Significant Increase In Survival Research Articles

Abstract 4205: Investigating micro-environmental changes in a syngeneic radio-recurrent prostate cancer model

Abstract Approximately 1 in 8 men in Canada will be diagnosed with prostate cancer (PCa) in their lifetime, with ~20% presenting with high-risk disease. A standard treatment is radiotherapy (RT) and while many patients respond well to RT, up to 40% of men with high-risk disease can recur, often within the prostate. It can be challenging to salvage prostate relapses due to the morbidity of re-irradiation and the high rate of subsequent progression. The tumor micro-environment (TME) is altered after radiotherapy with increased immune-suppressor and immune-stimulatory effects occurring concurrently. Therapies capitalizing on these changes in the TME might provide an advantage to tackling radiation-recurrent cancer. Identifying TME changes in radio-recurrent PCa is the first step; however, existing PCa animal models do not accurately model the TME. Our collaborator’s lab has created the DVL3 mouse model with a clinically relevant TME. It forms tumors with distinct glandular morphology and displays micro-environmental responses to RT like what is seen in high-risk PCa patients, making this model an innovative tool for investigating radio-recurrence. The objective of this study is to create a radio-recurrent DVL3 PCa cell line to explore changes in the TME both in vitro and in vivo via orthotopic injection. To generate the radio-recurrent model, the DVL3 Parental cells (DVL3-Par) underwent a conventionally fractionated RT schedule (78Gy/39fx), herein referred to as DVL3-CF cells. The first part of the project involves in vitro characterization. Radiation resistance was evaluated via clonogenic assay which demonstrated a significant increase in clonogenic survival in the DVL3-CF cells relative to DVL3-Par. Characterization of DVL3-CF cells demonstrates that they are more proliferative at baseline, they recover quicker following G2/M blocks, they have decreased senescence following high dose radiation, and they are more invasive in vitro. The second part of the project evaluates the radio-recurrent model in vivo using an orthotopic mouse model as this best emulates the TME and clinical tumor progression. Parental and CF cells will be injected orthotopically into the prostate of C57/BL-6 mice and monitored for growth by MRI which will provide information on tumor volume as well as vasculature. At the time of sacrifice, the prostate and draining lymph nodes will be cryo-preserved and formalin fixed for downstream histological analysis. Transcriptional analyses, MRI data, and histological analysis of the tumors and draining lymph nodes will be compared across the radio-recurrent model and its parental cell line to evaluate environmental changes and potentially implicated mechanisms. This study will elucidate some of the complex interactions that are occurring within the TME of radio-recurrent PCa, increasing our understanding of mechanisms of radio-recurrence. Citation Format: Stephanie D. White, Xiaoyong Huang, Ian Mills, Stanley K. Liu. Investigating micro-environmental changes in a syngeneic radio-recurrent prostate cancer model [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 4205.

A comprehensive analysis of CD47 expression in various histological subtypes of soft tissue sarcoma: exploring novel opportunities for macrophage-directed treatments

PurposeThe CD47 molecule, often referred to as the “do not eat me” signal, is frequently overexpressed in tumor cells. This signaling pathway limits phagocytosis by macrophages. Our objective was to determine CD47 abundance in various soft tissue sarcomas (STS) to investigate whether it could serve as a potential evasion mechanism for tumor cells. Additionally, we aimed to assess the prognostic value of CD47 expression by examining its association with different clinicopathological factors. This study aimed to elucidate the significance of CD47 in the context of emerging anti-tumor targeting approaches.MethodsIn this retrospective study, formalin-fixed paraffine-embedded (FFPE) tumor tissues of 55 treatment-naïve patients were evaluated by immunohistochemistry for the abundance of CD47 molecule on tumor cells. The categorization of CD47 positivity was as follows: 0 (no staining of tumor cells), 1 + (less than 1/3 of tumor area positive), 2 + (between 1/3 and 2/3 of tumor area positive), and 3 + (more than 2/3 of tumor area positive for CD47). Next, we compared CD47 abundance between different tumor grades (G1–3). We used Kaplan–Meier survival curves with log-rank test to analyze the differences in survival between patients with different CD47 expression. Moreover, we performed Cox proportional hazards regression model to evaluate the clinical significance of CD47.ResultsCD47 is widely prevalent across distinct STS subtypes. More than 80% of high grade undifferentiated pleiomorphic sarcoma (UPS), 70% of myxofibrosarcoma (MFS) and more than 60% of liposarcoma (LPS) samples displayed a pattern of moderate-to-diffuse positivity. This phenomenon remains consistent regardless of the tumor grade. However, there was a tendency for higher CD47 expression levels in the G3 group compared to the combined G1 + G2 groups when all LPS, MFS, and UPS were analyzed together. No significant associations were observed between CD47 abundance, death, and metastatic status. Additionally, high CD47 expression was associated with a statistically significant increase in progression-free survival in the studied cohort of patients.ConclusionThis study highlights the potential of the CD47 molecule as a promising immunotherapeutic target in STS, particularly given its elevated expression levels in diverse sarcoma types. Our data showed a notable trend linking CD47 expression to tumor grade, while also suggesting an interesting correlation between enhanced abundance of CD47 expression and a reduced hazard risk of disease progression. Although these findings shed light on different roles of CD47 in STS, further research is crucial to assess its potential in clinical settings.

Effectiveness of Vasopressin Against Cardiac Arrest: A Systematic Review of Systematic Reviews.

