Rare TP53 Research Articles

Botryoid-type Embryonal Rhabdomyosarcoma: A Comprehensive Clinicopathologic and Molecular Appraisal With Cross-comparison to its Conventional-type Counterpart.

Embryonal rhabdomyosarcoma (ERMS) is the most common subtype of RMS, occurring in soft tissue and visceral sites of young children, and is associated with favorable outcomes. A subset occurs in mucosal-lined luminal structures, displaying a unique grape-like growth termed as "botryoid-type." To further delineate the differences between conventional (cERMS) and botryoid-type (bERMS) RMS, we performed a comparative histologic review and comprehensive molecular profiling of 48 cases (25 bERMS and 23 cERMS). All tumors were subjected to a hybridization capture-based targeted matched tumor-normal DNA NGS assay. The mean age was 17 and 7 years for bERMS and cERMS, respectively. Most bERMS were female with a predilection for the gynecologic tract (75%), while cERMS had a slight male predominance and were preferentially located in abdominopelvic and paratesticular sites (30%, each). All bERMS exhibited an exophytic, bulbous architecture accompanied by a subepithelial "cambium layer." Distinctive germline alterations were detected, with DICER1 (18%) and FH (6%) mutations only in bERMS, and rare TP53, VHL, and APC mutations in cERMS. Similarly, contrasting somatic genomic landscapes were observed, with frequent DICER1 (52%, P**<0.0001) and TP53 (36%, P*<0.05) alterations exclusively in bERMS. Cartilaginous differentiation was only observed in DICER1-mutated bERMS. All patients had longitudinal follow-up. bERMS patients with somatic/germline DICER1 mutations showed significantly improved recurrence-free survival compared with that of DICER1-wild type patients (P*<0.05). Moreover, bERMS showed improved disease-specific survival compared with that of cERMS, with 8% versus 30% (P*<0.05) dead of disease, respectively. In summary, we compare the molecular underpinnings of the largest cohort of bERMS and cERMS with targeted DNA sequencing and long-term follow-up data. Our findings reveal divergent genomic topographies between the 2 groups, with bERMS showing unique germline and somatic abnormalities, including enrichment in DICER1 and TP53 alterations, and a trend towards improved survival.

A Comprehensive Clinicopathologic and Molecular Reappraisal of GLI1 -altered Mesenchymal Tumors with Pooled Outcome Analysis Showing Poor Survival in GLI1 - amplified Versus GLI1- rearranged Tumors.

GLI1 -altered mesenchymal tumor is a recently described distinct pathologic entity with an established risk of malignancy, being defined molecularly by either GLI1 gene fusions or amplifications. The clinicopathologic overlap of tumors driven by the 2 seemingly distinct mechanisms of GLI1 activation is still emerging. Herein, we report the largest series of molecularly confirmed GLI1 -altered mesenchymal neoplasms to date, including 23 GLI1- amplified and 15 GLI1 -rearranged new cases, and perform a comparative clinicopathologic, genomic, and survival investigation. GLI1- rearranged tumors occurred in younger patients (42 vs. 52y) and were larger compared with GLI1 -amplified tumors (5.6cm vs. 1.5cm, respectively). Histologic features were overall similar between the 2 groups, showing a multinodular pattern and a nested architecture of epithelioid, and less commonly spindle cells, surrounded by a rich capillary network. A distinct whorling pattern was noted among 3 GLI1 -amplified tumors. Scattered pleomorphic giant cells were rarely seen in both groups. The immunoprofile showed consistent expression of CD56, with variable S100, CD10 and SMA expression. Genomically, both groups had overall low mutation burdens, with rare TP53 mutations seen only in GLI1- amplified tumors. GLI1 -amplified mesenchymal tumors exhibit mostly a single amplicon at the 12q13-15 locus, compared with dedifferentiated liposarcoma, which showed a 2-peak amplification centered around CDK4 (12q14.1) and MDM2 (12q15). GLI1 -amplified tumors had a significantly higher GLI1 mRNA expression compared with GLI1 -rearranged tumors. Survival pooled analysis of current and published cases (n=83) showed a worse overall survival in GLI1 -amplified patients, with 16% succumbing to disease compared with 1.7% in the GLI1- rearranged group. Despite comparable progression rates, GLI1 -amplified tumors had a shorter median progression-free survival compared with GLI1 -rearranged tumors (25mo vs. 77mo). Univariate analysis showed that traditional histologic predictors of malignancy (mitotic count ≥4/10 high-power fields, presence of necrosis, and tumor size ≥5cm) are associated with worse prognosis among GLI1 -altered mesenchymal tumors.

Three novel rare TP53 fusion mutations in a patient with multiple primary cancers: a case report

Abstract As survival rates improve and detection technologies advance, the occurrence of multiple primary cancers (MPCs) has been increasing. Approximately 16% of cancer survivors develop a subsequent malignancy, with lung cancer often developing after esophageal cancer due to potential “field cancerization” effects. Despite this observation, the genetic heterogeneity underlying MPCs remains understudied. However, the recent emergence of genetic testing has expanded the scope of investigations into MPCs to investigate signatures underlying cancer predisposition. This report reveals 3 unprecedented TP53 fusion mutations in a Chinese patient afflicted by MPCs, namely, AP1M2–TP53 (A1;T11) fusion, TP53–ILF3 (T10;I13) fusion, and SLC44A2–TP53 (S5;T11) fusion. This patient exhibited an extended period of survival after diagnosis of extensive-stage small cell lung cancer, which occurred 6 years after the diagnosis of esophageal squamous cell cancer. This unique report may provide supplementary data that enhance our understanding of the genetic landscape of MPCs.

