PD-L1 Levels Research Articles

Association of tumor-infiltrating lymphocytes (TILs) and immune signatures with PAM50 intrinsic subtyping hormone receptor-positive (HR+)/HER2-negative (HER2-) early breast cancer (BC): A translational analysis of two multicentric neoadjuvant trials.

558 Background: HR+/HER2− BCs are generally described as immunologically cold, with low levels of TILs and PD-L1. Nevertheless, the addition of immunotherapy (ICI) to chemo has been reported to increase pCR rates in high-risk early HR+/HER2– BCs. Assessing the interaction between immune features and tumor biology may identify of a subset of patients (pts) with HR+/HER2− BC that would be the ideal candidates for the combination of chemo and ICI. Methods: Gene-expression data from two multicentric neoadjuvant phase II trials (GIADA, LETLOB) enrolling stage II-IIIA HR+HER2- BC pts was retrieved (Dieci, CCR 2022; Guarneri, JCO 2014). In the GIADA trial, 758 genes were assessed on baseline tumor samples by nCounter (N=43). In the LETLOB trial, gene-expression data (Affymetrix platform) from baseline core-biopsies was available for 66 pts. Intrinsic subtype was assigned using a research-based PAM50 predictor. TILs were evaluated following guidelines. Association of TILs and immune signatures with intrinsic subtyping was assessed by Kruskal-Wallis test. Results: PAM50 subtype distribution (N=109) was 44% Luminal B (LumB), 33% Luminal A (LumA), 18% Basal-like, 5% HER2-enriched (HER2-E). TILs were assessed on 101 samples: median 2 (range 0-100). Basal-like BCs showed higher TILs than other subtypes (p=0.008). No significant difference was observed between LumA and LumB BCs (Table). Basal-like tumors showed higher levels of previously published immune signatures associated with CD8 T-cells (p<0.001), Cytotoxic cells (p<0.001), IFN-gamma (p<0.001), and response to ICI in the GeparNuevo trial (p=0.003) (N=109). PD-1 (p=0.002), PD-L1 (p=0.001), and PD-L2 (p=0.001) genes were also more expressed in Basal-like BCs (evaluable in 43 samples). No significant difference was observed between LumA and LumB tumors. A significant weak to moderate positive correlation was observed between continuous TILs and immune signatures and basal-like PAM50 signature (p<0.001, Spearman coefficient=0.372 for TILs, from 0.354 to 0.648 for immune signatures). Conclusions: PAM50 intrinsic subtyping and TILs/immune signatures capture only partially overlapping information on the biology of HR+/HER2- BCs. Pts with HR+/HER2- BCs simultaneously showing features of biological aggressiveness and enhanced immunogenicity may represent the ideal candidates for the combination of ICI and chemo. [Table: see text]

A phase II, multicenter, open-label study of polyPEPI1018 in combination with atezolizumab in participants with relapsed or refractory microsatellite-stable metastatic colorectal (MSS mCRC) cancer (Oberto-301).

3594 Background: The efficacy of checkpoint inhibitor immunotherapy in MSI-H mCRC has not been replicated in MSS mCRC. Therefore, additional interventions are needed to convert immunologically “cold” MSS CRC to “hot” tumors resembling MSI-H tumors. PolyPEPI1018 is an off-the-shelf, multi-peptide vaccine containing 12 immunogenic epitopes derived from 7 shared tumor antigens which demonstrated early evidence of clinical activity in first-line MSS mCRC. Methods: Patients with MSS mCRC who have progressed on 2-3 lines of prior chemotherapy regimen received PolyPEPI1018 (1.2 mg, sc) and atezolizumab (1,200 mg, iv) Q3W until disease progression or unacceptable toxicity. The primary endpoint was safety. A Simon 2-stage design was applied. Data on objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and correlation studies will be presented. Results: 18 patients (66 % female) were enrolled for stage 1 of the study. Median age was 54 years (range 38–79), 50% had liver metastases and 67% received at least 3 lines of prior therapies. PD-L1 expression was <1% for 85% of patients. The combination was well-tolerated; most common side effect related to treatment was Grade (Gr) 1-2 local skin reaction (n=22). Gr 2 events (n=2) at least possibly related to treatment were nausea, pyrexia and increased blood alkaline phosphatase. There were no Gr 3-5 events or study discontinuation due to treatment AE. The ORR was 0% and the DCR was 61%. The mPFS was 2.7 months (95%CI 1.4-4.0) and the mOS was not reached (95%CI 8.75 – NE months) at the data cut-off date. Ex-vivo FluoroSpot identified 13/16 subjects with robust, vaccine-specific CD8+ and/or CD4+ T cell responses detected against multiple vaccine antigens. For patients with available biopsy pairs (n=8), both the average PD-L1 expression (IC%, p=0.029) and the density of CD8+ tumor-infiltrating lymphocytes (TILs, p=0.019) were significantly increased, post-treatment. Patients with increased PFS (> 12 weeks) had both higher levels of post-treatment PD-L1 (p=0.007) and TILs (p=0.016) than patients with PFS ≤ 12 weeks. Conclusions: PolyPEPI1018 in combination with atezolizumab was well-tolerated with no Grade 3 AEs observed. Despite no objective tumor responses could be detected, correlative data suggest contribution of PolyPEPI1018-induced immune responses to the modulation of tumor microenvironment and to improved disease control. The study did not proceed to stage 2; it is on-going for the collection of survival data. Clinical trial information: NCT05243862 .

