Wild-type Tumors Research Articles

Abstract 3720: Utility of oncolytic virus as an immunostimulants and potential for combination therapy with ICIs

Abstract Background: Anti-PD-1 antibodies have been approved for a variety of carcinomas, but their efficacy in gastrointestinal cancers is limited. CD8-positive T cells (CD8) of tumor-infiltrating lymphocytes (TILs) and resident memory T cells (TRMs) with long-term anti-tumor immune function have been reported to be associated with the therapeutic efficacy of anti-PD-1 antibodies. Among our oncolytic adenoviruses (OVs), OBP-301, in which the human telomerase reverse transcriptase (hTERT) promoter element drives the expression of the viral E1A and E1B genes, is currently undergoing clinical trials. In the present study, we investigated the usefulness of OVs as immunostimulants and their potential efficacy in combination with anti-PD-1 antibodies. Methods: The cell lines used were the mouse colon cancer cell line CT26 and the mouse pancreatic cancer cell line PAN02. In vitro, the cytotoxic activity of the OVs and extracellular ATP in the culture supernatant were determined. OBP-502, an OBP-301 variant with the gene cassette of the RGD peptide, and anti-PD-1 antibody were used to examine the antitumor effect on subcutaneous tumors. OBP-702, another OBP-301 variant with the gene cassette of the wild-type p53 tumor suppressor gene, was used to examine the effect of long-term anti-tumor immunity. The effects on memory T-precursor cells (MPEC), CD8 and TRM were immunologically assessed in a rechallenge study. Results: OBP-502 and OBP-702 showed cytotoxic activity against CT26 and PAN02 cells in vitro, and significantly increased ATP in the culture supernatant. In vivo, the anti-PD-1 antibody/OBP-502 combination therapy significantly increased intratumor CD8 in CT26 and PAN02 subcutaneous tumors compared to each monotherapy. Complete response was achieved in 4 out of 12 mice by the combination therapy against CT26 tumors. When CT26 cells was re-seeded into tumor-free mice cured by the combination therapy, 2 out of 4 mice remained tumor-free. In the CT26 and PAN02 subcutaneous tumor model, OBP-702 increased CD44-positive CD62L-negative MPECs that differentiated into TRMs in the spleen, and this effect was blocked by ATP receptor inhibition. In CT26 and PAN02 bilateral subcutaneous tumor models, OBP-702 had a significant antitumor effect on both OBP-702-treated and -non-treated sides, and TIL analysis showed a significant increase in CD8 and TRM. In a rechallenge study, OBP-702-pretreatment showed significant growth inhibition of re-inoculated tumors. Conclusion: The OVs showed potent and long-term immune activation by inducing CD8 and TRM in tumors as well as direct anti-tumor effects, and synergistic effects with ICI. Citation Format: Daisuke Kadowaki, Shinji Kuroda, Masashi Hashimoto, Nobuhiko Kanaya, Yoshihiko Kakiuchi, Satoru Kikuchi, Kazuhiro Noma, Hiroshi Tazawa, Shunsuke Kagawa, Yasuo Urata, Toshiyoshi Fujiwara. Utility of oncolytic virus as an immunostimulants and potential for combination therapy with ICIs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3720.

Abstract 3815: Clinical efficacy and safety of HER2 targeted therapy in patients with metastatic colorectal cancer: A systematic review and meta-analysis

Abstract Background: Therapies targeting HER2 have shown clear benefit in patients with breast or gastric cancer,; and their use in metastatic colorectal cancer (mCRC) has been increasingly explored in recent years. HER2 overexpressed or amplified (HER2-positive) mCRC is seen in 2-8% of all mCRC cases and is enriched in RAS wild-type (WT) tumors, representing a patient population that may benefit from anti-HER2 therapies. Although, several single-arm and some non-comparative randomized clinical trials have now tested the use of diverse dual anti-HER2 therapies in mCRC, no summative analysis have been performed. We conducted a systematic review and meta-analysis to evaluate the overall clinical benefit (efficacy and safety) of dual-HER2 inhibition in patients with RAS-WT HER2-positive mCRC. Methods: A systematic review of available literature was performed for clinical trials involving patients with mCRC treated with HER2 targeting therapy. Two independent reviewers selected studies and extracted data from PubMed and abstracts published at major international oncology meetings during the past 5 years. Case reports and other studies not meeting inclusion criteria were excluded, therefore a total of 13 studies were used for analysis. Patients with RAS-mutant tumors within each trial were excluded due to known limited benefit of dual HER2 inhibition in this subset. Inverse variance method with random-effects model was used for meta-analysis to determine the overall effect of the therapy on overall response rate (ORR), disease control rate (DCR), progression free survival (PFS) and treatment-related adverse events (TRAE). Results: Systematic review identified 9 study cohorts which were utilized in the meta-analysis, encompassing a total of 279 patients with mCRC who received Trastuzumab (T) in combination with either Pertuzumab (P), Tucatinib (Tu), Pyrotinib (Py), or Lapatinib (L). Collectively, dual HER2 inhibition showed an ORR of 35.1% (95% CI: 29.4-41.0%), a DCR of 68.3% (95% CI: 62.5-73.9%), and a PFS of 5.2 months (95% CI: 4.4-6.0 months). Pooled TRAE of grade ≥ 3 across studies was 29.9% (95% CI: 18.8-42.4%) but displayed high inter-study heterogeneity (p<0.0001, I2 = 79%), mainly driven by one trial (Pyro-HER2; TRAE = 75.0%, 95% CI: 50.9-91.3%). Conclusions: Overall, this comprehensive meta-analysis confirms that dual HER2 targeting therapies yield meaningful clinical efficacy, with favorable safety profiles, and improve outcomes in patients with RAS-WT HER2-positive mCRC. Citation Format: Oscar E. Villarreal, Lianchun Xiao, Joelle Allam, Alexey Sorokin, Ying Yuan, Scott Kopetz, Kanwal Pratap Raghav. Clinical efficacy and safety of HER2 targeted therapy in patients with metastatic colorectal cancer: A systematic review and meta-analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3815.