TRIM28 inactivation in epithelial nephroblastoma is frequent and often associated with predisposing TRIM28 germline variants.

Wilms tumors (WTs) are histologically diverse childhood cancers with variable contributions of blastema, stroma, and epithelia. A variety of cancer genes operate in WTs, including the tripartite-motif-containing-28 gene (TRIM28). Case reports and small case series suggest that TRIM28 mutations are associated with epithelial morphology and WT predisposition. Here, we systematically investigated the prevalence of TRIM28 inactivation and predisposing mutations in a cohort of 126 WTs with >2/3 epithelial cells, spanning 20 years of biobanking in the German SIOP93-01/GPOH and SIOP2001/GPOH studies. Overall, 44.4% (56/126) cases exhibited loss of TRIM28 by immunohistochemical staining. Of these, 48 could be further analyzed molecularly, revealing TRIM28 sequence variants in each case - either homozygous (~2/3) or heterozygous with epigenetic silencing of the second allele (~1/3). The majority (80%) of the mutations resulted in premature stops and frameshifts. In addition, we detected missense mutations and small deletions predicted to destabilize the protein through interference with folding of key structural elements such as the zinc-binding clusters of the RING, B-box-2, and PHD domains or the central coiled-coil region. TRIM28-mutant tumors otherwise lacked WT-typical IGF2 alterations or driver events, except for rare TP53 progression events that occurred with expected frequency. Expression profiling identified TRIM28-mutant tumors as a homogeneous subset of epithelial WTs that mostly present with stage I disease. There was a high prevalence of perilobar nephrogenic rests, putative precursor lesions, that carried the same biallelic TRIM28 alterations in 7/7 cases tested. Importantly, 46% of the TRIM28 mutations were present in blood cells or normal kidney tissue, suggesting germline events or somatic mosaicism, partly supported by family history. Given the high prevalence of predisposing variants in TRIM28-driven WT, we suggest that immunohistochemical testing of TRIM28 be integrated into diagnostic practice as the management of WT in predisposed children differs from that with sporadic tumors. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

P142 Functional impact of a rare germline TP53 variant (NM_001126114.2, c.1018A&gt;G, p.N340D) in a family with the Li-Fraumeni-like syndrome

P142 Functional impact of a rare germline TP53 variant (NM_001126114.2, c.1018A&gt;G, p.N340D) in a family with the Li-Fraumeni-like syndrome

Abstract 3890: Sequencing of 888 pediatric solid tumors informs precision oncology trial design and data sharing initiatives in pediatric cancer

Abstract Pediatric pan-cancer genome analyses do not capture the full range of diagnoses encountered in clinical practice. To inform basket trial design and real-world precision oncology practice, we classified diagnoses and assessed the landscape of mutations, including trial-matching, in an unselected cohort of pediatric solid tumors. Since 2013 all Dana-Farber/Boston Children’s patients have been offered participation in the Profile study. Participant tumor samples were sequenced with DFCI-OncoPanel, a targeted panel test sequencing exons of up to 447 cancer genes for single nucleotide variants, insertions and deletions and copy number alterations, and introns and exons of up to 60 genes for rearrangements. Patient diagnosis was classified according to ICD-O, version 3.2. Genomic alterations were analyzed for matching to the actionable mutation lists of precision oncology basket trials (NCI-COG Pediatric MATCH, NCI-MATCH, and the ASCO TAPUR Study v.3). Data will be shared with the Childhood Cancer Data Initiative. There were 888 pediatric patients with sequencing enrolled in Profile between January 2013 and March 2019; 512 (58%) with solid tumors and 376 (42%) with CNS tumors. Fifty-five percent (491/888) of patients had one of ten common pediatric cancer diagnoses: neuroblastoma (n=80), low-grade glioma (n=72), Wilms tumor (n=57), medulloblastoma (n=55), pilocytic astrocytoma (n=47), rhabdomyosarcoma (n=44), osteosarcoma (n=42), ependymoma (n=39), Ewing sarcoma (n=28) and glioblastoma (n=27). The remaining 45% (397/888) had one of 85 distinct rare malignancies with less than 25 cases per diagnosis. Most (80/85) of these rare diagnoses are not represented in prior pediatric pan-cancer sequencing studies. Recurrent (&amp;gt;5%) pathogenic alterations were, in common and rare diagnoses, TP53 mutations(m) and deletions(del) and BRAFm and rearrangements(r), in common diagnoses, MYC/MYCN amplification (amp) and EWSR1r and, in rare diagnoses, CTNNB1m, CDKN2A/Bdel and NF1m/del. We found that 31% (n=271/888) of patients had at least 1 variant matching a basket trial treatment arm. Genes with matching alterations include BRAF (10%), NF1 (4%), PI3KCA (3%), NRAS (2%), BRCA2 (2%), ALK (1%), and FGFR1 (1%). Sequencing of pediatric malignancies is increasing. This study highlights opportunities to use the resulting genomic data to inform genome-selected clinical trial design and uncover drivers in pediatric cancers. The proportion of cases in this cohort with genomic alterations meeting eligibility for basket trials is equivalent to that seen in the pediatric MATCH screening study. Due to the low prevalence of the diagnoses in the long tail of cancer types in this study, defining the genomic landscape of ultra-rare cancers will require data sharing. Classifying pediatric cancer diagnoses using the ICD-O standard ontology system is feasible and will facilitate data sharing. Citation Format: Suzanne J. Forrest, Hersh Gupta, Abigail Ward, Yvonne Li, Duong Doan, Alyaa Al-Ibraheemi, Sanda Alexandrescu, Pratiti Bandopadhayay, Suzanne Shusterman, Elizabeth A. Mullen, Natalie Collins, Susan N. Chi, Karen D. Wright, Priti Kumari, Tali Mazor, Keith L. Ligon, Priyanka Shivdasani, Phani Davineni, Monica Manam, Richard L. Schilsky, Suanna S. Bruinooge, Jaime M. Guidry Auvil, Ethan Cerami, Barrett J. Rollins, Matthew L. Meyerson, Neal I. Lindeman, Laura MacConaill, Bruce E. Johnson, Andrew D. Cherniack, Alanna J. Church, Katherine A. Janeway. Sequencing of 888 pediatric solid tumors informs precision oncology trial design and data sharing initiatives in pediatric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3890.

