High Incidence Of TP53 Mutations Research Articles

Lenalidomide-associated B-cell ALL: clinical and pathologic correlates and sensitivity to lenalidomide withdrawal.

Lenalidomide is an effective component of induction and maintenance therapy for multiple myeloma, though with a risk of secondary malignancies, including acute lymphoblastic leukemia (ALL). In contrast to therapy-related myeloid neoplasia, lenalidomide-associated lymphoblastic neoplasia remains poorly characterized. We conducted a dual institution retrospective study of 32 ALL cases that arose after lenalidomide maintenance (all B-lineage, 31/32 BCR::ABL-negative). B-cell ALL (B-ALL) was diagnosed at median 54months (range, 5-119) after first exposure to lenalidomide and after median 42months of cumulative lenalidomide exposure (range, 2-114). High incidence of TP53 mutations (9/19 evaluable cases) and low hypodiploidy (8/26 patients) were identified. Despite median age of 65 years and poor-risk B-ALL features observed in the cohort, rates of complete response (CR) or CR with incomplete hematologic recovery were high (25/28 patients receiving treatment). Median event-free survival was 35.4months among treated patients (not reached among those undergoing allogeneic hematopoietic cell transplantation [HCT]). Sixteen patients remain alive without evidence of B-ALL after HCT or extended maintenance therapy. We also describe regression of B-ALL or immature B-cell populations with B-ALL immunophenotype after lenalidomide discontinuation in 5 patients, suggesting lenalidomide may drive leukemic progression even after initiation of lymphoblastic neoplasia and that lenalidomide withdrawal alone may be an appropriate first-line intervention in selected patients. Monitoring for early B-ALL-like proliferations may offer opportunities for lenalidomide withdrawal to prevent progression. Established combination chemotherapy regimens, newer surface antigen-targeted approaches, and allogeneic HCT are effective in many patients with lenalidomide-associated B-ALL and should be offered to medically fit patients.

Comprehensive evaluation of genomic alterations in patients with gastric and gastroesophageal adenocarcinoma stratified according to TP53 mutation status.

238 Background: Gastric (GC) and gastroesophageal adenocarcinomas (GEA) are molecularly diverse. Molecular biomarkers of clinical significance have been identified that impact treatment decision making. TP53 is the most frequently altered gene with approximately 50% of patients harboring mutations. However, TP53 mutations have not yet been confirmed as a target of therapeutic benefit. This study aimed to identify distinct genomic alterations that are dominant in TP53 mutated (MUT) versus wild-type (WT) GC and GEA in order to elucidate alternative therapeutic targets within these subsets. Methods: De-identified data for 3741 patients with GC and GEA was obtained from Foundation Medicine. The data obtained were age, gender, tumor mutational burden (TMB), and the distinct genomic alterations noted on DNA sequencing. The dataset was sorted by TP53 mutation status. Differences in mutation frequency were detected using the Fisher’s exact test of independence with a p-value of < 0.01 designated as the cutoff value for statistical significance. Results: The dataset consisted of 2946 GCs and 795 GEAs. TP53 mutations were present in 65.8% of specimens. 61.6% of GCs and 81.4% of GEAs were TP53 MUT positive (p = < .001). Median TMB score and the frequency of tumors with a TMB score > 10 was similar in both TP53 MUT and WT groups. 49 genes had statistically different mutation frequencies in TP53 MUT vs. WT patients. Top co-occurring genetic alterations in TP53 MUT patients included amplification and point mutations in MYC, CCNE1, MET, ERBB2, and EGFR. Amplification and point mutations in MDM2, CDK4, ARID1A, PIK3CA, and ERBB3 were the top co-occurring genetic alterations in TP53 WT patients. Conclusions: There was a high frequency of TP53 mutations in this group of GC and GEA patients, with a higher incidence of TP53 mutations identified in GEA samples. The mutational profiles of these tumors differed according to TP53 mutation status. These differences may be able to serve as the foundation for future clinical investigations.

B-Cell Acute Lymphoblastic Leukemia Arising in Patients with a Preexisting Diagnosis of Multiple Myeloma Is a Novel Cancer with High Incidence of TP53 Mutations

