NSCLC Tumor Cells Research Articles

EP.06G.01 Prognostic Significance of Purinergic Receptor- and Ectonucleotidase- Expression in NSCLC Tumor Cells and Stroma

EP.06G.01 Prognostic Significance of Purinergic Receptor- and Ectonucleotidase- Expression in NSCLC Tumor Cells and Stroma

Mechanistic study of PD-L1 regulation of metastatic proliferation in non-small cell lung cancer through modulation of IRE1α/XBP-1 signaling pathway in tumor-associated macrophages.

To explore the related research of PD-L1 in IRE1α/XBP-1 signaling pathway on non-small cell lung cancer. The tumor model of mice was established and divided into four groups; after successful modeling, the tumor tissue of mice was removed for subsequent experiments; the bought THP-1 cells were grouped into four different groups, a control group, nivolumab intervention group, IRE1α inhibition group, and nivolumab intervention + IRE1α inhibition group; after co-culture of the four groups of THP-1 cells with A549, THP-1 cell protein levels in the four groups were analyzed using Western blot; A549 cell migration, invasion and proliferation were assessed using the scratch assay, Transwell method, monoclonal experiment and CCK-8 method. In vivo studies indicated that the stimulation of nivolumab could strongly check the progress of NSCLC (non-small cell lung); two groups treated with 4 μ8c showed obvious effects on check point of NSCLC; In vitro experiments including Western-blot experiment, Scratch experiment, Transwell method, Monoclonal experiment and CCK-8 experiment suggest that nivolumab could inhibit migration, invasion and proliferation of NSCLC tumor cells and it. PD-L1 is capable of controlling metastatic and proliferative potential of NSCLC by the way of the modification of IRE1α/XBP-1 signaling in tumor-associated macrophages.

Human NTHL1 expression and subcellular distribution determines cisplatin sensitivity in human lung epithelial and non-small cell lung cancer cells.

Base excision repair is critical for maintaining genomic stability and for preventing malignant transformation. NTHL1 is a bifunctional DNA glycosylase/AP lyase that initiates repair of oxidatively damaged pyrimidines. Our recent work established that transient over-expression of NTHL1 leads to acquisition of several hallmarks of cancer in non-tumorigenic immortalized cells likely through interaction with nucleotide excision repair protein XPG. Here, we investigate how NTHL1 expression levels impact cellular sensitivity to cisplatin in non-tumorigenic immortalized cells and five non-small cell lung carcinomas cell lines. The cell line with lowest expression of NTHL1 (H522) shows the highest resistance to cisplatin indicating that decrease in NTHL1 levels may modulate resistance to crosslinking agents in NSCLC tumors. In a complementation study, overexpression of NTHL1 in H522 cell line sensitized it to cisplatin. Using NTHL1 N-terminal deletion mutants defective in nuclear localization we show that cisplatin treatment can alter NTHL1 subcellular localization possibly leading to altered protein-protein interactions and affecting cisplatin sensitivity. Experiments presented in this study reveal a previously unknown link between NTHL1 expression levels and cisplatin sensitivity of NSCLC tumor cells. These findings provide an opportunity to understand how altered NTHL1 expression levels and subcellular distribution can impact cisplatin sensitivity in NSCLC tumor cells.

LINC00668 promoted non-small lung cancer progression by miR-518c-3p/TRIP4 axis.

Non-small lung cancer ranks first in the cancer-related death of all malignant tumors. Exploring novel biological targets is of great significance for diagnosis and therapy of NSCLC. In this study, we aimed to explore the effect of LINC00668 on the biological functions of NSCLC cells and the underlying mechanism. RT-qPCR assays and western blot assays were utilized to estimate the relative gene expression at mRNA and protein levels, respectively. CCK8, colony formation, wound healing, transwell, and cell apoptosis assays were employed to assess cell function. IHC and FISH assays were used to determine the gene expression in NSCLC tissues. RIP and dual-luciferase assays were conducted to validate the combination between LINC00668 and miR-518c-3p. The correlation of expression between miR-518c-3p and LINC00668 or TRIP4 was determined by Pearson correlation analysis. LINC00668 was aberrantly upregulated in NSCLC tumor tissues and cell lines. Inhibition of LINC00668 significantly suppressed tumor proliferation, migration, invasion and promoted cell apoptosis. Mechanistically, LINC00668 could bind to miR-518c-3p, thus targeting the 3'UTR of TRIP4. TRIP4 overexpression rescued the weakened cell function mediated by LINC00668 silencing. LINC00668 acted as an oncogene in NSCLC progression through miR-518c-3p/TRIP4 axis. Our study disclosed a new mechanism of LINC00668 functioned in NSCLC and may give a deeper insight of the targeted therapy of NSCLC in the future.

TROP2 expression and response to immune checkpoint inhibition in patients with advanced non-small cell lung cancer.

