TP53 Variants Research Articles

Genetic, Surgical and Oncological Approach to Breast Cancer, with BRCA1, BRCA2, CDH1, PALB2, PTEN and TP53 Variants.

The aim of this study was to determine the frequency of germline variants in BRCA1, BRCA2, CDH1, PALB2, PTEN and TP53 in patients admitted to a medical genetics clinic with breast cancer and to assess these identified variants according to published genetic, surgical and oncological perspectives. Medical history, and cancer diagnosis information for 195 independent probands with operated breast cancer were collected from requisition forms and medical records. The exonic regions and exon-intron junctions in BRCA1, BRCA2, CDH1, PALB2, PTEN and TP53 genes were sequenced. Analysis of fastq files was performed on the Qiagen Clinical Insight-Analyse Universal with panel-specific pipeline and vcf files were interpreted clinically using Qiagen Clinical Insight-Interpret. Gene variants (pathogenic, likely pathogenic and variants of unknown significance) were detected in 53 (27.2%). Detailed information about the patients (age of diagnosis, family history, gender), cancer stage, tumour characteristics (ER, PR, human epidermal growth factor receptor 2 status) and all information related to the detected variants (gene, location, nucleotide and amino acid change, exon number, impact, mutation classification, dbSNP number and HGMD variant class) were assessed. In total, 58 mutations were identified including 14 novel, previously unreported variants. Molecular characterization and identification of mutations have important implications for predictive, preventive, and personalized medicine, including genetic counseling and development of specific treatment protocols. We emphasize variants of unknown significance (VUS) as the clinical significance of VUS changes over time and variant classification is important for clinical molecular genetic testing and clinical guidance. This study may provide new insights into risk assessment for variants in CDH1, PALB2, PTEN and TP53, in addition to BRCA1 and BRCA2, which may prove useful for clinical management of breast cancer patients. Further studies are needed to identify the common gene variants in the Turkish population and evaluate the pathogenity of VUS.

Molecular heterogeneity of pediatric choroid plexus carcinomas determines the distinctions in clinical course and prognosis.

Choroid plexus carcinomas (CPCs) are rare aggressive pediatric tumors of the brain with no treatment standards. Genetic profiling of CPCs is often confined to possible association with Li-Fraumeni syndrome, though only about a half of CPCs develop from syndromic predispositions. Whole-chromosome gains and losses typical of CPCs reflect genomic instability of these tumors, but only partially explain the aggressive clinical course. This retrospective study enrolled 25 pediatric patients with CPC, receiving treatment between January 2009 and June 2022. Molecular-genetic testing was performed for 20 cases with available tumor tissue and encompassed mutational status, chromosomal aberrations, and gene expression profiles. We analyzed several factors presumably influencing the outcomes, including molecular profiles and clinical parameters. The median follow-up constituted 5.2 years (absolute range 2.8-12.6 years). All studied CPCs had smooth mutational profiles with the only recurrent event being TP53 variants, either germline or somatic, encountered in 13 cases. Unbalanced whole-chromosome aberrations, notably multiple monosomies, were highly typical. In 7 tumors, chromosome losses were combined with complex genomic rearrangements: segmental gains and losses or signs of chromothripsis. This phenomenon was associated with extremely low 5-year survival: 20.0 ± 17.9% vs 85.7 ± 13.2%; P = .009. Transcriptomically, the cohort split into 2 polar clusters Ped_CPC1 and Ped_CPC2 differing by survival: 31.3 ± 17.8% vs 100%; P = .012. CPCs split into at least 2 molecular subtypes distinguished both genomically and transcriptomically. Clusterization of the tumors into Ped_CPC1 and Ped_CPC2 significantly correlates with survival. The distinction may prove relevant in clinical trials for dedicated and patient-oriented optimization of clinical protocols for these rare tumors.

Genetic variants in African-American and Hispanic patients with breast cancer.

Breast cancer is a disease with significant health disparity affecting mortality in minority women. The present study examined the genetic makeup of breast cancers in African-American and Hispanic/Latinx patients to determine specific genetic mutations associated with breast cancer in the minority population from South Los Angeles, United States. Whole-exome sequencing was performed on DNA extracted from breast cancer tumor biopsies collected from 13 African-American and 15 Hispanic women and 8 matched-normal samples for each ethnic category. The results were analyzed using Ensemble Variant Effect Predictor and Mutation Significance. Additionally, a comparative analysis with The Cancer Genome Atlas data was provided. Our data revealed somatic mutations in genes such as SET domain containing (lysine methyltransferase) 8, serine protease 1 and AT-rich interaction domain 1B (ARID1B) and known breast cancer genes, such as BRCA1/2, TP53 and the DNA damage response genes across all ethnicities. Additionally, Hispanic patients had BRCA1 associated RING domain 1B (BARD1) variants, while African-American patients had higher numbers of nonsynonymous variants in the RAD51 paralog B (RAD51B), ARID1B and X-ray repair cross complementing 3 (XRCC3) genes. In addition, our patients exhibited mutational signature enrichment that indicated DNA homologous recombination repair deficiencies. Therefore, African-American and Hispanic breast cancer samples showed considerable overlap in breast cancer genetic mutations. However, there are differences in specific genetic variants in TP53, BRCA1/2, BARD1 or ARID1B, which will require further study of their role in tumorigenesis.

Deep DNA sequencing of MGMT, TP53 and AGT in Mexican astrocytoma patients identifies an excess of genetic variants in women and a predictive biomarker.

Astrocytomas are a type of malignant brain tumor with an unfavorable clinical course. The impact of AGT and MGMT somatic variants in the prognosis of astrocytoma is unknown, and it is controversial for TP53. Moreover, there is a lack of knowledge regarding the molecular characteristics of astrocytomas in Mexican patients. We studied 48 Mexican patients, men and women, with astrocytoma (discovery cohort). We performed DNA deep sequencing in tumorsamples, targeting AGT, MGMT and TP53, and we studied MGMT gene promoter methylation status. Then we compared our findings to a cohort which included data from patients with astrocytoma from The Cancer Genome Atlas (validation cohort). In the discovery cohort, we found a higher number of somatic variants in AGT and MGMT than in the validation cohort (10.4% vs < 1%, p < 0.001), and, in both cohorts, we observed only women carried variants AGT variants. We also found that the presence of either MGMT variant or promoter methylation was associated to better survival and response to chemotherapy, and, in conjunction with TP53 variants, to progression-free survival. The occurrence of AGT variants only in women expands our knowledge about the molecular differences in astrocytoma between men and women. The increased prevalence of AGT and MGMT variants in the discovery cohort also points towards possible distinctions in the molecular landscape of astrocytoma among populations. Our findings warrant further study.