Multiple Myeloma (MM) is a malignancy of plasma cells that affects over 30,000 Americans every year. Despite advances in the treatment of the disease, approximately 12,000 American patients will still die of MM in 2019. One of the mainstays of treatment for MM is the immunomodulatory and antiangiogenic drug lenalidomide; which is used in induction therapy, maintenance therapy and treatment of relapsed disease. Although not fully elucidated, lenalidomide's mechanism of action in MM involves the drug binding to Cerebelon (CBN) and leads to the subsequent degradation of the Ikaros (IKZF1) and Aiolos (IKZF3) transcription factors (TF). These TFs play important regulatory roles in lymphocyte development. Despite lenalidomide's importance in MM treatment, several groups have reported that MM patients treated with lenalidomide rarely go on to develop B-cell acute lymphoblastic leukemia (B-ALL). The genetics and clonal relationship between the MM and subsequent B-ALL have not been previously defined. Importantly, it is not clear if the MM and B-ALL arise from the same founding clone that has been under selective pressure during lenalidomide treatment. As deletions in IKZF1 are common in B-ALL, one could hypothesize that lenalidomide's mechanism of action mimics this alteration and contributes to leukemogenesis. We sequenced the tumors from a cohort of seven patients with MM treated with lenalidomide who later developed B-ALL. These data did not show any mutational overlap between the MM and ALL samples-the tumors arose from different founding clones in each case. However, several genes were recurrently mutated in the B-ALL samples across the seven patients. These genes included TP53, ZFP36L2, KIR3DL2, RNASE-L, and TERT. Strikingly, five of the seven patients had a TP53 mutations in the B-ALL sample that was not present in the matched MM sample. The frequency of TP53 mutations in our cohort was much higher than that reported in adult de novo B-ALL patients which can range between 4.1-6.4% (Hernández-Rivas et al. 2017 and Foa et al. 2013). Utilizing CRISPR-Cas9 gene editing, we disrupted the Zfp36l2 or Actb in murine hematopoietic stem cells (HSCs) of mice with or without loss of Trp53. We performed our first transplantation experiment in which the cohorts of mice have loss of Trp53 alone, loss of Zfp36l2 alone, loss of both Trp53 and Zfp36l2, or a control knockout (KO) of Actb. To characterize the disruption of Zfp36l2 alone and in combination with Trp53 we analyzed the hematopoietic stem and progenitor cell compartments in the bone marrow of the above transplanted mice. In mice with a loss of Zfp36l2 there is a decrease in Lin- Sca-1+ c-Kit+ (LSK), short term-HSC (ST-HSC), and multipotent progenitors (MPP). This decrease was not observed in the mice with a loss of both Trp53 and Zfp36l2, where instead we noted an increase in monocyte progenitors (MP), granulocytes-macrophage progenitors (GMP), and common myeloid progenitors (CMP) cells. In this Trp53 Zfp36l2 double loss model we also noted a decrease in B220+ B-cells that was not seen in the Zfp36l2 alone. In this cohort of Trp53 Zfp36l2 loss, we characterized B-cell development through hardy fraction flow cytometry, and identified a decrease in fractions A and B/C (pre-pro and pro-B-cells, respectively) as compared to Zfp36l2 or Actb alone. As lenalidomide does not bind to Cbn in mice, we used the human B-ALL NALM6 cell line to test if treatment with lenalidomide will lead to a selective growth advantage of cells with the same genes knocked out versus wild-type control cells grown in the same culture. We hypothesize that lenalidomide treatment selectively enriched for pre-existing mutated cell clones that evolved into the B-ALL. Preliminary data in NALM6 cells with a loss of TP53 demonstrate a slight increase in cell number at day 7 compared to a RELA control. These experiments will be repeated with concurrent ZFP36L2 and TP53 mutations as well as ZFP36L2 alone. Treatment-related disease is a key consideration when deciding between different treatment options, and this project aims to understand the relationship between MM treatment and B-ALL occurrence. It may be possible to identify MM patients who are at-risk for B-ALL. For example, MM patients who harbor low-level TP53 mutations prior to lenalidomide treatment could be offered alternative treatment options. Disclosures Barnell: Geneoscopy Inc: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Wartman:Novartis: Consultancy; Incyte: Consultancy.

Sodium cantharidinate, a novel anti-pancreatic cancer agent that activates functional p53.

Despite the use of many types of chemotherapies for pancreatic cancer, optimal efficacy has not been obtained so far. Pancreatic cancer shows a high incidence of TP53 mutations, inactivating its tumor suppressor activity. In this study, we identified sodium cantharidinate as a novel, potential anti-pancreatic cancer agent that activates p53 function. Sodium cantharidinate reduced the viability of pancreatic cancer cells, including the human primary pancreatic cancer cells, PANC-1, AsPC-1, SW1990 and BXPC-3, in a dose-dependent manner. Sodium cantharidinate induced apoptosis and DNA damage of pancreatic cancer cells. Furthermore, proteome-wide sequencing analysis detected a marked perturbation in p53 signaling pathway on PANC-1 cells upon sodium cantharidinate. Consistent with the previous results, sodium cantharidinate treatment decreased Bcl-2 and mitochondrial cytochrome-c protein expression, as well as phosphorylation of MDM2; meanwhile, it increased the levels of cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, Bax, and phosphorylated p53, thus inducing the apoptosis of pancreatic cancer cells. The p53-activating effect of sodium cantharidinate was strongly abrogated by treatment with TP53-targeting shRNA. Moreover, sodium cantharidinate inhibited neoplasm growth via the JAK2-STAT3 pathway, which was inhibited by shRNA-TP53 and triggered by combination with gemcitabine. Combination therapy indicated that sodium cantharidinate and gemcitabine synergistically reduced ex vivo and in vivo growth of pancreatic neoplasm. Further docking studies revealed the different binding fates of sodium cantharidinate to activate wild-type p53 function. Thus, sodium cantharidinate could be a potential agent with promising anti-pancreatic cancer efficacy.

