TP53 Gene Mutation Status Research Articles

The Prognostic Predictive Value of TP53 mutation Variant Allele Frequency in Diffuse Large B-Cell Lymphoma

To explore the effect of TP53 mutation variant allele frequency(VAF) on the prognosis of diffuse large B-cell lymphoma(DLBCL) patients. This study included 155 patients with DLBCL who were first diagnosed in the People's Hospital of Xinjiang Uygur Autonomous Region from March 2009 to March 2022. Complete clinical data and paraffin-embedded tumor tissue samples were obtained, and DNA was extracted from tumor tissues. The gene mutation profile of DLBCL patients was detected and analyzed by second-generation sequencing technology. Kaplan-Meier method was used to analyze the mutation status of TP53 gene and the relationship between mutation VAF and OS. Cox regression univariate and multivariate analysis was use to analyze the independent factors affecting OS. A nornogram model for predicting 1, 3, and 5 years OS in DLBCL patients were established to evaluated the performance of the model based on C-index and calibration curves. The average value of TP53 mutation VAF in male DLBCL patients was significantly higher than that in female patients (P < 0.05). Patients with TP53 mutantion had shorter OS than those with wild-type patients (P =0.030). The optimal VAF threshold for TP53 mutation based on OS stratification was 33.61% (P < 0.001), and patients with TP53 mutation VAF ≥34% had shorter OS than those with TP53 mutation VAF < 34% and wild-type patients (P < 0.001). Multivariate Cox analysis showed that TP53 mutation VAF≥34% was an independent poor predictor of OS ( HR =4.05, P < 0.001), and IPI score ≥3 was an independent predictor of OS poor ( HR =2.27, P =0.008). In combination with factors with independent prognostic significance obtained from multi-factor analysis, we constructed a nomogram model for predicting 1-year, 3-year, 5-year OS in DLBCL patients. The results showed that the C index of TP53 mutation VAF combined with IPI model was 0.743, which predicted the value of 1-year, 3-year, and 5-year OS in DLBCL patients. Calibration curves show that the model has good agreement between predicted and actual survival of DLBCL patients at 1-year, 3-year, and 5-year. TP53 mutation VAF has prognostic value in DLBCL patients, and TP53 mutation VAF≥34% is an independent risk factor for OS in DLBCL patients. The prognosis model of TP53 mutation VAF combined with IPI nomogram constructed in this study has good predictive performance for DLBCL patients.

Calcium ion delivery by microbubble-assisted sonoporation stimulates cell death in human gastrointestinal cancer cells

Ultrasound-mediated cell membrane permeabilization – sonoporation, enhances drug delivery directly to tumor sites while reducing systemic side effects. The potential of ultrasound to augment intracellular calcium uptake – a critical regulator of cell death and proliferation – offers innovative alternative to conventional chemotherapy. However, calcium therapeutic applications remain underexplored in sonoporation studies. This research provides a comprehensive analysis of calcium sonoporation (CaSP), which combines ultrasound treatment with calcium ions and SonoVue microbubbles, on gastrointestinal cancer cells LoVo and HPAF-II. Initially, optimal sonoporation parameters were determined: an acoustic wave of 1 MHz frequency with a 50 % duty cycle at intensity of 2 W/cm2. Subsequently, various cellular bioeffects, such as viability, oxidative stress, metabolism, mitochondrial function, proliferation, and cell death, were assessed following CaSP treatment. CaSP significantly impaired cancer cell function by inducing oxidative and metabolic stress, evidenced by increased mitochondrial depolarization, decreased ATP levels, and elevated glucose uptake in a Ca2+ dose-dependent manner, leading to activation of the intrinsic apoptotic pathway. Cellular response to CaSP depended on the TP53 gene's mutational status: colon cancer cells were more susceptible to CaSP-induced apoptosis and G1 phase cell cycle arrest, whereas pancreatic cancer cells showed a higher necrotic response and G2 cell cycle arrest. These promising results encourage future research to optimize sonoporation parameters for clinical use, investigate synergistic effects with existing treatments, and assess long-term safety and efficacy in vivo. Our study highlights CaSP's clinical potential for improved safety and efficacy in cancer therapy, offering significant implications for the pharmaceutical and biomedical fields.

Exploring p53 protein expression and its link to TP53 mutation in myelodysplasia-related malignancies-Interpretive challenges and potential field of applications.

TP53 alterations have a significant prognostic effect in myeloid neoplasms. Our objective was to investigate the TP53 gene mutation status, p53 protein expression and their relationship in dysplasia-related myeloid neoplasms with varying levels of myeloblast counts. A total of 76 bone marrow biopsy samples with different blast counts were analysed. Total and strong (3+) p53 expression was determined. Dual immunohistochemical staining was performed to determine the cell population associated with p53 expression. NGS analysis was performed using the Accel-Amplicon Comprehensive TP53 panel. Both p53 expression and TP53 VAF showed a significant correlation with the myeloblast ratio (P < 0.0001); however, p53 expression was also present in other cell lineages. The VAF value exhibited a significant correlation with p53 expression. A high specificity (0.9800) was observed for TP53 mutation using the ≥ 10% strong (3+) p53 cut-off value, although the sensitivity (0.4231) was low. Strong (3+) p53 expression using a ≥ 10% cut-off value accurately predicts TP53 mutation but does not reveal the allelic state. The p53 expression is significantly influenced by myeloblast count, and histological interpretation should consider the presence of intermixed non-neoplastic marrow cells with varying physiological p53 expression.

