Therapy-related Myeloid Neoplasms Research Articles

Abstract 5070: Landscape of clonal hematopoiesis prior to PARP inhibitor treatment in patients with ovarian cancer

Abstract Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) are an important tool for treatment of ovarian and other cancers, particularly for those with mutations in BRCA1 or 2 genes or homologous recombination deficiency. The risk of developing secondary hematologic malignancy, particularly myelodysplastic syndrome or acute myeloid leukemia (MDS/AML), is substantially higher (4-12%) after PARPi maintenance or treatment in the second line and beyond in patients with ovarian cancer. This elevated risk necessitates additional investigation into the pathogenesis of PARPi-related secondary malignancy. Clonal hematopoiesis of indeterminate potential (CHIP) has been associated with the development of therapy-related myeloid neoplasms. We aim to understand the patterns of CHIP in patients with high grade serous ovarian carcinoma who receive PARPi therapy. Methods: We performed ultra-high-depth whole exome sequencing of plasma derived cfDNA or white blood cells (WBCs) barcoded with unique molecular identifiers from patients with high grade serous ovarian carcinoma who were treated with multiple lines of chemotherapy (n=6) or with PARPi in addition to chemotherapy (n = 10). Using spike-in mutated DNAs as positive controls, we detected variant alleles as low as 1% variant allele frequency. Gene set enrichment analysis was performed on identified groups. Results: We identified 7162 recurrent variants among our patients. We began by comparing quantities of CHIP between the samples from patients who eventually receive PARPi and those who do not. Samples contained a median of 2030 recurrent CHIP variants and 4300 total variants. No differences in total CHIP quantity or mutation type was discernable among samples from patients who will receive PARPi (BRCA1/2 enriched) vs those who will not. Among our 16 patients, 14 ultimately developed secondary hematologic malignancy (t-MN+ Comparison of t-MN+ (n=14) and t-MN- (n=2) defined a group of 14 variants enriched in the t-MN+ population and a group of 85 variants depleted in the t-MN+ population. The t-MN+ enriched variants we identified were not previously associated with CHIP and had significant overlaps with alkyl transferase activity (FDR q val < 0.01) and nucleotide binding gene sets (FDR q val < 0.01). Conclusions: We used a novel methodology to identify unbiased low variant mutational changes in the hematopoietic system. These results suggest that baseline clonal hematopoiesis variants are similar among patients newly diagnosed with ovarian carcinoma. We have also identified a signature potentially related to future risk of leukemia requiring further validation. Citation Format: Amma Asare, Sara Corvigno, Jun Yao, Li Zhao, Nicole D. Fleming, Joseph Celestino, Richard A. Hajek, Mark S. Kim, Alejandra Flores Legarreta, Karen H. Lu, Koichi Takahashi, P Andrew Futreal, Amir A. Jazaeri, Shannon N. Westin, Anil K. Sood, Sanghoon Lee. Landscape of clonal hematopoiesis prior to PARP inhibitor treatment in patients with ovarian 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 5070.

Multiple Primary Cancers With Hematologic Malignancies and Germline Predisposition: A Case Series.

The term "multiple primary (MP) cancers" refers to the existence of more than one cancer in the same patient. The combination of MP cancers with hematological malignancies is relatively uncommon. In this study, we present five patients diagnosed with MP cancers concomitant with hematological malignancies. We comprehensively analyzed their clinical characteristics, cytogenetic profiles, and germline and somatic variants. As first primaries, two patients had solid cancer not followed by cytotoxic therapy and three had hematologic cancer, followed by cytotoxic therapy. The second primaries were all hematologic malignancies that did not meet the criteria for therapy-related myeloid neoplasm. Notably, two (40%) out of the five patients harbored pathogenic potential/presumed germline variants in cancer predisposition genes. Therefore, germline variant testing should be considered when MP cancers with hematological malignancies require consideration for related donor stem cell transplantation.

CPX-351 and allogeneic stem cell transplant for a therapy-related acute myeloid leukemia that developed after treatment of acute promyelocytic leukemia: a case report and review of the literature

Therapy-related myeloid neoplasms (t-MNs), which develop after cytotoxic, radiation, or immunosuppressive therapy for an unrelated disease, account for 7%–8% of acute myeloid leukemia (AML). Worse outcomes and consequently shortened survival are associated with t-MNs as compared with de novo AML. Therapy-related MNs are being reported with increasing frequency in successfully treated acute promyelocytic leukemia (APL), in particular, before the introduction of all-trans retinoic acid (ATRA) plus arsenic trioxide (ATO). Considering the high curability of APL, t-MNs represent one of the prognosis-limiting factors in this setting of leukemia. We report our experience with a patient who developed t-AML 15 years after treatment for APL. Treatment included three cycles of chemotherapy with CPX-351 (Vyxeos, Jazz Pharmaceuticals) followed, as in remission, by an allogeneic hematopoietic stem cell transplant. A review of available literature was also included.

Assessment of hematological toxicity in patients with advanced neuroendocrine tumors and extensive/innumerable bone metastases undergoing lutetium-177 DOTATATE treatment.

592 Background: The introduction of peptide receptor radionuclide therapy (PRRT) using Lutetium-177 (177Lu) DOTATATE has led to a paradigm shift in the treatment of neuroendocrine tumors (NETs). Hematological toxicity is a well-recognized adverse effect (AE) of 177Lu DOTATATE. Most cytopenias are transient, with an estimated incidence of 10-25%, typically mild to moderate in severity (grade G1-2). The most dreaded AE of PRRT is therapy-related myeloid neoplasm (t-MN), with an estimated 2-8% incidence and a higher incidence reported in patients who also received prior chemotherapy. We sought to evaluate the hematological safety of 177Lu PRRT in the setting of NETs with extensive (i.e., innumerable) bone metastases. Methods: We retrospectively reviewed the medical records of all Mayo Clinic patients (pts) with extensive/innumerable osseous metastases, defined as >50% skeletal involvement by positron emission tomography (PET) DOTATATE, who were treated with 177Lu DOTATATE. Pts characteristics and laboratory results were collected before, during, and after 177Lu DOTATATE treatment. Hematotoxicity was graded according to the NCI-CTCAE v5. Results: Out of 27 pts, 13(48%) developed cytopenia(s) of any grade after treatment with one or more cycles of 177Lu DOTATATE. In total, there were 13(48%) pts with anemia, 13(48%) with thrombocytopenia, and 6(22%) with neutropenia. Six (22%) pts had severe hematological toxicity (G3-4). Twelve months post-PRRT, 7(26%) pts continued to experience cytopenia(s) of any grade, of whom 4(15%) had G3-4 hematotoxicity. One pt developed t-MN, and two pts had myelophthisis on bone marrow biopsy from infiltrating NET. 16(59%) pts received cytotoxic chemotherapy before PRRT, and 7(25.9%) developed subsequent cytopenia(s). Overall, 15(55%) pts completed four cycles, while 3 pts didn’t complete four cycles of 177Lu DOTATATE due to hematotoxicity. Conclusions: Pts with advanced NETs and extensive/innumerable bone metastases treated with PRRT could be at higher risk for myelosuppression than historical controls. Our study highlights the importance of carefully monitoring and assessing hematological parameters in pts being considered for 177Lu DOTATATE with extensive/innumerable bone metastasis. [Table: see text]

The crossroads of cancer therapies and clonal hematopoiesis

The intricate interplay between Clonal Hematopoiesis (CH) and the repercussions of cancer therapies has garnered significant research focus in recent years. Previously perceived as an age-related phenomenon, CH is now closely linked to inflammation (“Inflammaging”) and cancer, impacting leukemogenesis, cancer progression, and treatment responses. This review explores the complex interplay between CH and diverse cancer therapies, including chemotherapy, targeted treatments, radiation, stem cell transplants, CAR-T cell therapy, and immunotherapy, like immune checkpoint inhibitors. Notably, knowledge about post-chemotherapy CH mutation/acquisition has evolved from a de novo incident to more of a clonal selection process. Chemotherapy and radiation exposure, whether therapeutic or environmental, increases CH risk, particularly in genes like TP53 and PPM1D. Environmental toxins, especially in high-risk environments like post-disaster sites or space exploration, are associated with CH. CH affects clinical outcomes in stem cell transplant scenarios, including engraftment, survival, and t-MN development. The presence of CH also alters CAR-T cell therapy responses and impacts the efficacy and toxicity of immunotherapies. Furthermore, specific mutations like DNMT3A and TET2 thrive under inflammatory stress, influencing therapy outcomes and justifying the ongoing tailored interventions in clinical trials. This review underscores the critical need to integrate CH analysis into personalized medicine, enhancing risk assessments and refining treatment strategies. As we progress, multidisciplinary collaboration and comprehensive studies are imperative. Understanding CH's impact, especially concerning genotoxic stressors, will inform screening, surveillance, and early detection strategies, decreasing the risk of therapy-related myeloid neoplasms and revolutionizing cancer treatment paradigms.

