TP53 Variants Research Articles

Double Heterozygous Pathogenic Variants in TP53 and CHEK2 in Boy with Undifferentiated Embryonal Sarcoma of the Liver

Undifferentiated embryonal sarcoma of the liver is a rare mesenchymal malignancy that predominantly occurs in children. The relationship between this tumor entity and germline pathogenic variants (PVs) remains undefined. Here, we present the clinical case of a male patient diagnosed with undifferentiated embryonal sarcoma of the liver. Both germline and tumor samples were analyzed using next-generation sequencing. In the tumor tissue, PVs in TP53 (NM_000546.5):c.532del p.(His178Thrfs*69) and CHEK2 (NM_007194.4):c.85C>T p.(Gln29*) were identified, with both confirmed to be of germline origin. Copy number analyses indicated a loss of the wildtype TP53 allele in the tumor, consistent with a second hit, while it was the variant CHEK2 allele that was lost in the tumor. Our data indicate that the germline TP53 PV acts as a driver of tumorigenesis in the reported case and support a complex interaction between the germline TP53 and CHEK2 PVs. This case highlights the dynamic interplays of genetic alterations in tumorigenesis and emphasizes the need for continued investigation into the complex interactions between TP53 and CHEK2 PVs and into the association of undifferentiated embryonal sarcoma of the liver and Li–Fraumeni syndrome.

Distinct Molecular Alterations in Hepatocellular Carcinoma (HCC) Arising in a Non-Fibrotic Liver Compared to a Cirrhotic Liver

Abstract Introduction/Objective HCC is known to more likely develop in a cirrhotic liver than a non-cirrhotic liver, irrespective of etiology, with TP53 and CTNNB1 being the most frequent driver mutations in this group. Whether there are distinctive molecular pathways for HCCs developing in the non-cirrhotic liver is not known. This study evaluated whether there is any difference in the mutational profile of HCCs arising in a non-fibrotic liver compared to a cirrhotic liver. Methods/Case Report Clinicopathologic and in-house next generation sequencing (NGS) data from HCC cases sequenced between 1/1/18-6/30/23 were reviewed. Cases from a targeted precision NGS panel were excluded. Results were correlated with publicly available data from The Cancer Genome Atlas (TCGA). Results (if a Case Study enter NA) 29 cases were available for review; 24 patients had background cirrhosis at the time of diagnosis, while 5 patients had no evidence of fibrosis. 21 of 24 patients (88%) patients in the cirrhosis group had a variant in TP53 and/or CTNNB1 that was considered to be disease associated or probably disease associated, whereas only 1 of 5 patients (20%) in the non-fibrotic group had a variant detected in one of these genes (Fisher’s exact: p < 0.01). BAP1 and ARID1A variants were detected in two patients in the non-fibrotic group. TP53 and CTNNB1 were generally mutually exclusive (86% of cases), however, 3 patients harbored both TP53 and CTNBB1 variants; interestingly all 3 had background cirrhosis and HCV infection. All 6 poorly differentiated tumors occurred in cirrhotic livers and were found to have TP53 variants (Figure 1). TCGA data (N = 210) similarly showed that patients with intermediate fibrosis or established cirrhosis (N = 138) were more likely than patients without background fibrosis (N = 72) to harbor TP53 or CTNNB1 variants (64% vs 42%; Chi-squared: p < 0.01; Figure 2). Aside from TP53 and CTNNB1, the most common variants seen in the TCGA non-fibrotic group were in TTN (22), ALB (10), FREM2 (9), LRP1B (9), and MUC16 (9). Conclusion Both in-house and TCGA data showed that TP53 and/or CTNNB1 variants were more likely to be seen in HCC cases arising in a cirrhotic liver compared to a non-fibrotic liver. This data suggests there may be unique and less-well characterized molecular pathways involved in the neoplastic progression of HCC in a non-fibrotic liver.

