Acquisition Of TP53 Mutations Research Articles

Abstract IA025: Decoding breast cancer evolution with single cell genomics

Abstract I will discuss our efforts to develop and apply single cell DNA sequencing technologies to study breast cancer progression and evolution. We have developed a method called Acoustic Cell Tagmentation (ACT) which can provide high-throughput single cell copy number profiling of hundreds of cells in parallel at single-molecule resolution. We have applied this approach to study copy number evolution in Triple-negative breast cancer, which has revealed a ‘transient instability’ model of early copy number evolution, after the acquisition of TP53 mutations and genome doubling. We have also developed a computational toolkit called CopyKit to analyze single cell DNA sequencing data, and applied it to study metastasis in breast and colon cancer, as well as the spatial organization of subclones in metastatic tumors. Collectively these studies and tools provide new insights into the evolution of chromosome aberrations in human tumors. Citation Format: Nicholas Navin. Decoding breast cancer evolution with single cell genomics [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr IA025.

Ywhae Located on Chromosome 17p13 Promotes Myeloma Cells Proliferation Via Induction of Anti-Apoptosis and Survival Signaling Pathways

Patients with multiple myeloma (MM) carrying 17p deletion (del17p) have poor outcome. Around a third of MM patients with del(17p) are also reported to have a TP53 mutation. The acquisition of TP53 mutations seems to occur after allelic loss of 17p, and confers worse clinical outcomes. However, a recent publication has raised possibility that other additional genes resident at 17p13 may play a role in the clinical outcome of this subgroup of MM patients.The YWHAE gene is located at 17p13.3 and encodes the 14-3-3ε protein, one of the mammalian 14-3-3 protein family members that are highly conserved in eukaryotes. Altered 14-3-3ε expression is associated with development and progression of cancer. It has a context dependent pleotropic effect both as oncogene or tumor suppressor gene. However, its precise role in tumorigenesis has yet to be fully discovered.Using a large cohort of MM patients (n=369) RNA sequencing and SNP array data with complete clinical annotation, we observed a significant positive correlation (r=0.7) between YWHAE and TP53 at gene and copy number levels. In MM patients with del17p, YWHAE expression was significantly lower compared to normal plasma cells. Importantly, low expression of YWHAE correlated with poor clinical outcome (both EFS and OS), suggesting that YWHAE may be important in this specific myeloma clinical setting. However, in 2 independent MM datasets, we observed that overall YWHAE expression is higher in CD138+ MM cells compared to normal plasma cells. Moreover, we confirmed high expression of 14-3-3ε at the protein level in a large panel of MM cell lines and primary MM cells.To clarify this apparent paradox and shed light on the role of 14-3-3ε in MM, we used gene-specific CRISPR screen as well as shRNA knock down (KD) to study the functional impact of perturbation of YWHAE in MM. For the loss-of-function studies, we selected a large panel of MM cell lines based on their p53 status (WT, HD, Mu). We found a significant inhibitory effect of YWHAE KD on MM cell growth and survival over time, and induction of apoptosis via PARP cleavage and BAX increase. The antiproliferative effects were even more pronounced in p53 WT myeloma cell lines, such as MM1S and H929.Gene expression profiling (GEP) analysis showed a significant number of differentially expressed genes in YWHAE KD cells compared to control cells. Gene enrichment analysis revealed a major impact of YWHAE KD on a large set of genes involved in the mTORC1 signaling pathway. WB analysis confirmed a reduction of p-mTOR along with a decreased of p-p70 S6 kinase and p-4E BP1 after YWHAE KD. Considering the crucial role of mTOR pathway in the regulation of autophagy, we are now in the process of analyzing the effect of YWHAE on the autophagic response. Moreover, we found that YWHAE KD exerted a significant suppression of known E2F1 gene targets. Notably, a significant decrease in the expression of genes involved in the unfolded protein response was observed upon silencing/knock-out of YWHAE. As a consequence, myeloma cell sensitivity to bortezomib was increased after 14-3-3ε KD.In conclusion, YWHAE and its coding protein 14-3-3ε are constitutively expressed in MM cells. Perturbation of 14-3-3ε expression impacts MM cell growth via induction of apoptosis and inhibition of mTOR pathway. 14-3-3ε KD increase cell sensitivity to bortezomib. Ongoing efforts will further elucidate the potential molecular mechanism of YWHAE/14-3-3ε action in MM. DisclosuresAnderson:Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