Pediatric primary extragonadal choriocarcinoma - A study on male patients at a single tertiary medical institution

BackgroundPrimary extragonadal choriocarcinoma (PEGCC) in male is rare. It is highly malignant, typically presents with distant metastasis at the time of diagnosis, and responds poorly to treatment. Because of its associated high levels of PD-L1, the PD-1/PD-L1 pathway is a likely therapeutic target. Herein, we report our experience of treating pediatric PEGCC in six boys at a tertiary hospital. MethodsWe analyzed the data of six boys with pathologically confirmed PEGCC between 2009 and 2021. Their clinicodemographic and histopathological characteristics as well as treatments and clinical outcomes were retrieved from their medical charts. ResultsThe patients' median age was 15 (range: 12–17) years. The most common primary tumor site was the mediastinum (67%, 4/6), with one case each in the retroperitoneum (16.7%) and brain (16.7%). Except for the patient with brain PEGCC, all presented with metastasis at the time of diagnosis. The following metastatic sites were observed: the lungs (100%, 5/5), brain (3/5, 60%), liver (3/5, 60%), kidneys (2/5, 40%), and spleen (1/5, 20%). Most patients had dry cough, dyspnea, and hemoptysis at initial presentation, likely due to lung metastasis. Serum human chorionic gonadotropin (HCG) levels were highly elevated in all patients. All patients received platinum-based cytotoxic chemotherapy. The patient with brain choriocarcinoma underwent surgical tumor resection; all others underwent only surgical biopsy. Strong positive PD-L1 immunohistochemical staining was noted for two patients. One patient received the PD-L1 inhibitor pembrolizumab and achieved a good response. Our cohort's 1-year survival rate was 33.3%, with a median survival of 4.34 months. Serum HCG levels remained normal in the two survivors during follow-up visits. ConclusionThe poor response to current platinum-based chemotherapy remains a major challenge in the management of pediatric PEGCC. Adding pembrolizumab to a conventional chemotherapy regimen may improve the outcomes in boys with PEGCC.

A phase 1, open-label, dose escalation study on the safety and tolerability of ANK-101 in advanced solid tumors.

TPS2689 Background: IL-12 is a cytokine that can stimulate both innate and adaptive tumor immunity. Clinical trials of intravenous IL-12 demonstrated anti-tumor activity limited by significant systemic toxicity, including patient deaths. ANK-101 is an anchored immunotherapy that links IL-12 to aluminum hydroxide through an alum-binding protein (ABP). The ANK-101 complex anchors IL-12 in the tumor microenvironment (TME), resulting in prolonged local retention and low levels of systemic absorption. Preclinical studies in murine tumor models showed that ANK-101 is retained at the injection site for up to 28 days, recruits and activates T and NK cells, promotes M1 myeloid cell differentiation, induces regression of both injected and un-injected tumors, and induces immunologic memory. Methods: This is a first-in-human, open-label dose escalation study of ANK-101 in advanced solid tumors with a planned sample size of 12-36 participants. The first three dose escalation cohorts include a single participant, and then study enrollment follows a standard 3+3 dose escalation design. If no dose limiting toxicity (DLT) is observed, the dose level will be escalated until ≥1/3 or ≥2/6 patients experience a DLT. An additional ten patients will be enrolled at the recommended dose for expansion (RDE). Participants will be treated with intratumoral ANK-101 every 3 weeks for 4 cycles. Imaging or clinical assessments will be performed at week 12. If there is no significant clinical deterioration or unacceptable toxicity at this visit, participants may receive four more cycles. Eligible patients must have an advanced solid malignancy refractory to standard treatment and be accessible for injection and biopsy, measurable disease by RECIST v1.1, and ECOG PS of 0-1. Key exclusion criteria include tumors close to vital structures, uncontrolled bleeding disorders, and active autoimmune disease. Primary objectives of the study are to determine the safety and tolerability of ANK-101 and identify the RDE. Secondary objectives include pharmacokinetics (PK) and immunogenicity (ADA) of ANK-101 and clinical activity as measured by ORR, DCR, DOR and PFS by RECIST v1.1. Exploratory objectives include assessment of QOL using FACT-G and profiling immune-based pharmacodynamic (PD) changes. PD assessments will include serum cytokines and circulating immune cells, and local levels of PD-L1, CD8+ T cells, and CD68+ macrophages, and changes in gene expression within the TME. This clinical trial is in progress. Clinical trial information: NCT06171750 .

Possibilities of long-term atezolizumab immunotherapy in patients with metastatic urothelial cancer.

e14628 Background: Results from various studies have indicated that atezolizumab can be an effective and well-tolerated treatment option for patients with advanced and metastatic urothelial cancer. However, the landscape of oncological practices in Uzbekistan reflects a scarcity of experience and data concerning the utilization of atezolizumab in the treatment of patients afflicted with urothelial cancer. This research endeavors to bridge this gap by contributing. Methods: In our retrospective study, a total of 44 patients were selected, with a mean age of 64.5±7.6 years, diagnosed with stage IV urothelial cancer and the presence of distant metastases in the lungs, bones, and liver. There were 28 (63.6%) males and 16 (36.4%) females. Group 1 included 18 (40.9%) patients with elevated PD-L1 levels SP142 – IC 2/3, receiving atezolizumab at 1200 mg intravenously (IV) on day 1 of each 21-day cycle + cisplatin at a dose of 70 mg/m2 body surface area (BSA), IV, on day 1 of each cycle, and gemcitabine at a dose of 1000 mg/m2 BSA, IV, on days 1 and 8 of each cycle. Group 2 included 26 (59.1%) patients receiving standard chemotherapy (cisplatin + gemcitabine) in the same regimens. Immunohistochemical analysis was performed using the Ventana Bench Mark Ultra automated stainer with Ventana PD-L1 SP142 monoclonal antibodies. Results: The research was conducted from September 2019 to May 2023, and the administered treatment was the first line for these patients. Treatment was administered until disease progression was registered or until the development of unacceptable toxicity. Grade I toxicity was not observed in both groups. Grade II toxicity in Group 1 was noted in 3 (16.7%), and in Group 2 - in 4 (15.4%). Grade III toxicity was identified in 9 patients (50.0%) in Group 1 and 14 patients (53.8%) in Group 2. Grade IV toxicity was encountered in 6 patients (33.3%) in Group 1 and 8 patients (30.8%) in Group 2. Hematological toxicity, regardless of atezolizumab use, predominated among the III and IV degrees of toxicity. The median overall survival in Group 1 was 14.8 months (95% CI: 12.8-16.2), while in Group 2, it was 11.9 months (95% CI: 9.3-13.5). Progression-free survival in Group 1 was 6.8 months (95% CI: 5.1-7.4), and in Group 2, it was 4.3 months (95% CI: 3.8-5.7). The objective response rate in Group 1 was 44.5%, and in Group 2, it was 38.5%. Conclusions: Atezolizumab, used in combination with standard platinum-based chemotherapy in the first line, improves treatment outcomes for metastatic urothelial cancer by increasing overall survival and progression-free survival without causing an increase in the toxicity of the administered therapy.