This systematic review (SR) of SRs evaluates the effectiveness of vasopressin alone or in combination with other drugs in improving the outcomes of cardiac arrest (CA). Using a three-step approach, we searched five databases to identify all relevant SRs. Two reviewers independently selected suitable studies, assessed study quality, and extracted relevant data. If an outcome was reported by multiple SRs, a re-meta-analysis was conducted as needed; otherwise, a narrative analysis was performed. Twelve SRs covering 16 original studies were included in this review. The meta-analysis results revealed a significant increase in survival to hospital admission for patients with in-hospital CA (IHCA) or out-of-hospital CA (OHCA) receiving vasopressin alone compared with that for those receiving epinephrine alone. Furthermore, the return of spontaneous circulation (ROSC) was significantly increased in patients with OHCA receiving vasopressin with epinephrine compared with that in those receiving epinephrine alone. Compared with patients with IHCA receiving epinephrine with placebo, those receiving vasopressin, steroids, and epinephrine (VSE) exhibited significant increases in ROSC, survival to hospital discharge, favorable neurological outcomes, mean arterial pressure, renal failure-free days, coagulation failure-free days, and insulin requirement. VSE is the most effective drug combination for improving the short- and long-term outcomes of IHCA. It is recommended to use VSE in patients with IHCA. Future studies should investigate the effectiveness of VSE against OHCA and CA of various etiologies, the types and standard dosages of steroids for cardiac resuscitation, and the effectiveness of vasopressin-steroid in improving CA outcomes.

Sex-related differences in serum biomarker levels predict the activity and efficacy of immune checkpoint inhibitors in advanced melanoma and non-small cell lung cancer patients

BackgroundImmune Checkpoint Inhibitors (ICIs) lead to durable response and a significant increase in long-term survival in patients with advanced malignant melanoma (MM) and Non-Small Cell Lung Cancer (NSCLC). The identification of serum cytokines that can predict their activity and efficacy, and their sex interaction, could improve treatment personalization.MethodsIn this prospective study, we enrolled immunotherapy-naïve patients affected by advanced MM and NSCLC treated with ICIs. The primary endpoint was to dissect the potential sex correlations between serum cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, GM-CSF, MCP-1, TNF-ɑ, IP-10, VEGF, sPD-L1) and the objective response rate (ORR). Secondly, we analyzed biomarker changes during treatment related to ORR, disease control rate (DCR), progression free survival (PFS) and overall survival (OS). Blood samples, collected at baseline and during treatment until disease progression (PD) or up to 2 years, were analyzed using Luminex xMAP or ELLA technologies.ResultsSerum samples from 161 patients (98 males/63 females; 92 MM/69 NSCLC) were analyzed for treatment response. At baseline, IL-6 was significantly lower in females (F) versus males (M); lower levels of IL-4 in F and of IL-6 in both sexes significantly correlated with a better ORR, while higher IL-4 and TNF-ɑ values were predictive of a lower ORR in F versus M. One hundred and sixty-five patients were evaluable for survival analysis: at multiple Cox regression, an increased risk of PD was observed in F with higher baseline values of IL-4, sPD-L1 and IL-10, while higher IL-6 was a negative predictor in males. In males, higher levels of GM-CSF predict a longer survival, whereas higher IL-1β predicts a shorter survival. Regardless of sex, high baseline IL-8 values were associated with an increased risk of both PD and death, and high IL-6 levels only with shorter OS.ConclusionsSerum IL-1β, IL-4, IL-6, IL-10, GM-CSF, TNF-ɑ, and sPD-L1 had a significant sex-related predictive impact on ORR, PFS and OS in melanoma and NSCLC patients treated with ICIs. These results will potentially pave the way for new ICI combinations, designed according to baseline and early changes of these cytokines and stratified by sex.

Abstract B075: Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity

Abstract Ovarian cancer (OvCa) is among the most aggressive and lethal cancers with a poor response to immune checkpoint blockade (ICB) despite its modest tumor mutational burden and expression of known cancer antigens. Consequently, it is critical to understand the molecular mechanisms of tumor immune escape and resistance to immunotherapies. Intercellular signaling between cancer cells and the tumor microenvironment (TME) is a crucial determinant of tumor immunity and given the immunosuppressive nature of OvCa TME, it is of utmost importance to dissect how this immunosuppressed TME is established. To begin to determine which cancer-cell genes may function cell externally in controlling of OvCa immunity, we employed a first-of-its-kind spatial functional genomics approach we developed, called Perturb-map, which enables dozens of CRISPR gene knock-out (KO) cells to be resolved in a tissue/tumor by imaging, along with cells in the local TME. We used Perturb-map to determine how 35 different genes, identified through ligand-receptor analysis of ovarian tumors, influenced tumor response to anti-PD-1 treatment in an OvCa mouse model. Strikingly, Il4 KO tumors were unique amongst the 35 gene KOs making the tumors significantly more responsive to anti-PD-1 treatment despite not impacting tumor growth in the absence of immunotherapy. Further experiments with single CRISPR KO tumors validated the Perturb-map finding, where Il4 KO combined with anti-PD-1 treatment resulted in a major reduction in tumor growth and a significant increase in survival. Cyclic immunofluorescence (CyCIF) multiplex imaging revealed that loss of cancer cell-derived IL-4 led to a significant reduction in pro-tumor macrophages and an increase in dendritic cell (DC) subsets as well as both progenitor and terminally exhausted CD8 T cells. Upon anti-PD-1 treatment, Il4 KO tumors had significant enrichment for activated CD8 T cells and B cells. Importantly, spatial analysis of the tumor samples identified enrichment of T follicular helper cells, B cells, and DCs in tertiary lymphoid structures in tumors with combined IL-4 loss and PD-1 inhibition. Further, we analyzed a human OvCa single-cell transcriptomics dataset and demonstrated that an IL-4 response signature is expressed in tumor-associated macrophages, indicating active IL-4 signaling in human ovarian tumors. Importantly, we employed a CRISPR-knock-in expression reporter in mouse tumors as well as immunohistochemistry of both human and mouse tumor samples to confirm that IL-4 is expressed by cancer cells. Thus, we identified cancer cell-secreted IL-4 as a potent regulator of OvCa TME and response to ICB. Given that an anti-IL-4-receptor antibody is already in clinical use to treat immune diseases, our findings have the potential to be translated to the clinical management of this dreadful disease. Citation Format: Gurkan Mollaoglu, Alexander Tepper, Hunter Potak, Luisanna Pia, Angelo Amabile, Chiara Falcomata, Jaime Mateus-Tique, Noam Rabinovich, Rachel Brody, Lindsay Browning, Jia-Ren Lin, Peter Sorger, Sandro Santagata, Miriam Merad, Alessia Baccarini, Brian Brown. Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B075.