Choosing Wisely between Radiotherapy Dose-Fractionation Schedules: The Molecular Graded Prognostic Assessment (molGPA) for Elderly Glioblastoma (eGBM-molGPA)

This study aimed to develop a graded prognostic assessment (GPA) model integrating genomic characteristics in patients with elderly glioblastoma (eGBM), and compare the efficacy between conventionally fractionated radiotherapy (CFRT) vs. hypofractionated radiotherapy (HFRT) in each risk group. Patients aged ≥65 years who underwent surgical resection followed by radiotherapy (RT) with or without temozolomide (TMZ) for newly diagnosed IDH-wildtype eGBM between 2006 and 2021 were included in this multicenter cohort study. Patients who were planned for a ≥6-week or ≤4-week radiotherapy were regarded as being treated with CFRT or HFRT, respectively. Based on the prognostic factors significantly identified through multivariate analysis for overall survival (OS), we developed the molecular GPA for eGBM (eGBM-molGPA) and assigned 0.0, 0.5, and 1.0 points in proportion to the corresponding hazard ratio (HR) of each factor. Then, the survival outcomes by treatment groups were evaluated according to the eGBM-molGPA scores. A total of 334 and 239 patients who underwent CFRT and HFRT were included, respectively, and 86% of patients were treated with TMZ-based chemoradiation. With a median follow-up of 17.4 months for survivors, the median OS was 18.7 months for CFRT plus TMZ group, 15.1 months for HFRT plus TMZ group, and 10.4 months for RT alone group, respectively (all p<0.001). In the multivariate analysis, Karnofsky performance scale, surgical extent, TMZ, and the methylation status of the MGMT promoter were identified as strong prognostic factors for OS, with an estimated HR of greater than 1.5 (all p<0.001). Additionally, subventricular zone involvement, temporalis muscle thickness, RT regimen, and the mutation status of TERT promoter and TP53 gene were found to be significant prognostic factors for OS, with an estimated HR of less than 1.5. The eGBM-molGPA was established based on these prognostic factors (Table 1) and patients were allocated to three risk groups, which included high risk (total score of 3.0-4.5), intermediate risk (1.5-2.5), and low risk (0.0-1.0). Patients treated with CFRT plus TMZ had significantly improved OS compared to those treated with HFRT plus TMZ or RT alone in the low and intermediate risk groups (p<0.001). However, in the high-risk group, there was no significant difference in OS between treatment options (p = 0.770). CFRT plus TMZ can be a more effective strategy for selected eGBM patients compared to HFRT. For high-risk patients, a protracted treatment schedule might not be beneficial. The novel eGBM-molGPA can be used as a clinical tool for choosing wisely among treatment options. Further prospective studies are warranted to establish optimal RT guidelines for eGBM patients.

Cost Effectiveness of Molecular Diagnostic Testing Algorithms for the Treatment Selection of Frontline Ibrutinib for Patients with Chronic Lymphocytic Leukemia in Australia.

Clinical indications for ibrutinib reimbursement in Australia should consider the inclusion of patients with chronic lymphocytic leukemia (CLL) harboring prognostically unfavorable TP53/IGHV genomic aberrations. This study assessed the cost effectiveness of five first-line treatment strategies in CLL for young (aged ≤65years), fit patients without significant comorbidities: (1) no testing (fludarabine, cyclophosphamide and rituximab [FCR] for all), (2) test for del(17p) only, (3) test for TP53 gene mutation status, (4) test for TP53 and IGHV gene mutation status and (5) no testing (ibrutinib for all). A decision analytic model (decision tree and partitioned survival model) was developed from the Australian healthcare system perspective with a lifetime horizon. Comparative treatment effects were estimated from indirect treatment comparisons and survival analysis using several studies. Costs, utility and adverse events were derived from public literature sources. Deterministic and probabilistic sensitivity analyses explored the impact of modeling uncertainties on outcomes. Strategy 1 was associated with 5.69 quality-adjusted life-years (QALYs) and cost 458,836 Australian dollars (AUD). All other strategies had greater effectiveness but were more expensive than Strategy 1. At the willingness-to-pay (WTP) threshold of 100,000 AUD per QALY gained, Strategy 1 was most cost effective with an estimated probability of 68.8%. Strategy 4 was cost effective between thresholds 155,000-432,300 AUD per QALY gained, and Strategy 5 >432,300 AUD per QALY gained. Population targeting using mutation testing for TP53 and IGHV when performed with del(17p) testing specifically in the context of frontline ibrutinib choice does not make a cost-ineffective treatment into a cost-effective treatment.