3033 – SINGLE-CELL MULTI-OMIC ANALYSIS UNRAVELS GENETIC AND MOLECULAR HETEROGENEITY IN TRANSFORMED MYELOPROLIFERATIVE NEOPLASMS

One out of five patients with myeloproliferative neoplasms (MPN) will progress to an aggressive and largely untreatable form of Acute Myeloid Leukaemia (post-MPN secondary AML, or sAML). Transformation is associated with acquisition of TP53 missense mutations in approximately 50% of cases, and markedly increased genetic and molecular heterogeneity. This makes it challenging to effectively eliminate all subpopulations of tumour cells, and likely underlies the mostly universal treatment failure in sAML. Characterizing the complex genetic and cellular hierarchies associated with TP53 transformation is crucial for the development of effective targeted therapies. To resolve these multiple layers of intratumoral heterogeneity, we recently developed TARGET-seq (Rodriguez-Meira et al, 2019), a single-cell multi-omic method that correlates genetic, transcriptional and cell-surface proteomic readouts from the same single cell with extremely high resolution. TARGET-seq analysis of over 7000 Lineage-CD34+ cells (indexed for CD38/90/45RA/123) from TP53-mutant sAML patients revealed striking patterns of clonal evolution in different immunophenotypically defined stem/progenitor cell types, and identified preleukemic and leukemic subclones with markedly distinct molecular signatures. Acquisition of TP53 mutations was invariably associated with highly complex clonal evolution, enrichment of cell-cycle associated transcription and downregulation of key stem cell regulators. Interestingly, preleukemic subclones from patients undergoing transformation were highly enriched in WNT-beta catenin signaling and depleted in DNA-repair genes, as compared to non-transformed MPN patients. This suggests that preleukemic clones could be primed for disease transformation, and could potentially be targeted to prevent disease progression. Ultimately, single-cell multi-omic analysis identified key molecular regulators of TP53 mediated transformation, providing unique insights into the evolution of these tumors and how they might be therapeutically targeted.

Detection of TP53 Mutations in Tissue or Liquid Rebiopsies at Progression Identifies ALK+ Lung Cancer Patients with Poor Survival.

Anaplastic lymphoma kinase (ALK) sequencing can identify resistance mechanisms and guide next-line therapy in ALK+ non-small-cell lung cancer (NSCLC), but the clinical significance of other rebiopsy findings remains unclear. We analysed all stage-IV ALK+ NSCLC patients with longitudinally assessable TP53 status treated in our institutions (n = 62). Patients with TP53 mutations at baseline (TP53mutbas, n = 23) had worse overall survival (OS) than patients with initially wild-type tumours (TP53wtbas, n = 39, 44 vs. 62 months in median, p = 0.018). Within the generally favourable TP53wtbas group, detection of TP53 mutations at progression defined a “converted” subgroup (TP53mutconv, n = 9) with inferior OS, similar to that of TP53mutbas and shorter than that of patients remaining TP53 wild-type (TP53wtprogr, 45 vs. 94 months, p = 0.043). Progression-free survival (PFS) under treatment with tyrosine kinase inhibitors (TKI) for TP53mutconv was comparable to that of TP53mutbas and also shorter than that of TP53wtprogr cases (5 and 8 vs. 13 months, p = 0.0039). Fewer TP53wtprogr than TP53mutbas or TP53mutconv cases presented with metastatic disease at diagnosis (67% vs. 91% or 100%, p < 0.05). Thus, acquisition of TP53 mutations at progression is associated with more aggressive disease, shorter TKI responses and inferior OS in ALK+ NSCLC, comparable to primary TP53 mutated cases.