Bihemispheric IDH-Wildtype Glioblastoma with Unilateral FGFR3-TACC3 Fusion in Patient with Breast Cancer History: A Case Report and Literature Review

Abstract Introduction/Objective Adult IDH-wild type glioblastomas comprise a heterogeneous spectrum of neoplasms, characterized by poor prognosis and resistance to the chemoradiotherapy. For WHO integrated diagnosis classification and to establish treatment options, molecular analysis is necessary. Herein, we present a case of a glioblastoma, IDH-wildtype with involvement of both cerebral hemispheres and an uncharacteristic in-frame fusion limited to one hemisphere. Methods/Case Report The patient is a 52-year-old woman with past medical history of metastatic breast cancer who presented with headache and worsening facial pain. Brain magnetic resonance imaging brain demonstrated a right frontal lobe lesion with vasogenic edema. The patient was treated with Gamma Knife radiosurgery. Scans revealed enlargement and a new left frontal lobe lesion. Bihemispheric tumor resection and GammaTile placement was undertaken. Results (if a Case Study enter NA) Histologically, the tumor was composed of hypercellular astrocytes, microvascular proliferation, and necrosis with no evidence of metastatic disease. Immunohistochemistically, neoplastic cells showed glial fibrillary acidic protein expression and elevated Ki-67 proliferation index. Molecular examination revealed in the right hemisphere lesion post-radiation (1) FGFR3::TACC3 (exon 17::exon 11) fusion and (2) TERT promoter, while the left hemisphere lesion which did not receive radiation showed clinically relevant variants in (1) TERT promoter, (2) TP53, (3) NF1, and (3) PIK3CA without identification of a fusion. An integrated diagnosis of glioblastoma, IDH- wildtype, CNS WHO grade 4 was established bilaterally. Conclusion The fibroblast growth factor receptor 3 (FGFR3)-transforming acidic coiled-coil 3 (TACC3) fusion is identified as a rare molecular feature in glioblastomas with an estimated prevalence of 4%. Clinical trials suggest that F3T3-positive tumors have a better prognosis than those without the translocation. The identification of the oncogenic FGFR3-TACC3 fusion highlights the possibility of identifying patients potentially responsive to targeted therapy with FGFR kinase inhibitors. The significance of the molecular differences in the right and left hemisphere lesions is uncertain; glioblastomas are heterogeneous tumors with intra-and intertumoral heterogeneity but it may suggest independent origins.

TP53 mutation status and consensus molecular subtypes of colorectal cancer in patients from Rwanda

BackgroundMutations in the TP53 tumor suppressor gene are well-established drivers of colorectal cancer (CRC) development. However, data on the prevalence of TP53 variants and their association with consensus molecular subtype (CMS) classification in patients with CRC from Rwanda are currently lacking. This study addressed this knowledge gap by investigating TP53 mutation status concerning CMS classification in a CRC cohort from Rwanda.MethodsFormalin-fixed paraffin-embedded (FFPE) tissue blocks were obtained from 51 patients with CRC at the University Teaching Hospital of Kigali, Rwanda. Exons 4 to 11 and their flanking intron-exon boundaries in the TP53 gene were sequenced using Sanger sequencing to identify potential variants. The recently established immunohistochemistry-based classifier was employed to determine the CMS of each tumor.ResultsSequencing analysis of cancerous tissue DNA revealed TP53 pathogenic variants in 23 of 51 (45.1%) patients from Rwanda. These variants were predominantly missense types (18/23, 78.3%). The most frequent were c.455dup (p.P153Afs*28), c.524G > A (p.R175H), and c.733G > A (p.G245S), each identified in three tumors. Trinucleotide sequence context analysis of the 23 mutations (20 of which were single-base substitutions) revealed a predominance of the [C > N] pattern among single-base substitutions (SBSs) (18/20; 90.0%), with C[C > T]G being the most frequent mutation (5/18, 27.8%). Furthermore, pyrimidine bases (C and T) were preferentially found at the 5ʹ flanking position of the mutated cytosine (13/18; 72.2%). Analysis of CMS subtypes revealed the following distribution: CMS1 (microsatellite instability-immune) (6/51, 11.8%), CMS2 (canonical) (28/51, 54.9%), CMS3 (metabolic) (9/51, 17.6%), and CMS4 (mesenchymal) (8/51, 15.7%). Interestingly, the majority of TP53 variants were in the CMS2 subgroup (14/23; 60.1%).ConclusionOur findings indicate a high frequency of TP53 variants in CRC patients from Rwanda. Importantly, these variants are enriched in the CMS2 subtype. This study, representing the second investigation into molecular alterations in patients with CRC from Rwanda and the first to explore TP53 mutations and CMS classification, provides valuable insights into the molecular landscape of CRC in this understudied population.

