TP53 Wt Research Articles

Precision medicine applied to rare malignancies: A single-center experience at University Hospital of Verona.

e16296 Background: Therapeutic resources are often limited in rare tumors. NGS (Next Generation Sequencing) represents a valuable tool that can provide biological and prognostic information and guide therapeutic options. Here we describe a case series of the University Hospital of Verona, a center of excellence for neuroendocrine and pancreatic neoplasms. Methods: Between October 2020 and December 2022, 75 patients (pts) with a neuroendocrine neoplasm (NEN) or mixed neuroendocrine non-neuroendocrine neoplasia (MiNEN) underwent NGS analysis with an in-house assay CORE panel (covering 174 genes; 42 pts) or FoundationOne CDx panel (33 pts). We reported ACMG/AMP class IV-V molecular alterations as single nucleotide variations (SNVs), copy number variations (CNVs) including amplifications, deletions, fusions and loss of heterozygosity (LOH). Results: We included 53 well-differentiated neuroendocrine tumors (NETs), 17 poorly differentiated neuroendocrine carcinomas (NECs) and 5 MiNENs. The majority originated from gastro-entero-pancreatic tract (n=67, 89%). The most frequent gene alterations were TP53 (n=30; 12%: 28 SNV and 2 LOH); MEN1 (n=14, 5%: all SNV), CDKN2A (n=12; 5%: 2 SNV, 4 CNV LOH and 5 CNV loss), DAXX (n=12, 5%: all SNV) and RB1 (n=11, 4%: 8 SNV, 4 CNV LOH and 5 CNV loss). In MiNEN pts 3 druggable alterations were found: 1 TRIM-BRAF fusion, 1 SCL4A4-ROS1 fusion and 1 ERBB2 amplification. All MiNENs were MSS; median TMB (mTMB) was 4.86 mut/Mb. Among NET pts the most often altered genes were MEN1 (n=14), DAXX (n=12), TP53 (n=10), PTEN (n=7), CDKN2A (n=5), NF1 (n=5), TSC2 (n=5). The most prevalent mutations in NEC pts involved TP53 (n=17), RB1 (n=9), KRAS (n=7), CDKN2A (n=6), CDKN2B (n=5), CCNE1 (n=5), APC (n=4), SMAD4 (n=3). The mutational rate in NETs was 2.7 with a mTMB of 6.5; the mutational rate in NECs was 5.3 with a mTMB of 7.3 mut/Mb. In NET and NEC 23 actionable mutations were reported. In pancreatic NENs, DAXX mutation was present in 29% of NET G3 and absent in NEC cases. Among G1-G2 NET pts treated with PRRT in 2nd or further lines with a follow-up longer than 60 months (n=17), 6 (35%) were DAXX mutated and all (100%) were still alive at 5 years, compared to 7/11 (64%) in the DAXX wild type (wt) group. Focusing on NET with Ki67% between 3% and 55% (n=45), TP53 wt pts (n=35, 78%) showed a better median overall survival (mOS) than TP53 mutated (n=10, 22%) pts (131.3 vs 56.7 months; p= 0.0433). Conclusions: Our data support the role of NGS in pancreatic rare histologies. This work highlighted the negative prognostic role of TP53 status in G2-G3 NETs, the potential predictive role of DAXX in G1-G2 NET treated with PRRT and its contribution to the differential diagnosis between NET and NEC. Our findings suggest that the pathogenesis in NET is mostly mediated by defects in tumor suppressor genes, while the role of oncogene alterations seem to acquire more importance with increasingly poor differentiation.

Impact of EML4-ALK fusion variant and co-occurring TP53 mutation on treatment duration of first-line next-generation ALK TKIs in ALK fusion+ NSCLC.

9029 Background: Molecular biomarkers such as EML4-ALK fusion variant 3 (V3) and TP53 mutation are associated with poor prognosis in patients (pts) with anaplastic lymphoma kinase fusion positive ( ALK+) non–small cell lung cancer (NSCLC). We evaluated the effect of EML4-ALK fusion variants and TP53 mutation status on time to treatment discontinuation (TTD) of first-line (1L) treatment with a next-generation ALK tyrosine kinase inhibitor (TKI) in pts with ALK+ NSCLC in the real-world setting. Methods: Pts with advanced/metastatic NSCLC who were ALK+ in circulating tumor DNA by Guardant360 (G360) liquid biopsy test from Jan 1, 2018, to Dec 31, 2021, and received 1L monotherapy with a next-generation ALK TKI (alectinib, brigatinib, ceritinib, lorlatinib) were identified in the Guardant INFORM clinical-genomics database. Median TTD of 1L ALK TKI treatment was determined by Kaplan-Meier (KM) methods. The impact of EML4-ALK fusion variant and TP53 mutation status on TTD was evaluated. Results: Database search identified 495 pts with advanced/metastatic NSCLC who had ALK fusion by G360; of these, 214 pts received 1L treatment with a next-generation ALK TKI; 164 had G360 testing before treatment (alectinib, n=147; brigatinib, n=7; ceritinib, n=2; lorlatinib, n=8). Among these 164 pts, median age was 58.6 y, 53% were female, and 29% had baseline brain metastases. Median follow-up from start of 1L treatment was 14.8 mo. Median TTD was 19.6 mo (95% CI: 13.6–not reached [NR]). TP53 mutations were detected in 66 pts (40%). Median (95% CI) TTD in pts with TP53 mutation vs WT was 17.1 (9.5–28.5) vs 21.9 mo (14.3–NR; log-rank P=0.1712). Of 130 pts with EML4-ALK fusion variant type detected, 63 (48%) had V1, 54 (42%) V3, 8 (6%) V2, 4 (3%) V5, and 1 (1%) V8. In pts with EML4-ALK fusion V1 vs V3, median (95% CI) TTD was 19.0 (13.6–NR) vs 21.8 mo (8.7–NR; HR: 0.73; log-rank P=0.3039), with the KM plot showing numerically longer TTD in pts with V1 vs V3 during the first 18 mo and lines crossing at the tail end. Of 117 pts with both an EML4-ALK fusion variant (V1/V3) and TP53 data, 21 (18%) had both V3 and a TP53 mutation. Pts with co-occurring EML4-ALK V3 and TP53 mutation had the shortest TTD (median TTD 8.7 [3.9–NR; HR: 2.59; P=0.0219]; Table). Conclusions: Among pts with NSCLC and ALK fusion, co-occurrence of EML4-ALK V3 and TP53 mutation is common and associated with poor prognosis and high risk of early discontinuation of 1L treatment with a next-generation ALK TKI, most likely due to disease progression. Additional novel or combination therapies are warranted for this subpopulation. [Table: see text]