LMAP-04 CHOOSING WISELY BETWEEN RADIOTHERAPY DOSE-FRACTIONATION SCHEDULES: THE MOLECULAR GRADED PROGNOSTIC ASSESSMENT (MOLGPA) FOR ELDERLY GLIOBLASTOMA (EGBM-MOLGPA)

Abstract This study aimed develop a prognostic model integrating genomic characteristics in patients with elderly glioblastoma (eGBM), and compare the efficacy between conventionally fractionated radiotherapy (CFRT) vs. hypofractionated radiotherapy (HFRT). Patients aged ≥65 years who underwent radiotherapy for IDH-wildtype eGBM between 2006 and 2021 were included. Patients planned for a ≥6-week or ≤4-week radiotherapy were regarded as being treated with CFRT (334 patients; median, 60 Gy in 30 fractions) or HFRT (239 patients; median, 45 Gy in 15 fractions), respectively. We developed the molecular graded prognostic assessment score for eGBM (eGBM-molGPA) and assigned 0.0, 0.5, and 1.0 points in proportion to the corresponding hazard ratio (HR) of each prognostic factor for overall survival (OS). Temozolomide-based chemoradiation was applied for 86% of patients. With a median follow-up of 17.4 months, the median OS was 18.7 months for CFRT plus temozolomide group, 15.1 months for HFRT plus temozolomide group, and 10.4 months for RT alone group. The eGBM-molGPAwas established based on prognostic factors from multivariate analysis (performance, extent of resection, temozolomide, methylation of the MGMT promoter, subventricular zone involvement, temporalis muscle thickness, and the mutation status of TERT promoter and TP53 gene) and patients were allocated to three risk groups (high risk, total score 3.0–4.5; intermediate risk, 1.5–2.5; low risk, 0.0–1.0). Patients treated with CFRT plus temozolomide had significantly improved OS compared to those treated with HFRT plus temozolomide or radiotherapy alone in the low and intermediate risk groups (p&amp;lt;0.001). However, in the high-risk group, there was no significant difference in OS between treatment options. CFRT plus temozolomide can be a more effective strategy for selected eGBM patients compared to HFRT. For high-risk patients, a protracted treatment schedule might not be beneficial. The novel eGBM-molGPA can be used as a clinical tool for choosing wisely among radiotherapy options.

Abstract 4995: Targeting PLK1 effectively suppresses growth of small cell lung cancer

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor characterized by rapid disease progression and poor patient survival. Key transcription factors implicated as drivers of unique biological phenotypes of SCLC include ASCL1, NEUROD1, YAP1, and POU2F3. Strategies to exploit these phenotypes for innovative precision medicine approaches will be impactful. Using an unbiased agnostic preclinical drug screen to uncover therapeutic opportunities in SCLC, we identified a strong signal with PLK1 inhibitors (PLK1i) with low nanomolar IC50. We extended the in vitro findings by testing the efficacy of PLK1i in vivo using traditional xenograft of SCLC H526 cell line and patient derived xenografts (PDX). Volasertib achieved significant tumor growth inhibition relative to control in H526 xenografts. Also, onvansertib significantly inhibited growth of platinum-resistant and platinum-sensitive PDXs. The combination of PLK1i with standard chemotherapeutic agents identified promising synergy of the combination of onvansertib and paclitaxel. We further interrogated for predictive biomarkers of PLK1i sensitivity using gene expression profile comparing highly sensitive to less sensitive cell lines. High expression of C-MYC but not PLK1, TP53 or RB was associated with resistance to PLK1i. Conversely, while TP53 expression level did not correlate, TP53 gene mutation status (inactivating disruptive mutations) correlated with cell sensitivity to PLK1i. We queried the publicly available CCLE and the Cancer Therapeutics Response Portal to evaluate whether any of the SCLC subtypes have therapeutic vulnerability to PLK1i. In general, high expression of YAP1 in SCLC cell lines correlated with greater sensitivity to PLK1i. A YAP1 positive cell line, SW1271, with strong TP53 expression was particularly resistant to PLK1i. CRISPR knockout of YAP1 in this cell line enhanced SW1271 sensitivity to PLK1i suggesting that YAP1 expression as a marker of vulnerability to PLK1i could be context dependent especially when co-occurring with TP53 mutations. The mechanism of this interaction will be discussed. Citation Format: John C. Schmitz, Guojing Zhang, Andrey A. Ivanov, Abbe Pannucci, Maya Ridinger, Taofeek K. Owonikoko. Targeting PLK1 effectively suppresses growth of small cell lung 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 4995.

Impaired Plasma B Cell Markers in CD138+ Cells Predict Prognosis in Multiple Myeloma