8659 Aldosterone-Secreting Adrenocortical Carcinoma: A Rare Cause Of Primary Hyperaldosteronism Associated With Dismal Outcome

Abstract Disclosure: S. Beeby: None. C.H. Huzita: None. F.Q. Aratani: None. L.S. Motomia: None. F. Coelho: None. M. Polo: None. C.R. Victor: None. A.O. Hoff: None. J. Chambo: None. V. Srougi: None. F. Ledesma: None. A. Latronico: None. M.Q. Almeida: None. B.B. Mendonca: None. M.C. Fragoso: None. Background: Adrenocortical carcinoma (ACC) is a rare and one of the most aggressive solid tumors, with an incidence of 1-2 cases per million. In adults, hormonal excess is usually manifested by ACTH-independent Cushing's syndrome or mixed endocrine syndrome due to cortisol and androgen production. Isolated aldosterone secretion is an extremely rare condition, with an estimated 2.5% of patients with ACC. We aimed to describe two patients with aldosterone-secreting ACC with poor outcomes. Case 1 - A 34-year-old male who was diagnosed with hypertension at 29 years of age, associated with hypokalemia needing intravenous potassium replacement therapy. Diagnosis of primary hyperaldosteronism was established with aldosterone levels of 72 ng/dL (RR < 23.1 ng/dL) and renin activity of 1.1 ng/mL/h (RR 0.3 to 5.8 ng/mL/h. An abdominal CT scan identified a tumor mass of 9.5 x 8.0 x 4.5 cm. He underwent a left adrenalectomy and histological and immunohistochemistry data suggested an adrenocortical carcinoma (modified Weiss score 5/7, Ki67 40%). Initial staging was ENSAT III and a Helsinki score of 48. There was transient improvement, however recent laboratory studies showed an increase in aldosterone (66,9 ng/dL) accompanied by new episodes of hypokalemia. CT scans on follow-up showed recurrence of disease, with the presence of nodules on the surgical site and abdominal wall. He received 3 lines of systemic chemotherapy and mitotane and underwent 2 other surgical procedures to treat the metastatic lesions. Currently, overall survival is 3 years. Case 2 - A 44-year-old female incidentally found a right adrenal mass (6.6 x 6.4 cm) on MRI during the investigation of elevated liver enzymes and abdominal pain. Laboratory studies showed hypokalemia, with potassium levels of 2 mEq/L (RR 3.5-5 mEq/L), aldosterone levels of 243 ng/dL (RR < 23.1 ng/dL), and renin of 12.2 uUI/mL (RR 4.4 - 46.1 uUI/mL). She underwent right adrenalectomy and histology and immunohistochemistry data showed a modified Weiss 7/7 and Ki67 of 80%. The patient had a germline pathogenic variant of TP53 (p.R337H). Initial staging was ENSAT III and a Helsinki score of 88. She received adjuvant mitotane and systemic chemotherapy. Even so, follow-up imaging showed hepatic metastatic disease, with an elevated aldosterone production surpassing 1000 ng/dL. The patient's overall survival was 2 years and she passed away during surgical procedure for resection of liver metastases. Discussion: ACC is a rare and aggressive tumor, and isolated aldosterone production by such tumors is extremely rare. We presented two cases with poor prognostic features and in which despite adjuvant mitotane and systemic chemotherapy there was disease progression. Due to the rarity of this condition, data on patient outcomes is limited, and further understanding of the molecular signature of these tumors could bring new insights on the tumorigenesis process. Presentation: 6/3/2024

Abstract C158: Contribution of multi-organ cancer predisposition genes for gastric cancer risk of different histological subtypes