Somatic mutations and clonal hematopoiesis in congenital neutropenia

AbstractSevere congenital neutropenia (SCN) and Shwachman-Diamond syndrome (SDS) are congenital neutropenia syndromes with a high rate of leukemic transformation. Hematopoietic stressors may contribute to leukemic transformation by increasing the mutation rate in hematopoietic stem/progenitor cells (HSPCs) and/or by promoting clonal hematopoiesis. We sequenced the exome of individual hematopoietic colonies derived from 13 patients with congenital neutropenia to measure total mutation burden and performed error-corrected sequencing on a panel of 46 genes on 80 patients with congenital neutropenia to assess for clonal hematopoiesis. An average of 3.6 ± 1.2 somatic mutations per exome was identified in HSPCs from patients with SCN compared with 3.9 ± 0.4 for healthy controls (P = NS). Clonal hematopoiesis due to mutations in TP53 was present in 48% (13/27) of patients with SDS but was not seen in healthy controls (0/17, P < .001) or patients with SCN (0/40, P < .001). Our SDS cohort was young (median age 6.3 years), and many of the patients had multiple TP53 mutations. Conversely, clonal hematopoiesis due to mutations of CSF3R was present in patients with SCN but was not detected in healthy controls or patients with SDS. These data show that hematopoietic stress, including granulocyte colony-stimulating factor, do not increase the mutation burden in HSPCs in congenital neutropenia. Rather, distinct hematopoietic stressors result in the selective expansion of HSPCs carrying specific gene mutations. In particular, in SDS there is enormous selective pressure to expand TP53-mutated HSPCs, suggesting that acquisition of TP53 mutations is an early, likely initiating event, in the transformation to myelodysplastic syndrome/acute myeloid leukemia in patients with SDS.

Synergistic activity and heterogeneous acquired resistance of combined MDM2 and MEK inhibition in KRAS mutant cancers.

There are currently no effective targeted therapies for KRAS mutant cancers. Therapeutic strategies that combine MEK inhibitors with agents that target apoptotic pathways may be a promising therapeutic approach. We investigated combining MEK and MDM2 inhibitors as a potential treatment strategy for KRAS mutant non-small cell lung cancers and colorectal carcinomas that harbor wild-type TP53. The combination of pimasertib (MEK inhibitor) + SAR405838 (MDM2 inhibitor) was synergistic and induced the expression of PUMA and BIM, led to apoptosis and growth inhibition in vitro, and tumor regression in vivo. Acquired resistance to the combination commonly resulted from the acquisition of TP53 mutations, conferring complete resistance to MDM2 inhibition. In contrast, resistant clones exhibited marked variability in sensitivity to MEK inhibition, which significantly impacted sensitivity to subsequent treatment with alternative MEK inhibitor-based combination therapies. These results highlight both the potential promise and limitations of combining MEK and MDM2 inhibitors for treatment of KRAS mutant NSCLC and CRC.

Impact of genotype on leukaemic transformation in polycythaemia vera and essential thrombocythaemia.

The influence of driver mutations on leukaemic transformation was analysed in 1747 patients with polycythaemia vera or essential thrombocythaemia. With a median follow-up of 7·2years, 349 patients died and 62 progressed to acute leukaemia or myelodysplastic syndrome. Taking death as a competing risk, CALR genotype was associated with a lower risk of transformation [subdistribution hazard ratio (SHR): 0·13, 95% confidence interval (CI): 0·2-0·9, P=0·039], whereas JAK2 V617F showed borderline significance for higher risk (SHR: 2·05, 95% CI: 0·9-4·6, P=0·09). Myelofibrotic transformation increased leukaemic risk, except in CALR-mutated patients. Next generation sequencing of 51 genes at the time of transformation showed additional mutations (median number: 3; range: 1-5) in 25 out of 29 (86%) assessable cases. Mutations (median: 1; range: 1-3) were detected in 67% of paired samples from the chronic phase. Leukaemia appeared in a JAK2 V617F negative clone in 17 (58%) cases, eleven of them being previously JAK2 V617F-positive. JAK2 V617F-mutated leukaemia was significantly associated with complex karyotype and acquisition of TP53 mutations, whereas EZH2 and RUNX1 mutations were more frequent in JAK2 V617F-negative leukaemia. Survival was longer in JAK2 V617F-unmutated leukaemia (343days vs. 95days, P=0·003). In conclusion, CALR genotype is associated with a lower risk of leukaemic transformation. Leukaemia arising in a JAK2 V617F-negative clone is TP53 independent and shows better survival.