9040 Background: Despite anti-monoclonal antibodies targeting programmed cell death-1 (PD-1) or its ligand PD-L1 revolution, most patients (pts) with advanced NSCLC displayed primary resistance to PD1/PD-L1 blockade. Identifying targetable alterations involved in resistance represent a promising approach to improve immunotherapy efficacy. TROP2 expression is associated with a poor prognosis in NSCLC, and offers a promising target for treatment with the development of new antibody drug conjugate. TROP2 has been suggested to play a role in innate immunity response, but, it's unclear whether TROP2 expression by tumor cells is detrimental specifically in the context of PD1/PD-L1 blockade. The aim of our study was to investigate the the role of TROP2 expression in outcome of NSCLC pts treated with ICI. Methods: We analyzed the largest NSCLC gene expression data set worldwide including the whole-transcriptome profile of 891 pre-treatment tumors from POPLAR and OAK trials, two randomized studies assessing the efficacy of atezolizumab versus docetaxel in NSCLC. We complemented this transcriptional approach with multiplex IF and digital pathology analysis to confirm our findings at the protein level We investigated whether a non-invasive approach focusing on the level of plasma levels of circulating TROP2 may be relevant to predict efficacy of ICI. Results: The PFS of pts with high expression of TROP2 was significantly lower than in pts with low expression 2.5 vs 4.1 months, p < 0.001. Strikingly, high TROP2 expression was also associated with lower clinical benefit rate 15% vs 28%, p = 0.009 and worse overall survival (OS) 12.6 vs 16.3 months, p = 0.007 When adjusting for covariates such as histological subtype, TLS signature, PD-L1 expression, TROP2 remained independently associated with PFS and OS. None of these correlations were observed in the docetaxel arm, suggesting a predictive value of TROP2 gene expression specifically for response to ICI. By using a deconvolution approach, we also observed that TROP2-high tumors were characterized by a significant lowest expression of genes specific to immune populations (T effector memory cells,Th17 cells). Multiplex IF on an independent cohort 53 NSCLC tumors confirmed the correlation between high TROP2, microenvironment features and poor response to PD1 inhibition. Analysis of circulating plasma levels of TROP2 in 97 NSCLC patients revealed a strong correlation with expression in tumor tissue. High plasma levels of circulating TROP2 was also significantly associated with worse ORR, PFS and OS on treatment with PD1/PDL1 antagonist. Conclusions: Our study revealed that TROP2 expression by tumor cells in NSCLC is associated with worse outcome in patients with NSCLC independently of PDL1 expression. Targeting TROP2 in combination with ICI may represent a promising strategy to improve ICI efficacy in pts with TROP2-high NSCLC.

Abstract 811: Development of a NOX-ROS nanoinhibitor for enhancing response to radiotherapy in lung cancer cells while mitigation radiation-induced inflammation and injury in the normal lung tissue

Abstract Objective: One of the unmet clinical challenges is that many tumors remain insensitive to radiotherapy owing to intrinsic resistance or acquired resistance that leads to tumor progression or recurrence. Moreover, radiation-induced acute injury and chronic inflammation in normal tissues limit the radiation dose that can be applied to the tumor, and tolerable doses are often linked to suboptimal tumor control—even accepting side effects that lead to decreased quality of life. Our goal of the study is to develop a nanoformulated radiosensitizer that inhibits NOX-ROS signal pathway to enhance therapeutic response in tumor cells while reducing radiotoxicity in normal tissues. Methods: We developed a biomaterial-based nanoparticle radiosensitizer that acts upon abnormal redox status and altered metabolism in tumor cells to trigger a cascade of changes in signal pathways to enhance response to radiotherapy. This CD44-targeted radiosensitizer consists of a biodegradable and bioactive hyaluronic acid nanoparticle (HANP) encapsulated with a dual NOX1/4 inhibitor, GKT831 (HANP/GKT831). Orthotopic and subcutaneous NSCLC tumor mouse models using a WRJ388 cell line that was isolated from a metastatic lung adenocarcinoma in a lymph node of a KrasG12D/Lkb1null GEMM mouse were established for evaluation of the effect on the inhibition of tumor growth and protection of normal tissue after the treatment with intravenous administrations of HANP/GKT831 at 5 mg/kg (twice per week for five times) combined with radiation (2 Gy/each dose, twice per week for five times). The anti-tumor effect, immune response, and inflammation in the normal lung tissues were investigated using histological and immunofluorescence analyses. Results: Our results showed that HANP/GKT831 had stronger inhibitory effects on ROS generation and cell proliferation than that of GKT831 in mouse NSCLC tumor cells in vitro. Systemic injection of HANP/GKT831 combined with radiation had the strongest inhibition on tumor growth (~79%) compared with the combination of non-nanoformulated GKT831 with radiation treated mouse group (<50%). HANP/GKT831 combined with radiation led to an increased tumor specific CD8+ T cells in tumors and markedly reduced the level of myeloid derived suppressive cells (CD11b+/GR1+) in tumors. Furthermore, the combination of radiation and HANP/GKT831 reduced inflammatory cytokines, such as IFN-γ and TNF-α, and cells (CD11b+ cells) in the irradiated normal lung tissues. Conclusion: HANP/GKT831 is a biomaterial-based nanoparticle radiosensitizer that acts upon abnormal redox status and altered metabolism in tumor cells to trigger a cascade of changes in signal pathways to enhance the response of tumor cells to radiotherapy. It also has the potential to reduce the radiation-induced inflammatory response in normal lung tissues. Citation Format: Lei Zhu, Lumeng Zhang, Tongrui Liu, Wei Ping Qian, Wei Zhou, Lily Yang. Development of a NOX-ROS nanoinhibitor for enhancing response to radiotherapy in lung cancer cells while mitigation radiation-induced inflammation and injury in the normal lung tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 811.