The performance of multi-gene panels for breast/ovarian cancer predisposition

BRCA1 and BRCA2 are the most mutated genes in breast cancer. We analyzed 48 breast cancer subjects using two methods that differ in terms of number of genes investigated and strategy used (primers: Panel A - 12 genes - vs probes: Panel B - 48 genes). Both the panels and procedures identified “pathogenic” or “likely pathogenic” variants in TP53, ATM, CHEK2 and BARD1 besides BRCA1 and BRCA2. Panel B identified two other putatively pathogenic variants in RNASEL and in RAD50. Identification of variants other than the BRCA genes can be useful in patient management. A total of 121 variants were distributed within the 12 genes and were correctly detected by both panels. However, the number of calls without divergence, namely ± 0.10 difference of allelic frequency, was 78.3%, while calls with a divergence below 0.10 was 16.7%, thus indicating that only 5% (n = 275) of 5,412 calls had a divergence above 0.10. Although these panels differ from each other, both are useful in different situations, particularly when patients should be tested for genes other than BRCA1/2 (as occurs in patients affected by a so called hereditary syndrome) or for therapeutic purposes.

Drimia calcarata Bulb Extracts Deactivate the PI3K Signalling Pathway in Cervical HPV-18 Positive HeLa Cells

• D. calcarata extracts promoted proliferation of HPV types 16 and 18 positive Ca-Ski cells through maintaining the expression of STAT3, CLB1 and CDC2 and maintaining the activated levels of PI3K. • Water extract induced apoptosis and G2/M cell cycle arrest of HeLa cells through increased expression of the tumour suppressor p53. • Both methanol and water extracts deactivated the PI3K signalling pathways in HeLa cells. • D. calcarata plant extracts could be useful for therapeutic purpose against human HPV-18 positive gynaecologic cancers. The efficacy of Drimia calcarata against the human cervical cancer remains unexplored and less understood. The present study focused on investigating the cytotoxic effect of D. calcarata bulb extracts on cervical cancer cells. The growth inhibitory effects of D. calcarata extracts were determined using the MTT, Ki67 and PI3K Activation assays. Apoptosis induction was assessed using fluorescence microscopy, the Muse Ⓡ Cell Analyser and gene expression analysis by RT-PCR. The cytotoxicity of the fractions was evaluated using MTT assay and the apoptosis induction using Muse Ⓡ Cell Analyser. Both methanol extract (ME) and water extract (WE) showed safety against noncancerous KMST-6 and HEK-293 cells. The extracts exhibited anticancer activity against HeLa, with no significant cytotoxic effect against the Ca-Ski cells. The WE increased the Ki67 positive Ca-Ski population, while both ME and WE arrested HeLa cells at G2/M phase, and Ca-Ski cells in G0/G1 phase. AO/EB staining, Annexin V and Caspase 3/7 Activation revealed that the extracts significantly induced apoptosis in HeLa cells. In HeLa cells, the ME downregulated TP53 variants, while WE upregulated both TP53 variants in HeLa cells. Both extracts decreased the STAT5A and STAT5B mRNA expression in HeLa cells; however, these extracts upregulated cancer-promoting STAT3 in Ca-Ski cells. Additionally, these extracts inactivated the PI3K signalling pathway in HeLa cells but not in Ca-Ski cells. The resistance of the Ca-Ski cells to the D. calcarata extracts may be due to the upregulation of STAT3 and activated PI3K signalling pathway. The cytotoxicity and apoptosis induction in HeLa cells by D. calcarata extracts may be attributed due to downregulation of STAT5A survival mechanisms. Water fractions 1, 2, 3 and 6 and methanol fractions 1 and 2 reduced cell viability of HeLa cells. Water fractions 2, 3 and 6 and methanol fractions 1 and 3 induced apoptosis, which was preceded by secondary necrosis. However, water fraction 1 and methanol fraction 2 led to most cells undergoing necrotic cell death. There are several compounds that can be credited with the anticancer activities of the ME and WE extracts since several fractions exhibited cytotoxicity against the HeLa cells. These findings suggest that the D. calcarata extracts have anticancer activities, and thus, could be useful for therapeutic purposes against human HPV-18 positive gynaecologic cancers.

A study of gene variation in All-RAS wild-type metastatic colorectal cancer and its correlation with cetuximab

This study sought to explore the biological significance of genetic variation in RAS wild-type metastatic colorectal cancer (mCRC) in the real world, the difference in the efficacy of cetuximab in the treatment of mCRC with different genetic variants and identify clinical features and new predictors of efficacy. A retrospective analysis of the data of 60 patients with stage IV mCRC who received cetuximab at The First and Second Affiliated Hospital of Soochow University from 2016 to 2020 was conducted. The patients were divided into the following 3 groups according to the genetic test results: (I) group A (the all-RAS wild-type group); (II) group B (the all-RAS wild-type group with the tumor suppressor gene mutation); and (III) group C (the all-RAS wild-type group with the oncogenic driver gene mutation). A subgroup analysis was conducted to examine left CRC and local intervention, and the progression-free survival (PFS) and overall survival (OS) of the patients were observed. The all-RAS wild-type mCRC patients were divided into group A (n=10), group B (including the TP53, APC, PTEN, BRCA2, and SMAD4 variants) (n=42), and group C (including the ERBB2, BRAF, PIK3CA, and RET variants) (n=8). The median PFS of groups A, B, and C were 15.0, 12.0, and 3.0 months, respectively (P=0.007). Fitting sex as a stratified variable to the Cox survival analysis model showed that only the PFS of groups B and C differed significantly (P=0.011). In the left-sided mCRC patients, the median PFS of groups A, B, C were 3.0, 13.0, and 3.0 months, respectively (P=0.009). Among the patients in group B, the median PFS of the metastatic local intervention subgroup was 14.0 months, and the non-local intervention subgroup was 12.0 months (P=0.55). Only the type of combined gene mutation was an independent factor affecting PFS. The PFS and OS of mCRC patients with all-RAS wild-type and no combined mutations treated with cetuximab were not better than those of patients with combined mutations. Compared to mCRC patients with all-RAS wild-type and oncogenic driver gene mutations, cetuximab significantly prolonged the PFS of all-RAS wild-type patients with the tumor suppressor gene mutations.

Recurrent missense variants in clonal hematopoiesis-related genes present in the general population.

Clonal hematopoiesis (CH) consists in an abnormal expansion of a hematopoietic stem cell bearing an advantageous somatic variant. A survey of known recurrent somatic missense variants in DNMT3A, SF3B1, SRSF2, and TP53, some of the most prominent genes underlying CH of indeterminate potential (CHIP), in gnomAD noncancer database shows the presence of 73 variants. Many of them reach frequencies higher than 0.01% in various populations and, in many cases, are enriched in specific populations. Consistent with a potential involvement in CHIP, we found that the age distribution of the carriers is shifted towards old ages. Moreover, the variant allele frequencies are on average lower than 50%, expected for germline heterozygous variants. The pervasive presence of some of such variants in blood DNA from elder individuals is compatible with CHIP of somatic origin. On practical grounds, CHIP can lead to misclassification of somatic variants in cancer-predisposition genes as inherited, which bear consequences for the affected individuals and their families.