Type of prior genotoxic insult determines the genomic characteristics of therapy-related acute myeloid leukemia.

7515 Background: Therapy-related AML (tAML) is a long-term complication of cytotoxic cancer therapy. It is characterized by adverse genetics and inferior survival outcomes when compared to de novo AML. A proposed mechanism in tAML pathogenesis includes treatment-induced selection of clones harboring pre-existing mutations (i.e. clonal hematopoiesis, CH). We hypothesize that genotoxic therapies used to treat prior malignancy drive leukemogenesis through different mechanisms leading to unique clonal compositions. Methods: AML patients (pts) treated at The Ohio State University between 2015-2018 were included. Genetic profiling was performed using Miseq Illumina platform with a 49-gene targeted sequencing panel at our clinical laboratory. Results: We studied 337 AML pts (Table), of whom 53% had smoking history. Mutations involving ASXL1 were more common in smokers vs non-smokers (14% vs 5.8%, p= .001), while JAK2 mutations were more common in non-smokers (8% vs 1.2%, p= .003). Regarding specific genotoxic therapies and mutations in tAML, we investigated common CH-associated mutations including DNMT3A, TET2, and ASXL1 (DTA mutations). In tAML pts, those exposed to radiotherapy experienced a higher frequency of DTA (52% vs 27%, p= .05), NPM1 (21% vs 0%, p= .002), and SRSF2 (15% vs 0%, p= .01) mutations, and conversely, a lower incidence of TP53 mutations (21% vs 46%, p= .04). Pts with history of cytotoxic chemotherapy had a lower incidence of DTA mutations, including those who received platinum agents (8% vs 49%, p= .005) and taxanes (7% vs 52%, p< .001), but had a higher incidence of TP53 mutations (75% vs 25%, p< .001 for platinum; 53% vs 25%, p= .04 for taxanes). Similarly, alkylators and anthracyclines were associated with lower incidence of DNMT3A (0% vs 20%, p= .009) and ASXL1 (0% vs 12.5%, p= .04) mutations. Conclusions: Different genotoxic agents demonstrate unique effects in leukemia development. Our data suggest that CH clones with DTA mutations may be enriched with smoking and radiotherapy, while cytotoxic chemotherapy may confer a higher incidence of TP53 mutations. Given the adverse prognosis of TP53 mutated AML, identification of pre-existing CH clones might influence treatment selection in solid tumor pts receiving anticancer therapy. [Table: see text]

Choice of Hypomethylating Agent (HMA) for Initial Treatment Does Not Influence Outcome or Survival for Therapy-Related Myeloid Neoplasms (t-MN)

