TP53 Variant Allele Frequency Research Articles

Abstract 2425: Pairwise analysis of plasma cell-free DNA before and after paclitaxel plus ramucirumab treatment in patients with metastatic gastric cancer

Abstract Background: Plasma cell-free DNA (cfDNA) analysis has emerged as an appealing alternative to detect somatic mutations. This study compared cfDNA at baseline (baseline-cfDNA) and progressive disease (PD-cfDNA) and tumor tissue DNA (ttDNA) in patients with metastatic gastric cancer who underwent palliative second-line paclitaxel+ramucirumab treatment. Methods: We conducted targeted sequencing of 106 cancer-related genes using germline DNA, baseline-cfDNA, and PD-cfDNA samples. The results were then compared with conventional cancer panel results using ttDNA. Results: Of 76 consecutively enrolled patients who received paclitaxel+ramucirumab treatment, 46 patients (27 males; median age 57.5 [range, 32-73] years) with access to all three samples were analyzed along with their ttDNA data. A total of 138, 145, and 80 mutations were detected in baseline-cfDNA, PD-cfDNA, and ttDNA respectively. Combined analysis of baseline-cfDNA and ttDNA revealed that TP53 (56.5%) was the most frequently mutated gene, followed by CDH1 (26.1%), KRAS (21.7%), and APC (13.5%). For the top four genes, the sensitivity and positive predictive value of baseline-cfDNA compared with ttDNA were 71.8% and 51.9%, respectively. Compared with ttDNA alone, 32 patients (70.0%) benefited from baseline-cfDNA in detecting novel mutations. When combined with baseline-cfDNA and PD-cfDNA, novel mutations were discovered in 34 patients (73.9%). Of note, PD-cfDNA analysis detected 9 novel pathogenic mutations in TP53, APC, PIK3CA, CDH1, RNF43, CTNNB1, and BRCA2 genes in 8 patients (17.4%) after treatment. In baseline-cfDNA, patients with a circulating ttDNA fraction concentration at 110-160 bp of >4.3777 ng/µL had significantly shorter progression-free survival (PFS) (median 3.5 vs. 5.3 months, P=0.016). In addition, maximal variant allele frequency (VAF) values of >0.1045 (median 3.4 vs. 5.2 months, P=0.022) and the sum of VAF values of >0.2071 (median 3.4 vs. 5.2 months, P=0.028) were significantly associated with shorter PFS. In patients with TP53 mutations, those with TP53 VAF values of >0.1014 significantly had worse PFS (median 4.6 vs. 6.4 months, P=0.022). Conclusions: Although cfDNA could not entirely take over the role of ttDNA, cfDNA analysis identified additional somatic mutations that were otherwise missed by ttDNA alone. Moreover, PD-cfDNA analysis detected novel pathogenic mutations that developed during treatment, implicating the clonal evolution of cancer. In addition, baseline cfDNA predicted PFS of patients receiving paclitaxel+ramucirumab therapy. Citation Format: Ji-Won Kim, Dong Soo Kyung, Won Yeong Ko, Hwang-Phill Kim, Sung-Hyun Hwang, Kui-Jin Kim, Ju Hyun Lee, Jeongmin Seo, Minsu Kang, Eun Hee Jung, Koung Jin Suh, Se Hyun Kim, Jin Won Kim, Yu Jung Kim, Jee Hyun Kim, Keun-Wook Lee. Pairwise analysis of plasma cell-free DNA before and after paclitaxel plus ramucirumab treatment in patients with metastatic gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2425.

TP53 variant allele frequency and therapy-related setting independently predict survival in myelodysplastic syndromes with del(5q).

Among 210 patients with myelodysplastic syndromes (MDSs) with del(5q), molecular information was available at diagnosis or at least 3 months before leukaemic transformation in 146 cases. Multivariate analysis identified therapy-related setting (p = 0.02; HR 2.3) and TP53 variant allele frequency (VAF) ≥22% (p < 0.01; HR 2.8), but not SF3B1 mutation (p = 0.65), as independent risk factors for survival. Median survival was 11.7 versus 4 years (5/10-year survival 73%/52% vs. 42%/14%) in the absence (N = 112) versus presence (N = 34) of ≥1 risk factors; leukaemia-free survival was affected by TP53 VAF ≥22% (p < 0.01). Such information might inform treatment decision-making in MDS-del(5q) regarding allogeneic stem cell transplant.

Clonal Dynamics and Deterministic Clinical Fate Mapping of Patients with Myelodysplastic Neoplasms and Acute Myeloid Leukemia with TP53 Disruption

Background: Myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML) with multi-hit TP53 comprise the highest risk subgroup of all myeloid malignancies. TP53 aberration is recognized as a diagnostic category in the 2022 International Consensus Classification (ICC) and the 5 th edition of the WHO. However, these systems do not discern among the subtypes of TP53 aberrations regarding response to therapy. In this study, we explore the clonal dynamics that underlie such prognostic heterogeneity within distinct subgroups of TP53-aberrant MDS and AML, with a specific focus on the effect of hypomethylating agent (HMA) vs. araC-based chemotherapy on TP53 variant allele frequency (VAF). Methods: The UMass Leukemia Registry from the UMass Data Lake identified 76 patients harboring TP53 aberration(s) plus ICD-10 code of either D46.9 (MDS and its subentities) or C92.00 (AML and its subentities) between 2011-2023. Data was integrated with hematopathology records. TP53 exome sequencing was performed with coverage depth 500X to 6500X, and VAFs were adjusted for copy number variation (CNV), as previously described (Patel SA et al., Leuk Lymphoma 2021; 62: 3348-60). Patients with serial bone marrow biopsy samples were included. We defined multi-hit TP53 status as per the ICC: two or more distinct TP53 mutations with VAF ≥ 10%, or a single TP53 mutation plus one of the following abnormalities: (1) del(17p13.1), (2) TP53 VAF &amp;gt;50%, (3) copy-neutral loss-of-heterozygosity, or (4) any complex karyotype. Clonal dynamics were assessed for MDS vs. AML and for varying TP53 allelic states as a function of therapeutic intervention. Clinical fate mapping was performed based on management decisions at various points of clinical care. Results: For patients with TP53-aberrant MDS, 17 patients underwent serial bone marrow biopsies during treatment. Of these, 11 (64.7%) had multi-hit status. Eight patients (47.1%) underwent hematopoietic cell transplant (HCT), and 7 of these 8 patients had received HMA-based front-line therapy. For patients with MDS, we assessed the effect of HMA-based therapy on TP53 VAF. The mean TP53 VAF (sum of VAFs) was significantly lower after HMA-based treatment (35% vs. 12.6%) ( p = 0.0075) (Panel A). For patients with TP53-aberrant AML, 19 patients underwent serial bone marrow biopsies. Of these, 14 (73.7%) had multi-hit status. Only 2 patients (10.5%) underwent HCT. Five patients (26.3%) received araC-based induction, while 11 patients (57.9%) initially received HMA-based induction. For patients with AML with multi-hit TP53, we assessed the effect of HMA-based vs. araC-based front-line therapy on TP53 VAF. HMA therapy led to mean fold reduction in TP53 VAF (pre-treatment to post-treatment) of 11.7 ± 5.8 ( p = 0.019), while araC-based therapy actually led to mean fold increase (but not statistically significant) in TP53 VAF (pre-treatment to post-treatment) of 2.6 ± 1.05 ( p = 0.319) (Panel B). HMA-based treatment resulted in significantly greater reduction in the VAF in aberrant clones and subclones compared to araC-based treatment. Compared to AML, most patients with MDS (62.9%) proceeded with palliative intent therapy at the time of diagnosis. Although patients with AML (53.7%) attempted to proceed with curative intent at the time of diagnosis, only 14.6% eventually proceeded with HCT, largely due to clinical decompensation or disease progression during front-line therapy. Among HCT recipients for MDS ( n =10), 7 (70%) remained in remission. Among HCT recipients for AML (n = 6), 4 (66.7%) remained in morphologic leukemia-free state. Conclusion: There is limited literature on clonal and subclonal diversity in patients with MDS or AML with TP53 aberrations. Such considerations constitute an important therapeutic issue, as certain interventions may only eliminate a fraction of a patient's mutant cells. In our study, HMA- and araC-based regimens had differential effects on various subclones: HMA was more effective in reducing the TP53 mutational burden and increasing patient ability to proceed with HCT. AraC-based therapy appeared less effective, perhaps due to selection pressure in favor of TP53-mutant cells. Prospective trials are required to identify optimal regimens that lead to the best long-term outcomes, and single-cell resolution might assist with more definitively assessing dynamics of clonal response to treatment.