Molecular Typing and Clinical Characteristics of Synchronous Multiple Primary Colorectal Cancer

Synchronous multiple primary colorectal cancer (sMPCC) is clinically rare, but its incidence has increased over the past decade. However, little is known about the molecular and clinical features of sMPCC, which may differ from those of single primary colorectal cancer (SPCRC). To evaluate the clinical characteristics and pathogenic variations in lesions and the molecular typing of sMPCC. From November 2012 to April 2021, patients with colorectal cancer (CRC) treated at the Sixth Affiliated Hospital of Sun Yat-sen University were enrolled in this cohort study. Follow-up ended on January 31, 2022. The primary outcome was mismatch repair (MMR) status of each lesion in all patients examined using immunohistochemistry (IHC). Microsatellite instability (MSI) and tumor mutation burden (TMB) were also calculated. A total of 13 276 patients with CRC were enrolled, and 239 patients with sMPCC (mean [SD] age, 63.3 [12.2] years; 173 men [72.4%]) with available clinical data were evaluated. Seventy-eight patients with sMPCC and 94 with SPCRC also underwent next-generation sequencing (NGS)-based molecular testing. The deficient MMR (dMMR)/MSI-H frequencies in sMPCC were significantly higher than those in SPCRC, which was confirmed by both IHC (50 of 239 patients vs 872 of 13 037 patients) and NGS (17 of 78 patients vs 5 of 94 patients). According to the MMR/MSI status of different lesions in patients with sMPCC, they were further divided into 3 subgroups: all dMMR/MSI-H, dMMR/MSI-H and proficient MMR (pMMR)/microsatellite stability (MSS), and all pMMR/MSS. The EGFR and PIK3CA variants were more common, whereas TP53 variants were less prevalent in patients with sMPCC than in those with SPCRC. Moreover, higher tumor mutation burden was associated with higher MSI in patients with sMPCC rather than in those with SPCRC. In this cohort study of sMPCC, the incidence of dMMR/MSI-H in patients with sMPCC was significantly higher than that in patients with SPCRC. These findings suggest that sMPCC can be classified into 3 subgroups according to the MMR/MSI status of each lesion, which might be applied to guide personalized therapies for better disease management.

Molecular characterization of AML-MRC reveals TP53 mutation as an adverse prognostic factor irrespective of MRC-defining criteria, TP53 allelic state, or TP53 variant allele frequency.

Acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) generally confers poor prognosis, however, patient outcomes are heterogeneous. The impact of TP53 allelic state and variant allele frequency (VAF) in AML-MRC remains poorly defined. We retrospectively evaluated 266 AML-MRC patients who had NGS testing at our institution from 2014 to 2020 and analyzed their clinical outcomes based on clinicopathological features. TP53 mutations were associated with cytogenetic abnormalities in 5q, 7q, 17p, and complex karyotype. Prognostic evaluation of TP53MUT AML-MRC revealed no difference in outcome between TP53 double/multi-hit state and single-hit state. Patients with high TP53MUT variant allele frequency (VAF) had inferior outcomes compared to patients with low TP53MUT VAF. When compared to TP53WT patients, TP53MUT patients had inferior outcomes regardless of MRC-defining criteria, TP53 allelic state, or VAF. TP53 mutations and elevated serum LDH were independent predictors for inferior OS and EFS, while PHF6 mutations and transplantation were independent predictors for favorable OS and EFS. NRAS mutation was an independent predictor for favorable EFS. Our study suggests that TP53MUT AML-MRC defines a very-high-risk subentity of AML in which novel therapies should be explored.

Diagnostic accuracy of mutational analysis along the Müllerian tract to detect ovarian cancer

ObjectiveOvarian cancer is known for its poor prognosis, which is mainly due to the lack of early symptoms and adequate screening options. In this study we evaluated whether mutational analysis...

Pathogenic TP53 mutations Are Associated with a Poor Prognosis in De Novo AML

Introduction: Mutations in TP53 are the most common mutations in human cancer and occur in almost every cancer subtype. In adult acute myeloid leukemia (AML), TP53 mutations, especially those associated with complex cytogenetics, are associated with a poor prognosis. However, given the rarity of TP53 mutations in pediatric AML, less is known about its prognostic significance. Methods:TP53 variant status was determined in patients with AML enrolled on 4 successive COG trials for de novo pediatric AML (NCT00002798, NCT00070174, NCT01407757, NCT01371981). Next generation sequencing was retrospectively employed on diagnostic specimens to screen for variants including single nucleotide variants (SNVs) and insertions and deletions (indels). Variants were classified as benign, likely benign, unknown significance, likely pathogenic, and pathogenic using ClinVar and ACMG/AMP guidelines. Results: Of 1790 patients (age: median 10.48 years, range 0.01-29.84 years), 68 (3.8%) had TP53 variants. Sixty-four (94.1%) of these were SNVs, 2 (2.9%) were in-frame indels, and 2 (2.9%) were frameshifts leading to early protein truncation. Twenty-six (38.2%) were considered likely pathogenic or pathogenic (TP53mut) (age: median 8.38 years, range 0.31-17.97 years), 33 (48.5%) considered likely benign or benign, and the remaining 9 (13.2%) were of unknown significance. Thirty-four (53.1%) TP53 SNVs fell within the DNA binding domain, including 100% of TP53mut SNVs (including many occurring at DNA binding sites). We compared disease characteristics and clinical outcomes between patients with TP53mut and patients without any TP53 variant (TP53wt). Patients with non-pathogenic variants were excluded from the analysis. There was a higher prevalence of complex cytogenetics (defined as 3 or more chromosomal aberrations, including at least one structural aberration on karyotype) in TP53mut patients compared to TP53wt patients (55% vs. 18%; p=<.0001). There was no difference in the prevalence of co-occurring contemporarily defined high-risk alterations (TP53mut 30.8% vs. TP53wt 32.1%; p=0.879) but TP53mut patients were less likely to have co-occurring low-risk alterations compared to TP53wt patients (15.4% vs. 41.1%; p=0.008). While there were no differences in complete remission rates (TP53mut 85% vs. TP53wt 77.6%; p=0.426) or their ability to clear minimal residual disease (TP53mut 55% vs. TP53wt 71.6%; p=0.132), TP53mut patients had a lower event-free survival (EFS) compared to TP53wt patients (5yr: 13.3% vs. 46.3%; p <.0001), which was largely driven by a higher relapse risk (RR) (5yr: 76.5% vs. 42.1%; p <.0001). Salvageability following an event was largely unsuccessful for TP53mut patients, including only 1 long-term survivor following an event, which contributed to a lower overall survival (OS) compared to TP53wt patients (5yr: 15.9% vs. 64.7%; p < .0001). Further, EFS (13.3% vs. 26.3%; p <.0001; Figure 1a), RR (5yr: 76.5% vs. 60.9%; p <.0001; Figure 1b) and OS (5yr: 15.9% vs. 42.1%; p <.0001; Figure 1a) were significantly inferior compared to contemporarily defined high-risk patients. When patients with co-occurring high-risk alterations were excluded from analysis, TP53mut patients maintained their poor 5-yr EFS (13.3%), RR (76.5%) and OS (15.9%). Conclusions: In the largest study of TP53 mutations in pediatric patients with de novo AML, we show that pathogenic TP53variants are rare but prognostically significant. TP53mut patients were more likely to have complex cytogenetics and less likely to have co-occurring low-risk alterations. Outcomes for TP53mut patients were significantly inferior compared to contemporarily defined high-risk patients. Finally, these poor outcomes were maintained even when TP53mut patients with co-occurring high-risk lesions were excluded from analysis. Taken together, these data would suggest TP53 pathogenic mutations should be considered high-risk defining lesions in pediatric AML. Since HSCT is the standard of care for relapsed disease, the lack of salvageability following an event may suggest this disease is less alloreactive than other subtypes of AML, but further investigation is needed. Given the dismal outcomes after an event, clinical trials and novel targeted agents should be prioritized for relapsed patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Predicting functional effect of missense variants using graph attention neural networks.