Bone Marrow Mesenchymal Stem Cells (BMSCs)-Originated miR-1298 Impedes the Aggressiveness of Non-Small Cell Lung Cancer by Hindering the Chemokine Receptor 4 (CXCR4)-Induced Epithelial-Mesenchymal Transition (EMT) Process

Exosomes are a subclass of extracellular vesicles, which are produced and secreted by various cells including bone marrow mesenchymal stem cells (BMSCs). BMSCs-originated exosomes can provide a beneficial microenvironment and manipulate tumor growth. However, whether BMSCs-derived miR-1298 exerts roles in NSCLC remains unclear. miR-1298 level was quantified in NSCLC tumor tissues and para-cancerous tissues and NSCLC cell lines. Cells were transfected with miR-136 mimics/miR-136 inhibitors or treatment with BMSCs-originated exosomes to measure cell biological behaviors. Our results found a diminished miR-1298 expression in NSCLC tumor specimens and cell lines. Meanwhile, miR-1298 overexpression or miR-1298 derived from BMSC-originated exosomes can restrain the proliferating feature of NSCLC cells, and impedes cell aggressiveness via hindering EMT process. Additionally, CXCR4 was a target of miR-1298. In conclusion, miR-1298 is served as a tumor suppressor gene in NSCLC and can retard the proliferating and invading behaviors of NSCLC cells by targeting CXCR4 expression, indicating that it might be a novel therapeutic target for treating NSCLC.

TDP1 and TOP1 as targets in anticancer treatment of NSCLC: Activity and protein level in normal and tumor tissue from 150 NSCLC patients correlated to clinical data

ObjectivesTopoisomerase 1 (TOP1) is a drug target used in anticancer treatment of various cancer types. The effect of the TOP1 drugs can be counteracted by the enzymatic activity of tyrosyl-DNA phosphodiesterase 1 (TDP1). Thus, to elucidate the relevance of combining TDP1 and TOP1 as drug targets for anticancer treatment in NSCLC, TDP1 and TOP1 was for the first time quantified in a large cohort of paired normal and tumor tissue from NSCLC patients, and data were correlated between the two enzymes and to clinical data. Materials and methodsTDP1 and TOP1 activity and protein concentration were measured in paired normal and tumor tissue from 150 NSCLC patients using TDP1 and TOP1 specific biosensors and ELISA. TDP1 and TOP1 activity and protein concentration were correlated to clinical data. ResultsTDP1 and TOP1 activity and protein concentration were significantly upregulated from normal to tumor tissue for the individual patients, but did not correlate to any of the clinical data. TDP1 and TOP1 activity were upregulated in 89.3% and 82.7% of the patients, respectively, and correlated in both normal and tumor tissue. The same tendency was observed for protein concentration with an upregulation of TDP1 and TOP1 in 73.0% and 84.4% of the patients, respectively. The activity and protein concentration correlated in normal and tumor tissue for both TDP1 and TOP1. ConclusionThe upregulations of TDP1 and TOP1 from normal to tumor tissue combined with the observation that TDP1 and TOP1 did not correlate to any of the clinical data indicate that both proteins are important for development or maintenance of the tumor cells in NSCLC. Correlations between TDP1 and TOP1 indicate a biological dependency and potential co-regulation of the enzymes. These observations is encouraging in relation to using TOP1 and TDP1 as targets in anticancer treatment of NSCLC.

P69.01 The Role of the Pregnancy Associated Protein Glycodelin and Its Influence on the Immune System in Non-Small-Cell Lung Cancer

Lung cancer is the leading cause of cancer related death worldwide and the challenge of developing effective therapies especially for advanced stages remains. Novel approaches concentrating on immunotherapies showed a favorable response in lung cancer patients, making this an interesting and promising field for future research. Glycodelin is an immunosuppressive glycoprotein in reproduction and pregnancy maintenance. Interestingly, high expression levels were also observed in different cancer types, e.g. in non-small-cell lung cancer. To investigate whether glycodelin has similar immunomodulatory characteristics in NSCLC, 22 lectins were used to compare the glycosylation pattern of glycodelin secreted by NSCLC tumor cells to immunosuppressive glycodelin A isolated from amniotic fluid. Furthermore, tumor cell line supernatant containing high amounts of glycodelin was used to treat different immune cell lines. Binding assays were performed and possible effects of the treatment have been evaluated using microarray gene expression analyses. To investigate its influence on the efficiency of immunotherapy, glycodelin was measured in the serum of inoperable immunotherapy-treated NSCLC patients (n = 139) and progression-free survival analysis was performed. We were able to show that the glycosylation of glycodelin from NSCLC is highly similar to glycodelin A and especially shares functional sialylation which is crucial for immune system regulation. In addition, we could validate that glycodelin binds to immune cells. The evaluation of microarray gene expression assays shall give hints for immune escape mechanisms induced by glycodelin. In patients, high serum concentrations of glycodelin were associated with a decreased progression-free survival (p = 0.048) of patients receiving an anti-PD-1-/ PD-L1 therapy. In conclusion, we demonstrate that glycodelin is a protein with a high potential of being a novel target in immuno-oncology especially for NSCLC patients.