TP53 Variant Allele Frequency and Therapy-Related Setting Independently Predict Survival in Myelodysplastic Syndromes with Del(5q)

Background: According to the international consensus classification (ICC), diagnosis of myelodysplastic syndromes (MDS) with del(5q) requires presence of i) del5q, alone or associated with no more than one other cytogenetic abnormality, exclusive of -7/del(7q), ii) &amp;lt;5% bone marrow (BM) and &amp;lt;2% circulating blasts, and iii) the absence of “multi-hit” TP53 ( Arber et al. Blood 2022;140: 1200). We have recently reported on TP53 variant allele frequency (VAF) ≥22% being the sole genetic predictor of inferior survival in MDS-del(5q) (Fleti. et al. AJH 2023;98:E76). In the current study, we examined the additional prognostic impact of therapy-related setting, in the context of TP53 VAF and other risk factors. Methods : The current study included patients with MDS-del(5q) who were seen at the Mayo Clinic or Moffitt Cancer Center, USA. Diagnosis of MDS-del(5q) and therapy-related qualification was according to ICC criteria ( Arber et al. Blood 2022;140: 1200), with the exception that patients with biallelic or multi-hit TP53 were included to allow unbiased analysis of impact on prognosis. Patient selection was based on availability of mutation information from next-generation sequencing (NGS). The latter was assessed either at diagnosis or at different time points during the disease course, but at least 3 months before leukemic transformation. Otherwise, all analyses, including estimation of overall and leukemia-free survival, were based on variables collected at time of initial diagnosis of MDS-del(5q). TP53 mutation VAF threshold of 22% was considered based on our previously published report cited above. Age cutoff at 67 years was determined by ROC analysis. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results: A total of 210 patients (median age 72 years; 65% females), meeting the above-stipulated criteria for MDS-del(5q), were considered: 172 primary and 38 therapy-related. Table 1 outlines clinical and laboratory characteristics obtained at the time of diagnosis: 42% were transfusion-dependent, 69% had hemoglobin level &amp;lt;10 g/dL, 30% platelet count &amp;lt;150 x 10(9)/L, and 15% carried additional cytogenetic abnormality. NGS data performed either at diagnosis or at least 3 months prior to leukemic transformation was available in 146 patients; the most frequent mutations were TP53 (21%), SF3B1 (16%), DNMT3A (12%), TET2 (12%), ASXL1 (10%), and JAK2 (7%) (Table 1). At a median follow-up of 3.9 years (range 0.002-18.5) for living patients, 95 (45%) deaths and 34 (16%) leukemic transformations were recorded; treatment undertaken included lenalidomide in 162 (77%) cases. NGS data-exclusive multivariable analysis that included all 210 patients identified therapy-related setting (p&amp;lt;0.01; HR 2.9; Figure 1a), age &amp;gt;67 years (p=0.01; HR 1.8), transfusion-dependency (p=0.04; HR 1.5), and platelet count &amp;lt;150 x 10(9)/L (p=0.03; HR 1.6), as independent predictors of inferior survival. The significant effect of age and platelet count was sustained when analysis was limited to patients with primary MDS-del(5q) but lost when applied to therapy-related disease. NGS data-inclusive multivariable analysis, which was applicable to the subset of 146 patients, identified, as risk factors, therapy-related setting (p&amp;lt;0.01; HR 2.3) and TP53 VAF ≥22% (p&amp;lt;0.01; HR 2.4; Figure 1b) while age &amp;gt;67 years sustained borderline significance (p=0.07; HR 1.8). Figure 1c depicts survival data based on the presence or absence of TP53 VAF ≥22% and therapy-related qualification; low-risk with no risk factors (N=112; median survival 11.7 years; 5/10-year survival 73%/52%) and high-risk with one or both risk factors (N=34; median survival 4 years; 5/10-year survival 42%/14%); leukemia-free survival was affected by TP53 VAF ≥22% (p&amp;lt;0.01) but not therapy-related setting (p=0.13). Conclusions : The current study highlights TP53 VAF and therapy-related setting as the most prominent risk factors for survival in MDS-del(5q) and proposes a simple risk model that distinguishes patients with a projected 10-year survival that exceeds 50%, in the absence of both risk factors, and plummets to 14%, in the presence of one of the two risk factors. Accordingly, allogeneic stem cell transplant might be deferred in the former instance while it should be pursued sooner than later, in the latter instance.