Abstract Approximately 10% of gastric cancer (GC) cases exhibit familial clustering, yet only 1-3% can be explained by known hereditary syndromes, being Hereditary Diffuse Gastric Cancer (HDGC) the most well-established disease predisposing for diffuse gastric cancer. Familial intestinal gastric cancer (FIGC) has been defined clinically, but it remains mostly genetically unexplained. Likewise, the heritability of mixed GC remains to be uncovered. We aimed to estimate the frequency of known cancer predisposition gene variants in GC cases with family history of cancer and/or early onset. We evaluated the contribution of pathogenic and likely pathogenic (P/LP) variants in well-established and multi-organ cancer predisposition genes for GC risk in a large multi-center study involving 750 GC patients, either by targeted sequencing or whole exome sequencing. We identified 45 patients (6%) with P/LP variants in ATM (17 cases), BRCA2 (10 cases), MLH1 (five cases), TP53 (three cases), BRCA1, PALB2, RAD51D and CHEK2 (two patients each), and RAD51C and PMS2 (one case each), all mutually exclusive. The P/LP variant prevalence was higher in intestinal (9.8%) than in diffuse (4.3%) or mixed GC (4.5%) (p-value=0.023), with no clear difference per mutated gene by histological subtypes. Only sixteen of the 45 variant carriers fulfilled the criteria for genetic testing of at least one cancer predisposition syndrome. Our findings indicate that multi- organ cancer predisposition genes should be included in gene panels used for investigating germline variants in GC patients, especially when there is family history of cancer but irrespective of histology subtype, as this would increase the chance of identifying patients that might benefit from prevention and targeted treatment strategies. Citation Format: Ana P. Estrada-Florez, Paul C. Lott, Luis G. Carvajal Carmona. Contribution of multi-organ cancer predisposition genes for gastric cancer risk of different histological subtypes [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C158.

Cancer associated variant enrichment CAVE, a gene agnostic approach to identify low burden variants in chronic lymphocytic leukemia

Intratumoral heterogeneity is an important clinical challenge because low burden clones expressing specific genetic alterations drive therapeutic resistance mechanisms. We have developed CAVE (cancer-associated variant enrichment), a gene-agnostic computational tool to identify specific enrichment of low-burden cancer driver variants in next-generation sequencing (NGS) data. For this study, CAVE was applied to TP53 in chronic lymphocytic leukemia (CLL) as a cancer model. Indeed, as TP53 mutations are part of treatment decision-making algorithms and low-burden variants are frequent, there is a need to distinguish true variants from background noise. Recommendations have been published for reliable calling of low-VAF variants of TP53 in CLL and the assessment of the background noise for each platform is essential for the quality of the testing. CAVE is able to detect specific enrichment of low-burden variants starting at variant allele frequencies (VAFs) as low as 0.3%. In silico TP53 dependent and independent analyses confirmed the true driver nature of all these variants. Orthogonal validation using either ddPCR or NGS analyses of follow-up samples confirmed variant identification. CAVE can be easily deployed in any cancer-related NGS workflow to detect the enrichment of low-burden variants of clinical interest.

Germline variant affecting p53β isoforms predisposes to familial cancer

Germline and somatic TP53 variants play a crucial role during tumorigenesis. However, genetic variations that solely affect the alternatively spliced p53 isoforms, p53β and p53γ, are not fully considered in the molecular diagnosis of Li-Fraumeni syndrome and cancer. In our search for additional cancer predisposing variants, we identify a heterozygous stop-lost variant affecting the p53β isoforms (p.*342Serext*17) in four families suspected of an autosomal dominant cancer syndrome with colorectal, breast and papillary thyroid cancers. The stop-lost variant leads to the 17 amino-acid extension of the p53β isoforms, which increases oligomerization to canonical p53α and dysregulates the expression of p53’s transcriptional targets. Our study reveals the capacity of p53β mutants to influence p53 signalling and contribute to the susceptibility of different cancer types. These findings underscore the significance of p53 isoforms and the necessity of comprehensive investigation into the entire TP53 gene in understanding cancer predisposition.

Germline p.R181H variant in TP53 in a family exemplifying the genotype-phenotype correlations in Li-Fraumeni syndrome.