Abstract 1216: Pan-RAS inhibition by a tumor-targeted biotherapeutic

Abstract Oncogenic RAS signaling drives tumor growth in about 30% of human malignancies. In recent years the first mutant-specific inhibitors of KRAS G12C (sotorasib and adagrasib) entered the clinic. While some durable responses were noted in KRAS-mutant non-small cell lung cancer, the clinical experience of KRAS G12C inhibitors is marked by rapid drug resistance, either intrinsic or acquired, causing re-activation of the MAPK pathway. Common resistance mechanisms include compensatory signaling through the related RAS isoforms NRAS and HRAS, as well as the selection of non-G12C KRAS mutations, among others. The bacterial peptidase RRSP potently cleaves all RAS mutants and isoforms in the conserved Switch I region. We have shown that receptor-mediated intracellular delivery of RRSP using an engineered protein delivery system (RASx) can inactivate all RAS signaling in a cell, causing apoptosis in RAS-addicted tumor cells in vitro and in vivo. Since systemic pan-RAS inhibition is not likely to be tolerated therapeutically, precise tumor targeting is a necessary feature of a true pan-RAS inhibitor. Here, we describe the unique ability of the RASx system to specify the cell surface receptor used by RRSP to enter cells, allowing exquisite targeting and killing of KRAS, NRAS and HRAS-driven tumor cells while sparing healthy tissues. We screened a panel of patient-derived tumor organoids (PDOs), to demonstrate broad tumor cell killing across pancreatic and colorectal cancer-derived PDOs. 97% of KRAS mutant organoids were sensitive to RASx, independent of the specific mutation. Intriguingly, 46% of wildtype RAS PDOs were sensitive to RASx, including organoids with activating mutations in EGFR. In contrast, PDOs with mutations downstream of RAS such as BRAF V600E were resistant to RASx. Indeed, RASx is effective not only against RAS-addicted tumors, but also wildtype RAS tumors driven by upstream receptor tyrosine kinases, potentially expanding the clinical utility of this platform. RASx is a first-in-class, tumor-targeted pan-RAS inhibitor that represents a unique entry into the growing armamentarium against oncogenic RAS. Citation Format: Greg L. Beilhartz, Huazhu Liang, Molly Udaskin, Christine Ng, Nikolina Radulovich, Roman A. Melnyk. Pan-RAS inhibition by a tumor-targeted biotherapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1216.

Abstract 1757: Pan-cancer analysis identifies signatures of HRD-positivity in germline BRCA1/2 mutated non-BRCA associated cancers

Abstract Introduction: BRCA-associated cancers (breast, ovarian, pancreas, and prostate) with germline pathogenic mutations in BRCA1/2 (gBRCA1/2) are associated with homologous recombination deficiency (HRD) and benefit from platinum/PARPi. However, the role of gBRCA1/2 in non-BRCA assoc. cancers is poorly understood. HRD tumors present with ‘genomic scars’ that can be quantified in tumor profiling (“HRD-sum” score). We reasoned that the HRD-sum score together with germline mutation status (including loss of wild-type) is a strong correlate of HRD-positivity. Leveraging 42,448 prospectively sequenced patients across 63 cancer types, we sought to characterize the prevalence of the HRD-positivity in non-BRCA-assoc. cancers. Methods: Allele-specific copy number data was inferred using FACETS. HRD-sum was calculated as a sum of the numbers of telomeric imbalances, large-scale transitions and genome-wide losses of heterozygosity. Calculating each score was iteratively optimized to improve detection of the scars using MSK-IMPACT targeted sequencing data using patient-matched WES data from 1841 tumors as reference. Germline mutations were identified by a clinically validated pipeline and annotated for pathogenicity using ClinVar. After excluding low quality tumors, 27,375 patients were retained for analysis. “HRD-positive” tumors are those with gBRCA1/2 mutations, loss of wild type, and an HRD-sum score >=42. Results: Expectedly, gBRCA1/2 mutations were more frequent in BRCA-assoc. cancers (7.2%, 558 of 7,737 patients) compared to non-BRCA-assoc. cancers (2.6%, 500 of 19,638) (p=5e-60). Among non-BRCA-assoc. cancers, gBRCA1/2 were most common in esophagogastric (3.9%), lung (3%), hepatobiliary (2.9%), and bladder (2.8%) cancers. Although biallelic loss in gBRCA1/2 patients was sig. higher in BRCA-assoc. cancers (80% vs. 37% in non-BRCA-assoc.), several cancer types showed high rates of loss of wild-type including gBRCA1 tumors in hepatobiliary (5/5) and endometrial (10/11) cancers. HRD-sum scores in all gBRCA1/2 biallelic tumors were sig. higher (median 58) compared to monoallelic (26) or wild-type (25) tumors. Notably, the non-BRCA assoc. cancers with biallelic gBRCA1/2 harbored higher HRD-sum scores compared to monoallelic (median 51 vs. 22, p<0.0001). These include cancer types such as endometrial (64 vs. 37, p= 0.0014) and esophagogastric (62 vs. 45, p=0.0017). In all, 68% of gBRCA1/2 patients in BRCA-assoc. cancers showed HRD-positivity. Interestingly, 18% (91/500) of gBRCA1/2 patients in non-BRCA-assoc. cancers also showed HRD-positivity. These included 52% of esophagogastric, 28% of lung, and 22% of bladder cancer patients with gBRCA1/2. Conclusion: Using a robust measure to detect HRD scars from MSK-IMPACT panel data, we demonstrate evidence for BRCA-mediated tumorigenesis in an expanded spectrum of cancers with potential therapeutic relevance. Citation Format: Daniel M. Muldoon, Miika Mehine, Samuel Tischfield, A. Rose Brannon, Marc Ladanyi, Zsofia K. Stadler, David B. Solit, Alexander Drilon, Michael F. Berger, Yonina Murciano-Goroff, Chaitanya Bandlamudi. Pan-cancer analysis identifies signatures of HRD-positivity in germline BRCA1/2 mutated non-BRCA associated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1757.