Introduction: T-MN is a serious complication of prior chemotherapy and/or radiation therapy. In 2008, the World Health Organization created a new category for t-MN due to the distinct variance in etiology when compared to de novo myelodysplastic syndromes (MDS) or acute myelogenous leukemia (AML). Compared to de novo MDS/AML, t-MN characteristically has poor risk cytogenetics and a higher incidence of TP53 mutations which are both associated with inferior survival. Small, retrospective analyses have evaluated the efficacy of front-line HMA therapy for t-AML and t-MDS, with response rates of approximately 38% (Klimek et al. Leukemia Res, 2012 and Bally et al, Leukemia Res, 2013). However, the response duration (RD), progression-free survival (PFS), and overall survival (OS) outcomes are poorly characterized in t-MN treated with HMA. This retrospective study evaluates the efficacy and survival of t-MN patients (pts) treated with azacitidine (AZA) or decitabine (DAC) in the largest cohort analyzed to-date. Methods: Our cohort included pts with t-MN at Memorial Sloan Kettering Cancer Center treated with either AZA or DAC from January 1, 2002 to September 30, 2016. The primary objective was to compare the best overall response rate (ORR) between AZA and DAC, including complete remission (CR), marrow CR (mCR), hematologic improvement (HI), and partial response (PR). The best ORR was then compared to stable disease (SD), treatment failure (TF), and progressive disease (PD). Secondary objectives included OS, PFS, and RD. Responses were assessed using the 2006 MDS International Working Group (IWG) guidelines and 2003 AML IWG guidelines. We also assessed the impact of TP53 mutations on response to HMA and survival outcomes. Results: Of the 118 pts (56% male), 76 pts received DAC and 42 pts received AZA. Median age was 70 years (yrs) (range, 25-90). DAC recipients were younger (median 67 yrs, range, 25-85) compared to AZA (median 73 yrs, range, 42-90). More pts with AML were treated with DAC (n=27, 35.5%) and more pts with MDS were treated with AZA (n=36, 85.7%). Most MDS pts had Poor (19.3%) or Very Poor (42%) risk cytogenetics, resulting in a High (23.9%) or Very High (54.5%) overall IPSS-R risk category. Time to response was faster with DAC (9.4 weeks) versus (vs.) AZA (13.3 weeks) with more pts proceeding to hematopoietic cell transplant (HCT) in the DAC group (n=19, 25%) vs. AZA (n=3, 7.1%). Overall, there were no statistical differences in response between AZA and DAC when we analyzed combined TF/PD (45.2% and 32.9%, respectively), SD (14.3% and 25%, respectively), and combined CR/HI/PR (40.5% and 42.1%, respectively) with a p-value of 0.28 (Table 1). However, more patients receiving DAC achieved CR and mCR (18.4% and 14.5%, respectively) vs. AZA (4.8% and 7.1%, respectively) and AZA had a higher HI rate (28.6%) vs. DAC (5.3%). DAC pts also appeared to have higher TF rates (25%) vs. AZA (19%) with more PD in the AZA group (26.2%) vs. DAC (7.9%). Median OS was 10.9 months (mo) and PFS was 5.3 mo (Figure 1). There was no difference in median OS between AZA (11.2 mo) and DAC (10.9 mo, p=0.61) and no difference in the median PFS between AZA (5.1 mo) and DAC (5.3 mo, p=0.83). Lastly, there was no difference in median RD between AZA (4.5 mo) and DAC (3.4 mo, p=0.39). Of the 58 pts with known TP53 mutation status, 29 (50%) had ≥1 TP53 mutation(s). There was no difference in OS and PFS between mutant and wildtype TP53 pts, and no difference in survival between AZA and DAC recipients in the mutant TP53 cohort (Figure 2). Conclusions: This large, retrospective study showed no difference in the ORR between AZA and DAC. The faster response and higher rates of CR and mCR may favor the use of DAC as a bridge to HCT in eligible pts. Conversely, the higher rates of HI seen with AZA can improve the quality of life in MDS pts who are ineligible for HCT. Our results confirm prior reports of poor OS seen in t-MN. In our cohort, we showed no difference in survival outcomes between AZA and DAC. There was also no statistical difference in OS/PFS between DAC and AZA in mutant TP53 pts, however point estimates may indicate a pattern favoring DAC over AZA that needs to be explored in a larger sample size. Although we confirmed the efficacy of HMA in this difficult to treat population, response duration and survival outcomes remain poor. Development of more effective therapies that specifically target the biology of t-MN are needed to improve survival outcomes.

Primary and secondary gliosarcomas: clinical, molecular and survival characteristics.

Gliosarcoma is classified by the World Health Organization as a variant of glioblastoma. These tumors exhibit biphasic histologic and immunophenotypic features, reflecting both glial and mesenchymal differentiation. Gliosarcomas can be further classified into primary (de novo) tumors, and secondary gliosarcomas, which are diagnosed at recurrence after a diagnosis of glioblastoma. Using a retrospective review, patients seen at MD Anderson Cancer Center between 2004 and 2014 with a pathology-confirmed diagnosis of gliosarcoma were identified. 34 patients with a diagnosis of gliosarcoma seen at the time of initial diagnosis or at recurrence were identified (24 primary gliosarcomas (PGS), 10 secondary gliosarcomas (SGS)). Molecular analysis performed on fourteen patients revealed a high incidence of TP53 mutations and, rarely, EGFR and IDH mutations. Median overall survival (OS) for all patients was 17.5 months from the diagnosis of gliosarcoma, with a progression free survival (PFS) of 6.4 months. Comparing PGS with SGS, the median OS was 24.7 and 8.95 months, respectively (from the time of sarcomatous transformation in the case of SGS). The median OS in SGS patients from the initial diagnosis of GB was 25 months, with a PFS of 10.7 months. Molecular analysis revealed a higher than expected rate of TP53 mutations in GS patients and, typical of primary glioblastoma, IDH mutations were uncommon. Though our data shows improved outcomes for both PGS and SGS when compared to the literature, this is most likely a reflection of selection bias of patients treated on clinical trials at a quaternary center.