Clinical and Molecular Characterization of Patients with Therapy Related Myeloid Neoplasms (TRMN)

Acknowledgements Chemotherapy and/or radiotherapy in cancer patients compose a future risk of developing a therapy related myeloid neoplasm (TRMN). However, the presence of a germline landscape might contribute and modify the overall risk. It has been suggested that around 18.5% of TRMN patients have a germline cancer mutation. In particular, cancer predisposing genes or syndromes involving alterations in the DNA repair mechanism such as CHEK2, BRCA1, BRCA2, MLH1, MSH2, MSH6 or PMS2 might be enriched in patients who develop a primary tumor (PT) and a subsequent secondary myeloid neoplasm. The role of the alterations in cancer susceptibility genes in association with the treatment for the PT in the development of TRMN is still unclear. To evaluate the clinical and genetic landscape of TRMN patients to understand the role of different risk factors and provide tools for differential diagnosis and clinical management. Clinical evaluation of 96 patients with TRMN and 12 patients with a secondary myeloid neoplasm after a non-treated with chemotherapy primary tumor (nTMN) was performed. In addition, germline landscape and somatic alterations were studied in the TRMN series by tNGS (N=49) and WES (N=47). Tumoral sample was obtained from bone marrow. CD3+ T lymphocytes from peripheral blood (PB) were enriched by immunomagnetic selection and used as germline/control sample. A minimum of 98% purity in CD3+ was selected to prevent myeloid contamination. Variant frequency (VAF) <30% found in tumor but not in CD3+ sample was annotated as somatic alteration. Only pathogenic/likely pathogenic variants were considered. The median age of onset was 62.3 and 71.1 years for the PT and the TRMN, respectively. 32.5% had complex karyotype. Similar positive correlation was found between the age of onset of the PT and the age of onset of the TRMN and nTNM. A negative correlation was found between the age of onset of the PT and the latency until the TRMN and nTMN diagnosis, which suggests that elderly age condition is more relevant than the cytotoxic treatment (Fig.1). No differences in latencies were found between MN subtypes, despite ring sideroblasts and del(5q) myeloid subtypes correlated with shorter and higher latencies, respectively. Different latencies were neither found when the PT was solid or hematologic, but interestingly, TRMN patients with previous colorectal cancer had shorter latencies. On the other hand, 91.3% of the TRMN patients presented a molecular alteration (considering both genetic or cytogenetic alterations), including 60.9% and 75% of patients with germline and acquired alterations, respectively. Germline mutations included 52.2% and 47.8% annotated in cancer predisposition genes of the DNA repair mechanisms and myeloid genes, respectively. Only 39.1% of TRMN patients were found with pathogenic somatic variants in myeloid genes while 68.75% were found with an abnormal karyotype. Finally, higher number of germline and somatic mutations was found in TRMN patients with an abnormal karyotype. However, a higher percentage of germline mutations in myeloid genes was found in TRMN with a normal karyotype (Fig.2). Higher percentage of molecular alterations (both germline and somatic) in myeloid genes was found in TRMN patients with a previous solid PT. However, higher percentage of mutations in cancer predisposition genes was found when the PT was hematologic. Our results highlight the role of mutations in cancer predisposition genes in the development of TRMN. Similar latency associations were found for TRMN and nTMN patients, which suggest that other risk factors different than treatment might contribute to the TRMN development. Altered karyotypes in TRMN patients are associated with acquired mutations in myeloid genes but not with germline mutations, which suggests two different entities with different prognosis. Molecular alterations in myeloid genes are more relevant in TRMN patients with solid PT, while molecular alterations in cancer predisposition genes are associated with TRMN patients with hematologic PT. This work was supported in part by a grant from the Instituto de Salud Carlos III, Ministerio de Economia y Competitividad, Spain (PI 20/00531)(Co-funded by European Regional Development Fund. ERDF, a way to build Europe). 2021 SGR 00560 (GRC) Generalitat de Catalunya; economical support from CERCA Programme and Fundació Adey.

Salvage Autologous Peripheral Blood Stem Cell Transplantation after the Development of tMDS or AML in Multiple Myeloma Patients

Multiple Myeloma (MM) patients are often treated with high-dose chemotherapy and autologous stem cell transplantation (ASCT) for disease control. Lenalidomide is typically used as maintenance but results in an estimated 3.3% chance of developing a therapy-related myeloid neoplasm (TMN) typically myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML). This complication is often devastating with a median survival for those with a TMN of just 13 months. Over the past 6 years, 4 patients with multiple myeloma who developed a TMN were treated with a salvage ASCT using banked stem cells collected prior to their initial ASCT and before lenalidomide maintenance. None of these patients qualified for an allogeneic transplant. Melphalan 140 - 200 mg/m2 was used as conditioning prior to infusion of the peripheral blood stem cells, resulting in successful MDS/AML disease control. Case 1 = 61-year-old man with MM initially treated with CyBorD then transplanted in 2010. Post transplant treatment included lenalidomide maintenance, followed by a variety of salvage regimens over the next 7 years including thalidomide, carfilzomib, daratumumab and pomalidomide. He developed cytopenias evaluated by bone marrow biopsy in May 2017. This showed tMDS (Karyotype 47-48, XY, der (5)t(5:20)(q11.1;q11.1), +19, der(21:22)(q10:q10),+2~3mar[cp11]/46-48,idem,-der(5),+20[cp5]/46,XY[4]. FISH confirmed deletion 5q in 76% and loss of chromosome 5 in 6.5% of cells. NGS showed a frame shift mutation of TP53 at a variant allele frequency of 92.5%. He underwent a second ASCT in Aug 2017 and recovered normal hematopoiesis within three weeks. Unfortunately, he developed an aggressive relapse of his multiple myeloma 9 months later and succumbed from refractory multiple myeloma 14 months after the salvage transplant, without evidence of MDS. Case 2 = 72-year-old man with MM initially treated with CyBorD then an ASCT in May 2015. Lenalidomide maintenance was continued until October 2017 when he developed severe thrombocytopenia and circulating blasts. A bone marrow biopsy showed tMDS EB-1. Cytogenetics 46, XY t(3:21) (q26.2,q22) [18]/46, XY[2]. He received a second ASCT after melphalan 140 mg/m2 in Oct 2017 followed by recovery of normal blood counts. Bone marrow biopsy showed complete remission. He remains in remission from both the multiple myeloma and MDS almost 6 years later without any further therapy. Case 3 = 65-year-old man with MM treated with bortezomib + dexamethasone followed by ASCT in Aug 2012. He received maintenance lenalidomide for 4 years but then progressed. In Jan 2018 he developed pancytopenia and bone marrow biopsy demonstrated MDS with ringed sideroblasts. Cytogenetics were normal but NGS showed mutations of ASXL1, RUNX1, SF3B1, SETBP1 and DNMT3A. He underwent a second ASCT using his banked stem cells after melphalan 140 mg/m2 in Jan 2018 followed by full hematopoietic recovery. A repeat bone marrow biopsy in March 2022 showed persistent MDS harboring ASXL1, SF3B1, and DNMT3A mutations, but no detectable RUNX1 or SETBP1 abnormalities. His multiple myeloma remains in remission >5 years later. He receives only intermittent erythropoietin to maintain a Hgb of 11. Case 4 = 58-year-old man with MM initially treated with CyBorD followed by an ASCT in May 2014. He received lenalidomide maintenance but relapsed multiple times after 4 years ultimately complicated by renal failure requiring permanent dialysis. In March 2022 he developed pancytopenia. Bone marrow biopsy showed 15% blasts harboring del(5q), del(17p) and trisomy 8 by FISH. NGS demonstrated a mutation of ASXL1, and a copy number loss of TP53. Six months of decitabine did not improve his blood counts. In Dec 2022 he underwent a second ASCT after melphalan 140 mg/m2. A repeat bone marrow biopsy in Feb 2023 showed a complete morphological remission with a normal NGS. He is off all treatment 8 months post-transplant other than erythropoeitic support due to ongoing dialysis-dependent renal failure. The development of TMN is an uncommon but devastating event in patients with multiple myeloma after ASCT and lenalidomide maintenance. These patients responded well to a salvage ASCT using banked stem cells. This approach was well tolerated and provided durable hematopoietic recovery. Second ASCT using banked autologous stem cells should be considered in such patients who are not otherwise candidates for allogeneic transplantation.