Li-Fraumeni syndrome (LFS) is a cancer predisposition syndrome associated with germline pathogenic/likely pathogenic variants in TP53. Genotype-phenotype correlations are progressively being characterized in LFS with certain TP53 variants associated with attenuated penetrance and phenotypes. We report on a family harboring a TP53 p.R181H variant presenting with a restricted cancer phenotype in adulthood. The proband was a female with breast cancer at the age of 71 years who had three first degree relatives also diagnosed with breast cancer after the age of 40 years (mother, two sisters). Of the nine individuals harboring the variant (6 genetically confirmed, 3 obligate heterozygous), six have not developed malignancies at this time (age range: 36-42). No childhood-onset cancers were reported in this family. A concomitant literature review identified 51 additional individuals harboring the p.R181H variant in TP53, presenting a tumor phenotype dominated by breast cancer. Rare occurrences of other adult-onset cancers (prostate, colorectal and thyroid) and only few childhood onset cancer were documented. These observations are consistent with functional analysis showing that p.R181H retains partial p53 function and suggesting possible reduced cancer penetrance, particularly in the pediatric setting.

Abstract IA004: Risk of therapy-related subsequent cancers in individuals with germline TP53 variants

Abstract Background. Based primarily on in vitro and animal studies, germline TP53 variants are considered to confer sensitivity to ionizing radiation-induced DNA damage, but data in humans are sparse. We sought to quantify risk of developing radiotherapy and chemotherapy-related subsequent neoplasms by pooling individual-level data from nine cohorts of individuals with confirmed pathogenic/likely pathogenic germline TP53 variants. Methods. 891 individuals were followed from six months after first cancer until diagnosis of a subsequent neoplasm, death or date of last follow-up. Radiotherapy and chemotherapy for each cancer were considered as time-dependent covariates in Cox regression models with age as the time-scale, adjusted for sex and occurrence of prior malignant or benign tumors. Subsequent cancers were classified as in- or out-of-field based on the body region of previous cancers. Results. Among 891 individuals, 643 (72.2%) were female and 297 (33.3%) were diagnosed with a first cancer before age 20 years. Breast cancer was the most commonly diagnosed first cancer (N=371), followed by soft tissue sarcoma (N=131) and central nervous system cancers (N=84). First cancer treatment included radiotherapy for 237 (26.6%) and/or chemotherapy for 421 (47.3%) individuals. Analyses are underway to define the characteristics of subsequent neoplasms (including age at diagnosis, latency, and distribution of diagnosis type) and to calculate relative and absolute risk estimates for radiotherapy and chemotherapy overall and by subsequent neoplasm type. Conclusions. This study will quantify the contribution of radiotherapy and chemotherapy to the risk of subsequent cancers among individuals with Li-Fraumeni syndrome. Understanding the magnitude of this risk will support health care providers and patients in making informed decisions about cancer management. Citation Format: Anita Villani, Payal Khincha, Jessica Hatton, Sara Schonfeld, Catherine Goudie, Leatrisse Oba, Kishan Pithadia, Birte Sänger, Christian Kratz, Anh N. Le, Caitlin Orr, Kara N. Maxwell, Anne Naumer, Jennie Vagher, Wendy Kohlmann, Joshua Schiffman, Elizabeth Santana dos Santos, Orli Michaeli, Jacob J. Carson, Derek S. Tsang, Raymond Kim, Jamie L. Maciaszek, Kim E. Nichols, Victoria E. Goodman, Suzanne P. MacFarland, Lara Reichman, Maria Isabel Achatz, Sharon Savage, David Malkin, Lindsay M. Morton. Risk of therapy-related subsequent cancers in individuals with germline TP53 variants [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr IA004.

Cell-free DNA from germline TP53 mutation carriers reflect cancer-like fragmentation patterns

Germline pathogenic TP53 variants predispose individuals to a high lifetime risk of developing multiple cancers and are the hallmark feature of Li-Fraumeni syndrome (LFS). Our group has previously shown that LFS patients harbor shorter plasma cell-free DNA fragmentation; independent of cancer status. To understand the functional underpinning of cfDNA fragmentation in LFS, we conducted a fragmentomic analysis of 199 cfDNA samples from 82 TP53 mutation carriers and 30 healthy TP53-wildtype controls. We find that LFS individuals exhibit an increased prevalence of A/T nucleotides at fragment ends, dysregulated nucleosome positioning at p53 binding sites, and loci-specific changes in chromatin accessibility at development-associated transcription factor binding sites and at cancer-associated open chromatin regions. Machine learning classification resulted in robust differentiation between TP53 mutant versus wildtype cfDNA samples (AUC-ROC = 0.710–1.000) and intra-patient longitudinal analysis of ctDNA fragmentation signal enabled early cancer detection. These results suggest that cfDNA fragmentation may be a useful diagnostic tool in LFS patients and provides an important baseline for cancer early detection.

