Effect of oral posaconazole on venetoclax plasma concentration and efficacy in patients with acute myeloid leukemia

Effect of oral posaconazole on venetoclax plasma concentration and efficacy in patients with acute myeloid leukemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of oral posaconazole on venetoclax plasma concentration and efficacy in patients with acute myeloid leukemia Mengqi Guo, Jingwen Du, Yingzhi He, Dezhi Qiu, Yuxian Huang, Yinjie Qin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4188474/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract BCL-2 was the first gene identified to have antiapoptotic effects and venetoclax is an oral selective BCL-2 inhibitor, which has great potential in the treatment of patients with acute myeloid leukemia (AML) who are not candidates for intensive therapy. Notably, posaconazole, an oral antifungal drug, is also a strong factor that can affect blood venetoclax concentrations. To the best of our knowledge, the relationship between BCL-2 expression, posaconazole and venetoclax, as well as the influence of them on treatment efficacy and the prognosis of patients with AML, has not been reported. Therefore, in the present study, the relationship between BCL-2 expression and blood venetoclax concentration was analyzed in 35 patients with AML. BCL-2 mRNA expression levels were examined by reverse transcription quantitative PCR. Blood venetoclax concentrations were measured using high-performance liquid chromatography-tandem mass spectrometry. The results revealed that among patients with AML, those with lower primary BCL-2 expression had a higher complete remission (CR) rate (P = 0.005), overall response rate (P < 0.0001) and progression-free survival time (P = 0.04). Posaconazole was revealed to be a strong factor that was able to increase blood venetoclax concentration (P < 0.001) and CR rate in the venetoclax plus posaconazole group compared with that in the venetoclax monotherapy group (P = 0.002); however, no significant difference was identified in the occurrence of adverse reactions between these groups. Among low and high blood venetoclax concentration groups, the event-free survival of the former group was significantly higher (P = 0.013). In conclusion, the results of the present study could be used to guide clinical practice in the treatment of AML. BCL-2 expression posaconazole venetoclax concentration acute myeloid leukemia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Acute myeloid leukemia (AML) is the most common among all types of diagnosed acute leukemia worldwide (1). Previous reports have revealed that AML is complex and aggressive, with a wide variety of clinical presentations, morphological features and immunophenotypes (1,2). Specific characteristics of AML include the inhibition of myeloid progenitor cell maturation and clonal expansion of bone marrow blasts (1). In particular, evasion of apoptosis is an important mechanism that increases leukemic cell survival rate. As an anti-apoptotic gene, BCL-2 increases the number of AML cells by prolonging cell survival (3,4). To the best of our knowledge, BCL-2 was the first gene to be identified as antiapoptotic. BCL-2 can prolong cell survival and has been shown to be overexpressed in a wide variety of tumors, such as colorectal cancer, breast cancer and non-small cell lung cancer (2-7). BCL-2 was first discovered in the t(14;18) chromosomal translocation in B-cell lymphoma (3,4). According to previous research, the upregulated expression of members of the BCL-2 family, such as BCL-2 and MCL-1, may be related to the pathogenesis, poor prognosis and treatment resistance of AML; therefore, BCL-2-targeted therapy could be considered a treatment strategy with great potential for patients with AML (8-13). Venetoclax is an orally selective BCL-2 inhibitor; its main mode of action is through BAK and BAX-induced activation of mitochondrial outer membrane permeabilization to induce apoptosis. Venetoclax has been shown to inhibit cell proliferation and induce apoptosis of tumor cell lines, and to slow down tumor progression to prolong overall survival (OS) (14,15). Venetoclax has a high specificity for BCL-2, low toxicity against healthy cells and cytotoxic activity against a variety of hematological tumors, including AML. It was approved by the US Food and Drug Administration (FDA) in 2016 for the treatment of chronic lymphocytic leukemia (14-17). Since 2018, the FDA gradually granted approval to venetoclax for the combined treatment of patients with newly-diagnosed AML who are aged ≥75 years, or those who cannot tolerate chemotherapy (15). The pharmacokinetics of venetoclax have been well characterized and are described in the literature (16,17). Notably, blood venetoclax concentrations exhibit a great deal of variation depending on factors, such as pharmacokinetic characteristics, population differences, diet habits and drug combinations. In the clinical treatment of AML, posaconazole, an antifungal drug commonly used in the clinic, is often used to prevent fungal infection because patients often suffer from myelosuppression. Previous studies have concluded that the combination of posaconazole can reduce the oral dose of venetoclax by ≥75% (16-21). The present study on the effect of posaconazole combined with venetoclax on the blood concentration of venetoclax is based on the results of a theoretical model calculation or strictly controlled variable, which lacks real-world data for comparison and verification (22). Due to various uncontrollable factors existing in actual treatment, it is expected that the real-world data will differ from the theoretical values to varying degrees, and the conclusion may be different. Therefore, it is of great significance to further explore the effect of combined posaconazole on the blood concentration of venetoclax in clinical practice, and to determine whether there are differences in the treatment efficacy and occurrence of adverse reactions in patients with AML with different concentration ranges of venetoclax, so as to provide a more specific reference for better balancing the safety and effectiveness of venetoclax in clinical use. In addition, to the best of our knowledge, the relationship between BCL-2 expression and the concentration of venetoclax has not been conclusively determined, and the relationship between BCL-2 expression and the outcome and prognosis of patients with AML has not been clearly defined. Therefore, further exploration may also provide novel information regarding clinical treatment. Based on data from 35 patients with AML, the objective of the present study was to characterize BCL-2 expression and venetoclax concentration, to explore the relationship between these two factors, and to assess whether they have an effect on the treatment efficacy and prognosis of patients with AML. In addition, the present study evaluated the exposure-efficacy and exposure-safety relationships in patients with AML with different venetoclax plasma concentrations. Materials and methods Reference standard. A total of 35 patients who were diagnosed with AML [excluding acute promyelocytic leukemia (AML-M3)] at Zhujiang Hospital (Guangzhou, China) between February 2018 and July 2022 were selected in this single-center, retrospective study. The mean age of the patients was 56 years (range, 10–72 years). The bone marrow morphology (according to the French American UK criteria) ( 22 ) and flow cytometry results of all patients were consistent with the diagnosis of AML. The inclusion criteria were as follows: i) ≥ 20% of peripheral blood or bone marrow primordial granules (or monocytes) were diagnosed with AML. ii) When a patient was confirmed to have clonal recurrent cytological abnormalities: t(8;21) (q22;q22), AML-ETO, t (15;17) (q22;q12), PML-RARA and its variants; t(16;16) (p23;q11) or inv ( 16 ) (p13;q22), CBFB-MYH11 or MLL gene rearrangement, even if the proportion of bone marrow primordial granules was < 20%, AML should also be diagnosed. The exclusion criteria were as follows: i) Patients who was diagnosed with AML-M3. ii) There was extramedullary infiltration (including active central nervous system