How a significant increase in survival in pancreatic cancer is achieved. The role of nutritional status and supportive care: A review

Pancreatic cancer (PC) is a serious public health problem. The mortality rate of patients with PC remains one of the highest among cancers. Early diagnosis of PC is challenging, so it is often diagnosed in the later stages. Current treatment approaches, including surgery, neoadjuvant and adjuvant chemotherapy, chemoradiotherapy, and supportive care, have demonstrated improved outcomes. A significant problem remains exocrine pancreatic insufficiency (EPI) in patients with PC, which requires enzyme replacement therapy. However, this is not given due attention in the Russian literature. This review addresses the survival trends of patients with PC, current therapies, and enzyme replacement therapy as an integral part of supportive care and improvement of nutritional status; also, the issues of routing patients with PC are addressed. It is emphasized that the diagnosis and treatment of EPI are mandatory to improve and maintain the nutritional status and quality of life; failure to treat EPI renders antitumor treatment ineffective.

Evaluating CD133 as a Radiotheranostic Target in Small-Cell Lung Cancer.

Despite decades of work, small-cell lung cancer (SCLC) remains a frustratingly recalcitrant disease. Both diagnosis and treatment are challenges: low-dose computed tomography (the approved method used for lung cancer screening) is unable to reliably detect early SCLC, and the malignancy's 5 year survival rate stands at a paltry 7%. Clearly, the development of novel diagnostic and therapeutic tools for SCLC is an urgent, unmet need. CD133 is a transmembrane protein that is expressed at low levels in normal tissue but is overexpressed by a variety of tumors, including SCLC. We previously explored CD133 as a biomarker for a novel autoantibody-to-immunopositron emission tomography (PET) strategy for the diagnosis of SCLC, work that first suggested the promise of the antigen as a radiotheranostic target in the disease. Herein, we report the in vivo validation of a pair of CD133-targeted radioimmunoconjugates for the PET imaging and radioimmunotherapy of SCLC. To this end, [89Zr]Zr-DFO-αCD133 was first interrogated in a trio of advanced murine models of SCLC─i.e., orthotopic, metastatic, and patient-derived xenografts─with the PET probe consistently producing high activity concentrations (>%ID/g) in tumor lesions combined with low uptake in healthy tissues. Subsequently, a variant of αCD133 labeled with the β-emitting radiometal 177Lu─[177Lu]Lu-DTPA-A″-CHX-αCD133─was synthesized and evaluated in a longitudinal therapy study in a subcutaneous xenograft model of SCLC, ultimately revealing that treatment with a dose of 9.6 MBq of the radioimmunoconjugate produced a significant increase in median survival compared to a control cohort. Taken together, these data establish CD133 as a viable target for the nuclear imaging and radiopharmaceutical therapy of SCLC.

Vitamin D Improves Klebsiella-Induced Severe Pneumonia in Rats by Regulating Intestinal Microbiota.

In the context of progressively uncontrolled drug resistance of bacteria, the difficulty of treating Klebsiella (KP)-induced pneumonia increases. Searching for drugs other than antibiotics has become an urgent task. Vitamin D (VD), meanwhile, is shown to be capable of treating pneumonia. Therefore, we aimed to explore the effects and mechanisms of VD on KP-infected rats. Male Sprague Dawley rats were divided into the Control, VD, KP and KP+VD groups. A rat pneumonia model was induced using an intratracheal drop of 2.4×108 CFU/mL KP. VD treatment was performed by gavage using 5 μg/kg. Subsequently, the survival of the rats was recorded, and the lungs, bronchoalveolar lavage fluid, and feces of the rats were collected 4 days after KP infection. Next, the water content of lung tissues was measured by the wet-to-dry weight ratio. Histopathological changes of lung tissues were observed by Hematoxylin and Eosin staining and the levels of inflammatory factors (TNF-α, IL-1β, MCP1) were detected using ELISA. The feces of rats in each group were also subjected to 16S rDNA gene analysis of intestinal microbiota. Compared with the KP group, the KP+VD group showed a significant increase in survival, a significant decrease in water content and bacterial counts in the lungs, a significant improvement in lung injury, and a significant decline in the levels of TNF-α, IL-1β, and MCP1. According to the 16S rDNA sequencing, VD altered the structure of the intestinal bacterial community in the KP-infected rats and made the species richness similar to that of healthy rats. Additionally, the abundance of Anaeroglobus was significantly increased in the KP+VD group. VD modulates intestinal microbiota to increase the resistance of rats to pneumonia caused by Klebsiella infection.

Abstract A055: Context-dependent role of acid sphingomyelinase in pancreatic ductal adenocarcinoma

Abstract Sphingolipids, a class of lipids that includes the bioactive molecules ceramide and sphingosine-1-phosphate (S1P), play a contrasting role in the regulation of cancer cell death and survival. Acid sphingomyelinase (SMPD1) is the key enzyme that breaks down sphingomyelin into ceramide, accounting for more than 50% of the cellular ceramide supply and influencing the development of cancer. Here, we studied the role of SMPD1 in different cellular compartments during pancreatic ductal adenocarcinoma (PDAC) carcinogenesis. We performed a targeted quantitative plasma metabolite analysis of 637 patients (PDAC, 356; nonpancreatic disease, 281). We completed the IHC multiplex staining in the TMA of 124 patients with PDAC. Using CRISPR-Cas9 gene editing, SMPD1 was deleted in PDAC cell lines. The influence of SMPD1-KO on proliferation, colony formation, and migration was studied. Tumor progression and carcinogenesis was monitored in an orthotopic PDAC model and metastasis models using SMPD1 null cells, as well as in KC and KPC mice. Plasma metabolite analysis of 637 patients (PDAC, 356; nonpancreatic disease, 281) revealed the most pronounced changes in the group of complex lipids with the highest prevalence of sphingomyelins and ceramides. Overall survival was significantly correlated with high SMPD1 expression in tumor tissue in two independent cohorts. Interestingly, IHC multiplex analysis of SMPD1 'high expression' in cancer cells was associated with poorer survival, while SMPD1 'high expression' in immune cells was associated with longer overall survival. Expression level of Smpd1 differed from preneoplastic to neoplastic lesions in PDAC mouse tissue. Smpd1-KO in PDAC cell lines resulted in decreased proliferation, colony formation, and migration. In an orthotopic mouse model, Smpd1-KO clones resulted in significantly lower tumor weight and size compared to WT clones. Furthermore, the injection of WT clones into global Smpd1het-KO mice resulted in a significant increase in tumor weight compared to the injection of WT clones into WT mice. In two different metastasis models, injecting Smpd1-KO led to a significantly reduced formation of metastases in the lung and liver compared to WT clones. Interestingly, injecting WT clones into global Smpd1het-KO mice resulted in a significant decrease in metastasis formation in the lung compared to the injection of WT clones into WT mice. In general, we show a significant increase in survival in an orthotopic PDAC model using PDAC Smpd1-KO cells. In conclusion, SMPD1 has a context-dependent role in cancer cells and immune cells in PDAC. Inhibition of SMPD1 specifically in cancer cells is a viable therapeutic option, which should be explored in future studies. Citation Format: Ahmed Alnatsha, Qi Li, Klea Ricku, Prince Allawadhi, Jianying Xu, Didem Saka, Theresa Weltermann, Quan Zhaou, Nicole Schreiner, Kirsten Lauber, Ivonne Regel, Ujjwal Mukund Mahajan, Julia Mayerle. Context-dependent role of acid sphingomyelinase in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A055.