Background: Multiple myeloma (MM), characterized by the proliferation of clonal plasma B-cell in the bone marrow, is the second most common hematologic malignancy in the US. MM represents the final stage in a continuum of plasma cell dyscrasias, arising from the premalignant conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Despite therapeutic advances, MM remains incurable with the prognostic improvement observed only in a subset of patients. The variations in clinical course are largely caused by the underlying intertumoral heterogeneity between different patients. To further improve the clinical outcome of MM patients, biomarkers that can effectively stratified patients are critically needed for directing personalized treatment. In this study, we hypothesize that the magnitude of plasma B cell malignancy is a primary factor that determines the clinical phenotypes of MM patients, which can be reflected by gene expression in CD138+ cells. Methods: Using single-cell RNA sequencing (scRNA-seq) data, we develop a plasma B gene signature and apply it to gene expression profiles of CD138+ cells from MM patients (Figure 1 A). The resulting score, denoted as plasma B cell malignancy (PBM) score, indicates the degree of plasma B cell malignancy of MM samples with a higher value indicating higher malignancy status. Genomic and/or transcriptomic data from five MM studies (i.e., MMRF CoMMpass, UAMS, MAQC-II and APEX phase 3 trial and Chng study) were used to examine the associations between PBM score and known MM driver genes, and its clinical impact on MM progression and prognosis. Univariate and multivariate Cox regression models were constructed to evaluate the prognostic value of patient specific PBM scores when used alone or combined with established clinical factors. Results: Survival analysis indicates that the PBM score defined based on plasma B cell markers are significantly associated with patient overall survival. In contrast, gene signatures charactering other immune cells are less (other three B cell types) or not prognostic (non-B immune cell types) (Figure 1 B). Then we investigated the effects of PBM score on MM development and prognosis. Overall, a higher PBM score was significantly associated with a more advanced stage in the spectrum of plasma cell dyscrasias (all P < 0.05, Figure 1C). Higher PBM score was also associated with shorter overall survival (OS) in patients with MM (HR = 1.72, P < 0.001) (Figure 1D) and its prognostic effect was independent of the currently available clinical prognostic model, such as the International Staging System (ISS) (Figure 1E). Mutation status of TP53 gene has been found to be associated with patient prognosis. Investigations on the relationships between PBM score and MM driver genes found that the PBM score was significantly increased in patients carrying TP53 mutations compared to those with mild-type alleles. Of note, PBM scores could predict patients’ survival even among those who don't carry TP53 mutations, with log-rank P values of 0.002 (Figure 1F). The model based on the PBM score alone had a c-index of 0.62 to predict the OS of MM. The clinical model based on age, sex, race, and (ISS) stage had a c-index of 0.67 to predict OS. Incorporating PBM score into the models based on clinical factors significantly improved the prognostic performance of the models, with the c-index increased to 0.70 for the model that combined PBM and clinical factors (P < 0.001) (Figure 1G).Conclusions: There may be a clinical utility of the PBM score to predict MM outcomes in combination with more standard clinical parameters. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Characteristics and Overall Survival Among Acute Myeloid Leukemia (AML) Patients with TP53 Gene Mutation (TP53m) or Chromosome 17p Deletion (17p del)

Introduction TP53m is observed in 5%-15% of newly diagnosed AML patients (pts) and represent a poor prognostic group that is highly refractory to available treatment, both intensive and non-intensive, including venetoclax-based therapies. Currently there are no established therapies specifically targeting TP53m in AML. Better understanding of the outcomes in TP53m AML pts with standard-of-care treatments can provide important insights for clinical management and drug development. Methods Adult (≥18 years [yrs]) pts with new AML diagnosis from January 1, 2014 to February 28, 2021 were included from the nationwide Flatiron Health electronic health record-derived de-identified database. Prior to first line (1L) therapy start, all pts had TP53m determined from molecular tests or 17p del identified by cytogenetic/fluorescent in situ hybridization. Pts who received prior anti-leukemic therapies (except hydroxyurea, oral etoposide) or who had concurrent malignancies were excluded. Pts receiving 1L therapy were divided into 3 cohorts: hypomethylating agents (HMA; azacitidine/decitabine) and venetoclax (Cohort A), intensive chemotherapy (Cohort B), or HMA without venetoclax (Cohort C). Median overall survival (mOS) was estimated using Kaplan-Meier method. Baseline and clinical characteristics can impact treatment choice and survival in each cohort. Multivariate Cox proportional hazards models (including age, time to 1L therapy, comorbidities, TP53m status and cytogenic risk level, presence of 17p del, secondary and therapy-related AML, healthcare practice type, ECOG score, and post-1L allogeneic stem cell transplantation [allo-SCT]) were used to compare OS among treatment cohorts. Results A total of 370 AML pts with either TP53m (n=124) or 17p del (n=166) or both (n=80) were included. Median age was 72 (range 24-84) yrs, and majority were male (59%) and white (69%). Most pts (85%) were from community oncology clinics. Overall median follow-up time for all pts was 6 (range 0-74) mos and was similar for the three cohorts (6, 7, and 5 mos for Cohort A, B, and C, respectively). Although cytogenetic risk category information was missing for more than one-third (34%) of pts, majority (65%) belonged to the ELN 2017 adverse cytogenetic risk category. Deletions in chromosomes 5, 7, or 17 were reported in 88% (n=324) of pts, with 68% having more than one deletion. Secondary AML was reported for 118 (32%) pts, of which 92 (78%) had prior myelodysplastic syndrome; 38 pts (10%) had therapy-related AML. Bone marrow (BM) blasts were ≤30%, 31-50%, and >50% in 41%, 24%, and 29% of pts, respectively. Treatment Cohort A, B, and C included 94 (25%), 135 (37%), and 141 (38%) unique AML pts, respectively. Pts in Cohort A and C were older (median 75 and 77 yrs vs 62 yrs for Cohort B) and had more comorbidities (65% and 60% vs 40% in Cohort B with at least one comorbidity). BM blast clearance post-1L was reported in 54% of pts (115/215) overall and 67% (38/57), 62% (68/110), and 19% (9/48), with median duration of blast clearance of 6.3, 6.9, and 5.4 mos for Cohort A, B, and C, respectively. Only 30 pts (8%) received allo-SCT (5%, 16%, and 3% in Cohort A, B, and C, respectively). Death during follow-up was reported in 80% (n=294) of pts with mOS of 7.3 mos (95% confidence interval [CI] 6.5-8.3). mOS estimate was 7.4 mos (95% CI 6.0-8.8) for Cohort A, 9.4 mos (95% CI 7.2-10.4) for Cohort B and 5.9 mos (95% CI 4.3-7.5) for Cohort C. Survival was much higher in pts receiving allo-SCT, which only few pts in each cohort received (Table). Addition of venetoclax to HMA (Cohort A) did not improve OS compared with HMA only (Cohort C) in an adjusted Cox model (adjusted hazard ratio [aHR]=0.9, 95% CI 0.7-1.3). In a similar analysis, no difference in OS was observed between Cohort A and B (aHR=0.9, 95% CI 0.6-1.4). Conclusions In this large cohort of AML pts with TP53m/17p del, poor OS is observed with current standard-of-care treatment options. Pts treated with venetoclax and HMA combination did not have significantly different OS compared with pts treated with HMA only or pts receiving intensive chemotherapy. Younger age, fewer comorbidities, and higher number of pts receiving allo-SCT could have influenced apparent longer OS in the intensive chemotherapy cohort that was no longer significant in an adjusted model. Despite recent advances, TP53m AML pts continue to experience poor outcomes highlighting the significant unmet medical need and the need for novel therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The spectrum of subclonal TP53 mutations in chronic lymphocytic leukemia: A next generation sequencing retrospective study.

