High Variant Allele Frequency Research Articles

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 < 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.

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.

Ivosidenib for Patients with Clonal Cytopenia of Undetermined Significance and Mutations in IDH1

Background Myeloid neoplasms (MN), including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), are hematologic malignancies that are highly resistant to therapy and confer a poor prognosis. Recent studies have revealed that hematopoietic stem cells and progenitor cells carrying preleukemic mutations, defined as clonal hematopoiesis (CH), are the cells of origin in MN. CH can occur many years before the diagnosis of MN. Multiple mutations, mutations with a high variant allele frequency (VAF), and mutations in TP53, spliceosome genes, and isocitrate dehydrogenase 1/2 ( IDH1/2) have been identified as high-risk features for CH progression to MN. Individuals with cytopenias who harbor acquired mutations in their blood yet do not meet criteria for hematologic malignancy, an entity called clonal cytopenia of undetermined significance (CCUS), have an even higher risk of developing MN (estimated at 95% over 10 years of follow-up). Thus, despite being almost inevitably destined for progression to malignancy, there are no established preventative therapies for patients with CCUS. The IDH1 inhibitor ivosidenib has been extensively evaluated in clinical studies and has been shown to effectively inhibit the gain-of-function activity of the mutated protein. Clinical data has shown that ivosidenib is safe and well-tolerated with excellent response rates even in patients with relapsed or refractory AML. Individuals with lower co-mutational burden are more likely to have a clinical response and achieve molecular remission with ivosidenib. Since CCUS is characterized by a lower mutation burden compared to AML or MDS, we hypothesize that the use of ivosidenib in IDH1-mutant CCUS will result in a higher and more prolonged clinical response. Study Design and Methods NCT05030441 is a decentralized open label, multicenter pilot study exploring the efficacy of ivosidenib in patients with IDH1-mutant CCUS. The study is offered as a decentralized trial (DCT) where patients across the US can participate with study procedures occurring in the patient's home using telemedicine, mobile phlebotomy services and with local providers. By reducing barriers to trial participation, we expect that the DCT design will improve accessibility for this rare patient population. This represents an important step toward making personalized medicine available to a socio-economically diverse patient population and enables rare mutations to be studied at scale. Patients with CCUS harboring the IDH1 R132 mutation are considered eligible if they do not have any concurrent active malignancy requiring treatment. Patients must have unexplained cytopenia lasting ≥6 months, defined as hemoglobin &amp;lt;10 g/dL, absolute neutrophil count &amp;lt;1.8 × 10 9/L and platelets &amp;lt;100 × 10 9/L, as well as adequate baseline organ function including renal and liver function, as per the National Institutes of Health Common Terminology Criteria for Adverse Events (NCI CTCAE) grading. Recruitment and data collection will be coordinated with local providers for patients that enroll remotely. A total of 15 patients will be accrued to receive ivosidenib 500 mg daily for up to 18 cycles. The primary endpoint is rate of improvement in hematologic parameters using specific cutoffs for erythroid, platelet and neutrophil responses based on the MDS response criteria. The study will be considered positive if at least 6 out of the 15 patients have a hematologic improvement. Secondary endpoints include change in mutant IDH1 VAF, measured at baseline and every 3 cycles on peripheral blood samples, safety of ivosidenib, and AML/MDS-free survival which will be evaluated by descriptive statistics and Kaplan-Meier methodology. Enrollment opened in one US center and by decentralized structure in April 2022, with 3 patients accrued, and is expected to open in other US centers in 2023. If the study endpoints are met, our aim is to expand to a larger population with the goal of determining whether ivosidenib can prevent the development of hematologic malignancy in patients with IDH1-mutant CCUS.

Mesenchymal Stromal Cells from Individuals with DNMT3A mut Clonal Hematopoiesis Instruct Young Healthy Hematopoietic Stem/Progenitor Cells Towards a Myeloid-Biased Aging Phenotype Resembling That of Patients with Clonal Cytopenia of Undetermined Significance (CCUS)