Abstract A059: Targeting pancreatic cancer metabolic dependencies through glutamine antagonism

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer. A factor that contributes to the poor prognosis of the disease is the complex tumor microenvironment (TME). The PDAC TME is composed of excessive fibrosis and desmoplasia, which creates a harsh environment resulting in hypoxia and altered nutrient availability. To promote survival and proliferation in this environment, PDAC cells can reprogram glutamine (Gln) metabolism. Previous studies have demonstrated that PDAC cells use Gln to support proliferation and redox balance. However, earlier attempts to inhibit Gln metabolism using glutaminase inhibitors resulted in rapid metabolic reprogramming and therapeutic resistance. We hypothesized that a Gln analogue, such as 6-Diazo-5-oxo-L-norleucine (DON), could broadly target Gln metabolism in PDAC and prevent rapid adaptation. Indeed, DON treatment led to a significant decrease in PDAC proliferation and a profound reduction of various metabolites involved in central carbon and nucleotide metabolism, suggesting that DON creates a metabolic crisis. In addition, we observed a significant decrease in tumor growth in various in vivo models (syngeneic, immunodeficient and PDXs) using DRP-104 (sirpiglenastat), a novel pro-drug version of DON that was designed to circumvent DON associated GI toxicity and allow the therapeutic exploration of broad Gln antagonism. Mechanistically, we found that ERK signaling is increased as a compensatory mechanism through the increased activity of receptor tyrosine kinases (RTKs). Combinatorial treatment of DRP-104 and Trametinib (MEK1/2) inhibitor led to a significant increase in survival in a syngeneic model PDAC. Taken together, these pre-clinical results suggest that broadly targeting Gln metabolism could provide a new therapeutic avenue for PDAC and that the combination with an ERK signaling pathway inhibitor could further improve the therapeutic outcome. Citation Format: Joel Encarnacion-Rosado, Alec C. Kimmelman, Robert Wild. Targeting pancreatic cancer metabolic dependencies through glutamine antagonism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A059.

Abstract B095: Leukemia inhibitory factor fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma

Abstract While inflammation is a known and well-validated risk factor for developing pancreatic ductal adenocarcinoma (PDA), it remains unclear how Leukemia inhibitory factor (LIF), a cytokine of the IL-6 class family, influences tumor initiation and progression in PDA. LIF is highly upregulated in PDA when compared to the normal healthy pancreas. Using a human single-cell RNAseq PDA data set, we identified pancreatic cancer cells as the main source of LIF, followed by fibroblasts and stellate cells. After confirming that Pdx1-expressing cells represent the majority of Lif-expressing cells in the pancreas, we crossed our newly developed Lif flox/flox allele into the well-established KrasLSL-G12D/+, Trp53flox/flox, Pdx1-Cre (KPC) mouse model. Lif flox/flox, Pdx1-Cre (LC) mice present with normal pancreas histology, mouse weight, and mouse size at 12 months, indicating that Lif is dispensable for mouse pancreas development and function. Lif flox/flox, Trp53flox/flox, Pdx1-Cre (LPC) mice also showed no differences in pancreas histology when compared to normal Lif-expressing PC mice. Lif flox/floxKPC (LKPC) mice develop PDA tumors. Cell lines from LKPC tumors (n=4) presented with undetectable Lif expression and no LIF protein secretion. Control KPC tumor cell lines (n=4) showed Lif expression and LIF secretion, confirming the successful deletion of Lif in our newly developed mouse model. LKPC mice (n=21) showed a significant increase in disease-specific overall survival at 72 days when compared to control KPC mice (n=26) at 62 days (p<0.05). When mice reached endpoint, LKPC mice presented with significantly smaller tumors with an average tumor weight of 0.7g compared to KPC control animals with an average tumor weight of 1.3g (p<0.05). In summary, we find that cancer-cell expressed LIF augments tumor growth and shortens disease-specific overall survival, nominating leukemia inhibitory factor depletion as a promising therapeutic target in KRAS-driven pancreatic cancer. Citation Format: Lisa Miller-Phillips, Imene Boukhalfa Hanafi, Sarah Umetsu, Adam Olshen, Ritu Roy, Ashley Kiemen, Man-Tzu Wang, Eric Collisson. Leukemia inhibitory factor fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B095.

