0.0032). Molecularly, the positive rate of BRAFV600E, NRASQ61R, CD117, PD-1 and PD-L1 was 40.00% (26/65), 43.08% (28/65), 70.77% (46/65), 46.67% (21/45) and 28.89% (13/45), respectively. Positive BRAFV600E was identified as an independent risk factor for DM (HR: 2.533; 95% CI: 1.046-6.136, p = 0.039). The expression level of BRAFV600E was positively correlated with vascular invasion (p = 0.01), as well as the expression levels of PD-1 (p = 0.038) and PD-L1 (p = 0.049). Tumour thickness ≥ 4 mm, ulceration, the coexistence of regression and TILs, and positive BRAFV600E were risk factors for poor prognosis of CoM patients. Besides, expression level of BRAFV600E was positively correlated with the expression levels of PD-1 and PD-L1.

The prognostic significance of programmed cell death protein 1 and its ligand on lymphoma cells and tumor-immune cells in diffuse large B-cell lymphoma, not otherwise specified.

Diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS) is the most common type non-Hodgkin's lymphoma, where the treatment of relapsed/refractory cases is the major challenge. Programmed cell death protein 1 (PD-1) and its ligand PD-L1 play a crucial role in the negative regulation of the immune response against the disease. The aim of the study was to analyze the expression of PD-1 and PD-L1 on lymphoma cells (LCs) and tumor-immune cells (TICs) and to investigate their correlation with outcome. Samples from 283 patients diagnosed with DLBCL, NOS (both germinal center B cell like [GCB] and non-GCB subtypes) were included in the study. Expression of PD-1 and PD-L1 was determined using double immunohistochemical staining (D-IHC) for PD-1/PAX5 and PD-L1/PAX5 on tissue microarrays. LCs were highlighted by D-IHC to obtain more accurate results. Clinical data and histologic diagnoses were obtained from electronic data records. We correlated clinical characteristics, and PD-1 and PD-L1 expression on LCs and TICs with progression-free survival (PFS) and overall survival (OS). Expression of PD-1 on TICs was observed in 38.4% and on LCs in 8.8% of cases, while PD-L1 was expressed on TICs in 46.8% and on LCs in 6.5% of cases. PD-L1 expression on LCs was more frequent in non-GCB subtype (p = 0.047). In addition, patients with PD-L1 expression on LCs had significantly shorter PFS (p = 0.015), and the expression retained significant in the multivariate model (p = 0.034). PD-L1 was more frequently expressed in LCs of the non-GCB subtype. Additionally, PD-L1 in LCs may predict shorter PFS time. D-IHC staining for PD-L1/PAX5 is a feasible method to assess PD-L1 expression on LCs of DLBCL, NOS patients and can be used to identify patients who may benefit from targeted immunotherapy with checkpoint inhibitors.

LncRNA XIST Interacts with Regulatory T Cells within the Tumor Microenvironment in Chronic Hepatitis B-Associated Hepatocellular Carcinoma.

Alterations in the expression of several long non-coding RNAs (lncRNAs) have been shown in chronic hepatitis B-associated hepatocellular carcinoma (CHB-HCC). Here, we aimed to investigate the association between the expression of inflammation-associated lncRNA X-inactive specific transcript (XIST) and the type of inflammatory cells within the tumor microenvironment. Twenty-one consecutive cirrhotic patients with CHB-HCC were included. XIST expression levels were investigated on formalin-fixed paraffin-embedded (FFPE) tumoral and peritumoral tissue samples by real-time polymerase chain reaction (RT-PCR). Immunohistochemical staining for CD3, CD4, CD8, CD25, CD163, CTLA4, and PD-1 were performed. The findings were statistically analyzed. Of the 21 cases, 11 (52.4%) had tumoral and 10 (47.6%) had peritumoral XIST expression. No significant association was found between the degree of inflammation and XIST expression. The number of intratumoral CD3, CD4, CD8 and CD20 positive cells was higher in XIST-expressing tumors, albeit without statistical significance. Tumoral and peritumoral XIST expression tended to be more common in patients with tumoral and peritumoral CD4high inflammation. The number of intratumoral CD25 positive cells was significantly higher in XIST-expressing tumors (p=0.01). Tumoral XIST expression was significantly more common in intratumoral CD25high cases (p=0.04). Peritumoral XIST expression was also more common among patients with CD25high peritumoral inflammation, albeit without statistical significance (p=0.19). lncRNA XIST is expressed in CHB-HCC and its expression is significantly associated with the inflammatory tumor microenvironment, particularly with the presence and number of CD25 (+) regulatory T cells. In vitro studies are needed to explore the detailed mechanism.

