DNA-binding Domain Of TP53 Research Articles

Abstract PO5-15-02: Integration of synonymous mutations and variants of unknown significance for basal and longitudinal characterization of metastatic breast cancer by circulating tumor DNA

Abstract Background: Despite how commonly synonymous mutations (SYN) and unknown significance (VUS) are detected in tissue and circulating tumor DNA (ctDNA) next-generation sequencing (NGS), the potential for these somatic changes to impact personalized approaches in metastatic breast cancer (MBC) is unclear. Emerging evidence suggests that SYN mutations may play a role in cancer biology through changes in mRNA stability, splicing, and gene expression. The aim of this study was to characterize SYN and VUS in a large multicenter consortium and to analyze their interplay with pathogenic variants (PATH). Methods: The study retrospectively analyzed a multi-institutional cohort comprising 1189 patients with MBC characterized for ctDNA using NGS (Guardant360™, Guardant Health) before treatment start (BL) and at disease progression (PD). Pathway classification was defined based on prior research (Sanchez-Vega F et al, Cell. 2018). Single Nucleotide Variations (SNVs) were annotated for oncogenicity (OncoKB) and protein domain (UniProtKB). Associations between clinical characteristics and pathway classifications for SYN and VUS were explored by multinomial logistic regression; survival was tested through Cox regression in terms of overall survival (OS). Results: Hormone-receptor positive (HR+)/HER2 negative (luminal-like) was the most represented subtype (68.7%) followed by HER2-positive (14.6%), and triple-negative (TNBC) (16.7%). PATH were mainly detected in the TP53 DNA binding domain, ESR1 ligand binding domain, helical and catalytic domains of PIK3CA, SYN in the ligand binding domain of ESR1, MET Sema, KIT protein kinase domain, and FGFR1 Ig-like C2-type 1. In contrast, VUS were mainly found in the ESR1 ligand binding domain, MET Sema, ATM PI3K/PI4K catalytic domain, DDR2 Protein kinase and PIK3CA C2 PI3K-type. Analyzing pathways, after multivariable multinomial logistic regression, RAS SYN, PI3K and ESR PATH were significantly associated with HER2+ MBC (respectively RRR=4.14, P=0.046; RRR = 0.474, P < 0.001 and RRR = 0.306, P < 0.001). TNBC was associated with PATH PI3K (RRR = 0.366, P < 0.001), PATH P53 (RRR = 4.71, P < 0.001) and PATH RAS (RRR = 0.362, P = 0.026, 95% CI 0.147 - 0.888). In luminal-like MBC, a significant impact on OS was observed for SYN P53 (HR = 2.53, P = 0.008) and PATH ER (HR = 1.54, P < 0.001), PATH P53 (HR = 1.56, P < 0.001), PATH cell-cycle (HR = 1.64, P = 0.0299), and PATH RAS (HR = 1.62, P = 0.010). An impact on OS was observed in TNBC for SYN cell-cycle (HR = 5.41, P = 0.006), VUS ESR (HR = 2.87, P = 0.018), PATH P53 (HR = 1.84, P = 0.012) and PATH cell-cycle (HR = 5.07, P < 0.001). VUS MYC (HR = 38.5, P = 0.001) and PATH PI3K3 (HR = 2.32, P = 0.022) were significant among HER2 positive. Differences in SYN, VUS and PATH alterations were then compared between baseline and progressive disease. While a significant increase in mutant allele frequency (MAF) was observed for PATH (P=0.0010) and VUS (P=0.0152) at PD, no differences were highlighted for SYN (P=0.8362). Conclusions: In our cohort we characterized the MBC for PATH, VUS and SYN utilizing the ctDNA. In this study, considering the complex genomic landscape of MBC, we demonstrated that both SYN and VUS mutations had an impact on the survival outcome. Therefore, further analysis to validate the prognostic and predictive role of SYN and VUS, with or without PATH mutations, are needed to better characterize and monitoring of MBC through ctDNA. Moreover, functional studies are needed to better understand their role in the nuanced biology of MBC. Citation Format: Elisabetta Molteni, Carolina Reduzzi, Andrew Davis, Lorenzo Foffano, Arielle Medford, Katherine Clifton, Whitney L. Hensing, Marko Velimirovic, Ami N. Shah, Laura Munoz Arcos, Charles S. Dai, Jennifer C. Keenan, Elyssa Denault, William Gradishar, Giuseppe Damante, Amir Behdad, Lorenzo Gerratana, Fabio Puglisi, Cynthia Ma, Aditya Bardia, Massimo Cristofanilli. Integration of synonymous mutations and variants of unknown significance for basal and longitudinal characterization of metastatic breast cancer by circulating tumor DNA [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-15-02.

Recurrent Mutations in Refractory/Relapsed Diffuse Large B-Cell Lymphoma by Targeted Gene Sequencing

Introduction: Whole-genome sequencing of diffuse large B-cell lymphoma (DLBCL) has identified recurrent mutations involved in pathogenesis and potentially affecting response to therapy. In this pilot study, a targeted gene panel was created to identify mutations associated with relapse/refractoriness. Material and Methods: A 14-gene targeted panel was designed to sequence thirteen patients who were in remission and 8 cases that had relapsed/refractory to treatment. A paired diagnostic biopsy and a relapse biopsy were sequenced to find genes repeatedly altered in relapse. Results: A total of 751 nonsynonymous and truncating mutations were identified. Truncated mutations in NOTCH1, TNFAIP3, and CD58 were associated with poor treatment outcomes. In cases that did not respond to treatment, a high number of mutations were found in the EZH2 gene, followed by the DNA-binding domain of TP53 and MYD88. Termination mutations in the intracellular domain of NOTCH were found in 75% of non-responsive cases. Co-occurrence of loss of function mutations of TNFAIP3 and missense mutations in MYD88 was associated with a non-responsive cohort. Discussion: The study highlights mutations associated with chemotherapeutic response in DLBCL with implications for initial diagnostic biopsy response prediction.

