High Variant Allele Frequency Research Articles

Circulating tumor DNA assisting lymphoma genetic feature profiling and identification.

Lymphoma tissue biopsies cannot fully capture genetic features due to accessibility and heterogeneity. We aimed to assess the applicability of circulating tumor DNA (ctDNA) for genomic profiling and disease surveillance in classic Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), and diffuse large B-cell lymphoma (DLBCL). Tumor tissue and/or liquid biopsies of 49 cHLs, 32 PMBCLs, and 74 DLBCLs were subject to next-generation sequencing targeting 475 genes. The concordance of genetic aberrations in ctDNA and paired tissues was investigated, followed by elevating ctDNA-based mutational landscapes and the correlation between ctDNA dynamics and radiological response/progression. ctDNA exhibited high concordance with tissue samples in cHL (78%), PMBCL (84%), and DLBCL (78%). In cHL, more unique mutations were detected in ctDNA than in tissue biopsies (P < 0.01), with higher variant allele frequencies (P < 0.01). Distinct genomic features in cHL, PMBCL, and DLBCL, including STAT6, SOCS1, BTG2, and PIM1 alterations, could be captured by ctDNA alone. Prevalent PD-L1/PD-L2 amplifications were associated with more concomitant alterations in PMBCL (P < 0.01). Moreover, ctDNA fluctuation could reflect treatment responses and indicate relapse before imaging diagnosis. Lymphoma genomic profiling by ctDNA was concordant with that by tumor tissues. ctDNA might also be applied in lymphoma surveillance.

Impact of EML4-ALK variants and co-occurring TP53 mutations on duration of first-line ALK tyrosine kinase inhibitor treatment and overall survival in ALK fusion–positive NSCLC: Real-world outcomes from the GuardantINFORM database

Tyrosine kinase inhibitors (TKIs) are first-line treatment options for ALK-positive (ALK+) non-small cell lung cancer (NSCLC). Factors such as variant allele frequencies (VAF), EML4-ALK fusion variant, and concurrent TP53 mutations (TP53mt) in circulating tumor DNA (ctDNA) may affect treatment outcomes. We evaluated their effects on time to discontinuation (TTD) of first-line treatment with next-generation ALK TKIs in a real-world setting. Adults with advanced/metastatic NSCLC and ctDNA-detected ALK fusion who received first-line next-generation ALK TKI monotherapy were identified in GuardantINFORM. Effects of ALK fusion VAF, EML4-ALK variants, and TP53mt detection on TTD were evaluated. 307 patients with ALK fusion in baseline ctDNA received first-line alectinib (n=280), brigatinib (n=15), lorlatinib (n=9), or ceritinib (n=3); 150 patients (49%) had ALK-fusion VAF ≥1%. Among 232 patients with EML4-ALK fusions (v1, 50%; v3, 36%), TP53mt co-occurred with v1 in 42 (18%) and v3 in 32 (14%). Patients with VAF <1% versus ≥1% had median TTD of 32.2 (95% CI: 20.7-NE) versus 14.7 months (10.4-19.9; HR: 1.57 [95% CI: 1.09-2.26]; P=0.0146). Median TTD was 13.1 (9.5-19.9) versus 27.6 months (17.3-not estimable [NE]) in patients with versus without TP53mt detected (HR: 1.53 [1.07-2.19]; P=0.0202) and 20.3 (14.4-NE) versus 11.5 months (7.4-31.1) in patients with v1 versus v3 (HR: 1.29 [0.83-2.01]; P=0.2641). Patients with TP53mt and v3 had median TTD of 7.4 months (95% CI: 4.2-31.1). High ctDNA VAF, EML4-ALK v3, and TP53mt were associated with early discontinuation of first-line ALK TKIs.

Bronchial Basaloid Papillary Tumor of Uncertain Malignant Potential: A Case Report.

Bronchial papillomas are benign tumors, which can be divided into different subtypes based on the cellular features. So far, no bronchial papilloma with basaloid cell features has been reported. We report a bronchial basaloid papillary tumor in a 67-year-old woman. Tumor recurrence and malignant transformation were observed after a long-term follow-up. The clinical, histological, immunohistochemical, and genetic features were reappraised. The primary tumor was characterized by papillary growth pattern and basaloid neoplastic cells, only a small amount of neoplastic cells showed mature characteristics. The tumor originated from respiratory epithelium and had a low proliferation index by Ki-67. Keratin (KRT) 5/6 and KRT7 showed patchy or partial positivity. Myoepithelial markers were negative. P63 was diffusely positive, but it was negative in the small amount of tumor cells with mature characteristics. The common genetic alterations (EGFR, KRAS, BRAF V600E, HER2, and ALK) of lung cancers were not detected. However, tumor recurrence was observed in the mediastinum and esophagus 12 years after surgery. The recurrent tumor had a morphology overlapping with that of the primary tumor; however, it displayed significantly malignant characteristics. The recurrent tumor was not related to high-risk HPV. A high variant allele frequency was observed in tumor suppressor gene BRCA1, TP53, oncogene GNA11, and KIT, which were all missense mutations. Considering the bland features of the primary tumor and the fact of tumor recurrence and undisputed malignant transformation, the basaloid papillary tumor was considered a tumor with uncertain malignant potential.

Observed germline BRCA1/2 and TP53 tumor genomic profiling allele frequencies.