Abstract 1934: The PLK1 inhibitor, onvansertib, is active as monotherapy and in combination with cetuximab in RAS wild-type colorectal cancer patient-derived xenografts

Abstract Background: Cetuximab and panitumumab are monoclonal antibodies targeting the epidermal growth factor receptor (EGFR), that provide clinical benefit to metastatic colorectal cancer (mCRC) patients with RAS wild-type (RASWT) tumors. Unfortunately, intrinsic or acquired resistance to these therapies limits their clinical effectiveness, necessitating the development of more efficient therapeutic strategies. Onvansertib is an oral, small molecule, selective inhibitor of the PLK1 kinase, currently in clinical development for KRAS-mutant mCRC. The aim of this study was to assess the anti-tumor activity of onvansertib monotherapy and in combination with cetuximab in RASWT CRC patient-derived xenograft (PDX) models. Methods: Twenty RASWT CRC PDXs were selected and engrafted in nude mice. Once tumors reached 200-350-mm3, mice were treated with vehicle, onvansertib (60mg/kg, QD), cetuximab (20mg/kg, BIW) or the combination for 18 days. PDX models were chosen based on their sensitivity to cetuximab, resulting in a selection of 7 cetuximab-sensitive (CetuxS) and 13 cetuximab-resistant (CetuxR) PDXs, including 7 with intrinsic resistance and 6 with acquired-resistance. Tumor volume change from baseline (TVC) was calculated as 100%x(Vt-Vo)/Vo and tumor growth inhibition (TGI) as 100%x(TVCcontrol-TVCtreated)/TVCcontrol. Tumor regression was defined as TVCD18<0 and tumor stasis 0≤TVCD18<100. Results: Cetuximab sensitivity was confirmed in the selected models, cetuximab induced tumor regression in all the CetuxS PDXs, while no or limited activity was observed in the resistant models (median TGI=29%, IQR 16-61). Onvansertib exhibited potent anti-tumor activity in 17 (85%) PDXs, resulting in tumor regression (n=11) or tumor stasis (n=6). Onvansertib TGI at Day 18 was not significantly different in CetuxS PDXs (median TGI 102, IQR 76-103) compared to CetuxR PDXs (median TGI 108, IQR 74-124), supporting that onvansertib anti-tumor activity is independent of the sensitivity/resistance to cetuximab. The combination of onvansertib and cetuximab induced tumor regression in 18 (90%) PDXs. The combination showed significantly improved efficacy compared to individual therapies in some of the models. Conclusions: Onvansertib monotherapy displayed potent anti-tumor activity in RASWT CRC PDX models, independently of their sensitivity to cetuximab. Additionally, onvansertib combined with cetuximab exhibited either comparable or superior anti-tumor activity than the monotherapies. Collectively, this data supports the clinical development of the PLK1 inhibitor onvansertib for RASWT mCRC. Citation Format: Maya Ridinger, Preeti Kanikarla Marie, Fengqin Gao, Zhensheng Liu, Giulia Maddalena, David Menter, Alexey Sorokin, Tod Smeal, Scott Kopetz. The PLK1 inhibitor, onvansertib, is active as monotherapy and in combination with cetuximab in RAS wild-type colorectal cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1934.

Abstract 4913: Morphological feature discrepancies in wild-type vs. BRCA1/BRCA2 mutated high-grade serous ovarian cancer

Abstract Introduction: The understanding of morphological heterogeneity at the cellular level in High-Grade Serous Ovarian Cancer (HGSOC), particularly in relation to BRCA1 and BRCA2 mutations, is still an ongoing area of research. This study aims to elucidate the morphological features that distinguish wild-type (WT) tumors from those with BRCA1/BRCA2 mutations using deep learning models. Methods: A total of 300 H&E stained HGSOC slides were collected in a patient-wise manner and tumor areas were annotated by an expert pathologist. We used a convolutional neural network model with spatial attention pre-trained on pathological images to extract features from slides, and the features were clustered into 96 groups. Subsequently, a nuclei segmentation model was employed for precise cell segmentation, followed by measurement of cellular features including cell shape (area, eccentricity) and cell texture (dissimilarity, correlation). The assessment of cell texture evaluates the variation and uniformity in gray-level intensity across the cell. We performed independent t-tests on samples of 100 patches from each cluster, comparing cells of selected patches from the top-N clusters representing the highest proportion of each label. Results: H&E slides (n=300) include 69 (23%) BRCA1 mutants and 50 (16.7%) BRCA2 mutants. In Table1, negative t-statistics suggest that WT carcinoma cancer cells exhibit lower values for the measured features compared to BRCA mutants, whereas positive values denote higher values in WT. BRCA1 and BRCA2 mutant carcinoma cancer cells showed significantly larger areas and higher eccentricity than WT carcinoma cancer cells in all top-N clusters settings (N=3, 5, 10, 30). Furthermore, WT carcinoma cancer cells showed higher dissimilarity and reduced correlation compared to BRCA1 mutant carcinoma cells in four settings. Conclusions: Our observations in BRCA-mutated HGSOC cells enrich our understanding of tumor cell morphology with BRCA mutations. Table 1. Comparison of cell features between WT and BRCA mutants cell feature comparison top-3 clusters (t-statistics, p-value) top-5 clusters (t-statistics, p-value) top-10 clusters (t-statistics, p-value) top-30 clusters (t-statistics, p-value) area WT vs BRCA1 -37.41, <0.001 -50.2, <0.001 -37.53, <0.001 -85.95, <0.001 WT vs BRCA2 -42.14, <0.001 -50.61, <0.001 -16.57, <0.001 -51.98, <0.001 eccentricity WT vs BRCA1 -24.40, <0.001 -34.14, <0.001 -38.02, <0.001 -37.40, <0.001 WT vs BRCA2 -26.77, <0.001 -30.46, <0.001 -29.47, <0.001 -47.64, <0.001 dissimilarity WT vs BRCA1 55.17, <0.001 52.73, <0.001 103.57, <0.001 166.78, <0.001 WT vs BRCA2 -38.15, <0.001 -24.75, <0.001 0.2, <0.001 107.73, <0.001 correlation WT vs BRCA1 1.94, <0.001 -2.46, <0.001 -50.24, <0.001 -65.96, <0.001 WT vs BRCA2 87.62, <0.001 90.83, <0.001 52.6, <0.001 -48.90, <0.001 Citation Format: JaeHeon Lee, Hyunil Kim, Yongeun Lee, Yoon-La Choi, Kyungsoo Jung, Tae-Yeong Kwak, Sun Woo Kim, Hyeyoon Chang. Morphological feature discrepancies in wild-type vs. BRCA1/BRCA2 mutated high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4913.