Donor Clonal Hematopoiesis and Recipient Outcomes After Transplantation.

Clonal hematopoiesis (CH) can be transmitted from a donor to a recipient during allogeneic hematopoietic cell transplantation. Exclusion of candidate donors with CH is controversial since its impact on recipient outcomes and graft alloimmune function is uncertain. We performed targeted error-corrected sequencing on samples from 1,727 donors age 40 years or older and assessed the effect of donor CH on recipient clinical outcomes. We measured long-term engraftment of 102 donor clones and cytokine levels in 256 recipients at 3 and 12 months after transplant. CH was present in 22.5% of donors, with DNMT3A (14.6%) and TET2 (5.2%) mutations being most common; 85% of donor clones showed long-term engraftment in recipients after transplantation, including clones with a variant allele fraction < 0.01. DNMT3A-CH with a variant allele fraction ≥ 0.01, but not smaller clones, was associated with improved recipient overall (hazard ratio [HR], 0.79; P = .042) and progression-free survival (HR, 0.72; P = .003) after adjustment for significant clinical variables. In patients who received calcineurin-based graft-versus-host disease prophylaxis, donor DNMT3A-CH was associated with reduced relapse (subdistribution HR, 0.59; P = .014), increased chronic graft-versus-host disease (subdistribution HR, 1.36; P = .042), and higher interleukin-12p70 levels in recipients. No recipient of sole DNMT3A or TET2-CH developed donor cell leukemia (DCL). In seven of eight cases, DCL evolved from donor CH with rare TP53 or splicing factor mutations or from donors carrying germline DDX41 mutations. Donor CH is closely associated with clinical outcomes in transplant recipients, with differential impact on graft alloimmune function and potential for leukemic transformation related to mutated gene and somatic clonal abundance. Donor DNMT3A-CH is associated with improved recipient survival because of reduced relapse risk and with an augmented network of inflammatory cytokines in recipients. Risk of DCL in allogeneic hematopoietic cell transplantation is driven by somatic myelodysplastic syndrome-associated mutations or germline predisposition in donors.

A first report of a rare TP53 variant associated with Li-Fraumeni syndrome manifesting as invasive breast cancer and malignant solitary fibrous tumor

BackgroundLi-Fraumeni is a rare autosomal dominant cancer predisposition syndrome. The basis is a germline mutation of TP53 gene which encodes tumor suppressor protein resulting in early onset of tumors, most often breast cancer, soft tissue sarcomas, brain tumors, adrenocortical carcinomas, and leukemia.Case reportWe present a case of a young woman with a positive family history for cancer diagnosed with malignant solitary fibrous tumor and luminal B-like invasive breast cancer. Breast cancer and sarcomas account for the majority of tumors associated with Li-Fraumeni syndrome, yet solitary fibrous tumor is a rare clinical entity with no established guidelines for treatment. Even though both primary tumors were successfully resected, the sarcoma relapsed in the form of lung metastases. The NGS analysis revealed single nucleotide variant (c.1101-1G>A) in TP53 gene, affecting the acceptor splice site at intron 10. Until now, only one case of this genetic variant has been documented with conflicting interpretations of pathogenicity.ConclusionsThe knowledge of TP53 mutation status is essential since the management of these patients requires different approach to avoid excessive toxicity due to the risk of developing secondary malignancy. Using the clinical criteria to screen for affected individuals facilitates appropriate early genetic counseling of patients and their families. Following the American College of Medical Genetics criteria, we believe that the reported single nucleotide variant (c.1101-1G>A) in TP53 gene should be considered pathogenic.