Myelodysplastic Syndromes with I(17)(q10) and Prognostic Implications of Mutations of TP53 and SETBP1

INTRODUCTIONMyelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid stem cell disorders highly prevalent in elderly populations. MDS are characterized by inefficient hematopoiesis, peripheral blood (PB) cytopenias, and increased risk of transformation to acute myeloid leukemia (AML; 20–30%). Around 50% of MDS patients carry at least one karyotoypic aberration, the most common being 5q-, -7/7q-, +8, 20q-, and isochromosome 17(q10) [i(17q)]. Isochromosome 17(q10) according to cytogenetic risk stratification is of intermediate prognostic significance when is observed as a single abnormality. As i(17q) has be postulated to be associated to recurrent mutational patterns we investigated the TP53 and SETBP1 mutational status in 31 untreated MDS patients harboring i(17q).METHODSGenomic DNA was isolated from fixed cells from bone marrow samples using QIAamp DNA mini-kit Qiagen. TP53 exons 5–9 and SETBP1 exon 3 were amplified using PCR. Amplification products were all purified and sequenced in an automated sequencer. Additionally, we studied the methylation status of TP53 in 21 of the 31 patients. Sequence analyses were performed with PyroQ-CpG analysis software.RESULTSSETBP1 mutational spectrum was characterized by the presence of non-synonmous point mutations, mainly located in residues 868-871 in 13 out of the 31 analyzed patients (41.9 %). In seven of the 13 positive cases, the mutations corresponded to heterozygous D868N, in 5 cases associated with an isolated i(17q) and with 1 additional abnormality in the remaining samples. Three of the 13 SETBP1 mutations were heterozygous G870S associated to i(17q) as a single abnormality. Another three patients had single heterozygous mutations S869G, and I871T along with an i(17q) as a single abnormality or D868Y in the context of a complex karyotype. Regarding TP53 mutations six of the 31 had non-synonymous point mutations. Two patients had mutations in exon 7, three had mutations in exons 5, 6, and 8, and one patient had an intronic mutation at the splicing recognition site.A statistically significant correlation was found between TP53 mutation and a complex karyotype (P=0.001), and between SETBP1 mutation and isolated i(17q) (P=0.001). Univariate analysis for overall survival (OS) found a statistically significant difference between non-mutated and TP53-mutated patients (14.1 months vs. 2.9 months, respectively; P=0.001), and between SETBP1-mutated and TP53-mutated patients (14.5 months vs. 2.9 months, respectively; P=0.002). Only one patient, with isolated i(17q), was found to have an aberrant TP53 methylation status.CONCLUSIONSMDS patients with i(17q) as a sole abnormality presented a higher incidence of SETBP1 mutations, whereas a higher incidence of TP53 mutations, were found in the presence of complex karyotypes. These data allowed us to conclude that a better informed and more accurate prognosis can be achieved in MDS patients with isolated i(17q) or i(17q) plus one additional abnormality, by studying the mutational status of SETBP1 as a first approach.ACKNOWLEDGEMENTS: This work has been partially supported by grants from Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02010); by Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER) (RD07/0020/2004; RD12/0036/0044); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Internacional Josep Carreras; Obra Social “la Caixa”; Sociedad Española de Hematología y Hemoterapia (SEHH) and Celgene Spain. DisclosuresSole:Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