leukemia, skin infiltration and myeloid sarcoma). iii) AML was treatment-related (including tumor radiotherapy and chemotherapy). iv) There was major organ dysfunction (mainly heart, liver, kidney function, etc.) that was not suitable for treatment with venetoclax. The assessment of the grade of adverse reactions was performed according to the Common Terminology Criteria for Adverse Events 5.0 ( 23 ). The standards of low blood venetoclax concentration (0-990 ng/ml), standard blood venetoclax concentration (991-3,210 ng/ml) and high blood venetoclax concentration (3,211-5,370 ng/ml) were based on related research ( 21 ). Combined with previous studies on the relationship between BCL-2 expression and AML, a binary classification of initial BCL-2 expression in 35 patients with AML was based on the measured median level of BCL-2 expression ( 4 , 8 , 9 ). The primary endpoint was disease progression or death from any cause, and the secondary endpoint was change or discontinuation of the treatment regimen for any reason. Method of administration. All 35 patients were treated with azactidine (AZA)/AZA + cytarabine. In addition, there were two treatment groups for venetoclax. Patients in the venetoclax monotherapy group were treated with 100 and 200 mg on the 1st and 2nd day, respectively; from the third day, they were treated with 400 mg venetoclax orally once a day. In the venetoclax plus posaconazole group, patients were treated with 100 mg venetoclax orally once a day and 5 ml (200 mg) posaconazole orally three times a day (Fig. 1 ). A related study was used as reference to determine dosage and frequency of both drugs ( 16 ). Sample collection. Each course of venetoclax treatment was recommended for 28 days. Bone marrow specimens were collected from 35 patients with AML at the time of primary diagnosis, and efficacy was assessed when patients stopped taking venetoclax (for patients who used venetoclax for < 28 days) or after the first course of venetoclax (for patients who used venetoclax for ≥ 28 days). Bone marrow samples used to measure BCL-2 expression were taken for the first time after initial diagnosis or recurrence, and when efficacy was assessed. For each case, steady-state venetoclax concentrations were measured 5–8 h after accepting venetoclax on the 6th day after patients started single dose therapy (the half-life of venetoclax was 26 h) ( 24 ). Efficacy was evaluated according to the results of bone marrow morphology and peripheral blood count, and the criteria were as follows: Complete remission (CR): Proportion of bone marrow primordial granules ≤ 5%; there were no original cells in peripheral blood circulation and no Auer rod-like bodies in the original cells. No extramedullary invasion; neutrophil count ≥ 1.0x10 9 /l (1,000/µl); platelet count ≥ 100x10 9 /l (100,000/µl). CR but incomplete recovery of blood count (CRi): On the basis of meeting the bone marrow and clinical criteria for CR, peripheral blood still had one of the following: i) Neutrophil count < 1.0x10 9 /l (1,000/µl); ii) platelet count < 100x10 9 /l (100,000/µl). Partial response (PR): When the peripheral blood cell count reached CR standard, the proportion of naive cells in bone marrow decreased by 5–25% and ≥ 50% compared with before treatment. If Auer clavicular bodies were observed in the original cells, even bone marrow primordial granules < 5% should be defined as PR. OR: The total number of patients with CR, CRi and PR after treatment. No remission (NR) : Bone marrow, blood and clinical test results did not meet the aforementioned criteria ( 25 ). Reverse transcription quantitative PCR (RT-qPCR) detection of BCL-2 mRNA expression in AML cells. BCL-2 mRNA expression levels were measured in bone marrow samples from 35 patients by RT-qPCR using GAPDH as an internal reference. The primers used were as follows: GAPDH, forward 5’-CAAGAGCACAAGAGGAAGAGAG-3’, reverse 5’-CTACATGGCAACTGTGAGGAG-3’; BCL-2, forward 5’- GGCCAGGGTCAGAGTTAAATAG-3’ and reverse 5’-GGAGGTTCTCAGATGTTCTTCTC-3’. RNA was extracted using RNAiso Plus (Takara Bio, Inc.). RT was performed using PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio, Inc.) according to the manufacturer’s protocol. The thermocycling conditions for qPCR (SYBR green PCR kit; Takara Bio, Inc.) were as follows: 95˚C for 30 sec, followed by 40 cycles at 95˚C for 5 sec and 60˚C for 34 sec, followed by 60˚C for 60 sec and 95˚C for 15 sec. Relative BCL-2 mRNA expression levels were calculated using the 2 −ΔΔCq method ( 26 ). The 2-ΔCq value was selected as the result of the relative expression levels of BCL-2 in the present study based on the experimental method. Determination of plasma venetoclax concentration by HPLC/MS/MS . Patient plasma samples were treated with 50% methanol (containing the internal standard Venetoclax-d8; (Clearsynth) to precipitate protein. The sample size (3 µl) underwent chromatography using the Ultimate@ XB⁃C18 chromatography column (4.6x50 mm; 5 µm; Welch Materials, Inc.); the column temperature was 60˚C. The mobile phase consisted of an aqueous solution containing 0.1% formic acid and 2 mmol/l ammonium acetate (phase A) and a methanol solution containing 0.1% formic acid (phase B), with a flow rate of 0.7 ml/min. The gradient elution method was used, from 0.01 min to 1.0 min, the concentration of phase B was increased from 60–95%; from 1.0 min to 2.5 min, the concentration of phase B was held at 95%; from 2.5 min to 2.6 min, the concentration of phase B was decreased from 95–60%; finally, the concentration of phase B was maintained at 60% from 2.6 min to 3.5 min. Electrospray ion source was selected for multi-ion reaction monitoring detection. Its ion source parameters were ( 27 ): ion spray injection voltage, 4,500 V; ion source temperature, 550˚C; spray gas, 50 psi; auxiliary heating gas, 55 psi; air curtain gas, 20 psi; collision gas, Medium. Simultaneous monitoring of venetoclax m/z 868.5 ~ 321.7 (quantitative ion), venetoclax m/z 868.5 ~ 177.6 (qualitative ion) and venetoclax -d8 m/z 876.6 ~ 329.4 (quantitative ion) was performed. The peak area ratio of Venetoclax and Venetoclax⁃d8 was used to calculate the plasma Venetoclax concentration. Jasper high-performance liquid chromatography (Shimadzu Corporation) in tandem with AB Sciex Triple Quad 4500 MD mass spectrometry (AB Sciex) were used in this determination. Analyst®1.6.3 software (AB Sciex) was used for result analysis ( 28 , 29 ). Statistical analysis . Statistical evaluation of the data was performed using SPSS 25.0 (IBM Corp.). GraphPad Prism 9.0 (Dotmatics) was used for the creation of figures. In the present study, the measured data do not obey a normal distribution and are presented as median (range). Count data are presented as number of cases (%). Comparisons of categorical variables between groups were made using Fisher's exact test or χ 2 test. Spearman’s test was used to explore the correlation between continuous variables, and Mann-Whitney U test was used to compare the differences of the same continuous variable between different groups. Wilcoxon signed-rank test was used to compare the difference in BCL-2 expression before and after venetoclax treatment. In the present study, patient prognosis was mainly explored through three indicators: OS, the time from randomization to death of any cause; progression-free survival (PFS), the time between enrollment and tumor progression or death; and event-free survival (EFS), the time from enrollment to the occurrence of any event, including disease progression, discontinuation of treatment for any reason or death. OS, PFS and EFS and were calculated using Kaplan-Meier survival curve and log-rank test, assessing whether the expression levels of BCL-2, the blood concentrations of venetoclax or whether posaconazole was used affected survival. Data missing from the survival analysis are included as truncated data. In all analyses, two-tailed tests were performed, and P < 0.05 was considered to indicate a statistically significant difference. Results Baseline data. A total of 35 patients with AML, including 15 women and 20 men, were involved in the analyses, with a median age of 56 years. Before accepting