e22533 Background: Tumor genomic profiling (TGP) is increasingly used to assist with cancer treatment planning. While these tests are not intended to find germline variants, they are a possible incidental outcome. Currently there are no clear guidelines on when to suspect if a somatic variant identified on TGP may need confirmatory germline genetic testing. Variant allele frequency (VAF) is one tool commonly used to assess if a variant may be germline. While some studies have demonstrated germline VAF ranges on TGP, the data is limited. The purpose of this study was to review VAF ranges of BRCA1/2 and TP53 variants at a large reference laboratory to better understand VAF ranges of confirmed germline variants. Methods: A retrospective review of test results from 184 patients who underwent TGP and hereditary cancer germline testing at a large reference laboratory was performed. Patients were included in the study if they had both TGP and germline testing performed, regardless of the order of testing. Results were analyzed to determine variants identified and VAF. Analysis of BRCA1/2 and TP53 variants included patients who had variants identified on TGP and had germline testing that would have confirmed the variants if present in the germline. The VAFs were obtained, and simple statistics were used to describe the findings. Results: Twenty-one patients were found to have 23 BRCA1/2 variants identified on TGP (2 patients had 2 BRCA1/2 TGP variants). Of these TGP variants, 16 were confirmed on germline testing (69.6%). The TGP VAF for these patients ranged from 38.2% to 85.1%. The mean was 61.0% and the median was 61.8%. The remaining 7 TGP variants that were not confirmed on germline testing ranged from 7.2% to 63.4%. The mean was 31.1% and the median was 28%. Thirty-eight patients were found to have 41 TP53 variants identified on TGP (3 patients had 2 TP53 TGP variants). Of these patients, 1 had a TP53 variant confirmed on germline testing with a VAF of 44.7%. The 40 remaining variants in this dataset were not confirmed on germline testing. The VAF for these 40 variants ranged from 1.3% to 95.3%. The mean was 38.4% and the median was 34.5%. Conclusions: In this study, 69.6% of BRCA1/2 TGP variants were confirmed on germline testing compared to 2.4% of TP53 TGP variants. Confirmed germline variants identified on TGP had VAFs that were &gt; 38%. It’s important to note that TP53 variants may have very high VAFs (up to 95.3% in this study) and not be.

Abstract PO1-14-01: Clonal Architecture of circulating tumor DNA predicts Early Progression on Tamoxifen With or Without Palbociclib in Hormone Receptor-Positive Advanced Breast Cancer: NCCH1607/PATHWAY trial

Abstract Objective: Some patients with poor prognosis hormone receptor-positive, human epidermal growth factor receptor 2-negative (HR+/HER2-) advanced breast cancer (ABC) experience early disease progression on the combination of CDK4/6 inhibitors and endocrine therapy. We aimed to determine whether clonal architecture assessed by circulating tumor DNA (ctDNA) could identify patients at higher risk of early progression on tamoxifen therapy with or without palbociclib. Methods: PATHWAY trial is a randomized, placebo-controlled, Asian international, double-blind, phase 3 study evaluating palbociclib plus tamoxifen (PAL+TAM) versus placebo plus tamoxifen (PLB+TAM) in pre/perimenopausal and postmenopausal women with HR+/HER2- ABC (NCT03423199). The study was conducted as a Clinical Research Collaboration with the National Cancer Center Hospital as the regulatory study sponsor and Pfizer providing drug and financial support. The primary endpoint was investigator assessed progression-free survival (PFS). Genomic profiling by next-generation sequencing was an exploratory endpoint to characterize molecular alterations in ctDNA and correlate these with clinical outcomes. Plasma samples were collected at cycle 1-day 1 (pre-treatment) and cycle 2-day 15 and sequenced with the Guardant360 assay (Guardant Health). Patients were included if they had a minimum of 14 days of treatment in the first cycle. The variant allele frequency (VAF) was defined as number of mutant molecules at a specific nucleotide location over the total number of molecules present in the background at a specific given genomic location. We assessed the gene with highest VAF as the dominant clone allele frequency (DCAF). The association of changes in ctDNA alterations with PFS was evaluated to identify biomarkers of early progression. Results: Pre-treatment plasma was successfully sequenced in 177 of the 184 patients; 88 patients treated with PAL+TAM, and 89 with PLB+TAM. ctDNA was detected in 144 (81.4%) patients; the median number of genetic abnormalities was 2 (range, 0-19). PIK3CA, TP53, ERBB2, ESR1, AKT1, EGFR and PTEN were the genes with highest VAF as the dominant clone (24.3%, 11.9%, 4.5%, 4.5%, 3.4%, 3.4%, 3.4%, respectively). The median DCAF was 1.79 (range, 0.1-81.23); PAL+TAM, 2.53 (range, 0.1-63.5) and PLB+TAM, 1.65 (range, 0.08-81.23). Patients with ≥ 2 detectable genomic alterations and DCAF ≥ 1.8% had worse PFS (hazard ratio (HR) 1.93 [95% confidence interval (CI): 1.375 to 2.721], p = 0.0003 and HR 1.95 [95% CI: 1.345 to 2.834], p &amp;lt; 0.0001, respectively) compared to all other patients. In addition, we evaluated whether changes of clonal architecture in ctDNA at early treatment time point (ie. Cycle 2 Day 15) are associated with PFS. Patients with increased DCAF had worse PFS on both PAL+TAM (HR 2.80 [95% CI: 0.552 to 14.15], p = 0.04) and PLB+TAM (HR 2.02 [95% CI: 1.022 to 3.990], p = 0.01). All patients with high pretreatment DCAF (≥ 1.8%) and increase in DCAF progressed within 1 year (n=5, 2 patients treated with PAL+TAM, and 3 with PLB+TAM). Conclusion: Evaluation of clonal architecture in ctDNA could support identification of HR+/HER2- ABC patients with poorer prognosis, who may be at higher risk of early progression, regardless of CDK4/6 inhibitor use. Citation Format: Yoon-Sim Yap, Yuki Kojima, Akinobu Hamada, Hirofumi Mukai, Yen-Shen Lu, Joo Hyuk Sohn, Yoshiko Umeyama, Emi Noguchi, Kazuki Sudo, Tomomi Hata, Aya Kuchiba, Taro Shibata, Shigehiro Yagishita, Masayuki Yoshida, Shinji Kohsaka, Kouya Shiraishi, Kenichi Nakamura, Kenji Tamura, Kan Yonemori. Clonal Architecture of circulating tumor DNA predicts Early Progression on Tamoxifen With or Without Palbociclib in Hormone Receptor-Positive Advanced Breast Cancer: NCCH1607/PATHWAY trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-14-01.