Folliculotropic Mycosis Fungoides Is Associated with Decreased PD1 Staining Compared with Classic Mycosis Fungoides.

Programmed cell death protein 1 (PD-1) plays a pivotal role in immune system regulation, with its expression levels linked to malignancy prognosis. However, existing reports on PD-1 staining in mycosis fungoides (MF) present conflicting findings, and little attention has been given to PD-1 staining in different MF variants. To address this, we conducted a retrospective study, employing immunohistochemistry to examine PD-1 expression in cases of folliculotropic MF and non-folliculotropic MF. We analyzed 24 cases of folliculotropic MF and 18 cases of non-folliculotropic MF, and recorded both the percentage of PD-1-labeled tumor cells and the intensity score (negative, weak, medium, or strong). Our results revealed significant disparity in PD-1 labeling between patch/plaque MF and folliculotropic MF (p = 0.028). Non-folliculotropic MF exhibited higher PD-1 labeling in tumor cells (58.3%) compared to folliculotropic MF (40.2%). Notably, there was no significant difference in PD-1 staining between folliculotropic MF and non-folliculotropic MF when both were in the early stage/indolent disease category. However, when considering the tumor stage, folliculotropic MF exhibited PD-1 staining in tumor cells at a rate of 21.1%, while non-folliculotropic MF showed PD-1 staining in tumor cells at a rate of 46.6% (p = 0.005). Additionally, among folliculotropic MF cases, 13 out of 24 cases displayed differing PD-1 expression patterns between epidermal and dermal components, with preserved PD-1 staining in the epidermal component and loss of staining in the dermal component. Furthermore, consistent with the prior literature, tumor cells with large cell transformations exhibited significantly lower PD-1 labeling (p = 0.017). Our findings showcase the unique PD-1 staining patterns in MF.

Investigation of the PD-1/PD-L1 Expression in the Lesional Skins of Patients With Psoriasis.

Psoriasis is an immune-mediated, chronic and inflammatory disease whose pathogenesis is affected by the interactions of several immune cells and cytokines. PD-1 is an inhibitor receptor that is expressed to a large extent in T lymphocytes and responsible for regulating autoimmunity and self-tolerance. In this study, we aimed to investigate the expression of PD-1/PD-L molecules in the lesioned skins of psoriasis patients. The study included 30 psoriasis patients, and 15 healthy volunteers as the control group. Anti PD-1 and PD-L1 antibodies were applied to the skin biopsy samples that were collected from the patient and control groups. Cytoplasmic and membranous staining of PD-1 and PD-L1 were considered positive. The number of stained immune cells that was examined for each case. The percentage of the tissues with high PD-1 (+) and PDL-1 (+) immune cell counts were significantly higher in the psoriasis patients compared to healthy controls (P values = 0.004 and 0.002, respectively). A negative and statistically significant correlation was detected between PDL-1(+) immune cell numbers and PASI scores (P = 0.033, r=-0.57). In the lesioned skin samples of psoriasis patients, the PD-1 and PD-L1 expressions were significantly higher in immune cells than that in the skin samples of the healthy controls. This study was the first investigation of the expression of PD-1/PD-L molecules in the immune cells in found the lesioned skins of psoriasis patients.