Prevalence of TP53 Mutations in Del(17p) Myeloma: Spectrum of Mutations and Effect on Survival

Background.The overall improvement in treatment outcomes and survival of multiple myeloma (MM) patients has highlighted the persistently poor outlook for patients with del(17p). Around a third of MM patients with del(17p) are also reported to have a TP53 mutation, while this mutation is rare in the absence of del(17p). The prognostic impact of TP53 mutations in the context of del(17p) is unresolved. We investigated the presence of TP53 mutations in a cohort of del(17p) patients to correlate mutation status with clinical outcome. We assessed the types of TP53 mutations, and the timing of mutations in relation to the acquisition of del(17p).Methods. CD138+ plasma cells were obtained from bone marrow of patients at diagnosis and prior to starting each new line of therapy, subjected to FISH analysis using standard probes, and surplus tumour cells banked. Between 2004-2015, we identified 52 patients who had at least one FISH result confirming del(17p) in ≥10% CD138+ cells, of whom 16 patients (30.8%) had del(17p) at diagnosis. In 22 patients, samples were available at more than one time point (2-4) for analysis. Clinical details, including treatment and disease response (2014 IMWG criteria) were collated. Adverse risk FISH was defined as t(4;14), t(14;16) and 1q+. Genomic DNA was extracted from CD138+ plasma cells using DNeasyBlood & Tissue Kit (Qiagen). 87 gDNAsamples were amplified by PCR and screened for TP53 mutations in exons 4-11 using DHPLC. TP53 mutations were verified by Sanger sequencing. Survival was estimated using Kaplan-Meier methods, and differences were assessed by log-rank test with p-values <0.05 considered significant.Results. TP53 mutations were identified in 19 (36.5%) patients, and there was no significant difference in median age at diagnosis between patients with or without mutations (61.3 vs 57.3 years, p=0.07). Of the 16 patients with del(17p) at diagnosis, 5 (31.3%) had a TP53 mutation in the diagnostic sample, while 14 (38.8%) of the 36 patients in the relapse subgroup had a mutation (p=0.8).Of the 18 TP53 variants identified, 17 were single nucleotide variants (SNV), of which 14 (82.4%) were missense mutations, 2 were nonsense mutations and 1 altered a splice site; all were documented on the IARC TP53 database. One patient had a novel in-frame indel. The majority of mutations occurred in exons 5, 6 and 8, which forms an integral part of the p53 DNA binding domain. Most mutations identified were predicted to produce a non-functional protein with less than 20% transcriptional activity, as specified by the IARC database. Eleven (57.9%) of the TP53 mutated patients also had at least one high risk genetic feature compared to 16 (48.5%) of the non-TP53 mutated patients (p=0.6).For relapse patients, median time from diagnosis to acquisition of del(17p) was 34.5 months (range 1-178), and median time from acquisition of del(17p) to TP53 mutation was 4 months (<1-45).In 3 patients with TP53 mutations detected at relapse, a preceding sample confirmed to harbour del(17p) was non-mutated, and occurrence of the mutation occurred at 24, 37 and 39 months after del(17p) was first detected. A further 2 patients had a sample preceding del(17p) acquisition, in which there was no TP53 mutation. For the remainder of patients, del(17p) and TP53 mutation were picked up simultaneously.Median overall survival (OS) from diagnosis was significantly shorter for TP53 mutated patients (23 vs 74 months, p=0.03). Median OS from time of del(17p) acquisition was also significantly shorter for TP53 mutated patients (14 vs 29 months, p=0.01), although PFS from next line of therapy was similar (7 vs 12 months, p=0.70). The median survival of TP53 mutated patients from first detection of mutant TP53 was 7 months.Summary. TP53 mutations are common in del(17p) MM with a broad range of variants, the majority of which are likely to produce non-functional variant proteins. The acquisition of TP53 mutations seems to occur after allelic loss of 17p, and to confer even worse clinical outcomes. In the era of genomic medicine, there is a case for routinely screening for TP53 mutations in these patients, to aid in management decisions and patient counselling. An urgent need exists to develop therapies to counteract the loss of this tumour suppressor pathway. [Display omitted] DisclosuresYong:Autolus Ltd: Equity Ownership, Patents & Royalties: APRIL based chimeric antigen receptor; Janssen: Research Funding.