Prevalence, Dynamics and Clinical Significance of Clonal Hematopoiesis of Indeterminate Potential (CHIP) in Newly Diagnosed Cancer Patients

INTRODUCTION Prevalence of CHIP in cancer patients (pts) is estimated at about 25%, its presence being associated with inferior outcomes and with increased risk of development of therapy-related myeloid neoplasms (TRMN). Despite the increased body of knowledge on cancer and CHIP, processes driving the selection of clones and their latter malignant transformation have not been fully elucidated. We hypothesized that CHIP in cancer pts might not only lead to TRMN but also affect the prognosis of the primary neoplasm and its treatment-related toxicity. Our study aims to describe the prevalence and dynamics of CHIP in treatment-naïve pts with cancer and to analyze its impact on clinical outcomes. METHODS This study included 103 pts with a first cancer diagnosis at age ≥ 60 years and eligible for anticancer treatment without prior exposure to cytostatic agents. Peripheral blood (PB) samples were collected at diagnosis and 6 months after treatment. A customized NGS panel covering common CHIP genes ( DNMT3A, TET2, ASXL1, JAK2, PPM1D, TP53, SF3B1, GNB1, SRSF2, CHEK2, CBL, GNAS, and NRAS) was used to identify CHIP-positive (VAF≥ 1%) and CHIP-negative cases. Clonal dynamics were assessed through NGS at 6 months after treatment, categorized as ‘growing’ when their VAF increased by more than 25% compared to the baseline, ‘shrinking’ when VAF decreased by more than 25% compared to the baseline and ‘stable’ if it remains unaltered. RESULTS Baseline characteristics are shown in table 1.The prevalence of CHIP in our cohort was 35%, with an average of 1.5 somatic variants per patient and 54 identified variants, and a median VAF of 4.7% (IQR 2.5-11.0%). Notably, mutations in DNMT3A (33%), TET2 (28%), and PPM1D (13%) were the most prevailing gene aberrations, accounting for nearly 75% of all variants, while other common CHIP genes such as ASXL1 (9%) or JAK2 (0%) were less frequent. TP53 variants represented 9% of all mutations, whereas SF3B1, GNB1, SRSF2, and CBL each accounted for 2%. Breast cancer pts displayed a significantly higher prevalence of CHIP compared to other primary neoplasms (66% vs. 36%, p=0.01) whereas no patient with bladder neoplasm presented CHIP at diagnosis (p=0.04). These observations were not warranted only by differences in the age or smoking habit of these subgroup of neoplasms. The mutational spectrum of CHIP across different cancer categories was comparable. Among 20 paired samples sequenced (baseline and post-genotoxic exposure), 41% of all variants exhibited a growing pattern, 31% a shrinking pattern, and 28% remained static (Figure 1). Platinum-based therapy exposition promoted clonal expansion in DNMT3A mutations (p=0.03), while this effect was not observed in PPM1D or other genes, likely due to the low sample size. Age, tobacco use, and type of primary neoplasm did not appear to influence clonal fitness. There were no significant differences in the incidence of infectious complications or chemotherapy-induced hematologic toxicity between CHIP-positive and CHIP-negative cohorts. With the current follow-up time (16.8 months), the overall response (ORR) and complete response rates (CR) appeared comparable (ORR: 94% in CHIP-positive vs. 85% in CHIP-negative; CR: 79% vs. 70%, respectively). A patient with diffuse large B-cell lymphoma and CHIP ( ASXL1, PPM1D, and TP53 variants) developed a TRMN (MDS-MD) 7 months after completing R-CHOP treatment. CONCLUSION The prevalence of CHIP in our cancer pts cohort is 35%, with breast cancer cases displaying a CHIP occurrence around 62% not previously reported. Our study highlights an enrichment of mutations in PPM1D in treatment-naïve cancer pts, surpassing the frequency of ASXL1 in contrast to prior literature. Genotoxic therapy promotes clonal expansion in 41% of variants in our cohort; although factors influencing CHIP fitness remain poorly understood, DNMT3A showed heightened susceptibility to platinum therapy. Finally, and in contrast contrast with our initial hypothesis, we found no evidence of impaired outcomes in the CHIP population. These results emphasize the need for further longitudinal follow-up. Acknowledgements: This work was supported by two grants from the Instituto de Salud Carlos III (PI20/00881 and PI 20/00531)(Co-funded by European Regional Development Fund. ERDF, a way to build Europe). 2021 SGR 00560 (GRC) Generalitat de Catalunya; economical support from CERCA Programme.

CUX1mut Acute Myeloid Leukemia As a Distinct Biological Entity: An Analysis of Clinical Outcomes and Implications

1. Introduction CUX1 is a tumor suppressor gene located on the long arm of chromosome 7. CUX1 mutations induce apoptosis evasion and are more commonly seen in therapy-related myeloid neoplasms. It is frequently mutated in myelodysplastic syndrome and less frequently in acute myeloid leukemia (AML). Preclinical investigations are ongoing to explore therapeutic approaches in CUX1-deficient leukemia cell lines. However, data on the clinical outcomes of patients with AML and mutated CUX1 are sparse. We aimed to characterize the biological and clinical outcomes of CUX1 mut AML. 2. Methods We analyzed next-generation sequencing from 318 patients in the Project ERIS database with newly diagnosed AML treated from January 1, 2013 to April 18, 2023 at VCU Massey Comprehensive Cancer Center. We identified 12 patients with CUX1 mut AML and compared them to a cohort of 306 CUX1 wild-type historical controls. We recorded baseline patient-related and disease characteristics, including age, ECOG, Charlson comorbidity index (CCI) scores, molecular profiling and ELN 2022 cytogenetic risk, dates of regimen initiation, and survival. We used the D'Agostino & Pearson method for normality testing and the t-test or Mann-Whitney (as applicable) tests for between-group comparisons. Categorical comparisons used Fischer's exact test. We applied the Bonferroni correction if multiple comparisons were made. The Wilson-Brown method was used for 95% confidence intervals. We analyzed survival by the Kaplan-Meier method with significance determined by the log-rank test. The event for calculating the overall survival (OS) was the date of death. Patients were otherwise censored at the date of last contact. 3. Results CUX1 mutations are found at a frequency of 3.9% (95% CI, 2.2-6.5) in AML. The median variant allele frequency of CUX1 mutations was 40.3% (range: 3.8-48.7), consistent with a heterozygous presentation. The median age at diagnosis of patients with CUX1 mut who received any anti-leukemic therapy was 57 years (range: 30-78 years), which was not significantly different from CUX1 wt at 65 years (range: 19-88, p = 0.312). Thirty-three (95% CI, 13.8-60.9) percent of patients had AML with myelodysplasia-related changes (AML-MRC) on the initial diagnostic bone marrow biopsy, compared with 29.2% (95% CI, 24.2-34.7, p=0.752) for CUX1 wt. CUX1 mut AML was found in cytogenetically normal specimens at a frequency of 30.0% compared with 49.3% for CUX1 wt (p = 0.337). The most frequent cytogenetic abnormality in CUX1 mut AML was -7 (20.0%), followed by +8 (10.0%) and complex cytogenetics (10.0%). The most frequent cooperating molecular lesions were RUNX1, SRSF2, STAG2, FLT3-ITD, and NRAS (25.0% each). Patients with CUX1 mutations were nonsignificantly more likely to have ELN 2022 adverse-risk disease (odds ratio: 2.013, p = 0.379). In patients treated with intensive cytarabine-based inductions, the median overall survival was 26.3 months in the CUX1 mut group and 17.6 months in the CUX1 wt cohort, which was not significantly different (p = 0.501). Two patients with CUX1 mut AML were treated with first-line venetoclax and a hypomethylating agent. The overall survival was 12.7 months in one patient and not reached at a follow-up time of 20.2 months in the second. 4. Discussion While definitive conclusions are limited by the rarity of the mutation, mutations in CUX1 are heterozygous and appear to be non-significantly more frequently associated with myelodysplastic-related changes and secondary-type mutations. These findings may explain a higher proportion of patients harboring CUX1 mutations that have ELN 2022 adverse-risk disease. While we did not detect any significant survival differences between patients with and without CUX1 mutations, larger cohorts are needed to confirm the prognostic impact of these rare mutations in AML.