venetoclax, 3 patients were in a state of no remission after chemotherapy, 2 patients had relapsed after chemotherapy, 4 patients had accepted treatment after allogeneic hematopoietic stem cell transplantation because of different reasons (in 3 patients minimal residual disease reverted to positive; 1 patient progressed from myelodysplastic syndrome), 1 patient had relapsed before treatment, and the remaining 26 patients were newly diagnosed. Among them, 4 patients had favorable risk, 10 had intermediate risk and 21 had poor risk AML. The range and median of bone marrow blasts was 2.04–94.6 and 26.98%, respectively. Blast data from 4 patients were missing. Regarding genetic mutations, there were 11 patients with the WT1 mutation, 4 with the NPM1 mutation, 5 with the RUNX1 mutation, 4 with the ASXL1 mutation, 4 with the TET mutation, 3 with the TP53 mutation, 2 with the BCOR mutation, 4 with the AML-ETO1 mutation and 3 with the NRAS mutation. The albumin level in the 35 patients fluctuated between 29 and 56 g/l, with a median albumin concentration of 37.6 g/l (normal albumin level, 35–50 g/l). A total of 17 patients received 100 mg venetoclax once a day + 5 ml posaconazole three times a day, and 18 patients took only venetoclax daily. The venetoclax concentration varied greatly, with the minimum and maximum concentrations at 250 and 5,370 ng/ml, respectively, and the median venetoclax concentration was 1,420 ng/ml. By the end of the follow-up, 3 patients relapsed after taking venetoclax. In total, 6 patients died: 1 from severe pneumonia, 2 from septic shock and 3 from AML. Baseline data and the grouping of patients are shown in Figs. 1 and 2 , and Table I. Analyses of the potential influential factors on blood venetoclax concentration . The present study assessed patient sex, age, albumin level and whether treatment was combined with posaconazole or not to explore their effects on venetoclax concentration. It was observed that age (P = 0.689; Fig. 3 A), albumin level (P = 0.945; Fig. 3 B) and sex (P = 0.771; Fig. 3 D) were not significantly associated with blood venetoclax concentration. However, patients who received venetoclax and posaconazole had higher blood venetoclax concentrations than those who used venetoclax only (P < 0.001; Fig. 3 C). The ranges in the venetoclax monotherapy group and venetoclax plus posaconazole group were 256-2,430 and 969-5,370 ng/ml, respectively. Furthermore, in the venetoclax monotherapy group, half of the patients (9/18) achieved a low concentration while the rest of the patients achieved high concentrations. In the venetoclax plus posaconazole group, the proportion of patients that achieved low concentrations, standard concentrations and high concentrations were 6% (1/17), 35% (6/17) and 59% (10/17), respectively (Fig. 1 ). Analyses of potential influential factors on BCL-2 expression. The present study revealed that BCL-2 expression in the 35 patients with AML at primary diagnosis was significantly correlated with bone marrow blast percentage (P < 0.001, r = 0.717; Fig. 3 F). However, BCL-2 expression was not significantly correlated with age (P = 0.943; Fig. 3 E), sex (P = 0.128; Fig. 3 G) and cytogenetic risk (P = 0.170; Fig. 3 H). In all patients, a statistically significant difference was seen in BCL-2 expression between primary diagnosis and after using venetoclax within 28 days (P < 0.001; Fig. 3 I). Statistically significant differences in BCL-2 expression before and after medication were also seen in the venetoclax monotherapy group (P < 0.001) and in the venetoclax plus posaconazole group (P < 0.001) (Fig. 3 I). Notably, significant differences in BCL-2 expression before and after medication were also detected in the low concentration group (P = 0.002), the standard concentration group (P = 0.031) and the high concentration group (P < 0.001) (Fig. 3 J). Efficacy analyses. Out of the 35 patients, 18 patients were in the low BCL-2 expression group and 17 patients were in the high BCL-2 expression group. A significant difference was observed in the number of patients with overall response (OR) (P < 0.0001) and in the number of patients that achieved CR (P = 0.005) (Fig. 4 A) The proportion of patients with OR and CR in the low-expression and high-expression BCL-2 groups was 100% (18/18) vs. 41% (7/17), and 61% (11/18) vs. 12% (2/17), respectively. (Fig. 4 A) There was no significant difference in the number of patients who achieved CRi (P = 0.658) and the number of patients who achieved PR (P = 1) between the two different BCL-2 expression groups (Fig. 4 A). There was no significant difference in the number of patients who achieved CRi (P = 0.177), PR (P = 0.177) or OR(P = 0.711) between the venetoclax monotherapy group and the venetoclax plus posaconazole group (Fig. 4 B). However, there was a significant difference in the number of patients who achieved CR (P = 0.002) between these two groups. In the venetoclax monotherapy group and venetoclax plus posaconazole group, 2 and 11 patients achieved CR, respectively (Fig. 4 B). In the low blood venetoclax concentration group, the numbers of patients with OR, and those that achieved CR, CRi and PR were 6, 1, 3 and 2 patients, respectively. In the standard concentration group, the number of patients were 5, 4, 1 and 0, respectively. Furthermore, in the high blood venetoclax concentration group the numbers were 14, 8, 2 and 4 patients, respectively. There was no significant difference in the number of patients in the three different venetoclax concentration groups (low venetoclax concentration vs. standard venetoclax concentration vs. high venetoclax concentration) that were OR(P = 0.680), achieved CR (P = 0.068), achieved CRi (P = 1) or achieved PR (P = 0.696) (Fig. 4 C). Safety analyses . Regarding adverse events, similar results were recorded between the venetoclax monotherapy group and the venetoclax plus posaconazole group, as follows: Group 1 (without agranulocytosis or fever), P = 0.603; group 2 (fever with agranulocytosis), P = 0.489; group 3 (only agranulocytosis), P = 0.711 ; group 4 (only fever), P = 0.264; 0–2 grade leukopenia, P = 0.489; 3–4 grade leukopenia, P = 0.489; 0–2 grade anemia, P = 1; 3–4 grade anemia, P = 1; 0–2 grade thrombocytopenia, P = 1; 3–4 grade thrombocytopenia, P = 1; alanine aminotransferase (ALT) increase, P = 1 (Fig. 5 A, C, E, G and S1 ). No significant difference was observed in the number of adverse-exposure reactions between the patients within different venetoclax concentration groups (low concentration vs. standard concentration vs. high concentration), as follows: Group 1 (without agranulocytosis or fever), P = 0.472; group 2 (fever with agranulocytosis), P = 0.510; group 3 (only agranulocytosis), P = 0.886; group 4 (only fever), P = 0.071; 0–2 grade leukopenia, P = 0.343; 3–4 grade leukopenia, P = 0.343; 0–2 grade anemia, P = 0.260; 3–4 grade anemia, P = 0.260; 0–2 grade thrombocytopenia, P = 0.307; 3–4 grade thrombocytopenia, P = 0.307; ALT increase, P = 0.208 (Figs. 5 B, D, F, H and S2 ). Throughout the study, no renal function impairments and tumor lysis syndrome were observed. Effect of BCL-2 expression and venetoclax concentration on prognosis. OS of the patients was not significantly associated with initial BCL-2 expression (high expression vs. low expression, P = 0.11; Fig. 6 A). In addition, no significant differences in OS were determined among the three venetoclax concentration groups (low concentration vs. standard venetoclax, P = 0.91; low concentration vs. high concentration, P = 0.32; standard venetoclax vs. high concentration, P = 0.40; Fig. 6 B), or between the venetoclax monotherapy group and the venetoclax plus posaconazole group (P = 0.50; Fig. 6 C). Regarding PFS, patients with low initial BCL-2 expression had a better trend than those in the high BCL-2 expression group (P = 0.04; Fig. 6 D). There were no obvious significant differences in PFS among the different venetoclax concentration groups and between the two treatments groups, respectively (low venetoclax concentration vs. standard concentration, P = 0.48; high concentration vs. standard concentration, P = 0.71; low concentration vs. high concentration, P = 