TP53 Germline Variations Influence the Predisposition and Prognosis of Acute Lymphoblastic Leukemia in Children

Germline TP53 mutations is the genetic basis of Li-Fraumeni syndrome (LFS), a highly penetrant cancer predisposition condition. Recent reports of germline TP53 variants in children with hypodiploid acute lymphoblastic leukemia (ALL) suggest that childhood leukemia is another manifestation of LFS. However, the pattern, prevalence, and clinical relevance of pathogenic variants in TP53 in childhood ALL remain unknown.To comprehensively characterize genetic variants in TP53 in childhood ALL, we performed targeted sequencing of TP53 coding regions in 3858 children from the Children's Oncology Group front-line ALL clinical trials (COG AALL0232 and P9900). In total, 49 different non-silent coding rare TP53 variants were identifiedin 77 (2.0%) patients sequenced, including 40 missense, 1 nonsense, 6 frameshift, and 2 inframe deletion variants. To determine which TP53 variants are potentially pathogenic and related to ALL risk, we examined their experimentally-validated p53 transcriptional activity, bioinformatically-predicted damaging effects on TP53 function, and also the variant frequency in non-ALL populations. Twenty-two TP53 variants were classified as ALL risk-related (VAR), including 7 protein-truncating and 15 missense variants, all of which were either absent or exceedingly rare in non-ALL populations (i.e., allele frequency < 0.006% in the ExAC dataset). Fourteen missense VAR reside in the p53 core DNA-binding domain, with two affecting residues essential for DNA contact and seven located at residues that are important for the overall architecture of the DNA-binding surface. The 27 remaining TP53 variants were classified as “variants of unknown significance” (VUS). Together, of 3858 children in this ALL cohort, 26 (0.67%) patients had a predicted VAR and 51 (1.3%) had a VUS in the TP53 gene.We next evaluated the association of germline TP53 variants with patient demographics and ALL tumor genetic characteristics. Children with a TP53 VAR were significantly older at diagnosis (P < 0.0001), and had significantly lower leukocyte count at presentation than those with a VUS or wild-type (WT) genotype (P= 0.006). Of 26 patients carrying a germline TP53 VAR, 17 (65.4%) exhibited hypodiploidy in ALL blasts (mostly low hypoploidy or masked hypoploidy). In contrast, hypodiploid ALL was only present in 3.9% and 1.2% of children with TP53 VUS or WT genotype, respectively. The prevalence of TP53 VAR did not differ by genetic ancestry (P= 0.9). We also examined the relationship between germline TP53 variants and treatment outcomes of ALL therapy. In the COG AALL0232 cohort, the presence of a TP53 VAR was associated with a significantly lower event free survival (EFS) and overall survival (OS) compared to patients without a risk variant (hazard ratio [HR] of 2.84, P= 0.0007 for EFS; and HR of 3.13, P= 0.003 for OS). In multivariate analyses, TP53 VAR remained prognostic after adjusting for ancestry, age, leukocyte count at diagnosis, and minimal residual disease at the end of remission induction therapy (HR of 3.36, P= 0.0002 for EFS; HR of 2.88, P= 0.007 for OS). Importantly, there was a markedly higher risk of second cancers among patients with TP53 VARs compared to those with TP53 VUS or WT TP53: 5-year cumulative incidence of 25.1% vs. 0.7%, P= 5.4 × 10-11. In the COG P9900 cohort, only 4 patients harbored TP53 VAR, of whom 3 cases experienced adverse events (one patient each with ALL relapse, second cancer, or death in remission), and both EFS and OS were significantly worse than patients with TP53 VUS or WT genotype (HR of 7.14, P= 0.0008 for EFS; HR of 14.19, P= 1.1 x 10-5 for OS). Taken together, 14 of 26 patients with a TP53 VAR experienced adverse events, with 5 ALL relapses (36% of all events) and 5 second cancers (36%). This pattern of adverse events was dramatically different from those in patients with WT TP53 or VUS, for whom ALL relapse accounted for 75% of all events with only 4% as second cancers (P= 1.2 × 10-7). In fact, even within hypodiploid ALL patients who experienced adverse events, the frequency of second cancer was significantly higher in those with a TP53 VAR than those without (50% vs 5%, P= 0.01), further suggesting that germline TP53 variation was the underlying cause of second cancers in these cases.In conclusion, our findings identified germline TP53 variants as an important determinant of ALL predisposition and adverse treatment outcomes, particularly the risk of second malignant neoplasms. DisclosuresBorowitz:Beckman Coulter: Honoraria; Becton-Dickinson Biosciences: Research Funding; HTG Molecular: Honoraria. Mullighan:Loxo Oncology: Research Funding; Amgen: Consultancy. Hunger:Jazz Pharmaceuticals: Honoraria; Novartis: Consultancy; Erytech Pharmaceuticals: Consultancy; Amgen: Consultancy, Equity Ownership.

A Signature-Based Classification of Gastric Cancer That Stratifies Tumor Immunity and Predicts Responses to PD-1 Inhibitors.