Abstract 6832: Gender specific role of LMTK3 in neuronal-tumor microenvironment crosstalk in CRC

Abstract Background: Lemur tail kinase 3 (LMTK3) is a serine/threonine protein kinase with roles in multiple cellular pathways, including Wnt signaling, KIT modulation, and the estrogen receptor pathway; identifying it as a potential target in various cancers. LMTK3 is highly expressed within the central nervous system (CNS) and has been demonstrated to influence neuronal signaling. This study aimed to investigate the potential connection between LMTK3 and neuronal signaling pathways in colorectal cancer (CRC). Methods: Using the Cre/loxP system, we generated a C57BL/6 mouse strain that lacks the expression of LMTK3. 1 × 106 MC38 cells were subcutaneously implanted into 8 weeks old male and female wildtype (WT) and knockout (KO) mice. Tumor growth was measured until endpoint was reached. Tumors were then processed for RNA sequencing. Differentially expressed genes (DEGs) for each comparison group: WT Female vs WT Male, WT Female vs KO Female, WT Male vs KO Male, and KO Female vs KO Male were analyzed. Ingenuity pathway analysis (IPA) was done for identification of upstream regulators involved and affected cellular pathways. Expression levels of DEGs for glutamate receptor subunits were validated using qRT-PCR. Results: Both male and female LMTK3 KO mice showed highly significant (p<0.01) reduced tumor size when compared to WT mice of the same sex. Additionally, the KO female tumors were significantly (p<0.05) smaller when compared to the KO males. RNA sequencing revealed 761 DEGs in KO and WT female and 133 DEGs in KO female and male cohort comparisons (q<0.05). IPA results showed significant (p<0.01, Z-score←2) alterations in pathways including glutaminergic receptor signaling, neurovascular coupling, CREB signaling in neurons, and synaptic long-term depression in the WT vs KO female tumor cohort comparison. Within these pathways DEGs: CACNA1E, CACNA2DA, CACNG2, GRIA1, GRIA2, GRIN1, and GRIN2B, were downregulated in KO females. Upstream regulator analysis identified transcription regulators such as OTX2 and NEUROD1 as well as beta-estradiol to be significantly inhibited (p<0.01, Z-score←2) in KO females. qRT-PCR analysis showed significant (p<0.05) decreased expression in GRIA1, GRIN1, and GRIN2B as well as GRIN2A and GRIN2C in KO females when compared to WT females or KO males, validating RNA seq results. Conclusions: These results showed the involvement of LMTK3 in the gut-brain axis and its effect on CRC tumor growth. Downregulation of multiple neuronal pathways and genes suggest that LMTK3 may influence neuron signaling and neurotransmitter release to produce stimulatory or inhibitory effects within the tumor environment. The concurrent inhibition of beta-estradiol in LMTK3 KO tumors allude to possible sex differences the kinase’s effect on the observed tumor growth. Further studies are needed to confirm the mechanism of interaction of LMTK3 within these pathways. Citation Format: Lesly Torres-Gonzalez, Shivani Soni, Yan Yang, Goar Smbatyan, Jae Ho Lo, Pooja Mittal, Francesca Battaglin, Indrakant K. Singh, Priya Jayachandran, Sandra Algaze, Alexandra Wong, Karam Ashouri, Wu Zhang, Georgios Giamas, Joshua Millstein, Heinz-Josef Lenz. Gender specific role of LMTK3 in neuronal-tumor microenvironment crosstalk in CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6832.

Abstract 5170: Novel dSTRIDE-HR assays can accurately assess DNA repair defects in human cancers and distinguish HR status of tumors

Abstract Accurate patient stratification is crucial for the successful application of targeted anti-cancer therapeutic approaches. It has been shown that PARP inhibitors are synthetic lethal with defective homologous recombination (HR) repair, with genes such as BRCA1 and BRCA2 identified as conferring therapy resistance or sensitivity, depending on their mutational status. Moreover, there is evidence of patient benefit of carboplatin-based therapy in HR deficient (HRD) tumors. With a growing emphasis on HRD in cancer treatment, efforts have been made to define the HRD phenotype. Nonetheless, the current approaches employed in the clinic, such as Next Generation Sequencing (NGS), HRD score or RAD51 assays have significant limitations and their interpretation can be unclear. To answer the need for a functional HR-specific biomarker, we have recently developed and introduced two new assays, called collectively dSTRIDE-HR. The assays are based on the STRIDE platform technology that enables direct and sensitive detection of single- or double-strand DNA breaks in situ in fixed cells. Novel dSTRIDE-HR explicitly detect double-strand DNA breaks localized in close proximity to RPA (dSTRIDE-RPA) or RAD51 (dSTRIDE-RAD51) proteins. The assays have been shown to be reliable reporters of HR activation in cellular models after exogenous DNA damage induction, yet their potential to predict HR status in an in vivo setting has not been tested so far. To assess the suitability of the dSTRIDE-HR assays for determining the HR status of patients’ tumor cells, we applied these assays in FFPE sections from biopsies derived from breast cancer patients with a different mutational status of BRCA1 and BRCA2 genes. The assays showed low dSTRIDE-RPA and dSTRIDE-RAD51 readouts in BRCA1/2 mutated tumors in comparison to wild type tumors, as well as a great concordance of readouts within a given genetic background. Additionally, the cell cycle analysis proved nearly twofold increase in foci numbers of dSTRIDE-RPA and dSTRIDE-RAD51 in G2/S phase. Our study unveils a potential of dSTRIDE-HR assays to become predictive biomarkers reporting on the status of HR repair in breast tumors, although more patient samples need to be tested to confirm initial findings. Citation Format: Monika Jarosz, Zsombor Prucsi, Franek Sierpowski, Karolina Stepien, Sophie Ostrowska-Paton, Szymon Koman, Nadeem Riaz, Simon Powell, Fresia Pareja, Edaise da Silva, Magdalena Kordon-Kiszala, Kamil Solarczyk. Novel dSTRIDE-HR assays can accurately assess DNA repair defects in human cancers and distinguish HR status of tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5170.