Chronic lymphocytic leukemia (CLL) is a hematological disorder with complex clinical and biological behavior. TP53 mutational status and cytogenetic assessment of the deletion of the corresponding locus (17p13.1) are considered the most relevant biomarkers associated with pharmaco-predictive response, chemo-refractoriness, and worse prognosis in CLL patients. The implementation of Next Generation Sequencing (NGS) methodologies in the clinical laboratory allows for comprehensively analyzing the TP53 gene and detecting mutations with allele frequencies ≤10%, that is, "subclonal mutations". We retrospectively studied TP53 gene mutational status by NGS in 220 samples from 171 CLL patients. TP53 mutations were found in 60/220 (27.3%) samples and 47/171 (27.5%) patients. Interestingly, subclonal mutations could be detected in 31/60 samples (51.7%) corresponding to 25 patients (25/47, 53.2%). We identified 44 distinct subclonal TP53 mutations clustered in the central DNA-binding domain of p53 protein (exons 5-8, codons 133-286). Missense mutations were predominant (>80%), whereas indels, nonsense, and splice site variants were less represented. All subclonal TP53 variants but one [p.(Pro191fs)] were already described in NCI and/or Seshat databases as "damaging" and/or "probably damaging" mutations (38/44, 86% and 6/44, 14%, respectively). Longitudinal samples were available for 37 patients. Almost half of them displayed at least one TP53 mutant subclone, which could be alone (4/16, 25%) or concomitant with other TP53 mutant clonal ones (12/16, 75%); different patterns of mutational dynamics overtimes were documented. In conclusion, utilization of NGS in our "real-life" cohort of CLL patients demonstrated an elevated frequency of subclonal TP53 mutations. This finding indicates the need for precisely identifying these mutations during disease since the clones carrying them may become predominant and be responsible for therapy failures.

Real-World Evidence on Therapeutic Strategies and Treatment-Sequencing in Patients with Chronic Lymphocytic Leukemia: An International Study of Eric, the European Research Initiative on CLL

Real-World Evidence on Therapeutic Strategies and Treatment-Sequencing in Patients with Chronic Lymphocytic Leukemia: An International Study of Eric, the European Research Initiative on CLL

Differences in genetics and microenvironment of lung adenocarcinoma patients with or without TP53 mutation

BackgroundDifferences in genetics and microenvironment of LUAD patients with or without TP53 mutation were analyzed to illustrate the role of TP53 mutation within the carcinogenesis of LUAD, which will provide new concepts for the treatment of LUAD.Methods In this study, we used genetics and clinical info from the TCGA database, including somatic mutations data, RNA-seq, miRNA-seq, and clinical data. More than one bioinformatics tools were used to analyze the unique genomic pattern of TP53-related LUAD.ResultsAccording to TP53 gene mutation status, we divided the LUAD patients into two groups, including 265 in the mutant group (MU) and 295 in the wild-type group (WT). 787 significant somatic mutations were detected between the groups, including mutations in titin (TTN), type 2 ryanodine receptor (RYR2) and CUB and Sushi multiple domains 3(CSMD3), which were up-regulated in the MU. However, no significant survival difference was observed. At the RNA level, we obtained 923 significantly differentially expressed genes; in the MU, α-defensin 5(DEFA5), pregnancy-specific glycoprotein 5(PSG5) and neuropeptide Y(NPY) were the most up-regulated genes, glucose-6-phosphatase (G6PC), alpha-fetoprotein (AFP) and carry gametocidal (GC) were the most down-regulated genes. GSVA analysis revealed 30 significant pathways. Compared with the WT, the expression of 12 pathways in the mutant group was up-regulated, most of which pointed to cell division. There were significant differences in tumor immune infiltrating cells, such as Macrophages M1, T cells CD4 memory activated, Mast cells resting, and Dendritic cells resting. In terms of immune genes, a total of 35 immune-related genes were screened, of which VGF (VGF nerve growth factor inducible) and PGC (peroxisome proliferator-activated receptor gamma coactivator) were the most significant up-regulated and down-regulated genes, respectively. Research on the expression pattern of immunomodulators found that 9 immune checkpoint molecules and 6 immune costimulatory molecules were considerably wholly different between the two groups.ConclusionsTaking the mutant group as a reference, LUAD patients in the mutant group had significant differences in somatic mutations, mRNA-seq, miRNA-seq, immune infiltration, and immunomodulators, indicating that TP53 mutation plays a crucial role in the occurrence and development of LUAD.