Abstract A08: Targeting HSP90 epichaperome in TP53 mutant AML

Abstract The Bcl-2 inhibitor venetoclax (VEN)/hypomethylation agent (HMA) combination achieves high response rates, has improved outcomes for many patients with AML and is now considered standard of care for patients who are older or unfit to receive intensive chemotherapy. However, the median overall survival is only 14.7 months on this regimen and only 2.5 months post relapse. Molecular analysis demonstrates that mutations in TP53 and oncogenic kinases are key determinants of lower response rates and early relapse. Preclinical studies also show that increased kinase signaling in AML stem/progenitor cells in TP53 mutant AML. The heat shock protein 90 (HSP90) chaperone, a key regulator of proteostasis, is responsible for the correct folding of kinases and transcription factors. HSP90-associated-epichaperomes, formed in malignant cells, are complexes consisting of HSP90, co-chaperones, and associated proteins that support the maturation, activity, and stability of many cancer-associated kinases and transcription factors including mutated TP53. Hence, HSP90 epichaperome inhibition has the potential of targeting TP53 mutant AML. In contrast to other HSP90 ATP inhibitors, PU-H71 (zelavespib) is a competitive inhibitor specific for the ATP binding site of HSP90 epichaperomes. We here investigate the therapeutic potentials of targeting HSP90 epitherachorme with PU-H71 in TP53 mutated AML. Western blot analysis found increased HSP90 and several signaling proteins in TP53 knockout and mutant Molm13 cells generated by CRISPR/cas-9 or by exposing to idasanutlin, compared to the isogeneic wild-type controls. Using a fluorochrome-labelled PU-H71 and flow cytometry, we demonstrate the presence of HSP90 epichaperomes in AML cells and AML stem/progenitor cells with TP53 mutations, but not in normal bone marrow and bone marrow stem/progenitor cells. PU-H71 effectively kills AML cells and AML stem/progenitor cells with various TP53 mutations, and prolongs survival in TP53-mutant AML xenograft mice with minimal effects on normal CD34+ bone marrow cells and hematopoiesis. PU-H71 increased Bim expression and enhanced VEN activity in AML cells and AML stem/progenitor cells with TP53 mutations. Importantly, in a mixture of TP53 wild-type/R248W Molm13 cells (1000:1), nutlin3a selectively killed TP53 wild-type but enriched TP53 mutant Molm13 cells; VEN treatment favored the outgrowth of TP53-mutant cells, while PU-H71 effectively killed TP53 wild-type and mutant cells. Furthermore, PU-H71 exhibited anti-leukemia activity against both TP53 WT and mutant AML cells, which was further enhanced by VEN in vivo in a xenograft model of mixed TP53 WT and mutated Molm13 cells (10:1). Our data support that the HSP90 epichaperome is essential for the growth and survival of AML and AML stem/progenitor cells harboring mutant TP53. Inhibition of HSP90 by PU-H71 targets AML cells/stem/progenitor cells enhances VEN activity and prevents outgrowth of VEN-resistant TP53 mutant AML cells. This concept warrants clinical evaluations. Citation Format: Bing Carter, Po Yee Mak, Wenjing Tao, Lauren B Ostermann, Yuki Nishida, Steffen Boettcher, Michael Andreeff. Targeting HSP90 epichaperome in TP53 mutant AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A08.

Abstract 1412: Co-existence of TP53 mutation confers endocrine resistance in PIK3CA-mutated ER+HER2- breast cancer

Abstract Background: PIK3CA mutation is commonly found in estrogen receptor (ER)+ human epidermal growth factor receptor-2 (HER2)- breast cancer (BC) and can be therapeutically targeted by alpelisib, an alpha-specific PIK3CA inhibitor. While previous studies presented conflicting results on the prognostic influence of PIK3CA mutation among ER+HER2- breast cancer, few have been able to address causes of the prognostic discrepancy on a clinical level. In this study, we investigated clinical and genomic features according to a coexistence of TP53 mutation in PIK3CA mutated ER+HER2- breast cancer. Methods: Tumor tissue based next generation sequencing (NGS) was analyzed for 224 HR+ HER2- BC patients from April 2017 to August 2021 in Gangnam Severance Hospital, Seoul, South Korea. Matching clinical records with patient information were reviewed retrospectively. Clinicopathologic features including age, tumor size, histologic grade (HG), TNM stage, estrogen receptor (ER) expression, progesterone (PgR) expression, Ki-67, and Oncotype Dx Recurrence Score (RS) were analyzed. Further analysis was carried out using The Cancer Genome Atlas (TCGA) to explore the relationship of mutation status with intrinsic subtype and risk of recurrence (ROR) score. Results: We identified PIK3CA mutation (PIK3CA m) in 105 (46.9%) of 224 patients, and of these, TP53 co-mutation was found in 25 (23.8%). None of clinicopathologic features were different according to PIK3CA mutation. When PIK3CA m cohort was analyzed according to TP53 mutation (TP53 m) status, tumors with TP53 m presented unfavorable features such as higher HG (p<0.001), lower PgR expression (TP53 wt: Median 7 versus TP53 m: Median 5, p=0.027), and higher N stage (p=0.008). It also displayed a significantly higher proliferative profiles with Ki-67 (TP53 wt: Median 15 versus TP53 m: Median 20, p<0.001) and Oncotype Dx RS (TP53 wt: Median 15 versus TP53 m: Median 26.5, p=0.002). In the 539 ER+HER2- breast cancer from the TCGA database, PIK3CA and TP53 co-mutation was found in 33 (6.1%). Distribution of intrinsic subtypes revealed a higher rate of Luminal B in PIK3CA and TP53 co-mutant tumors (51.5%) than in PIK3CA m and TP53 wt tumors (14.3%, p<0.001). The ROR score was found to be higher in the PIK3CA-TP53 co-mutation compared to PIK3CA m alone (p<0.001). Conclusion: Unlike PIK3CA mutation, PIK3CA and TP53 co-mutation clearly divided multiple clinicopathologic features of HR+ HER2- BC including with. Poorer prognosis of co-mutation was also corroborated by TCGA analysis of intrinsic subtype and ROR score. These findings indirectly suggest that co-mutation of TP53 gene may be associated with less sensitivity to PIK3CA targeted therapy in PIK3CA m ER+HER2- breast cancers. Further research is warranted to improve an efficacy of PIK3CA targeted therapy for PIK3CA m ER+ breast cancer with endocrine-resistant factors. Citation Format: Yoonwon Kook, Kyungsoo Kim, Ji Soo Jang, Seung Ho Baek, Soong June BAe, Joon Jeong, Sung Gwe Ahn. Co-existence of TP53 mutation confers endocrine resistance in PIK3CA-mutated ER+HER2- breast cancer [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 1412.

Abstract 251: Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids

Abstract The promise of precision medicine is based on the idea that genetic alterations present only in tumor cells create vulnerabilities that can be targeted for therapeutic intent. In some instances, somatically mutated driver genes such as KRAS, BRAF or PIK3CA can be targeted with small molecule inhibitors specific for mutant oncoproteins. In other cases therapeutic vulnerability is an indirect consequence, such as response to immune checkpoint inhibitors caused by somatic inactivation of mismatch repair genes and subsequent creation of neo-antigens in tumors. Synthetic lethal vulnerabilities dependent on common somatic alterations would be highly specific therapeutic targets applicable to a large number of cancer patients. We sought to identify potential therapeutic vulnerabilities for colon cancer by performing a genome wide CRISPR knockout screen in a colon cancer organoid in both TP53-Wild-Type and TP53-Knockout backgrounds. We identified 1784 gene knockouts with TP53-dependent effects on Darwinian fitness. Examples include MDM2 and PPM1D knockouts, both of which selectively harmed the TP53 WT organoids. 250 gene knockouts selectively harmed the TP53 KO organoids and represent novel avenues for development of TP53 synthetic lethal targeted therapeutics. We also identified ~1000 gene knockouts under significant negative Darwinian selection in both TP53 WT and TP53 KO organoids which are not known to be common essential genes. Several of these organoid model specific vulnerabilities are in pathways that are downstream of the somatic mutations present in the tumor and are therefore candidates for highly-specific targeted therapeutic intervention. Examples include Werner’s pathway dependencies (WRN, EME1, MUS81) which are known to result from mismatch repair deficiency, WNT pathway dependencies (CTNNB1, PORCN) which result from RNF43 inactivating somatic mutation, and BRAF dependency resulting from its own activating oncogenic somatic mutation. In summary, genome-wide CRISPR knockout library screening of human colon cancer organoids can provide a comprehensive overview of dependencies interacting with either natural or engineered mutations and are a promising novel platform for discovering personalized or pathway based therapeutic targets. Future work will focus on target validation and additional primary screens using our biobank of tumor-normal organoid pairs. Citation Format: Phillip J. Buckhaults, Sana Khalili, Carolyn Banister, Prashanth R. Gokare, Dave Pocalyko, Kurtis Bachman. Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids [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 251.