Functional pri-miR-34b/c rs4938723 and KRAS 3′UTR rs61764370 SNPs: Novel phenotype modifiers in Li-Fraumeni Syndrome?

PurposeLi-Fraumeni Syndrome (LFS) is a rare cancer predisposing condition caused by germline pathogenic TP53 variants, in which core tumors comprise sarcomas, breast, brain and adrenocortical neoplasms. Clinical manifestations are highly variable in carriers of the Brazilian germline founder variant TP53 p.R337H, possibly due to the influence of modifier genes such as miRNA genes involved in the regulation of the p53 pathway. Herein, we investigated the potential phenotypic effects of two miRNA-related functional SNPs, pri-miR-34b/c rs4938723 and 3′UTR KRAS rs61764370, in a cohort of 273 LFS patients from Southern and Southeastern Brazil. MethodsThe genotyping of selected SNPs was performed by TaqMan® allelic discrimination and subsequently custom TaqMan® genotyping results were confirmed by Sanger sequencing in all SNP-positive LFS patients. ResultsAlthough the KRAS SNP showed no effect as a phenotype modulator, the rs4938723 CC genotype was significantly associated with development of LFS non-core tumors (first tumor diagnosis) in p.R337H carriers (p = 0.039). Non-core tumors were also more frequently diagnosed in carriers of germline TP53 DNA binding domain variants harboring the rs4938723 C variant allele. Previous studies described pri-miR-34b/c rs4938723 C as a risk allele for sporadic occurrence of thyroid and prostate cancers (non-core tumors of the LFS spectrum). ConclusionWith this study, we presented additional evidence about the importance of analyzing miRNA genes that could indirectly regulate p53 expression, and, therefore, may modulate the LFS phenotype, such as those of the miR-34 family.

Abstract C005: TP53 wildtype status can predict sensitivity to XPO1 inhibitors in Patient-Derived Cancer Models

Abstract Introduction: Selinexor (SEL) and eltanexor (ELT) are selective inhibitors of exportin 1 (XPO1), that lead to the nuclear accumulation and activation of key tumor suppressors (e.g. p53, p21, RB, and FOXO), resulting in selective cancer cell death. In the SIENDO trial of SEL maintenance for patients with advanced stage or relapsed endometrial cancer (EC), SEL reduced disease progression compared to placebo, with a more pronounced reduction seen in patients with TP53 wild-type (WT) EC (Makker et al, ASCO 2022). As TP53 is the most commonly mutated gene in human cancers, and p53 is a cargo of XPO1, we investigated whether TP53 wild type (WT) status could serve as a mechanistic predictor for cancer cell sensitivity to XPO1 inhibitors. Methods: PDO and PDX models representing 17 cancer types, each with duplicate TP53 WT and MUT models were used (4 models per cancer type), with an emphasis on models with common hotspot MUTs. Tumor types were included if biological replicate models with TP53 wild-type or TP53 mutations were available for comparison. SEL, ELT and a control agent (CDDP or staurosporine) IC50 values after 5 days of exposure were calculated using CellTiter Glo assay. Sensitivity to SEL and ELT was considered TP53 WT dependent for each cancer type if the IC50 values for the TP53 MUT models were at least 1.5x higher than the average IC50 for the TP53 WT models. Similarly, the IC50 of SEL at 72h was determined for HCT116 WT and R175H cells to directly evaluate the association between TP53 MUT and resistance to XPO1 inhibitors in a syngeneic background. Results: In PDX/PDO models, sensitivity to SEL and/or ELT was determined to be TP53 WT dependent in 9 cancer types (endometrial, ovarian, kidney, liver, esophageal, lung adeno and squamous, pancreatic, and bladder). For example, the 2 TP53 WT EC PDX tested were ~20 fold more sensitive to SEL and ELT than 2 EC PDX with mutations within the TP53 DNA binding domain (TP53 WT SEL IC50 = 31nM and 61nM; p.Y220C SEL IC50= 333nM; p.R213* SEL IC50 = 1542nM). Interestingly, the IC50 of SEL and ELT were lower than CDDP for the TP53 WT EC models, as well as in the kidney cancer models. For the remaining 8 indications (head and neck, gastric​, gallbladder​, cholangiocarcinoma, cervical​, breast​, mixed mullerian​, prostate​​), XPO1 inhibitor sensitivity was TP53 WT independent. Reduced sensitivity to SEL was also observed in syngeneic HCT116 cells with the TP53 R175H (IC50=343 nM) MUT compared to WT (IC50=174 nM). Notably, TP53 MUT status conferred resistance to the control agents in only models of two cancer types (esophageal and lung adenocarcinoma), demonstrating specificity between TP53 MUT status and XPO1 inhibitors. Conclusions: Using TP53 WT status as a biomarker of sensitivity to XPO1 inhibitors SEL and ELT is cancer-type specific. Further experiments are ongoing to validate these findings in CRISPR-engineered SNG-M EC cells with clinically relevant R175H and R248Q TP53 hotspot mutations, including experiments related to cancer-type specific SEL/ELT mechanism of action. Citation Format: Marie Maloof, Andrea Ellero, Pietro Taverna, Christopher J Walker. TP53 wildtype status can predict sensitivity to XPO1 inhibitors in Patient-Derived Cancer Models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C005.