0.06; venetoclax monotherapy vs. venetoclax plus posaconazole, P = 0.38). However, the PFS trend in the high concentration group was higher than that in the low concentration group, but this was not significant (Fig. 6 E and F). There were no significant differences in EFS between patients in the high and low initial BCL-2 expression groups (P = 0.79; Fig. 6 G). Furthermore, the differences in EFS were not significant between the standard concentration and low concentration (P = 0.75), and standard concentration and high concentration (P = 0.10) groups. However, EFS was significantly different between the high concentration and low concentration groups (P = 0.01; Fig. 6 H). There was no evident difference in EFS between patients treated with venetoclax and those treated with venetoclax plus posaconazole (P = 0.31; Fig. 6 I). Discussion Previous studies have suggested that upregulation of BCL-2 is association with the occurrence and development of AML ( 8 ). BCL-2 can accelerate the progression of AML, and high BCL-2 expression has been detected in both relapsed AML cells and treatment-resistant AML cells ( 28 , 30 , 31 ). The present study revealed that BCL-2 expression among individuals was highly variable, which is consistent with other previous research ( 4 , 8 – 10 ). The current study demonstrated that BCL-2 expression was correlated with the proportion of bone marrow blasts in patients with AML at initial diagnosis. It has previously been reported that, compared with in healthy individuals, the expression of BCL-2 is upregulated in patients newly diagnosed with AML; however, BCL-2 expression was not shown to be related to the proportion of bone marrow blasts, which differs from the present conclusion ( 8 ). Based on the present results, irrespective of the different treatment groups or different venetoclax concentration groups, there were significant differences in the expression of BCL-2 before and after medication. This suggests that venetoclax is a factor that may reduce BCL-2 expression in patients with AML. Through analysis of the low and high BCL-2 expression groups, it was revealed that BCL-2 expression levels at initial onset may have a negative impact on whether patients with AML exhibit CR after treatment, which may be related to the upregulation of BCL-2 leading to relapse and venetoclax drug resistance ( 32 ). Since most patients who responded to the treatment achieved CR, the significant difference in the number of patients achieving OR in the low BCL-2 expression group and the high BCL-2 expression group may be related to the fact that the majority of patients that achieved OR in the two groups had achieved CR. That is, the significant difference in the number of patients achieving OR in different BCL-2 expression groups may be caused by the significant difference in the number of patients achieving CR. Furthermore, it was revealed that patients with low BCL-2 expression had better PFS than those who had high BCL-2 expression; however, there was no significant difference in OS and EFS between the two groups. Although the present findings are similar to those of previous studies ( 33 – 35 ), some other studies that have questioned whether BCL-2 upregulation can really affect the prognosis of patients with AML ( 35 , 36 ). Notably, a previous study did confirm the hypothesis that BCL-2 upregulation could lead to relapse and drug resistance in patients with AML ( 32 ); thus, BCL-2 may affect the prognosis of patients with AML in this manner. The present study indicated that venetoclax may improve treatment efficacy and prognosis of patients with AML by reducing BCL-2 expression. According to a previous study and clinical practice, posaconazole can be used for antifungal prophylaxis in patients with AML ( 16 ). Furthermore, it has been reported to significantly increase blood venetoclax concentration; therefore, the venetoclax dose can be reduced by ≥ 75% due to posaconazole ( 16 ). In addition, this previous study revealed that, relative to 400 mg venetoclax alone, coadministration of 100 mg venetoclax with posaconazole (300 mg) can significantly increase mean venetoclax peak concentration and area under the concentration time curve of 0–24 h. The results of the present study revealed that, compared with in the 400 mg venetoclax dose group, the combined venetoclax and posaconazole group appeared to have a higher concentration of venetoclax in the blood. Almost all of the patients who were treated with venetoclax and posaconazole met or even exceeded the standard concentration. Notably, venetoclax concentrations in the combined venetoclax and posaconazole group were higher than those in the venetoclax monotherapy group. This is consistent with the conclusions from a previous study, thus suggesting that posaconazole can significantly increase blood venetoclax concentrations ( 16 ). One of the concerns of the present study was that some of the patients (7/18) who received 400 mg venetoclax at the beginning had to decrease their venetoclax dose due to other reasons (such as severe agranulocytosis, anemia and thrombocytopenia) during treatment. However, the blood venetoclax concentrations of the patients fluctuated greatly in the two groups with different administration regimens, which was consistent with the conclusions from a previous study ( 16 ). It may be hypothesized that this, on the one hand, is due to the differences in bioavailability of venetoclax among different individuals (based on factors such as ethnicity, liver condition and dietary habits), and on the other hand, this could be related to the high heterogeneity in posaconazole concentrations among patients ( 37 ). Notably, the CR rate in the present study (11%) was better than previously reported in patients with AML given only venetoclax (6%) ( 8 ). It may be hypothesized that the reason for this difference could be that the subjects included in previous study were almost all patients with relapsed refractory AML with a poor prognosis. In particular, different outcomes were identified in different medication groups. In the present study, it appears that in the venetoclax monotherapy group and the venetoclax plus posaconazole group, there was a significant difference in the number of patients achieving CR. Since a higher proportion of patients achieved CR in the venetoclax plus posaconazole group, this may be related to the higher venetoclax concentration in this group. Notably, there were no obvious differences in the occurrence of adverse events between the venetoclax monotherapy group and the venetoclax plus posaconazole group. The present study compared the prognosis of the two groups and revealed that, although there was no significant difference in OS, PFS and EFS between the two groups, the EFS trend of the venetoclax monotherapy group was higher than that of the venetoclax plus posaconazole group. This may be because of the fact that a larger proportion of patients met or exceeded the standard venetoclax concentration in blood in the combination group. Even though there was no significant difference in the number and proportion of adverse events between the two groups, a higher venetoclax concentration may lead to an earlier onset of adverse reactions. Since only one study ( 16 ) has explored the effect of two drug administration regimens on the treatment efficacy and prognosis of patients with AML, and the sample size of this study was small, this finding should be further explored in a study with an expanded sample size. The present results are similar to the results of a previous study on the effect of venetoclax concentration with different combination regimens on the treatment efficacy in patients with AML ( 11 ); there were no significant differences in the number of patients who responded to treatment, achieved CR, achieved CRi or achieved PR among the low concentration, standard concentration and high concentration groups in the present study. As for the occurrence of adverse events, there was no apparent difference among the different concentration groups, which is also similar to the results from recent literature ( 11 ). When comparing the three different venetoclax