Accurate prediction of damaging missense variants is critically important for interpreting a genome sequence. Although many methods have been developed, their performance has been limited. Recent advances in machine learning and the availability of large-scale population genomic sequencing data provide new opportunities to considerably improve computational predictions. Here we describe the graphical missense variant pathogenicity predictor (gMVP), a new method based on graph attention neural networks. Its main component is a graph with nodes that capture predictive features of amino acids and edges weighted by co-evolution strength, enabling effective pooling of information from the local protein context and functionally correlated distal positions. Evaluation of deep mutational scan data shows that gMVP outperforms other published methods in identifying damaging variants in TP53, PTEN, BRCA1 and MSH2. Furthermore, it achieves the best separation of de novo missense variants in neuro developmental disorder cases from those in controls. Finally, the model supports transfer learning to optimize gain- and loss-of-function predictions in sodium and calcium channels. In summary, we demonstrate that gMVP can improve interpretation of missense variants in clinical testing and genetic studies.

Molecular Landscape of Chronic Lymphocytic Leukemia Using Targeted Gene Panel Sequencing

Introduction Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease with various genetic abnormalities that can affect the treatment and survival of patients. Recently, by the advent of next-generation sequencing (NGS) techniques recurrently mutated genes have been described in CLL. Driver gene mutations such as NOTCH1, SF3B1, TP53, and MYD88 have been considered to have prognostic impact in CLL. The aim of this study was to detect pathogenic variants of commonly mutated genes in CLL patients and to explore their associations with IGHV mutational status and cytogenetic abnormalities. Patients and Methods The samples were collected from bone marrow or peripheral blood of 124 CLL patients. Genomic DNA was isolated using QIAamp Blood Mini kit. NGS analysis was performed using Twist Custom Panel and bidirectional sequencing with a minimum coverage of 1000x on Illumina NextSeq according to the manufacturers’ instructions. Data were analyzed by NextGene software. The following 27 recurrently mutated CLL genes were studied: PIK3CD, PIK3CA, SF3B1, MYD88, FBXW7, BRAF, NOTCH1, PTEN, BIRC3, PTPN6, MAP2K1, TP53, STAT5B, STAT3, CD79B, TCF3, MAPK1, DDX3X,ATM, BCL2, BTK, KRAS, NRAS, CARD11, CXCR4, PLCG2, POT1. Variants below 5% variant allele frequency (VAF) were filtered out from the analysis. Variants were considered subclonal with 5-10% VAF and clonal when VAF was >10%. Classification of variants were performed according to the American College of Medical Genetics and Genomics (ACMG) criteria. Results Our NGS study revealed a total of 96 variants in 124 CLL patients. Overall, 63/124 patients (50.8%) harbored at least one mutation, the majority of them (60/96, 62.5%) was clonal. In 36 patients only one pathogenic variant was detected, while 88 patients carried two or more (2-4) genetic alterations. The most frequently mutated gene was the NOTCH1, followed by TP53, SF3B1, FBXW7, and BRIC3. Subclonal variants were also detected in 36/124 patients (29%), particularly in NOTCH1,FBXW7, BIRC3, and PIK3CA genes. TP53 and NOTCH1 variants occured mostly in unmutated IGHV CLL cases. Patients with mutated IGHV harbored mainly FBXW7 and BIRC3 mutationss. FBXW7 variants were detected in 9/125 (7%) patients, that is a higher frequency compared to other studies. Fluorescence in situ hybridization (FISH) was performed in 113 cases. The majority of these patients (84 cases, 74%) had one or more cytogenetic abnormality. In this cohort, 13q deletion was the most common aberration (65%, 55/84 patients), followed by 11q deletion (26%, 22/84 patients) and 17q deletion (14%, 12/84 patients). Deletion of 17p co-occured with TP53 single nucleotide variants in 10 of 12 patients. SF3B1, NOTCH1, BIRC3 and TP53 variants occured with 11q deletion in 10 cases. Trisomy 12 was detected with NOTCH1, BIRC3 and FBXW7 variants, and showed negative association with unfavorable SF3B1 mutations. Conclusions Our results confirmed the presence of high number of clonal and subclonal variants in the most frequently mutated genes (NOTCH1, TP53, SF3B1, FBXW7, BIRC3). TP53, NOTCH1 and SF3B1 mutations were enriched with unfavorable biologic factors, as they were found to be associated with unmutated IGHV and cytogenetic aberrations, such as del17p, del11q and trisomy 12. Cases with NOTCH1 variants harbored all types of the investigated cytogenetic alterations (del13q, del11q, de17p, trisomy 12) and were associated exclusively with unmutated IGHV. TP53 variants occured mostly in unmutated IGHV CLL cases together with del17p. Patients with SF3B1 variants showed del11q, del13q and mostly unmutated IGHV. FBXW7 variants enriched in both of unmutated and mutated IGHV cases and harbored del13q, de17p and trisomy 12. FBXW7 and NOTCH1 pathogenic variants have the same biological consequences in CLL, therefore presence of FBXW7 mutations may have clinical relevance regarding anti-CD20 therapy. Taken together, these NGS results complemented with the study of IGHV mutational status and cytogenetic data can contribute to better prognostic workup and management of our CLL patients.