Clonal hematopoiesis of indeterminate potential (CHIP) describes the age-related acquisition of somatic mutations in hematopoietic stem/progenitor cells (HSPC) leading to clonal blood cell expansion and variable risk of progression to myeloid malignancy. DNMT3A is the most commonly mutated gene and while single mutations do not raise the likelihood of progression, high variant allele frequencies (VAF) and presence of co-mutations do. In myeloid diseases, the bone marrow (BM) microenvironment exhibits alterations promoting malignant transformation. However, it is unknown whether similar alterations exist in the pre-malignant niche. We set out to analyze whether human mesenchymal stromal cells (MSC) from CHIP individuals with a DNMT3A mutation ( DNMT3A mut) are altered and whether they are able to remodel the HSPC compartment in healthy hematopoiesis. From our published cohort of previously sequenced BM samples from hematologically healthy individuals undergoing hip replacement surgery, we selected samples with either an isolated DNMT3A mut at a low VAF (2.47-6.8%, n=4, median age 73.5y, termed low-risk CHIP), or DNMT3A mut samples with a high VAF (17.5-28.5%) and at least one co-mutation (n=5, median age 73y, termed high-risk CHIP) as well as 12 age-matched samples without any mutation (n=12, median age 72y). HSPC were isolated from 3 younger healthy donors (median age 50y). MSC were expanded by plastic adherence and subjected to multilevel characterization. Compared to age-matched healthy controls, MSC from DNMT3A mut BM samples (CHIP-MSC) exhibited higher proliferation rates, lower colony-forming-unit (CFU) fibroblast capacity, as well as reduced adipogenic and increased osteogenic differentiation potential. To understand how altered CHIP-MSC influence the hematopoietic compartment, younger healthy CD34+ HSPC were co-cultured with the different MSC subgroups for 42 hours in a low cytokine-containing medium, followed by bulk RNAseq on subsequently sorted cell populations (MSC: CD45-/CD90+; HSPC: CD45+/CD34+). We found 31 differentially expressed genes (DEG) (logFC±0.5, FDR&amp;lt;0.05) in CHIP-MSC compared to their age-matched counterparts. Functional analyses suggested an activation of communication towards HSPC via extracellular vesicles. Strikingly, differential gene expression of sorted young healthy HSPC after co-culture revealed 375 DEG (322 up-regulated, 53 down-regulated; logFC±0.5, FDR&amp;lt;0.05) in HSPC cultured with CHIP compared to non-CHIP age-matched MSC (Figure panel A). We then used gene set variation analysis (GSVA) to analyze HSPC compartment composition after co-culture. Based on enrichment scores, unsupervised clustering identified two distinctive profiles that correlated with HSPC cultured with either non-CHIP or high-risk CHIP-MSC, while those cultured with low-risk CHIP-MSC showed an intermediate phenotype (Figure panel B). Co-culture of HSPC with high-risk CHIP-MSC resulted in enrichment of hematopoietic stem cells (HSC) and multipotent progenitors (MPP), as well as myeloid skewing represented by higher scores for common-myeloid (CMP) and granulocyte-monocyte (GMP) progenitors. Conversely, in contrast to the control group, megakaryocyte-erythroid progenitors (MEP) were underrepresented. CFU capacity was increased in healthy HSPC cultured with CHIP-MSC and multiparameter flow cytometry analysis showed a trend towards increased CMP/GMP and decreased MEP numbers, consistent with the RNAseq data. Finally, we evaluated the BM HSPC compartment composition of a cohort of 49 CCUS patients with DNMT3A mut (VAF 1-49%, median age 76y) compared to 56 control BM samples using whole transcriptome sequencing data. Intriguingly, we observed the same profile with a myeloid shift in progenitor populations as in our healthy young HSPC exposed to high-risk CHIP-MSC. Taken together, our results indicate that MSC from individuals harboring a DNMT3Amutation are able to instruct young healthy HSPC and influence their fate, thereby promoting a differentiation bias that shifts the hematopoietic compartment toward that associated with an aged pre-malignant state, even in the absence of DNMT3A mut HSPC. These findings suggest that focusing on the interaction between MSC and HSPC is a promising therapeutic strategy to intercept disease progression. Functional analysis of identified deregulated pathways in DNMT3Amut CHIP is ongoing.

Selinexor Plus Ruxolitinib in JAK Inhibitor (JAKi)-Naïve Patients with Myelofibrosis: Long Term Follow up from XPORT-MF-034 Suggestive of Disease Modification