Gastric cancer is a leading cause of cancer-related deaths with considerable heterogeneity among patients. Appropriate classifications are essential for prognosis prediction and individualized treatment. Although immunotherapy showed potential efficacy in a portion of patients with gastric cancer, few studies have tried to classify gastric cancer specifically based on immune signatures. In this study, we established a 3-subtype cluster with low (CLIM), medium (CMIM), and high (CHIM) enrichment of immune signatures based on immunogenomic profiling. We validated the classification in multiple independent datasets. The CHIM subtype exhibited a relatively better prognosis and showed features of “hot tumors”, including low tumor purity, high stromal components, overexpression of immune checkpoint molecules, and enriched tumor-infiltrated immune cells (activated T cells and macrophages). In addition, CHIM tumors were also characterized by frequent ARID1A mutation, rare TP53 mutation, hypermethylation status, and altered protein expression (HER2, β-catenin, Cyclin E1, PREX1, LCK, PD-L1, Transglutaminase, and cleaved Caspase 7). By Gene Set Variation Analysis, “TGFβ signaling pathway” and “GAP junction” were enriched in CLIM tumors and inversely correlated with CD8+ and CD4+ T cell infiltration. Of note, the CHIM patients showed a higher response rate to immunotherapy (44.4% vs. 11.1% and 16.7%) and a more prolonged progression-free survival (4.83 vs. 1.86 and 2.75 months) than CMIM and CLIM patients in a microsatellite-independent manner. In conclusion, the new immune signature-based subtypes have potential therapeutic and prognostic implications for gastric cancer management, especially immunotherapy.

A Rare TP53 Mutation Predominant in Ashkenazi Jews Confers Risk of Multiple Cancers.

Germline mutations in TP53 cause a rare high penetrance cancer syndrome, Li-Fraumeni syndrome (LFS). Here, we identified a rare TP53 tetramerization domain missense mutation, c.1000G>C;p.G334R, in a family with multiple late-onset LFS-spectrum cancers. Twenty additional c.1000G>C probands and one c.1000G>A proband were identified, and available tumors showed biallelic somatic inactivation of TP53. The majority of families were of Ashkenazi Jewish descent, and the TP53 c.1000G>C allele was found on a commonly inherited chromosome 17p13.1 haplotype. Transient transfection of the p.G334R allele conferred a mild defect in colony suppression assays. Lymphoblastoid cell lines from the index family in comparison with TP53 normal lines showed that although classical p53 target gene activation was maintained, a subset of p53 target genes (including PCLO, PLTP, PLXNB3, and LCN15) showed defective transactivation when treated with Nutlin-3a. Structural analysis demonstrated thermal instability of the G334R-mutant tetramer, and the G334R-mutant protein showed increased preponderance of mutant conformation. Clinical case review in comparison with classic LFS cohorts demonstrated similar rates of pediatric adrenocortical tumors and other LFS component cancers, but the latter at significantly later ages of onset. Our data show that TP53 c.1000G>C;p.G334R is found predominantly in Ashkenazi Jewish individuals, causes a mild defect in p53 function, and leads to low penetrance LFS. SIGNIFICANCE: TP53 c.1000C>G;p.G334R is a pathogenic, Ashkenazi Jewish-predominant mutation associated with a familial multiple cancer syndrome in which carriers should undergo screening and preventive measures to reduce cancer risk.

TP53 fusions with next-generation sequencing (NGS) in Chinese non-small cell lung cancer (NSCLC): A multicenter study.

e21742 Background: Currently, with the advances in detection techniques, such as next generation sequencing (NGS), more and more rare or atypical TP53 fusions had been identified. Such as TP53-PSMD14, the importance of EGFR signaling in the pathogenesis of lung cancer and the efficacy of EGFR-TKI treatment had been demonstrated. The aim of this study was to evaluate the prevalence of TP53 fusions in Chinese NSCLC populations, which had not been reported earlier, and to describe targeting potential in Chinese NSCLC populations. Methods: A multicenter study in China was initiated from February 2014, and NSCLC patients have been enrolled as of December 2018. A total of 2743 patients with NSCLC were screened by using next-generation sequencing (NGS)-based 381 genes panel assay for detecting TP53 fusions. Results: Of this entire cohort, just four (0.15%) patients were identified with a TP53 fusion, including DNAH2-TP53 (1), TP53-MPDU1 (1), TP53-FXR2 (1), TP53-VEZF1 (1). Of the TP53 fusion NSCLC patients, 50.00% were detected in female patients. Biopsies were obtained from primary lung tumor (25.00%) and metastatic sites (75.00%). Overall TMB in the TP53 fusion was high, which had more than 20 mut/Mb. Of the TP53 fusion NSCLC, two cases (50.00%) featured EGFR SV alterations. Conclusions: The frequency of TP53 fusions in Chinese populations with NSCLC is extremely rare (0.17%). TP53 fusions may reduce responsiveness to TKIs and worsen prognosis in EGFR-mutated NSCLC patients, mainly those carrying exon 19 deletions or exon 21 L858R.