Strategies for Alleviating Tyrosine Kinase Resistance in Acute Myeloid Leukemia By Modulating p53 Signaling Pathway with Small Molecules

Background:FLT3-ITD can be found in up to 30% of acute myeloid leukemia (AML) patients and confers a poor prognosis. While midostaurin and gilteritinib are FDA-approved for these high-risk patients, they only result in a temporary response without transplantation, suggesting the importance of identifying the acquired resistance. Previously, we demonstrated that cabozantinib, an oral multi-target tyrosine kinase inhibitor, was selectively cytotoxic to AML cells with FLT3-ITD (MV4-11 and Molm13). Subsequently, we established cabozantinib-resistant human leukemic MV4-11 derivative (MV4-11-XR) and Molm13 derivative (Molm13-XR) after gradual escalating concentration of cabozantinib incubation from parental MV4-11 (MV4-11-P) and Molm13 (Molm13-P) cells, respectively. Subsequently, cell cytotoxicity assay showed these two cabozantinib-resistant cells were also resistant to quizartinib, sorafenib and gilteritinib. RNA-seq analysis revealed that MV4-11-XR and Molm13-XR cells shared 87 identical differentially down-regulated genes compared to their respective parental cells. Further Metascape for gene enriched pathway analysis demonstrated the most affected was p53 signaling pathway. Aim: To investigate whether modulation with p53 activator RITA or MDM2 inhibitor RG7388 could alleviate the drug resistance and their underlying molecular mechanism. Materials and Methods: Human leukemia MV4-11 and Molm13 cells were used in this study. The differential expression genes (DEGs) were examined using RNA-seq (Illumina NextSeq-500). Metascape enrichment pathway analyses were preformed to predict the biological functions of DEGs. Cabozantinib, RITA, and RG7388 (Idasanutlin) were used. MTS assay was performed to the effects of various drug on cell growth. Western blotting, flow cytometry and quantitative real-time PCR were applied to elucidate potential molecular mechanism. Fluorescent leukemic cell xenografts in zebrafish and high content imaging system were used for investigating the efficacy of various drugs in vivo. Results: The gene expression profile in MV4-11-P and Molm13-P cells was used as a baseline to establish the up- and down-regulated genes in MV4-11-XR and Molm13-XR cells. The FPKM were estimated with the selection criteria of p value <0.05 and [log2 (fold change)] <1 for significantly differential expression for downregulation. We identified a total of 747 DEGs between the MV4-11-P and -XR cells and 407 DEGs between the Molm13-P and -XR cells. Among them, 87 DEGs were shared by these two cohorts. Metascape enrichment analysis revealed that those down-regulated DEGs were enriched in p53-signaling pathway, in line with decreased expression of P53-associated genes, BBC3, CCNG2, CKDN1A, ZMAT3, confirmed by using q-RT-PCR. IGV (Integrative Genomics Viewer) analysis showed that the variant allele frequency of R248W progressed from 57% in MV4-11-P to 100% in MV4-11-XR cells, a similar phenomenon also appeared in P72R (65% to 98%). Microsatellite analysis confirmed the loss of heterozygosity in the TP53 gene of MV4-11-XR. On the other hand, there were not TP53 variants in both Molm13-P and Molm13-XR cells, but increased level of MDM2 in Molm13-XR cells was noted. Further study revealed that p53 activator RITA with 77.6nM could reduce IC50 of cabozantinib from 1026nM to 310.7nM, coupled with increased gene expression and protein levels of BBC3 (PUMA), CCNG2 (cyclin G2), CDKN1A (p21) and ZMAT3. In addition, RITA had synergistic effect with cabozantinib on cell cycle arrest on G2/M phase, increased apoptosis and decreased colony formation and proliferation. On the other hand, MDM2 inhibitor RG7388 reduced the IC50 of cabozantinib from 517nM to 101nM with only 50.6 nM in Molm13-XR cells. Similarly, RG7388 could increase those P53-associated gene expression and protein levels, and had synergistic effect with cabozantinib on cell cycle arrest on G0/G1 phase, increased apoptosis and decreased colony formation and proliferation. In addition, in vivo zebrafish experiment demonstrated that the combination of RG7388 and cabozantinib had a synergistic and significant inhibitory effect compared with single use. Conclusion: These findings suggested that reactivation of p53 is a potential therapeutic option to alleviate drug resistance in FLT3-mutated AML, provided that AML patients are tested for p53 dysfunction, including TP53 mutational status and downregulation.