Mocravimod, a S1P receptor modulator, increases T cell counts in bone marrow biopsies from patients undergoing allogeneic hematopoietic stem cell transplantation

Graft-versus-leukemia (GvL) effects are critical to prevent relapses after allogeneic hematopoietic cell transplantation (allo-HCT). However, the success of allo-HCT is limited by graft-versus-host disease (GvHD). Both, CD4+ and CD8+ T cells contribute to GvHD and GvL. The sphingosine-1-phosphate receptor (S1PR) signaling plays a crucial role in lymphocyte trafficking. Mocravimod is an S1PR modulator and its administration leads to blocking lymphocyte egress from lymphoid organs. We hypothesized that this applies to the bone marrow (BM) too, and analyzed BM biopsies from the clinical study with mocravimod (phase I trial in allo-HCT patients; NCT01830010) by immunohistochemical staining for CD3, CD4, CD8, TIA1, FoxP3, PD1, T-Bet, GATA3, and ROR-γt to identify and quantify T cell subsets in situ. Allo-HCT patients without receiving mocravimod were used as controls. BM from 9 patients in the mocravimod group and 10 patients in the control group were examined. CD3+ T cells were found to accumulate in the BM of mocravimod-treated patients compared to controls, both on day 30 and 90 post-transplant. The effect was stronger for CD4+ T cells, than CD8+ T cells, which is in line with data from murine studies showing that CD4+ T cells are more sensitive to mocravimod treatment than CD8+ T cells. Clinically-relevant acute GvHD events (grade II-IV) were slightly lower, but comparable to controls when mocravimod was administered. Taken together, data are supportive of mocravimod’s mode of action and bring additional evidence of fewer relapses for allo-HCT patients treated with S1PR modulators.

The prognostic value of tumour-infiltrating lymphocytes, programmed cell death protein-1 and programmed cell death ligand-1 in Stage I-III triple-negative breast cancer.

Tumour-infiltrating lymphocytes (TILs) represent a robust biological prognostic biomarker in triple-negative breast cancer (TNBC); however, the contribution of different subsets of immune cells is unclear. We investigated the prognostic value of immune markers, including stromal TILs (sTILs), CD8+T and FOPX3+T cells, PD-1 and PD-L1 in non-metastatic TNBC. In total, 259 patients with Stage I-III TNBC were reviewed. The density of sTILs along with the presence of total (t), stromal (s), and intratumoral (i) CD8+T cells and FOPX3+T cells were evaluated by haematoxylin and eosin and immunohistochemical staining. Immunohistochemical staining of PD-1, PD-L1 was also conducted. All immune markers were positively correlated with each other (P < 0.05). In the multivariate analysis, sTILs (P = 0.046), tCD8+T cells (P = 0.024), iCD8+T cells (P = 0.050) and PD-1 (P = 0.039) were identified as independent prognostic factors for disease-free survival (DFS). Further analysis showed that tCD8+T cells (P = 0.026), iCD8+T cells (P = 0.017) and PD-1 (P = 0.037) increased the prognostic value for DFS beyond that of the classic clinicopathological factors and sTILs. In addition to sTILs, inclusion of tCD8+T, iCD8+T cells, or PD-1 may further refine the prognostic model for non-metastatic TNBC beyond that including classical factors alone.

Abstract P5-02-11: Immune cell profile of tumors from patients with metastatic (met) HER2+ breast cancer (BC) with &amp;lt; 30 months overall survival (OS)