The Role Of Early TP53 Mutations On The Evolution Of Therapy-Related AML

Therapy-related acute myeloid leukemia (t-AML) and therapy-related myelodysplastic syndrome (t-MDS) are well-recognized complications of cytotoxic chemotherapy and/or radiotherapy. Compared to de novo AML, there is a higher incidence of TP53 mutations, abnormalities of chromosomes 5 or 7, and complex cytogenetics in t-AML. The response to chemotherapy is also reduced compared to de novo AML, and long-term remissions are rare. From a pathogenesis perspective, the key difference between de novo AML and t-AML is the prior exposure of t-AML patients to cytotoxic therapy during treatment of their primary cancer. We previously reported the initial findings of our study to sequence the genome of 22 cases of t-AML (Ramsingh et al, Abstract #784, 2012). These data were compared to the genomic sequence of 49 cases of de novo AML (TCGA, NEJM, 2013). Surprisingly, the data showed that the total number of somatic single nucleotide variants (SNVs) and the percentage of transversions (which are associated with chemotherapy-induced DNA damage) were similar in t-AML and de novo AML. Thus, there is no evidence that chemotherapy induces genome-wide DNA damage in t-AML.We extended this data set by sequencing 149 genes of interest in an additional 89 patients with t-AML/t-MDS. Mutations of TP53 were the most frequent (31.5%), and TP53 was the only gene mutated at a higher frequency in t-AML or t-MDS compared with de novo AML or MDS respectively. The mechanism by which TP53 mutations are selectively enriched in t-AML/t-MDS is unclear. Since t-AML/t-MDS has a similar mutation burden compared to de novo AML, it is not likely that chemotherapy directly induces TP53 mutations. We recently reported that individual HSCs accumulate somatic mutations as a function of age. By age 50, there are on average 5 coding gene mutations per HSC clone (Welch et al, Cell, 2012). As it is estimated that there are 10,000 HSCs in humans, it is possible that HSC clones harboring aging-related TP53 mutations are present in a subset of healthy individuals. Based on these observations, we propose a model in which rare TP53 mutant-bearing HSC clones have a selective growth advantage in patients undergoing chemotherapy. This results in their clonal expansion, with additional mutations in the HSC clone undoubtedly required for full leukemic transformation. This model predicts that TP53 mutations are present in many patients years prior to the development of t-AML/t-MDS. To test this prediction, we developed a next generation sequencing technique able to detect rare subclones harboring TP53 mutations to as low as 1 in 1,000 cells. We identified 7 cases of t-AML/t-MDS with specific TP53 mutations for whom we had blood or bone marrow banked 3-10 years prior to the development of t-AML/t-MDS. The specific TP53 mutations were detected in two cases. In patient UPN 530447, bi-allelic mutations of TP53 were identified in mobilized peripheral blood leukocytes 6 years prior to the development of t-AML at frequencies of 0.5 and 0.7%. In patient UPN 341666, a heterozygous TP53 mutation was identified in mobilized peripheral blood leukocytes 3 years prior to t-MDS diagnosis at a frequency of 0.1%. In at least some cases, we have clear evidence that the TP53 mutations occurred prior to the acquisition of other driver mutations or the development of cytogenetic abnormalities. To directly test the hypothesis that TP53 mutations confer a clonal advantage after chemotherapy, we generated mixed bone marrow chimeras containing wild type and Tp53+/- cells. No clonal advantage of Tp53+/- cells was identified in untreated chimeras. However, upon treatment with N-ethyl-N-nitrosourea, Tp53+/- HSCs had a significant growth advantage. Collectively, these data suggest that HSCs that acquire heterozygous TP53 mutations as a function of normal aging may be selected for in the presence of cytotoxic therapy. This provides a potential mechanism for the high incidence of TP53 mutations in t-AML/t-MDS. The early acquisition of TP53 mutations in the founding clone likely contributes to the frequent cytogenetic abnormalities and poor response to chemotherapy that are typical of t-AML/t-MDS. Disclosures:Welch:Eisai: Research Funding.

The proline TP53 variant stimulates likely lymphangiogenesis in an orthotopic mouse model of pancreatic cancer

Background:Pancreatic cancer is a deadly disease characterised by high incidence of TP53 mutations. Restoration of TP53 function is perceived as a highly attractive therapeutic strategy, whose effects are not well characterised.Methods:The current work adapted an inducible strategy of stage-specific reexpression of wild-type (wt) TP53 in an in vivo orthotopic mouse model of pancreatic cancer.Results:The reconstitution of wt TP53 function in TP53-mutant DanG and MiaPaCa-2 cells caused G1 cell cycle arrest but no evidence of apoptosis induction. Consistent with subcutaneous xenograft models, we found that wt TP53 reduced primary tumour growth. Wt TP53 reexpression during early tumour growth led to significant increase in vascularisation. This correlated with an unexpectedly high rate of micro-metastases in lymph nodes of animals with wt TP53 induction, despite the 90% decrease in median primary tumour weight. Reexpression of wt TP53 later in tumour development did not significantly affect the number of CD31-reactive vessels, but increased lymphatic vessel density.Conclusion:The increased number of lymphatic vessels and micro-metastases suggests that wt TP53 induction complexly affected the biology of different tumour constituents of pancreatic cancer. Our observation suggests that combination of the inducible system with an orthotopic model can yield important insights into in vivo pancreatic cancer biology.