Clinicopathological significance of the expression of PD-L1 in non-small cell lung cancer

IntroductionPD1/PD-L1 pathway targeting therapies are nowadays an established treatment option for patients with NSCLC. We assessed whether PD-L1 expression in NSCLC tumor cells was associated with specific clinical features or overall survival using four different clones. Methods and resultsA retrospective study included formalin-fixed paraffin embedded (FFPE) surgical tumors from 482 patients. PD-L1 status was assessed with immunohistochemistry in tumor cells on tissue microarrays using clones 28-8, 22C3, SP263 and SP142. Associations with OS were assessed by Kaplan-Meier and multivariate Cox's regression analysis.Patients' median age: 68 years (39–86); histology: adenocarcinoma (AdCa) 61%, squamous-cell carcinoma (SqCC) 33%, and large cell carcinoma (LCC) 6%; p-stage: IA (46%), IB (30%), IIA (10%), IIB (11,4%), IIIA (1,2%), IIIB – IV (0,4%). PD-L1 positivity (≥1%) in NSCLC for clones 28-8, 22C3, SP263, SP142 was 41.5%, 34.2%, 42.7%, 10.4%, respectively (Pearson Chi-square p < 0.0001). PD-L1 expression was correlated with histology, tumor size and grading. Statistically significant association between PD-L1 expression and OS in NSCLC and Non-AdCa was observed with clone SP142 (log-rank p = 0.045 and p = 0.05, respectively). Statistically significant association between PD-L1 expression and OS in LCC was observed with clones 22C3 (log-rank p = 0.009) and SP263 (log-rank p = 0.050). ConclusionsOverexpression of the PD-L1 clone SP142 was associated with poor overall survival in NSCLC and Non-AdCa. Clones 22C3 and SP263 were associated with poor prognosis in LCC. PD-L1 status might serve as a prognostic marker in NSCLC.

Eliminating Radiation Resistance of Non-Small Cell Lung Cancer by Dihydroartemisinin Through Abrogating Immunity Escaping and Promoting Radiation Sensitivity by Inhibiting PD-L1 Expression.

Radiation resistance is linked to immune escaping and radiation sensitivity. In this study, we found that the PD-L1 expressions of non-killed tumor cells in NSCLC were enhanced after radiotherapy, and dihydroartemisinin (DHA) could synergistically enhance the antitumor effect of radiotherapy in NSCLC. A total of 48 NSCLC patients with sufficient tumor tissues for further analyses were enrolled. The PD-L1 expressions of NSCLC were evaluated by immunohistochemistry. Cell apoptosis was measured by flow cytometry, and the relationship between the PD-L1 expression and radiation resistance was investigated in patient specimens, xenograft model, and cell lines. First, the results indicate that the PD-L1 expression of NSCLC was positively related with the radiation resistance. Second, we found that DHA could eliminate the radiation resistance and synergistically enhance the antitumor effect of radiotherapy in the NSCLC cells lines and xenograft model. Finally, mechanistically, DHA could inhibit the PD-L1 expression to avoid immune escaping by inhibiting TGF-β, PI3K/Akt, and STAT3 signaling pathways. In addition, DHA could activate TRIM21 and regulate the EMT-related proteins by inhibiting the PD-L1 so as to enhance the radiation sensitivity and eliminate radiation resistance to NSCLC. Collectively, this study established a basis for the rational design of integrated radiotherapy and DHA for the treatment of NSCLC.

48. DEVELOPING TUMOR-HOMING CYTOTOXIC HUMAN INDUCED NEURAL STEM CELLS AS AN ADJUVANT TREATMENT FOR RADIATION THERAPY OF BRAIN METASTASES

INTRODUCTIONNon-small cell lung cancer (NSCLC) is the most common primary cancer to metastasize to the brain. Radiation is first-line for multifocal brain metastases, but recurrence is observed in 40% of patients. An adjuvant treatment to radiation is needed to effectively treat post-radiation tumor. Genetically engineered neural stem cells (NSCs) have the unique ability to seek out tumors and deliver therapeutic payloads that significantly reduce tumor burden. Here we have transdifferentiated human fibroblasts into induced neural stem cells (hiNSC) and explored the efficacy of hiNSCs therapy for NSCLC brain metastases.METHODShiNSCs were infused intracerebroventricularly (ICV) into mice with bilateral intracranial H460 NSCLC tumors. Bioluminescent imaging (BLI) was used to determine hiNSCs persistence while fluorescent analysis of brain sections characterized tumor-homing migration. In vitro co-culture assays and isobologram analysis were used to determine the synergistic effect of the cytotoxic protein TRAIL and radiation therapy on NSCLC tumor cells. To determine efficacy in vivo, H460 cells were implanted in the brains of mice and treated with either hiNSC-TRAIL alone or in combination with 2 Gy radiation. Tumor volumes were then tracked via BLI.RESULTS/CONCLUSIONhiNSCs persisted in the brain >1 week after ICV injection, and hiNSCs were found to co-localize with both bilateral tumor foci. Isobologram analysis showed a combination index of 0.64, suggesting radiation and TRAIL have a synergistic cytotoxic effect on NSCLC tumors. In vivo, radiation and hiNSC-TRAIL therapy reduced tumor volumes 90% compared to control-treated animals, while each therapy alone only reduced tumors 21% and 52%, respectively. While neither monotherapy significantly impacted survival, combination therapy demonstrated a 40% extension in survival, with treated mice surviving a median of 28 days while controls animals only survived 20 days. Together, these results demonstrate the therapeutic potential of hiNSC-TRAIL as an adjuvant to radiation for treatment of NSCLC brain metastases.