Abstract 3361: Clinical and genomic correlates of liquid biopsy (LB) derived variant allele frequency (VAF) in advanced NSCLC patients

Abstract Background: Initially developed as an alternative to tissue-based molecular assessment, LB is expanding its role in cancer care, with quantitative measure of blood ctDNA being tested for response prediction, response assessment and minimal residual disease detection. Its use for molecular classification has been extensively validated, but little is known about determinants of shedding parameters such as VAF. While it is correlated with tumor burden, characteristics of the tumor such as site of metastasis or particular mutations may have a role. Scope of this study is to elucidate these factors. Methods: Data from advanced NSCLC patients included in two prospective cohorts who received a LB with 36-genes InVisionFirst-Lung (INI) and the 324-gene FoundationOne Liquid CDx (FMI) assays were reviewed and patients with available 18F FDG positron emission tomography scan (PET) performed within 30 days from LB were included. Total Metabolic Tumor Volume was calculated for each PET with a 42% SUVmax threshold. The higher VAF (mVAF) was calculated for each LB, excluding germline mutations and clonal hematopoiesis-related variants. Patients should be either untreated or with progressive disease at the time of LB. Regression analysis was performed according to a rank-based estimation model. Results: Overall 301 LB were included, 119 with InVisionFirst-Lung and 182 with FoundationOne Liquid CDx. LB were contributive in 154 (85%) FMI and 102 (86%) INI. MaxVAF was correlated with tMTV in FMI ( rho 0.3571, p 0.0004) and IVI (rho 0.3387, p = 0.0003) cohort. In FMI cohort, median mVAF was higher for TP53 mutant vs wt (n= 111, mVAF 21.1 % vs 8.0%, p < 0.0001), RB1 mutant (n = 11, mVAF 26.3% vs 4.7%, p 0.0103) and in those with liver metastasis ( p 0.0089). LDH was correlated to mVAF (rho 0.280, p = 0.0037). In multivariate model, tMTV (p < 0.0001), TP53 mutation ( p 0.0039), RB1 mutation (p 0.0003) and liver metastasis ( p 0.0371) remained significantly associated with mVAF. When LDH was added to the model (LDH available for 119 pts), tMTV lost its significance (p 0.561). In INI cohort, median mVAF was also higher in patients with TP53 mutation ( 7.1% vs 3%, p 0.0038), in patients with liver metastasis ( 10.1% vs 4.6%, p 0.0299), and it was correlated with tMTV (rho 0.502, p < 0.0001). It was confirmed in a multivariate model (p < 0.0001 for tMTV and 0.0049 for TP53). Correlation between mVAF and LDH was also seen (LDH available for 54 pts, rho 0.591, p < 0.0001). Among patients with liver metastasis, mVAF was correlated with MTV of liver lesions in FMI (n = 30, rho = 0.212, p = 0.0069) and INI cohort (n= 26, rho 0.204, p 0.0313). Overall, in patients with LDH < ULN, 18/103 were non contributive, vs 4/70 in those with LDH > ULN. Conclusion: In patients with aNSCLC, mVAF depends on disease burden, on the molecular characteristics of the disease and on the presence and extent of liver disease. LDH levels above ULN predict the presence of ctDNA and percentage of mVAF. Citation Format: Filippo Gustavo Dall'Olio, Damien Vasseur, Camilo Garcia, Arianna Marinello, Mihaela Aldea, Laura Mezquita, Jordi Remon, Anas Gazzah, Marco Tagliamento, Kristi Beshiri, Pernelle Lavaud, Maud Ngocamus, David Planchard, Etienne Rouleau, Antoine Italiano, Benjamin Besse. Clinical and genomic correlates of liquid biopsy (LB) derived variant allele frequency (VAF) in advanced NSCLC patients [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 3361.

Abstract P2-17-03: Preclinical study of trastuzumab deruxtecan (T-Dxd; DS-8201a) in combination with DNA damage response pathway inhibitors in HER2-low/Hormone receptor negative breast cancer patient-derived xenograft models

Abstract Background: Triple negative breast cancer (TNBC), defined by negative hormone receptor (HR) status and the lack of HER2 gene amplification, is a significant clinical challenge because of its aggressive clinical course and association with a poor prognosis. Recent studies demonstrated that approximately a third of TNBC has low expression of HER2, so called HER2-low TNBC, which could be targeted by the new generation HER2-directed antibody-drug conjugate (ADC). Trastuzumab deruxtecan (also known as T-Dxd, DS-8201a) is a novel HER2-directed ADC with a topoisomerase 1 inhibitor payload and a tetrapeptide-based cleavable linker, which has been FDA approved for patients with metastatic HER2-positive, or amplified breast cancer. T-Dxd has also shown to improve survival outcomes when compared to physicians’ choice chemotherapy in patients with metastatic HR-negative/HER2-low breast cancer in the Phase 3 DESTINY-Breast04 trial. However, resistance is invariable. We hypothesize that small molecule inhibitors against DNA damage response (DDR) pathways could potentiate the DNA damaging effect of T-Dxd and improve its anti-tumor activity in HER2-low TNBC. Method: We conducted preclinical experiments to assess the anti-tumor activity of T-Dxd and each of the DDR pathway inhibitors, including the ATR inhibitor AZD6738, the ATM inhibitor AZD1390, and the PARP inhibitor olaparib, as well as the Wee 1 inhibitor AZD1775, either alone or in combination, compared to the vehicle treatment, in a panel of HER2-low TNBC PDX models in vivo, as well as mechanistic studies using HER2-low TNBC cell lines in vitro. HER2-low was defined as 1+ or 2+ on HER2 immunohistochemistry. Results: Four HER2-low TNBC PDX models, including WHIM2 (TP53 mutated, basal-like), WHIM6 (TP53 WT, basal-like), WHIM12 (TP53, PIK3CA, and PTEN mutated, claudin-low), and WHIM30 (BRCA1 and TP53 mutated, basal-like), as well as a panel of 4 TNBC cell lines with variable HER2 expression were included in the study. We demonstrated that the addition of DDR pathway inhibitors to T-Dxd led to further tumor shrinkage and delayed tumor progression in HER2-low TNBC PDX models in vivo and induced synergistic anti-tumor effects in vitro. Enhanced DNA damaging effect and apoptosis induction were observed with combination therapies in vitro. Biomarker analysis on xenograft tumors harvested 3 days following treatment with either Vehicle, T-Dxd, olaparib, or T-Dxd plus olaparib in WHIM6, was also performed, which demonstrated that the addition of olaparib enhanced DNA damage (increased pH2AX), and promoted apoptosis (increased cleaved PARP). Further mechanistic studies in HER2-low TNBC cell lines demonstrated the formation of DNA topoisomerase 1 covalent complexes (TopoIcc) following treatment with T-Dxd. Interestingly, while the formation of Topolcc was reduced with the addition of either ATR or ATM inhibitor, no reduction was observed with the addition of olaparib. Additionally, we observed that combination therapies reduced cell migration in vitro. The combination of olaparib with T-Dxd, in particular, reduced mammosphere formation and Epithelial Mesenchymal Transition (EMT) modulation markers, including SNAIL, SLUG and/or TWIST. Conclusion: Our study demonstrated that the combination of T-Dxd and DDR pathway inhibitors induced additive or synergistic anti-tumor effect in HER2-low TNBC. Our data from the studies of cell lines and PDX models provided preclinical rationale for the combination of T-Dxd and DDR pathway inhibitors in patients with HER2-low TNBC. The olaparib combination showed unique mechanistic characteristics compared with other DDR inhibitors, which are being further examined in additional HER2-low TNBC models. Citation Format: Adrian Gonzalez-Gonzalez, Zhanfang Guo, Alice Meroni, Emily Cybulla, Jeremy Hoog, Alessandro Vindigni, Cynthia Ma. Preclinical study of trastuzumab deruxtecan (T-Dxd; DS-8201a) in combination with DNA damage response pathway inhibitors in HER2-low/Hormone receptor negative breast cancer patient-derived xenograft models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-17-03.