Dual-Recognition-Mediated Autocatalytic Amplification Assay for the Subpopulations of PD-L1 Positive Extracellular Vesicle.

The PD-L1 protein on extracellular vesicles (EVs) is a promising biomarker for tumor immunotherapy. However, PD-L1+ EVs have various cell origins, so further analysis of the subpopulations is essential to help understand better their relationship with tumor immunotherapy. Different from the previous work which focus on the level of total PD-L1+ EVs expression, we, herein, report a dual-recognition mediated autocatalytic amplification (DRMAA) assay to detect the PD-L1 derived from tumors (EpCAM+), immune T cells (CD3+), and total (Lipids) EVs, respectively. The DRMAA assay employed proximity hybridization to construct a complete trigger sequence and then catalyzed the cross-hybridization of three hairpin probes, producing a three-way DNA junction (3-WJ) structure carrying the newly exposed trigger sequence. The 3-WJ complex subsequently initiated an autocatalytic amplification reaction and higher sensitivity than the traditional catalytic hairpin assembly assay was obtained. It was found that the EpCAM+ and PD-L1+ EVs were more effective than others in distinguishing lung cancer patients from healthy people. Surprisingly, the CD3+ and PD-L1+ EVs in lung cancer patients were also upregulated, indicating that immune cell-derived PD-L1+ EVs are also non-negligible marker in a tumor microenvironment. Our results suggested that the DRMAA assay would improve the study of subpopulations of PD-L1+ EVs to provide new insights for cancer immunotherapies.

DRG2 is required for surface localization of PD-L1 and the efficacy of anti-PD-1 therapy.

More than half of tumor patients with high PD-L1 expression do not respond to anti-PD-1/PD-L1 therapy, and the underlying mechanisms are yet to be clarified. Here we show that developmentally regulated GTP-binding protein 2 (DRG2) is required for response of PD-L1-expressing tumors to anti-PD-1 therapy. DRG2 depletion enhanced IFN-γ signaling and increased the PD-L1 level in melanoma cells. However, it inhibited recycling of endosomal PD-L1 and reduced surface PD-L1 levels, which led to defects in interaction with PD-1. Anti-PD-1 did not expand effector-like T cells within DRG2-depleted tumors and failed to improve the survival of DRG2-depleted tumor-bearing mice. Cohort analysis revealed that patients bearing melanoma with low DRG2 protein levels were resistant to anti-PD-1 therapy. These findings identify DRG2 as a key regulator of recycling of endosomal PD-L1 and response to anti-PD-1 therapy and provide insights into how to increase the correlation between PD-L1 expression and response to anti-PD-1 therapy.

A digital microfluidic device integrated with electrochemical sensor and 3D matrix for detecting soluble PD-L1

PD1/PD-L1 checkpoint inhibitors are at the forefront of cancer immunotherapies. However, the overall response rate remains only 10–30%. Even among initial responders, drug resistance often occurs, which can lead to prolonged use of a futile therapy in the race with the fatal disease. It would be ideal to closely monitor key indicators of patients’ immune responsiveness, such as circulating PD-L1 levels. Traditional PD-L1 detection methods, such as ELISA, are limited in sensitivity and rely on core lab facilities, preventing their use for the regular monitoring. Electrochemical sensors exist as an attractive candidate for point-of-care tool, yet, streamlining multiple processes in a single platform remains a challenge. To overcome this challenge, this work integrated electrochemical sensor arrays into a digital microfluidic device to combine their distinct merits, so that soluble PD-L1 (sPD-L1) molecules can be rapidly detected in a programmed and automated manner. This new platform featured microscale electrochemical sensor arrays modified with electrically conductive 3D matrix, and can detect as low as 1 pg/mL sPD-L1 with high specificity. The sensors also have desired repeatability and can obtain reproducible results on different days. To demonstrate the functionality of the device to process more complex biofluids, we used the device to detect sPD-L1 molecules secreted by human breast cancer cell line in culture media directly and observed 2X increase in signal compared with control experiment. This novel platform holds promise for the close monitoring of sPD-L1 level in human physiological fluids to evaluate the efficacy of PD-1/PD-L1 immunotherapy.

Statins inhibit paclitaxel-induced PD-L1 expression and increase CD8+ T cytotoxicity for better prognosis in breast cancer.

In recent years, the widespread use of lipid-lowering drugs, especially statins, has attracted people's attention. Statin use may be potentially associated with a reduced risk of breast cancer. To explore the relationship between statin use and cancer risk. And further explore the potential role of statins in the adjuvant treatment of breast cancer. Data for the Mendelian randomization portion of the study were obtained from genome-wide association studies of common cancers in the UK Biobank and FinnGen studies and from the Global Lipid Genetics Consortium's low density lipoprotein (LDL). In addition, the impacts of statins and chemotherapy drugs on breast cancer were examined using both in vitro and in vivo models, with particular attention to the expression levels of the immune checkpoint protein PD-L1 and its potential to suppress tumor growth. Data from about 3.8 million cancer patients and ~1.3 million LDL-measuring individuals were analyzed. Genetically proxied HMGCR inhibition (statins) was associated with breast cancer risk reduction (P=0.0005). In vitro experiments showed that lovastatin significantly inhibited paclitaxel-induced PD-L1 expression and assisted paclitaxel in suppressing tumor cell growth. Furthermore, the combination therapy involving lovastatin and paclitaxel amplified CD8+ T-cell infiltration, bolstering their tumor-killing capacity and enhancing in vivo efficacy. The utilization of statins is correlated with improved prognoses for breast cancer patients and may play a role in facilitating the transition from cold to hot tumors. Combination therapy with lovastatin and paclitaxel enhances CD8+ T-cell activity and leads to better prognostic characteristics.