Abstract 3: Deep learning-based integration of esophageal cancer morphology with genomics

Abstract Esophageal cancer is a major cause of cancer mortality world-wide. Even with advanced treatment options available, patients with esophageal cancer have a poor 5-year survival rate of 16%. Genomic studies of esophageal cancer have revealed frequently mutated genes involved in different oncogenic pathways. In this study, we attempted to predict genomic alterations, pathological and clinical features directly from H&amp;E-stained whole slide images (WSIs) of individual esophageal cancer cases by utilizing modern deep learning methods. We used WSIs with esophageal cancer obtained from TCGA and annotated cancerous epithelial and stromal components separately for esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) (n= 25 cases for each) to create a training set. Subsequently, patches of size 256×256 pixels were extracted, and a U-Net convolutional neural network (CNN) was trained for semantic segmentation of tumor epithelium and stromal pixels. Tumor epithelium regions identified by the first U-Net CNN were fed into a Squeezenet model to learn to distinguish between EAC and ESCC in the training phase. Finally, for each subtype (EAC and ESCC), separate Densenet models were trained to predict gene mutation status. We also trained independent Densenet models to predict tumor stage, grade, and survival outcome from the tumor epithelial patches. After training, we tested these models on an independent validation set obtained from TCGA (n = 99) and UCSF (n = 25) patients, ensuring patient level separation between the training and the validation sets. We obtained an average AUC of 0.90 and 0.87 for epithelium vs. stromal and EAC vs. ESCC classification, respectively, on the validation datasets. These models enabled us to predict tumor stage, grade, and survival outcome with an accuracy of &amp;gt; 0.9. TP53 gene alterations were detected in over 50% of esophageal cancer cases, and our model was able to predict TP53 gene mutation status on WSIs with an AUC of 0.91. Overall, this study demonstrates how deep learning algorithms can aid in better understanding and predicting key biological and clinical features of aggressive malignancies at both population and individual patient level. This integration of artificial intelligence with molecular and histopathologic features could also help to accelerate precision cancer diagnosis and treatment in the clinical setting. Citation Format: Ruchika Verma, Wei Wu, Neeraj Kumar, Elizabeth Yu, Won-Tak Choi, Sarah Umetsu, Trever Bivona. Deep learning-based integration of esophageal cancer morphology with genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3.

Distinctive Clinicopathologic Features of Monomorphic B-cell Post-transplant Lymphoproliferative Disorders in Children.

Post-transplant lymphoproliferative disorders (PTLDs) comprise a heterogeneous group of Epstein-Barr virus (EBV)-positive or negative lymphoid or plasmacytic lesions in solid organ or hematopoietic stem cell (HSC) transplant recipients. Although PTLDs in adults have been extensively studied, the clinicopathologic features of monomorphic B-cell PTLD in children, particularly EBV-negative forms, are still poorly understood. We retrospectively reviewed all our pediatric cases of monomorphic B-cell PTLDs diagnosed in the past 10 years. Clinical data were reviewed. Pathologic data including histologic types and EBV status were analyzed. Additional immunohistochemical stains, FISH studies, and TP53 gene mutational status were performed. 4 of 18 cases were EBV-negative. All 4 EBV-negative cases were strikingly confined to the gastrointestinal (GI) tract or abdominal lymph nodes, while tumors in EBV-positive cases were found at various anatomic sites; 2 of 4 EBV-negative cases carried mutations in TP53 gene. Our cohort also included 2 rare types of PTLD, one plasmablastic lymphoma and one high-grade B-cell lymphoma, not otherwise specified (HGBL, NOS). We report that monomorphic B-cell PTLDs in children have distinctive clinical and pathological features. More studies are needed to clarify whether and how much these pediatric PTLDs differ from their adult counterparts.

Integrative prognostic models predict long-term survival after immunochemotherapy in chronic lymphocytic leukemia patients.

Chemoimmunotherapy with fludarabine, cyclophosphamide and rituximab (FCR) can induce long-term remissions in patients with chronic lymphocytic leukemia. Treatment efficacy with Bruton's tyrosine kinase inhibitors was found similar to FCR in untreated chronic lymphocytic leukemia patients with a mutated immunoglobulin heavy chain variable (IGHV) gene. In order to identify patients who specifically benefit from FCR, we developed integrative models including established prognostic parameters and gene expression profiling (GEP). GEP was conducted on n=337 CLL8 trial samples, “core” probe sets were summarized on gene levels and RMA normalized. Prognostic models were built using penalized Cox proportional hazards models with the smoothly clipped absolute deviation penalty. We identified a prognostic signature of less than a dozen genes, which substituted for established prognostic factors, including TP53 and IGHV gene mutation status. Independent prognostic impact was confirmed for treatment, β2-microglobulin and del(17p) regarding overall survival and for treatment, del(11q), del(17p) and SF3B1 mutation for progression-free survival. The combination of independent prognostic and GEP variables performed equal to models including only established non-GEP variables. GEP variables showed higher prognostic accuracy for patients with long progression-free survival compared to categorical variables like the IGHV gene mutation status and reliably predicted overall survival in CLL8 and an independent cohort. GEP-based prognostic models can help to identify patients who specifically benefit from FCR treatment. The CLL8 trial is registered under EUDRACT-2004-004938-14 and clinicaltrials gov. Identifier: NCT00281918.