A Genomic Approach for High-Grade B Cell Lymphoma with MYC and BCL2 and / or BCL6 Rearrangements: Two Clusters Stood out on Next Generation Sequencing Results

Introduction High-grade B cell lymphomas with double or triple hit rearrangement (HGBL DH/TH), which appeared on the 4th revised edition WHO classification of lymphoid neoplasms, are aggressive mature B-cell lymphomas with high-grade cytology (Burkitt-like, blastoid or DLBCL-like) that have a MYC (8q24) rearrangement in combination with a BCL2 (18q21) and / or a BCL6 (3q27) rearrangement. There are currently no treatment guidelines for this subtype, and the usual immunochemotherapy leads to few complete remissions. While the NGS technology has been used in diffuse large B cell lymphoma, there has been no specific study on additional genetic events which would predict the clinical outcome in HGBL DH/TH. Here, we report our preliminary results using a targeted DNA capture panel approach (NGS) to identify subgroups with different clinical outcomes among HGBL DH/TH. Methods We conducted a retrospective study on 44 patients diagnosed with DH or TH HGBL and treated at Centre Hospitalier Lyon Sud or Centre Léon Bérard starting in 2013. We collected the medical information regarding the treatment, time to response to treatment or relapse, progression free survival, and overall survival. They all benefited from a DNA-targeted NGS analysis covering 54 genes. Results The cohort included 20 females and 24 males, with a median age of 65 (31-87). Among them, 22 patients were MYC/ BCL2 DH (50%), 12 were MYC/ BCL6 DH (27,3%) and 10 were TH (22,7%). Eleven patients had B symptoms (25%). The Ann Arbor stage was high (III-IV) in 97.5%. The WHO Performance Status was low (0-1) for most of the patients (70%). The majority of them presented with an IPI above to 2 (82,5%) and an elevated LDH level (80%). The Ki67 index was above 70% (33/42) in 78,5% of the cases and above 90% (19/42) in nearly half of them. The major finding of the NGS analysis was the identification of 2 groups based on the TP53/ CDKN2A_p14ARF mutational status. The CDKN2A locus encodes for 2 alternative open reading frames leading to 2 unrelated proteins p14ARF and p16INK4A. P14ARF is involved in the regulation of the TP53 pathway whereas p16INK4A is not. We therefor defined 2 groups : the first group included patients with either TP53 or CDKN2A_p14ARF-only alterations (group A, n=10) while the second group had no alteration in these two coding sequences (group B, n=34) although 12 patients had a CDKN2A_p16INK4A loss or mutation affecting both CDKN2A transcripts. Most of TP53 mutations were missense mutations mostly in TP53 DNA-binding domain and 2 cases appeared to be single hit mutations with an allele frequency below 50%. The patient with a p14ARF only mutation had a start codon mutation p.(M1*) with an allele frequency of 96% without CDKN2A copy number loss. The median overall survival (OS) (Figure 1) was 6,2 months for group A, and 55,5 months for group B (p = 0,0076). The median follow-up time was 14 months (7,5 months in the group A, 18 in group B). Only 2 patients in group A survived and are still in complete remission, one was DH BCL6 and the other DH BCL2 Conclusion Our preliminary results indicate a strong negative prognostic impact of either CDKN2A_p14ARF or TP53 gene alterations on the OS of HGBL DH/TH patients. The biological basis for the difference between groups would be in favour of the alteration of the p14ARF-TP53 tumor suppressive pathway as major contributor of chemoresistance. A more detailed analysis is on-going to further identify mutation profiles of double hit and triple hit lymphomas and compare our finding with existing data. Our results confirm the more favourable outcome of DH BCL6 patients (data not shown), which is consistent with the 5 th revised OMS classification that removed that specific entity. Our results also indicate that routine NGS analysis of HGBL DH/TH patients offers new perspectives for patient stratification. Regarding their survival, patients with TP53 pathway alterations should probably not be treated with the standard RCHOP chemo-immunotherapy. Instead, a more intensive immunochemotherapy regimen or novel strategies such as first-line bispecific antibodies or CARTCells might be considered.

Plasma cell-free tumor DNA, PIK3CA and TP53 mutations predicted inferior endocrine-based treatment outcome in endocrine receptor-positive metastatic breast cancer

PurposeHow to factor both tumor burden and oncogenic genomic mutations as variables to predict the outcome of endocrine-based therapy (ET) in ER-positive/HER2-negative metastatic breast cancer patients (MBC) remains to be explored.MethodBlood samples prospectively collected from 163 ER-positive/HER2-negative female MBC patients, before ET, were used for cell-free tumor DNA (cfDNA) analysis. cfDNA was subjected to next-generation sequencing (NGS) to interrogate oncogenic PIK3CA hotspot and TP53 DNA-binding domain (DBD) mutations, including single nucleotide variants (SNVs) or small insertions and deletions (InDels). The variant calling threshold was set at 0.5%. Progression-free survival (PFS) was measured from the start of the ET treatment to the time of disease progression of the same treatment regimen.ResultsOverall, the median PFS was 8.3 months (95% CI 5.7–11.1 months). The median cfDNA was 38.5 ng (range 4.4–1935 ng). The proportion of patients with PIK3CA and TP53 alterations were 25.1 and 15.3%, respectively. Patients with high total cfDNA (HR 1.74, p = 0.003), PIK3CA mutation (HR 1.74, p = 0.007), and TP53 mutation (HR 1.64, p = 0.047) in liquid biopsy conferred worse outcome after ET. Even for patients with low tumor burden, the detrimental effect of PIK3CA or TP53 mutation remained significant (p < 0.001). For patients with either PIK3CA (p < 0.001) or TP53 mutation (p = 0.004), there was significant positive correlation between allele frequency (AF) and total cfDNA.ConclusionAfter adjustment of cfDNA level, PIK3CA and TP53 mutations observed in liquid biopsy exerted detrimental effects on the outcome of ET-based regimens. The AF of PIK3CA or TP53 may be a surrogate marker for PFS.