concentrations, no effect of venetoclax concentration on the OS or PFS of the 35 patients with AML was determined, which is consistent with a previous study ( 8 ). In addition, the EFS of the low concentration group was markedly longer than that of the high concentration group. It could be hypothesized that the increase in blood venetoclax concentration may aggravate myelosuppression, leading to increased and premature adverse reactions. However, the difference in EFS between the low concentration group and the standard concentration group was not significant; therefore, the standard venetoclax concentration may have similar safety to the low concentration. Based on the results regarding the occurrence of adverse reactions and changes in treatment strategies, we believe that in the real world, because of the occurrence of adverse reactions, not all patients are suitable for the standard venetoclax combined with low-dose azacytidine regimen recommended by the guidelines. ( 38 ). In a number of cases, venetoclax and posaconazole were not initiated at the same time, which can also be used as a less toxic option than traditional “3 + 7” chemotherapy regimen. However, the selection of administration order, dose, interval time and timing of administration are all issues that need to be further solved. As a treatment option that may be less toxic than traditional “3 + 7” chemotherapy regimen, its efficacy, association with the occurrence of adverse reactions and the prognosis of patients should be further compared with those of recommended or traditional therapies in future studies. In addition, the number of patients in the present study was small and the conclusions drawn need to be further validated in a larger population. Furthermore, the results may be biased due to the small sample size, thus a subgroup analysis of patients receiving AZA/AZA + cytarabine was not conducted in this study, which is a limitation of the present study that needs to be addressed in a larger sample in the future. In conclusion, primary BCL-2 expression may be associated with the proportion of bone marrow blasts, but not with the cytogenetic risk stratification of AML. BCL-2 expression in samples from patients with AML was revealed to be significantly different before and after receiving venetoclax, regardless of blood venetoclax concentration and treatment group. Compared with in the high BCL-2 expression group, patients with AML and low BCL-2 expression had a better CR rate, ORR and PFS. Venetoclax concentration varied greatly among patients and posaconazole was revealed to be a strong factor in increasing blood venetoclax concentration. Blood venetoclax concentration also affected the EFS of patients with AML, but it was not found to have a significant effect on treatment efficacy. Notably, BCL-2 expression, venetoclax concentration and posaconazole were shown to have no obvious effects on the OS of patients with AML. Declarations Acknowledgements This work was supported by the Basic and Applied Basic Research Fundation of Guangdong Province (grant no. 2023A1515010249) and the President Foundation of ZhuJiang Hospital, Southern Medical University (grant no. yzjj2022ms2). Thanks to all the organizations and groups that funded this research. Funding This work was supported by the Guangdong Provincial Basic and Applied Basic Research Fund of China (grant no. 2023A1515010249) and the President Foundation of ZhuJiang Hospital, Southern Medical University (grant no. yzjj2022ms2) Yuxian Huang. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors’ contributions Yuxian Huang conceived and designed the experiments. Mengqi Guo, Yingzhi He and Jingwen Du collected data. Mengqi Guo, Dezhi Qiu and Yinjie Qin analyzed data. Mengqi Guo, Yingzhi He and Jingwen Du wrote the paper. Mengqi Guo responsible for data visualization and use of related softwares. Mengqi Guo and Yuxian Huang confirm the authenticity of all the raw data. All authors read and approved the final manuscript. Ethics approval and consent to participate Written informed consent was obtained from the patients for data collection and use. The present study was approved by the Ethics Committee of Zhujiang Hospital of Southern Medical University and is in accordance with the guidelines of The Declaration of Helsinki (approval no. 2023-KY-086-01). Patient consent for publication Not applicable. Declaration of Interest Statement All authors disclosed no relevant relationships. References Goel H, Rahul E, Gupta I et al (2021) Molecular and genomic landscapes in secondary & therapy related acute myeloid leukemia[J]. Am J Blood Res 11(5):472–497 Weinberg OK, Porwit A, Orazi A et al (2023) The International Consensus Classification of acute myeloid leukemia[J]. 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J Mol Diagn Ther 13(03):413–417 (In Chinese) Vogeser M, Seger C (2008) A decade of HPLC-MS/MS in the routine clinical laboratory–goals for further developments. Clin Biochem 41(9):649–662 O'Halloran S, Ilett KF (2008) Evaluation of a deuterium-labeled internal standard for the measurement of sirolimus by high-throughput HPLC electrospray ionization tandem mass spectrometry. Clin Chem 54(8):1386–1389 Maung ZT, Maclean FR, Reid MM et al (1994) The relationship between bcl-2 expression and response to chemotherapy in acute leukaemia[J]. Br J Haematol 88(1):105–109 Bincoletto C, Saad ST, Da SE et al (1999) Haematopoietic response and bcl-2 expression in patients with acute myeloid leukaemia[J]. Eur J Haematol 62(1):38–42 Zhang H, Nakauchi Y, Köhnke T et al (2020) Integrated analysis of patient samples identifies biomarkers for venetoclax efficacy and combination strategies in acute myeloid leukemia[J]. Nat Cancer 1(8):826–839 Karakas T, Miething CC, Maurer U et al (2002) The coexpression of the apoptosis-related genes bcl-2 and wt1 in predicting survival in adult acute myeloid leukemia[J]. Leukemia 16(5):846–854 Bilbao-Sieyro C, Rodríguez-Medina C, Florido Y et al BCL2 Expression at Post-Induction and Complete Remission Impact Outcome in Acute Myeloid Leukemia[J]. Diagnostics (Basel), 2020,10(12). Naumovski L, Martinovsky G, Wong C et al (1998) BCL-2 expression does not not correlate with patient outcome in pediatric acute myelogenous leukemia[J]. Leuk Res 22(1):81–87 El-Shakankiry NH, El-Sayed GM, El-Maghraby S et al (2009) Bcl-2 protein expression in egyptian acute myeloid leukemia[J]. J Egypt Natl Canc Inst 21(1):71–76 Mattiuzzi G, Yilmaz M, Kantarjian H et al (2015) Pharmacokinetics of posaconazole prophylaxis of patients with acute myeloid leukemia[J]. J Infect Chemother 21(9):663–667 Sekeres MA, Guyatt G, Abel G (2020) American Society of Hematology 2020 guidelines for treating newly diagnosed acute myeloid leukemia in older adults. Blood Adv 4(15):3528–3549 Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files TableI.docx S1.tif Figure S1. Number of patients with elevated ALT in the ven group and ven plus posaconzole group. ven, venetoclax; ALT, alanine transaminase. S2.tif Figure S2. Number of patients with elevated ALT in the low blood ven concentration group, standard blood ven concentration group and high blood ven concentration group. ven, venetoclax; ALT, alanine transaminase. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4188474","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285637412,"identity":"50fe5ff3-e58b-406d-b2c7-a7a9f5d1febe","order_by":0,"name":"Mengqi Guo","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengqi","middleName":"","lastName":"Guo","suffix":""},{"id":285637413,"identity":"53268635-02eb-4de2-8705-df58934c22d3","order_by":1,"name":"Jingwen Du","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingwen","middleName":"","lastName":"Du","suffix":""},{"id":285637414,"identity":"fafbfd03-2c47-48d5-a985-3e0a3c0c5659","order_by":2,"name":"Yingzhi He","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yingzhi","middleName":"","lastName":"He","suffix":""},{"id":285637415,"identity":"f6c0c3c1-70c1-4ff3-a8e2-e4122099f82c","order_by":3,"name":"Dezhi Qiu","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dezhi","middleName":"","lastName":"Qiu","suffix":""},{"id":285637416,"identity":"ce2ae65a-1c2c-4c35-b03f-3fca3aef9eb2","order_by":4,"name":"Yuxian Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYBACAwYGxgcfDCTk2NjbDxCthdlwRoGNMR/PmQSitbAJc3xIS5wn4WBAnBZziRwzZgaDw+ltEgwJDD8qthHWYjkjLe1xgcHh3DbpxgOMPWduE+GwG8nHjWeAtMgcSGBmbCNKS2KbNA/QYWwSCQbEakk+BtSSlkCCljPPkg1nGNgYtgED+SBxfjmeY/jgwx8Jefn29oMPflQQoYVBIAHBPkCEeiDgJ1LdKBgFo2AUjGAAAOHTPldgdE8vAAAAAElFTkSuQmCC","orcid":"","institution":"Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yuxian","middleName":"","lastName":"Huang","suffix":""},{"id":285637417,"identity":"1e3fda28-7512-4fb5-b3d2-01067e1f4c02","order_by":5,"name":"Yinjie Qin","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yinjie","middleName":"","lastName":"Qin","suffix":""}],"badges":[],"createdAt":"2024-03-29 14:35:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4188474/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4188474/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54115255,"identity":"d362d11c-30bb-4756-ba39-3cca292e26c3","added_by":"auto","created_at":"2024-04-04 19:30:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51270,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of patients with different primary BCL-2 expression levels and blood ven concentrations in the two therapy regimen groups. ven, venetoclax; pos, posaconazole.\u003c/p\u003e","description":"","filename":"Onlinefig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4188474/v1/22e736d452726fb73e959212.png"},{"id":54115258,"identity":"654a89b6-7c80-4795-969e-6bf74434039c","added_by":"auto","created_at":"2024-04-04 19:30:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":67916,"visible":true,"origin":"","legend":"\u003cp\u003ePrimary BCL-2 expression and blood ven concentration in 35 patients with AML. (A) Blood ven concentration of 35 patients. (B) Primary BCL-2 expression of 35 patients. Each column represents a patient with AML. AML, acute myeloid leukemia; ven, venetoclax.\u003c/p\u003e","description":"","filename":"Onlinefig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4188474/v1/59b1b5bc9875162b496383c3.png"},{"id":54115261,"identity":"807cb232-d68c-44e4-81bf-fef834965481","added_by":"auto","created_at":"2024-04-04 19:30:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":108081,"visible":true,"origin":"","legend":"\u003cp\u003eAnalyses of potentially influential factors on blood ven concentration, BCL-2 expression, and the difference in BCL-2 expression before and after treatment. Correlation analyses between (A) age or (B) serum albumin and ven blood concentration. Analyses of the influence of (C) different treatment methods and (D) sex on ven blood concentration. Correlation analyses between (E) age or (F) percentage of bone marrow primordial granules and BCL-2 expression. Analyses of the influence of (G) sex and (H) cytogenetic risk on BCL-2 expression. Analyses of the influence of (I) different treatment regimens and (J) different blood ven concentrations on the difference in BCL-2 expression before and after treatment. \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05. ven, venetoclax; NS, not significant.\u003c/p\u003e","description":"","filename":"Onlinefig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4188474/v1/d58042255c5e52677152e197.png"},{"id":54115253,"identity":"944dd811-7cbf-410d-aa4a-31aac92b5475","added_by":"auto","created_at":"2024-04-04 19:30:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":136939,"visible":true,"origin":"","legend":"\u003cp\u003eAnalyses of the effect of BCL-2 expression, ven blood concentration and medication regimen on patients with AML. Analyses of the difference in the number of patients that responded to treatment, achieved CR, achieved CRi, achieved PR or were in the NR state (A) between the BCL-2 high expression group and BCL-2 low expression group;\u003cstrong\u003e \u003c/strong\u003e(B) between the ven group and ven plus posaconzole group; and (C) among the low blood ven concentration group, standard blood ven concentration group and high blood ven concentration group.\u003csup\u003e ***\u003c/sup\u003eP\u0026lt;0.001, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01. CR, complete remission; CRi, CR but incomplete recovery of blood count; NR, no remission; NS, not significant; OR, overall response; PR, partial remission.\u003c/p\u003e","description":"","filename":"Onlinefig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4188474/v1/2da1b8ecee00a9ca2094cb53.png"},{"id":54115252,"identity":"c76cd958-8574-41d0-bbf3-3f0cc6c46d17","added_by":"auto","created_at":"2024-04-04 19:30:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":107281,"visible":true,"origin":"","legend":"\u003cp\u003eAnalyses of the relationship between ven and adverse events. Agranulocytosis and/or fever in patients (A) treated with different medication regimens and (B) with different blood venetoclax concentration gradients. Leukopenia in patients (C) treated with different medication regimens and (D) with different blood venetoclax concentration gradients. Anemia in patients (E) treated with different medication regimens and (F) with different blood venetoclax concentration gradients. Thrombocytopenia in patients (G) treated with different medication regimens and (H) with different blood ven concentration gradients. ven, venetoclax; NS, not significant.\u003c/p\u003e","description":"","filename":"Onlinefig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4188474/v1/bb7bb958650c70d8742b3387.png"},{"id":54115254,"identity":"3e36efcd-6d8c-4764-93ca-05af79866f9c","added_by":"auto","created_at":"2024-04-04 19:30:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":87346,"visible":true,"origin":"","legend":"\u003cp\u003eAnalyses of the effect of BCL-2 expression and ven concentration on prognosis. Kaplan-Meier survival curves of OS of (A) patients with high or low initial BCL-2 expression, (B) patients with different blood ven concentration levels, and (C) patients treated with different medication regimens. Kaplan-Meier survival curves of EFS of (D) patients with high or low initial BCL-2 expression, (E) patients with different blood ven concentration levels, and (F) patients treated with different medication regimens. Kaplan-Meier survival curves of PFS of (G) patients with high or low initial BCL-2 expression, (H) patients with different blood ven concentration levels, and (I) patients treated with different medication regimens. ven, venetoclax; mEFS, median EFS; mOS, median OS; mPFS, median PFS; EFS, event-free survival; OS, overall survival; PFS, progression-free survival.\u003c/p\u003e","description":"","filename":"Onlinefig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4188474/v1/903d67d90227212156666276.png"},{"id":54563697,"identity":"67705013-1731-4cc0-a46c-e1b4c9c9f0bd","added_by":"auto","created_at":"2024-04-12 10:41:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1178194,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4188474/v1/a6eeb046-04e4-467a-8af2-e85cd2f4befb.pdf"},{"id":54115259,"identity":"28093783-bd83-42d2-8919-689c260de108","added_by":"auto","created_at":"2024-04-04 19:30:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"TableI.docx","url":"https://assets-eu.researchsquare.com/files/rs-4188474/v1/672ed245121034723a81ecd5.docx"},{"id":54115257,"identity":"d684ecd0-fc53-465e-9904-476023de7a3a","added_by":"auto","created_at":"2024-04-04 19:30:28","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":475792,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1. Number of patients with elevated ALT in the ven group and ven plus posaconzole group. ven, venetoclax; ALT, alanine transaminase.\u003c/p\u003e","description":"","filename":"S1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4188474/v1/611d9efc39166e55a17c3b45.tif"},{"id":54115260,"identity":"22414826-62b3-4798-bfb2-59de4a26dc25","added_by":"auto","created_at":"2024-04-04 19:30:28","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":478292,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S2. Number of patients with elevated ALT in the low blood ven concentration group, standard blood ven concentration group and high blood ven concentration group. ven, venetoclax; ALT, alanine transaminase.