Abstract PO2-14-06: CIRCULATING TUMOR DNA DYNAMICS IN THE FIRST TREATMENT LINE OF THE SONIA TRIAL

Abstract Background and objective: CDK4/6 inhibitors (CDK4/6i) improve the outcome of patients with ER+/HER2- advanced breast cancer when added to endocrine therapy in the first line or second line of treatment. The recently reported SONIA trial shows no difference in progression free survival after two lines of treatment (PFS2), overall survival (OS) or quality of life benefit of CDK4/6i in first-line compared to second-line, while adverse events are substantially higher when CDK4/6i is added in the first-line. However, a subgroup of patients shows early progression on endocrine mono-treatment and not all patients respond equally well to CDK4/6i. This highlights the need for early response markers during the first weeks of treatment to reduce unnecessary toxicity while providing the most optimal care. In this SONIA side study, we aim to investigate whether we could identify patients who will progress rapidly during first line of therapy by using circulating tumor DNA (ctDNA) dynamics. Methods: Blood samples were obtained from patients included in the SONIA trial in both the first line CDK4/6i + aromatase inhibitor (AI) combination arm (arm A) and the first line AI monotherapy arm (arm B) at three time points: before start, after two weeks and after three months of first line treatment. Cell free DNA (cfDNA) was isolated from all samples and subsequently baseline cfDNA was sequenced with the Oncomine™ Breast cfDNA Assay v2 (Thermofisher), a focused NGS panel of 10 genes. Dedicated dPCR assays were used to track an identified driver mutation over time. Results: Blood samples of 332 patients (206 in arm A and 126 in arm B) before start of first line treatment and after two weeks of treatment were obtained. At baseline, in 142 of the 332 (42.7%) patients at least one somatic mutation was detected. In 30 patients two or more mutations were identified, in which case the mutation with the highest variant allele frequency (VAF) was followed over time. The most frequently mutated genes were PIK3CA (102 patients (30.7% of the total included cohort)), TP53 (32 patients (9.6%)), SF3B1 (8 patients (2.4%) and ESR1 (6 patients (1.8%)). The median observed VAF at baseline was 4.1% (IQR: 1.5%-13.1%). Of the 142 patients with detectable ctDNA at baseline, 141 patients and 100 patients, respectively, had blood samples taken after 2 weeks and after 3 months of treatment. After two weeks, the VAF of the tracked mutation was increased in 15.2% (14/92) of the patients in arm A and in 18.4% (9/49) of the patients in arm B. At the same time 40.2% (37/92) of the patients in arm A and 34.7% (17/49) of the patients in arm B had cleared ctDNA. No difference was observed between the arms for both an increase in VAF (P=0.63, Chi-square test) and ctDNA clearance (P=0.52, Chi-square test). After 3 months an increased VAF of the tracked mutation compared to baseline was observed in 6.3% (4/64) of the patients in arm A and 8.3% (3/36) of the patients in arm B. At this timepoint 59.4% (38/64) of the patients in arm A and 61.1% (22/36) of the patients in arm B had cleared ctDNA. No difference was observed between the arms for both ctDNA clearance (P=0.86, Chi-square test) and increase in VAF (P=0.70, Chi-square test) after three months. Conclusions: ctDNA was detected at baseline in almost half of the patients in the SONIA study. Compared to baseline, a decreasing number of patients had an elevation of the VAF of the tracked mutation and an increasing number of patients showed ctDNA clearance from 2 weeks to 3 months after start of treatment. There was no difference between the two arms observed regarding ctDNA dynamics. Interestingly, irrespective of treatment arm, about 1/3rd of patients remained ctDNA-positive even after 3 months of treatment. Associations of these ctDNA data with the clinical outcome data are currently being evaluated and will be presented at the meeting. Citation Format: Elisabeth M Jongbloed, Stefania Stella, Lotte van Bergen, Corine Beaufort, Jean Helmijr, Vanja de Weerd, Frans Erdkamp, Joan Heijns, Yvonne Kamm, Anne-Marie van Riel, Quirine van Rossum-Schornagel, Maurice Jansen, Inge RHM Konings, Gabe Sonke, John WM Martens, Agnes Jager, Saskia M Wilting. CIRCULATING TUMOR DNA DYNAMICS IN THE FIRST TREATMENT LINE OF THE SONIA TRIAL [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-06.

Abstract PO5-06-14: Genomic Landscape and Clinical Outcomes of Triple-Negative Invasive Lobular Carcinoma

Abstract Background: Triple-negative (TN) invasive lobular carcinoma (ILC) of the breast is a rare entity, comprising roughly 2% of primary ILC. Clinicopathologic studies have shown that TN ILC is associated with more pleomorphism, higher nuclear grade, older patient age, and worse disease-related outcomes compared to estrogen receptor-positive (ER+) ILC. Intrinsic subtyping indicates that TN ILC less frequently expresses basal markers than TN invasive ductal carcinoma (IDC). This prompted investigation of the genomic landscape and clinical management of TN ILC to provide insight into potential therapeutic approaches. Methods: Twenty patients with TN ILC were included in this study. Three patients were treated at a single academic center and underwent comprehensive genomic profiling (CGP) of breast or metastatic tissue; clinicopathologic details were obtained from their electronic health records. Seventeen patients with TN ILC who underwent CGP of breast or metastatic biopsies were included from the Foundation Medicine, Inc. database. PD-L1 testing for all patients was determined by immunohistochemistry (IHC) using the VENTANA SP142 or Dako 22C3 commercial assays. HER2 expression was determined by IHC. Negative HER2 expression was defined as an IHC score of 0, or IHC 1+ or 2+ with non-amplified fluorescence in situ hybridization (FISH). Results: In the Foundation Medicine database of patients with TN ILC, the most frequent genomic alterations were in CDH1 (15/17 patients [88.2%]), TP53 (10/17 patients [58.8%]), and PIK3CA (10/17 patients [58.8%]). Short variant mutations in ERBB2 were found in 3/17 patients (17.6%). Fourteen patients were HER2-low (HER2 IHC 1+ or 2+ with non-amplified FISH). No cases were MSI high or expressed PD-L1 (8 of 17 cases underwent PD-L1 assessment by SP142). Median tumor mutational burden (TMB) was 5 muts/Mb. Among the three patients with TN ILC treated at a single center, the most common somatic mutations were in CDH1 and TP53, and no cases were MSI high or expressed PD-L1 (by SP142 or 22C3). All three patients had pleomorphic ILC, histologic grade 2 or 3, and HER2 IHC 0. One patient with mpT1cN1a(sn) ER+/HER2- ILC of the right breast in 2011 received adjuvant TAC (docetaxel, doxorubicin, cyclophosphamide), breast/axillary radiation, and ten years of adjuvant endocrine therapy. She was diagnosed with TN ILC of the left supraclavicular nodes in 2022; she did not exhibit a response to docetaxel and carboplatin with pembrolizumab, and she subsequently received palliative radiation followed by capecitabine. Another patient with locally advanced TN ILC progressed on the KEYNOTE-522 regimen, immediately developed metastases, and is currently receiving palliative sacituzumab govitecan. The third patient with cT3N1 TN ILC progressed on neoadjuvant therapy (through the I-SPY 2 trial) with oral paclitaxel, encequidar, and dostarlimab, followed by dostarlimab with dose-dense doxorubicin and cyclophosphamide. Her surgical pathology demonstrated ypT3N3a disease with 10 × 7 cm of tumor, and she developed skin metastases on the chest wall within weeks post-operatively. She received comprehensive chest wall and nodal radiation and is currently receiving palliative capecitabine with neratinib given a high variant allele frequency driver somatic ERBB2 mutation. Conclusions: The most common somatic mutations among TN ILC patients included in this study were in CDH1, TP53, and PIK3CA. Currently the management of TN ILC is extrapolated from that of TN IDC, however our patients did not respond to chemoimmunotherapy. The presence of HER2-low status or somatic ERBB2 mutations may provide opportunities for treatment with an anti-HER2 antibody-drug conjugate or tyrosine kinase inhibitor. Studies with larger sample sizes of TN ILC are needed for molecular profiling and assessment of outcomes with specific therapeutic approaches. Citation Format: Hemali Batra-Sharma, Smruthy Sivakumar, Prashanth Ashok Kumar, Ethan Sokol, Rebecca Shatsky, Jeffrey Ross. Genomic Landscape and Clinical Outcomes of Triple-Negative Invasive Lobular Carcinoma [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-14.