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin β4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4+ CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling.

Comparative genomic analysis and its prognostic impact on survival between viral hepatitis-related and non-viral hepatitis intrahepatic cholangiocarcinoma.

4120 Background: Intrahepatic cholangiocarcinoma (IHCC) is hard-to-treat cancer with a high mortality rate worldwide. Hepatitis B virus (HBV) or hepatitis C virus (HCV) is involved in the development of IHCC, especially in Asian countries. Despite rapidly growing genomic profiling studies of IHCC in Western and Eastern populations in recent years, less on genomic heterogeneity of viral hepatitis-related and non-viral hepatitis IHCC been reported. This study aims to provide a comprehensive genomic analysis of IHCC and its prognostic value in IHCC populations with or without viral hepatitis infection. Methods: FFPE tissues from 157 patients with IHCC were subject to next-generation sequencing using the FoundationOne CDx (n = 52) or ACTOnco + comprehensive genomic profiling panels (n = 105). Genomic alterations and features, including single nucleotide variations (SNVs), short insertions and deletions (InDels), copy-number variations (CNVs), fusion genes (only FoundationOne CDx), tumor mutations burden (TMB), and microsatellite instability (MSI) status, were analyzed. The prevalence of genetic mutations and their prognostic values were compared between patients with a history of hepatitis B/C infection (BC; n = 71, 45.2%) and those without hepatitis B/C infection (NBNC; n = 79, 50.3%). Seven patients had no medical record of HBV or HCV infection. Results: The most frequently mutated genes in BC- and NBNC-related IHCC populations were TP53, KRAS, and IDH1. The genetic alternations related to PI3K/AKT/mTOR signaling pathway and copy number gain of receptor tyrosine kinase were relatively dominant in BC-related IHCC. In contrast, the Ras/Raf/MAPK pathway alterations were frequently common in NBNC-related IHCC. In addition, we investigated the correlation between commonly altered genes or pathways and overall survival (OS). Interestingly, the prognostic biomarkers in BC- and NBNC-related IHCC were significantly different. The results showed that TP53 DNA-binding domain (DBD) and TET2 mutations were associated with poor prognosis in BC-related IHCC. On the contrary, CDKN2A deletion and Ras/Raf/MAPK pathway alteration were associated with inferior prognosis in NBNC-related IHCC. However, neither the PI3K/AKT/mTOR signaling alteration nor IDH1/2 mutation affected the OS in BC- or NBNC-related IHCC. Notably, we found that SWI/ SNF complex involved in ARID1A, ARID1B, ARID2, PBRM1, and SMARCA4 alterations exhibited a beneficial prognosis in BC-related IHCC patients, which has not been discussed in previous studies. Conclusions: This study provides comparative genomic profiling between BC- and NBNC-related IHCC and shows genomic heterogeneity and different dominant prognostic biomarkers and activated signaling pathways. Different treatment strategies should be considered in these two subpopulations based on these results.

Sex- and mutation-specific p53 gain-of-function activity in gliomagenesis.

In cancer, missense mutations in the DNA-binding domain of TP53 are common. They abrogate canonical p53 activity and frequently confer gain-of-oncogenic function (GOF) through localization of transcriptionally active mutant p53 to non-canonical genes. We found that several recurring p53 mutations exhibit a sex difference in frequency in patients with glioblastoma (GBM). In vitro and in vivo analysis of three mutations, p53R172H, p53Y202C, and p53Y217C revealed unique interactions between cellular sex and p53 GOF mutations that determined each mutation's ability to transform male versus female primary mouse astrocytes. These phenotypic differences were correlated with sex- and p53 mutation- specific patterns of genomic localization to the transcriptional start sites of upregulated genes belonging to core cancer pathways. The promoter regions of these genes exhibited a sex difference in enrichment for different transcription factor DNA-binding motifs. Together, our data establish a novel mechanism for sex specific mutant p53 GOF activity in GBM with implications for all cancer.

Non-disruptive mutation in TP53 DNA-binding domain is a beneficial factor of esophageal squamous cell carcinoma.

BackgroundTP53 is frequently altered in esophageal squamous cell carcinoma (ESCC). However, the landscape of TP53 mutation and its effects on patients remain controversial.MethodsSomatic mutations of TP53 in 161 patients with resectable ESCC were identified by next-generation sequencing (NGS) and verified by immunohistochemistry (IHC). Patients were stratified into seven TP53 mutations, and depending on the extent of the effect on the encoded protein, it was divided into “disruptive” and “non-disruptive” types. The association of TP53 mutation with clinicopathological properties and disease outcome was investigated.ResultsTP53 mutations were discovered in 85.7% patients, of which 68.9% carried mutations in the DNA-binding domain (DBD). A total of 47.8% and 37.9% patients had disruptive and non-disruptive TP53 mutations, respectively. Most patients carried only one TP53 mutation, but 15.5% had double mutations. TP53 mutations were dominant in exons 5 to 8. Missense mutation was the most frequent (97/163, 59.5%), and the top five frequently occurring variations included R273X, Y220X, H193, H179X, and R175H. Multivariable analysis revealed non-disruptive mutation in TP53 DBD as the independent prognostic predictor for progression-free survival (PFS) and overall survival (OS). The expression of p53 positively correlated with non-disruptive mutation in DBD. Patients with high p53 protein expression showed better outcomes.ConclusionsNon-disruptive mutation in TP53 DBD serves as an independent beneficial prognostic factor of prolonged survival in resectable ESCC.