Abstract P6-10-11: Engineered neoantigen-specific T cell receptors to treat metastatic breast cancer

Abstract T cell receptor engineered T cell (TCR T) therapy has emerged as a promising therapeutic modality for solid cancer following recent trials demonstrating the safety and efficacy of TCR T therapies against some types of metastatic solid cancers. However, the broader application of TCR T towards many solid tumors, including metastatic breast cancer (MBC), has been limited by several factors, chiefly among them the current scarcity of tumor selective target antigens. Neoantigens, which are expressed exclusively in cancer cells, are currently underrepresented in TCR T development, being targeted in only about 7% of trials conducted to date, and thus represent a relatively untapped source of potentially safe and effective novel targets. Driver mutations in AKT1, ESR1, PIK3CA, and TP53 are common in patients with MBC, and could serve as ideal neoantigen targets for TCR T therapies. We hypothesized that we could generate MBC driver mutation-specific T cells from which we could isolate and clone neoantigen-specific TCRs to generate TCR T products for MBC. We identified 13 driver missense mutations that are among the most frequent in patients with MBC, which included AKT1 (E17K), ESR1 (K303R, Y537S, D538G), PIK3CA (E542K, E545K, H1047L, H1047R), and TP53 (R175H, R248Q, R248W, R273C, R273H), then designed peptide libraries consisting of 15-mer overlapping peptides that contain these mutations. To determine if these neopeptides could elicit T cell responses, we isolated T cells from 15 healthy donors and 11 MBC patients who expressed at least one of the targeted mutations and performed successive stimulations with neopeptide pulsed dendritic cells, then screened the resulting T cell lines for neoantigen specificity using an IFN-γ ELISpot assay. We observed neopeptide T cell responses in 8/16 lines generated from healthy donors and 7/11 lines generated from MBC patients, which were collectively directed against 11/13 of the targeted driver mutations. To isolate neoantigen-specific TCRs from one of these lines, we performed IFN-γ capture, limiting dilution, and 5’ RACE, and isolated an HLA-B*35 restricted TP53 R248W-specifc TCR. Gene transfer of this TCR conferred edited T cells with potent activity towards the TP53 R248W and not the TP53 WT peptide as assessed by ELISpot (1036 vs 46 SFU/1 × 105 cells, respectively) and chromium release cytoxicity assay targeting peptide pulsed autologous PHA blasts (37.5% vs 0% lysis at E:T 40:1, respectively). To increase the throughput of TCR discovery, we next used a single cell RNA sequencing based TCR discovery approach whereby we stimulated T cells from one of the generated lines with ESR1 WT or neopeptide and identified responsive T cell clones through upregulation of IFN-γ and/or TNF-α. This strategy has so far enabled us to identify and validate two ESR1 mutant-specific TCRs. This includes an HLA-C*01 restricted TCR that confers edited T cells with dual activity towards both ESR1 Y537S and D538G, but not WT peptide as determined by both ELISpot (2094, 3194, and 79 SFU, respectively) and chromium release cytotoxicity (31.3%, 77.8%, and 9.1% lysis, respectively), as well as an HLA-B*40 restricted TCR that confers high ESR1 Y537S specificity (5039 vs 138 SFU in response to ESR1 Y537S vs WT peptide, respectively). In summary, we have demonstrated responses of T cells derived from both healthy donors and MBC patients towards neopeptides derived from common MBC driver mutations. We have so far isolated neoantigen specific TCRs from two of the neoantigen-specific T cells lines, including TCRs specific towards TP53 R248W, ESR1 Y537S, dual ESR1 Y537S+D538G that are restricted to three different HLA alleles, and have successfully used these TCRs to generate TCR T products with high neoantigen activity. These results encourage further efforts to identify TCRs recognizing these MBC driver mutations, with our ultimate aim to translate neoantigen-targeted TCR T therapies to clinical trials of MBC. Citation Format: Paul Shafer, Wingchi K. Leung, Mae L. Woods, Carlos Rodriguez-Plata, Arushana Ali, Saisha Nalawade, Lauren M. Kelley, Jarrett Joubert, Anthony Manliguez, Spyridoula Vasileiou, Suzanne A. Fuqua, Premal Lulla, Cliona Rooney, Ann Leen, Valentina Hoyos. Engineered neoantigen-specific T cell receptors to treat metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-11.

Correlation Analysis of the TP53 Mutation With Clinical Characteristics in the Prognosis of Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) with TP53 mutations has a worse prognosis. It was generally more resistant to chemotherapy and radiation. Our aim was to investigate the correlation between the TP53 co-mutated gene and clinical features, and prognostic value in patients with NSCLC. Seventy-three patients with a diagnosis of NSCLC at our hospital were recruited. They were divided into the TP53 mutation status (minor) (TP53 MU) and TP53 wild-type (major) (TP53 WT) groups according to their clinical characteristics after their mutation data and clinical information were collected. Serum markers were compared between groups using Mann-Whitney U test. Other clinical factors were compared between groups using χ2 test and Fisher exact test. The log-rank test was used to compare survival curves. Of the 73 patients with NSCLC, 37 (50.68%) were found to carry TP53 mutation. TP53 MU and TP53 WT groups (n = 36) showed a significant difference in the number of smokers, incidence of squamous cell carcinoma, EGFR mutation, and number of advanced patients (P < .05), while gender, age, lymph node metastasis, and KRAS mutation did not differ significantly between the 2 groups. The survival curves in the TP53/KRAS and the TP53/EGFR co-mutation groups suggest that patients with NSCLC may have a shorter progression-free survival (PFS) if they carry one of the 2 types of co-mutation. TP53 gene mutations are more common in patients with NSCLC and squamous cell carcinoma. New predictive markers for NSCLC prognosis may be TP53/KRAS and TP53/EGFR co-mutations.

ELN Risk Stratification Is Not Predictive of Outcomes for Treatment-Naïve Patients with Acute Myeloid Leukemia Treated with Venetoclax and Azacitidine

Background: Venetoclax (Ven), a highly selective oral BCL-2 inhibitor, in combination with hypomethylating agents (HMAs) such as azacitidine (Aza), is approved for the treatment of patients (pts) with newly diagnosed acute myeloid leukemia (AML) who are ineligible for intensive chemotherapy (IC). An open-label, non-randomized phase 1b trial (NCT02203773) and the confirmatory VIALE-A phase 3 randomized trial (NCT02993523), both enrolling pts with intermediate and poor risk cytogenetics per NCCN Guidelines, demonstrated that the combination of Ven+Aza led to high rates of complete remission and longer overall survival (OS) than Aza alone (median OS of 14.7 months [mo] vs 9.6 mo), with similar safety profiles. In this exploratory post hoc analysis of the pooled treatment-naïve, IC ineligible pts treated with Ven+Aza in the phase 1b and phase 3 trials, patients were stratified according to the 2017 ELN risk classification system (which is based on data from patients receiving IC and utilizes cytogenetic and molecular determinants commonly associated with AML). We report clinical outcomes for these patients per ELN 2017 risk group, which will also be updated using the newly published ELN 2022 criteria. Methods: For the phase 1b trial, our analysis includes pts who were enrolled to evaluate safety and efficacy of Ven+Aza at a daily dose of 400 mg for VEN. For the phase 3 trial, pts were randomly assigned in a 2:1 ratio to the Ven+Aza group or placebo (Pbo)+Aza group. All pts received a standard dose of Aza (75 mg/m2 SC or IV on days 1-7 every 28-day cycle). Ven (400 mg) or Pbo was administered orally, once daily, in 28-day cycles. Pts with favorable risk cytogenetics such as t(8;21), inv(16), t(16;16) or t(15;17) as per the NCCN Guidelines Version 2 were excluded from the study. The pooled group of pts from the two trials having both site-reported cytogenetic data and bone marrow aspirate with centrally determined molecular data allowing for ELN genetic risk classification were included in this analysis. Pts were retrospectively classified per the ELN 2017 criteria as favorable-, intermediate-, or adverse-risk AML. Results: Inclusion of central molecular data allowed the reclassification of patients according to ELN 2017 recommendations (Figure 1). 63 of the 67 Ven 400mg + Aza pts enrolled in the phase 1b trial, and 329 of the 431 total pts enrolled in the VIALE-A trial were evaluable for inclusion in this analysis (279/353 in the pooled Ven+Aza group and 113/145 in the Pbo+Aza control group from the phase 3 study). In the Ven+Aza and Pbo+Aza groups, 46 (16.5%) and 20 (17.7%) pts had favorable-risk AML, 65 (23.3%) and 19 (16.8%) had intermediate-risk AML, and 168 (60.2%) and 74 (65.5%) had adverse-risk AML, respectively, by ELN. In favorable-risk AML pts, CR+CRi was achieved in 69.6% with VEN+AZA vs in 20% with Pbo+Aza; for intermediate-risk AML, CR+CRi was achieved in 75.4% vs 42.1%; for adverse-risk AML, CR+CRi was achieved in 61.3% vs 25.7%. Median OS was longer with Ven+Aza in all three risk categories when compared to Pbo+Aza: 21.09 vs. 12.96 mo, HR = 0.45 [95% CI: 0.24, 0.83] in the favorable-risk group; 23.26 vs. 13.14 mo, HR = 0.62 [95% CI: 0.35, 1.08] in the intermediate-risk group; 11.53 vs. 7.43 mo, HR = 0.59 [95% CI: 0.43, 0.82] in the adverse-risk group. The median OS was similar for favorable and intermediate risk groups who received Ven+Aza (21.09 mo and 23.26 mo, respectively; Figure 2). While the median OS of the Ven+Aza adverse risk group was lower (11.53 mo), two subgroups with distinct outcomes are apparent: adverse risk pts with a TP53 mutation (36.3% [61/168]) and adverse risk pts with a RUNX1 mutation (40.5% [68/168]) (one pt had both TP53 and RUNX1). The median OS for adverse risk pts with a RUNX1 mutation and TP53 WT was 22.9 mo while the median OS for adverse risk pts with a TP53 mutation and RUNX1 WT was 5.42 mo. Conclusions: ELN 2017 risk groups, based on younger patients who received IC regimens, are not prognostic in patients receiving Ven+Aza. This exploratory analysis suggests little OS distinction between favorable and intermediate risk groups. Furthermore, adverse risk ELN groups can be further refined to determine mutationally-defined cohorts with more favorable outcomes. A modified risk stratification system is needed for pts receiving newer therapies, such as Ven+Aza, that uses determinants that are prognostic for pts treated in the context of these regimens with distinct mechanisms of actions from IC. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Application of NGS molecular classification in the diagnosis of endometrial carcinoma: A supplement to traditional pathological diagnosis.