Stability of rare TP53 co-mutations in AML patients.

7528 Background: Acute Myeloid Leukemia (AML) is a devastating disease with poor overall survival. Access to precision medicines, is revolutionizing AML care and is driving an increase in Next Generation Sequencing (NGS) utilization to determine the genomic profile of patients with AML. Advanced analysis into the interplay between mutational status for multiple genes is granting access to new targeted and precision medicine treatment options with improved outcomes. Methods: Rate of NGS adoption and TP53 mutation status were determined for a cohort of 984 AML patients tested by NGS from the Diaceutics Diagnostic Index between 2017 and 2019. Mutation rate of other genes on the same NGS panel used to determine if TP53 status was determined and compared using the z-score test for two population proportions. The stability of TP53 and co-mutation status at time of diagnosis and relapse/refractory was also analyzed. Results: We have seen a 2-fold increase in the average routine clinical practice uptake of NGS in a representative real-world patient cohort (Q3 2017, n = 144; Q3 2019, n = 290). In a cohort of 984 AML patients tested by NGS 151 (15.3%) had a TP53 mutation (Exon 4-9). Of those that had a TP53 mutation, significant negative associations were observed with mutations in seven other genes (ASXL1 p = 0.00308, CEBPA p = 0.0027, FLT3 p &lt; 0.00001, IDH1 p = 0.04338, NRAS p = 0.0012, RUNX1 p = 0.01878 and TET2 p = 0.0251). There were no statistically significant differences in TP53 mutation and other gene mutation rates between diagnosis and relapse/refractory timepoints. Conclusions: This data suggests that TP53 and other gene co-mutants may act in similar activation pathways resulting in rare detection. One possibility is that double mutants result in synthetic lethality leading to a low clonal population. Second, when co-mutated clones escape immune surveillance and regulation, it results in particularly aggressive leukemias with lower overall survival. The stability of TP53 and co-mutation is relatively stable, which has implications for testing algorithms and clinical utility as a marker of MRD. Although a clinical trial may be difficult due to low numbers of patients, a TP53 antagonist and targeted therapy may be a valuable treatment option in rare cases where co-mutation does exist. [Table: see text]

379P - Study of germline mutations in high risk cancer patients from a tertiary care center in India

BackgroundAbout 10% cancers are due to cancer susceptibility genes. Testing of high risk patients according to NCCN guidelines is important for detection of hereditary cases and at-risk relatives for secondary prevention of cancer. The exact burden of germline mutations in Indian population is not known because of lack of education and unaffordability. We undertook this study for detection of hereditary cancer cases from Western India. MethodsAll patients who underwent testing from June 2015 to June 2019 for extended germline mutation panel of 96 genes using next generation sequencing (NGS) on Illumina platform were selected. Clinical data was analysed and correlated with the results. ResultsOut of total 290 patients counselled, 84 (28.9%) underwent genetic testing. Mutations were found in 63 % (53/84) patients whose median age was 44 years. 24.5% (13/53) had no positive family history of cancer. Most common malignancies were breast, 31; of which 15 were triple negative breast cancers, followed by ovary, 13 and colon, 4 cases. BRCA1/BRCA2 mutations were seen in 66% (35/53), MSH2 in 3 and MLH1 and RAD51D mutation in 2 patients each. 38 mutations were pathogenic, 4 likely pathogenic and 11 were Variant of Unknown Significance (VUS). Three novel BRCA1 mutations (c.1039delC, c.42_44dupGCT, c.68-5_77del) and one novel BRCA2 mutation (c.7342A>T) was identified. A rare TP53 mutation (c.(-29 + 1_-28-1)_(672 + 1_673-1)dup) was also identified in one patient, who had three metachronous malignancies. ConclusionsHereditary breast and ovarian cancer syndrome due to BRCA1/2 mutations was commonest in our study followed by Lynch syndrome due to MSH2 mutation. 20% VUS, 4 novel and 1 rare mutation were identified. Negative family history does not rule out hereditary predisposition. The knowledge shared would assist in further genetic testing and enable detection of more hereditary cancer cases. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Rare TP53 variant associated with Li-Fraumeni syndrome exhibits variable penetrance in a Saudi family

Li-Fraumeni syndrome (LFS) is an inherited, autosomal-dominant condition that predisposes individuals to a wide-spectrum of tumors at an early age. Approximately 70% of families with classic LFS have pathogenic variants in the tumor suppressor gene TP53 that disrupt protein function or stability. While more than 70% of pathogenic variants in TP53 are missense variants, the vast majority occur very infrequently, and thus their clinical significance is uncertain or conflicting. Here, we report an extremely rare TP53 missense variant, c.799C > T (p.Arg267Trp), identified in a 2-year-old Saudi proband diagnosed with choroid plexus carcinoma (CPC) and six of his first- and second-degree relatives. CPC is frequently found in families with LFS, and this is the first detailed report of a family with this variant. Intriguingly, the proband’s father is homozygous for TP53 c.799C > T and phenotypically normal at 39 years of age. While loss of TP53 heterozygosity is often observed in tumors from individuals with LFS, homozygous germline TP53 pathogenic variants are rare. Based on our analysis of this single family, we hypothesize that TP53 c.799C > T has low or variable penetrance for LFS, with predisposition to the development of CPC. The observations from this family have furthered our understanding of the phenotypic variability that may be caused by one variant of TP53, even in the same family, and suggest that other factors (genetic and/or environmental) may play a role in mechanism of disease manifestation in LFS.