\u003c/p\u003e","description":"","filename":"S2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4188474/v1/569bb8120fc27fe9829db80e.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of oral posaconazole on venetoclax plasma concentration and efficacy in patients with acute myeloid leukemia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute myeloid leukemia\u0026nbsp;(AML) is the most common among all types of diagnosed acute leukemia worldwide (1). Previous reports have revealed that AML is complex and aggressive, with a wide variety of clinical presentations, morphological features and immunophenotypes (1,2). Specific characteristics of AML include the inhibition of myeloid progenitor cell maturation and clonal expansion of bone marrow blasts (1). In particular, evasion of apoptosis is an important mechanism that increases leukemic cell survival rate. As an anti-apoptotic gene, BCL-2 increases the number of AML cells by prolonging cell survival (3,4).\u0026nbsp;To the best of our knowledge, BCL-2 was the first gene to be identified as antiapoptotic. BCL-2 can\u0026nbsp;prolong cell survival and has been shown to be overexpressed in a wide variety\u0026nbsp;of\u0026nbsp;tumors, such as colorectal cancer, breast cancer and non-small cell lung cancer\u0026nbsp;(2-7). BCL-2 was first discovered in the t(14;18) chromosomal translocation in B-cell lymphoma (3,4).\u003c/p\u003e\n\u003cp\u003eAccording to previous research, the upregulated\u0026nbsp;expression of members of the BCL-2 family, such as BCL-2 and MCL-1, may be related to the pathogenesis, poor prognosis and treatment resistance of AML; therefore, BCL-2-targeted therapy could be considered a treatment strategy with great potential for patients with AML (8-13). Venetoclax is an orally selective BCL-2 inhibitor; its main mode of action is through BAK and BAX-induced activation of mitochondrial outer membrane permeabilization to induce apoptosis. Venetoclax has been shown to inhibit cell proliferation and induce apoptosis of tumor cell lines, and to slow down tumor progression to prolong overall survival (OS) (14,15). Venetoclax has a high specificity for BCL-2, low toxicity against healthy cells and cytotoxic activity against a variety of hematological tumors, including AML. It was approved by the US Food and Drug Administration (FDA) in 2016 for the treatment of chronic lymphocytic leukemia (14-17). Since 2018, the FDA gradually granted approval to venetoclax for the combined treatment of patients with newly-diagnosed AML who are aged \u0026ge;75 years, or those who cannot tolerate chemotherapy (15).\u003c/p\u003e\n\u003cp\u003eThe pharmacokinetics of venetoclax have been well characterized and are described in the literature (16,17). Notably, blood venetoclax concentrations exhibit a great deal of variation depending on factors, such as pharmacokinetic characteristics, population differences, diet habits and drug combinations. In the clinical treatment of AML, posaconazole, an antifungal drug commonly used in the clinic, is often used to prevent fungal infection because patients often suffer from myelosuppression. Previous studies have concluded that the combination of posaconazole can reduce the oral dose of venetoclax by \u0026ge;75% (16-21). The present study on the effect of posaconazole combined with venetoclax on the blood concentration of venetoclax is based on the results of a theoretical model calculation or strictly controlled variable, which lacks real-world data for comparison and verification (22). Due to various uncontrollable factors existing in actual treatment, it is expected that the real-world data will differ from the theoretical values to varying degrees, and the conclusion may be different. Therefore, it is of great significance to further explore the effect of combined posaconazole on the blood concentration of venetoclax in clinical practice, and to determine whether there are differences in the treatment efficacy and occurrence of adverse reactions in patients with AML with different concentration ranges of venetoclax, so as to provide a more specific reference for better balancing the safety and effectiveness of venetoclax in clinical use. In addition, to the best of our knowledge, the relationship between BCL-2 expression and the concentration of venetoclax has not been conclusively determined, and the relationship between BCL-2 expression and the outcome and prognosis of patients with AML has not been clearly defined. Therefore, further exploration may also provide novel information regarding clinical treatment.\u003c/p\u003e\n\u003cp\u003eBased on data from 35 patients with AML, the objective of the present study was to characterize BCL-2 expression and venetoclax concentration, to explore the relationship between these two factors, and to assess whether they have an effect on the treatment efficacy and prognosis of patients with AML. In addition, the present study evaluated the exposure-efficacy and exposure-safety relationships in patients with AML with different venetoclax plasma concentrations.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cem\u003eReference standard.\u003c/em\u003e A total of 35 patients who were diagnosed with AML [excluding acute promyelocytic leukemia (AML-M3)] at Zhujiang Hospital (Guangzhou, China) between February 2018 and July 2022 were selected in this single-center, retrospective study. The mean age of the patients was 56 years (range, 10\u0026ndash;72 years). The bone marrow morphology (according to the French American UK criteria) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and flow cytometry results of all patients were consistent with the diagnosis of AML. The inclusion criteria were as follows: i)\u0026thinsp;\u0026ge;\u0026thinsp;20% of peripheral blood or bone marrow primordial granules (or monocytes) were diagnosed with AML. ii) When a patient was confirmed to have clonal recurrent cytological abnormalities: t(8;21) (q22;q22), AML-ETO, t (15;17) (q22;q12), PML-RARA and its variants; t(16;16) (p23;q11) or inv (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) (p13;q22), CBFB-MYH11 or MLL gene rearrangement, even if the proportion of bone marrow primordial granules was \u0026lt;\u0026thinsp;20%, AML should also be diagnosed. The exclusion criteria were as follows: i) Patients who was diagnosed with AML-M3. ii) There was extramedullary infiltration (including active central nervous system leukemia, skin infiltration and myeloid sarcoma). iii) AML was treatment-related (including tumor radiotherapy and chemotherapy). iv) There was major organ dysfunction (mainly heart, liver, kidney function, etc.) that was not suitable for treatment with venetoclax.\u003c/p\u003e \u003cp\u003eThe assessment of the grade of adverse reactions was performed according to the Common Terminology Criteria for Adverse Events 5.0 (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The standards of low blood venetoclax concentration (0-990 ng/ml), standard blood venetoclax concentration (991-3,210 ng/ml) and high blood venetoclax concentration (3,211-5,370 ng/ml) were based on related research (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Combined with previous studies on the relationship between BCL-2 expression and AML, a binary classification of initial BCL-2 expression in 35 patients with AML was based on the measured median level of BCL-2 expression (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe primary endpoint was disease progression or death from any cause, and the secondary endpoint was change or discontinuation of the treatment regimen for any reason.\u003c/p\u003e \u003cp\u003e\u003cem\u003eMethod of administration.\u003c/em\u003e All 35 patients were treated with azactidine (AZA)/AZA\u0026thinsp;+\u0026thinsp;cytarabine. In addition, there were two treatment groups for venetoclax. Patients in the venetoclax monotherapy group were treated with 100 and 200 mg on the 1st and 2nd day, respectively; from the third day, they were treated with 400 mg venetoclax orally once a day. In the venetoclax plus posaconazole group, patients were treated with 100 mg venetoclax orally once a day and 5 ml (200 mg) posaconazole orally three times a day (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A related study was used as reference to determine dosage and frequency of both drugs (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eSample collection.