Clonal Hematopoiesis and Bone Marrow Infiltration in Patients With Follicular Helper T-Cell Lymphoma of Angioimmunoblastic Type

Follicular helper T-cell (TFH) lymphoma harbors recurrent mutations of RHOAG17V, IDH2R172, TET2, and DNMT3A. TET2 and DNMT3A mutations are the most frequently affected genes in clonal hematopoiesis (CH). The aim of our study was to investigate the frequency of CH in bone marrow biopsies (BMB) of TFH/angioimmunoblastic T-cell lymphoma (TFH-AITL) patients and its association with myeloid neoplasms. A total of 29 BMB from 22 patients with a diagnosis of TFH–AITL were analyzed by next-generation sequencing (NGS) with a custom panel. Morphologically, 5 BMB revealed that TFH–AITL infiltrates of >5% of bone marrow (BM) cellularity confirmed in 4 cases by NGS-based T-cell clonality. IDH2R172 was demonstrated only in 1 (3%) of 29, and RHOAG17V in 2 (7%) of 29 samples. TET2 and DNMT3A were identified in 24 (83%) of 29 and 17 (59%) of 29 BMB, respectively. In the parallel lymph node the frequencies of mutations were 27% (IDH2R172), 64% (RHOAG17V), 86% (TET2), and 50% (DNMT3A). TET2 and/or DNMT3A mutations identical in lymph node and BMB were present in 18 (82%) of 22 patients, regardless of BM infiltration. In 3 cases the CH mutations were detected 13, 41, and 145 months before TFH–AITL diagnosis. Cases with TET2/DNMT3A mutations and BM variant allele frequencies >40% (7/18, 39%) showed lower blood counts. However, only low platelet count was statistically significant (P = .024). Myeloid neoplasms and/or myelodysplastic syndrome-related mutations were identified in 4 cases (4/22; 18%); all with high TET2 variant allele frequencies (>40%; P = .0114). In conclusion, CH is present in 82% of TFH–AITL and can be demonstrated up to 145 months before TFH–AITL diagnosis. NGS T-cell clonality analysis is an excellent tool to confirm TFH–AITL BM infiltration. Concurrent myeloid neoplasms were identified in 18% of the cases and were associated with TET2 mutations with high allelic burden (>40%). We demonstrated that myeloid neoplasms might occur simultaneously or precede the diagnosis of TFH lymphoma.

Towards accurate indel calling for oncopanel sequencing through an international pipeline competition at precisionFDA

Accurately calling indels with next-generation sequencing (NGS) data is critical for clinical application. The precisionFDA team collaborated with the U.S. Food and Drug Administration’s (FDA’s) National Center for Toxicological Research (NCTR) and successfully completed the NCTR Indel Calling from Oncopanel Sequencing Data Challenge, to evaluate the performance of indel calling pipelines. Top performers were selected based on precision, recall, and F1-score. The performance of many other pipelines was close to the top performers, which produced a top cluster of performers. The performance was significantly higher in high confidence regions and coding regions, and significantly lower in low complexity regions. Oncopanel capture and other issues may have occurred that affected the recall rate. Indels with higher variant allele frequency (VAF) may generally be called with higher confidence. Many of the indel calling pipelines had good performance. Some of them performed generally well across all three oncopanels, while others were better for a specific oncopanel. The performance of indel calling can further be improved by restricting the calls within high confidence intervals (HCIs) and coding regions, and by excluding low complexity regions (LCR) regions. Certain VAF cut-offs could be applied according to the applications.

Abstract CT163: FIT-001: A phase 1 clinical trial of the farnesyl transferase inhibitor KO-2806 alone or as part of combination therapy for advanced solid tumors