Multi-Hit TP53 Mutations in Myeloid Neoplasms: Prognostic Impact of Morphologic Subtype Designation and Variant Allele Frequency

Background: Biallelic TP53 alterations result from either sequence variations or deletions involving TP53 and are not necessarily synonymous with “multi-hit TP53”. According to the 2022 international consensus classification (ICC) for myeloid neoplasms (MN), multi-hit TP53 signifies i) the presence of two or more distinct TP53 mutations, each with variant allele frequency (VAF) ≥10%, ii) a single TP53 mutation with VAF ≥50%, or iii) a single TP53 mutation with VAF ≥10% accompanied by a cytogenetically-apparent del(17p13.1), copy-neutral loss of heterozygosity (LOH) at the 17p TP53 locus, or, in the absence of LOH information, complex karyotype ( Arber et al. Blood 2022;140: 1200). The current study is focused on MN with multi-hit TP53 and examines the additional prognostic impact of morphologic subtype designation, bone marrow (BM) or peripheral blood (PB) blast percentage, TP53 VAF, and MN with diagnostic qualifiers (i.e., therapy-related, or secondary progressing from myelodysplastic/myeloproliferative/overlap syndromes/neoplasms). Methods : The current study was conducted under an institutional review board approved minimum risk protocol that allowed retrospective collection and analysis of data from Mayo Clinic patient records. Multi-hit TP53 was defined as per ICC criteria as outlined above ( Arber et al. Blood 2022;140: 1200). Morphologic subtype designations of MN were according to ICC criteria and assigned at the time of TP53 detection. NGS and cytogenetic information was available in all study patients. Survival analyses were calculated from time of TP53 mutation detection. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results : Initial screening flagged 143 patients with biallelic TP53 abnormalities derived from formal laboratory reports of NGS data and cytogenetic studies; of these, 130 met ICC criteria for multi-hit TP53: pure erythroid leukemia (PEL; N=24), acute myeloid leukemia (AML)-not PEL (N=54), myelodysplastic syndromes (MDS; N=36), MDS/AML (N=11), and other MN (N=5). Of these 130 informative cases, 128 (98%) harbored complex/monosomal karyotype (CK/MK). Further analysis excluded patients with “other” MN (N=5) and those without CK/MK (N=2), the latter to mitigate the confounding effect of CK/MK on survival and the former because of small sample size and disease heterogeneity. Clinical and laboratory characteristics of the 124 study patients are outlined in table 1. Survival analysis stratified by ICC-defined MN subtypes revealed the prognostic relevance of morphological distinction between PEL vs “AML-not PEL” (p&amp;lt;0.01; HR 2.4) and PEL vs “ TP53-muated MDS/AML” (p=0.02; HR 2.3) while survival was similar between “AML-not PEL” and “ TP53-mutated MDS/AML” (p=0.9; Figure 1a). Survival in “ TP53-mutated MDS” was significantly longer, compared to PEL (p&amp;lt;0.01; HR 0.2), “ TP53-mutated AML-not PEL” (p=0.01; HR 0.5), and “ TP53-mutated MDS/AML” (p=0.07; HR 0.5), the latter with borderline significance (Figure 1a). Multivariable analysis confirmed the independent prognostic relevance of ICC subtype designation (p&amp;lt;0.01) and also revealed additional negative prognostic contribution from advanced age (p=0.02), male gender (p=0.02), MN, secondary (p&amp;lt;0.01), MN, therapy-related (p=0.03), and DNMT3A mutation (p=0.02). Significance was retained in all instances, with the exception of MN, therapy-related (p=0.15), when analysis was repeated after excluding PEL cases (Figure 1b). Conclusions : The current study confirms the prognostic validity of the ICC morphologic subtype designation and TP53 VAF classification threshold, in the context of multi-hit TP53. The study also highlights the prognostic distinction between PEL and “ TP53-mutated AML-not PEL” and the prognostic alignment between the latter and “ TP53-muated MDS/AML”, both of which displayed inferior survival, compared to “ TP53-mutated MDS”. The study also suggests additional prognostic contribution from secondary and therapy-related qualification while the observation regarding DNMT3A mutation requires validation with higher number of informative cases.

Heterogeneity of TP53 Mutations in Mantle Cell Lymphoma- Challenges in Risk Stratification and Subclassification

Introduction: Somatic mutations in TP53 occur in approximately 10-20% of mantle cell lymphoma (MCL) patients (pts) at diagnosis. Eskelund and colleagues (Blood 2017) demonstrated poor survival among 20 pts with TP53-mutated MCL following intensive induction and transplant, and reported sequence-level variant data. However, TP53 mutations are heterogeneous, and few studies in MCL provide detailed sequencing data or variant allele frequency (VAF). Furthermore, the clinical impact of TP53 mutations may vary, and there is no algorithm shown to predict outcome for a given variant. We hypothesized that high VAF, or presence of a “disruptive” TP53 mutation as reported in squamous cell carcinoma (SCC; Poeta NEJM 2007), may adversely impact prognosis in MCL. To address this, we analyzed all MCL pts with available TP53 sequencing data at our center, including exact TP53 variant data and VAF, sMIPI score, and classification as disruptive vs not, and impact on survival from time of testing. Methods: Institutional records were analyzed with IRB approval, identifying 65 MCL pts who underwent sequencing for TP53 mutation from any tumor sample, as a single-gene assay or part of a panel. We analyzed VAF, type of mutation, and sMIPI score at time of TP53 testing. We classified TP53 mutations as “disruptive” or not according to method of Poeta and colleagues; these mutations result in a STOP codon, or an amino acid polarity change in the L2-L3 DNA binding domain of the TP53 protein (Poeta NEJM 2017). The Kaplan-Meier method was used to estimate overall survival (OS) and progression-free survival (PFS) from time of TP53 testing to death or last follow up, and the log-rank test was used to compare outcomes based on TP53 mutational status. Among TP53 mutated cases, Cox proportional analyses were performed to explore the impact of the following covariates on survival: disruptive status, VAF, sMIPI score at time of TP53 testing, and presence of complex cytogenetics. Results: Sixty-five pts underwent TP53 sequencing, and 23 (35%) had a TP53 mutation (mTP53+). mTP53+ pts had inferior PFS (p=0.03) compared to TP53 wild-type, and a trend toward inferior OS (p=0.07); median OS was 26 months (95% CI: 8.1, NA) from time of TP53 testing. Ki-67 index, blastoid histology, and age were similar between mTP53+ and wild-type status, but mTP53+ pts had a higher rate of complex cytogenetics (&amp;gt; 3 lesions; 48% vs 26%). SMIPI was also higher in mTP53+ pts (median score 6, interquartile range (IQR) range 4-7) vs TP53 wild-type pts (median 4, IQR range 3-5.2). MTP53+ pts were more likely to have received a BTK-inhibitor (BTK-I) (91 % vs 64%) and CAR-T therapy (39% vs 18%). Details of the mTP53+ subset are shown in Table 1; 4 pts had &amp;gt;1 TP53 mutation, median VAF was 40%, and 30% of pts had a disruptive mutation. Among mTP53+ pts, on univariate analysis, higher sMIPI at time of TP53 testing (p=0.003) was associated with worse OS (Table 2). In a multivariable Cox model including VAF &amp;gt;= 40%, presence of disruptive mutation, sMIPI, and complex cytogenetics, only sMIPI at time of TP53 testing was associated with inferior OS (p=0.02). Conclusion: TP53 variants in MCL are diverse, and in our cohort were largely unique from those reported by Eskelund and colleagues (Blood 2017). Despite now-routine application of sequencing and improvements in therapy, both of which may attenuate the negative prognostic impact of TP53 mutation, we confirmed poor outcomes in mTP53+ MCL. Among mTP53+ pts, only sMIPI predicted inferior OS on both univariate and multivariate analysis. Higher VAF and adoption of a clinical algorithm from SCC failed to discriminate among TP53 variants in terms of prognosis. Larger datasets of uniformly tested MCL pts are needed to elucidate prognosis among TP53 mutation variants, and improve understanding and management of mTP53+ MCL.