Tracking the origins and drivers of subclonal metastatic expansion in prostate cancer

Tumour heterogeneity in primary prostate cancer is a well-established phenomenon. However, how the subclonal diversity of tumours changes during metastasis and progression to lethality is poorly understood. Here we reveal the precise direction of metastatic spread across four lethal prostate cancer patients using whole-genome and ultra-deep targeted sequencing of longitudinally collected primary and metastatic tumours. We find one case of metastatic spread to the surgical bed causing local recurrence, and another case of cross-metastatic site seeding combining with dynamic remoulding of subclonal mixtures in response to therapy. By ultra-deep sequencing end-stage blood, we detect both metastatic and primary tumour clones, even years after removal of the prostate. Analysis of mutations associated with metastasis reveals an enrichment of TP53 mutations, and additional sequencing of metastases from 19 patients demonstrates that acquisition of TP53 mutations is linked with the expansion of subclones with metastatic potential which we can detect in the blood.

Molecular pathogenesis of IDH mutations in gliomas

The isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) genes are mutated in 50-80% of astrocytomas, oligodendrogliomas or oligoastrocytomas of grades II and III, and secondary glioblastomas; they are, however, seldom mutated in primary glioblastomas and never in other types of glioma. Gliomas with IDH1/2 mutations always harbor either TP53 mutations or total 1p/19q loss. This suggests these two types of tumor may arise from common progenitor cells that have IDH1/2 mutations, subsequently evolving into each tumor type with the acquisition of TP53 mutations or total 1p/19q loss. Survival is significantly longer for patients with IDH-mutated gliomas than for those with IDH-wild type tumors. This observation indicates that IDH status defines biologically different subgroups among gliomas. The molecular pathogenesis of IDH1/2 mutations in the development of gliomas is unclear. The mutated IDH1/2 enzyme generates D-2-hydroxyglutarate. Several theories have been proposed, including: increased angiogenesis because of accumulation of HIF-1α; a glioma CpG island methylator phenotype (G-CIMP) induced by inhibition of TET2; and increased vulnerability to oxidative stress because of depletion of antioxidants. Elucidating the pathogenesis of IDH mutations will aid better understanding of the molecular mechanisms of gliomagenesis and may lead to the development of novel molecular classification and therapy.

Richter syndrome: molecular insights and clinical perspectives

Richter syndrome (RS) represents the clinico-pathologic transformation of chronic lymphocytic leukaemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). The clinical definition of RS is heterogeneous, and encompasses at least two biologically different conditions: (i) CLL transformation to a clonally related DLBCL, that accounts for the majority of cases; (ii) development of a DLBCL unrelated to the CLL clone. In clonally related RS, the pathogenetic link between the CLL and the DLBCL phases is substantiated by the acquisition of novel molecular lesions at the time of clinico-pathologic transformation. RS is not a rare event in the natural history of CLL, since the cumulative incidence of RS at 10 years exceeds 10%. Prompt recognition of RS is known to be clinically useful, and may be favoured by close monitoring of CLL patients harbouring clinical and/or biological risk factors of RS development. Conventional risk factors that are independent predictors of RS development at the time of CLL diagnosis include: (i) expression of CD38; (ii) absence of del13q14 and (iii) lymph node size > or =3 cm. Other risk factors of RS development include CD38 genotype and usage of specific immunoglobulin variable genes. The molecular pathogenesis of RS has been elucidated to a certain extent. Acquisition of TP53 mutations and/or 17p13 deletion is a frequent molecular event in RS, as it is in other types of transformation from indolent to aggressive B-cell malignancies. Additional molecular alterations are being revealed by genome wide studies. Once that transformation has occurred, RS prognosis may be predicted by the RS score, based on performance status, LDH, platelet count, tumour size and number of prior therapies. Depending on patient's age and RS score, the therapeutic options for RS may range from conventional immunochemotherapy to allogeneic bone marrow transplantation.