Abstract Background: The “Thousand Patient HER-2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin has been used to identify HER2+ BC patients with durable complete response (never relapsed) to trastuzumab-based therapy. ~10% of met HER2+ BC patients achieve a durable complete response to trastuzumab, meaning the majority of patients progress on treatment. Higher stromal tumour immune infiltrate has been associated with longer OS in met HER2+ BC. There is limited tumor immune profile and PD-1 expression data available for patients with met HER2+ BC with short OS. Using the SVUH database, we have identified a preliminary cohort of 21 met HER2+ BC patients that received trastuzumab and had an OS &amp;lt; 30 months. This study examines the levels of pan T cell marker CD3, cytotoxic T cell marker CD8, Natural Killer (NK) cell marker CD56 and immune checkpoint PD-1 by immunohistochemistry (IHC) in this preliminary cohort. Methods: Formalin-fixed, paraffin-embedded (FFPE) biopsy specimens (n=21 primary, n=7 matched metastatic biopsies) and associated clinico-pathological data were curated. Tumor biopsies were processed for IHC staining of CD8 (Agilent IR62361-2), CD3 (Agilent IR50361-2), CD56 (Agilent IR62861-2) and PD-1 (Roche 07099029001). PD-1 staining was available for 20/21 samples. Staining was performed using the DAKO Link 48 Autostainer as per the manufacturer’s instructions using positive (tonsil tissue) and negative controls (isotype controls). Slides were processed using the Aperio AT2 Digital Slide Scanner (Leica Biosystems), reviewed using Aperio ImageScope 12.4 software (Leica Biosystems) and analyzed in QuPath (University of Edinburgh). Images were annotated to outline tumor areas and an algorithm was trained to identify cells and classify them as either tumor or stromal. Data was expressed as number of positively stained cells/mm2 breast tumor or stromal tissue. Survival studies utilized the Kaplan Meier method. The paired Student’s T test was utilized for primary vs metastatic site comparisons. Results: Designating samples with &amp;gt; 1 stained cell/mm2 breast tumor as positive (pos) and zero stained cells as negative (neg), 19/21 (90.5%) primary samples were pos for CD3, 15/21 (71.4%) for CD56, 14/21 (66.6%) for CD8, and 10/20 (50%) for PD-1. Within the stromal compartment, 20/21 (95.2%) primary samples were pos for CD8, 18/21 (85.7%) for CD56, 16/21 (76.2%) for CD3 and 8/20 (40%) for PD-1. PD-1 expression in the primary tumor (median OS PD-1pos 7.85 mo vs PD-1neg 5.39 mo, hazard ratio (HR) 0.642 (95% CI 0.256-1.613), p=0.346) or the stroma (median OS PD-1pos 8.84 mo vs PD-1neg 5.39 mo, HR 0.495 (95% CI 0.197-1.244), p=0.135) was not significantly associated with OS. When comparing matched primary and metastatic samples (n=7), increased stromal levels of CD3 (4/7), CD8 (4/7), CD56 (5/7) and PD-1 (4/7) were observed. Increased levels of CD3 and CD8 were observed for 2/7 samples, and increased levels of CD56 and PD-1 for 4/7 samples. With the exception of tumor CD8 levels which decreased, mean values for tumor and stromal CD3, CD56, PD-1 and stromal CD8 levels were higher in metastatic sites but all differences were not found to be significant (p&amp;gt;0.05). Conclusions: Our results suggest that met HER2+ BC patients with &amp;lt; 30 months OS have significant T cell and NK cell presence in the tumor and stromal compartments in both primary and metastatic sites. Further expansion of this limited dataset is planned to gain greater insight in to the immune cell profiles and PD-1 status of met HER2+ BC patients with short OS. Citation Format: Denis M. Collins, Janet McCormack, Laura P. Ivers, Jose Javier Berenguer Pina, Jo Ballot, Cecily Quinn, Darko Skrobo, Alex J. Eustace, Naomi Walsh, Aurelie Fabre, John Crown. Immune cell profile of tumors from patients with metastatic (met) HER2+ breast cancer (BC) with &amp;lt; 30 months overall survival (OS). [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-11.

PD-1 and PD-L1 Expression in Indian Women with Breast Cancer.