Predicted Functional Consequences of TP53 Alterations and Prognosis in TP53-Mutated Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) cases with TP53 mutations ( TP53mut) are associated with poor responses to chemoimmunotherapy (Eskelund Blood 2017). Across myeloid and solid tumors, predicted functional consequences and pathogenicity of specific TP53 mutations have been linked to differential patient outcomes (Pollock Breast Cancer Res Treat 2022, Yabe Cancers 2021). We aimed to characterize the impact of these features and associated molecular alterations on treatment outcome in TP53mut MCL patients treated with standard frontline regimens and on the ongoing phase 2 BOVEN trial. The BOVEN trial aims to assess the efficacy of frontline treatment with zanubrutinib, obinutuzumab, and venetoclax in TP53mut MCL (Kumar Blood 2021). Methods: The BOVEN trial enrolled 25 patients with TP53mut MCL. Retrospective chart review was performed for 22 patients with TP53mut MCL treated at frontline with standard chemoimmunotherapy regimens (Joffe Blood 2019). Tumor histology and FISH were obtained for each case. Mutational profiling (SNV/indels, CNA) was obtained prior to therapy with various CLIA-certified targeted sequencing platforms (Cheng J Mol Diagn 2015, Dias-Santagata EMBO Mol Med 2010). Mutations in TP53 were annotated using predicted phenotypes and pathogenicity from the TP53 Database (de Andrade Cell Death Differ 2022) and from phenotypic annotation of TP53 mutations (Giacomelli Nat Genet 2018). The primary outcome measures were overall and progression-free survival (OS/PFS). Results: Each patient included in the BOVEN cohort featured variants in TP53 with 26 SNV/indels across all 25 patients. There were 25 SNV/indels across all 22 patients treated with standard regimens. Within the trial cohort 14/25 cases had evidence of biallelic inactivation of TP53 with co-occurring SNV/indels in the gene and 17p loss by FISH or TP53 copy losses by SNParray; the comparator cohort featured 11/22 cases with biallelic losses. Across cohorts, most mutations were missense (&amp;gt;80% in both), with the majority occurring in the DNA binding domain of the p53 protein (76% and 91%, respectively), and with &amp;gt;30% at known hotspot codons in both cohorts. Most mutations (&amp;gt;70% in both cohorts) were predicted to adversely impact WT p53 function, including nonfunctional transactivation activity and dominant negative effect on the WT allele. The majority were also predicted to be highly pathogenic by bioinformatic methods (AGVGD, SIFT, PolyPhen2, PHANTM, REVEL, BayesDel). However, none of these features correlated with outcome. In the BOVEN cohort the variant allele frequency (VAF) of the TP53 SNV/indels approached significant association with poor PFS (HR 33.2, p=0.05) but not OS (HR 28.6, p=0.09). In contrast, in the comparator cohort TP53 VAF appeared to have an opposite effect direction, though this was not significant for PFS (HR 0.98, p=0.2) or OS (HR 0.97, p=0.06). We next investigated the impact of co-occurring molecular alterations on outcome. Besides TP53, the most frequently mutated genes in the trial cohort were KMT2D (24%), ATM (16%), NOTCH2 (12%), and CCND1 (12%). Alterations in these genes did not correlate with outcome. Within the comparator cohort the recurrently mutated genes were SMARCA4 (23%), ARID1A (18%), ARID1B (14%), ATM (14%), and CCND1 (14%). As described previously in this cohort (Joffe Blood 2019), alterations in SMARCA4 were associated with worse PFS (HR 4.2, p=0.02) but not OS (HR 2.2, p=0.27). Mutational burden (SNV and CNA load) was not prognostic in either 12/25 BOVEN samples sequenced with the same panel or the comparator cohort. There was no significant difference in outcome between patients with single mutations in TP53 vs. those with biallelic losses in TP53 in either cohort. Conclusion: Across patients with TP53mut MCL treated either with standard frontline chemoimmunotherapy or on the BOVEN trial, predictions of intrinsic pathogenicity and functional consequences of the TP53 alterations were not prognostic. The impact of TP53 mutation VAF remains unclear, with seemingly opposite effect directions between the two cohorts, though neither finding was significant. As previously described in the comparator cohort, alterations in SMARCA4 are associated with worse PFS in TP53mut MCL, though this was not observed in the BOVEN cohort. There were no significant differences in outcome among patients with biallelic inactivations in TP53 or with higher burdens of somatic alterations.

Inflammatory Rhabdomyoblastic Tumor: Clinicopathologic and Molecular Analysis of 13 Cases