TP53 variants underlying pediatric low-hypodiploidy B-cell acute lymphoblastic leukemia demonstrate diverse origins and may persist as a hematopoietic clone in remission.

Pediatric low-hypodiploidy B-cell acute lymphoblastic leukemia (LH-ALL) with TP53 variants has been proposed to be considered a manifestation of Li-Fraumeni syndrome (LFS). However, our study demonstrates that of the majority the pathogenic variants in the TP53 gene are somatic (70.5%), and only 12.5% of patients with germline fulfilled the criteria of LFS. We also describe the first case of hypodiploid BCP-ALL with a mosaic pathogenic mutation in TP53 and the first case of the persistence of clonal hematopoiesis with the TР53 gene mutation in the child during 3-year minimal residual disease-negative remission, similar to what has been described in adults.

Variation characteristics and clinical significance of TP53 in patients with myeloid neoplasms

ABSTRACT Objectives: MDS and AML characterized by TP53 variations have a poor prognosis in general. However, specifically, differences in prognosis have also been observed in patients with different TP53 variants and VAFs. Methods: Here, we retrospectively analyzed datasets of patients with MDS, MPN, and AML who underwent targeted DNA sequencing from February 2018 to December 2023, and patients with reportable TP53 variations were screened. Demographic data and clinical data were collected, and the relationship between TP53 alterations and patient prognosis (AML/MDS) was analyzed using the cBioPortal and Kaplan–Meier Plotter databases. The relationship between the VAFs of TP53 variations and prognoses was analyzed using data from the present study. Results: Sixty-two variants of TP53 were identified in 58 patients. We mainly identified single mutations (79.31%, 46/58), followed by double (17.24%, 10/58) and triple (3.45%, 2/58) mutations. The variations were mainly enriched in exon4–exon8 of TP53. Missense (72.58%, 45/62) mutations were the main type of variations, followed by splice-site (9.68%, 6/62), nonsense (9.68%, 6/62), frameshift (6.45%, 4/62), and indel (1.61%, 1/62) mutations. In this study, p.Arg175His and p.Arg273His were high-frequency TP53 mutations, and DNMT3A and TET2 were commonly co-mutated genes in the three types of myeloid neoplasms; However, we reported some new TP53 variants in MPN that have not been found in the public database. Moreover, MDS or AML characterized by altered TP53 had a shorter OS than patients in the unaltered group (P<0.01), low TP53 mRNA levels were associated with shorter OS in patients with AML (P<0.01). Data from our center further found higher VAF (≥10%) associated with shorter OS in patients with MDS (median 2.75 vs. 24 months) (P<0.01). Conclusion: TP53 mutations are mainly enriched in exon4–exon8, are missense and single mutations in myeloid neoplasms, and are associated with poor prognosis of MDS/AML, and higher VAF (≥10%) of TP53 mutations associated with a shorter OS in patients with MDS.

Cell-free DNA from ascites identifies clinically relevant variants and tumour evolution in patients with advanced ovarian cancer.

The emergence of targeted therapies has transformed ovarian cancer treatment. However, biomarker profiling for precision medicine is limited by access to quality, tumour-enriched tissue samples. The use of cell-free DNA (cfDNA) in ascites presents a potential solution to this challenge. In this study, next-generation sequencing was performed on ascites-derived cfDNA samples (26 samples from 15 human participants with ovarian cancer), with matched DNA from ascites-derived tumour cells (n = 5) and archived formalin-fixed paraffin-embedded (FFPE) tissue (n = 5). Similar tumour purity and variant detection were achieved with cfDNA compared to FFPE and ascites cell DNA. Analysis of large-scale genomic alterations, loss of heterozygosity and tumour mutation burden identified six cases of high genomic instability (including four with pathogenic BRCA1 and BRCA2 mutations). Copy number profiles and subclone prevalence changed between sequential ascites samples, particularly in a case where deletions and chromothripsis in Chr17p13.1 and Chr8q resulted in changes in clinically relevant TP53 and MYC variants over time. Ascites cfDNA identified clinically actionable information, concordant to tissue biopsies, enabling opportunistic molecular profiling. This advocates for analysis of ascites cfDNA in lieu of accessing tumour tissue via biopsy.