Effective delivery of anti-PD-L1 siRNA with human heavy chain ferritin (HFn) in acute myeloid leukemia cell lines.

Because of the high biocompatibility, self-assembly capability, and CD71-mediated endocytosis, using human heavy chain ferritin (HFn) as a nanocarrier would greatly increase therapeutic effectiveness and reduce possible adverse events. Anti-PD-L1 siRNA can downregulate the level of PD-L1 on tumor cells, resulting in the activation of effector T cells against leukemia. Therefore, this study aimed to produce the tumor-targeting siPD-L1/HFn nanocarrier. Briefly, the HFn coding sequence was cloned into a pET-28a, and the constructed expression plasmid was subsequently transformed into E. coli BL21. After induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG), HFn was purified with Ni-affinity chromatography and dialyzed against PBS. The protein characteristics were analyzed using SDS-PAGE, Western Blot, and Dynamic light scattering (DLS). The final concentration was assessed using the Bicinchoninic acid (BCA) assay. The encapsulation was performed using the standard pH system. The treatment effects of siPD-L1/HFn were carried out on HL-60 and K-562 cancer cell lines. The RT-PCR was used to determine the mRNA expression of PD-L1. The biocompatibility and excretion of siPD-L1/HFn have also been evaluated. The expression and purity of HFn were well verified through SDS-PAGE, WB, and DLS. RT-PCR analyses also showed significant siRNA-mediated PD-L1 silencing in both HL-60 and K-562 cells. Our study suggested a promising approach for siRNA delivery. This efficient delivery system can pave the way for the co-delivery of siRNAs and multiple chemotherapies to address the emerging needs of cancer combination therapy.

Asiaticoside promoted ferroptosis and suppressed immune escape in gastric cancer cells by downregulating the Wnt/β-catenin pathway

BackgroundOur previous study has revealed that asiaticoside (AC) promotes endoplasmic reticulum stress and antagonizes proliferation and migration of gastric cancer (GC) via miR-635/HMGA1 axis. However, the effect and mechanism of AC on other progressions of GC, such as ferroptosis and immune escape, are still unknown. MethodsAGS and HGC27 cells were incubated with 1, 2 and 4 μM of AC for 24 h. Mice xenografted with AGS cells were intragastrically injected with AC. The effect and mechanism of AC on GC were determined by the measurement of the ferrous iron level, the ROS level and the glutathione peroxidase (GSH) content, flow cytometry, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and western blotting assays. ResultsAC increased the Fe2+ level and the ROS level, but decreased the expression of GPX4 and SLC7A11 and the GSH level. Besides, AC enhanced the percent of CD8+ T cells and the IFN-γ concentration, but reduced the PD-L1 expression and the IL-10 level. Mechanically, AC downregulated the relative levels of β-catenin, active-β-catenin, p-GSK3β/GSK3β, cyclin D1 and c-Myc in GC cells, which were rescued with the application of LiCl (an activator of Wnt/β-catenin pathway) in AGS cells. Moreover, activation of Wnt/β-catenin pathway by LiCl or the β-catenin overexpression inverted the effect of AC on ferroptosis and immune escape in GC cells. In vivo, AC treatment declined the tumor size and weight, the level of GPX4, SLC7A11, PD-L1 and IFN-γ, and the expression of Wnt/β-catenin pathway. ConclusionAC enhanced ferroptosis and repressed immune escape by downregulating the Wnt/β-catenin signaling in GC.

Abstract PO1-25-03: Programmed Death-Ligand 1 and Receptor Tyrosine Kinases in Breast Cancer

Abstract Programmed death-ligand 1 (PD-L1) is an immune checkpoint expressed in a wide range of malignancies leading to immune tolerance and cancer cell immune evasion. Receptor tyrosine kinases (RTKs) are key regulators of cancer cell proliferation, survival, and invasion. The Hepatocyte Growth Factor (HGF) receptor, MET, and the Epidermal Growth Factor Receptor (EGFR) are RTKs known for their tumorigenic potential in a variety of human malignancies. This study aimed to evaluate the effect of the small-molecule tyrosine kinase inhibitors (TKIs) on the expression of PD-L1 in breast cancer cells and the effect of their combination with inflammatory cytokines. The study also assessed the association of the expression of the tumoral PD-L1 mRNA with each of MET and EGFR mRNA expression and the tumor features and treatment outcomes in breast cancer patients using the METABRIC dataset publicly available from cBioPortal for Cancer Genomics. The TKIs crizotinib [a MET inhibitor] and gefitinib [an EGFR inhibitor] were used as a single treatment and in combination with the cytokines; tumor necrosis factor-α (TNF-α) or interferon-γ (INF-γ) in MCF7 and MDA-MB-231 breast cancer cells in vitro. The cells were also treated with the mitogens HGF and EGF. The viability of cells after the different treatments was determined using the MTT viability assay. The effects of the combination treatments were analyzed using combination index (CI) analysis. The expression level of PD-L1 in cancer cells was assessed using Western blotting. The combination treatment of crizotinib with TNF-α and INF-γ produced a synergistic growth inhibition of MCF7 and MDA-MB-231 cells with CI values of 0.31 and 0.55, respectively. Alternatively, combined crizotinib and TNF-α produced an antagonist effect on the viability of MCF7 cells (CI=2.49). The combination of gefitinib with TNF-α produced a synergistic growth inhibition in both MCF7 and MDA-MB-231 cells with CI values of 0.39 and 0.78, respectively. Alternatively, gefitinib resulted in an additive effect when combined with INF-γ (CI=0.99) and an antagonistic effect when combined with TNF-α (CI=2.68) in MCF7 and MDA-MB-231 cells, respectively. Treatment with HGF (50 and 100 ng/ml) increased the protein levels of PD-L1 while EGF (50 and 100 ng/ml) reduced its levels in MDA-MB-231 cells. Treatment with the TKIs crizotinib (0.1-4 µM) and gefitinib (1-40 µM) significantly reduced PD-L1 levels in MDA-MB-231 cells compared to vehicle-treated cells. The analysis of the METABRIC dataset revealed that the mRNA expression of PD-L1 was positively correlated with the mRNA expression of the EGFR gene (r= 0.081, p< 0.001) but not the MET gene. A double-high PD-L1/MET expression was significantly associated with younger age at diagnosis, high-grade carcinoma, greater tumor size, hormone receptor-negative status, HER2-positivity, and non-luminal disease compared to patients with a double-low PD-L1/MET expression. Similar findings were reported for patients with a double-high PD-L1/EGFR expression compared to patients with a double-low PD-L1/EGFR expression. Nevertheless, the mRNA expression of the PD-L1, MET, and EGFR genes as well as the co-expression of PD-L1/MET or PD-L1/EGFR genes did not affect the overall survival of breast cancer patients. Collectively, MET and EGFR TKIs could modulate the effects of inflammatory cytokines differently based on the type of cytokine and the molecular subtype of breast cancer cells. The expression of PD-L1 in breast cancer cells was upregulated by HGF while TKIs reduced its expression. The co-expression of the PD-L1 gene with MET and EGFR genes in breast cancer was associated with advanced disease presentation and worse prognosticators in patients. Together, TKIs that target MET or EGFR could be an appealing therapeutic target in breast cancer, particularly in younger patients with the non-luminal disease. Citation Format: Nehad Ayoub, Muhsen Al-Diabat, Moath Al-Shorman, Laith Al-Eitan. Programmed Death-Ligand 1 and Receptor Tyrosine Kinases in 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 PO1-25-03.