The interaction between programmed cell death protein 1 (PD-1) on activated T-lymphocytes and programmed death-ligand 1 (PD-L1) on tumor cells or antigen-presenting cells sends immunosuppressive signals leading to the escape of tumor cells from the host anti-tumor immune response. Inhibiting this interaction with antibodies against PD-1 or PD-L1 is emerging as a valuable therapeutic strategy. However, tissue distribution patterns for PD-L1 and PD-1 in breast cancer patients from India are not reported, yet many clinical trials are underway. In this study the expression of PD-1 and PD-L1 in breast cancer patient samples from India was characterized. The study included 392 cases of operated breast cancer (2012-2017) from a tertiary cancer care center in Bangalore, Karnataka, India. Paraffin blocks were retrievable and receptor status was known. Immunohistochemistry (IHC) was performed using anti-PD-L1 and anti-PD-1 antibodies. RNA was isolated from 76 fresh tumors and nine adjacent normal tissues (2019). PD-L1 transcript levels were measured by RT-qPCR using Hypoxanthine-guanine phosphoribosyl transferase (HPRT) as a reference gene. Based on IHC, PD-1 expression within tumor-infiltrating immune cells (TIICs) was observed in 55/385 cases (14%) across all breast cancer types. In triple-negative breast cancer (TNBC), 21/132 cases (16%) showed PD-1 staining in TIICs. The overall expression of PD-L1 in breast tumor cells across all breast cancer subtypes and TIICs was 11% (41/378) and 39% (151/385), respectively. A relatively higher proportion of TNBC cases had PD-L1 expression in tumor cells (17/132 cases, 13%) and immune cells (68/132 cases, 52%). We also detected PD-L1 transcript expression by qRT-PCR in freshly isolated tumor samples. These findings show that around 52% (68/132) of the TNBC cases express PD-L1 in TIICs. Hence, anti-PD-1/PD-L1 therapy alone or combined with chemotherapy may be a promising treatment for TNBC in Indian patients.

Relationship Between Immunohistochemical CD3, CD4, CD5, CD8, and PD1 Staining and Histopathological Diagnosis of Cervical Lesions in Patients With Abnormal Colposcopic Findings.

This study aimed to analyse the relationship between clinicopathological factors in cervical intraepithelial lesions and abnormal colposcopic findings. Thirty high-grade squamous intraepithelial lesion (HSIL) and thirty low-grade squamous intraepithelial lesion (LSIL) patients who underwent biopsy due to abnormal colposcopic findings were included in the study. The immunoreactivity of CD3, CD4, CD5, CD8, and PD-1 was analysed immunohistochemically in tumor-infiltrating lymphocytes (TILs) and stromal lymphocytes. In TILs, CD3, CD4, CD5, CD8, and PD-1 were highly stained in 20/30 (66.6%), 16/30 (53.3%), 15/30 (50.0%), 24/30 (80.0%), and 13/30 (43.3%) of the cases for the HSIL group, while 7/30 (23.3%), 4/30 (13.3%), 5/30 (16.6%), 9/30 (30.0%), and 5/30 (16.6%) were in the LSIL group, respectively. CD3, CD4, CD5, CD8, and PD-1 immunostainings for TILs were higher in the HSIL group (p=0.001, p=0.001, p=0.006, p˂0.001, p=0.024, respectively). Only PD-1 was significantly higher in lymphocytes in the stroma (p=0.001). CD3, CD4, and CD8 also show a positive correlation with the Ki-67 proliferation index. CD3, CD4, CD5, and CD8 may contribute to PD-1-mediated tumour control. Immunohistochemical staining plays a key role in evaluating the tumour microenvironment.