Interobserver Reliability of Programmed Cell Death Ligand-1 Scoring Using the VENTANA PD-L1 (SP263) Assay in NSCLC.

The VENTANA PD-L1 (SP263) Assay isapproved for use with anti-programmed cell death-1/programmed cell death ligand-1 (PD-1/PD-L1) therapies in NSCLC and urothelial carcinoma. Here, we investigate interobserver reliability of the SP263 assay, applied to PD-L1 scoring of tumor cells (TCs) in NSCLC. Six practicing European pulmonary pathologists independently scored the proportion of TCs expressing PD-L1 (TC score) from 200 archival, commercially sourced, formalin-fixed paraffin-embedded NSCLC resections stained using the SP263 assay. Agreement in scores was analyzed using the intraclass correlation coefficient and concordance in patient's classification using Fleiss' kappa. Results from 172 samples showed strong pair-wise correlations between pathologists (R2 >0.89) for TC scoring with an intraclass correlation coefficient of 0.96. Overall agreement was greater than 90% for TC of 1% and above, and greater than 94% for TCs of at least 25% and at least 50%. Fleiss' kappa showed substantial agreement for TC of 1% and above, and almost perfect agreement for TCs of at least 25% and at least 50%. Assessment of TC score in NSCLC was highly reproducible using the SP263 assay, building confidence in the accuracy of this assay in selection of patients for anti-PD-1/PD-L1 therapy.

Synergistic anticancer effect of combined use of Trichosanthes kirilowii with cisplatin and pemetrexed enhances apoptosis of H1299 non-small-cell lung cancer cells via modulation of ErbB3

BackgroundLung cancer is one of the most common malignancies worldwide. To treat lung cancer, various anticancer drugs were developed and tested, but they failed because of drug resistance. In the present study, we tested herbal medicines, such as TK and CuD, as anticancer drugs to decrease side effects and resistance. MethodsCell viability was measured by an MTT assay. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by cucurbitacin D was measured by an annexin V-FITC/PI assay. We performed RTK kit analysis. Levels of p-ErbB3, p-STAT3, p-NF-κB, and caspases were measured by western blot analysis. Nuclear staining of ErbB3 was measured by immunocytochemistry. Transcriptional activity of STAT3 and NF-κB was detected by STAT3 and NF-κB luciferase reporter gene assays. ResultsWe found a synergistic effect of TK with CDDP and PXD in primary culture of human NSCLC tumor cells. The combination of CDDP/PXD and TK or CuD inhibited the proliferation of H1299 cells. The combination of CDDP/PXD and TK or CuD induced sub-G1 and G2/M cell cycle arrest in H1299 cells. The combination of CDDP/PXD and TK or CuD induced apoptosis, regulated apoptotic molecules, caused morphological changes and inhibited colony formation in H1299 cells. We found that TK suppresses p-ErbB3 expression and signaling. The combination of CDDP/PXD and TK or CuD inhibited p-AKT, p-Erk, and p-JNK signaling and suppressed Stat3 and NF-κB transcriptional activity in H1299 cells. More importantly, the combination of CDDP/PXD and TK or CuD inhibited p-ErbB3 and downstream molecules in H1299 cells. The combination of CDDP/PXD and TK or CuD inhibited ErbB2/ErbB3 dimerization. Our results clearly demonstrate that the synergistic effect of CDDP/PXD and TK or CuD inhibits cell growth and induces apoptosis by inhibiting ErbB3 signaling. ConclusionThe combination of CDDP/PXD and TK or CuD decreases cell proliferation and induces apoptosis by inhibiting ErbB3 signaling in H1299 lung cancer cells. TK or CuD could be useful as a compound to treat lung cancer. Additionally, targeting ErbB3 may also be useful for treating lung cancer.