Abstract PO5-06-06: Tumor/stromal expression of CD3/CD8/PD-1/PD-L1 and overall survival (OS) in patients (pts) with metastatic (met) HER2+ breast cancer (BC)

Abstract Background: The “Thousand Patient HER-2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin contains a cohort of met HER2+ BC pts treated with trastuzumab, with OS ranging from short term to durable complete response (never relapsed). While the majority of pts progress on treatment, higher stromal tumor immune infiltrate has been associated with longer OS in met HER2+ BC. Using the SVUH database, we have identified a cohort of met HER2+ BC pts that received trastuzumab and have a broad OS range (0.3 months (mos) to 200.9 mos). This study examines the tumor and stromal levels of pan T cell marker CD3, cytotoxic T cell marker CD8, and immune checkpoints PD-L1 and PD-1 by immunohistochemistry (IHC) in primary and met samples from this cohort. Methods: Clinico-pathological data was available for formalin-fixed, paraffin-embedded biopsy specimens (n=45 primary, n=25 matched met biopsies). IHC staining was conducted for CD3 (Agilent IR50361-2), CD8 (Agilent IR62361-2), PD-L1, (Agilent M365329-2), and PD-1 (Roche 07099029001). A DAKO Link 48 Autostainer was utilised for staining, incorporating positive (tonsil tissue) and negative controls (isotype controls). Slide processing used an Aperio AT2 Digital Slide Scanner (Leica Biosystems), Aperio ImageScope 12.4 software (Leica Biosystems) and QuPath analysis software (University of Edinburgh). Following annotation of tumor areas, an algorithm was trained to identify immune cells and designate them as tumor or stromal. Data was expressed as the number of positively stained cells/mm2 tissue. A median cut-off was applied to denote “high” and “low” expression for CD3 and CD8. For PD-1 and PD-L1, samples with ≥ 1 stained cell/mm2 were designated as positive (pos) and samples with zero stained cells as negative (neg). The Kaplan Meier method was utilised for survival studies. A paired Student’s t test was utilised for primary vs metastatic site comparisons. Results: 51.1% of primary samples displayed high CD3 (23/45) and CD8 (23/45) expression in both the stromal and tumor compartments. Within the tumor compartment, 18.6% (8/43) of samples were pos for PD-L1 expression, and 57.8% (26/45) were pos for PD-1 expression. In the stromal compartment, 30.2% (13/43) of samples were pos for PD-L1 expression, and 68.9% (31/45) were pos for PD-1 expression. High tumor levels of CD3 (median OS CD3 high 113.4 mos vs CD3 low 7.9 mos, HR 0.2938 (95% CI 0.130-0.618), p=0.0012) and CD8 (median OS CD8 high 73.9 mos vs CD8 low 10.8 mos, HR 0.4764 (95% CI 0.238-0.954), p=0.0365), but not stromal levels of these markers, were associated with improved OS. There were no significant differences in CD3, CD8, PD-1 and PD-L1 levels between matched primary and met samples (p >0.05). Tumor, but not stromal, PD-L1 expression was associated with longer OS (HR 0.3120 (95% CI 0.146-0.667), p=0.0027). Interestingly, stromal PD-1 expression, but not tumor PD-1 expression, was also associated with longer OS (median OS PD-1 pos 64.7 mos vs. PD-1 neg 5.6 mos, HR 0.158 (95% CI 0.060-0.411), p=0.0002). Conclusion: Our results report a link between OS and tumor (CD3+, CD8+ and PD-L1+)/stromal (PD-1+) immune cell infiltrate in met HER2+ BC patients treated with trastuzumab. Further expansion of this preliminary dataset is warranted. Citation Format: Denis Collins, Janet McCormack, Laura Ivers, Jose Javier Berenguer-Pina, Jo Ballot, Cecily Quinn, Darko Skrobo, Alex Eustace, Naomi Walsh, Aurelie Fabre, John Crown. Tumor/stromal expression of CD3/CD8/PD-1/PD-L1 and overall survival (OS) in patients (pts) with metastatic (met) HER2+ breast cancer (BC) [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 PO5-06-06.