Clinical Characteristics and Outcomes of Therapy-Related Chronic Myelomonocytic Leukemia: Analysis of a Large Cohort

Introduction: Therapy-related myeloid neoplasms are associated with a previous exposure to cytotoxic agents or ionizing radiation, and account for 10-20% of all myeloid neoplasms. They are associated with a worse outcome, usually with high risk genetic abnormalities and poor response to therapies. Chronic myelomonocytic leukemia (CMML) is a rare myeloid neoplasm with persistent monocytosis, bone marrow dysplasia and an inherent risk of acute myeloid leukemia (AML) transformation. Due to its rarity, it is difficult to establish the specific impact of previous therapy in CMML. We previously reported the characteristics and outcomes of patients with therapy-related CMML (tCMML). Herein we report an update, including molecular characteristics, being the largest tCMML cohort reported to date. Methods: We retrospective evaluated a cohort of patients diagnosed with CMML from 2005 to 2022. Data regarding previous exposure to treatment was captured and tCMML diagnosis was established for patients with a documented prior history of radiation therapy or cytotoxic chemotherapy due to an antecedent malignancy prior to the diagnosis of CMML. Those non-tCMML were defined as de novo CMML (dnCMML). Overall survival (OS) was calculated from diagnosis to death, and leukemia-free survival (LFS) was calculated from diagnosis to death or AML transformation. Results: A total of 532 patients with CMML were included in the study, of whom 71 (13%) were tCMML. When comparing tCMML with dnCMML, there were no differences in median age (73 vs 70 years old) or sex (67% vs 69% male). No differences were noted in white blood cell count, absolute monocyte count, hemoglobin or platelet count. According to the WHO 2016 classification, 38% vs 44% were CMML-0, 40% vs 36% were CMML-1, and 22% vs 20% were CMML-2, for patients with dnCMML and tCMML, respectively. According to the FAB classification, 42% vs 52% were dysplastic and 58% vs 49% were proliferative, for patients with dnCMML and tCMML, respectively. Chromosome 7 abnormalities where more common among tCMML compared to dnCMML (14% vs 4%, p=0.006), with no differences in frequency of complex karyotype (8% vs 3%, p=0.1). In tCMML patients, 35% received only radiotherapy, 31% only chemotherapy and 34% both. In patients that received chemotherapy, the most common therapies were alkylating agents (45%), agents targeting microtubules (37%) and antimetabolite agents (35%). The median latency from therapy to tCMML was 6.5 years (range, 0.1-24). Most common neoplasms prior to the diagnosis of tCMML were prostate (25%), lymphoma (23%) and breast cancer (14%). Most frequently detected mutations at diagnosis were TET2 (55% vs 61%, p=0.5), ASXL1 (44% vs 53%, p=0.3), and SRSF2 (43% vs 30%, p=0.1), in patients with dnCMML and tCMML, respectively. Patients with tCMML had a lower incidence of NRAS (22% vs 6%, p=0.007) and CBL (18% vs 5%, p=0.04) mutations compared to dnCMML, but these mutations were present at a higher median VAF among patients with tCMML (48% vs 18% [p=0.04] for NRAS and 91% vs 20% [p=0.02] for CBL) (Figure 1). TP53 mutations were more frequent in the tCMML although differences were not significant (12% vs 4%, p=0.06). Among all patients, 352 patients received treatment and were evaluable for response (305 patients with dnCMML and 47 patients with tCMML). More than 90% of patients received an hypomethylating agent-based therapy, with an ORR of 59% for dnCMML and 60% for tCMML. The median follow-up was 58 months, and the median OS and LFS for the entire cohort were 36 and 29 months, respectively. No differences in OS (36 vs 35 months, p=0.3) or LFS (29 vs 28 months, p=0.8) were observed between dnCMML and tCMML, However, when comparing by risk groups according to CPSS-Mol, patients with tCMML classified as intermediate-1 (Int-1) risk had a significant lower OS (38 vs 88 months, p=0.02) and LFS (38 vs 85 months, p=0.03), compared to Int-1 dnCMML (Figure 2). In a multivariate model for OS including CPSS-mol, tCMML stratified by type of therapy and age, patients with tCMML with a previous exposure to chemotherapy had a hazard ratio of 1.76 (1.07-2.89, p=0.03). Conclusion: Patients with tCMML have a higher proportion of chromosome 7 abnormalities and a lower incidence of NRAS and CBL mutations. Although responses to HMA were similar, patients with tCMML with a previous exposure to chemotherapy are associated with a lower survival, especially patients classified as CPSS-Mol intermediate-1.

Mutational Profile and Dynamics of PPM1D-Mutant Clones in the Spectrum of Myeloid Disorders

Introduction Mutations in PPM1D a regulator of DNA damage response, are enriched in patients with clonal hematopoiesis (CH) exposed to cytotoxic treatment or with therapy-related myeloid neoplasm. However, their role in leukemic transformation is unclear. Here, we describe a large cohort of patients with PPM1D mutations from CH to acute myeloid leukemia (AML) to understand the molecular landscape of mutant PPM1D related disease and the longitudinal dynamics of CH under treatment. Methods We included, in a non-sequential cohort, 96 PPM1D mutated patients treated at Gustave Roussy with CH, clonal cytopenia of unknown significance (CCUS), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) or AML. A 77 gene panel NGS analysis was performed. An additional 16 PPM1D mutated AML patients from ALFA trials (1200, 0701,0702) were included. 10 patients with ovarian cancer from this cohort had sequential blood samples (OvBIOMark trial) available prior to hematologic evaluation, from the diagnosis or first relapse of their cancer through the course of therapy. Those samples were analyzed with a UMI based 18 gene panel (HaloplexHS, Agilent). Overall survival (OS) analyses were performed with the Kaplan-Meier method from the time of diagnosis until death from any cause. Results Among the 112 patients with PPM1D mutations, 23 (21%) had CH, 36 (32%) CCUS, 19 (17%) MDS, 25 (22%) AML (including 4 relapses), and 9 (8%) MPN. Median age was 65 [range, 21-88] years with 67% of females. Only 18% of the patients had no previous cancer history. Most frequent primary cancers were gynecological cancer (27%), lymphoid malignancies (22%), and breast cancer (17%). Median time between primary cancer diagnosis and hematologic assessment was 5.3 [range, 1.2-8.5] years. Eighty six percent of patients had one PPM1D mutation, 9% had 2, and 5% had 3 or more (restricted to CH and CCUS). Median variant allele frequency (VAF) was 3% [0.2-38], 3% [1-31], 2.4% [1-41], 23% [1-50], and 4% [1-42] in CH, CCUS, MDS, AML and MPN, respectively. Among CH/CCUS patients, PPM1D was the sole detected somatic mutation in 39% (23/59), compared to 9% (5/53) in MDS/AML/MPN patients (odds ratio=6, p=0.0004); 3/5 of the latter had complex karyotype. The most frequently co-mutated genes were DNMT3A (29%) and TP53 (25%), uniformly across all conditions ( Fig 1A). MDS-related gene mutations, RUNX1 (7%), ASXL1 (4%), SF3B1 (4%), SRSF2 (4%), U2AF1 (4%), were specific to AML/MDS/MPN. Among 28 patients with both PPM1D and TP53 mutations, PPM1D mutations were dominant or co-dominant in 64% (18/28), and secondary in 36% (10/28). IPSS-M risk of evaluable MDS was high/very high in 54% (7/13) of patients. ELN 2022 classification of AML was adverse in 68% (17/25) of patients. AML treatment options included best supportive care for 8% of patients (2), 5-Azacytidine for 32% (8), intensive chemotherapy for 60% (15). With a median follow up of 2.4 years, the median OS was 4.6 (CI95%; 4.1-NA), 1.31 (CI95%; 0.53-NA), 1.27 (0.43-NA), 0.66 (CI95%; 0.42-1.26) and 20.4 (CI95%; 20.4-NA) years for CH, CCUS, MDS, AML and MPN, respectively. TP53 mutation status did not stratify OS. Four ovarian cancer patients with CH/CCUS transformed to MDS/AML with a median of 5.3 [1-12] years. At transformation, 3/4 had a stable PPM1D mutation, 1/4 an increase in PPM1D VAF (2 to 28%), and 3/4 had TP53 mutations before and at transformation. We analyzed 67 timepoints from 10 ovarian cancer patients during therapy (median 7 per patient). 10/10 received alkylating agents and 5/10 PARPi. The median number of mutations per patient was 4 and 6 at baseline and last follow-up, respectively. PPM1D-mutated clone size increased from 0.4% [0.1-3] at baseline to 7% [2-30] at last follow-up. Beyond clonal expansion, PPM1D VAF dynamics showed non-linear changes related to alkylating agents exposure: 2/10 patients had a continuous expansion, 2/10 had expansion then contraction (GR-1/3, Fig 1B), and 6/10 had expansion then stabilization (GR-2, Fig 1B). Conclusion We described a large cohort of PPM1D mutated patients from CH to AML. Their prognosis was poor independently of TP53 mutation at AML/MDS stage. PPM1D mutations were frequently part of the dominant clone. To confirm the clonal architecture, single cell sequencing analysis in 8 AML/MDS patients are ongoing. Trajectory of PPM1D mutations in ovarian cancer patients revealed a non-linear alkylating agent dependency which warrants further investigation.