Combining Ramachandran plot and molecular dynamics simulation for structural-based variant classification: Using TP53 variants as model

The wide application of new DNA sequencing technologies is generating vast quantities of genetic variation data at unprecedented speed. Developing methodologies to decode the pathogenicity of the variants is imperatively demanding. We hypothesized that as deleterious variants may function through disturbing structural stability of their affected proteins, information from structural change caused by genetic variants can be used to identify the variants with deleterious effects. In order to measure the structural change for proteins with large size, we designed a method named RP-MDS composed of Ramachandran plot (RP) and Molecular Dynamics Simulation (MDS). Ramachandran plot captures the variant-caused secondary structural change, whereas MDS provides a quantitative measure for the variant-caused globular structural change. We tested the method using variants in TP53 DNA binding domain of 219 residues as the model. In total, RP-MDS identified 23 of 38 (60.5%) TP53 known Pathogenic variants and 17 of 42 (41%) TP53 VUS that caused significant changes of P53 structure. Our study demonstrates that RP-MDS method provides a powerful protein structure-based tool to screen deleterious genetic variants affecting large-size proteins.

Functional Classification of TP53 Mutations in Acute Myeloid Leukemia

In more than 50% of human cancers, somatically acquired aberrations of the tumor suppressor gene TP53 are encountered. Approx. 10% of patients with acute myeloid leukemia (AML) reveal TP53 mutations with higher incidences in therapy-related subtypes and erythroleukemias. These mutations are regarded early events of leukemogenesis. In contrast to mere deletions at the TP53 locus, TP53 mutations confer an exceedingly adverse prognosis in AML even when occurring at a subclonal level. However whether different TP53 mutations in AML exhibit a different functional impact on disease progression or outcome remains unknown. In the present study, we have investigated this issue using four TP53 specific, functional scoring systems in a large cohort of patients of the German-Austrian AML study group (AMLSG). The AMLSG cohort consisted of a total of 1537 patients with newly diagnosed AML who were intensively treated within three multicenter, clinical trials. A total of 108 TP53 mutations were detected in 98 patients using targeted amplicon sequencing - 88 (81.4%) missense, 8 (7.4%) nonsense and 6 (5.6%) splice site mutations as well as 6 small insertions and deletions. For each of the four functional TP53 scores, we have assessed their impact on overall survival (OS) and event-free survival (EFS). First, we compared the impact of TP53 missense mutations in 84 patients with all other types of mutations (n= 14). In a next approach, TP53 mutations were grouped into disruptive (n=42) and non-disruptive (n=56) ones, based on the impact of the particular mutations on the protein structure predicted from the crystal structure of p53-DNA complexes (Poeta et al. New Engl J Med 2007). We then classified missense TP53 mutations (n=84) based on their "Evolutionary Action Scores (EAp53)" (Neskey et al. Cancer Res 2015). This algorithm takes evolutionary sensitivity and amino acid conservation into account and scores missense TP53 mutations from 0 to 100. Mutations with the high EAp53 score are considered high risk whereas wild type TP53 has an EAp53 score of zero. We extracted the EAp53 score of those AMLSG patients showing missense mutations from the respective server (http://mammoth.bcm.tmc.edu/EAp53) and used the threshold of 75 from the initial publication to divide the patients into low-risk (&lt;75, n=49) and high-risk groups (≥75, n=35). However, with these three functional scoring systems, no difference regarding OS and EFS could be shown between the mutational groups. The "Relative Fitness Score (RFS)" was recently developed for the TP53 DNA binding domain (DBD) mutants as an indicator of their functional impact (Kotlar et al. Molecular Cell 2018). A catalogue of 9833 TP53 DBD mutants were generated and their selective growth was assessed in p53 null cancer cell lines. The RFS score for each mutant is the median of its relative enrichment or depletion in culture, calculated at 3 time points and depicted as a log (base 2) value. A high RFS indicates selective growth of the mutant corresponding to its higher fitness. We extracted the RFS for the TP53 DBD mutations of the AMLSG cohort (n=83) using the online data resource (GSE115072) and performed a receiver-operating characteristics (ROC) analysis to calculate the optimal threshold of RFS separating deceased from survivors most efficiently. Thereby, the best RFS cut-off value according to the Youden index was -0.135. Applying this threshold (low-risk RFS ≤ -0.135, n=25; high-risk RFS &gt; -0.135, n= 58) we demonstrated a significantly better OS (P=0.009) and EFS (P=0.037) for patients with a low-risk RFS in multivariable analyses adjusting for age, white blood cell count, cytogenetics and type of AML. Using the AML-specific TP53 RFS score, we could show that more than 30% of patients with TP53 mutations (25/83) reveal a significantly better survival indicating that this score is of prognostic value. Based on these findings, we propose that - along with clinical parameters - RFS values of TP53 mutations should also be considered for a comprehensive risk assessment of TP53 mutated AML patients. Disclosures Zebisch: Celgene: Honoraria; Novartis: Honoraria, Other: Advisory board; AbbVie: Other: Advisory board; Roche: Honoraria. Bullinger:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Daiichi Sankyo: Honoraria; Gilead: Honoraria; Hexal: Honoraria; Menarini: Honoraria; Novartis: Honoraria; Astellas: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Seattle Genetics: Honoraria; Bayer: Other: Financing of scientific research; Abbvie: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Jazz Pharmaceuticals: Honoraria. Döhner:Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria; CTI Biopharma: Consultancy, Honoraria. Sill:Novartis: Other: Advisory board; AbbVie: Other: Advisory board; Astellas: Other: Advisory board; Astex: Other: Advisory board.