P2.09-14 Evaluation on Inter-Assay Consistency and Inter-Reader Precision for PD-L1 Assays: Ventana SP263 and Dako 22C3 in Non-Small Cell Lung Cancer

FDA has approved several anti-PD1/PD-L1 checkpoint inhibitors for the treatment of a variety of cancers. It has been shown the expression level of PD-L1 in NSCLC tumor cells is associated with the response to anti-PD-1/PD-L1 treatment. Currently, five anti-PD1/PD-L1 inhibitors have been approved for either as a companion diagnostic (e.g., pembrolizumab) or as complementary diagnostic (e.g., nivolumab, atezolizumab and durvalumab). The blueprint studies and several others have reported the concordance on different PD-L1 IHC assays, however, few Chinese samples were included for assay consistence study; in addition, it remains unclear how these two assays perform in inter-reader variability of pathologists in China. Surgical specimens from 48 NSCLC patients were selected for PD-L1 IHC by Ventana SP263 and Dako 22C3. Tumor proportion scores (TPS) were interpreted by three pathologists from three centers. Inter-assay consistency were evaluated by Kappa test at different cut-off value (1%, 25%, and 50%) for each pathologist; and inter-reader precision were performed by Kendall’s W test for Ventana SP263 and Dako 22C3, respectively. 1) Inter-assay consistency: When TPS were categorized as <1%, 1% to 24%, ≥25%, the overall agreement percentage (OPA) between two IHC assays was 81.25% observed by Pathologist A (κ=0.710), 89.6% observed by Pathologist B (κ=0.84) and 79.2% observed by Pathologist C (κ=0.684); If the TPS were categorized as <1%, 1% to 50%, ≥50%, the OPA were 81.25%, 93.75% and 85.4% (κ= 0.675, 0.899 and 0.760), respectively. 2) Inter-reader precision: The interpretation results on Ventana SP263 by three pathologists were consistent, when TPS was categorized as <1%, 1% to 49%, ≥50% (p=0.017) or <1%, 1% to 24%, ≥25% (p=0.047) respectively; the consistency was not optimal, when TPS was further categorized with narrower groups as <1%, 1% to 24%, 25% to 49%, ≥50% (p=0.065). The interpretation results of Dako 22C3 were inconsistent among three observers in all categories above, with all p values greater than 0.05. 1) The inter-assay consistency of PD-L1 assays is high between Ventana SP263 and Dako 22C3; 2) The interpretation results of Ventana SP263 is more consistent among different observers.

Exploiting Conditional Vulnerabilities Caused by the Systems Level Effects of Tumor Treating Fields

Tumor Treating Fields (TTFields) delivers low-intensity, intermediate frequency, alternating electric fields non-invasively to a tumor. Optune, a TTFields delivery device, has been approved for glioblastoma and clinical trials are ongoing for other cancers. Other mechanisms of action besides interference with mitosis have been identified and suggest alternative clinical strategies to the use of TTFields. Human NSCLC and pancreatic cell lines were used to examine TTFields effects in vitro while an tumor explant system was used for ex vivo studies. The Inovitro system was used to generate TTFields. Quantification of DNA damage was done by using 53BP1 and γ-H2AX foci. The DNA fiber assay was used to quantify newly synthesized DNA to examine replication fork dynamics. R loop detection was done by dot-blot and immunofluorescence using the S9.6 antibody. Clonogenic cell survival was performed with TTFields combined with specific agents (ionizing radiation (IR), cisplatin, olaparib, IR plus cisplatin, IR plus olaparib). We have developed an ex vivo model for lung tumors that are exposed to to TTFields. TTFields exposure decreased the expression of FANC/BRCA1 pathway genes resulting in the downregulation of DNA DSB repair of IR-induced DSBs. Most importantly, TTFields alone increased the number of γH2AX foci and the incidence of chromatid aberrations as a function of TTFields exposure time. Furthermore, TTFields exposure decreased the length of newly replicated DNA and increase R-loop formation as a function of exposure time, suggesting that TTFields induce replication stress. We hypothesized that by applying TTFields, a systems level conditional vulnerability develops (inhibition of DNA repair, increased replication stress, production of DSBs, reduced mitophagy, and other stresses) rendering cells more susceptible to agents that cause DNA damage or replication stress or interfere with its repair. In agreement with our hypothesis, NSCLC cell susceptibility to radiation increased when cells were exposed to TTFields prior to IR treatment compared to IR treatment followed by TTFields. TTFields also enhanced cisplatin toxicity in a synergistic manner. Furthermore, TTFields exposure of NSCLC and pancreatic tumor cells concomitant with the PARP inhibitor Olaparib followed by radiation resulted in synergistic cell killing compared to radiation or Olaparib alone or in combination. Our initial results of ex vivo tumor explants exposed to TTFields supports our in vitro results in that we see the down-regulation of key DNA repair pathway proteins as was seen in our in vitro studies. Our data identifies systems level perturbations to key molecular and biochemical pathways that render tumor cells vulnerable to a number of different agents. We suggest that TTFields would serve well as neoadjuvant therapy prior to radiation treatment and either prior to or concomitant with chemotherapy agents.