Oncostatin M expression and TP53 mutation status regulate tumor-infiltration of immune cells and survival outcomes in cholangiocarcinoma.

In this study, we used bioinformatics tools to analyze transcriptome data from cholangiocarcinoma (CCA) patients in multiple datasets (Sun Yat-sen University, TCGA and GSE32225 cohorts) to identify mechanisms that regulate tumor infiltration by immune cells and survival outcomes. We identified 96 differentially expressed genes (DEGs), including 13 upregulated and 83 downregulated genes, in CCA tissues as regulatory T cells were significantly higher and the proportions of activated natural killer cells and monocytes were significantly lower in CCA tissues than the precancerous tissues. The survival outcomes of CCA patients were associated with the TP53 gene mutation status, levels of Oncostatin M (OSM) expression, and the proportions of tumor-infiltrating immune cell types, including dendritic cells, monocytes, and T follicular helper cells. Functional enrichment analysis of the DEGs in the high OSM-expressing CCA tissues showed that pathways related to tumor progression and immune response were significantly upregulated. Our study demonstrates that OSM expression and TP53 mutation status regulate the tumor infiltration by immune cells and survival outcomes in CCA. OSM is thus a potential prognostic biomarker and therapeutic target in cholangiocarcinoma.

Transcriptional profiling reveals a subset of human breast tumors that retain wt TP53 but display mutant p53-associated features.

TP53 gene mutations are very common in human cancer. While such mutations abrogate the tumor suppressive activities of the wild‐type (wt) p53 protein, some of them also endow the mutant (mut) protein with oncogenic gain of function (GOF), facilitating cancer progression. Yet, p53 may acquire altered functionality even without being mutated; in particular, experiments with cultured cells revealed that wtp53 can be rewired to adopt mut‐like features in response to growth factors or cancer‐mimicking genetic manipulations. To assess whether such rewiring also occurs in human tumors, we interrogated gene expression profiles and pathway deregulation patterns in the METABRIC breast cancer (BC) dataset as a function of TP53 gene mutation status. Harnessing the power of machine learning, we optimized a gene expression classifier for ER+Her2‐ patients that distinguishes tumors carrying TP53 mutations from those retaining wt TP53. Interestingly, a small subset of wt TP53 tumors displayed gene expression and pathway deregulation patterns markedly similar to those of TP53‐mutated tumors. Moreover, similar to TP53‐mutated tumors, these ‘pseudomutant’ cases displayed a signature for enhanced proliferation and had worse prognosis than typical wtp53 tumors. Notably, these tumors revealed upregulation of genes which, in BC cell lines, were reported to be positively regulated by p53 GOF mutants. Thus, such tumors may benefit from mut p53‐associated activities without having to accrue TP53 mutations.

Abstract LB-244: Prevention of ovarian cancer tumor establishment with mortalin targeting and p53 reactivator drugs

Abstract Background: The majority of high-grade serous ovarian cancer patients are diagnosed at late stage and recur after primary chemotherapy and cytoreductive surgery. Our objective was to test low-toxicity drug combinations to prevent ovarian cancer tumor establishment. SHetA2 is a novel small molecule drug that exhibited in vivo colorectal cancer prevention activity, reasonable oral bioavailability and pharmacokinetics and lack of toxicity in extensive preclinical studies. SHetA2 binding to its HSPA9 target (mortalin) causes release and nuclear accumulation of p53, which contributes to the mechanism of cancer cell death because ovarian cancer cells with missense p53 mutations are more resistant to SHetA2. Thus, we hypothesized that combination of SHetA2 with a p53 reactivator drug, PRIMA1MET would synergistically prevent ovarian cancer tumor establishment as a model of recurrence. Methods: Effects of SHetA2 and PRIMA1MET alone and in combination were evaluated in ovarian cancer cell lines with different TP53 gene mutation status. Immunocompromised mice were injected IP with MES-OV TP53 R282W ovarian cancer cells. Drug treatment was initiated 5 weeks prior to development of ascites as observed in a pilot study and in this intervention experiment. Animals were randomized to treatment groups (N=12: Controls, PRIMA1MET, SHetA2, SHETA2+PRIMA1MET) based on body weight. After 5 weeks treatment, animals were sacrificed and necropsied. Tissues are being evaluated for toxicity and to verify drug mechanism. Statistical Analysis (SAS 9.4) used tumor development as the primary outcome. Univariate Fisher’s exact test was used to compare outcome among groups. Multiple logistic regression models with, or without, interaction was used to assess synergistic and additive effects between the two drugs. Results: In cell culture studies, combinations of SHetA2 and PRIMA1MET exhibited synergy in the presence of missense p53 mutations and additive or synergistic activity in cultures with wild type or null p53. In the animal model, tumor-free rates were 0% in the control, 25% in the PRIMA1MET, 42% in the SHetA2, and 67% in the combination group. Univariate Fisher’s exact test revealed a significant difference between the control and SHetA2 groups (p=0.037) only. Logistic regression indicated that the interaction between SHetA2 and PRIMA1MET was not significantly synergistic (p=0.973). However, a subsequent additive model showed that SHetA2 (p=0.004, OR=10.384, 95% CI: 2.158, 48.965) and PRIMA1MET (p=0.048, OR=4.464, 95% CI: 1.014, 19.655) functioned additively in preventing tumor development. No evidence of gross toxicity was observed. Conclusion: SHetA2 alone, and in combination with PRIMA1MET, significantly prevented ovarian cancer tumor establishment without evidence of toxicity warranting further study in clinical trials. Funding: R01 CA196200, FWC-Ovarinnovate: Ovarcome Research Excellence Award Citation Format: Satish Kumar Ramraj, Zitha Redempta Isingizwe, Sugantha Priya Elayapillai, Amy L. Bosley, Yan Daniel Zhao, Doris M. Benbrook. Prevention of ovarian cancer tumor establishment with mortalin targeting and p53 reactivator drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-244.