Tall fescue tiller survival over summer in a subtropical environment: The role of the size and depth of root systems

AbstractPasture persistence is a key determinant of the economic and environmental performance of pastoral animal production systems. Large and deep root systems that help resist summer water stress have been proposed as a relevant trait for vegetative persistence of perennial temperate forage species growing in subtropical climates or under future climatically challenging scenarios. In a previous study [Jauregui et al., 2017. Persistence of tall fescue in a subtropical environment: Tiller survival over summer in response to flowering control and nitrogen supply. Grass and Forage Science 72, 454–466] we have shown that nitrogen fertilization and grazing management aimed at ‘control flowering’ increased the survival of tall fescue tillers during harsh summers in Uruguay (lat. 32°S). Here we assessed: (i) to what extent tiller survival is mediated by root system size in spring and (ii) what consequences tiller survival entails for root mass, depth and morphology the following autumn. In two field experiments, significant increases in tiller survival in response to nitrogen fertilization and grazing management (+60% and +80% in 2011/12 and 2012/13, respectively) were not related to concomitant effects on the size or depth of the root system in spring (p > .10). Even when six‐fold within‐treatment variation in root mass was observed, within‐treatment variation in summer tiller survival was little affected (<15%, p = .08). In turn, differences in tiller survival over summer affected little root system characteristics the following autumn. Therefore, we found scant support for the hypothesis that large and deep root systems contribute to survival of tall fescue tillers in this subtropical humid climate. Except for soils with less than 30 mm of plant available water holding capacity, summer water deficits did not induce severe tiller mortality in tall fescue in this climate.

Early discontinuation of immune checkpoint inhibitor therapy prior to disease progression in patients with metastatic non-small cell lung cancer: a survival analysis.

Limited survival data are available for patients with metastatic non-small cell lung cancer (mNSCLC) who stop immune checkpoint inhibitor therapy (ICI) early for reasons other than progression of disease (POD), such as immune-related adverse events (irAEs). We conducted a retrospective observational study of all patients with mNSCLC treated with ICIs, with or without combination chemotherapy, at 3 Mayo Clinic sites between 2011 and 2022. Separate analyses were conducted at 6- and 12-month intervals. Patients who discontinued ICI due to POD prior to these time points were excluded from the analysis. A total of 246 patients with stage IV NSCLC used ICIs. Patients were then excluded if they had experienced POD prior to 6 or 12 months, resulting in 81 and 63 patients, respectively, for each timepoint. Sixty-four patients continued treatment beyond 6 months and were found to have longer progression-free survival (PFS) compared to the 17 patients who discontinued treatment (22.8 months vs 11.8 months, P =1.1E-04), as well as a significant increase in overall survival (OS) (33.9 months vs 14.4 months, P =7.2E-08). Forty patients continued treatment beyond 12 months and had longer PFS compared to the 23 patients that discontinued treatment (27.9 months vs 14.8 months, P =1.1E-04), as well as a significant increase in OS (39.7 months vs 18.0 months, P =2.0E-07). The most common reason for ICI discontinuation was irAEs. Other common reasons for stopping ICI were non-irAEs and stable disease. At both time points, 12 patients continued or restarted ICI after experiencing an irAE, and 2 patients experienced recurrent/new grade 1-2 irAEs. More patients continued/rechallenged with ICI after experiencing an irAE in the groups that continued ICI compared to those that discontinued ICI. Patients with mNSCLC and no POD who continued ICI beyond 6 months and 12 months, experienced significantly increased PFS and OS compared to patients who discontinued ICI, with larger increases in those who continued ICI past 12 months. Oncology providers should discuss the survival benefits of continuing ICI and offer support to overcome obstacles to continuation of treatment, if possible, particularly management of grade 1 and 2 irAEs.

Dose-Dependent Effects of Radiation on Mitochondrial Morphology and Clonogenic Cell Survival in Human Microvascular Endothelial Cells.

To better understand radiation-induced organ dysfunction at both high and low doses, it is critical to understand how endothelial cells (ECs) respond to radiation. The impact of irradiation (IR) on ECs varies depending on the dose administered. High doses can directly damage ECs, leading to EC impairment. In contrast, the effects of low doses on ECs are subtle but more complex. Low doses in this study refer to radiation exposure levels that are below those that cause immediate and necrotic damage. Mitochondria are the primary cellular components affected by IR, and this study explored their role in determining the effect of radiation on microvascular endothelial cells. Human dermal microvascular ECs (HMEC-1) were exposed to varying IR doses ranging from 0.1 Gy to 8 Gy (~0.4 Gy/min) in the AFRRI 60-Cobalt facility. Results indicated that high doses led to a dose-dependent reduction in cell survival, which can be attributed to factors such as DNA damage, oxidative stress, cell senescence, and mitochondrial dysfunction. However, low doses induced a small but significant increase in cell survival, and this was achieved without detectable DNA damage, oxidative stress, cell senescence, or mitochondrial dysfunction in HMEC-1. Moreover, the mitochondrial morphology was assessed, revealing that all doses increased the percentage of elongated mitochondria, with low doses (0.25 Gy and 0.5 Gy) having a greater effect than high doses. However, only high doses caused an increase in mitochondrial fragmentation/swelling. The study further revealed that low doses induced mitochondrial elongation, likely via an increase in mitochondrial fusion protein 1 (Mfn1), while high doses caused mitochondrial fragmentation via a decrease in optic atrophy protein 1 (Opa1). In conclusion, the study suggests, for the first time, that changes in mitochondrial morphology are likely involved in the mechanism for the radiation dose-dependent effect on the survival of microvascular endothelial cells. This research, by delineating the specific mechanisms through which radiation affects endothelial cells, offers invaluable insights into the potential impact of radiation exposure on cardiovascular health.

Improvement of Endothelial Cell-Polycaprolactone Interaction through Surface Modification via Aminolysis, Hydrolysis, and a Combined Approach

Polycaprolactone (PCL) is a promising material for the fabrication of alternatives to autologous grafts used in coronary bypass surgery. PCL biodegrades over time, allowing cells to infiltrate the polymeric matrix, replacing the biodegrading graft, and creating a fully functional vessel constituted of autologous tissue. However, the high hydrophobicity of PCL is associated with poor cell affinity. Surface modification of PCL can increase this cell affinity, making PCL an improved scaffold material for acellular vascular grafts. In this study, the surface of PCL films was modified by hydrolysis, aminolysis, and the combination thereof to introduce carboxyl, hydroxyl, and amino groups on the surface. Only the hydrolyzed films exhibited a significant increase in their hydrophilicity, although further testing showed that all aminolysis conditions had amino groups on the surface. Furthermore, in vitro experiments with human umbilical endothelial cells (HUVECs) were performed to assess changes in cell affinity for PCL due to the surface treatments. PCL treated with sodium hydroxide (NaOH), a hydrolysis reaction, showed a significant increase in endothelial cell adhesion after 24 hours with a significant increase in cell survival after 72 hours. Thus, NaOH treatment improves the biocompatibility and endothelialization of PCL, creating a competent candidate for artificial, acellular, biodegradable vascular grafts.