Abstract 4295: Apoptotic DNase DFFB-induced DNA damage within cancer persister cells underlies acquired resistance to targeted therapies across multiple tumor types

Abstract Cancer acquired drug resistance contributes to a large proportion of patient deaths. A major obstacle to the development of effective therapies which prevent resistance is our lack of understanding of the relevant fundamental molecular adaptive processes. Here we report the discovery that tumor cells rely upon sublethal engagement of apoptotic death machinery and apoptotic DNase DFFB to acquire resistance to targeted therapies in multiple tumor types in cell culture and in vivo. We found that while DFFB wild type tumor cells readily acquire resistance to targeted therapies by escaping from the quiescent “persister” state into proliferating resistant cells, persister cells lacking functional DFFB remain indefinitely in a quiescent state on treatment. Mechanistically, we found that DFFB, which is activated by apoptotic caspases in surviving cancer persister cells, induces persistent DNA damage which promotes resistance through acquired mutations and nongenetic resistance through suppression of growth-arresting interferon signaling. During drug treatment acute interferon induction arises from sublethal mitochondrial outer membrane permeabilization and cytoplasmic exposure of mitochondrial nucleic acids which activate pattern recognition receptors. However, during longer treatments we found that DFFB knockout persister cells exhibit strikingly elevated levels of STAT1 and STAT2 and interferon stimulated gene sets. This interferon signaling is growth arresting because treatment of DFFB knockout persister cells with a JAK1/2 inhibitor rescues their ability to regrow. Furthermore, in DFFB wild type persister cells we found that DFFB-dependent DNA damage activates stress response transcription factor ATF3 which blocks the transcriptional activation of interferon stimulated genes following initial interferon induction. This is consistent with prior data in other contexts demonstrating that ATF3 is induced by DNA damage and that ATF3 directly binds to promoters of multiple interferon genes repressing their expression. These findings reveal that DFFB activation in persister cells prevents chronic interferon signaling and enables escape into proliferating drug resistant tumor cells. Therefore, DFFB-mediated interferon suppression represents a novel mechanism that is fundamental to acquired drug resistance. In addition, high expression of DFFB and ATF3 correlates with worse overall survival in a pan-cancer analysis and DFFB knockout mice develop normally indicating DFFB is a nonessential gene in normal tissue. Therefore, we propose that DFFB is a promising therapeutic target to prevent acquired resistance to targeted therapy. Citation Format: August Finley Williams, David A. Gervasio, Claire E. Turkal, Anna E. Stuhlfire, Michael X. Wang, Brandon E. Mauch, Ariel H. Nguyen, Michelle H. Paw, Mehrshad Hairani, Cooper P. Lathrop, Sophie H. Harris, Jennifer L. Page, Matthew J. Hangauer. Apoptotic DNase DFFB-induced DNA damage within cancer persister cells underlies acquired resistance to targeted therapies across multiple tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4295.

Abstract 7548: Role of bromodomain extra terminal inhibitor (BETi) in combination with radiotherapy for the treatment of NF1-associated malignant peripheral nerve sheath tumor

Abstract Introduction: Neurofibromatosis Type 1 (NF-1) associated malignant peripheral nerve sheath tumors (MPNST) harbor mutations in the polycomb repressive complex 2 (PRC2) in 70% of tumors. PRC2 loss results in epigenetic changes, which increase histone acetylation and make the DNA vulnerable to binding by bromodomain proteins. This interaction could be targeted to uncouple histone acetylation from transcription using bromodomain extra terminal inhibitors (BETi). As radiation therapy (RT) is a cornerstone of MPNST treatment, we analyzed the effects of BETi with and without RT. Methods: Mice were implanted with patient-derived xenografts (PDX) of PRC2-wildtype model developed in the Torres laboratory, MPNST007. Mice were segregated into vehicle and I-BET 762 treatment groups and treated with either vehicle or drug for one week before the RT was initiated. Half of the mice in each treatment group (n = 10) received radiation (2 Gy per fraction for 5 days for a total of 10 Gy). The study endpoints were (1) tumor growth rates, (2) tumor size and weight at study termination, (3) immunohistochemical (IHC) staining for biomarkers of proliferation (Ki67), apoptosis (cleaved caspase 3, CC3), and DNA damage (γH2AX), and (4) RNA sequencing of PDXs from each experimental arm. Results: Significant tumor regression was observed in PRC2-mutant PDX tumors treated with I-BET 762 based on tumor growth kinetics over 45 days of treatment (p < 0.001). Tumor volume was significantly reduced by I-BET 762 compared to vehicle (p < 0.01). Combining I-BET 762 and RT resulted in significant tumor volume reduction (p <0.001). RNA sequencing and intersection of differentially expressed genes related to “DNA damage repair” and those repressed by I-BET 762 was performed for the MPNST007 PDXs. The expression of 259 genes related to DNA damage repair decreased after I-BET 762 treatment, including XRCC1, ERCC4, RAD50, RAD51D, and LIG4 (data not shown). IHC analysis prompted by the RNA sequencing results of the I-BET 762-treated PDXs revealed higher levels of γH2AX. An increase in necrotic and fibrotic tissue with increased CC3 in the BETi plus radiation group was observed. Conclusions: Genetic mutations in NF-1-associated MPNST tumors result in susceptibility to BETi treatment. In a PDX model of PRC2 wild-type tumors, BETi treatment resulted in tumor regression and sensitized MPNST xenografts to RT. Combining BETi and RT enhanced tumor death and increased expression of genes associated with DNA damage and apoptosis. Citation Format: Alcia A. Gingrich, Sharon Landers, Angela Bhalla, Cristian Rodriguez-Aguayo, Rossana Lazcano, Suresh Satpati, Alines Lebron-Torres, Emily Z. Keung, Christopher P. Scally, Christina L. Roland, Kelly K. Hunt, Heather Lyu, Heather Lillemoe, Ashleigh Guadagnolo, Alexander Lazar, John Slopis, Ian McCutcheon, John A. Livingston, Ahsan Farooqi, Kunal Rai, Keila E. Torres. Role of bromodomain extra terminal inhibitor (BETi) in combination with radiotherapy for the treatment of NF1-associated malignant peripheral nerve sheath tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7548.