Molecular Diagnostics for TP53 Is Recommended in B-Cell Lymphomas

Introduction Multiple studies in patients with B-cell lymphomas (BCL) have shown that TP53 is associated with inferior therapeutic efficacy and survival outcomes. Current treatment algorithms for chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are tailored for patients harboring a TP53 mutation using a different therapeutic strategy. Regardless, targeted next generation sequencing (tNGS) is not standard practice in the diagnostic workup of lymphomas. In solid malignancies, e.g., ovarian cancer (PMID: 35752743), our institute has shown that p53 immunohistochemistry (IHC) is a good and widely used marker to assess TP53 mutations. In lymphomas however, this correlation is not yet as clearly established. Here we evaluate the accuracy of p53 IHC as a surrogate marker for TP53 mutational analysis in a large cohort of BCL. Method For this retrospective study 278 consecutive patients diagnosed with BCL between 2014-2022 were selected. The cases were diagnosed according to the revised 4th edition WHO classification (2016), and included diffuse large B-cell lymphoma (DLBCL) not otherwise specified (NOS, n=145), primary cutaneous DLBCL leg type (PCDLBCL LT, n=13), high grade B-cell lymphoma (HGBCL, n=8), primary central nervous system lymphoma (PCNSL, n=11), primary mediastinal B-cell lymphoma (PMBCL, n=3), Burkitt lymphoma (n=4), mantle cell lymphoma (MCL, n=18) and a diverse group of indolent mature B-cell neoplasms (MBCN, n=76, specified in figure 1). P53 IHC staining was performed using the Dako Autostainer Link 48 (Dako Omnis, Glostrup, Denmark), according to standard procedures. For each case, p53 IHC was blindly and independently assigned by one junior (LMH) and one senior (PMJ) hematopathologist to one of the following categories: abnormal (complete absence OR staining > 50% of tumor cells) or wild type (WT) (staining 1-50% of tumor cells). All discordant cases were discussed at a consensus meeting attending both pathologists and assigned a definitive category. The inter-rater reliability was measured with Cohen's kappa coefficient. Pathogenic variants of TP53 for all cases were identified with an in-house developed tNGS panel (LYMFv1), including 52 B-cell lymphoma-relevant genes and covering all exons of TP53, as described before (PMID: 34478526). Results IHC showed 213 p53 WT and 65 abnormal. Kappa correlation of the p53 IHC assessment between the two pathologists was 0,82, indicating a strong level of agreement. tNGS identified a TP53 mutation in 75 patients (26,9%), of which DLBCL NOS was the largest subgroup (n=43), followed by MBCN (n=14) and MCL (n=7). Overall, most TP53 mutations were non-synonymous (80,5%) and localized in exon 7 (36%), as shown in the Oncoprintplot (figure 1). In MBCN the most frequent location was exon 5 (33%), while in DLBCL as the largest subgroup, mainly exon 7 (38%) was affected. The overall accuracy of p53 IHC was 85,6% (95% CI 80,9-89,5%) for the presence of TP53 mutation. The corresponding sensitivity and specificity were 66,7% (95% CI 56,0-77,4%) and 92,2% (95% CI 88,5-95,9%), respectively. 25 BCLs showed a false-negative (FN) p53 WT IHC, despite detection of TP53 mutation. MBCN was the least accurate performing group (8/14 FN), followed by DLBCL NOS (13/43 FN). On the other hand, there were 15 BCLs with a false-positive (FP) abnormal IHC staining without identification of a TP53 mutation (figure 2). Again, MBCN was the lowest performing group (7/62 FP), but the overall specificity was high. Conclusion Our data indicate that the performance of p53 IHC is not sufficient as a surrogate marker for TP53 mutations in the diagnostic workup of lymphoma patients. Although the specificity is high with a low amount of FP, the sensitivity is relatively low with a high number of FN. It seems that misinterpretation of the p53 IHC is not an adequate explanation for the low sensitivity, considering the strong inter-rater reliability and the re-assessment of all discordant cases. By utilizing only p53 IHC there is a significant chance a TP53 mutation is missed, resulting in misevaluation of a high-risk patient. Therefore, molecular analysis is recommended in all patients with BCL, especially for further development of risk-directed therapies based on TP53 mutation status. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Residual Disease Kinetics Among Patients with High-Risk Factors Treated with First-Line Fixed-Duration Ibrutinib Plus Venetoclax (Ibr+Ven) Versus Chlorambucil Plus Obinutuzumab (Clb+O): The Glow Study

Background: In the GLOW study (NCT03462719), fixed-duration Ibr+Ven demonstrated superior progression-free survival (PFS), deeper, better sustained undetectable minimal residual disease (uMRD) responses, and fewer patients (pts) requiring subsequent anti-cancer treatment versus Clb+O in older and/or comorbid pts with previously untreated chronic lymphocytic leukemia (CLL; Kater AP, et al. NEJM Evidence. 2022). Unmutated IGHV (uIGHV) and TP53 mutations are risk factors associated with worse outcomes for both chemoimmunotherapy and venetoclax plus anti-CD20 therapies for CLL. Here we investigate MRD kinetics and outcomes of Ibr+Ven in GLOW according to these risk factors. Methods: Pts aged ≥ 65 years or 18 to 64 years with cumulative illness rating scale score > 6 or creatinine clearance < 70 mL/min were enrolled and randomized 1:1 to Ibr+Ven (3 cycles of Ibr lead-in, followed by 12 cycles of Ibr+Ven) or 6 cycles of Clb+O, with a cycle defined as 28 days. Pts with del(17p) or known TP53 mutations at screening were excluded, and central evaluation of TP53 mutational status was performed during the study. The primary end point was PFS by independent review committee. Among pts with partial response or better, MRD in peripheral blood was evaluated using next-generation sequencing via clonoSEQ on-treatment and at 3-6 month intervals post-treatment. uMRD results are reported in peripheral blood (PB) at < 10-4 unless otherwise noted. Since the primary analysis, a post hoc retrospective reclassification of the IGHV status of baseline samples was conducted to reduce the number of unknowns. We used clonoSEQ data as part of CLL clonal testing (Adaptive Biotechnologies, Seattle, WA). Results: There were 106 pts randomized to Ibr+Ven and 105 to Clb+O. In the Ibr+Ven arm, uMRD rates increased from 46.2% after 6 cycles of the combination to 54.7% at 3 months after end of treatment (EOT+3), demonstrating that the majority of disease clearance in the PB occurred early, during the first 6 months of combination treatment. Post-treatment, 77.6% (45/58) of pts in the Ibr+Ven arm sustained their uMRD status from EOT+3 to EOT+18, compared with 12.2% (5/41) in the Clb+O arm. In the Ibr+Ven arm, 17/24 patients with intermediate MRD ≥ 10-4 to < 10-2 at EOT+3 did not worsen through EOT+18. Among pts with detectable MRD (≥ 10-4) at EOT+3, 6.5% (2/31) clinically progressed by EOT+18 in the Ibr+Ven arm versus 68.1% (32/47) in the Clb+O arm. After reclassification of baseline samples, uIGHV / mutated IGHV (mIGHV) / unknown status was 63.2%/30.2%/6.6% in the Ibr+Ven arm and 54.3%/33.3%/12.4% in the Clb+O arm. In the Ibr+Ven arm, rates of uMRD after 6 cycles of combination treatment and at EOT+3, respectively, were 52.2% and 59.7% in pts with uIGHV and 31.3% and 40.6% in pts with mIGHV (Fig A). On-treatment MRD kinetics differed by IGHV status in the Ibr+Ven arm, with uMRD achieved at a higher rate and earlier in pts with uIGHV. In the Ibr+Ven arm, uMRD was sustained post-treatment (from EOT+3 and EOT+18) in 80.0% (32/40) of pts with uIGHV and 76.9% (10/13) of pts with mIGHV. Among pts with detectable MRD (≥ 10-4) at EOT+3, 2/16 with uIGHV and 0/14 with mIGHV had clinically progressed by EOT+18 in the Ibr+Ven arm; corresponding rates were 83.3% (25/30) and 42.9% (6/14) in the Clb+O arm. Five of 7 pts with TP53-mutated CLL achieved uMRD at EOT+3 in the Ibr+Ven arm; TP53 variant allele frequencies ranged between 5.9% and 29.6% for these 5 pts and all maintained uMRD through EOT+18. With a median study follow-up of 34.1 months, PFS rates after IGHV reclassification were well sustained post-treatment regardless of IGHV status in the Ibr+Ven arm, while uIGHV pts in the Clb+O arm relapsed more quickly (Fig B). PFS, overall survival, and subgroup analyses with longer study follow-up will be presented. Conclusion: All-oral, once-daily, fixed-duration Ibr+Ven achieved uMRD responses that were better sustained than Clb+O during the first 18 months post-treatment. Notably, with Ibr+Ven, uMRD rates were higher and achieved earlier in pts with uIGHV CLL versus mIGHV CLL, while uMRD was similarly sustained post-treatment irrespective of IGHV status. Clinical progressions in the first 18 months post-treatment with Ibr+Ven were uncommon even among pts with detectable MRD ≥ 10-4. MRD kinetics and sustained responses demonstrate strong efficacy of the fixed-duration Ibr+Ven combination in older pts with high-risk genomic features. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular, Epigenetic, and Immune Landscape of TP53-mutated (TP53-M) Acute Myeloid Leukemia (AML) and Higher Risk Myelodysplastic Syndromes (HR-MDS)