This study aims to demonstrate the advantages of NGS molecular classification in EC diagnosis and to assess whether molecular classification could be performed on curettage specimens and its concordance with subsequent hysterectomy specimens. 80 patients with hysterectomy specimens and 35/80 patients with paired curettage specimens were stratified as POLE mut, MSI-H, TP53 wt, or TP53 abn group by NGS panel. Histotype, tumor grade, IHC results, and other pathological details were taken from original pathological reports. The correlation analysis of 80 patients with hysterectomy specimens between NGS molecular classification and clinicopathological characters displayed that the POLE mut group was associated with EEC (87.5%) and TP53 abn subtype was correlated to a later stage (Stage II-IV, 47.6%), G3 (76.2%), serous histology (61.9%) and myometrial invasion ≥50% (47.6%). A favorable concordance (31/32, 96.9%) was shown in MSI analysis and MMR IHC results, and the agreement rate of p53 IHC and TP53 mutation was 81.5% (53/65). Compared with the p53 IHC abnormal group, the TP53 mutation group had a higher correlation with high-risk factors. A high level of concordance (31/35, 88.0%) of NGS molecular classification was achieved between curettage specimens and hysterectomy specimens while grade and histotype (including unclassified group) from curettage specimens and hysterectomy specimens showed only moderate levels of agreement, 54.3% (19/35) and 68.6% (24/35), respectively. NGS molecular classification achieved on curettage samples showed high concordance with the final hysterectomy specimens, demonstrating superior to the conventional pathological assessment of grade and histotype and potential utilization in clinical practice.

Abstract CT123: A phase 2 study of the MDM2 inhibitor milademetan in patients with TP53-wild type and MDM2-amplified advanced or metastatic solid tumors (MANTRA-2)

Abstract Background: Murine double minute 2 (MDM2) is a potent negative regulator of the tumor suppressor p53. MDM2 induces degradation of p53 and promotes tumorigenesis in solid tumors, and preclinical models have shown that inhibition of MDM2 can restore p53 tumor suppressor activity in TP53-wild type (WT), MDM2-amplified tumors. We performed a mutual exclusivity analysis of patients with solid tumors (n=42,125; AACR Project GENIE) and found that the frequency of co-occurring TP53 mutations decreased with increasing MDM2 copy number. An MDM2 copy number of 12 was chosen as the threshold. An estimated 1.1% of solid tumors meet this molecular criteria, excluding glioblastomas, de-differentiated liposarcomas, and intimal sarcomas where this signature is enriched. Milademetan (RAIN-32), an oral, selective MDM2 inhibitor, inhibits growth of TP53-WT/MDM2-amplified cell lines and patient-derived xenograft models from varying tumor types. Furthermore, tumor regression was observed in 3/3 non-liposarcoma patients with MDM2 copy number &amp;gt;12 in a phase 1 trial of milademetan. MANTRA-2 (RAIN-3202) is a phase 2, multicenter, single-arm, open-label, basket trial designed to evaluate the efficacy or clinical benefit of milademetan in TP53-WT solid tumors with MDM2 amplification (copy number ≥12). Methods: Eligible patients must be ≥18 years of age with histologically and/or cytologically confirmed locally advanced, incurable or metastatic solid tumors refractory to standard therapy. Local testing demonstrating TP53 WT and MDM2 amplification is required, defined as a MDM2 copy number ≥12 or 6-fold increase. Patients with well-differentiated/de-differentiated liposarcomas, intimal sarcomas, or primary central nervous system tumors are excluded. Prior treatment with an MDM2 inhibitor is not permitted. Patients receive milademetan 260 mg orally once daily on Days 1-3 and 15-17 of a 28-day cycle. Tumor response is evaluated by RECIST v1.1 at Weeks 8, 16, 24, and 32, and then every 12 weeks. Primary endpoint: objective response rate. Secondary endpoints include: duration of response; progression-free survival; growth modulation index; disease control rate; overall survival; safety; health-related quality of life scores. Exploratory endpoints include: biomarkers in blood and/or tumor tissue; pharmacodynamics; pharmacokinetics. Enrollment of 65 patients is planned to ensure that 57 patients have centrally confirmed TP53 WT and MDM2 copy number ≥12. The trial opened in November 2021 and is actively enrolling patients. ClinicalTrials.gov: NCT05012397. Citation Format: Ecaterina I. Dumbrava, Glenn J. Hanna, Gregory M. Cote, Thomas E. Stinchcombe, Melissa L. Johnson, Christopher T. Chen, Siddhartha H. Devarakonda, Naisargee Shah, Feng Xu, Robert C. Doebele, Mrinal M. Gounder. A phase 2 study of the MDM2 inhibitor milademetan in patients with TP53-wild type and MDM2-amplified advanced or metastatic solid tumors (MANTRA-2) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT123.

Abstract 6301: DNA methyltransferase inhibitors increase ERV reactivation and STING-dependent interferon/inflammasome signaling in TP53 mutant AML