Inflammatory rhabdomyoblastic tumors (IRMTs) are newly recognized skeletal muscle tumors with uncertain malignant potential. We investigated 13 IRMTs using clinicopathologic, genetic, and epigenetic methods. The cohort included 7 men and 6 women, aged 23 to 80 years (median, 50 years), of whom 2 had neurofibromatosis type 1. Most tumors occurred in the deep soft tissues of the lower limbs, head/neck, trunk wall, and retroperitoneum/pelvis. Two tumors involved the hypopharyngeal submucosa as polypoid masses. Eight tumors showed conventional histology of predominantly spindled cells with nuclear atypia, low mitotic activity, and massive inflammatory infiltrates. Three tumors showed atypical histology, including uniform epithelioid or plump cells and mitotically active histiocytes. The remaining 2 tumors demonstrated malignant progression to rhabdomyosarcoma; one had additional IRMT histology and the other was a pure sarcoma. All 11 IRMTs without malignant progression exhibited indolent behavior at a median follow-up of 43 months. One of the 2 patients with IRMTs with malignant progression died of lung metastases. All IRMTs were positive for desmin and PAX7, whereas myogenin and MyoD1 were expressed in a subset of cases. Targeted next-generation sequencing identified pathogenic mutations in NF1 (5/8) and TP53 (4/8). All TP53 mutations co-occurred with NF1 mutations. TP53 variant allele frequency was much lower than that of NF1 in 2 cases. These tumors showed geographic (subclonal) strong p53 immunoreactivity, suggesting the secondary emergence of a TP53-mutant clone. DNA methylation–based copy number analysis conducted in 11 tumors revealed characteristic flat patterns with relative gains, including chromosomes 5, 18, 20, 21, and/or 22 in most cases. Widespread loss of heterozygosity with retained biparental copies of these chromosomes was confirmed in 4 tumors analyzed via allele-specific profiling. Based on unsupervised DNA methylation analysis, none of the 11 tumors tested clustered with existing reference entities but formed a coherent group, although its specificity warrants further study.

Prognostic heterogeneity and clonal dynamics within distinct subgroups of myelodysplastic syndrome and acute myeloid leukemia with TP53 disruptions.

TP53 aberrations constitute the highest risk subset of myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). The International Consensus Classification questions the blast threshold between MDS and AML. In this study, we assess the distinction between MDS and AML for 76 patients with TP53 aberrations. We observed no significant differences between MDS and AML regarding TP53 genomics. Median overall survival (OS) was 223 days for the entire group, but prognostic discrimination within subgroups showed the most inferior OS (46 days) for AML with multihit allelic state plus TP53 variant allele frequency (VAF) > 50%. In multivariate analysis, unadjusted Cox models revealed the following variables as independent risk factors for mortality: AML (vs. MDS) (hazard ratio [HR]: 2.50, confidence interval [CI]: 1.4-4.4, p = 0.001), complex karyotype (HR: 3.00, CI: 1.4-6.1, p = 0.003), multihit status (HR: 2.30, CI 1.3-4.2, p = 0.005), and absence of hematopoietic cell transplant (HCT) (HR: 3.90, CI: 1.8-8.9, p = 0.0009). Clonal dynamic modeling showed a significant reduction in TP53 VAF with front-line hypomethylating agents. These findings clarify the impact of specific covariates on outcomes of TP53-aberrant myeloid neoplasms, irrespective of the diagnosis of MDS versus AML, and may influence HCT decisions.

Low TP53 variant allele frequency as a biomarker for anti-programmed death (ligand) 1 monotherapy in lung adenocarcinoma.

TP53 mutation heterogeneity should be considered when using TP53 as a predictive biomarker for anti-programmed death (ligand) 1 (PD-(L)1) monotherapy in lung adenocarcinoma (LUAD). However, whether TP53 variant allele frequency (VAF) should also be considered remains unknown. Patients with LUAD from both published research and the local cohort were included to discover and validate the relationship between TP53 VAF and the efficacy of PD-(L)1inhibitors. The Cancer Genome Atlas (TCGA) LUAD data were included for genomic, transcriptomic, and tumor microenvironment analysis. Among 159 patients in the discovery cohort, low TP53 VAF patients (VAF ≤ 25%) experienced significantly longer progression-free survival (PFS) than both high TP53 VAF (5.4 vs. 3.3months; p=.021) and TP53-wild-type patients (5.4 vs. 2.5months; p=.011). Multivariate Cox regression revealed low TP53 VAF as an independent biomarker of better efficacy. Among 50 patients in the combined validation cohort, median PFS of low TP53 VAF patients was also significantly longer than that of high TP53 VAF patients (12.0 vs. 2.1months; p=.037). Analyzed with 469 TCGA LUAD samples, low TP53 VAF is associated with significantly higher PD-L1 expression, enrichment of gene sets related to T-cell activation, T cell-mediated immunity, and interferon-γ signaling pathways, and independently associated with more tumor-infiltrating CD8+ T cells compared with both high TP53 VAF and TP53-wild type. TP53 VAF should also be considered when using TP53 as a predictive biomarker. Only low TP53 VAF is independently associated with better efficacy of anti-PD-(L)1 monotherapy, which may result from higher PD-L1 expression and more tumor-infiltrating CD8+ T cells.

TP53 Mutations in AML Patients Are Associated with Dismal Clinical Outcome Irrespective of Frontline Induction Regimen and Allogeneic Hematopoietic Cell Transplantation

TP53 mutations are associated with extremely poor outcomes in acute myeloid leukemia (AML). The outcomes of patients with TP53-mutated (TP53MUT) AML after different frontline treatment modalities are not well established. Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative procedure for AML; however, long-term outcomes among patients with TP53MUT AML after allo-HCT are dismal, and the benefit of allo-HCT remains controversial. We sought to evaluate the outcomes of patients with TP53MUT AML after treatment with different frontline induction therapies and allo-HCT. A total of 113 patients with TP53MUT AML were retrospectively evaluated. Patients with TP53MUT AML who received intensive or azacitidine-venetoclax induction had higher complete remission rates compared to patients treated with other hypomethylating-agent-based induction regimens. However, OS and EFS were not significantly different among the induction regimen groups. Allo-HCT was associated with improved OS and EFS among patients with TP53MUT AML; however, allo-HCT was not significantly associated with improved OS or EFS in time-dependent or landmark analysis. While the outcomes of all patients were generally poor irrespective of therapeutic strategy, transplanted patients with lower TP53MUT variant allele frequency (VAF) at the time of diagnosis had superior outcomes compared to transplanted patients with higher TP53 VAF. Our study provides further evidence that the current standards of care for AML confer limited therapeutic benefit to patients with TP53 mutations.