Background: Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by splenomegaly, constitutional symptoms, and inflammation due to unregulated, clonal proliferation of hematopoietic stem cells in the bone marrow. Common genetic drivers include JAK2, CALR, and MPL. Although improvements in splenomegaly and symptoms occur with the current approved therapies, additional assessments are needed to understand the impact of therapies on disease modification. Increased burden of variant allele frequency (VAF) of driver mutations is associated with poor disease outcomes, and increased expression of pro-inflammatory cytokines is implicated in disease pathophysiology and symptoms burden. Selinexor (SEL) is an investigational oral XPO1 inhibitor that may inhibit multiple MF-relevant pathways including STAT, ERK, and AKT. Preclinical studies have shown potential synergy between SEL and ruxolitinib (RUX) treatment in vivo. Safety and efficacy analysis of SEL plus RUX in JAKi treatment-naïve MF in a Phase 1 study supported a 60 mg SEL dose as the recommended dose in a Phase 3 study. Here we include exploratory analyses of a range of biomarkers in the Phase 1 study of SEL and RUX in JAKi-naïve myelofibrosis. Methods: Phase 1 of XPORT-MF-034 (NCT04562389) is an open-label study evaluating the safety and efficacy of SEL at 40mg and 60mg once weekly plus RUX with JAKi-naïve MF. Assessments included safety and rates of spleen volume reduction of ≥35% (SVR35) and total symptom score reduction of ≥50% (TSS50). Platelet and hemoglobin levels were also assessed. Longitudinal clinical biomarker assessments included %VAF change at week 24 for driver genes and plasma cytokine levels at week 4 (Cycle 2 Day 1). Bone marrow (BM) biopsies were obtained at screening and week 24, then evaluated for bone marrow fibrosis (BMF) and other BM cellular markers (CD61, CD71). Results: As of April 10, 2023, a total of 24 patients (pts) received at least one dose of SEL (40 mg: n=10; 60 mg n=14). The most common adverse events (AEs) overall of any grade were nausea (75% majority grade 1-2), fatigue (58%), anemia (54%), and thrombocytopenia (54%). Those receiving a prophylactic anti-emetic reported lower incidence and severity of nausea AEs compared to those that did not; pts experienced a median weight increase of 3.3 kg at week 24. Two pts discontinued treatment due to treatment-related AEs (thrombocytopenia and neuropathy). Generally stable hemoglobin levels were observed in 48% of transfusion-independent pts. Median hemoglobin levels were 10.1 g/dL at baseline, 9.3 g/dL at week 12, and 10.2 g/dL at week 24. Median platelet counts remained generally stable with median values of 258 k/uL at baseline, 164 k/uL at week 12, and 193 k/uL at week 24. SVR35 at week 24 in the intent-to-treat (ITT) population was achieved by 40% of the 40 mg cohort and 79% of the 60 mg cohort and TSS50 was achieved by 10% and 58%, respectively. Mutation analysis showed no pts were triple negative, with 18 harboring JAK2 mutations, 5 with CALR, and 1 with MPL. Of 13 pts with available data at week 24, 5 pts (38%) had a reduction of VAF that was ≥20%, and 3/5 of these pts had high VAF (&amp;gt;50%) driver mutations at baseline and were also characterized as HMR. A panel of 74 cytokines was evaluated in 21 MF pts with baseline and week 2 samples (including 10 with end-of-treatment [EOT] samples), and in 10 healthy donors. Comparing baseline MF to healthy samples, 50% of the analyzed cytokines were elevated. A rapid and durable decrease of pro-inflammatory MF-relevant cytokines including TGF-β1, IFNɣ, TNF-α, IL-7, IL-1Ra, and IL-16 was observed in the majority of pts at week 2 and continued into EOT. Notably, the reduction in IL-18 correlated to SVR and TSS response at week 24, with R 2 values of 0.29 and 0.28, respectively. BM analysis at week 24 showed an increased number of erythroid CD71+ elements in 3/14 pts. Conclusions: SEL plus RUX was generally well tolerated and manageable.Evidence of potential disease modification in pts with JAKi-naïve MF patients was seen via rapid stabilization of platelets and maintaining hemoglobin levels, VAF reduction, and pro-inflammatory cytokine reduction. Promising biomarker and efficacy data suggests that SEL in combination with RUX has the potential to become a novel, first-line treatment for pts with MF. Updated clinical data with longer follow-ups and additional subgroup analyses will be available at the time of presentation.

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.

Genetic Evolution from Chronic Myeloproliferative Neoplasms to Acute Myeloid Leukemia: An Analysis of Forty-Six Paired Samples