Clinicopathological features of angioimmunoblastic T-cell lymphoma pattern Ⅰ

Objective: To investigate the clinicopathological features of angioimmunoblastic T-cell lymphoma pattern Ⅰ (AITL Pattern Ⅰ). Methods: The clinicopathological data of 11 AITL Pattern Ⅰ cases that were diagnosed at the Beijing Friendship Hospital Affiliated to Capital Medical University (10 cases) and Beijing Lu Daopei Hospital (1 cases) from January 2019 to October 2021 were retrospectively collected. Immunophenotype, Epstein-Barr virus infection status and T cell receptor (TCR) clonality of the tumor cells were tested, and clinicopathological features of cases were analyzed. Results: Among the 11 AITL Pattern Ⅰ cases, the male to female ratio was 1.2∶1.0. The median age was 59 years (range 47-78 years). Seven cases had B symptoms, while eleven cases presented with systemic lymphadenopathy. According to Ann Arbor system staging, two cases were classified as stage Ⅰ-Ⅱ, and 9 cases as stage Ⅲ-Ⅳ. Hepatosplenomegaly was present in two cases (2/11), three cases (3/11) had skin rash and pruritus, and two cases (2/11) had pleural effusion. Previously, 6 cases (6/11) were diagnosed as reactive hyperplasia, 1 case (1/11) as EBV-associated lymphoproliferative disorder, and 4 cases (4/11) as hyperplasia of lymphoid tissue, which was unable to exclude lymphoma. Histologically, all the 11 cases showed hyperplastic follicles in the paracortical regions with well-formed germinal centers. The hyperplastic follicles showed ill-defined borders and attenuated mantle zones in 7 cases. Mantle zones completely disappeared in 4 cases. The follicles were surrounded by a thin layer of atypical lymphocytes with bright or faintly stained cytoplasm. In 2 cases, the clear cells were located between the germinal centers and the thin residual mantle cell layers, showing a circular growth pattern. The cells were medium in size, with irregular karyotype, coarse chromatin and indistinct nucleoli. Immunohistochemically, CD21 staining showed that the meshworks of follicular dendritic cells(FDC)were mainly confined to the follicles. There was a subtle expansion of the meshworks of FDC in 4 cases with ill-defined borders. The atypical cells surrounding the follicles expressed CD3 (11/11), CD4 (11/11), PD-1 (11/11), CXCL13 (6/11), ICOS (10/11) and CD10 (7/11). PD-1 staining showed a strong perifollicular pattern, and a small number of positive cells were scattered around the high endothelial veins in the interfollicular region. CXCL13, ICOS and CD10 showed similar distribution patterns. EBV-encoded small RNA probe (EBER) in situ hybridization showed that EBER positive B lymphocytes were scattered in the interfollicular region (5-20/HPF) in all cases. T cell receptor gene rearrangement was monoclonal in all cases. Conclusions: Diagnosing AITL Pattern Ⅰ may be challenging and requires comprehensive analysis of clinical manifestations, histological morphology, immunophenotype and gene rearrangement results.

Multiomics profiling and association with molecular and immune features in association with benefits from immunotherapy for patients with previously treated stage IV or recurrent squamous cell lung cancer from the phase III SWOG LungMAP S1400I trial.