Abstract DNA methyltransferase inhibitors (DNMTis), which transcriptionally activate hypermethylated genes in cancers and leukemias, also activate endogenous retroviruses (ERVs), leading to increased cytosolic double-stranded (ds) RNA and interferon (IFN) signaling, in a process termed viral mimicry. The tumor suppressor TP53 has been reported to cooperate with DNA methylation and IFN signaling to maintain transcriptional silencing of various repeat sequences, including ERVs. We now report that DNMTis, used to treat acute myeloid leukemia (AML) patients unfit for chemotherapy, robustly induce expression of ERVs and STING-dependent IFN/inflammasome signaling in AML with mutations in TP53(approximately 10%), a subset with poor prognosis. First, our studies of ERV transcripts (N=13) levels in TP53 mutant (KASUMI, KG1-1A, U937) and wild-type (WT; MOLM-14, OCI/AML2, and OCI/AML3) AML cell lines and primary cells (N=6 mutant, N=6 WT) show that, while TP53 mutant cells have low baseline ERV expression, DNMTi treatment significantly increases ERV expression, compared with WT TP53 cells (p&amp;lt;0.05). Moreover, inhibiting TP53 in WT TP53 AML cell lines with the specific TP53 inhibitor pifithrin significantly upregulates ERVs, validating the role of TP53 in suppressing ERVs in AML cells. Second, while TCGA data analysis showed that baseline STING transcript are low in TP53 mutant vs WT AML, phospho-STING (activated) is actually increased in TP53 mutant AML cells and DNMTi treatment further activates STING. Increased expression of downstream IFN/inflammasome genes in TP53 mutant AML treated with DNMTi, compared with results in WT cells, validates these results. Moreover, DNMTi combination with poly (ADP-ribose) polymerase inhibitors (PARPis), which we previously reported to activate STING in breast and ovarian cancer though increasing cytosolic dsDNA and dsRNA, further augments STING activation and IFN/inflammasome signaling (p&amp;lt;0.05). Third, the IFN/inflammasome signaling response to DNMTI/PARPI signaling is directly linked with decreased expression of DNA ds break repair (DSBR) genes and activity, leading to homologous recombination defects (HRD). Fourth, the mechanism for both IFN/inflammasome signaling and HRD induction is dependent on STING, since both CRISPR/CAS KO of STING or treatment with the STING inhibitor H-151 can rescue these effects in TP53 mutant and WT AML cells. Our findings increase understanding of the therapy potential for combining DNMTi+PARPi treatment in AML and suggest use of this paradigm to treat mutant TP53 AML. These results also suggest that activating STING may be a key strategy for increasing IFN/inflammasome signaling in treatment of TP53 mutant AML and that immune responses may be further augmented with immune therapy in AML and other cancers. Citation Format: Aksinija A. Kogan, Michael J. Topper, Lora Stojanovic, Lena J. McLaughlin, Tammy J. Kingsbury, Maria R. Baer, Michael Kessler, Stephen B. Baylin, Feyruz V. Rassool. DNA methyltransferase inhibitors increase ERV reactivation and STING-dependent interferon/inflammasome signaling in TP53 mutant AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6301.

Abstract 3364: Diagnosing Li-Fraumeni syndrome from the somatic genome

Abstract Background: Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in the tumor suppressor gene TP53. LFS is estimated to occur in 1:1000 - 1:5000 people and is associated with a 80% lifetime cancer risk. This syndrome is diagnosed using familial cancer history and germline TP53 sequencing; however, clinical heterogeneity and variants of unknown significance limit diagnostic precision. Accurate diagnosis is imperative to implement surveillance for secondary malignancies and familial genetic testing. Methods: We hypothesized that LFS cancers evolve uniquely from sporadic cancers, implying that the somatic genomes of LFS patients exhibit distinct characteristics that can infer the predisposition syndrome. To investigate this, we interrogated mutational signatures, TP53 copy number, TP53 loss of heterozygosity, ploidy, and the incidence of chromothripsis in tumor compared to normal samples (blood or fibroblast) of individuals with germline TP53 mutations (n=27), somatic TP53 mutations (n=17) and WT for TP53 (n=158). We created a random forest model with 10-fold cross validation to determine if somatic features could diagnose LFS. Results: No signatures were significantly associated with LFS (Wilcoxon rank-sum test, Benjamin-Hochberg FDR correction). As previously reported in the literature, LFS compared to non-LFS cancers in our cohort were more likely to: be hyperdiploid (odds ratio (OR) = 11.83, FDR &amp;lt; 0.0001, Fisher exact test), have undergone TP53 loss of heterozygosity (OR = 23.15, FDR &amp;lt; 0.0001, Fisher exact test) and experience chromothripsis (OR = 7.76, FDR &amp;lt; 0.001, Fisher exact test). The area under the receiver operating curve (AUROC) for our random forest model with 10-fold cross validation was 0.90, the area under the precision recall curve (AUPRC) was 0.59, the positive predictive value (PPV) was 0.70, the negative predictive value (NPV) was 0.93 and the F1-score was 0.52. This implies that the somatic genomic features are reliable indicators of this germline syndrome. We have obtained access to a future 50 LFS samples from the Pediatric Cancer Genome Project dataset, which we hope will improve our model’s performance. Conclusion: We have developed a machine learning tool that uses somatic features to identify LFS, a germline cancer predisposition syndrome. As the importance of precision oncology becomes apparent, a tool to identify LFS patients from the somatic genome will facilitate early diagnosis. This will allow individuals to enter a surveillance program for early detection of secondary tumors, leading to improved outcomes. Citation Format: Brianne Laverty, Vallijah Subasri, Nicholas Light, David Malkin. Diagnosing Li-Fraumeni syndrome from the somatic genome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3364.

A phase 2 study of the MDM2 inhibitor milademetan in patients with TP53-wild type and MDM2-amplified advanced or metastatic solid tumors (MANTRA-2).

TPS3165 Background: Murine double minute 2 (MDM2) is a potent negative regulator of the tumor suppressor p53. MDM2 induces degradation of p53 and promotes tumorigenesis in solid tumors, and preclinical models have shown that inhibition of MDM2 can restore p53 tumor suppressor activity in TP53-wild type (WT), MDM2-amplified tumors. We performed a mutual exclusivity analysis of patients with solid tumors (n = 42,125; AACR Project GENIE) and found that the frequency of co-occurring TP53 mutations decreased with increasing MDM2 copy number. An MDM2 copy number of 12 was chosen as the threshold. An estimated 1.1% of solid tumors meet this molecular criteria, excluding glioblastomas, dedifferentiated liposarcomas, and intimal sarcomas where this signature is enriched. Milademetan (RAIN-32), an oral, selective MDM2 inhibitor, inhibits growth of TP53-WT/ MDM2-amplified cell lines and patient-derived xenograft models from varying tumor types. Furthermore, tumor regression was observed in 3/3 non-liposarcoma patients with MDM2 copy number &gt; 12 in a phase 1 trial of milademetan. MANTRA-2 (RAIN-3202) is a phase 2, multicenter, single-arm, open-label, basket trial designed to evaluate the efficacy or clinical benefit of milademetan in TP53-WT solid tumors with MDM2 amplification (copy number ≥ 12). Methods: Eligible patients must be ≥ 18 years of age with histologically and/or cytologically confirmed locally advanced, incurable or metastatic solid tumors refractory to standard therapy. Local testing demonstrating TP53 WT and MDM2 amplification is required, defined as a MDM2 copy number ≥ 12 or 6-fold increase. Patients with well-differentiated/de-differentiated liposarcomas, intimal sarcomas, or primary central nervous system tumors are excluded. Prior treatment with an MDM2 inhibitor is not permitted. Patients receive milademetan 260 mg orally once daily on Days 1–3 and 15–17 of a 28-day cycle. Tumor response is evaluated by RECIST v1.1 at Weeks 8, 16, 24, and 32, and then every 12 weeks. Primary endpoint: objective response rate. Secondary endpoints include: duration of response; progression-free survival; growth modulation index; disease control rate; overall survival; safety; health-related quality of life scores. Exploratory endpoints include: biomarkers in blood and/or tumor tissue; pharmacodynamics; pharmacokinetics. Enrollment of 65 patients is planned to ensure that 57 patients have centrally confirmed TP53 WT and MDM2 copy number ≥ 12. The trial opened in November 2021 and is actively enrolling patients. Clinical trial information: NCT05012397.