Introduction: Mutations in TP53 confer an adverse prognosis in patients (pts) with AML and HR-MDS. Comprehensive analyses of the genetic, epigenetic, and immunologic landscape of TP53-M AML and HR-MDS are lacking, especially associations of allelic status of TP53 with immune and epigenetic disease phenotype. Methods: Using a large, randomized study with extensive and prospective clinical, histopathological, cytogenetic, molecular, epigenetic, and immunologic annotations, we compared data from 61 TP53-M (37 AML, 24 HR-MDS) and 144 TP53 wild-type (TP53-WT; 89 AML, 55 HR-MDS) pts. Pts were randomized to azacitidine (AZA) monotherapy or AZA + anti-PDL1 antibody durvalumab, which did not improve clinical outcomes (Zeidan et al. Blood Advances 2022). A 38 gene-targeted myeloid mutation analysis from screening bone marrow (BM) was performed at Munich Leukemia Laboratory. Only pathogenic TP53 variants with variant allele frequency (VAF) ≥2% were included. Pts with a common SNP or VUS in the TP53 gene were classified as TP53-WT. Multi-hit TP53 mutations were defined as previously published by Grob et al. (Blood 2022) which confirmed that TP53-M AML and HR-MDS can be grouped as one entity. BM aspirates from screening and cycle 3 day 22 (C3D22) were used to study gene expression profiles by RNA-sequencing and immune phenotyping by flow cytometry. Gene set enrichment analysis (GSEA) using the MSigDB and H (Hallmark) gene sets as provided by the msigdbr R package were performed. Flow cytometry data were analyzed into a collection of 170 reportable subpopulations in the immune cell and immune checkpoint panel and 68 reportables in the tumor/blast panel. DNA methylation of peripheral blood samples was assessed using Illumina's Infinium Human Methylation EPIC methylation array pre-treatment and at C2D1. Global DNA methylation scores (GDMS) and focal DNA demethylation in PD-1, PD-L1 and PD-L2 regulatory regions were analyzed. Results: Of the 61 TP53-M and 144 TP53-WT pts, 85.4% and 10.4% had complex karyotype, respectively. TP53-M pts had fewer co-occurring mutations compared with TP53-WT (mean number of mutations: 1.8 [SD: 0.77] vs 2.9 [SD: 1.4]; P <0.001). Distinct mutational patterns by TP53 mutational status were noted (Figure 1). The median VAF for TP53 mutations was 40%, and 90% had a VAF ≥10%. 22 (11.0%) and 41 (20.5%) pts were mono-hit and multi-hit, respectively. Global DNA methylation was independent of TP53 mutation status at baseline when comparing mono-hit, multi-hit and wild-type. AZA therapy led to statistically significant decrease in GDMS independent of TP53 mutation status (Figure 2). Various loci of PD-1, PD-L1 and PD-L2 genes showed differences in methylation status by TP53 mutation status, which were reversed on treatment. However, methylation of 2 PD-L2 gene loci (cg07211259 and cg11299543) remained significantly lower for multi-hit TP53-M compared to TP53-W at C2D1. Patterns of differentially expressed genes were similar for multi-hit TP53-M vs wild-type and for all TP53-M vs wild-type. The number of differentially expressed genes was higher in the AML cohort compared to MDS pts both at baseline (1887 vs 78) and after C3 (116 vs 0) with TP53-M samples having more differentially expressed genes. ZNF560 was the gene with the strongest differential expression by TP53 mutation status. TP53-M pts had higher expression of T-cell genes (e.g., IL7R), markers of proliferation (MKI67), and PD-L1 (CD274) expression compared to TP53-W at baseline. Genes in hallmark clusters of heme metabolism, inflammatory response, cell cycle regulation, and TGF-β signaling pathways were enriched in TP53-M pts in GSEA. TP53-M pts had higher median levels of PD-L1-positive progenitors (5.6% vs 2.4%; p=0.006) and less myeloid progenitor cells (58% vs 75%; p=0.005) than TP53-WT at baseline. However, there was no shift towards a more exhausted T-cell phenotype among TP53-M pts (CD3+CD4+ PD1+ TIM3+ T-cells and CD3+CD8+PD1+TIM3+ T-cells as % of CD3+CD4+ T-cells and CD3+CD8+ T-cells, respectively). Conclusion: Although an increased population of cytotoxic T-cells suggest an activated immune system, we also found the upregulation of inhibitory immune checkpoints such as PD-L1 in TP53-mut pts suggesting an immunosuppressive microenvironment as a potential contributor to the poor prognosis of TP53-M AML and MDS with similar findings across disease type (HR-MDS vs AML) and mono- vs multi-hit TP53 mutation status. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Growth Kinetics of Clonal Hematopoiesis Inform Risk of Blood Cancer Progression