Clinical and molecular characteristics of acute myeloid leukemia and the dismal prognosis of TP53 mutations in a real-world setting

ABSTRACTObjectives:This study aimed to evaluate the incidence and prognostic significance of common cytogenetic and molecular abnormalities in patients with TP53-mutated and non-TP53-mutated acute myeloid leukemia (AML).Methods:We retrospectively analyzed the clinical data of 326 patients with newly diagnosed AML hospitalized in our institution between October 2015 and June 2021. Classification variables were reported as percentages and compared by χ2 tests. Survival rates were evaluated by the Kaplan-Meier method.Results:The incidence of TP53 mutations in AML patients in this clinic was 9.8%, of whom 87.5% patients were over 50 years old. The common concurrent mutations of TP53 were DNMT3A, IDH2, NRAS and TET2. Patients with a TP53 variant allele frequency (VAF) ≤ 40% had better overall survival (OS) than patients with a VAF >40%. Compared with non-TP53-mutated patients, significantly more TP53-mutated patients were gene-fusion negative, +mar, – 7/del (7q), – 5/del (5q), – 17/17p-, – 12/12p-, incomplete (inc) karyotype, or complex karyotype (CK), and had FLT3-ITD or IDH2 mutations, as well as a lower complete remission (CR) rate (31.3%) and higher recurrence rate (80.0%). The 2-year OS rates of TP53-mutated and non-TP53-mutated patients were 18.8% and 47.3%, respectively (P < 0.001). Univariate analysis showed that non-TP53-mutated patients with MLL family gene fusion, +mar or – 17/17p – karyotype, and FLT3-ITD mutations had a poor prognosis, while t(8; 21) karyotype was associated with a better prognosis. TP53-mutated patients with – 7/del (7q) or – 5/del (5q) karyotype had a poor prognosis.Conclusions:The cytogenetic and molecular landscapes differed between TP53-mutated and non-TP53-mutated patients, and some abnormalities had different values between them.

FLAG-IDA or venetoclax in previously treated TP53mut acute myeloid leukemia.

e19047 Background: Patients with del17p or TP53mut acute myeloid leukemia (AML) have short durations of remission and dismal overall survival. The benefit of fludarabine-based intensive chemotherapy (IC), such as FLAG-IDA or FLAG, remains unclear. Additionally, while venetoclax has revolutionized the treatment of AML, the comparative efficacy of venetoclax with a hypomethylating agent (HMA; decitabine or azacitidine) remains unknown in patients with del17p or TP53mut AML. We aimed to compare FLAG-based regimens to venetoclax + HMA in this subgroup of disease while controlling for IC candidacy. Methods: We retrospectively analyzed 79 patients with AML and TP53mut or del17p. Of these, 25 met study inclusion criteria: age 70 or younger, treated with front-line IC, and treated with either fludarabine-based therapy (with or without idarubicin, N = 15) or venetoclax + HMA (N = 10) in the subsequent line. To further reduce confounding effects, no patients in the FLAG group received venetoclax + HMA, and no patients in the venetoclax group received FLAG. We recorded baseline patient and disease characteristics, cytogenetic risk, response, and survival. We used Fisher’s exact test for nominal data and Mann-Whitney for ordinal data. We analyzed survival by the Kaplan-Meier method and compared groups with the Mantel-Cox test. Results: We analyzed 25 patients with TP53mut or del17p AML initially treated with IC and subsequently underwent salvage therapy with FLAG or venetoclax + HMA. The salvage regimen in each cohort was initiated after a median of one line of therapy, with no difference between groups ( p = 0.089). Patients in the FLAG cohort were significantly younger than the venetoclax cohort (54 y. vs 62 y., p = 0.046), but there were no significant differences in the Charlson Comorbidity Index scores (4 vs 4, p = 0.127) or ECOG scores (1 vs 2, p = 0.053) at salvage. There were also no differences in the median TP53 variant allele frequency (VAF) between the cohorts ( p = 0.095). The composite complete remission (CRR: CR + CRi) rate was 28.6% in the venetoclax cohort, compared to 46.7% in the FLAG cohort ( p = 0.648). Patients that received FLAG had significantly prolonged median overall survival compared to venetoclax + HMA (13.1 m. vs 4.5 m., p = 0.004). Conclusions: In patients with del17p or TP53mut AML, FLAG-based salvage appears to offer a significant improvement in overall survival following IC failure. Prospective trial designs are needed to confirm these findings. [Table: see text]

The clinical picture of ERCC6L2 disease: from bone marrow failure to acute leukemia

AbstractBiallelic germ line excision repair cross-complementing 6 like 2 (ERCC6L2) variants strongly predispose to bone marrow failure (BMF) and myeloid malignancies, characterized by somatic TP53-mutated clones and erythroid predominance. We present a series of 52 subjects (35 families) with ERCC6L2 biallelic germ line variants collected retrospectively from 11 centers globally, with a follow-up of 1165 person-years. At initial investigations, 32 individuals were diagnosed with BMF and 15 with a hematological malignancy (HM). The subjects presented with 19 different variants of ERCC6L2, and we identified a founder mutation, c.1424delT, in Finnish patients. The median age of the subjects at baseline was 18 years (range, 2-65 years). Changes in the complete blood count were mild despite severe bone marrow (BM) hypoplasia and somatic TP53 mutations, with no significant difference between subjects with or without HMs. Signs of progressive disease included increasing TP53 variant allele frequency, dysplasia in megakaryocytes and/or erythroid lineage, and erythroid predominance in the BM morphology. The median age at the onset of HM was 37.0 years (95% CI, 31.5-42.5; range, 12-65 years). The overall survival (OS) at 3 years was 95% (95% CI, 85-100) and 19% (95% CI, 0-39) for patients with BMF and HM, respectively. Patients with myelodysplastic syndrome or acute myeloid leukemia with mutated TP53 undergoing hematopoietic stem cell transplantation had a poor outcome with a 3-year OS of 28% (95% CI, 0-61). Our results demonstrated the importance of early recognition and active surveillance in patients with biallelic germ line ERCC6L2 variants.

Molecular characterization and prognosis of mutant TP53 acute myeloid leukemia and myelodysplastic syndrome with excess blasts.

Myeloid tumors typically harbor TP53 mutations, which are linked to a dismal prognosis. There are fewer studies on whether TP53-mutated acute myeloid leukemia (AML) and myelodysplastic syndrome with excess blasts (MDS-EB) differ in molecular characteristics and should be considered as separate entities. Between January 2016 and December 2021, a retrospective analysis was done on a total of 73 newly diagnosed AML patients and 61 MDS-EB patients from the first affiliated hospital of Soochow University. We described a survival profile and a thorough characterization of newly found TP53-mutant AML and MDS-EB and investigated the relationship between these characteristics and overall survival (OS). 38 (31.1%) were mono-allelic, and 84 (68.9%) were bi-allelic. There is no significant difference between TP53-mutated AML and MDS-EB (median OS 12.9 verse 14.4months; p=.558). Better overall survival was linked to mono-allelic TP53 than bi-allelic TP53(HR=3.030, CI:1.714-5.354, p < .001). However, the number of TP53 mutations and comutations were not significantly associated with OS. TP53 variant allele frequency cutoff of 50% is significant correlation with OS (HR: 2.177, 95% CI: 1.142-4.148; p=.0063). Our data revealed that allele status and allogeneic hematopoietic stem cell transplant independently affect the prognostic of AML and MDS-EB patients, with a concordance of molecular features and survival between these two disease entities. Our analysis favors considering TP53-mutated AML/MDS-EB as a distinct disorder.