TP53 Germline Variations Influence the Predisposition and Prognosis of B-Cell Acute Lymphoblastic Leukemia in Children.

Purpose Germline TP53 variation is the genetic basis of Li-Fraumeni syndrome, a highly penetrant cancer predisposition condition. Recent reports of germline TP53 variants in childhood hypodiploid acute lymphoblastic leukemia (ALL) suggest that this type of leukemia is another manifestation of Li-Fraumeni syndrome; however, the pattern, prevalence, and clinical relevance of TP53 variants in childhood ALL remain unknown. Patients and Methods Targeted sequencing of TP53 coding regions was performed in 3,801 children from the Children's Oncology Group frontline ALL clinical trials, AALL0232 and P9900. TP53 variant pathogenicity was evaluated according to experimentally determined transcriptional activity, in silico prediction of damaging effects, and prevalence in non-ALL control populations. TP53 variants were analyzed for their association with ALL presenting features and treatment outcomes. Results We identified 49 unique nonsilent rare TP53 coding variants in 77 (2.0%) of 3,801 patients sequenced, of which 22 variants were classified as pathogenic. TP53 pathogenic variants were significantly over-represented in ALL compared with non-ALL controls (odds ratio, 5.2; P < .001). Children with TP53 pathogenic variants were significantly older at ALL diagnosis (median age, 15.5 years v 7.3 years; P < .001) and were more likely to have hypodiploid ALL (65.4% v 1.2%; P < .001). Carrying germline TP53 pathogenic variants was associated with inferior event-free survival and overall survival (hazard ratio, 4.2 and 3.9; P < .001 and .001, respectively). In particular, children with TP53 pathogenic variants were at a dramatically higher risk of second cancers than those without pathogenic variants, with 5-year cumulative incidence of 25.1% and 0.7% ( P < .001), respectively. Conclusion Loss-of-function germline TP53 variants predispose children to ALL and to adverse treatment outcomes with ALL therapy, particularly the risk of second malignant neoplasms.

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.

P53 deregulation in Epstein-Barr virus-associated gastric cancer

TP53 is a tumour suppressor gene frequently mutated in human cancers; nevertheless, in EBV-associated malignancies mutations are uncommon despite frequent deregulation of the p53 pathway. In this study, we aimed to investigate p53 expression, TP53 mRNA levels and TP53 mutations in EBV-associated gastric carcinoma (EBVaGC). A case-control study was performed using 46 patients: 15 EBVaGC and 31 EBV-negative GC (EBVnGC) cases. p53 expression was detected by immunohistochemistry (IHC), the evaluation of p53 mRNA levels was performed by RT-qPCR and TP53 mutations were investigated only in EBVaGC cases using the DNA sanger sequencing method. p53 expression was found in 97.8% (45/46) of all gastric cancer cases (including EBVaGC and EBVnGC groups). Despite the high frequency of p53 expression in both groups, the percentages of cells are significantly higher among EBVaGC cases (p = 0.027). Regarding the mRNA levels, we found a significantly increased expression of p53 mRNA in EBVnGC (2−ΔΔCt = 13.4 ± 2.4; p = 0.0029) when compared with EBVaGC. Furthermore, the sequencing analysis of TP53 gene revealed that only one of the 15 EBVaGC cases presented a missense mutation. Our results demonstrated that EBV-associated gastric carcinomas are characterized by a significant decrease of TP53 mRNA levels with a strong p53 expression and rare TP53 mutations when compared with EBV-negative cancers. Considering these results, EBV seems to induce a stabilization of p53 in the EBVaGC independently of the presence of mutations, which remains to be explained.

Abstract AP05: ULTRA–DEEP SEQUENCING DETECTS OVARIAN CANCER CELLS IN PERITONEAL FLUID AND REVEALS SOMATIC TP53 MUTATIONS IN NON–CANCEROUS TISSUES