Clinicopathological significance and prognostic value of intratumoral and peritumoral lymphocytes in endometrial cancer patients.

e17116 Background: The characterization and prognostic relevance of immune cells in the tumor microenvironment of endometrial cancer (EC) remain unknown. Our aims are to analyze the presence of tumor infiltrating lymphocytes (TIL) and peritumoral lymphocytes (PTL) in tumoral tissue of patients with EC, and to identify the correlation between TILs and PTLs subsets with clinicopathologic features and its prognostic value Methods: CD3, CD4, CD8, CD20, and FOXP-3 was determined by immunohistochemestry (IHQ). A 4-point score was defined based on TIL counts per highpowered field: (negative, low, moderate, and high). We used 10% positive peritumoral lymphocytes as a low-high cutoff value. POLE mutation was identified by Sanger sequencing of the exonuclease domain (exons 9-14). Analysis of mismatch repair expression and TP53 gene mutation status were assessed. Results from IHQ and analysis mutation were correlated with clinicopathological parameters and survival. Results: We recovered tumor samples from 68 FIGO stage I–IV EC patients. POLE mutations were identified in 5 of 44 (11.4%) EC analyzed. Microsatellite instability (MSI) and TP53 mutation were found in 45% and 25 % of cases respectively. According PTL, MSI tumors were significantly associated with low CD4+ (p = 0.01). High CD8+ was significantly associated with endometrioid grade 1-2 tumors (p = 0.04). Low FOXP3+ was significantly associated with endometrioid grade 1-2 (p = 0.02), MSS tumors (p &lt; 0.01), FIGO stage I-II (p &lt; 0.01), POLE WT (p &lt; 0.01), TP53 WT (p &lt; 0.01), and negative lymphovascular space invasion (p &lt; 0.01). Negative CD20+ TIL was associated with endometrioid grade 1-2 tumors (p &lt; 0.01) and ≥50% myometrial invasion (p = 0.03). Moderate CD8+ TIL was associated with lower tumor stage (p = 0.01). High CD8+ TIL was associated with better 5-year overall survival (OS) rate (high: 100 % vs. low: 53%; p = 0.003). No significant association was observed between POLE status, TP53 status, MMR expression and survival. Conclusions: Regulatory and cytotoxic T cells subsets differs in EC patients. High CD8+ TILs was significantly associated with better 5-year OS.

Time series analysis of TP53 gene mutations in recurrent HPV-negative vulvar squamous cell carcinoma

The impact of TP53 gene mutations in recurrent HPV-negative vulvar squamous cell carcinomas is unclear. TP53 gene mutations were analyzed in archival tissues of 24 primary squamous cell carcinoma and local vulvar recurrences arising in chronic inflammatory dermatoses by analyzing the full coding sequence of the TP53 gene and correlated with disease-free survival. After resection of the primary squamous cell carcinoma with clear margins 19/24 patients had one and 5/24 had multiple recurrences. The first recurrence occurred after median of 46 months (range 12–180 months). In all, 17/24 (71%) primary squamous cell carcinomas had TP53 gene mutations and recurred after median disease-free intervals of 33 months (range 12–180). 14/17 (88%) recurrent squamous cell carcinomas carried again TP53 gene mutations, five with identical and nine with different, more complex TP53 gene mutations. 7/24 (29%) patients with a p53 wild-type primary SCC had the first recurrence after median 65 months (range 14–144) featuring p53 wild-type in 3/7 (43%) and TP53 gene mutations in 4/7 (57%) recurrent squamous cell carcinomas. Disease-free intervals of > 5 years (60–180 months) were observed in 10/24 patients total (42%; equally divided among p53 wild-type (5/7; 71%) and TP53 gene mutated (5/17; 29%) squamous cell carcinomas). In summary, squamous cell carcinomas recurred in the residual vulvar dermatosis independent of TP53 gene mutational status of the primary squamous cell carcinoma. The majority of TP53 gene mutated cancers recurred with different TP53 gene mutations, some of them more complex, and patients with p53 wild type developed TP53 gene mutations in the recurrent squamous cell carcinomas, possibly indicating increased genetic instability in longstanding chronic inflammatory dermatoses. A change of TP53 gene mutational status after > 5 years suggests de novo oncogenic events/carcinogenesis. Longer disease-free intervals in patients with p53 wild-type primary squamous cell carcinoma suggest that TP53 gene mutational status may serve as a prognostic marker for disease-free intervals.