Abstract PO3-13-05: PD-L1 and PD-L2 Protein Expression is Frequently Discordant in Breast Cancer

Abstract Background: In neoadjuvant setting, PD-1 inhibitors have shown significantly higher pathological responses in patients (pts) with early-stage triple-negative breast cancer (TNBC) irrespective of PD-L1 status. However, durable responses are less common in estrogen receptor-positive (ER+) pts. Most breast cancer (BC) clinical trials of PD-1 inhibitors have thus far focused on PD-L1 expression for patient eligibility. The alternative PD-1 ligand, PD-L2, with reported ~4-fold greater affinity for PD-1, has been largely understudied. We recently reported that high cancer cell protein levels of PD-L2 in pts with treatment-naïve ER+ BC was an independent predictor of shorter progression-free survival. PD-L2 expression was significantly high in ER- group as well, however, given the low numbers of ER- pts, the study was not powered enough to determine the correlation of PD-L2 with PFS in the ER- subgroup. These findings suggest that PD-L2 has an important role in BC and combined PD-L1/PD-L2 status may help improve BC pt selection for PD-1 inhibitors. We therefore initiated efforts to determine baseline expression patterns of PD-L1 and PD-L2 in BC. Methods: PD-L1 and PD-L2 protein levels in cancer cells and tumor-infiltrating immune cells were prospectively analyzed by immunohistochemistry (IHC) using validated antibodies in diagnostic core biopsies of 31 consecutive pts diagnosed with localized or locoregional ER+/HER2- BC or TNBC. Percent positivity of PD-L1 and PD-L2 in cancer cells and immune cells was determined by our breast pathologist. Detectable PD-L1 or PD-L2 expression in ≥1% of cancer cells or stromal immune cells was considered positive. Spearman correlation and Wilcoxon paired analyses were used to measure and correlate PD-L1 and PD-L2 protein expression in cancer and immune cells. Results: There was no significant correlation between PD-L1 and PD-L2 expression, neither across all cases (N=31), nor within ER+ (N=22) or TNBC (N=9) cases. However, PD-L1 and PD-L2 expression patterns in BC differed in several ways. PD-L1-positivity in immune cells was higher than in cancer cells (median=5.0% vs. 0.0%; p=0.001), whereas PD-L2-positivity was higher in cancer cells than in immune cells (median=30% vs. 5.0%; p< 0.001). PD-L1 positivity in cancer cells and immune cells were positively correlated in TNBC (rho=0.69, p=0.04) but not in ER+ BC. Conversely, PD-L2 positivity in cancer cells and immune cells were positively correlated in ER+ BC (rho=0.68, p=0.001) but not in TNBC. TNBC diverged from ER+ BC by displaying higher PD-L1 positivity in immune cells (median=20.0% vs. 1.0%; p=0.004). By the conventional cut point for positivity of ≥1% for PD-L1 and PD-L2 in cancer cells or immune cells, all TNBC tumors were PD-L1-positive (9/9), with 8 also being PD-L2-positive. Of the 22 ER+ cases, 16 were PD-L2-positive, of which only 9 were also PD-L1-positive. Table 1. shows cross tabulation data for PD-L1 and PD-L2 status by BC subtype. Conclusions: PD-L1 and PD-L2 proteins show divergent expression and are not correlated in BC. Discordant PD-L2 and PD-L1 expression may be more common in ER+ BC than in TNBC. This finding justifies efforts to explore PD-L2 as a complementary marker to PD-L1 for improved prediction of response to PD-1 inhibitors, which may benefit patients with aggressive ER+ BC that are eligible for chemotherapy. Table 1. PD-L1 vs. PD-L2 Status by Breast Cancer Subtype Citation Format: Lubna Chaudhary, Julie Jorns, Yunguang Sun, Sailaja Kamaraju, Yee Chung Cheng, Amanda Kong, Tina Yen, Caitlin Patten, Chandler Cortina, Inna Chervoneva, Christopher Chitambar, Hallgeir Rui. PD-L1 and PD-L2 Protein Expression is Frequently Discordant in 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 PO3-13-05.

Abstract PO5-24-01: Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models

Abstract Despite improvement of outcomes for HR+, HER2- metastatic breast cancer by cyclin dependent kinase 4 and 6 (CDK4/6) inhibitors, safety concerns and disease progression raise a critical need to identify novel approaches. Narazaciclib (ON123300), a novel CDK4/6i, designed to enhance efficacy and safety by its multi-targeted kinase inhibitor activity. Narazaciclib is in Ph I trials; NCT04739293 and CXHL1900340; studying different administration regimens and in endometrial cancer in combination with letrozole (NCT05705505). Our study explored the activity of narazaciclib and its metabolite ON1232580 in comparisons to the FDA approved CDK4/6i and identify additional targets engaged by narazaciclib. To identify direct and secondary targets engaged by narazaciclib and palbociclib, proteome wide Cellular Thermal Shift Assay (CETSA) and Integrative Inferred Kinase Activity (INKA) analysis were performed. Both CETSA and INKA analysis identified additional targets engaged by narazaciclib compared to palbociclib in both MDA-MB-231 lysates and intact cells such as BUB1, CHEK1, AURKA, GSK3α and GSK3β. In TNBC patients with BUB1 overexpression, bioinformatics analysis indicates a low survival correlation. Molecular docking data showed a higher affinity of narazaciclib with BUB1 compared to palbociclib and abemaciclib. The differential activity of narazaciclib was explored by using cohorts of human and mouse breast cancer cell lines and cell based assays. Comparison of narazaciclib’s in vitro IC50 profile to other CDK4/6i was studied against a panel of 370 kinases (HotSpot) and Kd values were determined by KINOMEscan. Intracellular IC50 kinase values were determined by NanoBret technology. Narazaciclib and abemaciclib were found to be the most promiscuous in vitro kinase inhibitors and ribociclib the most specific. Abemaciclib and narazaciclib had similar profiles against the CDK family members. Kd values of CDK4/cyclinD1 binding show similar trends; abemaciclib (0.08 nM), narazaciclib (0.18 nM), palbociclib (0.75 nM) and ribociclib (1.3 nM). Although narazaciclib displayed nM IC50 values in the in vitro assays against many CDKs, narazaciclib was very specific in cellular kinase assays with highest activity against CDK4/6, CSF1R and NUAK1. A stronger inhibition of cell proliferation, as well as induction of apoptosis and senescence were detected in narazaciclib and its metabolite ON1232580 treated PYMT cells, a murine mammary carcinoma model which recapitulates luminal BC subtype, compared to the other CDK4/6i. Both narazaciclib and ON1232580 enhanced CCL5 and CXCL10 mRNA levels, while a higher reduction of PD-L1 and pRb protein levels and a promoting effect on the H2D1 and B2M mRNA levels was shown in narazaciclib and its metabolite treated PYMT cells. Narazaciclib showed significant synergy with anti-PD1 in the EMT-6 syngeneic breast cancer model. A differential effect of narazaciclib on the expression levels of 62 cytokines was detected compared to abemaciclib and palbociclib. Lastly, inhibition of autophagy sensitizes cancer cells to both ON123300 and ON1232580, and induces irreversible cell proliferation inhibition, providing a novel therapeutic approach. Our findings identify important differences generated from assay models studying kinase inhibition, binding and pathway engagement. Understanding the role of the differential targets engaged by narazaciclib, the potential enhanced antitumor immunity and the sensitization by autophagy inhibitors to cell death, will guide future clinical studies. Citation Format: Petros Kechagioglou, Debomita Chakraborty, Camille Dupont, Hajime Yurugi, Ute Distler, Stefan Tenzer, Alexey Chernobrovkin, Kristina Riegel, Julianne Mooz, Mahil Lamber, Volker Dötsch, Stephen Cosenza, Steven Fruchtman, Krishnaraj Rajalingam. Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models [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 PO5-24-01.