Abstract Background: Farnesyl transferase inhibitors (FTIs) block post-translational modification of RAS and other farnesylated proteins. HRAS-driven tumors are highly sensitive to FTI treatment. Recent clinical trials (NCT03719690, NCT02383927) of the FTI, tipifarnib, in patients with HRAS-mutant (HRAS-m) head and neck squamous cell carcinoma harboring high variant allele frequency mutations (VAF ≥20%), showed objective response rates of up to 50% and favorable long-term outcomes. KO-2806 is a next-generation FTI that has increased potency and improved pharmacokinetic properties. In preclinical studies, KO-2806 has been shown to: (1) enhance tumor growth inhibition of tyrosine kinase inhibitors, including cabozantinib, in multiple clear cell renal cell carcinoma (ccRCC) cell line- and patient-derived xenograft models; and (2) enhance activity of KRAS inhibitors, including adagrasib, in KRAS mutant non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and pancreatic ductal adenocarcinoma (PDAC) mouse models. These preclinical data support clinical investigation of KO-2806 alone and in combination therapy. Study Design: FIT-001 is a first-in-human, multicenter, open-label clinical trial that will evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics (Pd), and preliminary antitumor activity of KO-2806 as monotherapy or in combination therapy in advanced solid tumors (KO-2806-001; NCT06026410). Up to 270 patients will be enrolled in phase 1a and phase 1b combined across 50 sites. Phase 1a will have separate monotherapy and combination dose-escalation arms. As monotherapy, the study will enroll patients with N/K/HRAS alterations in specific solid tumor types, such as NSCLC, CRC, and PDAC, who are refractory to standard-of-care therapies. KO-2806 and cabozantinib will be combined in patients with advanced or metastatic ccRCC who progressed on ≥1 prior line of immunotherapy-based systemic therapy; KO-2806 and adagrasib will be combined in patients with KRAS-G12C mutant locally advanced or metastatic NSCLC who received ≥1 prior systemic therapy. On the basis of emerging data from phase 1a, two Pd cohorts (n≤12) with mandatory pre- and on-treatment tumor biopsies may be enrolled. In each Phase 1b dose expansion, patients will receive the recommended phase 2 dose [RP2D] of KO-2806 with cabozantinib (in ccRCC) or adagrasib (in NSCLC), or will be randomized by dose if 2 potential KO-2806 RP2Ds are identified for a combination. Other combination arms may also be considered. The study began accrual in October 2023. Citation Format: Glenn J. Hanna, Douglas R. Adkins, Jacob S. Thomas, Justine Y. Bruce, Manish R. Patel, Guru Sonpavde, Jason Henry, Nawal Bendris, Zijing Zhang, Amitava Mitra, Amaya Gascó, Andrew Saunders, Stephen Dale. FIT-001: A phase 1 clinical trial of the farnesyl transferase inhibitor KO-2806 alone or as part of combination therapy for advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT163.

Abstract 5029: VAF in ctDNA correlated with advanced breast cancer tumor burden and prognosis

Abstract Purpose: In patients with first-line advanced breast cancer (ABC), the correlation between ctDNA variant allele frequency (VAF) and tumor disease burden, and its prognostic value remains poorly investigated. Methods: This study included patients with ABC diagnosed at Peking University Cancer Hospital who performed ctDNA test before receiving first-line treatment. Baseline plasma samples were collected for assessing ctDNA alterations and VAF with next-generation sequencing. The sum of tumor target lesion diameters (SLD) was measured with imaging methods according to RECIST 1.1 criteria. Results: The final cohort included 184 patients. The median age of the cohort was 49.4 (IQR: 42.3-56.8) years. The median VAF was 15.6% (IQR: 5.4%-33.7%). VAF showed positive correlation with SLD in patients with relatively large tumor lesions (r = 0.314, p = 0.003), but not in patients with small tumor lesion (p = 0.226). VAF was associated with multiple metastasis sites (p = 0.001). Multivariate Cox regression analysis showed that high VAF was associated with shorter overall survival (OS) (HR: 3.519, 95% confidence interval (CI): 2.149-5.761), and first-line progression-free survival (PFS) (HR: 2.352, 95%CI: 1.462-3.782). Conclusion: ctDNA VAF associated with tumor disease burden, and was a prognostic factor for patients with ABC. A combination of ctDNA test and radiographic imaging might enhance tumor burden evaluation, and improve prognosis stratification in patients with ABC. Citation Format: Jianxin Zhong, Huiping Li, Bin Shao, Hanfang Jiang, Xiaoran Liu, Haoran Tang, Shidong Jia. VAF in ctDNA correlated with advanced breast cancer tumor burden and prognosis [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 5029.

Abstract 3934: Smoking's influence on divergent paths to lung cancer

Abstract Lung cancer is the leading cause of cancer mortality in the world. Many studies have highlighted how mutagens from cigarette smoke promote specific mutations such as G12 mutations in KRAS, contributing to clonal evolution that leads to lung cancer. Despite the indisputable correlation between smoking and lung cancer, people who have never smoked account for the 7th leading cause of cancer mortality. Given the differences in predominant exposures, we hypothesize that the clonal evolution between people with and without smoking histories are markedly different. Therefore, we analyzed lung samples from people with different cigarette smoking exposure to elucidate smoking’s effect on the somatic mutational landscape in the progression to lung cancer. To observe somatic mutations subjected to different exposures of smoking, we used a rare-mutation detection technique called DuplexSeq to analyze somatic variants from 200+ lung samples. We acquired DNA from cells from bronchoscopy brushings and lung punches of histologically normal tissue from people with and without lung cancer and who currently, formerly, or never smoked cigarettes. Through collaborations, we characterized mutations using bioinformatic pipelines, various databases, and statistical methods. In samples from people who smoke, we observe an enhancement of mutations with higher variant allele frequencies and pervasive positive selection acting on mutations in many cancer-associated genes. From our bronchoscopy brushings, NOTCH1 mutations show the most definitive increase of protein-altering variants, while NF1 and KRAS show a predominance of driver mutation in lung punches. Unexpectedly, people without lung cancer who never smoked had a notable presence of pathogenic mutations in PTEN and PIK3CA. Furthermore, multiple punches from the same individual highlight how expansions of mutations have stronger similarity within an individual than across individuals regardless of smoking status. This association points to the importance of the inherent lung microenvironment in influencing the mutational landscapes in lung cancer progression. Altogether, these results show how smoking increases the oncogenicity of observable mutations in the lung but does not preclude the presence of oncogenic clones, emphasizing the value of precision diagnoses and prevention. Ongoing research will be centered around functional assays to determine the impact of observed mutations on tissue function and longitudinal studies using mutational landscapes as a proxy for lung cancer risk. Citation Format: Edward James Evans, Fabio Marongiu, Emilia Lim, Oriol Pich, Yonghua Zhuang, Dexiang Gao, William Hill, Cian Murphy, Ryan Turner, Moumita Ghosh, York Miller, Mariam Jamal-Hanjani, Charles Swanton, James DeGregori. Smoking's influence on divergent paths to lung 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 3934.

Rim enhancement of pancreatic ductal adenocarcinoma: investigating the relationship with DCE-MRI-based radiomics and next-generation sequencing.