Optimized molecular classification of endometrial carcinoma-based on the combination of POLE mutations with homologous recombination deficiency (HRD).

e17628 Background: Molecular analysis of endometrial carcinoma (EC) has identified four clinically significant molecular subgroups with different prognoses: POLE mutations, microsatellite instability-high (MSI-H/dMMR), copy number low (CN-L/p53-normal), and copy number high (CN-H/p53-aberrant). However, the convenience and repeatability of detection methods for molecular classification, as well as the evaluation of their prognostic significance, need to be further explored. Recently, homologous recombination deficiency (HRD) has been studied to show the prognostic value in EC. Here, we aimed to establish a next-generation sequencing (NGS)-based one-stop method with the combination of HRD and POLE mutations for molecular classification of EC. Methods: Paraffin-embedded tissues of 143 eligible EC patients were collected from Beijing Cancer Hospital between 2011 and 2020 and sequenced using a panel of 520 cancer-related genes which could also detect MSI status (OncoScreen Plus, Burning Rock Biotech). HRD score was calculated as the sum of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST) scores based on the sequencing data. Results: The median follow-up time in this cohort was 38.77 months. We firstly classified EC patients into four subgroups: POLE mutations (mut) (n = 9, 6.3%), MSI-H (n = 45, 31.5%), TP53 wildtype (wt) (n = 67, 46.9%), and TP53 mut (n = 22, 15.4%). POLE mut subgroup had the excellent prognosis as no patient in this subgroup relapsed after surgical resection. Compared with TP53 mut subgroup, the other two subgroups had better prognosis [disease free survival (DFS): TP53 wt: hazard ratio (HR), 0.2, 95% CI, 0.07-0.57, P = 0.003; MSI-H: HR, 0.2, 95% CI, 0.06-0.68, P = 0.01; overall survival (OS): TP53 wt: HR; 0.11, 95% CI, 0.02-0.56, P = 0.008; MSI-H: HR, 0.25, 95% CI, 0.06-1.03, P = 0.055]. Except for the patients with POLE mut, the others were then further optimized and divided into two groups based on HRD score with the cutoff of 13: HRD-high (n = 15, 11.5%) and HRD-low (n = 116, 88.5%). Patients in HRD-low subgroup showed much improved survival than those in HRD-high subgroup (DFS: HR, 0.06, 95% CI, 0.02-0.15, P &lt; 0.001; OS: HR, 0.06, 95% CI, 0.02-0.21, P &lt; 0.001). Most notably, the postoperative recurrence rate was 36.4% in the TP53 mut subgroup with the worst prognosis in the traditional classification, and 66.7% in the HRD-high subgroup with the worst prognosis in the optimized classification. Conclusions: The method of classifying EC patients into four subgroups by NGS large panel provides similar clinical features to previous reports classified by other methods but is more convenient and stable. Furthermore, the method of combination of POLE mutations with HRD offers more accurate disease risk stratification and demonstrates the potential for directing therapy for EC patients.

Abstract PO-077: Establishment of a novel living biobank of patient-derived pancreatic cancer organoids with genomic and drug response characterization

Abstract Advanced pancreatic cancer has a dismal prognosis and current treatment options (FOLFIRINOX, Gemcitabine/nab-paclitaxel [GnP]) are associated with toxicity. Although some patients achieve partial responses, most progress rapidly and become chemorefractory. While RNA subtypes, genomic alterations, and protein biomarkers have prognostic value, predictive biomarkers to guide therapy are needed. Patient-derived organoids (PDOs) are an increasingly popular model for predicting patient responses to standard-of-care therapy and investigating personalized therapy options. We present a novel biobank of 42 PDOs and drug profiling data with 5 standard of care agents and 3 kinase inhibitors. Tissue was processed from n=103 biopsies from 97 patients with a confirmed pathologic diagnosis of advanced (Stage III-IV) pancreatic ductal adenocarcinoma who presented to a single Canadian tertiary care centre between 2017-2020. Matched WGS was available in all cases. Our PDO generation success was 42/103 (41%). We observed a trend towards decreased establishment in tumors that were KRAS WT, TP53 WT, or had higher HRDetect scores. Conversely, polyploidy, SMAD4 WT, and major imbalances in mutant KRAS were associated with successful PDO establishment. These associations were not statistically significant after multiple comparisons correction, but suggest selection for success with more aggressive tumors. Drug profiling was performed on all 42 PDOs with the individual agents of FOLFIRINOX (5-FU, irinotecan, oxaliplatin), GnP (gemcitabine, paclitaxel), and three targeted agents (afatinib, trametinib, and talazoparib). Combination testing was also performed for gemcitabine + paclitaxel. Drug responses were measured through both viability and growth rate (GRMetrics). We found that GRMetrics minimized effects from different PDO growth rates. Matched clinical data were available for 23 patients who received FOLFIRINOX, 11 patients who received GnP, and one patient who received gemcitabine monotherapy. Similar to previous studies, we found that in vitro PDO responses to 5-FU, irinotecan, and GnP were correlated with patient responses based on RECIST criteria. Interestingly, and similar to previous reports in colorectal cancer PDOs, we found that oxaliplatin responses were not predictive of RECIST response. As expected, PDOs were resistant to afatinib (EGFRi), which reflects negative clinical trials, and may also be masked by use of EGF in growth media. A range of responses to trametinib (MEKi) were seen but were not correlated with KRAS allelic dosage. A similar range of response was seen to talazoparib (PARPi), but did not correlate with oxaliplatin response or HRDetect scores. In summary, we have established a novel biobank of PDOs from advanced pancreatic cancer patients. Notably, PDOs were less likely to establish from tumors that were KRAS WT or HR-deficient, even though these patients are likely to benefit from targeted approaches. Further investigation is required to develop PDO use in clinical drug prediction and drug discovery. Citation Format: Irene Y. Xie, Laura Tamblyn, Karen Ng, Eugenia Flores-Figueroa, Julie M. Wilson, Gun Ho Jang, Amy X. Zhang, Stephanie Ramotar, Anna Dodd, Nikolina Radulovich, Jennifer J. Knox, Grainne M. O'Kane, Steven Gallinger, Faiyaz Notta. Establishment of a novel living biobank of patient-derived pancreatic cancer organoids with genomic and drug response characterization [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-077.

Outcomes in Patients with Poor-Risk Cytogenetics with or without TP53 Mutations Treated with Venetoclax Combined with Hypomethylating Agents

Outcomes in Patients with Poor-Risk Cytogenetics with or without TP53 Mutations Treated with Venetoclax Combined with Hypomethylating Agents

An Enterohepatic Recirculation Population Pharmacokinetic and MIC-1 Pharmacokinetic-Pharmacodynamic Model for the MDM2 Inhibitor Navtemadlin (KRT-232) in Healthy Subjects