Molecular and Cytogenetic Characteristics of Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) in Patients Exposed to Chemotherapy (CT) and/or Radiation (XRT)

Background: Therapy-related myeloid neoplasm (t-MNs) are caused by exposure to CT and/or XRT and present with high rates of adverse cytogenetics (CG). However there are pts who present with non-adverse CG making the definition of T-MN controversial. Correlation between exposure to specific CT, molecular profile, and impact on overall survival (OS) has not been established. Methods: 171 CT/XRT exposed pts with AML/MDS from Weill Cornell and Northwestern University were evaluated for disease characteristics, CT/XRT treatment, and survival. Study is ongoing and we plan report on appx 500 CT/XRT exposed pts with AML/MDS. CT exposure was categorized as: Alkylators, Topoisomerase inhibitors (TI), Anthracyclines, Taxanes, Anti-metabolites and Platinum compounds. Mutations were classified as: Signaling/kinase pathway ( FLT3, KRAS, NRAS, PTPN11, NF1), Epigenetic modifiers ( DNMT3A, IDH1, IDH2, TET2, ASXL1, EZH2), NPM1, Transcription factors ( CEBPA, RUNX1, GATA2), Tumor suppressors ( TP53), Spliceosome ( SRSF2, U2AF1, SF3B1, ZRSR2), Cohesin ( RAD21, STAG1, STAG2, SMC1A, SMC3) and secondary type mutations ( ASXL1, BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2). Analyses: Chi-square and Fisher's Exact test was used for comparisons of categorical and continuous factors. Cox Regression model was used to evaluate the effect of gene mutations and CG risk factors on OS. All p-values are two-sided with significance at 0.05 alpha level (SAS Version 9.4, SAS Institute, Inc., Cary, NC). Results: Among 171 with CT/XRT exposure, median age at diagnosis was 70 yrs (AML: 76%; MDS 24%). 56%, 20% and 24% had exposure to CT only, XRT only, and CT+XRT, respectively. In t-AML, 52% and 48% had adverse and non-adverse CG, respectively, with no difference in t-MDS (50% in both). Pts (AML/MDS) with previous CT/CT+XRT were more likely to have adverse CG compared to XRT-treated pts (56% vs. 33%, HR 4.16, p <0.0001). With regards to specific CT exposure, presence of adverse CG was higher in pts exposed to alkylators (OR 3.11, p=0.0002), anti-metabolites (OR 2.55, p=0.02), TI (OR 1.9, p=0.05) and platinums (OR 2.40, p=0.01); this was not observed with anthracyclines and taxanes. Characteristics of pts presenting with adverse CG vs. not are shown in Table 1 along with OS with mutation classes. Age-adjusted 5-year OS of adverse CG vs. non-adverse-risk pts exposed to CT/XRT was less than 5% vs. 30%. Median OS of pts exposed to CT/XRT vs XRT alone was 14 months (95% C.I. 11-18) vs 16 months (95% C.I 10-44). Only 25.88 % of CT/XRT exposed AML pts with non-adverse CG were classified as ELN adverse (2022). Mutation frequencies by CT exposure are detailed in Table 2. TP53 mutations were more common in pts exposed to anti-metabolites (OR 2.71, C.I. 1.28-5.72, p=0.01) and platinums (OR 2.12, C.I. 1.09-4.13, p=0.03) (comparison with other CTs) while epigenetic modifiers were less likely with anthracyclines, alkylators, platinums, TI, and taxanes. Secondary type mutations were not enriched in pts exposed to any particular CT class (alkylators 25%, TI 30%, antimetabolites 28%, cisplatin 24%, and taxanes 26%). When stratified by latency from CT/XRT exposure (<5 vs > 5 yrs; Table 1), we found that adverse CG were seen in 47.22 % and 54.55 % of pts when disease was diagnosed <5 and >5 yrs, respectively. Frequency of TP53 mutations were higher with latency <5 yrs, while epigenetic mutations were more likely for >5 yrs latency. Finally we looked at treatment in a subset of pts (n=98). 57.5% of pts were treated with HMA+venetoclax, 20.6% with 7+3, 8.8% with HIDAC regimen, 14.7% with CPX-351, 17.5% with HMA alone. CR/CRi was attained by 46.4% (53% in HMA+V vs. 39% others) and 28.8% were primary refractory. Among pts <75 yrs (n=118), 38.98% were consolidated with HSCT. Analyses comparing to pts w/o CT/XRT exposure will be presented. Conclusions: In our pts with AML/MDS exposed to previous chemotherapy/XRT, we found (i) True adverse CG, TP53 and secondary type mutations were present in a subset of AML/MDS, thus suggesting that not all exposure has a t-MN like signature and therapy relatedness needs to be redefined by biological subgroups rather than exposure alone. (ii) OS of non-adverse CG (AML) was comparable to historical data in denovo AML again suggesting that not all AML/MDS after chemo/XRT exposure behaves like t-MN (iii) We found an association of shorter latency and antimetabolite and platinum exposure to TP53 mutations and needs further validation in a larger dataset.

Single-Cell Whole-Genome Sequencing after Chemotherapeutic Treatment Reveals Cell Type-Specific Mutational Burdens

Introduction The hematopoietic system is especially vulnerable to chemotherapeutic treatment-related long-term consequences, including clonal and malignant hematopoiesis. These diseases are caused by the acquisition of driver mutations in hematopoietic stem and progenitor cells (HSPCs). An increased rate of mutation accumulation, as identified in HSPCs of patients who receive chemotherapeutic treatment, can therefore result in disrupted blood production. Accordingly, childhood cancer survivors have a five-fold increased risk of secondary cancers, amongst which therapy-related myeloid neoplasms (t-MN) are one of the most prevalent. Here, we aim to increase the understanding of the origin of t-MN through the characterization of the short- and long-term mutational consequences of chemotherapy in different hematopoietic stem and progenitor cell types. Methods We studied the genome-wide mutation burden in the HSPC compartment at the time of an initial cancer diagnosis, following chemotherapeutic treatment, and upon a second cancer diagnosis. We performed whole-genome sequencing (WGS) of single HSPCs, including hematopoietic stem cells (HSCs, CD34+CD38-CD45RA-CD90+), multipotent progenitor cells (MPPs, CD34+CD38-CD45RA-CD90-), common myeloid progenitor cells (CMPs, CD34+CD38+CD45RA-), and granulocyte-monocyte progenitor cells (GMPs, CD34+CD38+CD45RA+). In order to obtain enough DNA for WGS, we performed clonal expansion of single cells or primary template-directed amplification of the DNA of one cell. Next, we normalized the mutation burden in each cell to the expected number of mutations (Osorio et al. Cell Reports 2018). In addition, we performed both whole genome and transcriptome sequencing of individual HSCs/MPPs. Results First, we investigated pediatric ten t-MN patients with a latency between the initial and second cancer diagnosis spanning 1-12 years. We applied WGS to clonally expanded HSPCs (CD34+CD38-CD45RA-) and found that the relative increase in mutation burden in HSPCs is restricted to the first five years after initial treatment. To gain more insight into the heterogeneous HSPC compartment, we subsequently investigated cell-type specific mutational loads based on immunophenotypic markers. So far, we have included three pediatric cancer patients with comparable treatment regimens and diagnoses, namely medium risk acute lymphoblastic leukemia. Here, we found that the mutation burden is lower in HSCs when compared to the progenitor cell types (Figure 1A). When comparing the mutation load in the HSCs to the linear mutation accumulation observed in HSCs and MPPs of healthy donors, we identified a fraction of cells overlapping with the expected mutation load based on the age of the patient (Figure 1B). These data suggest that HSCs are more protected against chemotherapy-induced mutagenesis than progenitor cells, although this protective effect varies between individual cells. Therefore, we also performed both whole genome and transcriptome sequencing from a single cell, to characterize the transcriptional state of HSCs/MPPs showing a relatively high or low mutation load within their respective cell type. Conclusions Our results indicate that primitive hematopoietic stem cells are more protected against chemotherapy-induced mutations than the more proliferative progenitor cells. In addition, we find that in t-MN patients with a long latency (>5 years), the mutation load in HSPCs returns to the expected baseline. Based on our data, we hypothesize that the more damaged progenitor cells may be replaced over time, fueled by the undamaged primitive stem cell pool. Thus, our results may explain why the risk of t-MN drops 5-7 years after treatment end.