Abstract 3900: The Safety and efficacy profile of a PD-L1 directed, Engineered Toxin Body, as a novel targeted direct-cell kill approach for the treatment of PD-L1 expressing cancers

Abstract Engineered Toxin Bodies (ETBs) are comprised of a deimmunized Shiga-like toxin subunit A (SLTA) genetically fused to an antibody-like targeting domain. The antibody targeting domain allows for specific targeting of cancer cells while the SLTA component promotes self-internalization of ETBs, an activity that allows for the delivery of an enzymatic and permanent ribosomal destruction against targeted cells even in the context of non-or-poorly internalizing receptor targets. Molecular Templates has developed PD-L1-targeting ETBs as an approach to directly target tumor cells and overcome resistance mechanisms against PD-1 and PD-L1 antibodies. The cytotoxicity delivered by PD-L1-specific ETBs is engineered to be independent of a requirement for tumor infiltrating lymphocytes (TILs), high tumor mutational burden, or modulatory effects of the tumor microenvironment. Further, the activity is not dependent on blockade of the PD-1/PD-L1 checkpoint axis. Thus, PD-L1 targeting ETBs represent a distinct class of therapeutics with direct cell-kill mechanism of action and ability for activity in patients who have progressed on current standard of care or checkpoint therapy. In this study, we highlight the efficacy and safety profile of MT-6020, a human and cynomolgus cross-reactive, PD-L1 targeted, ETB. MT-6020 retains potent catalytic activity and mediates enzymatic destruction of ribosomes at comparable levels to wild-type SLTA in a cell free model. In addition, MT-6020 binds to human NSCLC, Melanoma, and TNBC tumor cell lines with nM affinity and mediates cellular cytotoxicity via ribosomal destruction at low nM to sub-nM potency. MT-6020 binds to cell lines expressing non-human primate (NHP)-PD-L1 and elicits cytotoxic responses comparable to those observed on human tumor target cells. MT-6020 demonstrated pharmacodynamic and pharmacokinetic effects and displayed a favorable tolerability profile in a repeat dose NHP study at doses that are above the presumed therapeutically active concentration. Further our lead PD-L1 ETB, MT-6035, is built upon the MT-6020 scaffold and can deliver a viral peptide for cell surface presentation to and targeting by a specific antiviral CTL population (antigen seeding technology (AST)) for a second and complementary mechanism for tumor cell destruction. MT-6020 and MT-6035 represent a novel approach to targeting and destroying tumors expressing PD-L1 that is unlikely to be inhibited by resistance mechanisms to current checkpoint inhibitors, is well tolerated in relevant toxicity models, and has the capacity for activity in indications where standard of care has failed. Molecular Templates is poised to initiate clinical development of the PD-L1 targeted-ETB (AST), MT-6035, in 2H - 2019. Citation Format: Hilario J. Ramos, Asis K. Sarkar, Sara Le Mar, Brigitte Brieschke, Joseph D. Dekker, Veronica R. Partridge, Pablo A. Maceda, Michaela M. Sousares, Garrett L. Robinson, Aimee Iberg, Shaoyou Chu, Jensing Liu, Jack P. Higgins, Erin K. Willert. The Safety and efficacy profile of a PD-L1 directed, Engineered Toxin Body, as a novel targeted direct-cell kill approach for the treatment of PD-L1 expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3900.

Abstract 353: A novel molecule with profound tumor killing activity

Abstract LY3343544: A novel MET antibody drug conjugate that shows profound pre-clinical in vivo anti-tumor activities, irrespective of MET pathway dependence MET is over-expressed in many types of human tumors. Due to the heterogeneity of human tumors, MET antibodies or small molecule inhibitors have benefited only small subsets of patients with tumors driven by signaling through the c-Met pathway. The patient selection strategies to identify those tumors with MET activation dependence are helpful in predicting sensitivity to many of these inhibitors. It was reported previously that Lilly’s MET antibody, emibetuzumab, showed clinical activity in selective NSCLC patients with high MET IHC staining (90% to 100% 3+ positive) when it was combined with erlotinib in Phase II clinical Trials. In searching for a better treatment for patients carrying the MET overexpression tumors regardless other co-existing mutations, we developed LY3343544, a novel antibody drug conjugate (ADC) molecule that consists of emibetuzumab conjugated with the potent microtubule inhibitor MMAE using a unique lysine conjugation approach. Upon binding to MET, LY3343544 is internalized via receptor-mediated endocytosis. LY3343544 maintains the similar binding and internalization activities to the cell surface MET as compared to emibetuzumab in the competitive cell binding assay and the internalization assay. We reported here that LY3343544 showed profound anti-tumor activity in a preclinical mouse models, and overcome intrinsic resistance mechanisms including KRAS, BRAF, PI3K and TP53 mutations. LY3343544 kills tumor cells expressing a wide range of MET levels on the cell surface and is capable of killing a variety of MET-overexpressing tumor cells including pancreatic, cholanglocarcinoma, colorectal, NSCLC, gastric, head and neck tumor cells in vitro. In contrast, LY3343544 does not kill human normal endothelial cells and normal epithelial cells, no activity on human peripheral blood mononuclear cells with or without activation as well as in cell-based assays. Moreover, LY3343544 is more stable in rodent PK studies than typical inter chain Cys VC-MMAE conjugates and showed tumor regressions in colorectal, NSCLC, gastric and pancreatic mouse xenograft models. Furthermore, LY3343544 shows profound tumor regression in &amp;gt;50% of PDAC PDX models (n=40): 20% complete response (CR); 22.5% partial response (PR); and 17.5% stable disease (SD); overall disease control rate (DCR) is 60%. In addition, LY3343544 shows tumor growth inhibition in cholangiocarcinoma PDX model that is resistant to emibetuzumab. In summary, LY3343544 is highly potent in killing a variety of tumor cells in cell-based killing assays. It demonstrated good stability in vivo and profound anti-tumor efficacy in multiple mouse xenograft models and patient-derived xenograft models thus is a promising agent to treat many types of cancers. Citation Format: Ling Liu, Aaron D. Wrobleski, Yin Yin, Wei Zeng, Xianming Chen, David J. Stokell, Sheng-bin Peng, Amita Datta-Mannan, Gregory P. Donoho, Philip W. Iversen, Philip Hipskind, Yiqing Feng. A novel molecule with profound tumor killing activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 353.