Abstract 5555: Lung squamous cancer cell mTORC2 signaling suppresses antitumor immunity by up-regulation of PSGL-1, a ligand of VISTA/PD-1H checkpoint receptor

Abstract Lung squamous cell carcinoma (LUSC) represents 30% of non-small cell lung cancer (NSCLC) cases and has a high mortality rate with limited targeted therapies. Unlike lung adenocarcinoma (LUAD), which has seen advancements in targeted therapies, LUSC has not benefited from similar breakthroughs despite having similar genetic abnormalities. While immune checkpoint inhibitor therapies have shown promise in some LUSC patients, a significant portion does not respond effectively, leaving limited treatment options. In this study, we investigated vulnerabilities in NSCLC and found that a substantial number of LUSC patients have genetic alterations in the PI3K-mTORC2-AKT signaling pathway. By using CRISPR/CAS-9, we showed that disrupting mTORC2 function in LUSC cells reduced glycolysis and lactate secretion, which in turn reduced tumor growth in vivo. Remarkably, the loss of mTORC2 also enhanced the activity of CD4+ and CD8+ T cells within the tumors. To further understand the metabolic and immune changes associated with mTORC2 loss of function, we performed RNA sequencing comparing wild-type (WT) and mTORC2-deficient cells. Our results confirmed a link between mTORC2 and HIF-2α, while regulating PSGL-1, a ligand of the VISTA/PD-1H checkpoint receptor. Furthermore, introducing a stable, non-degradable form of HIF-2α in mTORC2-deficient tumor cells restored PSGL-1 expression in vitro and in vivo. These results suggest that the interaction between PSGL-1 and VISTA plays a role in suppressing the anti-tumor immune response in LUSC tumors. Indeed, anti-VISTA treatment inhibited the growth of LUSC WT tumors in vivo, suggesting that mTORC2 plays a role in regulating PSGL-1 expression in the tumor microenvironment. Together, these data demonstrate that targeting mTORC2 or inhibiting VISTA checkpoint receptor may serve as a potential therapeutic strategy for improving immunotherapy in LUSC. Citation Format: Verra M. Ngwa, Yoonha Hwang, Deanna N. Edwards, Wenqiang Song, Jin Chen. Lung squamous cancer cell mTORC2 signaling suppresses antitumor immunity by up-regulation of PSGL-1, a ligand of VISTA/PD-1H checkpoint receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5555.

Evaluation of the clinical significance of long non-coding RNA MALAT1 genetic variants in human lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most frequent histological subtype of lung cancer, which is the most common malignant tumor and the main cause of cancer-related mortality globally. Recent reports revealed that long non-coding RNA (lncRNA) of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays a crucial role in tumorigenesis and metastasis development in lung cancer. However, the contribution of MALAT1 genetic variants to the development of LUAD is unclear, especially in epidermal growth factor receptor (EGFR) mutation status. In this study, 272 LADC patients with different EGFR status were recruited to dissect the allelic discrimination of the MALAT1 polymorphisms at rs3200401, rs619586, and rs1194338. The findings of the study showed that MALAT1 polymorphisms rs3200401, rs619586, and rs1194338 were not associated to LUAD susceptibility; however, rs3200401 polymorphisms was significantly correlated to EGFR wild-type status and tumor stages in LUAD patients in dominant model (p=0.016). Further analyses using the datasets from The Cancer Genome Atlas (TCGA) revealed that lower MALAT1 mRNA levels were associated with the advanced stage, and lymph node metastasis in LADC patients. In conclusion, our results showed that MALAT1 rs3200401 polymorphisms dramatically raised the probability of LUAD development.

Critical appraisal of surgical margins according to KRAS status in liver resection for colorectal liver metastases: Should surgical strategy be influenced by tumor biology?

BackgroundKRAS mutation is a negative prognostic factor for colorectal liver metastases. Several studies have investigated the resection margins according to KRAS status, with conflicting results. The aim of the study was to assess the oncologic outcomes of R0 and R1 resections for colorectal liver metastases according to KRAS status. MethodsAll patients who underwent resection for colorectal liver metastases between 2010 and 2015 with available KRAS status were enrolled in this multicentric international cohort study. Logistic regression models were used to investigate the outcomes of R0 and R1 colorectal liver metastases resections according to KRAS status: wild type versus mutated. The primary outcomes were overall survival and disease-free survival. ResultsThe analysis included 593 patients. KRAS mutation was associated with shorter overall survival (40 vs 60 months; P = .0012) and disease-free survival (15 vs 21 months; P = .003). In KRAS-mutated tumors, the resection margin did not influence oncologic outcomes. In multivariable analysis, the only predictor of disease-free survival and overall survival was primary tumor location (P = .03 and P = .03, respectively). In KRAS wild-type tumors, R0 resection was associated with prolonged overall survival (74 vs 45 months, P < .001) and disease-free survival (30 vs 17 months, P < .001). The multivariable model confirmed that R0 resection margin was associated with prolonged overall survival (hazard ratio = 1.43, 95% confidence interval: 1.01–2.03) and disease-free survival (hazard ratio = 1.42; 95% confidence interval: 1.06–1.91). ConclusionsKRAS-mutated colorectal liver metastases showed more aggressive tumor biology with inferior overall survival and disease-free survival after liver resection. Although R0 resection was not associated with improved oncologic outcomes in the KRAS-mutated tumors group, it seems to be of paramount importance for achieving prolonged long-term survival in KRAS wild-type tumors.