TP53 DNA Binding Domain Mutations Predict Progression-Free Survival of Bevacizumab Therapy in Metastatic Colorectal Cancer.

(1) Background: Bevacizumab-based regimens are a standard treatment for metastatic colorectal cancer (mCRC) patients, however meaningful clinical biomarkers for treatment benefit remain scarce. (2) Methods: Tumor samples from 36 mCRC patients treated with bevacizumab-based chemotherapy underwent comprehensive genomic profiling. Alterations in frequently altered genes and important signaling pathways were correlated with progression-free survival (PFS). (3) Results: Overall genetic alteration analysis of investigated genes and pathways did not identify promising new predictors of PFS. However, when considering mutation subtypes, TP53 DNA binding domain (DBD) missense mutations were associated with prolonged PFS (HR, 0.41; 95% CI, 0.13−0.65; p = 0.005). In contrast, TP53 truncating mutations were associated with short PFS (HR, 2.95; 95% CI, 1.45−27.50; p = 0.017). Importantly, neither TP53 mutation subtype was associated with overall response rate. In multivariate analysis, TP53 DBD missense mutations remained an independent PFS predictor (HR, 0.31; 95% CI, 0.13–0.77; p = 0.011). The other genetic factor independently associated with PFS were PTPRT/PTPRD deleterious alterations, which we previously identified in a screen for biomarkers of bevacizumab response. (4) Conclusions: TP53 DBD missense mutations may predict prolonged PFS in mCRC patients treated with bevacizumab-based therapy. Analyses of TP53 mutations as clinical biomarkers should take the biological impact of different mutation subtypes into consideration to improve patient stratification.

Abstract 4511: Prostate cancer-specific enrichment of TP53 missense mutations elicits differential, context-dependent biochemical and biologic outcomes

Abstract Prostatic adenocarcinoma (PCa) remains the second leading cause of cancer death in men in the United States. While organ-confined disease can be successfully treated with surgery or radiation, patients with advanced disease undergo androgen deprivation therapy (ADT), which targets the androgen receptor (AR) signaling axis. This approach effectively treats the disease as PCa cells are exquisitely dependent on AR signaling for proliferation and survival. Unfortunately, cancer progression resumes after 2-3 years and results in a lethal stage of disease, termed castration-resistant prostate cancer (CRPC). While PCa typically demonstrates low overall mutational burden, TP53 is frequently mutated in both primary and advanced disease. These mutations are most commonly missense mutations in the DNA binding domain of TP53, and can occur in the presence or absence of a wildtype TP53 allele. Interestingly, specific enrichment of certain TP53 mutations frequently occurs in PCa when compared to other cancers, and similar to many other cancers, these mutations occur in the presence or absence of wildtype TP53. Using hormone therapy sensitive and CRPC cells, a panel of cell lines was generated to model these mutations in the presence or absence of wildtype TP53 expression. Defining the TP53 missense mutant-sensitive transcriptomes demonstrated context dependent, differential gene expression between TP53 missense mutants, related to the expression of wildtype TP53 in those cells. This also applied to cistrome analysis, illustrating an expansion of the p53 cistrome upon expression of mutant TP53. Furthermore, expression of p53 mutants also elicited context dependent effects on canonical p53 functions, thereby modulating distinct, downstream biological outcomes. Consequently, these data define the distinct and context dependent roles that mutant TP53 plays in PCa progression. Citation Format: Jennifer J. McCann, Irina Vasilevskaya, Neermala PoudelNeupane, Jeffry Dean, Amy Mandigo, Renee De Leeuw, Chris McNair, Matthew Schiewer, Karen Knudsen. Prostate cancer-specific enrichment of TP53 missense mutations elicits differential, context-dependent biochemical and biologic outcomes [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 4511.

Droplet Digital PCR Monitoring of TP53 Mutated Circulating Tumor DNA Levels in Lymphoma Patients