Retrospective Correlation between First Drug Treatment Duration and Survival Outcomes in Sequential Treatment with Regorafenib and Trifluridine/Tipiracil in Refractory Metastatic Colorectal Cancer: A Real-World Subgroup Analysis.

Background: Patients with refractory metastatic colorectal cancer (mCRC) rarely receive third-line or further treatment. In this context, regorafenib (R) and trifluridine/tipiracil (T) are two important novel therapeutic choices with statistically significant increases in overall survival (OS), progression-free survival (PFS), and disease control, with different toxicity profiles. This study is a subgroup analysis of our larger retrospective study, already published, whose objective was to assess the outcomes of patients when R and T were given sequentially. Patients and Methods: The study involved thirteen Italian cancer centers on a 10-year retrospective observation (2012-2022). In this subgroup analysis, we focused our attention on the correlation between the first drug treatment duration (<3 months, 3 to <6 months and ≥6 months) and survival outcomes in patients who had received the sequence regorafenib-to-trifluridine/tipiracil, or vice versa. Results: The initial study included 866 patients with mCRC who received sequential T/R, or R/T, or T or R alone. This analysis is focused on evaluating the impact of the duration of the first treatment in the sequence on clinical outcomes (OS, PFS) and includes 146 and 116 patients of the T/R and R/T sequences, respectively. Based on the duration of the first drug treatment, subgroups for the T/R sequence included 27 patients (18.4%) who received T for <3 months, 86 (58.9%) treated for 3 to <6 months, and 33 (22.6%) treated for ≥6 months; in the reverse sequence (R as the first drug), subgroups included 18 patients (15.5%) who received their first treatment for <3 months, 62 (53.4%) treated for 3 to <6 months, and 35 (31.0%) treated for ≥6 months. In patients who received their first drug treatment for a period of 3 to <6 months, the R/T sequence had a significantly longer median OS (13.7 vs. 10.8 months, p = 0.0069) and a longer median PFS (10.8 vs. 8.5 months, p = 0.0003) than the T/R group. There were no statistically significant differences between groups with first drug treatment durations of <3 months and ≥6 months. Conclusions: Our analysis seems to suggest that the administration of R for a period of 3 to <6 months before that of T can prolong both OS and PFS, as compared to the opposite sequence.

Abstract B014: Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity

Abstract Ovarian cancer (OvCa) is among the most aggressive and lethal cancers with a poor response to immune checkpoint blockade (ICB) despite its modest tumor mutational burden and expression of known cancer antigens. Consequently, it is critical to understand the molecular mechanisms of tumor immune escape and resistance to immunotherapies. Intercellular signaling between cancer cells and the tumor microenvironment (TME) is a crucial determinant of tumor immunity and given the immunosuppressive nature of OvCa TME, it is of utmost importance to dissect how this immunosuppressed TME is established. To begin to determine which cancer-cell genes may function cell externally in controlling of OvCa immunity, we employed a first-of-its-kind spatial functional genomics approach we developed, called Perturb-map, which enables dozens of CRISPR gene knock-out (KO) cells to be resolved in a tissue/tumor by imaging, along with cells in the local TME. We used Perturb-map to determine how 35 different genes, identified through ligand-receptor analysis of ovarian tumors, influenced tumor response to anti-PD-1 treatment in an OvCa mouse model. Strikingly, Il4 KO tumors were unique amongst the 35 gene KOs making the tumors significantly more responsive to anti-PD-1 treatment despite not impacting tumor growth in the absence of immunotherapy. Further experiments with single CRISPR KO tumors validated the Perturb-map finding, where Il4 KO combined with anti-PD-1 treatment resulted in a major reduction in tumor growth and a significant increase in survival. Cyclic immunofluorescence (CyCIF) multiplex imaging revealed that loss of cancer cell-derived IL-4 led to a significant reduction in pro-tumor macrophages and an increase in dendritic cell (DC) subsets as well as both progenitor and terminally exhausted CD8 T cells. Upon anti-PD-1 treatment, Il4 KO tumors had significant enrichment for activated CD8 T cells and B cells. Importantly, spatial analysis of the tumor samples identified enrichment of T follicular helper cells, B cells, and DCs in tertiary lymphoid structures in tumors with combined IL-4 loss and PD-1 inhibition. Further, we analyzed a human OvCa single-cell transcriptomics dataset and demonstrated that an IL-4 response signature is expressed in tumor-associated macrophages, indicating active IL-4 signaling in human ovarian tumors. Importantly, we employed a CRISPR-knock-in expression reporter in mouse tumors as well as immunohistochemistry of both human and mouse tumor samples to confirm that IL-4 is expressed by cancer cells. Thus, we identified cancer cell-secreted IL-4 as a potent regulator of OvCa TME and response to ICB. Given that an anti-IL-4-receptor antibody is already in clinical use to treat immune diseases, our findings have the potential to be translated to the clinical management of this dreadful disease. Citation Format: Gurkan Mollaoglu, Alexander Tepper, Hunter Potak, Luisanna Pia, Angelo Amabile, Chiara Falcomata, Jaime Mateus-Tique, Noam Rabinovich, Rachel Brody, Lindsay Browning, Jia-Ren Lin, Peter Sorger, Sandro Santagata, Miriam Merad, Alessia Baccarini, Brian Brown. Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B014.