Abstract B099: A common WNT4 polymorphism drives gynecologic cancer phenotypes and health disparities

Abstract Introduction: Beyond BRCA1/2 mutations, there is a limited understanding of gynecologic cancer etiology and risk. An underexplored risk factor is Wnt ligand WNT4, central to ovarian and uterine organogenesis. A single nucleotide polymorphism (SNP) at a key WNT4 regulatory site (rs3820282) is associated with 10-25% increased risk for gynecologic pathologies, including ovarian cancer (OvCa), but the mechanism is unknown. Importantly, rs3820282 variant allele frequency (VAF) is ~0% in African populations, ~15% in Caucasians, 20-40% in Latinx populations, and 45-55% in Asian populations, paralleling increased incidence of aggressive OvCa subtypes in Asian populations. We hypothesize the rs3820282 variant drives OvCa health disparities by aberrant activation of WNT4, mediating distinct cancer etiology, therapy resistance, and poor patient outcomes. Methods: To address our hypothesis, we employed global untargeted metabolomics, SNP genotyping, WNT4 overexpression, WNT4 siRNA-mediated knockdown, cell viability assays, and Reverse Phase Protein Array (RPPA). We assessed both OvCa cell lines, murine syngeneic tumor models (ID8 Tp53-/- and ID8 Tp53-/-, Brca2-/-), formalin-fixed pre- and post-chemotherapy primary human tumors (18 pairs), and snap frozen primary gynecologic tumors (n=103). Statistical tests performed in Prism GraphPad and include multi-comparison ANOVA, paired t-test, and unpaired t-test. Results: WNT4-overexpression in the ID8 model drove cisplatin resistance in vitro, and resistance and metastatic outgrowth in vivo (n=5 mice/model; post-treatment metastatic nodule mass: control = 5±8mg; WNT4 = 42±18mg; p=0.0078). In paired OvCa tumors pre-/post-treatment with chemotherapy (n=18 pairs), we found WNT4 in the top 10 most-strongly induced genes post-chemotherapy (&amp;gt;4-fold, p =0.0005). In OvCa cell lines, WNT4 siRNA only suppressed growth in WNT4 variant lines (60% reduced growth in n=4 variants, vs n=4 wild-type p=0.010). WNT4 siRNA dysregulated mitochondrial function only in WNT4 variant lines, linking the rs3820282 variant to WNT4-driven metabolic remodeling. RPPA analysis of gynecologic tumors (52% het.+variant); note this is one of the largest proteomic studies enriched for patient diversity (47% from non-White). We identified differential metabolic signaling; AMPK activation was increased in variant tumors (AMPKα1-pT172 + AMPKα2-pS345; p=0.031) along with AMPK signaling targets. Conversely, glucose metabolism proteins were increased in wild-type tumors, and inversely correlated with AMPK activation (Spearman ρ=-0.34, p=0.0005), suggesting that WNT4 genotype underpins AMPK activation and metabolic remodeling. Conclusions: The rs3820282 variant permits aberrant activation of WNT4, driving AMPK-mediated metabolic remodeling and therapy resistance. As the rs3820282 VAF differs widely across populations, WNT4 polymorphism may drive disparities in gynecologic cancer risk and outcomes. Understanding WNT4 signaling and regulation via the rs3820282 variant can lead to precision treatments exploiting cellular dependencies on WNT4 signaling. Citation Format: Benjamin G. Bitler, Madeleine T. Shackleford, Lindsey Trevino, Linda S. Cook, Matthew J. Sikora. A common WNT4 polymorphism drives gynecologic cancer phenotypes and health disparities [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B099.

Challenges and Opportunities of Gene Therapy in Cancer

Challenges and Opportunities of Gene Therapy in Cancer

Abstract B016: Novel curcumin analogues as therapies for endometrial cancer with TP53 mutations

Abstract The protein p53 is designated as the guardian of the genome because it is the principal tumor suppressor and functions to surveil the genome, enforce cell cycle checkpoints, and trigger apoptosis when genomic instability is beyond repair. Mutations in TP53 are found in &amp;gt;50% of human tumors and in 25% of the deadliest endometrial malignancies. Yet, few studies have targeted the mutant protein to restore its wild type (WT) tumor suppressor functions. Curcumin, a turmeric derivative, has been shown to help return WT function to mutant p53. However, curcumin is not bioavailable to most of the body because of its water insolubility, hydrophobic nature and rapid metabolization. Our laboratory is testing novel, more bioavailable analogs of curcumin including HO-3867 and AKT-100. In these studies, we tested these agents in preclinical models of serous endometrial cancer to assess their effects on cell growth and p53-dependent signaling. We used CyQUANT cell proliferation assay to identify IC50 values for HO-3867 and AKT-100 in ECC-1 and KLE endometrial cancer cell lines. We performed RNA sequencing to elucidate mechanisms of action and verified p53 expression changes via Western blotting. IC50 values ranged from 100 nanomolar (AKT-100) to low micromolar (HO-3867). RNA sequencing data revealed the re-enforcement of cell cycle checkpoints including the upregulation of CDKN1A (p21) and GADD45 (Gadd45). Proliferative signaling downstream of ATM, an important protein kinase stimulating cell growth, was downregulated by both agents. Western blotting confirmed the classic negative feedback loop of p53, where total p53 levels decreased after drug treatment. Taken together, our findings show that AKT-100 and HO-3867 are able to restore WT tumor suppressor function in otherwise oncogenic mutant p53 by reducing cell replication and promoting apoptosis. Thus, curcumin analogues such as AKT-100 and HO-3867 show promise in the treatment of a subset of endometrial tumors expressing mutant p53 by returning WT p53 functionality, and future studies will move to in vivo analyses of curcumin analogue function. Citation Format: Kimberly K. Leslie, Alex Goss, Jamie Padilla, Lane E. Smith. Novel curcumin analogues as therapies for endometrial cancer with TP53 mutations [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B016.