INTRODUCTION: Circulating tumor DNA (ctDNA), a portion of circulating cell-free DNA (cfDNA), is released from tumor cells into the circulatory system, which contains mutations corresponding to the patient's tumour alleles. Detection of ctDNA may noninvasively signal the presence of minimal residual disease (MRD) and predict prognosis. In recent years, droplet digital PCR (ddPCR) has emerged as a sensitive and effective tool for detection of point mutations in cfDNA. In this work, by application of ddPCR, we monitored the mutated TP53 ctDNA levels in serial plasma samples of lymphoma patients with identified TP53 hotspot mutations in their tumor biopsies. These results may give clues about the prognostic implications of different mutation locations and therapeutic strategies.METHODS: Lymphoma-focused next-generation sequencing were performed in tumor biopsies from over 200 lymphoma patients. A total of 134 sequential plasma samples from 32 lymphoma patients with TP53 hotspot mutations were subsequently monitored. cfDNA was extracted from a median sample volume of 4.8 ml (range 3.5-7.0 ml) of plasma. Specific MGB probes were designed for each TP53 mutation site and were then validated by tumor biopsies as well as healthy plasma samples as positive and negative controls respectively. Patient samples were considered to be mutation-positive if the mutant concentration in the sample was higher than the 95% confidence interval of the assay-specific false positive rate. The ctDNA sequential samples were quantitatively tracked using the ddPCR system QX200 (Bio-Rad Laboratories).RESULTS: 30 different TP53 mutations in 134 plasma samples from 32 lymphoma patients were monitored. All the mutations are hotspot in tumors according to COSMIC database, and are predicted to make damaging effect on TP53 protein by SIFT prediction. The average TP53 mutation frequency in tumor biopsies is 40.41% (2.30~92.21%). A total number of 30 specific MGB probes were designed for each TP53 mutation site. Their average false positive rate is 0.0004 copies/ul (0~0.002 copies/ul).In our results, 6 out of 20 (30%) patients with mutations in Loop3 and LSH motifs within the DNA binding domain of TP53 had a clearance of ctDNA, while 6 of 12 (50%) patients with mutations outside of the Loop3 and LSH motifs had a clearance of ctDNA. Literatures reported that patients with non-detectable ctDNA in plasma have negative minimal residual disease (MRD). So these results suggested that Loop3 and LSH motifs, which were reported to interact with DNA groove directly, are more critical than other structures.To investigate the association between different Ann Arbor stages and prognosis in TP53 mutated patients, we divided all the patients according to their stages. We found that patients with earlier Ann Arbor stages were more likely to have their ctDNA cleared (stage I/II: 83.33%; stage III/IV: 28%).In addition, we also analyzed the prognostic implication of cytogenetic abnormality. We found that, in patients with del (17p) as well as TP53 mutation, 7 of 8 had persistent detectable TP53 mutated ctDNA, only 1 of 8 (12.5%) had a clearance of ctDNA, While among the patients carrying TP53 mutations but not accompanied del (17p), 11 of 24 (45.83%) patients had a clearance of ctDNA. These results suggested that it is more difficult to achieve remission in patients with no wide-type TP53 allele compared to those have one wide-type TP53 allele.We further evaluated CAR-T cell immunotherapy and chemotherapy alone to 30 refractory recurrent TP53 mutated lymphoma patients. After CAR-T cell immunotherapy, 11 of 21 (52.38%) patients have non-detectable TP53 mutated ctDNA, while only 2 of 9 (22.22%) patients who received chemotherapy alone have non-detectable ctDNA. It suggested that CAR-T cell immunotherapy is more effective than chemotherapy alone to clear TP53 mutated ctDNA.CONCLUSIONS: Circulating tumor DNA is a promising biomarker to noninvasively monitor tumor load and quantify MRD. By applying droplet digital PCR, ctDNA could be measured and monitored sensitively, specifically and fast. In this work, we designed and validated 30 TP53 probes and tracked 134 sequential plasma from 32 patients. We found that it is easier to clear ctDNA for earlier stages of lymphoma patients with TP53 mutations. CAR-T cell immunotherapy is more effective than chemotherapy alone to continuously clear ctDNA.DISCLOSURE: No relevant conflicts of interest to declare. DisclosuresNo relevant conflicts of interest to declare.

Resistance to Fas-Mediated Apoptosis Does Not Correlate to Structural Alterations or Expression Changes of the Death Receptor in Papillary Thyroid Carcinomas

Background: Malignant cells exhibit significant resistance to FAS-mediated cell death, through different processes, including FAS mutations, soluble FAS expression, or FAS transcriptional dysregulation by P53, eventually escaping from immune surveillance. Since thyroid carcinomas were shown to be resistant to FAS-mediated apoptosis, we investigated the above mechanisms in thyroid carcinoma samples. Methods: Thirty-seven thyroid carcinoma samples were analyzed for mutations in FAS exon 9 and TP53 exons 5–8 and protein expression by means of immunohistochemistry. Moreover, thyroid carcinoma mRNA samples were subjected to reverse transcription – PCR, to evaluate the relative expression of transmembrane FAS versus its soluble form. Results: Analysis revealed indications for TP53 mutations in the anaplastic carcinomas, but not in the other thyroid specimens examined for TP53 or FAS exon 9 mutations. FAS receptor expression was observed in almost all thyroid specimens (97%) with significant up-regulation in papillary carcinomas. P53 nuclear staining was observed only in anaplastic carcinomas. Full-length FAS mRNA was detected in all specimens examined, with soluble FAS mRNA being either absent or present in very low amounts. Conclusions: Our results denote that FAS death domain or TP53 DNA-binding domain mutations, down-regulation of FAS receptor expression, or expression of FAS soluble isoform are not responsible for the seeming inhibition of FAS-mediated apoptosis in papillary thyroid carcinoma cells.

Mutant TP53 G245C and R273H promote cellular malignancy in esophageal squamous cell carcinoma

BackgroundTP53 gene mutations occur in more than 50% of human cancers and the vast majority of these mutations in human cancers are missense mutations, which broadly occur in DNA binding domain (DBD) (Amino acids 102–292) and mainly reside in six “hotspot” residues. TP53 G245C and R273H point mutations are two of the most frequent mutations in tumors and have been verified in several different cancers. In the previous study of the whole genome sequencing (WGS), we found some mutations of TP53 DBD in esophageal squamous cell carcinoma (ESCC) clinical samples. We focused on two high-frequent mutations TP53 p.G245C and TP53 p.R273H and investigated their oncogenic roles in ESCC cell lines, p53-defective cell lines H1299 and HCT116 p53−/−.ResultsMTS and colony formation assays showed that mutant TP53 G245C and R273H increased cell vitality and proliferation. Flow cytometry results revealed inhibition of ultraviolet radiation (UV)- and ionizing radiation (IR)- induced apoptosis and disruption of TP53-mediated cell cycle arrest after UV, IR and Nocodazole treatment. Transwell assays indicated that mutant TP53 G245C and R273H enhanced cell migration and invasion abilities. Moreover, western blot revealed that they were able to suppress the expression of TP53 downstream genes in the process of apoptosis and cell cycle arrest induced by UV, which suggests that these two mutations can influence apoptosis and growth arrest might be due, at least in part, to down-regulate the expression of P21, GADD45α and PARP.ConclusionsThese results indicate that mutant TP53 G245C and R273H can lead to more aggressive phenotypes and enhance cancer cell malignancy, which further uncover TP53 function in carcinogenesis and might be useful in clinical diagnosis and therapy of TP53 mutant cancers.