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.

Prognostic Discrimination within a Heterogeneous Population of TP53-Aberrant Myelodysplastic Syndromes and Acute Myeloid Leukemia

Background:TP53-aberrant myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) constitute the highest risk molecular subgroup of myeloid neoplasms, and there are currently no FDA-approved targeted therapies for this subset of patients. We have recently explored the reasons for these distinctly adverse outcomes using clonal dynamic modeling. However, prognostic discrimination within this heterogeneous group of patients has not been validated in large-scale efforts. More recently, efforts to gain clarity on this disease subset include the IPSS-M model for MDS, the European LeukemiaNet (ELN) 2022 classification for AML, and the 2022 International Consensus Classification (ICC) for both MDS and AML. For example, the IPSS-M model has shown an adjusted hazard ratio of 3.27 (95% CI 2.38 - 4.48) for multi-hit TP53 aberration; the ELN 2022 classification now includes a new category termed "AML with mutated TP53" for patients with TP53 variant allele frequency (VAF) ≥ 10%; and the 2022 ICC has designated categories for MDS and AML with TP53 mutation. However, these systems do not discern among the subtypes of TP53 genomic aberrations with regard to clinical outcomes. Methods: Identification of patients from the UMass Chan Leukemia Registry was performed via UMass Clarity, a Microsoft SQL Server database comprised of 23,000 tables, in conjunction with the main reporting environment in EPIC. The Registry identified patients harboring one or more aberrations in TP53 and with ICD-10 code of either D46.9 (MDS, including its subentities) or C92.00 (AML, including its subentities) between 2011-2022. A total of 63 patients were identified. Patients with TP53-aberrant MDS or AML were stratified by the presence or absence of multi-hit status and/or TP53 VAF >50%. Multi-hit status was defined as per the 2022 ICC: two or more distinct TP53 mutations with VAF ≥ 10%, or a single TP53 mutation plus one of the following abnormalities: (1) del(17p13.1), (2) TP53 VAF >50%, (3) copy-neutral loss-of-heterozygosity, or (4) any complex karyotype. Monoallelic status was defined as non-multi-hit. Kaplan-Meier survival with log rank p value analysis was performed on subgroups based on the TP53 genomic state. Results: Median age of the UMass cohort (n = 63) was 69.8 ± 11.3 years. There were 35 patients (55.6%) with AML. We identified 58 patients who met the 2022 ICC definition for myeloid neoplasm with mutated TP53 (after exclusion of 5 patients with VAF < 10%) then stratified patients based on hit status and VAF. Median overall survival (OS) was similar among patients with monoallelic TP53-mutant MDS, multi-hit TP53-mutant MDS, and monoallelic TP53-mutant AML (median OS 440 days vs. 437 days vs. 440 days, respectively) (Panel A). In contrast, patients with multi-hit TP53-mutant AML had distinctly shorter survival (median 46 days) (Panel A). For patients with multi-hit status MDS or AML per the 2022 ICC (n = 41), we sub-stratified patients based on TP53 VAF. OS for patients with MDS with VAF ≤ 50% and VAF > 50% was similar (median 376 days vs. 435 days, respectively) (Panel B). Patients with AML with TP53 VAF > 50% had significantly inferior OS (median 43 days) (Panel B). Twelve (19.0%) of 63 patients underwent allogeneic hematopoietic cell transplant (allo-HCT) (2 had VAF >50%). Allo-HCT significantly improved median OS (732 days with allo-HCT vs. 161 days for n = 51 without allo-HCT; p = 0.0033) (data not shown). Only 3 (25%) of the 12 allo-HCT recipients achieved MRD-free remission lasting beyond 1000 days; VAF was ≤ 10% in all of these long-term survivors with TP53-mutant MDS or AML. Conclusion:TP53-aberrant MDS and AML comprise a prognostically heterogeneous group of patients with inferior clinical outcomes, though the highest risk subgroup appears to be AML with multi-hit TP53 status or AML with TP53 VAF > 50%. There is high value in determining the allelic state and hit status at the time of diagnosis to help prognosticate and develop a long-term management plan. Allo-HCT improves median OS, though durable MRD-negative remission is rare. Our data suggest that there may be a limited window of VAF ≤ 10% for the success of allo-HCT for TP53-aberrant myeloid neoplasm. We acknowledge that there is a selection bias for proceeding with allo-HCT. Large-scale systematic studies are needed to comprehensively profile patients classified as myeloid neoplasm with mutated TP53 such that risk-adapted management strategies can implemented. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcomes of Patients with TP53-Mutated Myeloid Neoplasms (TP53-MN) and the Role of Allogenic Blood or Marrow Transplantation (alloBMT)

Background: TP53 mutations are present in up to 20% of patients with myeloid malignancies and are frequently associated with a complex karyotype (CK) and poor responses to therapy. Even though overall response rates have improved significantly with less intensive induction modalities such as hypomethylating (HMA) agents alone or in combination with venetoclax, most patients succumb to their disease. AlloBMT remains the only curative option and even though the outcomes after alloBMT are disappointing, some patients achieve long-term survival. In the current study, we aimed to determine the clinical outcome of patients with TP53-MN and the impact of therapies including alloHCT on outcomes. Methods: We performed a single-center retrospective review of adult patients with TP53-MN evaluated at Johns Hopkins between November 2015 and February 2020. The analysis of somatic mutations was performed using a 59-gene targeted next-generation sequencing (NGS) assay. Cytogenetic analysis was performed on bone marrow or peripheral blood using standard methods including G-banding by trypsin with Giemsa staining. Results: We identified a total of 93 patients with TP53-MN. Disease subtypes included de novo AML (n = 22), AML arising from MDS/MPN (n = 15), MDS (n = 23), therapy-related myeloid neoplasm (n = 29), MDS/MPN overlap syndrome (n = 2) and MPN (n = 2). The median (Q1, Q3) age was 67 years (59 - 73 years). Cytogenetics was available for 89/93 (95.7%) patients. Of those, 73 (82.0%) patients had complex karyotype (CK). The median maximum TP53 variant allele frequency (VAF) per patient was 42.5%. Sixty-two (66.6%) patients had TP53 null phenotype: 14 (15.1%) patients had multiple TP53 mutations, 37 (41.6%) had deletion 17p and 11 (12.0%) had copy-neutral loss-of-heterozygosity (CnLOH). Overall, 79/93 (85%) patients received therapy: 20 (25.3%) were treated with HMA alone, 30 (38.0%) with HMA+venetoclax, 10 (12.7%) with HMA+others, 15 (19.0%) with intensive chemotherapy, 2 (2.5%) with an immunomodulatory drug, 1 (1.3%) with ivosidenib and 1 (1.3%) with a JAK2 inhibitor. Of those, 29 (36.7%) patients received non-myeloablative alloBMT with post-transplant cyclophosphamide (10 (34.5%) bone marrow and 19 (65.5%) mobilized peripheral blood grafts (PBSCT)). Median overall survival (OS) in patients receiving alloBMT, any therapy, or supportive care was 18.9, 4.1, and 2.5 months respectively (p < 0.001, Figure 1). Among patients in the alloBMT group, there was a significant OS difference between TP53-MN CK vs. non-CK (median OS 13.7 months vs not reached, p = 0.01, Figure 2). Also, patients with TP53-MN CK demonstrated better OS when treated with alloBMT compared to any therapy or supportive care. However, most TP53-MN CK patients ultimately succumbed to their disease regardless of the therapeutic modality. Therapeutic modalities prior to alloBMT did not appear to affect post-alloBMT outcomes but among patients who received PBSCT, OS was significantly better in patients receiving 400cGy vs 200 cGy total body irradiation (median OS 8.9 vs. 21 months, p=0.01). Conclusions: TP53-MN appears to be a heterogeneous disease with a variable prognosis. While OS of patients with TP53 mutations and non-CK who underwent alloBMT resembled that of intermediate-risk AML, patients with TP53 null phenotype associated with CK had a worse outcome. Even though OS appears to be significantly improved with alloBMT, particularly patients receiving PBSCT+400cGy TBI, most patients eventually relapse. Novel therapeutic strategies are urgently needed to improve the outcomes of TP53 CK patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic Impact of Clonal Hierarchy of Myelodysplasia-Related Gene Mutations in AML Patients