To identify the clinical and genetic variables associated with rim enhancement of pancreatic ductal adenocarcinoma (PDAC) and to develop a dynamic contrast-enhanced (DCE) MRI-based radiomics model for predicting the genetic status from next-generation sequencing (NGS). Patients with PDAC, who underwent pretreatment pancreatic DCE-MRI between November 2019 and July 2021, were eligible in this prospective study. Two radiologists evaluated presence of rim enhancement in PDAC, a known radiological prognostic indicator, on DCE MRI. NGS was conducted for the tissue from the lesion. The Mann-Whitney U and Chi-square tests were employed to identify clinical and genetic variables associated with rim enhancement in PDAC. For continuous variables predicting rim enhancement, the cutoff value was set based on the Youden's index from the receiver operating characteristic (ROC) curve. Radiomics features were extracted from a volume-of-interest of PDAC on four DCE maps (Ktrans, Kep, Ve, and iAUC). A random forest (RF) model was constructed using 10 selected radiomics features from a pool of 392 original features. This model aimed to predict the status of significant NGS variables associated with rim enhancement. The performance of the model was validated using test set. A total of 55 patients (32 men; median age 71 years) were randomly assigned to the training (n = 41) and test (n = 14) sets. In the training set, KRAS, TP53, CDKN2A, and SMAD4 mutation rates were 92.3%, 61.8%, 14.5%, and 9.1%, respectively. Tumor size and KRAS variant allele frequency (VAF) differed between rim-enhancing (n = 12) and nonrim-enhancing (n = 29) PDACs with a cutoff of 17.22%. The RF model's average AUC from 10-fold cross-validation for predicting KRAS VAF status was 0.698. In the test set comprising 6 tumors with low KRAS VAF and 8 with high KRAS VAF, the RF model's AUC reached 1.000, achieving a sensitivity of 75.0%, specificity of 100% and accuracy of 87.5%. Rim enhancement of PDAC is associated with KRAS VAF derived from NGS-based genetic information. For predicting the KRAS VAF status in PDAC, a radiomics model based on DCE maps showed promising results.

GATA2 mutant variant allele frequency may reflect prognosis in Chinese adult patients with de novo cytogenetically normal acute myeloid leukemia.

In this study, we analyzed GATA2 mutations (GATA2mut) and co-mutations in 166 Chinese patients with cytogenetically normal acute myeloid leukemia. This was done through targeted next-generation sequencing of 34 genes associated with myeloid leukemia. GATA2mut was identified in 17 (10%) patients being significantly correlated with co-mutations in CCAAT/enhancer-binding protein alpha (CEBPA) double mutation (P = 0.001). We observed that the N-terminal zinc finger domain (ZF1) was linked to CEBPA mutations, while the C-terminal zinc finger domain (ZF2) was associated with Wilms' tumor 1 (WT1) mutations. It was also noted that patients with GATA2mut had lower platelet counts at diagnosis (P = 0.032). In the entire cohort, GATA2mut had no significant prognostic impact on overall survival (OS) (P = 0.762) and relapse-free survival (RFS) (P = 0.369) compared to patients with GATA2wt. The OS (P = 0.737) and RFS (P = 0.894) of the ZF1 mutation were similar to those of the ZF2 mutation. Most patients with GATA2 mutations were classified in the ELN2022 favorable- and intermediate-risk groups. GATA2mut patients in the favorable-risk group were divided into GATA2High and GATA2Low groups using a median cutoff variant allele frequency (VAF) of 40.13%. GATA2High patients were associated with worse OS (P = 0.031) and RFS (P = 0.021) than GATA2Low patients. In the intermediate-riskgroup, the high median VAF ofGATA2 (≥38.51%) had no significant effect in OS and RFS compared with the low median VAF (<38.51%). This study offers new insights on the prognosis of GATA2mut in the favorable-risk group, where VAF can be used as a guide.

Abstract A007: Representative sampling of invasive breast carcinomas: Interim report from a prospective study (HOLST-F)

Abstract Background: Although a range of therapeutic options are available in the management of invasive breast carcinomas, spatial segregation of tumour subclones has hampered biomarker identification in single-region samples. Representative sampling (RS) overcomes spatial bias by sampling from a homogenized and well-mixed cancer specimen. The Homogenization of Leftover Surgical Tissue Feasibility Study (HoLST-F - NCT03832062) is a prospective trial aiming to assess the feasibility of RS in tumor tissue leftover after pathology sampling. An interim analysis of the breast cancer cohort is presented. Methods: In the context of the HoLST-F study and pre-specified end-points, representative samples derived from 75 leftover invasive breast carcinoma specimens underwent flow cytometric analysis of CK8/18, CD3, Ki67 and DAPI. Tumor cell enrichment by CK8/18 positivity and ploidy (in aneuploid carcinomas) was performed for downstream DNA extraction and whole exome sequencing. Somatic variants were determined using a bespoke pipeline to remove artefacts associated with fixation. Variant oncogenicity and therapeutic evidence levels were assigned by OncoKB variant annotation. Quantitative image analysis was applied to tissue sections from diagnostic FFPE blocks stained with Ki67 immunohistochemistry (n=78) and H&amp;E for lymphocytic infiltration scores (n=175) to correlate with flow cytometry. Results: In enriched representative samples, oncogenic mutations were commonly identified at high variant allele frequency (VAF) in known breast carcinoma driver genes including PIK3CA, CDH1, TP53, KMT2C and GATA3. Other clinically relevant oncogenic mutations were identified in ESR1, PTEN, ERBB2, RB1, AKT1, BRCA1, NF1 and FOXA1 which have been associated with resistance to various anti-cancer therapies. These mutations occurred at low and high VAFs indicative of both of clonal and sub-clonal resistance mechanisms. Recurrent variants in ESR1 (e.g. D538G) and PIK3CA (e.g. H1047R/L) were associated with Level 1 evidence for use as predictive biomarkers, while variants in twelve other genes across the cohort were associated with Level 2-4 evidence. Lymphocyte infiltration scores and Ki67 expression varied by tumor region, however RS by flow cytometry showed strong correlation with a weighted average across multiregional quantification of Ki67 expression (R=0.81, p=2.8 × 10-8) and lymphocyte infiltration (R=0.61, p&amp;lt;2.2 × 10-16). Conclusions: RS of invasive breast carcinoma in the HoLST-F trial has recapitulated the expected genomic driver landscape in breast cancer, in addition to identifying both clonal and subclonal genomic mechanisms of therapy resistance. Many of these mutations are either current or emerging therapeutic targets. Flow quantification of cells expressing phenotypic biomarkers (Ki67 and CD3) is feasible through RS and early analysis indicates that RS correlates with a weighted average across multiple regions. Leftover surgical tissue is an underutilized resource for biomarker assessment in breast carcinomas and can be examined by RS. Citation Format: Brian Hanley, Lisa Gallegos, Lavinia Spain, Husayn Pallikonda, Zayd Tippu, Samantha Hill, Aoune Barhoumi, Fiona Byrne, Yulia Dogva, Ashley Gilchrist, Glenn Noel-Storr, Hannah Veloz, Stacey Stanislaw, Harold Sansano, Kim Edmonds, Eleanor Carlyle, Nicholas Turner, James Larkin, Nelson Alexander, Samra Turajlic. Representative sampling of invasive breast carcinomas: Interim report from a prospective study (HOLST-F) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A007.