Abstract PO2-03-07: Immune Landscape in PD-L1-positive and TROP2-positive Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) tends to be more aggressive, and more likely to recur than other subtypes of breast cancer, and patients with metastatic TNBC have a poor prognosis. The recent approvals and data of pembrolizumab (anti-PD-1 inhibitor) in combination with chemotherapy in advanced-metastatic and early high-risk TNBC, and sacituzumab govitecan (an ADC that targets the tumor cell surface antigen TROP2 coupled with the irinotecan metabolite SN-38) in metastatic TNBC, has shed light on some of the immune and cancer signaling associated with TNBC. Although exciting progress has been made in the treatment of TNBC, there are still important gaps to fill to understand the biology of TNBC and identify additional therapies. Materials and Methods: We identified a total of 1500 patients whose tumors were negative for the expression of ER and HER2 receptors by gene array. PD-L1 and TROP2 expression data were available for 802 early TNBC patients, of whom 657 were treated with adjuvant chemotherapy. A comprehensive identification and analysis of immune-related genes (n= 1169) was conducted. Mann-Whitney test comparing the gene expression for all immune related-genes was evaluated in patients expressing PD-L1 and TROP2 at low and high expression levels. Immune gene modules resembling key immune gene signaling were analyzed in all subgroups identified. Fold changes and Mann-Whitney P values were calculated in each subgroup. Results: Patients were stratified based on high and low expression levels of PD-L1 and TROP-2 mRNA. PD-L1 highly expressing (PD-L1+) tumors showed a marked difference in immune-related genes as compared to PD-L1 low-level tumors (PD-L1-) with PD-L1+ tumors demonstrating a significantly higher proportion of correlated immune genes (CXCL9/10/11, IFN-γ, CCL5 and STAT1). TROP2 highly and low expressing tumors (TROP2+ and TROP2-, respectively) showed a relatively limited difference in terms of immune-related gene expression with key immune-related genes having statistical differential expression in the two subgroups. TROP2+ tumors showed a high presence of several Mucin family gene members (MUC1, -5AC, -4). In addition, CLDN 4, VTCN1, and MET were among the key immune genes significantly expressed in TROP2+ tumors (P = 5.73E-20; P = 6.2E-13, P = 2.1E-11). Overall, PD-L1+ tumors and TROP2+ tumors showed an immune suppressive genomic landscape but were potentially regulated by different signaling. Of note, tumors with very high expression of PD-L1 showed an inverse correlation with VTCN1 gene expression. Conclusions: This preliminary analysis confirmed that only a limited number of early TNBC highly expressed PD-L1 and that a different immune suppressive landscape for PD-L1± and TROP2± tumors exists. In addition, our data suggest that early TNBC cases lacking PD-L1 might escape immune surveillance by virtue of the upregulation of key alternate signaling. Ongoing efforts investigating the expression of such markers on stroma/epithelial tumor components will help to clarify their biological, clinical, and potential therapeutic role in TNBC. Citation Format: Balazs Gyorffy, Libero Santarpia. Immune Landscape in PD-L1-positive and TROP2-positive 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 PO2-03-07.

Novel ultrasensitive automated kinetic exclusion assay for measurement of plasma levels of soluble PD-L1, the predictive and prognostic biomarker in cancer patients treated with immune checkpoint inhibitors