Background: Murine double minute 2 (MDM2) is the primary negative regulator of the tumor suppressor protein, p53. Navtemadlin (KRT-232), a potent and selective, orally available MDM2 inhibitor restores p53 activity to drive apoptosis of cancer cells in TP53 WT malignancies. Navtemadlin is currently being evaluated in a phase 3 trial of patients with relapsed or refractory myelofibrosis, as well as in numerous phase 1b/2 trials in various hematologic malignancies and solid tumors. Serum macrophage inhibitor cytokine-1 (MIC-1) is a pharmacodynamic (PD) marker of p53-mediated activity in patients treated with navtemadlin (Allard et al. HemaSphere. 2020). Using pharmacokinetic (PK) and PD data from a healthy subject food effect study (Wong et al. Blood. 2020), we developed a population PK (PPK) model that characterized enterohepatic recirculation (EHR) as a half-life extending element in the PK profiles of navtemadlin and its major acyl glucuronide metabolite M1. MIC-1 PD data were incorporated into the model to quantify plasma concentration-driven MIC-1 excursions and to simulate PK and PD across time and dose in healthy subjects.Methods: PPK and PK-PD models were developed using the first-order conditional estimation with interaction (FOCE-I) method in NONMEM 7.4, with model covariates selected using a stepwise forward addition and backward elimination method based on a 5% significance level. Model quality was checked by inspecting model parameters and confidence intervals, as well as standard residual-based and simulation-based diagnostics, and prediction-corrected visual predictive checks. Navtemadlin plasma concentration and MIC-1 serum concentration-time data from the food effect study (KRT-232-105) were modeled (N=30 subjects after a single 60 mg navtemadlin dose). Candidate PPK semi-mechanistic models that described EHR with multi-compartment structures (gut, central, and peripheral compartments for navtemadlin, and central and gallbladder [GB] compartments for M1), first-order elimination, and mealtime effects on GB emptying were tested. Post hoc parameter estimates from the final PPK model were used to generate individual predicted navtemadlin plasma concentrations for the PK-PD model. Based on exploratory plots, the pharmacological mechanism of action of navtemadlin, and a bile acid recycling model (Guiastrennec et al. CPT Pharmacometrics Syst Pharmacol. 2018), an indirect response equation was selected for the MIC-1 effect compartment (Figure 1a).Results: Navtemadlin and M1 plasma concentrations, including a second peak attributed to EHR at ~8-12 h, were well described by a model with central and peripheral compartments, constant basal M1 release rate into bile (KBR BASAL), and incremental mealtime GB emptying rate (KBR MEAL, Figure 1a). Figure 1b shows simulated navtemadlin and M1 amounts in various compartments over time. Median oral clearance of navtemadlin was estimated at 36.35 L/h. Estimated median apparent oral clearance of navtemadlin in healthy subjects was higher than PPK estimates for patients with advanced solid tumors (24.9 L/h [Ma et al. Blood. 2019]). The median central and peripheral volumes of navtemadlin were 159 L and 390 L, respectively. Navtemadlin exposure was higher in healthy female subjects relative to male subjects. Between-subject variability in clearance was 31%. Typical MIC-1 maximum stimulatory effect (S max) was estimated at 6.82, close to the median maximum ratio of MIC-1 to baseline MIC-1 (7.29) in the observed data. SC 50 was estimated at 85.22 ng/mL, with a Hill coefficient of 2.02, indicating a relatively steep increase in MIC-1 serum concentration with increasing navtemadlin concentration. For both PPK and PK-PD models, diagnostic plots confirmed an adequate fit. Subjects with lower baseline MIC-1 had a larger response and reached a maximum MIC-1 concentration later. Older subjects had the largest covariate impact, with a higher MIC-1 response.Conclusion: A two-compartment PPK model with basal and incremental mealtime GB emptying rates captured concentration-time data for navtemadlin and its metabolite M1. EHR was evident and navtemadlin reabsorption following hydrolysis of biliary M1 in the intestine contributed to navtemadlin half-life. An indirect stimulatory PK-PD model effectively described the relationship between navtemadlin and MIC-1 in healthy subjects. [Display omitted] DisclosuresZhang: Certara, Inc.: Current Employment; Milad Pharmaceutical Consulting, LLC.: Ended employment in the past 24 months. Wong: Kartos Therapeutics: Current Employment; AbbVie Biotherapeutics: Current equity holder in publicly-traded company. Slatter: Telios Pharma: Current holder of stock options in a privately-held company; Kartos Therapeutics: Current Employment, Current holder of stock options in a privately-held company; AstraZeneca: Current equity holder in publicly-traded company; Amgen: Divested equity in a private or publicly-traded company in the past 24 months. OffLabel Disclosure:Yes, navtemadlin (KRT-232) is an investigational small molecule MDM2 inhibitor.

Pathway Analysis Provides Mechanistic Insights into Navtemadlin (KRT-232) Activity and Synergy with Decitabine in Acute Myeloid Leukemia Preclinical Models

Background: Murine double minute 2 (MDM2) is the primary negative regulator of the tumor suppressor protein, p53. Navtemadlin (KRT-232), a potent, selective, orally available MDM2 inhibitor, restores p53 activity to drive apoptosis of cancer cells in TP53 WT malignancies through the intrinsic (mitochondrial) pathway. Navtemadlin is being evaluated in a phase 3 trial in relapsed or refractory myelofibrosis, and in phase 1b/2 trials in various hematologic malignancies and solid tumors. Because p53 is infrequently mutated and MDM2 is often overexpressed in acute myeloid leukemia (AML), navtemadlin represents a rational therapeutic candidate (Prokocimer et al. Blood. 2017). Synergy between navtemadlin and the hypomethylating agent decitabine has been demonstrated in preclinical AML models (Canon et al. AACR. 2016), although the mechanisms mediating this effect remain unclear. The present study aims to identify mechanisms of navtemadlin-mediated apoptosis within AML cell lines, timing of pathway activation, and effects of combination treatment with navtemadlin + decitabine.Methods: MOLM13 or MV-4-11 AML cell lines were treated with a vehicle control, 0.5-1.0 µM navtemadlin, 1.0 µM decitabine, or with a combination of navtemadlin + decitabine. Cells were pretreated with decitabine for 42-48 hours followed by navtemadlin + fresh decitabine for 0, 5, 10, or 24 hours, and were then harvested for RNA-Seq and immunoblotting analyses of apoptotic pathway proteins. Gene set enrichment analysis (GSEA) and signaling pathway impact analysis (SPIA) were performed on RNA-Seq data to determine the most differentially regulated pathways (using KEGG curated signaling pathways) between controls, single-agent navtemadlin or decitabine, or combination treatment. Protein expression analyses were conducted to validate RNA-Seq data.Results: GSEA of MOLM13 and MV-4-11 cells at 10 hours showed that, relative to controls, several signaling pathways were differentially regulated to a statistically significant level (P≤0.05) with navtemadlin alone, including activation of apoptosis and p53 signaling pathways, and suppression of DNA replication and cell cycle pathways, in line with previous reports (Figure 1a). In MOLM13 cells treated with single-agent decitabine, lipid and proteoglycan-related signaling pathways were activated, whereas several metabolic pathways were suppressed. With combination treatment in both cell lines, unique activated pathways relative to controls included immune-related and cancer-signaling pathways; and unique suppressed pathways included base excision repair and cellular senescence pathways. SPIA analysis of both cell lines at 10 hours produced higher perturbation scores for apoptosis and p53 signaling pathways with combination treatment vs navtemadlin alone (Figure 1b), indicating more robust pathway activation. Additionally, tumor microenvironment (TME) and extracellular matrix (ECM) remodeling pathways showed more robust perturbation when combined with navtemadlin vs decitabine alone. At 24 hours, only SPIA was conducted, which showed less apoptotic pathway induction relative to 10 hours in both cell lines, suggesting time-dependent signaling effects. Cellular senescence pathways were suppressed, whereas central carbon metabolism and HIF-1 signaling were induced by single-agent navtemadlin relative to controls in both cell lines, with combination treatment further enhancing these effects. Combination treatment also induced significant changes in the expression of ECM remodeling genes including MMP9, MMP2, and ITGB7. Protein expression analyses in both cell lines confirmed early upregulation of key apoptosis regulators including p53, p21, PUMA, cleaved caspase-3 and cleaved-PARP with navtemadlin alone, and the DNA-damage response protein, yH2AX, with decitabine alone. The combination enhanced the induction of p53, p21, and yH2AX vs controls or single agents alone.Conclusion: Initial results from this study suggest that navtemadlin + decitabine enhanced apoptosis and p53 signaling pathways to a greater degree than either single agent in AML cell lines. Combination treatment also induced several unique DNA damage response, ECM-remodeling, and TME-related pathways. Activation of both proapoptotic and tumor-stromal interaction pathways suggest a unique mechanism of navtemadlin + decitabine for inhibiting AML cell growth. [Display omitted] DisclosuresValmeekam: Telios Pharmaceuticals: Current Employment, Current holder of stock options in a privately-held company, Other: travel, accommodations, expenses. Canon: Oncovalent Therapeutics: Current Employment; Amgen, Inc.: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months. Krejsa: Acerta Pharma: Current holder of individual stocks in a privately-held company; AstraZeneca: Current equity holder in publicly-traded company; Seattle Genetics: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months; Kartos Therapeutics: Current Employment, Current holder of stock options in a privately-held company, Other: travel, accommodations, expenses. Kelly: AstraZeneca: Consultancy; Bayer: Speakers Bureau; Verastem: Consultancy; Amgen: Consultancy; Berkley Lights: Current equity holder in publicly-traded company; Denovo Biopharma: Consultancy; Takeda: Consultancy; Sanofi-Aventis: Consultancy; Novartis: Speakers Bureau; Janssen: Speakers Bureau; Agios: Current equity holder in publicly-traded company; Celgene: Speakers Bureau; Epizyme: Speakers Bureau; Pharmacyclics: Speakers Bureau; Karyopharm: Speakers Bureau; Gilead: Speakers Bureau. OffLabel Disclosure:Yes, navtemadlin (KRT-232) is an investigational small molecule inhibitor.