TP53 Y220C Mutations in Patients with Myeloid Malignancies

Background: TP53 gene mutations lead to inactivation of the protein p53, causing genome instability, oncogenesis, and treatment resistance conferring an unfavorable prognosis. TP53 Y220C missense mutations in exon 6 (p.659A>G) are described as a frequent ‘hot spot’ mutation in solid tumors. A novel Y220C-targeted small molecule (PC14586) has shown preliminary efficacy and safety in patients with solid tumors (Dumbrava, E., ASCO 2022). No data has been reported about TP53 Y220C in hematologic malignancies to date. We set out to evaluate the clinicopathologic characteristics of TP53 Y220C in hematologic malignancies and particularly, myeloid neoplasms. Methods: We identified all patients with hematologic neoplasms with TP53 Y220C mutations at University of Texas MD Anderson Cancer Center and Memorial Sloan-Kettering Cancer Center, detected by standard next generation sequencing assays, and focused on patients with myeloid malignancies; summarizing the patient characteristics, disease features and outcomes for patients with this specific TP53 mutation. Overall survival (OS) was assessed by the Kaplan-Meier method from date of diagnosis to date of death from any cause or date of last follow-up. Leukemia-free survival (LFS) was calculated from time to diagnosis of TP53 Y220C mutated myelodysplastic syndrome (MDS) to date of transformation to acute myeloid leukemia (AML) or death. Survival analysis was conducted only for newly diagnosed patients with available follow-up data. Results: We identified 143 patients (pts) with hematologic malignancies harboring a TP53 Y220C mutation. MDS and AML were the most frequent diagnoses (MDS, n=66; AML, n=48). Median age was 69 years (range, 32-92) and 56% of patients were male (Table 1). We then analyzed in more detail patients with MNs, including CHIP/CCUS, MDS, AML and MDS/MPN. Among those, in 76 (64%) pts the TP53 Y220C mutation was detected at initial diagnosis and in 43 (36%) pts, TP53 Y220C was identified in the relapsed/refractory setting. Therapy-related myeloid neoplasms (MNs) accounted for 51 of 119 pts (41%). Complex karyotype was frequent (n=88/119, 74%) in the setting of TP53 Y220C. Median TP53 Y220C variant allele frequency (VAF) was 32% (range, 1%-96%). Eighty-two patients (69%) had additional co-mutations: 24 (20%) pts had additional (non-Y220C) TP53 mutations only; 34 (29%) pts had only non- TP53 co-mutations (most frequently DNMT3A and TET2) and 24 (20%) pts had additional TP53 mutations in the setting of other co-mutations. With a median follow-up of 9 months, median OS for patients with newly diagnosed TP53 Y220C MDS (n=39) and AML (n=23) was 13.1 months (95% CI, 7.6 to 18.7) and 8 months (95% CI, 5.6 to 13.4), respectively ( p=0.06) (Figure 2). For the TP53 Y220C MDS cohort, AML transformation occurred in 13 (33%) patients, after a median of 14.3 months. Median LFS for these patients was 12.2 months (95% CI, 7.9 to 16.5). Modest and non-statistically significant differences in median LFS for MDS patients who did and did not receive stem cell transplant (SCT) were identified; MDS+SCT (n=12) 14.5 months (95% CI, 4 to 25) vs MDS+no-SCT (n=27) 9.3 months (95% CI, 4.6 to 14), p=0.18. Interestingly, four patients diagnosed with TP53 Y220C mutated CHIP/CCUS with a median VAF of 2% were identified, and with a median follow-up of 34.6 months, none have developed dysplastic changes or progression to MDS or AML at the time of cut off. Additional outcomes data will be forthcoming in the analysis of this cohort of patients with TP53 Y220C mutated myeloid neoplasms. Conclusions: TP53 Y220C mutations are present in malignant blood disorders and are particularly more common in myelodysplastic syndromes and acute myeloid leukemia, with associated poor outcomes. Novel targeted therapies for this TP53 variant (Y220C) would be of interest for this patient population.

Ongoing Phase III Randomized Trial of Salvage DFP-10917 Vs. Non-Intensive Reinduction or Intensive Reinduction for Acute Myelogenous Leukemia (AML)

Background: DFP-10917 is a deoxycytidine analogue with a novel structure. It contains a side chain cyanide (CN) group at the 2 carbon of deoxyribose that induces DNA damage in a manner unique from structurally related nucleoside analogues (eg, cytarabine, decitabine, or gemcitabine). When administered at high doses, DFP-10917 inactivates deoxyribonucleic acid (DNA) polymerase leading to S-phase arrest similar to cytarabine. When administered at low concentrations by continuous infusion, it displays an alternative, potentially advantageous, mechanism of action - DNA strand breakage leading to G2/M phase arrest and ultimately cell apoptosis. The strand breakage occurs via a spontaneous beta elimination reaction that requires no additional enzymatic activity. There is no current standard treatment for patients with relapsed/refractory (R/R) AML. Results of a completed Phase I/II study in R/R AML (D11-11002; NCT01702155) demonstrated preliminary efficacy of DFP-10917 in this indication, with a ~50% complete response (CR) rate in Phase II. Five of 14 patients who achieved CR proceeded to stem cell transplantation. Based on completed studies, risks associated with DFP-10917 are predominantly hematological (eg, neutropenia, anemia, febrile neutropenia, leukopenia, lymphopenia, thrombocytopenia). Study Design and Methods: Based on promising Phase II results, the Sponsor initiated Study D18-11141 (NCT03926624), a Phase III, randomized, controlled study to compare the rate and duration of CR in patients with AML R/R to 2-4 prior induction regimens. Patients receive either DFP-10917 or non-intensive reinduction (low-dose cytarabine [LoDAC], azacitidine, decitabine, venetoclax + LoDAC or azacitidine or decitabine) or intensive reinduction (high and intermediate dose cytarabine regimens). Patients assigned to DFP-10917 receive 6 mg/m 2/day by continuous infusion for 14 days followed by a 14-day rest period. Control treatment is administered per physician's decision. Adults aged ≥18 years with histologically or pathologically confirmed diagnosis of AML based on World Health Organization classification and adequate performance status (≤2) and organ function are eligible. Study Status: The study is ongoing at 34 study centers in the United States. As of July 2023, 162 patients were enrolled, with 82 assigned to DFP-10917. A majority of the patients are male (57.4%) and aged <75 years (83.3%). Overall, 44% have AML with myelodysplasia-related changes; 36% have AML not otherwise specified; and <20% have AML with recurrent genetic abnormalities or therapy-related myeloid neoplasms. Enrollment and management of patients were affected from 2020 to 2021 by COVID-19 restrictions. A prospectively-planned interim analysis will occur after the 150 th patient randomized is followed for >2 months. Based on recruitment projections, this analysis is scheduled for 09/2023. At that time, the Data Safety Monitoring Board will review an unblinded estimate of the treatment effect for the primary endpoint of CR, with the study either stopped for superiority or futility or continuing to a final analysis. If continued, the number of patients required for the final analysis will range from 300 to 450, in increments of 50, with the number determined based on the size of the interim treatment effect for the primary endpoint of CR or the secondary endpoint of overall survival.