Introduction: The 2022 ICC classification identifies mutations in nine genes that classify AML with myelodysplasia-related gene mutations (AML-MRG). AML-MRG is associated with an unfavourable prognosis according to the 2022 ELN classification. However, it is unknown whether the clonal hierarchy of those mutations impacts prognosis. Methods: 485 adult newly-diagnosed AML patients (median age 54) with available genetic and follow-up data were included. Mutations present at diagnosis were identified by Illumina myeloid panel sequencing covering 56 AML-associated genes. Patients received standard induction and consolidation chemotherapy or underwent allogeneic hematopoietic cell transplantation (alloHCT). MRG mutations were classified as subclonal if their variant allele frequency (VAF) was ≥ 10% below the mutation with the highest VAF of other co-occuring mutations. Otherwise, MRG mutations were considered part of the dominant clone. Results: 177 (36.5%) patients carried at least one of the nine MRG mutations ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2. The MRG mutation was found in the dominant clone in 134 (75.7%) and in a subclone in 43 patients (24.3%). BCOR, RUNX1, SRSF2, STAG2 and U2AF1 were more likely found in the dominant clone, whereas ASXL1, EZH2, SF3B1 and ZRSR2 were more often found in a subclone. Patients with MRG mutations in the dominant as well as a subclone were significantly older than patients without MRG mutations (p < 0.001 and p = 0.03, respectively). Patients with MRG mutations in the dominant or a subclone and patients without MRG mutations achieved CR/CRi in 80.6%, 83.7% and 87.3%, respectively (p = n.s.). In line with the ELN classification, patients with MRG mutations in the dominant clone had a worse OS than patients without MRG mutations (p = 0.004, HR = 1.505, 95%CI 1.136-1.995); however, patients with MRG mutations in a subclone had a similar OS compared to patients with MRG mutations in the dominant clone and to patients without MRG mutations (Figure 1A). TP53 mutations with ≥10% VAF are defined as AML with mutated TP53 by ICC 2022. 39 (8%) patients carried a TP53 mutation. One third of the cases had a subclonal TP53 mutation. Median TP53 VAF was 39.6% and 15.5% in patients with dominant or subclonal TP53 mutation (p < 0.001). OS was significantly worse in patients with dominant TP53 mutation compared to TP53 wildtype patients (p = 0.002, HR = 2.047, 95%CI 1.276-3.284), whereas OS was similar in patients with subclonal TP53 mutation compared to TP53 wildtype patients (Figure 1B). Conclusion: In our study, 76% of MRG mutations occur in the dominant clone in newly diagnosed AML patients. Dominant MRG mutations have a worse prognosis compared to patients without MRG mutations, while prognosis of subclonal MRG mutations should be evaluated in larger cohorts. AML patients with subclonal TP53 mutation have a similar outcome to patients without TP53 mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Venetoclax and idasanutlin in relapsed/refractory AML: a nonrandomized, open-label phase 1b trial

AbstractThis phase 1b trial (NCT02670044) evaluated venetoclax-idasanutlin in patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) ineligible for cytotoxic chemotherapy. Two-dimensional dose escalation (DE, n = 50) was performed for venetoclax daily with idasanutlin on days 1 to 5 in 28-day cycles, followed by dosing schedule optimization (n = 6) to evaluate reduced venetoclax schedules (21-/14-day dosing). Common adverse events (occurring in ≥40% of patients) included diarrhea (87.3% of patients), nausea (74.5%), vomiting (52.7%), hypokalemia (50.9%), and febrile neutropenia (45.5%). During DE, across all doses, composite complete remission (CRc; CR + CR with incomplete blood count recovery + CR with incomplete platelet count recovery) rate was 26.0% and morphologic leukemia-free state (MLFS) rate was 12%. For anticipated recommended phase 2 doses (venetoclax 600 mg + idasanutlin 150 mg; venetoclax 600 mg + idasanutlin 200 mg), the combined CRc rate was 34.3% and the MLFS rate was 14.3%. Pretreatment IDH1/2 and RUNX1 mutations were associated with higher CRc rates (50.0% and 45.0%, respectively). CRc rate in patients with TP53 mutations was 20.0%, with responses noted among those with co-occurring IDH and RUNX1 mutations. In 12 out of 36 evaluable patients, 25 emergent TP53 mutations were observed; 22 were present at baseline with low TP53 variant allele frequency (median 0.0095% [range, 0.0006-0.4]). Venetoclax-idasanutlin showed manageable safety and encouraging efficacy in unfit patients with R/R AML. IDH1/2 and RUNX1 mutations were associated with venetoclax-idasanutlin sensitivity, even in some patients with co-occurring TP53 mutations; most emergent TP53 clones were preexisting. Our findings will aid ongoing/future trials of BCL-2/MDM2 inhibitor combinations. This trial was registered at www.clinicaltrials.gov as #NCT02670044.