Supratentorial Primitive Neuroectodermal Tumors of the Central Nervous System in Adults

Advances in understanding the molecular basis of primitive neuroectodermal tumors of the central nervous system (CNS-PNET) biology are critical to improve patient outcome. Recently, new data on their molecular features have been reported, suggesting that supratentorial PNET (s-PNET) in adult patients may represent a specific tumor entity among CNS-PNETs. In this study, we analyzed the clinicopathologic and molecular features of 12 cases of s-PNET in adult patients. The follow-up analysis showed that these tumors have an aggressive clinical behavior. At the histopathologic level, they resembled their pediatric counterpart, showing a variable spectrum of neuronal differentiation. These cases did not show astrocytic differentiation; therefore, they did not qualify for the differential diagnosis of glioblastoma variants. The tumors were also screened for mutation of TP53, IDH1, IDH2, and β-catenin, using single strand conformation polymorphism-based and sequencing assays, and were analyzed for c-myc/N-myc gene copy numbers with a quantitative polymerase chain reaction-based method. The strand conformation polymorphism-based mutational analysis showed that 5 tumors harbored TP53 mutations. In 2 cases, a mutation at codon 132 of the IDH1 gene was also found. No mutations of the β-catenin or IDH2 genes were identified. No cases presented c-myc or N-myc amplifications. Only 1 case presented overexpression of epidermal growth factor receptor. In conclusion, our data show a high incidence of TP53 mutations in this group of tumors and show, in comparison with pediatric s-PNET, the absence of amplification of the c-myc/N-myc genes, indicating that s-PNET in adult patients may represent a specific subset of tumors among CNS-PNETs.

Wild-type p53 in cancer cells: When a guardian turns into a blackguard

The tumor suppressor p53 controls a broad range of cellular responses. Induction of a transient (cell cycle arrest) or a permanent (senescence) block of cell proliferation, or the activation of cell death pathways in response to genotoxic stress comprise the major arms of the survival-death axis governed by p53. Due to these biological properties, inactivation of p53 is a crucial step in tumor development and progression, reflected by the high incidence of TP53 mutations in different types of human cancers. The remarkable potency of p53 in suppressing tumorigenic outgrowth has promoted the expectation that tumor cells expressing wild-type p53 (wtp53) should be more prone to elimination by cytotoxic treatments than tumor cells expressing mutant p53 (mutp53) with defunct wtp53 activities. However, recent findings yielded somewhat unexpected insights concerning the preponderance of the survival-promoting effects of wtp53 in cancer cells, a rather undesired property from the therapeutic point of view. In this commentary we will discuss the possibility that the developmentally established distinct patterns of wtp53 mediated responses in different tissues are an important factor in determining the ultimate outcome of cellular responses mediated by wtp53 in different types of tumor cells, with a particular focus on the divergent impact of wtp53 in malignant tumors of the central nervous system. We infer that a selective gain of pro-survival functions of wtp53 in cancer cells will confer a survival advantage that counteracts tumor therapy.

Mutations of the TP53 gene in acute myeloid leukemia are strongly associated with a complex aberrant karyotype

In acute myeloid leukemia (AML) with complex aberrant karyotype, a loss of one TP53 allele is frequently observed. We analyzed the incidence of TP53 mutations and deletions in 107 AML with complex aberrant karyotype. In 50 of 57 cases showing a loss of one TP53 allele, a TP53 mutation was detected in the remaining allele. In addition, in 33 of 50 cases with two TP53 copies, a TP53 mutation was found. Therefore, the frequency of TP53 mutations in AML with complex aberrant karyotype was 78%. In a second step, we analyzed TP53 mutations in a cohort of AML comprising different cytogenetic subgroups. TP53 mutations were detected in 33 of 235 cases (14%). Coincidences with other molecular mutations were rare. We confirmed a high incidence of TP53 mutations in AML with a complex aberrant karyotype (29/42, 69%) and demonstrated that TP53 mutations are very rare in AML without a complex aberrant karyotype (4/193, 2.1%).

Mutations of the TP53 Gene Occur in 13.4% of Acute Myeloid Leukemia and Are Strongly Associated with a Complex Aberrant Karyotype.