Recently, the blood plasma or serum levels of soluble programmed death protein 1 (PD-L1), but not tissue PD-L1 expression level, have been proposed as an effective predictive and prognostic biomarker in patients treated with immune checkpoint inhibitors for different types of cancers. The quantification of soluble PD-L1 in blood will provide a quick evaluation of patients’ immune status; however, the available assays have limitations in their sensitivity, reproducibility, and accuracy for use in clinical settings. To overcome these problems, this study was dedicated to developing an ultrasensitive automated flow-based kinetic exclusion assay (KinExA) for the accurate and precise measurement of soluble PD-L1 in plasma. The assay was developed with the assistance of KinExA™ 3200 biosensor. In this assay, PD-L1 in its calibrator or plasma sample solution was pre-equilibrated with anti-PD-L1 monoclonal antibody. The equilibrated mixture solution was then passed rapidly over PD-L1 protein that has been coated onto polymethylmethacrylate beads consolidated as a microcolumn in the observation cell of the KinExA™ biosensor. The free anti- PD-L1 antibody was bound to the immobilized PD-L1, however, the unbound molecules were removed from the beads microcolumn by flushing the system with phosphate-buffered saline. Fluorescein-labeled secondary antibody was passed rapidly over the beads, and the fluorescence signals were monitored during the flow of the labeled antibody through the beads. The calibration curve was generated by plotting the binding percentages as a function of PD-L1 concentrations in its sample solution. The working range of the assay with very a good correlation coefficient on a 4-parameter equation (r = 0.9992) was 0.5 − 100 pg mL─1. The assay limit of detection and quantitation were 0.15 and 0.5 pg mL─1, respectively. The recovery values of plasma-spiked PD-L1 were in the range of 96.4–104.3 % (±3.7–6.2 %). The precision of the assay was satisfactory; the values of the coefficient of variations did not exceed 6.2 % for both intra- and inter-day precision. The automated analysis by the proposed KinExA facilitates the processing of many specimens in clinical settings. The overall performance of the proposed KinExA is superior to the available assays for plasma levels of soluble PD-L1. The proposed assay is anticipated to have a great value in the measurement of PD-L1 where a more confident result is needed.

B cell-intrinsic IL-27 and IFNγ receptor signaling drive PD-L1 expression, IgG2a autoantibody production, VEGF-dependent tumor angiogenesis and cancer progression

Abstract B effector cells produce Abs and autoantibodies, while B regulatory cells use cytokines and transmembrane proteins to fine tune immune functions. Our lab recently revealed that tumor-infiltrating B cells produce IL-27 to promote immune evasion, partly by upregulating tumoral PD-L1, including that expressed by B cells per se. To investigate the autocrine activity of IL-27 in tumor progression, we generated μMT:Il27ra–/– bone marrow chimera mice lacking the IL-27 receptor specifically in B cells. These mice displayed substantially reduced tumor growth from orthotopically engrafted ER+ E0771 breast cancer cells, as associated with only moderately decreased PD-L1 levels but, unexpectedly, severely compromised blood vessel integrity. The residual PD-L1 levels and tumor growth were virtually abolished by additional B cell-specific deficiency in IFNγ receptor signaling, as occurring in double KO μMT:Il27ra–/–Ifngr1–/– mice, in which the microvessel permeability was also worsened. Mechanistically, this aligned with our findings that IL-27-producing B cells expressed high levels of VEGFA, whose transcription in activated B cells could be further enhanced by IL-27 and IFNγ. Double KO mice also lacked anti-dsDNA IgG2a autoantibodies, as elicited by tumor engraftment. Thus, B cell-intrinsic IL-27 and IFNγ receptor signaling synergize to promote B-cell PD-L1 expression, IgG2a autoantibody production, and vascular integrity, all of which may contribute to cancer development.

Impacts of tumor microenvironment during neoadjuvant chemotherapy in patients with esophageal squamous cell carcinoma.

With the advent of immune checkpoint inhibitors (ICIs), a better understanding of tumor microenvironment (TME) is becoming crucial in managing esophageal squamous cell carcinoma (ESCC) patients. We investigated the survival impact of TME status and changes in patients with ESCC who underwent neoadjuvant chemotherapy (NAC) followed by surgery (n = 264). We examined immunohistochemical status (CD4+, CD8+, CD20+, Foxp3+, HLA class-1+, CD204+, and programmed death ligand-1 [PD-L1+]) on 264 pre-NAC and 204 paired post-NAC specimens. Patients were classified by their pre- and post-NAC immune cell status and their changes following NAC. Our findings showed that pre-NAC TME status was not significantly associated with survival outcomes. In contrast, post-NAC TME status, such as low level of T cells, CD4+ T cells, and high PD-L1 combined positive score (CPS), were significantly associated with poor overall survival (OS). Notably, TME changes through NAC exerted significant survival impacts; patients with consistently low levels of T cells, low levels of CD4+ T cells, or high levels of PD-L1 (CPS) had very poor OS (3-year OS: 35.5%, 40.2%, and 33.3%, respectively). Tumor microenvironment changes of consistently low T cells, low CD4+ T cells, and high PD-L1 were independent predictors of poor OS in multivariate Cox hazards analyses, while factors indicating post-NAC status (T cells, CD4+, and PD-L1 [CPS]) alone were not. Therefore, we suggest that the consistently low T/high PD-L1 group could benefit from additional therapies, such as ICIs, and the importance of stratification by the TME, which has recently been recognized.

Type I IFN regulates neutrophils responses to murine beta-coronavirus infection

Abstract Introduction: Type I IFNs play a critical role in the immune response to viral infections. Suppressed type I IFN levels are predictive of severe SARS-CoV-2 infections. Given the critical role of neutrophils in viral-mediated lung injury, we investigated how type I IFNs regulated neutrophil responses during pulmonary coronaviral infections. Methods: IFNAR-/- and C57BL/6 (WT) mice were infected with 2,500PFU of murine beta-coronavirus (MHV-1) and survival determined. At day 3 post infection, cellular recruitment, cytokine profiles and markers of lung injury were evaluated. Neutrophils were evaluated for activation markers, ROS generation and NET formation. Results: All IFNAR-/- mice succumbed within 5 days versus 100% survival for WT mice. IFNAR-/- mice had higher pulmonary levels of GM-CSF (p=0.0079) and 3-fold more recruited neutrophils (p=0.0159) and monocytes (p=0.016). IFNAR-/- lung neutrophils had significantly lower TLR3, TLR4, TLR7, TLR9, CD88, CXCR2, CD69 (p<0.05 for all) but increased levels of PDL-1. IFNAR-/- neutrophils isolated during infection showed significantly decreased ROS and NETs compared to WT. Conclusion:IFNAR-/- mice are highly susceptible to MHV-1, displaying elevated neutrophil recruitment with lower frequency of activation markers, reduced ROS and NETs, and increased expression of PDL-1. Our findings demonstrate mechanisms by which inadequate type I IFN levels subvert critical aspects of neutrophil responses during severe viral respiratory infections.