Exomic and epigenomic analysis of pulmonary blastoma

ObjectivePulmonary blastoma is a rare, biphasic, adult-onset lung tumor. In this study, we investigate whether DICER1 pathogenic variants are a feature of pulmonary blastomas through in-depth analysis of the molecular events defining them. MethodsWe performed exome-wide sequencing and DNA methylation profiling of 8 pulmonary blastomas from 6 affected persons. ResultsWe identified biallelic somatic DICER1 pathogenic variants in 7 of 8 cases. The remaining case had a solitary missense pathogenic variant in the RNase IIIb domain of DICER1. Six of 8 cases carried a CTNNB1 hotspot variant and 4 of 8 had a somatic pathogenic variant in TP53. Methylation analysis showed that the pulmonary blastomas clustered with other DICER1-mutated tumors and not with other more common types of lung cancer. ConclusionWe conclude somatic DICER1 pathogenic variants are the major driver of pulmonary blastoma and are likely to act in conjunction with CTNNB1 hotspot variants that are often present.

Repeat Next-Generation Sequencing (15-Gene Panel) in Unifocal, Synchronous, and Metachronous Non-Small-Cell Lung Cancer-A Single-Center Experience.

In advanced non-squamous non-small-cell lung cancer (NSCLC), routine testing with next-generation sequencing (NGS) is recommended to identify actionable genomic alterations (AGAs). The therapeutic implications of repeated NGS testing on synchronous and metachronous tumors are unclear. Between February 2017 and October 2020, NSCLC samples from a single institution were reflex-tested using a targeted 15-gene NGS panel (TruSight Tumor 15, Illumina). Thirty-eight patients were identified with multiple NGS results from 82 samples: 11% were from single unifocal, 51% were from synchronous, and 38% were from metachronous tumors. Changes in EGFR, KRAS, PI3KCA, and TP53 variants were found in 22 patients' samples (58%). No changes were seen with longitudinal testing of multiple samples from single unifocal tumors, while changes were observed in 60% of synchronous and 71% of metachronous tumors. Of these, 26% of patients had AGA differences between samples. Acknowledging the limited sample size, a significant difference in overall survival was observed between synchronous separate primaries and metastasis. Repeat NGS testing of synchronous and metachronous NSCLC tumors may identify differing variants in >50% of patients. These changes may reflect separate primary lung carcinomas, tumor heterogeneity among intrapulmonary metastases, and clonal evolution. NGS testing of multiple tumors may enhance the identification of therapeutic targets for treatment decisions.

Co-observation of germline pathogenic variants in breast cancer predisposition genes: Results from analysis of the BRIDGES sequencing dataset

Co-observation of a gene variant with a pathogenic variant in another gene that explains the disease presentation has been designated as evidence against pathogenicity for commonly used variant classification guidelines. Multiple variant curation expert panels have specified, from consensus opinion, that this evidence type is not applicable for the classification of breast cancer predisposition gene variants. Statistical analysis of sequence data for 55,815 individuals diagnosed with breast cancer from the BRIDGES sequencing project was undertaken to formally assess the utility of co-observation data for germline variant classification. Our analysis included expected loss-of-function variants in 11 breast cancer predisposition genes and pathogenic missense variants in BRCA1, BRCA2, and TP53. We assessed whether co-observation of pathogenic variants in two different genes occurred more or less often than expected under the assumption of independence. Co-observation of pathogenic variants in each of BRCA1, BRCA2, and PALB2 with the remaining genes was less frequent than expected. This evidence for depletion remained after adjustment for age at diagnosis, study design (familial versus population-based), and country. Co-observation of a variant of uncertain significance in BRCA1, BRCA2, or PALB2 with a pathogenic variant in another breast cancer gene equated to supporting evidence against pathogenicity following criterion strength assignment based on the likelihood ratio and showed utility in reclassification of missense BRCA1 and BRCA2 variants identified in BRIDGES. Our approach has applicability for assessing the value of co-observation as a predictor of variant pathogenicity in other clinical contexts, including for gene-specific guidelines developed by ClinGen Variant Curation Expert Panels.

Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li–Fraumeni syndrome

Germline TP53 pathogenic variants can lead to a cancer susceptibility syndrome known as Li–Fraumeni (LFS). Variants affecting its activity can drive tumorigenesis altering p53 pathways and their identification is crucial for assessing individual risk. This study explored the functional impact of TP53 missense variants on its transcription factor activity. We selected seven TP53 missense variants (c.129G > C, c.320A > G, c.417G > T, c.460G > A, c,522G > T, c.589G > A and c.997C > T) identified in Brazilian families at-risk for LFS. Variants were created through site-directed mutagenesis and transfected into SK-OV-3 cells to assess their transcription activation capabilities. Variants K139N and V197M displayed significantly reduced transactivation activity in a TP53-dependent luciferase reporter assay. Additionally, K139N negatively impacted CDKN1A and MDM2 expression and had a limited effect on GADD45A and PMAIP1 upon irradiation-induced DNA damage. Variant V197M demonstrated functional impact in all target genes evaluated and loss of Ser15 phosphorylation. K139N and V197M variants presented a reduction of p21 levels after irradiation. Our data show that K139N and V197M negatively impact p53 functions, supporting their classification as pathogenic variants. This underscores the significance of conducting functional studies on germline TP53 missense variants classified as variants of uncertain significance to ensure proper management of LFS-related cancer risks.

Comprehensive classification of TP53 somatic missense variants based on their impact on p53 structural stability.

Somatic variation is a major type of genetic variation contributing to human diseases including cancer. Of the vast quantities of somatic variants identified, the functional impact of many somatic variants, in particular the missense variants, remains unclear. Lack of the functional information prevents the translation of rich variation data into clinical applications. We previously developed a method named Ramachandran Plot-Molecular Dynamics Simulations (RP-MDS), aiming to predict the function of germline missense variants based on their effects on protein structure stability, and successfully applied to predict the deleteriousness of unclassified germline missense variants in multiple cancer genes. We hypothesized that regardless of their different genetic origins, somatic missense variants and germline missense variants could have similar effects on the stability of their affected protein structure. As such, the RP-MDS method designed for germline missense variants should also be applicable to predict the function of somatic missense variants. In the current study, we tested our hypothesis by using the somatic missense variants in TP53 as a model. Of the 397 somatic missense variants analyzed, RP-MDS predicted that 195 (49.1%) variants were deleterious as they significantly disturbed p53 structure. The results were largely validated by using a p53-p21 promoter-green fluorescent protein (GFP) reporter gene assay. Our study demonstrated that deleterious somatic missense variants can be identified by referring to their effects on protein structural stability.

Case series of Li-Fraumeni syndrome: carcinogenic mechanisms in breast cancer with TP53 pathogenic variant carriers

BackgroundLi-Fraumeni syndrome (LFS), a hereditary condition attributed to TP53 pathogenic variants,(PV), is associated with high risks for various malignant tumors, including breast cancer. Notably, individuals harboring TP53 PVs are more likely (67–83%) to develop HER2 + breast cancer than noncarriers (16–25%). In this retrospective study, we evaluated the associations between TP53 variants and breast cancer phenotype.MethodsWe conducted a retrospective review of the medical records of patients with LFS treated at a single institution and reviewed the literature on TP53 functions and the mechanisms underlying HER2 + breast cancer development in LFS.ResultsWe analyzed data for 10 patients with LFS from 8 families. The median age at the onset of the first tumor was 35.5 years. Only case 2 met the classic criteria; this patient harbored a nonsense variant, whereas the other patients carried missense variants. We observed that 9 of 10 patients developed breast cancer. Immunohistochemical analyses revealed that 40% of breast cancers in patients with LFS were HR − /HER2 + . The median age at the onset of breast cancer was slightly younger in HR − /HER2 + tumors than in HR + /HER2 − tumors (31 years and 35.5 years, respectively).ConclusionsThe occurrence of HER2 + breast cancer subtype was 40% in our LFS case series, which is greater than that in the general population (16–25%). Some TP53 PVs may facilitate HER2-derived oncogenesis in breast cancer. However, further studies with larger sample sizes are warranted to clarify the oncogenic mechanisms underlying each subtype of breast cancer in TP53 PV carriers.