The cross talk between the molecular alterations and tumor immunity in the microenvironment in non-small-cell lung carcinoma.

e20043 Background: The previous study indicated that the biomarkers of molecular alterations and tumor immunity in the microenvironment could predict the response of immunotherapy in NSCLC patients. However, the cross talk between the two factors is unclear. This study was designed to investigated the features of tumor immunity in the microenvironment of deferential molecular characterization in NSCLC patients. Methods: Tumor tissue and matched blood samples from 62 patients with NSCLC were collected. The mutation profiles were sequenced by a cancer gene-targeted NGS panel. The PD-L1 expression and immune cells infiltrated in tumor microenvironment were detected by multiplex immunofluorescence staining. Results: The distinct of molecular alterations showed the different tumor microenvironments in terms of immune cell composition and of PD-L1 expression by tumor cells in NSCLC. KRAS-mutated tumors were characterized by higher level of PD-L1 expression and lower NK cells infiltrated in tumor area than KRAS-wild type tumor. TP53-mutated tumors were characterized by higher CD8+T cell and lower NK cells infiltrated in tumor area. In contrast, EGFR-mutated tumors were characterized by lower level of PD-L1 expression and higher NK cells. Moreover, we found that the PD-L1 expression increased in MET gene CNV gain tumors than MET wild type tumors. Conclusions: The features of tumor immunity in the microenvironment changed in deferential molecular characterization in NSCLC. it may help to explain the phenomenon that the efficacy of immunotherapy changed in deferential molecular characterization of NSCLC.

Abstract 5529: PD-L1 and CD8 IHC dual assessment in NSCLC using the Halioseek® assay: A multicentric study

Abstract Background Immune checkpoint inhibitors (ICI) have revolutionized the treatment of NSCLC, but only a small proportion of treated patients exhibit a long term response. Improved selection of the patients that will respond to ICI is a major unmet need. Despite its relatively limited predictive value, the percentage of PD-L1+ tumor cells supports their application. It is proposed that NSCLC patient stratification might be improved by additional biomarkers, in particular, the presence of tumor infiltrating lymphocytes (TILs) which play a crucial role in tumor immune response. Halioseek® PD-L1 staining allows accurate detection of positive tumor cells in NSCLC, is comparable to other IVD assays, and is CE-IVD marked (In Vitro Diagnostic Medical Device compliant with European Directive 98/79). This standardized assay also provides complementary characterization of the tumor micro-environment without requiring additional precious tumor samples. The dual staining of Halioseek® allows simultaneous evaluation of PD-L1+ and CD8+ cells on the same tissue section. The assay also includes a Digital Pathology (DP) analysis module to determine CD8+ cell density and a proximity index between CD8+ and PD-L1+ cells. Material and methods A multi-centric study including 5 laboratories was performed in order to 1) Further demonstrate the inter-laboratory reproducibility of the Halioseek® assay; 2) Compare Halioseek® results to routine PD-L1 laboratory tests; 3) Explore Halioseek® Analyzer DP tools to further characterize NSCLC samples. 10 NSCLC FFPE samples (50 slides) were stained using Halioseek® by 5 independent laboratories and 66 NSCLC FFPE samples were assessed using both Halioseek® and the laboratory's routinely used PD-L1 assay by 4 independent centers. The Halioseek® assay staining was performed according to manufacturer's instructions. Percentage of PD-L1+ tumor cells was estimated by pathologists; DP analysis was performed using Halioseek® Analyzer. Results For the 50 slides stained using Halioseek® and evaluated independently by 5 different laboratories, a concordance of 94% and 90% was observed using the 1% and 50% cut-offs of PD-L1 positive tumor cells, respectively. When analyzed by the same pathologist, concordance increased to 100% and 94%, respectively. For the 66 samples analyzed using both Halioseek® and laboratory PD-L1 routine test, concordances were 92% and 97%. Digital pathology tools allowed further characterization of patient samples in terms of CD8+ cells density and proximity indexes. Conclusion Halioseek® is a new robust assay leveraging the advantages of DP to combine TILs and PD-L1 quantification within the tumor and its microenvironment. Halioseek® gives reproducible and accurate results in independent routine laboratories and its DP solution allows better sample characterization. The predictive performance of Halioseek® for the response to ICI needs to be further investigated. Citation Format: Luciana Batista, Nicolas Brandone, Stéphane Garcia, Yanis Boumber, Essel Dulaimi, Philippe Tanière, Paul Hofman, Marius Ilié, Julien Adam, Jérôme Galon, Catherine Gerbon, Jacques Fieschi. PD-L1 and CD8 IHC dual assessment in NSCLC using the Halioseek® assay: A multicentric study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5529.