Relevance of mutations in protein deubiquitinases genes and TP53 in corticotroph pituitary tumors.

Corticotroph pituitary neuroendocrine tumors (PitNETs) develop from ACTH-producing cells. They commonly cause Cushing's disease (CD), however, some remain clinically silent. Recurrent USP8, USP48, BRAF and TP53 mutations occur in corticotroph PitNETs. The aim of our study was to determine frequency and relevance of these mutations in a possibly large series of corticotroph PitNETs. Study included 147 patients (100 CD and 47 silent tumors) that were screened for hot-spot mutations in USP8, USP48 and BRAF with Sanger sequencing, while 128 of these patients were screened for TP53 mutations with next generation sequencing and immunohistochemistry. USP8 mutations were found in 41% CD and 8,5% silent tumors, while USP48 mutations were found in 6% CD patients only. Both were more prevalent in women. They were related to higher rate of biochemical remission, non-invasive tumor growth, its smaller size and densely granulated histology, suggesting that these mutation may be favorable clinical features. Multivariate survival analyses did not confirm possible prognostic value of mutation in protein deubiquitinases. No BRAF mutations were found. Four TP53 mutations were identified (2 in CD, 2 in silent tumors) in tumors with size >10mm including 3 invasive ones. They were found in Crooke's cell and sparsely granulated tumors. Tumors with missense TP53 mutations had higher TP53 immunoreactivity score than wild-type tumors. Tumor with frameshift TP53 variant had low protein expression. TP53 mutation was a poor prognostic factor in CD according to uni- and multivariate survival analyses in spite of low mutations frequency. We confirmed high prevalence of USP8 mutations and low incidence of USP48 and TP53 mutations. Changes in protein deubiquitinases genes appear to be favorable prognostic factors in CD. TP53 mutations are rare, occur in both functioning and silent tumors and are related to poor clinical outcome in CD.

NEAT1 repression by MED12 creates chemosensitivity in p53 wild-type breast cancer cells.

Breast cancer is often treated with chemotherapy. However, the development of chemoresistance results in treatment failure. Long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been shown to contribute to chemoresistance in breast cancer cells. In studying the transcriptional regulation of NEAT1 using multi-omics approaches, we showed that NEAT1 is up-regulated by 5-fluorouracil in breast cancer cells with wild-type cellular tumor antigen p53 but not in mutant-p53-expressing breast cancer cells. The regulation of NEAT1 involves mediator complex subunit 12 (MED12)-mediated repression of histone acetylation marks at the promoter region of NEAT1. Knockdown of MED12 but not coactivator-associated arginine methyltransferase 1 (CARM1) induced histone acetylation at the NEAT1 promoter, leading to elevated NEAT1 mRNAs, resulting in a chemoresistant phenotype. The MED12-dependent regulation of NEAT1 differs between wild-type and mutant p53-expressing cells. MED12 depletion led to increased expression of NEAT1 in a wild-type p53 cell line, but decreased expression in a mutant p53 cell line. Chemoresistance caused by MED12 depletion can be partially rescued by NEAT1 knockdown in p53 wild-type cells. Collectively, our study reveals a novel mechanism of chemoresistance dependent on MED12 transcriptional regulation of NEAT1 in p53 wild-type breast cancer cells.

Clinical and biomarker results from a phase II trial of combined cabozantinib and durvalumab in patients with chemotherapy-refractory colorectal cancer (CRC): CAMILLA CRC cohort

CAMILLA is a basket trial (NCT03539822) evaluating cabozantinib plus the ICI durvalumab in chemorefractory gastrointestinal cancer. Herein, are the phase II colorectal cohort results. 29 patients were evaluable. 100% had confirmed pMMR/MSS tumors. Primary endpoint was met with ORR of 27.6% (95% CI 12.7-47.2%). Secondary endpoints of 4-month PFS rate was 44.83% (95% CI 26.5-64.3%); and median OS was 9.1 months (95% CI 5.8-20.2). Grade≥3 TRAE occurred in 39%. In post-hoc analysis of patients with RAS wild type tumors, ORR was 50% and median PFS and OS were 6.3 and 21.5 months respectively. Exploratory spatial transcriptomic profiling of pretreatment tumors showed upregulation of VEGF and MET signaling, increased extracellular matrix activity and preexisting anti-tumor immune responses coexisting with immune suppressive features like T cell migration barriers in responders versus non-responders. Cabozantinib plus durvalumab demonstrated anti-tumor activity, manageable toxicity, and have led to the activation of the phase III STELLAR-303 trial.

NF1 mutations as biomarker of response to immune checkpoint blockades for lung adenocarcinoma patients

Little is known about immune checkpoint inhibitors (ICI) response of NF1-mutated lung adenocarcinomas. 341/4,181 (8.2%) TCGA lung adenocarcinomas samples have a somatic NF1 mutation. NF1-mutated tumors have higher TMB (p < 0.0001), higher expression of immune genes (“hot phenotype”) and higher CD8 + T cell (p = 0.03) and macrophage (p = 0.02) infiltrations compared to NF1 wild-type tumors. NF1 mutation status appears as a candidate predictive biomarker for ICI response in lung adenocarcinoma patients.