Establishment and characterization of three stable Basal/HER2-positive breast cancer cell lines derived from Chinese breast carcinoma with identical missense mutations in the DNA-binding domain of TP53

BackgroundBasal/human epidermal growth factor receptor (HER)2-positive (HER2+) breast cancer is resistant to monoclonal antibody (herceptin) treatment. There are currently only three basal/HER2+ breast cancer cell lines available, but they are not from Chinese populations.MethodsThree immortalized cell lines (ZJU-0327, ZJU-0725, and ZJU-1127) were established from invasive ductal breast carcinoma tissue of two patients treated by surgical resection at our center. The cell lines were characterized in terms of histology, therapeutic response, and biomarker expression. Their tumorigenic potential was evaluated in an athymic nude (BALB/C nu) mouse xenograft model. Cell authentication testing by the techniques of short tandem repeat.ResultsZJU-0327, ZJU-0725, and ZJU-1127 cell lines were maintained for more than 110 passages in vitro. The cells grew as monolayers; showed typical epithelial morphology and ultrastructure; were polyploid; had doubling times of 18, 57.5, and 18 h, respectively; had a near-tetraploid (ZJU-0327 and ZJU-1127) or aneuploid (ZJU-0725) karyotype with structural aberrations and tumor protein 53 mutation; insensitive to chemotherapeutic drugs and/or radiation; show high invasiveness and tumorigenicity in mice; and had no mycoplasma contamination. The cell lines were basal/HER2+, expressed cluster of differentiation, and were associated with poor prognosis. Cell authentication testing by the American Type Culture Collection confirmed the human origin of the cell lines, which did not match those in existing databases.ConclusionsThe three novel basal/HER2+ breast cancer cell lines recapitulating the malignant characteristics of the parent tumor’s, and can be useful for clarifying the molecular pathogenesis of basal/HER2+ breast cancer.

Treatment-associated TP53 DNA-binding domain missense mutations in the pathogenesis of secondary gliosarcoma.

BackgroundGliosarcoma is a rare variant of glioblastoma (GBM) that exhibits frequent mutations in TP53 and can develop in a secondary fashion after chemoradiation of a primary GBM. Whether temozolomide (TMZ)-induced mutagenesis of the TP53 DNA-binding domain (DBD) can drive the pathogenesis of gliosarcoma is unclear.MethodsWe identified a case of a primary GBM that rapidly progressed into secondary gliosarcoma shortly after chemoradiation was initiated. Bulk tumor was collected and gliomasphere cultures derived from both the pre- and post-treatment tumors. We performed targeted DNA sequencing and transcriptome analyses of the specimens to understand their phylogenetic relationship and identify differentially expressed gene pathways. Gliomaspheres from the primary GBM were treated with TMZ and then analyzed to compare patterns of mutagenesis in vivo and ex vivo.ResultsThe pre- and post-treatment tumors shared EGFR, CDKN2A, and PTEN mutations, but only the secondary gliosarcoma exhibited TP53 DBD missense mutations. Two mutations, R110C, and R175H, were identified, each in distinct clones. Both were base transitions characteristic of TMZ mutagenesis. Gene expression analysis identified increased JAK-STAT signaling in the gliosarcoma, together with reduced expression of microRNAs known to regulate epithelial-mesenchymal transition. Ex vivo treatment of the GBM spheres with TMZ generated numerous variants in cancer driver genes, including TP53 and CDH1, which were mutated in the post-treatment tumor.ConclusionsTMZ-induced TP53 gain-of-function mutations can have a driving role in secondary gliosarcoma pathogenesis. Analysis of variants identified in ex vivo TMZ-treated gliomaspheres may have utility in predicting GBM evolutionary trajectories in vivo during standard chemoradiation.

Higher-than-expected population prevalence of potentially pathogenic germline TP53 variants in individuals unselected for cancer history.

Li-Fraumeni syndrome (LFS) is an autosomal-dominant cancer predisposition disorder associated with pathogenic germline variants in TP53, with a high penetrance over an individual's lifetime. The actual population prevalence of pathogenic germline TP53 mutations is still unclear, most likely due to biased selection of cancer affected families. The aim of this study was to estimate the population prevalence of potentially pathogenic TP53 exonic variants in three sequencing databases, totaling 63,983 unrelated individuals. Potential pathogenicity was defined using an original algorithm combining bioinformatic prediction tools, suggested clinical significance, and functional data. We identified 34 different potentially pathogenic TP53 variants in 131 out of 63,983 individuals (0.2%). Twenty-eight (82%) of these variants fell within the DNA-binding domain of TP53, with an enrichment for specific variants that were not previously identified as LFS mutation hotspots, such as the p.R290H and p.N235S variants. Our findings reveal that the population prevalence of potentially pathogenic TP53 variants may be up to 10 times higher than previously estimated from family-based studies. These results point to the need for further studies aimed at evaluating cancer penetrance modifiers as well as the risk associated between cancer and rare TP53 variants.