Longitudinal Screening of Mutant Hematopoietic Progenitor Cells (HPC) in Multiple Myeloma (MM) and Its Association with Secondary Primary Malignancies (SPM)

Background: The incidence of SPM in transplant eligible MM patients (pts) receiving lenalidomide maintenance is ≥ 12% and is associated with inferior overall survival (OS). Recent evidence suggests that while clonal hematopoiesis does not increase the risk of SPM, specific TP53 mutant HPC may expand under lenalidomide treatment and give rise to therapy related myeloid neoplasms and acute lymphoblastic leukemia. Genetic screening may facilitate personalized treatment to minimize the risk of SPM. However, there are no longitudinal studies in purified HPC to inform on the evolution of specific gene mutations and its association with clinical outcomes. Aim: Analyze the mutational landscape of HPC at diagnosis and throughout lenalidomide based regimens. Methods: This study included 335 pts: 43 with high risk smoldering MM, 223 transplant eligible and 69 ineligible active MM respectively enrolled in the CESAR, GEM2012MENOS/GEM2014MAIN and CLARIDEX clinical trials. Of the 335 pts analyzed at diagnosis, 60 were further investigated after induction, 30 at day 100 after autologous transplant (ASCT) and 47 during maintenance. Two or more studies were available throughout treatment in 36 pts. Fluorescence activated cell sorting of CD34+ HPC was performed in a total of 500 bone marrow aspirates. DNA was analyzed with a panel of 55 genes recurrently mutated in myeloid neoplasms. Sequencing depth was >1000x in 95% of nucleotides. Criteria to filter out included synonymous, intronic, invalid-transcript, 5'UTR, panel error and single nucleotide polymorphisms. The criteria to filter in was a AMP/ACMG classification of uncertain significance, likely pathogenic or pathogenic. Only the latter two were included in downstream analysis. Results: HPC from 184 of the 335 (55%) pts were mutated. The frequency in high-risk smoldering MM, transplant eligible and ineligible active MM was 46%, 26% and 35%, respectively. The genes more frequently mutated were DNMT3A (n=94, 52%), TP53 (n=29, 16%) and TET2 (n=28, 15%). The median variant allele frequency was 4.9, 7.5 and 7.0, respectively. In a pt-paired analysis between diagnosis and induction, 8 mutations were present in both time points, 25 became undetectable and 22 were acquired during treatment. The latter (eg, PPM1D and SF3B1) were observed in 14 of the 60 (23%) pts. In the 30 pts with paired samples at diagnosis and at day 100 after ASCT, 4 mutations were present in both time points, 48 became undetectable and only 3 were acquired during treatment ( ATRX, MPL and SAMDL9). Thus, the frequency of mutant HPC was higher at diagnosis vs ASCT (23% vs 5%, P <.001). In a pt-paired analysis between diagnosis and maintenance, 13 mutations were present in both time points, 12 became undetectable and 30 were acquired during treatment. The latter (eg, PPM1D and SF3B1) were observed in 24 of the 47 (51%) pts screened at both time points, and after a median of 5 years since diagnosis. With a median follow-up of 6 years, 17 of the 335 (5%) pts developed SPM. Mutant HPC were detected in 13 of the 17 pts. Accordingly, presence of mutant HPC was associated with a 2-fold increased risk of SPM (OR: 2.3; P = .03). Of the 13 pts, 3 showed mutations at diagnosis and in 8 these emerged during maintenance. The only case developing a myeloid neoplasm had a TP53 mutation present during maintenance that was undetectable at diagnosis. Pts with mutant vs non-mutated HPC at diagnosis showed similar OS (6y rates of 86% and 81%, respectively; P =.46). Similarly, there were no differences in OS of pts with emerging mutations vs those without mutant HPC after treatment (6y rates of 85% and 83%, respectively; P =.65). Specific mutations in DNMT3A, TP53 and TET2 and were not associated with inferior OS. Conclusions: To our knowledge this is the first study analyzing the mutational landscape of purified HPC throughout lenalidomide based regimens in high risk smoldering MM as well as transplant eligible and ineligible active MM. Deep sequencing of CD34+ HPC uncovered that the frequency of mutations is similar between precursor and malignant disease, and that TP53 is amongst the top mutated genes. There was considerable volatility with presence of both transient and emerging mutations throughout treatment. Importantly, the detection of mutant HPC was associated with increased risk of SPM without differences in survival. Thus, this study supports the longitudinal screening of HPC for the identification of patients at risk of developing SPM.

Identification, Monitoring and Reversibility of PARP Inhibitor Associated Clonal Haematopoiesis and Myelodysplastic Syndrome: A Case Series

Background: Poly ADP ribose polymerase inhibitors (PARPi) are widely used as maintenance treatment for ovarian cancer (OC) after response to platinum chemotherapy in the first line setting and recurrent disease. Their use has significantly improved outcomes, particularly in patients with BRCA mutations in whom first-line PARPi maintenance therapy increases progression-free survival from 13.8 to 56.0 months (Hazard Ratio, 0.33; 95% CI, 0.25-0.43). However, a recent meta-analysis of clinical trial adverse events and pharmacovigilance data showed that PARPi therapy significantly increases the risk of therapy related myeloid neoplasms (tMN), myelodysplastic syndrome (MDS) and acute myeloid leukaemia with poor outcomes. Platinum based chemotherapy is the mainstay of OC treatment and is associated with clonal haematopoiesis (CH) with mutations in DNA damage response genes. CH is recognised as a premalignant myeloid state but real world data on clonal dynamics during PARPi therapy are lacking. We present a case series of OC patients receiving PARPi-therapy referred for haematological review for beyond-expected HT or unexplained full blood count (FBC) abnormalities with serial FBC and myeloid next generation sequencing (mNGS) clonal monitoring in relation to PARPi therapy. Methods: Seven patients with stage III/IV high-grade OC were referred to haematology at the discretion of the treating oncologist between February 2017 and May 2022 (mean age (SD): 68 (7.0) years, median PARPi duration: 18 months (range: 4-86)). All patients had received prior platinum-based chemotherapy and were screened with mNGS in peripheral blood (PB) or bone marrow aspirate and trephine (BMAT) at initial assessment. Results: Reasons for referral are detailed in Table 1 and include two patients with isolated elevated nucleated red blood cell (NRBC) counts. Six patients had clonal pathology (CP), five of whom had single-nucleotide variants typical of platinum-exposure defined by the involved gene or platinum-associated mutational signatures. Clinical details including BRCA-status, haematological diagnosis and mNGS results are detailed in Table 1. PARPi-therapy was discontinued in four patients with CP, with resolution or improvement of FBC and m-NGS abnormalities (variant allele frequencies (VAFs) and/or copy number variations (CNVs)) in three patients. These findings were most marked in Patient 1, referred with grade 3 thrombocytopenia and transfusion dependence and in whom PARPi cessation resulted in transfusion independence and resolution of morphological and cytogenetic abnormalities (clinical course including BMAT results and haematological parameters illustrated in Figure 1), and Patient 3, who demonstrated selective regression of a TP53 mutated clone on PARPi cessation. Two patients with germline BRCA mutations continued PARPi therapy with 3 monthly PB mNGS monitoring after discussion at multidisciplinary team meetings. Conclusions: We show using sequential molecular and haematological monitoring before and after PARPi cessation that morphological, FBC and clonal abnormalities can be reversed on PARPi discontinuation, potentially mitigating the risk of life-threatening tMNs in some patients. We also highlight that, in addition to cytopenias, persistently elevated NRBCs may be an indicator of clinically relevant CP not well described in pharmacovigilance data based on common terminology criteria for adverse events criteria. Large scale prospective data on clonal dynamics in PARPi therapy are needed to inform mNGS screening and tMN risk stratification as scoring systems based on age associated clonal haematopoiesis of indeterminate potential may not be applicable. Until these data are available we demonstrate the value in MDT discussion and serial PB mNGS monitoring in formulating case-by-case therapeutic decisions regarding CP and PARPi therapy, particularly patients with high risk BRCA mutated OC.

A Phase IB/II Study of Blinatumomab in Patients with B-Cell Acute Lymphoblastic Leukemia (ALL) and B-Cell Non-Hodgkin Lymphoma (NHL) As Post-Allogeneic Blood or Marrow Transplant (alloBMT) Remission Maintenance