Population-Based Studies on Incidence, Survival Rates, and Genetic Alterations in Astrocytic and Oligodendroglial Gliomas

Published data on prognostic and predictive factors in patients with gliomas are largely based on clinical trials and hospital-based studies. This review summarizes data on incidence rates, survival, and genetic alterations from population-based studies of astrocytic and oligodendrogliomas that were carried out in the Canton of Zurich, Switzerland (approximately 1.16 million inhabitants). A total of 987 cases were diagnosed between 1980 and 1994 and patients were followed up at least until 1999. While survival rates for pilocytic astrocytomas were excellent (96% at 10 years), the prognosis of diffusely infiltrating gliomas was poorer, with median survival times (MST) of 5.6 years for low-grade astrocytoma WHO grade II, 1.6 years for anaplastic astrocytoma grade III, and 0.4 years for glioblastoma. For oligodendrogliomas the MSTwas 11.6 years for grade II and 3.5 years for grade III. TP53 mutations were most frequent in gemistocytic astrocytomas (88%), followed by fibrillary astrocytomas (53%) and oligoastrocytomas (44%), but infrequent (13%) in oligodendrogliomas. LOH 1p/19q typically occurred in tumors without TP53 mutations and were most frequent in oligodendrogliomas (69%), followed by oligoastrocytomas (45%), but were rare in fibrillary astrocytomas (7%) and absent in gemistocytic astrocytomas. Glioblastomas were most frequent (3.55 cases per 100,000 persons per year) adjusted to the European Standard Population, amounting to 69% of total incident cases. Observed survival rates were 42.4% at 6 months, 17.7% at one year, and 3.3% at 2 years. For all age groups, survival was inversely correlated with age, ranging from an MST of 8.8 months (<50 years) to 1.6 months (>80 years). In glioblastomas, LOH 10q was the most frequent genetic alteration (69%), followed by EGFR amplification (34%), TP53 mutations (31%), p16INK4a deletion (31%), and PTEN mutations (24%). LOH 10q occurred in association with any of the other genetic alterations, and was the only alteration associated with shorter survival of glioblastoma patients. Primary (de novo) glioblastomas prevailed (95%), while secondary glioblastomas that progressed from low-grade or anaplastic gliomas were rare (5%). Secondary glioblastomas were characterized by frequent LOH 10q (63%) and TP53 mutations (65%). Of the TP53 mutations in secondary glioblastomas, 57% were in hot-spot codons 248 and 273, while in primary glioblastomas, mutations were more evenly distributed. G:C-->A:T mutations at CpG sites were more frequent in secondary than primary glioblastomas, suggesting that the acquisition of TP53 mutations in these glioblastoma subtypes may occur through different mechanisms.

Genetic pathways to glioblastoma: a population-based study.

We conducted a population-based study on glioblastomas in the Canton of Zurich, Switzerland (population, 1.16 million) to determine the frequency of major genetic alterations and their effect on patient survival. Between 1980 and 1994, 715 glioblastomas were diagnosed. The incidence rate per 100,000 population/year, adjusted to the World Standard Population, was 3.32 in males and 2.24 in females. Observed survival rates were 42.4% at 6 months, 17.7% at 1 year, and 3.3% at 2 years. For all of the age groups, younger patients survived significantly longer, ranging from a median of 8.8 months (<50 years) to 1.6 months (>80 years). Loss of heterozygosity (LOH) 10q was the most frequent genetic alteration (69%), followed by EGFR amplification (34%), TP53 mutations (31%), p16(INK4a) deletion (31%), and PTEN mutations (24%). LOH 10q occurred in association with any of the other genetic alterations and was predictive of shorter survival. Primary (de novo) glioblastomas prevailed (95%), whereas secondary glioblastomas that progressed from low-grade or anaplastic gliomas were rare (5%). Secondary glioblastomas were characterized by frequent LOH 10q (63%) and TP53 mutations (65%). Of the TP53 mutations in secondary glioblastomas, 57% were in hotspot codons 248 and 273, whereas in primary glioblastomas, mutations were more equally distributed. G:C-->A:T mutations at CpG sites were more frequent in secondary than primary glioblastomas (56% versus 30%; P = 0.0208). This suggests that the acquisition of TP53 mutations in these glioblastoma subtypes occurs through different mechanisms.