Pre-Clinical Study on the Dual BCL2/BCL-XL Inhibitor AZD0466 for the Treatment of Chronic Lymphocytic Leukemia

Targeting BCL2 with venetoclax has proven successful for treatment of chronic lymphocytic leukemia (CLL), but resistance develops in a subset of patients. Among other mechanisms, mutations in BCL2 and increased BCL-XL expression confer venetoclax refractoriness in patient subgroups. It is therefore important to identify alternative treatment options targeting BCL-XL and BCL2 for combating venetoclax resistance. AZD4320 was developed as a dual BCL-2/BCL-XL inhibitor. AZD0466 is a dendrimer-drug conjugate of AZD4320, designed to optimize drug release and improve drug tolerability. Hence, we used the nano drug AZD0466 for in vivo experiments and AZD4320 for in vitro and ex vivo experiments. Firstly, we studied the efficacy of AZD0466 in vivo in the Eµ-TCL1 murine CLL adoptive transfer model. Due to prevalence of primary resistance, murine tumors with the best ex vivo response to AZD4320 were selected and 10 million cells were transferred by i.v. in syngeneic recipient mice. Upon reaching a tumor load of 10% CD19+ CD5+ cells in peripheral blood, mice were randomized and treated with 30mg/kg ibrutinib in drinking water, 34mg/kg or 70mg/kg of AZD0466 once weekly (QW) by i.v, or the vehicle. Treatments were continued until reaching a predefined humane endpoint. AZD0466 at 70mg/kg led to a significant increase in survival (median 72 days vs. 38 days, P=0.0009) similar to ibrutinib (67 days, P=0.0026) when compared to vehicle (Fig. 1). Analysis of blood parameters at euthanasia did not reveal significant difference in platelet counts between AZD0466, ibrutinib and vehicle treatments. To evaluate if combination treatment of AZD0466 with BTK inhibitors would improve efficacy, we transplanted murine Eµ-TCL1 tumors into syngeneic recipient mice and randomized them for treatment with vehicle, ibrutinib (30mg/kg in drinking water), acalabrutinib (25mg/kg, p.o. QD), AZD0466 (70mg/kg, i.v., QW) and combination of AZD0466 with ibrutinib or acalabrutinib. AZD0466 and vehicle treatments showed similar platelet and RBC counts measured 2 or 3 days after the 2 nd and 4 th dosing but, a significant increase in platelet and RBC counts were observed with the combination treatments. A significant decrease in WBC count after 2 weeks, and in bone marrow tumor load after 4 weeks was observed with combined AZD0466 and acalabrutinib treatment compared to the single drugs. Though spleen weights did not differ after 4 weeks between vehicle and AZD0466 monotherapy, a strong decrease in spleen weight and tumor cell number in spleen was observed with the combination of AZD0466 with ibrutinib or acalabrutinib compared to vehicle. To validate the findings in human CLL, we performed dynamic BH3 profiling on primary CLL samples with mutated (N=5) and unmutated IGHV (N=7). Exposure of CLL cells to 1µM acalabrutinib for 24 hours significantly increased BCL2 dependency in all CLL samples with unmutated IGHV and in a subset of mutated IGHV samples. These tumors also showed an increased response to venetoclax and AZD4320 upon exposure to acalabrutinib. To study the efficacy of the dual BCL2/BCL-XL inhibitor in the context of venetoclax resistance, we stably expressed the BCL2 mutations A113G, D103E, D103V, D103Y, G101V, R129L, V156D and wildtype (WT) in the venetoclax sensitive mantle cell lymphoma cell line MAVER-1. Response to AZD4320 or venetoclax was analyzed using Annexin V/7AAD staining and flow cytometry after 48 hours of treatment. The BCL2 mutants showed varying degrees of resistance to venetoclax. MAVER-1 expressing WT had an IC50 of 17.7nM while D103E, D103V, D103Y, G101V showed the strongest resistance to venetoclax with 5, 13.8, 4.5 and 7.3 fold increase in IC50 compared to WT. Of note, compared to WT (IC50 43.9nM), D103E, D103V and D103Y were sensitive to AZD4320 with a fold change in IC50 of 0.6, 1.8 and 1.3, respectively. G101V mutant cells showed a 5 fold increase in IC50 to AZD4320 compared to WT (Fig. 2). Moreover, AZD4320 was highly efficacious in MAVER-1 and MINO cell line models where resistance to venetoclax mediated by BCL-XL upregulation was modelled by an in vitro dose escalation method. In summary, our pre-clinical study shows that the dual BCL2/BCL-XL inhibitor could represent an important treatment option for venetoclax resistance mediated by specific BCL2 mutations or BCL-XL upregulation and that its efficacy could be further improved upon combination treatment with BTKi.