The TP53 gene is the most frequently mutated gene in human tumors identified so far. In a prior study we demonstrated that 78% of AML with complex aberrant karyotype show a mutation of the TP53 gene. The aim of this study was to determine the frequency of TP53 mutations in an unselected cohort of AML and to analyze the relation to cytogenetic and molecular genetic aberrations. In total 149 AML cases were examined by chromosome banding analysis and screened for FLT3-length mutations (FLT3-LM), MLL partial tandem duplication (MLL-PTD), NPM1 mutations, and TP53 mutations. The cohort included cases with t(8;21) (n=10), t(15;17) (n=6), inv(16) (n=4), 11q23/MLL-rearrangement (n=6), trisomy 8 sole (n=13), AML with normal karyotype (n=46), AML with complex aberrant karyotype defined as showing 3 and more clonal abnormalities but no balanced rearrangement leading to a leukemia specific fusion gene (n=26), and AML with other abnormalities (n=38). FLT3-LM were observed in 21, MLL-PTD in 4, and NPM1-Mutations in 26 cases. TP53 mutation screening of exons 3–9 was performed by denaturing high performance liquid chromatography (DHPLC). All mutations detected were verified by direct sequencing. Overall, TP53 mutations were detected in 20 of the 149 cases (13.4%). Within this cohort of TP53 mutated cases, coincidences of FLT3-LM and MLL-PTD, respectively, with TP53 mutation were detected in one case each. A complex aberrant karyotype was present in 17 of 20 cases (85%) with TP53 mutation. The remaining 3 cases with TP53 mutation showed a normal karyotype, a trisomy 8, and t(8;21) as the sole abnormality, respectively. Therefore, we confirmed a high incidence of TP53 mutations in AML with complex aberrant karyotype (17/26, 65.4%). On the other hand TP53 mutations are very rare in AML without a complex aberrant karyotype (3/123, 2.4%). Furthermore, we divided AML with complex aberrant karyotype into two subgroups:AML with “typical” complex aberrant karyotype showing a deletion of at least one of the following regions: 5q31, 7q31, 17p13 (definition according to Schoch et al. GCC, 2005) andAML with “untypical” complex aberrant karyotype comprizing all others.Interestingly, the frequency of TP53 mutations within the “typical” complex aberrant karyotype group was 75% (15/20) while in the “untypical” group it was 33% (2/6) (p=0.138). In conclusion, the overall incidence of TP53 mutations is low in AML. TP53 mutations are highly associated with AML and complex aberrant karyotype and occur very infrequently in all other cytogenetic subgroups (p<0.001). They occur frequently in particular in the subgroup showing a typical pattern of chromosomal deletions (5q, 7q, 17p). TP53 mutations might explain in part the chemoresistance of AML with complex aberrant karyotype. In addition to cytogenetics a rapid diagnostic screening for TP53 mutations could be a valuable tool to identify a subgroup of AML with poor prognosis. This would allow the early assignment of patients to alternative treatment strategies using also options targeting the TP53 pathway.

Molecular genetic analysis of deep-seated glioblastomas

Glioblastoma can be divided into genetic subsets. The most prominent criterion for dividing glioblastomas into subsets is the dichotomy between TP53 mutation and EGFR amplification, two genetic alterations that almost never coincide in the same tumor. Approximately one third of glioblastomas have TP53 mutations, one third have EGFR amplification, and one third have neither. When viewed in terms of tumor progression, secondary glioblastomas have a much higher incidence of TP53 mutations than do primary glioblastomas. When viewed in terms of the age of tumor onset, glioblastomas in young adults are likely to have TP53 mutations. However, no correlations have yet been found between the tumor locations and the genetic subsets. In this study, we evaluated the associations between the glioblastoma sites and the genetic subsets defined by the presence of the TP53 mutation or EGFR amplification in nine deep-seated glioblastomas of the thalamus and basal ganglia. All nine tumors were clinically defined as primary glioblastomas. Our investigation revealed that all tumors had TP53 mutations and none had EGFR amplifications. These findings suggest that glioblastomas deep-seated in the thalamus and basal ganglia can be grouped into a subset of glioblastomas with TP53 mutations, akin to the subsets of secondary and younger adult glioblastomas. The locations where the glioblastomas originate may be associated with the genetic features.

TP53 Tumor Suppressor Gene and Skin Carcinogenesis.

The tumor suppressor gene TP53 encodes for a nuclear phosphoprotein involved in the control of cell proliferation, particularly in stressed cells. TP53 gene mutations are the most frequent genetic event found in human cancers. Most mutations locate in the highly conserved domains of the gene. Their localizations vary according to the tissue and tumor type, but define some hot spot regions that may have a certain degree of tissue specificity. In certain cases, the type of nucleotide substitutions observed can help to find the carcinogenic agent. In recent years, TP53 gene mutations have been frequently observed in human skin tumors. In epithelial carcinomas, they involve mainly exons 5, 7, and 8. Interestingly, many are C to T transitions at dipyrimidine sites; particularly, one can find CC to TT double-base changes that are known to be specific to ultraviolet radiation. These data confirm at the molecular level the role of ultraviolet radiation as an important etiologic factor in the genesis of these lesions. The high incidence of TP53 mutations suggest that they play a role in keratinocyte transformation. Nevertheless, this event has not yet been defined as an early or late event. In melanomas, most studies have shown the detection of the p53 protein by immunohistochemistry, suggestive of the presence of a mutation in the gene prolonging the protein half-life. Anti-p53 reactivity is frequent in these tumors and seems to correlate with tumor aggressiveness. Confirmation and characterization of TP53 gene mutation at the DNA level would help to precisely define the role of this gene in the development of these tumors.