INTEGRAL‐ILCCO cohort data analysis revealed the association of clonal haematopoiesis with an increased risk of lung cancer

AbstractTo investigate the association between clonal haematopoiesis (CH) and lung cancer risk, we identified CH mutations in 1 059 lung cancer cases and 899 controls using the blood whole‐exome sequencing data generated from the Integrative Analysis of Lung Cancer Etiology and Risk project of the International Lung Cancer Consortium (INTEGRAL‐ILCCO). Based on the variant allele frequency (VAF) of these mutations, we stratified CH carriers into two groups, low VAF (1%–10%) and high VAF (≥10%), respectively. We observed a significant association between the presence of CH mutations and the risk of lung cancer after adjusting for known risk factors (odd ratio, OR = 1.37, 95% confidence interval, CI = 1.02–1.85). Such an association was largely driven by CH mutations with high‐VAF, the OR for high‐VAF CH and low‐VAF CH were 2.54 (1.38–4.93) and 1.14 (0.82–1.6), respectively. Trend analysis indicated a significant dose–response relationship (P trend = 0.004). This association between high‐VAF CH and lung cancer risk remained consistent when subjects were stratified by risk factors or lung cancer histological subtypes. A combination of results from INTEGRAL‐ILCCO, UKBB, and MGBB cohorts resulted in a meta‐analysed OR of 1.36 (95% CI = 1.14–1.62) for all CH carriers and of 1.76 (95% CI = 1.34–2.31) for high‐VAF CH carriers, respectively. In conclusion, our analysis revealed a significant association between CH and increased risk of lung cancer as supported by three independent cohorts.

Variant allele frequency in circulating tumor DNA correlated with tumor disease burden and predicted outcomes in patients with advanced breast cancer

PurposeIn patients with first-line advanced breast cancer (ABC), the correlation between ctDNA variant allele frequency (VAF) and tumor disease burden, and its prognostic value remains poorly investigated.MethodsThis study included patients with ABC diagnosed at Peking University Cancer Hospital who performed ctDNA test before receiving first-line treatment. Baseline plasma samples were collected for assessing ctDNA alterations and VAF with next-generation sequencing. The sum of tumor target lesion diameters (SLD) was measured with imaging methods according to RECIST 1.1 criteria.ResultsThe final cohort included 184 patients. The median age of the cohort was 49.4 (IQR: 42.3–56.8) years. The median VAF was 15.6% (IQR: 5.4%-33.7%). VAF showed positive correlation with SLD in patients with relatively large tumor lesions (r = 0.314, p = 0.003), but not in patients with small tumor lesions (p = 0.226). VAF was associated with multiple metastasis sites (p = 0.001). Multivariate Cox regression analysis showed that high VAF was associated with shorter overall survival (OS) (HR: 3.519, 95% confidence interval (CI): 2.149–5.761), and first-line progression-free survival (PFS) (HR: 2.352, 95%CI: 1.462–3.782). Combined VAF and SLD improved prediction performance, both median OS and PFS of patients in VAF(H)/SLD(H) group were significantly longer than VAF(L)/SLD(L) group (mOS: 49.3 vs. 174.1 months; mPFS: 9.6 vs. 25.3 months).ConclusionctDNA VAF associated with tumor disease burden, and was a prognostic factor for patients with ABC. A combination of ctDNA test and radiographic imaging might enhance tumor burden evaluation, and improve prognosis stratification in patients with ABC.

A multicenter retrospective comparison between systemic mastocytosis with t(8;21) AML and KIT mutant t(8;21) AML

Concomitant systemic mastocytosis (SM) has been detected in a proportion of patients with AML with t(8;21)(q22;q22.1)/RUNX1::RUNX1T1 with KIT gene mutations [KITpos t(8;21) AML]. However, there are few reports summarizing characteristics of SM-t(8;21) AML due to its rarity. In this multi-center, retrospective study, we made the largest-to-date comparison of the clinical characteristics, molecular features and prognosis between SM-t(8;21) AML (n=24) and KITpos t(8;21) AML (n=212). In addition to higher mast cell burdens (p<0.001), lower platelet levels (p=0.001), higher variant allele frequency (VAF) in KIT mutations (34.00% vs 16.40%, p=0.008) and more KIT exon 17 mutations (34.70% vs 21.05%, p=0.025) were observed in SM-t(8;21) AML patients. Moreover, mutations at KITD816 (69.57% vs 45.75%, p=0.030), KITD816V (45.83% vs 27.83%, p=0.046) and DNMT3A (8.70% vs 0.96%, p=0.050) were more common in SM-t(8;21) AML. Compared to non-D816 KITpos t(8;21) AML, SM-t(8;21) AML and KITD816 t(8;21) AML were at higher risk of refractory/relapsed leukemia (26.4% vs 50.0% vs 50.0%, p=0.002) with dismal prognosis [2-year OS: 79.2% vs 34.7% vs 60.7%, p=0.004; 2-year EFS: 61.7%vs 36.2% vs 39.7%, p=0.012]. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) could only improve prognosis of SM-t(8;21) AML patients. For all patients in our cohort, age > 60 years, KITD816 mutations and BCOR mutations were showed to be independent unfavorable predictors for OS and EFS. Our results revealed that SM-t(8;21) AML shared more similar clinical characteristics, molecular features and clinical outcomes with KITD816 t(8;21) AML.