Background: AlloBMT can be curative as consolidation for high risk B ALL and NHL. However, transplant-related toxicity and disease relapse limit survival. Post-transplantation cyclophosphamide (PTCy) as graft-versus-host disease (GVHD) prophylaxis limits GVHD and facilitates alternative allograft sources. Following PTCy, cellular immune reconstitution is favorable for strategies to augment anti-tumor immunity. Blinatumomab (blina) is effective in the treatment of CD19+ ALL and NHL. Blina leads to T cell activation that may enhance post-transplant tumor-specific T cell responses, leading to a more potent graft-versus-tumor effect. A study of 21 B ALL pts demonstrated the feasibility of post-transplant blina but did not improve survival when pts universally remained on immunosuppression for at least 1 cycle of treatment (Gaballa. Blood. 2022). We present results of a phase Ib/II trial to assess the tolerability and preliminary efficacy of blina as post-alloBMT remission maintenance in B-cell ALL and NHL in pts off immunosuppression. Methods: Pts ≥1 month-old with high risk CD19+ B ALL or ≥18 years-old with NHL who underwent alloBMT with PTCy were eligible. Pts had to be 60-180 days post-transplant with count recovery and in remission. Pts had to be off immunosuppression for ≥4 weeks prior to treatment, and without a history of grade ≥3 acute GVHD or severe chronic GVHD. Pts could receive 2 cycles of blina if they had evidence of disease (including MRD) at their pre- and/or post-transplant evaluations but otherwise received 1 cycle. Blina was given as a continuous infusion at 9 mcg/day on C1D1-7 and 28 mcg/day on C1D8-28 and C2D1-28. Results: The study closed to accrual on January 31, 2023 following enrollment of 42 pts (26 male/16 female) with a median age of 54 (Range 30-73). Among enrolled pts, 19 had Ph-negative B ALL and 23 had B-cell NHL. All pts underwent reduced-intensity conditioning alloBMT using a regimen of fludarabine, cyclophosphamide, and total body irradiation. Median prior lines of therapy were 2 (range 1-6). Additional transplant details are presented in Table 1. All B ALL pts were in an MRD-negative CR by flow cytometry at a sensitivity of 0.01% at alloBMT. All pts received a single cycle of therapy. Pts started blina a median of 137 days post-transplant (range, 90-182). Four pts failed to complete a full cycle due to G4 transaminitis (1), G4 neutropenia (1), relapse (1), and patient preference in the setting of tremors (1). Grade 3 or 4 adverse events felt to be at least possibly related to the study drug included G3 neutropenia (7%), G4 neutropenia (19%), anemia (5%), G3 ALT (5%), G4 AST (2%), G3 AST (2%), and neurotoxicity (7%). Two pts (5%) developed chronic GVHD following blina requiring the resumption of immunosuppression. At a median follow-up of 40 months post-alloBMT, the 3-year relapse-free survival is 73% (95% CI 54-85%) due to a 24% (95% CI 12-39%) incidence of relapse and a 4% (95% CI 0-17%) incidence of non-relapse mortality, as shown in Figure 2. All 3 (100%) relapsed B ALL pts and 2/6 (33%) relapsed B NHL pts presented with CNS involvement. Four pts with CNS relapse (80%) had CNS involvement prior to alloBMT. Among 2 pts who died without relapse, 1 was unrelated to alloBMT, while the other died of therapy-related myeloid neoplasm. Data on biomarkers including changes in T cell subpopulations in both BM and PB, and co-signaling molecule expression will be presented. Conclusions: Post-alloBMT maintenance therapy with blina is feasible with minimal toxicity in pts off immunosuppression. Initial survival outcomes are promising with the majority of relapses involving the CNS. A randomized trial of maintenance with blina after alloBMT with PTCy is needed to confirm efficacy in this high-risk population.

The Peripheral Blood Stem Cell Immunome Demonstrates Abnormal Immune Effector Cells Early in the Disease Course of Multiple Myeloma and Contributes to Inferior Outcomes and Secondary Myeloid Neoplasms

Background: Upfront autologous stem cell transplant (ASCT) is the standard-of-care for newly diagnosed multiple myeloma (MM) patients. However, relapse and the development of therapy-related myeloid neoplasms (t-MN) post ASCT results in considerable morbidity and mortality. We hypothesized that enrichment of abnormal myeloid progenitors and immune effector cells (IEC) in peripheral blood stem cells (PBSCs) of patients with MM is associated with a higher risk of relapse and development of t-MN. Methods: After institutional review board approval, we screened patients with active MM that were evaluated at Mayo Clinic, Rochester, MN between 01/01/2003 and 12/31/2020. Patients that underwent an ASCT and had cryopreserved PBSC product available for research were included. Using mass cytometry, we performed a comprehensive immunophenotyping of mobilized cryopreserved PBSCs from 54 MM patients undergoing 1 st ASCT, using validated antibody panel of 37 lymphoid- and myeloid-based markers each. Flow cytometry standard files were normalized and concatenated using the Fluidigm acquisition software. Flow cytometry standard files were uploaded to the OMIQ software from Dotmatics (www.omiq.ai, www.dotmatics.com), where transformation and cleaning (doublets, debris) were performed. To correct for between-sample technical variations (“batch effects”), we used fdaNorm within the omiq.ai platform. Clustering and visualization of normalized and concatenaded flowcytometry files were performed within the omiq.ai platform using PhenoGraph and UMAP, respectively. We performed principal component analysis (PCA) to identify major immunomic drivers of variability across patients. We explored the associations between immune subsets and the dichotomized clinical outcomes of interest: progression-free (PFS), myeloid neoplasm-free (MNFS) and overall survival (OS) from ASCT. A 2-sided false discovery rate adjusted P-value of <0.05 was considered significant when multiple comparisons were performed; otherwise, a P-value of <0.05 was considered significant. Results: The most abundant cell populations were T-cells (49% of the CD45 +) and myeloid cells (43% of the CD45 + cells). Clustering on the CD45 + cells revealed 30 unique lymphoid clusters and 27 unique myeloid clusters. Subclustering on NK-cells identified an additional 16 NK-cell clusters. On PCA, principal components-1 (PC-1) and 2 (PC-2) explained 15.2% and 12.5% of the variability in the data, respectively. A high PC-2 score was associated with inferior median PFS (20 vs. 52 months; P=0.005, Figure 1A), MNFS (52 vs. 85 months, P=0.03, Figure 1B) and OS (64 vs. 106 months, P=0.01) from ASCT. Immune subsets enriched in the high PC-2 cohort included the ‘NKT-like’ (CD56 +) T-cell subset which expressed the inhibitory CD159a receptor and TIGIT; a terminally differentiated (CD27/CD28 -, CD57/KLRG1 +) and exhausted (TIGIT/PD-1 +) T cell subset, 2 exhausted (TIGIT/PD-1 +) T cell subsets; an immunosenescent (CD27/CD28 -, CD57/KLRG1 +) T cell subset as well as a terminally exhausted (C27/C28 -, KLRG1/PD-1/TIGIT +) TCRgamma-delta subset. Immune subsets with high loadings within PC-1 (associated with improved clinical outcomes) consisted exclusively of CD4 + cells with early memory phenotypes (CD27/CD28/CD127 +). Abnormal expression of CD7 and HLA-DR expression on myeloid progenitor cell subsets was associated with an inferior PFS, MNFS, and OS. A NK-cell subcluster, characterized by an immature (CD56 high), inhibitory (CD159a +, NKG2A) phenotype, was enriched in patients developing t-MN (0.33% vs. 0.17%, P=0.006). Conclusion: We identify abnormal myeloid and immune effector cells in the PBSCs of patients with MM, even prior to ASCT. Patients with a PBSC product that is enriched in immunosuppressive T and myeloid cell subsets, and with a lower proportion of antigen presenting cells, progenitor populations, and anti-tumor macrophages have a shorter PFS and MNFS. This suggests that the predisposition to t-MN and inferior outcomes for MM are possibly determined early in the disease course. These findings need external validation and have the potential to guide sequencing of future treatment modalities.

Combined Deficiency of Chromosome 7 Myeloid Tumor Suppressors Enhances Chemotherapy Resistance

Monosomy 7 and del(7q) [-7/del(7q)] are recurrent in myeloid neoplasms and associated with chemoresistance. -7/del(7q) is identified in up to half of therapy-related myeloid neoplasms (t-MN), high-risk secondary malignancies arising after prior exposure to chemotherapy or radiation. -7/del(7q) is also detected in clonal hematopoiesis, suggesting chromosome 7 aberrations can be early events in disease etiology. Despite this prevalence, the pathogenesis of -7/del(7q) in leukemogenesis remains unclear. We previously reported that deficiency of the transcription factor CUX1, a 7q-encoded tumor suppressor gene, promotes hematopoietic stem and progenitor cell drug resistance and t-MN transformation. Herein, we determined the combined impact of CUX1 loss with additional 7q tumor suppressor genes. To this end, we established a CRISPR/Cas9-based murine model of del(7q) clonal hematopoiesis and drug resistance. After targeting four 7q genes simultaneously, combined deficiency of CUX1 and the histone methyltransferase EZH2 uniquely promoted clonal outgrowth under genotoxic pressure in vivo and in vitro. Mechanistically, clonal selection is due, in part, to decreased apoptosis after chemotherapy exposure. RNA-seq in the absence of genotoxic insult revealed that Cux1 and Ezh2 loss has an additive transcriptional impact that is enriched for -7/del(7q) patient-derived gene signatures. Overall, we reveal a previously unknown genetic interaction between the 7q genes CUX1 and EZH2, supporting the concept of 7q as a contiguous gene syndrome region . A refined understanding of the molecular pathways driving del(7q) pathogenesis and drug resistance will enable development of therapies designed to counter or prevent these high-risk malignancies. In addition, we report a tractable approach for interrogating the pathogenesis of aneuploid events more broadly in cancer.