Regulation of Alternative Splicing in B-Cell ALL By DYRK1A

DYRK1A, located in the Down syndrome critical region of chromosome 21, is a serine and threonine kinase that controls multiple cellular processes including apoptosis, cell cycle, transcription and signal transduction. We previously demonstrated that DYRK1A is required for maturation of B cells and is a therapeutic target in B-cell ALL, including both DS and non-DS subtypes. Key substrates within the B-cell lineage include D-type cyclins, FOXO1, and STAT3. Given that several studies have reported that DYRK1A phosphorylates various splicing factors and participates in splicing of Tau in neuronal cells, we investigated the contribution of DYRK1A to splicing in malignant B cells. We first treated B-ALL cell lines with two DYRK1A inhibitors, EHT 1610 and GNF 2133 and performed RNA-sequencing to detect changes in alternative splicing (AS). GO analysis of AS events showed RNA binding proteins among top differentially spliced genes. Of note, mis-splicing of RBM39 was one of the top differentially AS events associated with DYRK1A inhibition; this event involves inclusion of a “poison” exon that contains a premature termination codon and leads to a transcript predicted to be degraded by nonsense-mediated mRNA decay (NMD). We confirmed NMD by knocking down an essential NMD factor, UPF1, which stabilized the NMD isoform. The NMD isoform was also detected in B-ALL patient samples treated with DYRK1A inhibitors and in NALM6 cells in which DYRK1A was inducibly degraded. RBM39 has been reported to be a therapeutic target in AML, where cells with splicing factor mutations are more sensitive to loss of RBM39 than cells lacking splicing factor mutations. To determine whether RBM39 is a therapeutic target in B-ALL, we silenced RMB39 with shRNAs in vitro and in vivo. Genetic knockdown of RBM39 impeded B-ALL cell growth both in vitro and in xenograft models. Furthermore, inducible silencing of RBM39 hampered B-ALL progression in vivo evidenced by a significant increase in survival of mice engrafted with human B-ALL cells. We also found that indisulam and its analogs, which lead to DCAF15-mediated degradation of RBM39, suppressed the growth of B-ALL cells in vitro and in vivo. Importantly, the anti-tumor effect of RBM39 disruption extended beyond DS-ALL to Ph-like B-ALL, which continues to be an area of unmet medical need. We next analyzed the contributions of splicing factors that are known DYRK1A substrates to AS of RBM39. Knockdown of SRSF1 and SRSF2 led to increased inclusion of the poison exon even in the absence of DYRK1A inhibition, indicating that they normally function to exclude the exon. By contrast, knockdown of SF3B1 led to a reduction of the NMD isoform in the presence of EHT1610. Surprisingly, phosphorylation of SF3B1 by DYRK1A was not essential for the change in RBM39 splicing, as cells engineered to harbor two SF3B1 T434A phospho-deficient mutant alleles, did not show aberrant splicing. These results indicate that SF3B1, SRSF1, and SRSF2 are not the key substrates that mediate the effect of DYRK1A on alternative splicing. Next, given that DYRK1A is known to phosphorylate the C-terminal domain (CTD) of RNA polymerase II (Pol II) on Ser2 and Ser5, we investigated the contribution of Pol II phosphorylation to RBM39 splicing. IP-MS revealed an interaction between RNA Pol II and SF3B1, consistent with previous reports of co-transcriptional splicing. We found that inhibition of DYRK1A disrupted the colocalization of SF3B1 and RNA Pol II as well as with RBM39 RNA. However, DYRK1A inhibition had a transient effect on Ser2 and Ser5 phosphorylation, suggesting that cells have alternative pathways for Ser2/5 phosphorylation. Combining DYRK1A inhibition with inhibition or loss of CDK9, which also phosphorylates the CTD of RNA Pol II, led to a persistent decrease in RNA Pol II phosphorylation, marked increased NMD isoform and a profound reduction in RBM39 protein. Finally, we found that combining the CDK9 inhibitor dinaciclib with EHT 1610 synergistically inhibited growth of B-ALL patient cells in vitro and in vivo shown by significantly improved survival. Ectopic expression of RBM39 partially rescued the growth inhibition upon dinaciclib and EHT 1610 treatment suggesting downregulation of RBM39 is the key event in drug targeting. Together, our results reveal that DYRK1A controls RNA splicing by regulating the SF3B1-RNA Pol II interaction and that RBM39 is a therapeutic target in B-ALL.

The FLT3-like Gene Expression Signature Predicts Response to Quizartinib in Wild-Type FLT3 Acute Myeloid Leukemia: An Analysis of the Pethema Quiwi Trial

Background The identification of predictive biomarkers is crucial for guiding treatment decisions in acute myeloid leukemia (AML). Previously, we identified a FLT3-like gene expression signature in FLT3 wild-type AML patients, which clustered a variable proportion of FLT3 wild-type patients with FLT3-ITD and TKD mutated patients. The QUIWI trial was a randomized, placebo-controlled, phase II study preliminary showing a significant increase in overall survival (OS) in wild-type FLT3 AML patients treated with the FLT3 inhibitor quizartinib (Quiza) plus standard chemotherapy. This preplanned correlative study was designed to assess the value of the FLT3-like signature to predict responses to Quiza. Methods We performed RNA sequencing (RNAseq) analysis on a subset of patients from the clinical trial. RNA was extracted using standard methods, followed by assessment of nucleic acid integrity (TapeStation) and quantification (Qubit). Total mRNA sequencing was performed using polyA RNAseq with TruSeq technology. A total of 206 adequate samples from bone marrow and peripheral blood were sequenced (161 from FLT3-ITD negative enrolled in QUIWI; and 55 from FLT3-ITD positive patients who were screen failure of the QUIWI trial by this reason). The sequences were aligned to the GRCh37 reference genome using the Hisat algorithm. Gene expression quantification was performed using the Bioconductor workflow, and gene expression estimates (FPKM) were obtained. The gene expression estimates were log2 normalized. Finally, those genes mapping to the original FLT3-like signature (595 genes) were selected for downstream analysis. Clustering was based on the hierarchical method, using standard euclidean distance metrics. OS was defined as time from start of screening to death. Event-free survival (EFS) was defined as time from randomization to failure to achieve CR/CRi after 1 or 2 cycles, death in CR/CRi, or relapse (whichever occurred the first). Relapse-free survival was defined as time from randomization to disease relapse or death by any cause. Results Among the total 206 patients, a cluster of 54.37% (N=112) was enriched in FLT3-mutant cases (71.11% of cases, Figure 1A). This subgroup comprised 49.67% of wild-type FLT3 cases (N=80), hereafter FLT3-like patients. In the group of not FLT3-like patients, no differences were identified between the placebo and Quiza group in the total number of deaths (Fisher's p-value, 0.63), EFS (cox p-value, 0.83; HR 1.07 [0.56-2.06]), RFS (cox p-value 0.76; HR 0.88 [0.38-2.01]) and OS (cox p-value, 0.62; HR 1.22 [0.55-2.67]). Among FLT3-like patients, significant differences were identified in the total number of deaths (Fisher's p-value, 0.004), EFS (cox p-value, 0.009; HR 0.45 [0.25-0.82], RFS (cox p-value 0.01, HR 0.37 [0.18-0.79]) and OS (cox p-value 0.01, HR, 0.41 [0.20-0.84]) ( Figure 1B). No statistically significant association was observed between the FLT3-like pattern and the ELN-17 classification: 30.4% were low risk, 40.5% intermediate risk, 29.1% high risk. Conclusion The FLT3-like gene expression signature successfully identified a subset of patients who derived the most benefit from Quiz, while patients without the FLT3-like signature did not demonstrate a benefit compared with placebo. These findings support the use of the FLT3-like signature as a potential biomarker to identify those wild-type FLT3 AML patients who may benefit from Quiz, providing a valuable insight for personalized treatment in AML.