9046 Background: Immune checkpoint blockade (ICB) has become a standard pillar of treatment for lung cancer. However, only ̃20% of unselected patients can achieve durable clinical benefits. We performed immunogenomic profiling of tissue specimens from a randomized Phase III trial S1400I on metastatic lung squamous cell carcinoma (SCC) to evaluate if there were factors associated with better prognoses with ICB from single-agent versus combined targeting PD-1/CTLA-4 and evaluate if any differentiated between the treatments. Methods: We utilized FFPE tumor tissue submitted for Lung-MAP screening provided by the SWOG bank. SCC samples from 82 eligible patients treated with combined nivolumab+ipilimumab (N+I) or single agent nivolumab (N) were subjected to multiplex immunofluorescence (mIF, n = 82) and NanoString (ncounter PanCancer Immune Profiling Panel, n = 32). Cell density phenotypes (cells/mm2) were defined using image analysis of staining for cytokeratin, CD3, CD8, granzyme B, CD45RO, FOXP3, PD1, PD-L1, and CD68. Immunogenomic features were associated with response, PFS, and OS derived from data provided by the LungMap team to the CIDC portal. For statistical analyses, non-parametric tests were utilized to assess associations of cell phenotypes versus continuous or categorical variables, and log-rank test analysis was performed to identify cell phenotypes or genes correlated with survival. Results: In both arms higher densities of total CD3+CD45RO+ T cells ( P= 0.041), CD3+PD-1+ T cells ( P= 0.024) and CD3+CD8+PD-1 T cells in stroma ( P= 0.042) and CD3+CD8+GZMB+ T cells in the tumor compartment ( P= 0.011) were positively associated with PFS. In the N+I arm but not in the N arm, higher densities of CD3+CD8+GZMB+ T cells in the tumor compartment were associated with better PFS ( P= 0.015) and higher densities of stroma CD3+CD8-FOXP3+ T cells with worse OS. Spatial analysis showed that the presence of CD8+GZMB+ T cells close to malignant cells (median, ≤19.27 µm) was associated with better PFS ( P= 0.037) in N+I arm and cluster analysis showed low clustering of cells in TMB-high vs. TMB-low tumors (P &lt; 0.01). Gene expression profiling demonstrated that myeloid infiltration, immune recruitment, and inflammation genes were associated with a positive clinical outcome ( P&lt; 0.05). In both arms, BLNK, CD163, FCGR2A were associated with better OS ( P&lt; 0.01), IRF1 and BLNK were associated with increased PFS ( P&lt; 0.01). In the N+I arm but not in the N arm, we observed significantly higher CD45 immune cell scores, including CD8 T cells and neutrophils, in responders versus non-responders. Conclusions: Our findings suggest a potential advantage in PFS and OS with an increased presence of cytotoxic immune cells and genes associated with the recruitment and proliferation of these cell types before therapy.

Clinicopathological Features of Gastric Cancer with Autoimmune Gastritis.

Most gastric cancers develop in patients with chronic gastritis. Chronic gastritis can be classified into two major subtypes: Helicobacter pylori (H. pylori)-induced gastritis and autoimmune gastritis (AIG). Whereas H. pylori-related gastric cancers are more common and have been extensively investigated, the clinicopathological features of gastric cancer with autoimmune gastritis are unclear. Patients diagnosed with gastric cancer and hospitalized in the University Tokyo Hospital from 1998 to 2017 were enrolled. Diagnosis of autoimmune gastritis was based on positivity for serum anti-parietal cell antibody (APCA). We evaluated mucin expression and immune cell infiltration by immunohistochemical staining for MUC5AC, MUC6, PD-L1, CD3, CD11, Foxp3, and PD1. We also examined the presence of bacterial taxa that are reportedly enriched in AIG. Survival analyses of recurrence and 5-year mortality were also performed. In total, 261 patients (76 APCA-positive and 185 APCA-negative) were analyzed. Immunohistochemical staining in the matched cohort showed that AIG-related gastric cancer had higher MUC5AC expression (p = 0.0007) and MUC6 expression (p = 0.0007). Greater infiltration of CD3-positive (p = 0.001), Foxp3-positive (p < 0.001), and PD1-positive cells (p = 0.001); lesser infiltration of CD11b-positive (p = 0.005) cells; and a higher prevalence of Bacillus cereus (p = 0.006) were found in AIG-related gastric cancer patients. The cumulative incidences of gastric cancer recurrence were 2.99% at 2 years, 15.68% at 6 years, and 18.81% at 10 years in APCA-positive patients; they were 12.79% at 2 years, 21.35% at 6 years, and 31.85% at 10 years in APCA-negative patients. The cumulative incidences of mortality were 0% at 3 years and 0% at 5 years in APCA-positive patients; they were 1.52% at 3 years and 2.56% at 5 years in APCA-negative patients. We identified molecular differences between AIG and non-AIG gastric cancer. Differences in T-cell populations and the gastric microbiota may contribute to the pathogenesis of gastric cancers and potentially affect the response to immunotherapy.