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) is a frequent consequence of aging associated with increased risk of blood cancers, although overall individual risk is < 1% per annum. Proposed risk factors for blood cancer progression include a variant allele frequency (VAF) > 10%, specific gene mutations or the presence of more than one CHIP clone. However, the relative value of monitoring CHIP clone growth with respect to blood cancer risk is unknown. To address this evidence gap, we measured CHIP at baseline and 3 years later in 11,298 healthy individuals aged ≥70y from the ASPREE randomized clinical trial (www.aspree.org). Methods: A custom, high sensitivity (0.5%) and reproducible amplicon-based targeted sequencing assay was designed to assess CHIP in 19 genes, except for SRSF2, which had poor coverage. CHIP was defined as a VAF ≥ 2% with a minimum of 5 variant reads for previously reported CHIP or 20 for variants not previously reported. The analysis involved measuring CHIP variants in two serial blood samples collected at baseline and year 3 of follow-up for longitudinal analysis. Associations between CHIP and blood cancers were assessed using Cox proportional hazards models. Results: CHIP at baseline was present in 34% of participants who had a corresponding year 3 sample with adequate sequencing depth. Unexpectedly, 1,245/3,110 (40%) CHIP clones present at baseline fell below 2%, while 1,520 new CHIP clones appeared at 3 years. This equated to an incident CHIP rate of 7% per annum. CHIP genes more likely to expand and contract were TP53 and ASXL1 (odds ratio 1.25 and 1.22 respectively). To measure growth characteristics of CHIP clones, we calculated a Fitness Score, FS (Log2(VAF3-year/VAFBaseline) for the 3,110 clones present at baseline. We observed no significant mean fitness advantage for any recurrent CHIP variant other than PPM1D R552* (Figure 1). CHIP variants of DNMT3A (including DNMT3A R882), TET2 and ASXL1 had a statistically significant negative FS. Even variants generally considered most pathogenic (SF3B1 K700E, JAK2 V617F, U2AF1 Q157, TP53 R273) had no significant fitness advantage. Some CHIP clones had a significant growth disadvantage (FS < -1). For example, only 1/28 DNMT3A S393N clones present at baseline remained above 2% and almost half of TP53 variants reduced in size by more than 2-fold at 3 years. We observed 299 blood cancers over a median time of 6.9 years. Individuals with CHIP clones >10% VAF (n= 652) had increased incidence of blood cancers (adjusted hazard ratio, aHR 2.14, confidence interval, CI 1.34-3.42, p=0.002). Genes associated with increased risk for blood cancers were SF3B1/U2AF1 (aHR 3.02, CI 1.63-5.61, p<0.001) and TET2 (n=aHR 1.89, CI 1.18-3.02, p=0.008). Participants with small clones (VAF 2-10%) at baseline or year 3 had no increased risk for blood cancers compared to those without CHIP. To investigate if the growth kinetics of CHIP predicts blood cancer risk, we subdivided CHIP clones into expanding (FS > +1, n= 104), stable (FS -1 to +1, n= 2043) and contracting (FS < -1, n= 963). Baseline VAF of expanding clones were smaller than stable clones: geometric mean 3.9% and 5.0%, respectively (p<0.001). Expanding clones were enriched 2-fold for PPM1D, SF3B1 and JAK2 V617F. Participants with expanding clones (n=99) were more likely to have more than one CHIP variant (50% compared with 39% for stable and 37% for contracting), although 90% of these additional variants were stable or contracting, indicating that the expanding clone was dominant. For similar VAF levels at 3 years, participants with expanding CHIP clones had a higher risk of blood cancer than a stable or contracting one (Figure 2). Conclusions: This is the largest longitudinal study of CHIP variants and associated blood cancer outcomes to date. In contrast to other longitudinal studies, there was little evidence for selective growth advantage of CHIP clones with a VAF ≥ 2%, including those with pathogenic mutations of DNMT3A, SF3B1, U2AF1, TP53 and JAK2. While clone size (VAF > 10%) and gene mutation (SF3B1, U2AF1 and TET2) were associated with increased risk of blood cancer progression, our study cautions clinical intervention of CHIP based upon a single timepoint analysis irrespective of the gene mutation. Instead, we propose monitoring of clone size may be a clinically more useful strategy to inform counselling and patient selection for early intervention studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High Incidence of Clonal Hematopoiesis in Transfusion-Dependent Thalassemia Patients

Introduction: Despite improvement in survival in patients with transfusion-dependent thalassemia (TDT), patients experience significant morbidities, including iron overload, malignancies, thromboembolic events and stroke, and cardiac complications like heart failure and arrhythmias. It is established that global chain imbalance and excess iron leads to significant oxygen stress and to the formation of oxygen free radicals resulting in an increased risk of mutations. Clonal Hematopoiesis of Indeterminate Potential (CHIP) is characterized by the presence of somatic variants in the peripheral blood with a variant allele frequency (VAF) ≥2% in genes associated with hematologic malignancies. These variants occur more frequently with advanced age (~ 10% at age> 65 years). That said, recent data show that young patients with CHIP are at increased risk for developing not only hematological malignancies, but also coronary heart disease, cardiovascular events and stroke. In this study we explored if TDT patients have an increased risk of clonal hematopoiesis and if this observation correlates with their underlying cardiovascular status, thromboembolic complications and history of malignancy. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from ninety five patients with TDT just before their regular transfusion schedule (43 men and 52 women, mean age 39 years old, range 4-55 years ). 40 patients had a history of splenectomy, 32 patients had a history of cardiac disorder (13 patients with atrial fibrillation or other arrhythmias, 19 patients with valvular dysfunction). Genomic DNA was isolated from PBMCs from all patients and Next-Generation Sequencing analysis (NGS) was performed for the mutational status of 58 recurrently mutated genes in myeloid malignancies using a custom-designed Illumina sequencing panel (Illumina, San Diego, CA). Identification of mutations with a VAF ≥2% were reported after bioinformatics analysis and variant interpretation using the Varsome, ClinVar (v.2021) and Franklin databases. Results: Fifteen of 95 patients analyzed (15.8%) displayed mutations in the genes implicated in CHIP. The age-adjusted prevalence of clonal hematopoiesis in patients with TDT vs the reported in the general population is significantly higher. Overall, 22 somatic variants were identified in 15 patients with a median VAF of 40% (range 2.5%-52%): variants were detected in various genes including ATM, FLT3, ASXL1, MPL, SF3B1, BRAF, BTK, TP53, JAK2, RUNX1, ETV6 and EZH2. Four patients carried variants in 4 different genes (TP53, TET2, CEBPA, NPM1) with a mean VAF of 3% (range 2,45% - 4,14%). The patient carrying the TP53 variant is a 44-year old male with a history of splenectomy and arrhythmia (ectopic atrial tachycardia). The patient carrying the TET2 mutation is 55-year old female with a history of colon cancer ten years ago, but no evidence of malignancy relapse as of today. A 5-year old male patient carried a clone in the NPM1 gene with VAF of 2.7%, with no history of splenectomy or cardiac complications. Finally, a 20 year-old male patient carried a clone in the CEBPA gene (VAF 2.7%) along with a clone in the ATM gene (VAF 50.3%). No history of splenectomy, or thromboembolic complications were noted in this patient. Bioinformatics analysis revealed 18 variants in 11 patients with a VAF frequency within the germline range (VAF range 47-52%); germline DNA analysis is ongoing. Seventy TDT patients (73,7%) carried the FLT3 C680T variant that is higher than the incidence reported in gnomAD exome for the European population (60.7%). Ninety three patients carried the ASXL1 T2444C variant, comparable to the observed frequency in the general population. The TET2 C100T variant was observed in 3,1% of the analyzed population compared to the 1.5% reported in the general population. Further analysis revealed that none of the patients had a history of stroke and only two patients had a history of mitral valve regurgitation. Conclusion: We report for the first time that patients with TDT display a significantly higher frequency of variants in genes associated with myeloid malignancies. Life-long stress in the hematopoiesis in these patients may contribute to the increased risk of clonal mutations observed, however further analysis is required to better understand the impact of these observations.