Identification and surveillance of rare relapse-initiating stem cells during complete remission after transplantation

AbstractRelapse after complete remission (CR) remains the main cause of mortality after allogeneic stem cell transplantation for hematological malignancies and, therefore, improved biomarkers for early prediction of relapse remains a critical goal toward development and assessment of preemptive relapse treatment. Because the significance of cancer stem cells as a source of relapses remains unclear, we investigated whether mutational screening for persistence of rare cancer stem cells would enhance measurable residual disease (MRD) and early relapse prediction after transplantation. In a retrospective study of patients who relapsed and patients who achieved continuous-CR with myelodysplastic syndromes and related myeloid malignancies, combined flow cytometric cell sorting and mutational screening for persistence of rare relapse-initiating stem cells was performed in the bone marrow at multiple CR time points after transplantation. In 25 CR samples from 15 patients that later relapsed, only 9 samples were MRD-positive in mononuclear cells (MNCs) whereas flowcytometric-sorted hematopoietic stem and progenitor cells (HSPCs) were MRD-positive in all samples, and always with a higher variant allele frequency than in MNCs (mean, 97-fold). MRD-positivity in HSPCs preceded MNCs in multiple sequential samples, in some cases preceding relapse by >2 years. In contrast, in 13 patients in long-term continuous-CR, HSPCs remained MRD-negative. Enhanced MRD sensitivity was also observed in total CD34+ cells, but HSPCs were always more clonally involved (mean, 8-fold). In conclusion, identification of relapse-initiating cancer stem cells and mutational MRD screening for their persistence consistently enhances MRD sensitivity and earlier prediction of relapse after allogeneic stem cell transplantation.

The Frequency of Clonal Hematopoiesis Prior to AML and MDS Varies Among the Different Molecularly Defined Who Subtypes

Background: AML and MDS cells accumulate mutations over years or even decades. In the current WHO 2022 classification, some mutations are listed as “defining genetic abnormalities” (WHO DGA). A long phase of clonal hematopoiesis may precede some, but not all, AML or MDS. There are multiple scenarios in which order clonal hematopoiesis, WHO DGAs, and other mutations can occur. We analyzed whole-genome sequencing data from 583 patients with AML or MDS subtypes defined by a mutation and elucidated their role in the hierarchical sequence of mutations. Aim: (1) We compared patients with AML or MDS arising from clonal hematopoiesis to those with a founder mutation in a WHO DGA gene (e.g. NPM1). (2) We analyzed which other mutations may occur before WHO DGAs. Methods and patients: We analyzed 583 patients with AML or MDS who were classified into a WHO 2022 DGA class: MDS with low blasts and SF3B1 mutation (MDS- SF3B1, n=160), MDS with biallelic TP53 inactivation (MDS-bi TP53, n=40), AML, myelodysplasia-related (AML-MR with focus on mutations, n=151), AML with NPM1 (n=171), and AML with CEBPA (n=61). Amplification-free WGS was performed (median coverage &amp;gt; 100x). Genes with known relevance to myeloid malignancies were analyzed (n=47). We assumed that the mutation with the highest variant allele frequency (VAF) evolved first in the malignant process (+/- 5%). VAF was corrected for copy number changes (including the presence of one X chromosome in males) and copy-neutral loss of heterozygosity (CN-LOH). The following variant callers were used: Strelka2 (mutations and VAF), Pindel (for FLT3-ITD), HadoopCNV (CN-LOH) and GATK4 (CNV). Results: We classified patients into three groups (Figure 1a): (A) 322/583 (55%) of malignancies originate from a cell with a WHO DGA; (B) 174/583 (30%) of malignancies originate from a clonal hematopoietic cell with a DNTM3A or TET2 mutation (DT origin); (C) the remaining 87/583 (15%) of AML and MDS originate from a cell with a different initial mutation (“other”). The frequency of patients with the highest VAF in a WHO DGA varied between subtypes: MDS-bi TP53: 88%, MDS- SF3B1: 72%, AML- CEPBA: 64%, AML-MR: 57% and AML- NPM1: 27%. The highest number of patients with a disease putatively arising from DNMT3A or TET2 mutated clonal hematopoiesis was found in AML- NPM1 (49%) and the lowest in MDS-bi TP53 (5%) (for details, see Figure 1b). The group of patients with the highest VAF in neither WHO DGA nor DNMT3A/ TET2 (“other”) accounted for 17-23% of AML and only 4% and 8% of MDS- SF3B1 andMDS-bi TP53 respectively. IDH1 or IDH2 had the highest VAF in 17/583 patients (3%), especially AML- NPM1 (n=8) and AML-MR (n=7). Mutations in the spliceosome genes had the highest VAF in 12/583 patients (2%), with the combination of SRSF2 in AML- NPM1 in particular (n=6). In the following, we compared DT origin and WHO DGA origin. The differences did not affect all AML and MDS subtypes to the same extent. Patients with AML- CEBPA and DT origin were a median of 19 years older than patients with an initial CEBPA mutation (75 vs. 56 years). In AML- NPM1, the difference in median age was 11 years (71 vs. 60 years). In both groups, patients with DT origin are also predominantly male (73% vs. 44% for AML- CEBPA and 54% vs. 45% for AML- NPM1). Age and sex differences were not observed in AML-MR, MDS- SF3B1 and MDS-bi TP53 with or without DT origin. Overall survival (OS) was shorter in AML patients with DT origin compared to WHO DGA (0.7 vs. 1.8 years; p=0.073), but not in MDS (7.4 vs. 6.1 years; p=0.669). However, in a multivariate Cox analysis, including DT origin and age, age remained the only significant factor for OS. Conclusion: Using VAF to model the hierarchy of mutation evolution shows that for the majority of patients, WHO-defining mutations are early events, supporting their role as defining genetic abnormalities and (potential) drivers. However, other initial mutations occur in DNMT3A and TET2, indicating AML or MDS arising from clonal hematopoiesis,but also IDH1/ IDH2 or spliceosome genes. How these diseases will be classified in the future should be actively discussed, and aspects such as targeted therapies (with IDH inhibitors) or a second hematologic neoplasm after clonal hematopoiesis should be considered.