Abstract High–grade serous ovarian carcinoma (HGSOC), the most common and most aggressive type of ovarian cancer, is characterized by near–universal prevalence of TP53 mutations, even at the early stages of the disease. Thus, the detection of tumor–specific TP53 mutations in clinical samples could potentially enable early cancer detection, monitoring, and screening. A main limitation, however, is the high error rate of conventional DNA sequencing technologies (estimated at 1 in 1000 nucleotides), which precludes the identification of low frequency tumor mutant molecules from technical error. Duplex Sequencing is a next–generation sequencing technology that uniquely tags each strand of a DNA molecule and quantifies mutations only when present in both strands of DNA. This reduces the error rate of sequencing to 1 in 10 million nucleotides, an unprecedented sensitivity that could enable the detection of extremely low frequency tumor mutant molecules by ultra–deep sequencing. The goal of this study was to provide proof–of–principle of the ability of Duplex Sequencing to detect extremely rare TP53 mutated cancer cells disseminated into the peritoneal cavity of women with HGSOC. We analyzed 17 peritoneal fluid samples from women with HGSOC and known TP53 tumor mutation (cases) and 20 peritoneal fluid samples from women without cancer (controls). The tumor–specific TP53 mutation was detected in matched peritoneal fluid from 94% of women with HGSOC (16/17), including 2 patients with occult tubal intraepithelial neoplasia, 7 patients with early stage HGSOC, and 8 with negative peritoneal fluid cytopathology. Tumor–specific alleles were detected as low as 1 mutant molecule per 24,737 normal genomes. Additionally, we detected extremely low–frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cases from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age–associated, low frequency TP53 mutations were also found in 100% of matched peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic cancer). Our results demonstrate the ability of Duplex Sequencing to detect very rare cancer cells and provide evidence of widespread, low frequency, age–associated somatic TP53 mutation in non–cancerous tissue. These mutations likely represent a premalignant mutational background that accumulates in cancer and aging. Citation Format: Krimmel JD, Schmitt MW, Kennedy SR, Harrell MI, Agnew KJ, Loeb LA, Swisher EM, Risques RA. ULTRA–DEEP SEQUENCING DETECTS OVARIAN CANCER CELLS IN PERITONEAL FLUID AND REVEALS SOMATIC TP53 MUTATIONS IN NON–CANCEROUS TISSUES [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP05.

Prognostic Impact of Subclonal TP53 Aberrations in Chronic Lymphocytic Leukemia Validated By a Robust Targeted Next Generation Sequencing Assay

Introduction/background:Clonal TP53 aberrations (del(17p) and/or TP53 mutations) in patients with chronic lymphocytic leukemia (CLL) correlates with a poor prognosis. Similar outcome has been demonstrated for patients with subclonal TP53 aberrations (Rossi, Blood, 2014). In order to guide novel targeted therapies approved in frontline treatment of TP53 aberrated (TP53+) CLL, development and clinical validation of robust assays for subclonal TP53 aberrations is warranted.Methods:DNA extracted from peripheral blood of CLL patients were diluted 1:5 in DNA derived from a cell line containing a known, but rare TP53 point mutation. TP53 exons 2-10 were PCR amplified from undiluted and diluted DNA in parallel using Phusion proofreading DNA polymerase and subsequently sequenced by targeted Next Generation Sequencing (tNGS) on Illumina MiSeq and HiSeq 2500. Sensitivity and specificity of the assay was tested by serial dilution of patient samples with known TP53 insertions, deletions or substitutions. Consecutive biobanked samples from CLL patients at a single institution were used for validation of the clinical impact of subclonal TP53 aberrations. Nucleotide variants were called by CLC Biomedical Genomics Workbench 3.0. An algorithm for detection of true mutations was developed based on comparison of the diluted and undiluted samples analyzed in parallel. Overall survival (OS) was analyzed using Kaplan-Meier.Results:The sensitivity and specificity of the assay was validated by detection of all 8 known TP53 aberrations in serial dilutions with the threshold of the assay established at 0.2% allelic burden. Known mutations were still detectable at 0.02% at the highest dilution. A test sample of patients with known del(17p) demonstrated TP53 mutations in 6 out of 7 patients (5 clonal and 1 subclonal TP53+) in accordance with previously reported frequencies of TP53 mutations among patients with del(17p).In total, 92 samples from 46 consecutive patients were analyzed. With a median coverage of 93,272 reads (98% above 20,688 reads, range: 5,153-720,025), 27 TP53 aberrations were found in 10 (22%) of the patients. Twenty-six (96%) mutations were subclonal with a median allelic burden of 0.9% (range: 0.2-97.5%). Seven patients had a single aberration. In two previously treated patients, 8 and 10 subclonal aberrations were detected. One patient with a known del(17p) also had subclonal TP53 mutations. Three patients had solely subclonal mutations below 1% allelic burden. Considered the hot spot region of TP53 in CLL, exons 4-8 harbored 93% of the detected mutations.The median (IQR) survival for TP53+ patients and patients with wild type TP53 (wt-TP53) was 37.5 (range: 2-106) and 104 (range: 9-113) months, respectively. In Kaplan-Meier analyses, TP53+ patients had a significantly poorer OS versus patients with wt-TP53 (p=0.00002, Figure 1). The two TP53+ long-term survivors both had an allelic burden below 1%, and the only other TP53+ patient with less than 1% allelic burden survived for 53 months, indicating that the prognostic impact of very low allelic burden TP53 aberrations needs further investigation.Conclusion:We have developed a robust assay for TP53 aberrations with a sensitivity of 0.2% allelic burden based on an algorithm including a dilution step. In an initial test cohort of 46 patients, 10 patients demonstrated TP53 mutations as low as 0.2% allelic burden that significantly affected OS. Validation of the clinical impact of subclonal TP53 aberrations is ongoing based on our consecutive biobank with 600+ patients. Establishment of a new cut-off for clinical treatment decisions based on subclonal TP53 aberrations is warranted. [Display omitted] DisclosuresNiemann:Gilead: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Janssen: Consultancy.