Background: Secondary acute myeloid leukemia (sAML) may arise from chronic myeloproliferative neoplasm (MPN). The genetic evolution pattern may be understood by paired sample analysis. Knowledge of genetic change in MPN/sAML is still limited and more extensive studies should be done. Patients and Methods: Forty-six paired samples (27 males) at diagnosis of MPN (12 polycythemia vera, 6 essential thrombocythemia, 25 primary myelofibrosis and 3 MPN-unclassified) and sAML transformation were available. Mutational analyses of 27 genes ( JAK2V617F, CALR, MPL, ASXL1, IDH1, IDH2, TET2, DNMT3A, EZH2, SRSF2, U2AF1, SF3B1, ZRSR2, SMC1, STAG2, SMC3, RUNX1, SETBP1, IKZF1, WT1, K-RAS, N-RAS, C-CBL, TP53, RAD21, BCOR and BCORL1) were performed. Mutations and their variant allele frequencies (VAF) were measured by next generation sequencing. Results: At baseline (MPN), the mutation number was 2 (maximal 5). Forty-five samples harbored driver gene mutations (31 JAK2V617F, 13 CALR and 1 MPL). Twenty-eight samples harbored at least one epigenetic regulator mutations (11 TET2, 8 DNMT3A,7 ASXL1, 5 EZH2, 2 IDH2, 1 IDH1). Twelve samples harbored spliceosome mutations which were mutually exclusive (6 SRSF2, 3 SF3B1, 2 U2AF1 1 ZRSR2). Other mutations were RUNX1, BCORL1 (n=2 each), CBL and BCOR (n=1 each). In sAML, the median mutation number was 3 (maximal 7). Loss of mutation was found for JAK2V617F (9/31), TET2 (1/11) and SRSF2 (1/6). Acquisition was most common for RUNX1 (n=9), followed by TP53, ASXL1 (n=5 each), K-RAS (n=4) , ZRSR2, STAG2 (n=3 each). The median number of acquired mutation was 1 (maximal 5). The frequencies of gene mutations are summarized in Figure 1A. The baseline VAF of JAK2V617F was high (&amp;gt;35%) in 80.6% (25/31). In progression to sAML, VAF was constant in 29.0% (9/31, VAF changes &amp;lt;10%), increased in 20.0% (9/31, increased VAF&amp;gt; 10%), decreased in 41.9% (13/31, decreased VAF&amp;gt;10%) (Figure 1B). The baseline VAF in CALR mutation was high (&amp;gt;35%) in 91.3% (12/13). VAF remained stable in 76.9% (10/13), and loss of CALR mutation was not observed (Figure 1B). TET2 mutations tended to be stable (7/16), but clonal expansion (5/16 of all mutation events at MPN), or acquisition (n=2) could be found during transformation to sAML. DNMT3A mutations remained stable (5/8), exhibited clonal expansion (2/8), or acquisition (n=1) in sAML progression. ASXL1 mutations remained stable (6/7), with clonal expansion (1/7), or acquisition (n=5) during MPN/sAML progression. All EZH2 mutants had clonal expansion (5/5) and one acquired in sAML (Figure 1B). We found that CALR mutationwas often the founding clone with high VAF at MPN phase, stable clone during sAML, and might acquire mutations in signaling pathways (1 CBL, 1 KRAS), epigenetic regulators (2 ASXL1, 1 IDH2), transcription factors (2 RUNX1, 1 BCOR), spliceosome (2 ZRSR2, 1 SF3B1), and cohesin complex (1 STAG2). JAK2 mutationwas often preceded by other ancestral mutations such as TET2, ASXL1, DNMT3A, EZH2, IDH2, SRSF2 or ZRSR2. During evolution, leukemia arose from non- JAK2 mutated clones with mutations in EZH2, DNMT3A, RUNX1, EZH2, SETBP1, or acquisition of TP53, SF3B1, BCOR and STAG2 mutations) in sAML. In outcome, the median time from MPN to sAML was 82 (range 3.7 to 338.5) months. Female gender, loss of JAK2V617F, presence of U2AF1 and absence of IDH1 were associated with shorter time to sAML transformation. No other genetic mutation in our gene panel or the number of mutations harbored by patients in our cohort had significant impact on time to sAML. Marked increase of VAF was observed for JAK2 (7/22) , DNMT3A (1/7) , IDH1 (2/3), IDH2 (1/3), TET2 (3/10) , EZH2 (4/5), C-CBL (1/1) and SRSF2 (1/4). Marked decrease of VAF was observed for JAK2 (4/22), MPL (1/1), DNMT3A (1/7) , ASXL1 (1/8), SF3B1 (2/3), SRSF2 (1/4) and U2AF1 (1/2). Multiple mutational clones were detected for TET2 (two clones in 4 samples and 3 clones in 1). Clonal expansion and reduction were observed in two pairs of samples upon sAML transformation. Conclusions: At the MPN stage, most patients had driver mutations with frequent co-mutations of epigenetic modifier or spliceosome genes. CALR mutant clones were stable while for JAK2, sAML often arose from clones without JAK2V617F which acquired RUNX1, TP53, ASXL1 and K-RAS. Research funding:Ministryof Science and Technology Council,Taiwan (NSTC 112-2314-B-182-055) and Ministry of Health and Welfare, Taiwan (112-TDU-B-222-124001)