\u003c/em\u003e Each course of venetoclax treatment was recommended for 28 days. Bone marrow specimens were collected from 35 patients with AML at the time of primary diagnosis, and efficacy was assessed when patients stopped taking venetoclax (for patients who used venetoclax for \u0026lt;\u0026thinsp;28 days) or after the first course of venetoclax (for patients who used venetoclax for \u0026ge;\u0026thinsp;28 days). Bone marrow samples used to measure BCL-2 expression were taken for the first time after initial diagnosis or recurrence, and when efficacy was assessed. For each case, steady-state venetoclax concentrations were measured 5\u0026ndash;8 h after accepting venetoclax on the 6th day after patients started single dose therapy (the half-life of venetoclax was 26 h) (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Efficacy was evaluated according to the results of bone marrow morphology and peripheral blood count, and the criteria were as follows: Complete remission (CR): Proportion of bone marrow primordial granules\u0026thinsp;\u0026le;\u0026thinsp;5%; there were no original cells in peripheral blood circulation and no Auer rod-like bodies in the original cells. No extramedullary invasion; neutrophil count\u0026thinsp;\u0026ge;\u0026thinsp;1.0x10\u003csup\u003e9\u003c/sup\u003e/l (1,000/\u0026micro;l); platelet count\u0026thinsp;\u0026ge;\u0026thinsp;100x10\u003csup\u003e9\u003c/sup\u003e/l (100,000/\u0026micro;l). CR but incomplete recovery of blood count (CRi): On the basis of meeting the bone marrow and clinical criteria for CR, peripheral blood still had one of the following: i) Neutrophil count\u0026thinsp;\u0026lt;\u0026thinsp;1.0x10\u003csup\u003e9\u003c/sup\u003e/l (1,000/\u0026micro;l); ii) platelet count\u0026thinsp;\u0026lt;\u0026thinsp;100x10\u003csup\u003e9\u003c/sup\u003e/l (100,000/\u0026micro;l). Partial response (PR): When the peripheral blood cell count reached CR standard, the proportion of naive cells in bone marrow decreased by 5\u0026ndash;25% and \u0026ge;\u0026thinsp;50% compared with before treatment. If Auer clavicular bodies were observed in the original cells, even bone marrow primordial granules\u0026thinsp;\u0026lt;\u0026thinsp;5% should be defined as PR. OR: The total number of patients with CR, CRi and PR after treatment. No remission (NR) : Bone marrow, blood and clinical test results did not meet the aforementioned criteria (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eReverse transcription quantitative PCR (RT-qPCR) detection of BCL-2 mRNA expression in AML cells.\u003c/em\u003e BCL-2 mRNA expression levels were measured in bone marrow samples from 35 patients by RT-qPCR using GAPDH as an internal reference. The primers used were as follows: GAPDH, forward 5\u0026rsquo;-CAAGAGCACAAGAGGAAGAGAG-3\u0026rsquo;, reverse 5\u0026rsquo;-CTACATGGCAACTGTGAGGAG-3\u0026rsquo;; BCL-2, forward 5\u0026rsquo;- GGCCAGGGTCAGAGTTAAATAG-3\u0026rsquo; and reverse 5\u0026rsquo;-GGAGGTTCTCAGATGTTCTTCTC-3\u0026rsquo;. RNA was extracted using RNAiso Plus (Takara Bio, Inc.). RT was performed using PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio, Inc.) according to the manufacturer\u0026rsquo;s protocol. The thermocycling conditions for qPCR (SYBR green PCR kit; Takara Bio, Inc.) were as follows: 95˚C for 30 sec, followed by 40 cycles at 95˚C for 5 sec and 60˚C for 34 sec, followed by 60˚C for 60 sec and 95˚C for 15 sec. Relative BCL-2 mRNA expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCq\u003c/sup\u003e method (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The 2-ΔCq value was selected as the result of the relative expression levels of BCL-2 in the present study based on the experimental method.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDetermination of plasma venetoclax concentration by HPLC/MS/MS .\u003c/em\u003e Patient plasma samples were treated with 50% methanol (containing the internal standard Venetoclax-d8; (Clearsynth) to precipitate protein. The sample size (3 \u0026micro;l) underwent chromatography using the Ultimate@ XB⁃C18 chromatography column (4.6x50 mm; 5 \u0026micro;m; Welch Materials, Inc.); the column temperature was 60˚C. The mobile phase consisted of an aqueous solution containing 0.1% formic acid and 2 mmol/l ammonium acetate (phase A) and a methanol solution containing 0.1% formic acid (phase B), with a flow rate of 0.7 ml/min. The gradient elution method was used, from 0.01 min to 1.0 min, the concentration of phase B was increased from 60\u0026ndash;95%; from 1.0 min to 2.5 min, the concentration of phase B was held at 95%; from 2.5 min to 2.6 min, the concentration of phase B was decreased from 95\u0026ndash;60%; finally, the concentration of phase B was maintained at 60% from 2.6 min to 3.5 min. Electrospray ion source was selected for multi-ion reaction monitoring detection. Its ion source parameters were (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e): ion spray injection voltage, 4,500 V; ion source temperature, 550˚C; spray gas, 50 psi; auxiliary heating gas, 55 psi; air curtain gas, 20 psi; collision gas, Medium. Simultaneous monitoring of venetoclax m/z 868.5\u0026thinsp;~\u0026thinsp;321.7 (quantitative ion), venetoclax m/z 868.5\u0026thinsp;~\u0026thinsp;177.6 (qualitative ion) and venetoclax -d8 m/z 876.6\u0026thinsp;~\u0026thinsp;329.4 (quantitative ion) was performed. The peak area ratio of Venetoclax and Venetoclax⁃d8 was used to calculate the plasma Venetoclax concentration. Jasper high-performance liquid chromatography (Shimadzu Corporation) in tandem with AB Sciex Triple Quad 4500 MD mass spectrometry (AB Sciex) were used in this determination. Analyst\u0026reg;1.6.3 software (AB Sciex) was used for result analysis (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical analysis\u003c/em\u003e. Statistical evaluation of the data was performed using SPSS 25.0 (IBM Corp.). GraphPad Prism 9.0 (Dotmatics) was used for the creation of figures. In the present study, the measured data do not obey a normal distribution and are presented as median (range). Count data are presented as number of cases (%). Comparisons of categorical variables between groups were made using Fisher's exact test or χ\u003csup\u003e2\u003c/sup\u003e test. Spearman\u0026rsquo;s test was used to explore the correlation between continuous variables, and Mann-Whitney U test was used to compare the differences of the same continuous variable between different groups. Wilcoxon signed-rank test was used to compare the difference in BCL-2 expression before and after venetoclax treatment.\u003c/p\u003e \u003cp\u003eIn the present study, patient prognosis was mainly explored through three indicators: OS, the time from randomization to death of any cause; progression-free survival (PFS), the time between enrollment and tumor progression or death; and event-free survival (EFS), the time from enrollment to the occurrence of any event, including disease progression, discontinuation of treatment for any reason or death. OS, PFS and EFS and were calculated using Kaplan-Meier survival curve and log-rank test, assessing whether the expression levels of BCL-2, the blood concentrations of venetoclax or whether posaconazole was used affected survival. Data missing from the survival analysis are included as truncated data. In all analyses, two-tailed tests were performed, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eBaseline data.\u003c/em\u003e A total of 35 patients with AML, including 15 women and 20 men, were involved in the analyses, with a median age of 56 years. Before accepting venetoclax, 3 patients were in a state of no remission after chemotherapy, 2 patients had relapsed after chemotherapy, 4 patients had accepted treatment after allogeneic hematopoietic stem cell transplantation because of different reasons (in 3 patients minimal residual disease reverted to positive; 1 patient progressed from myelodysplastic syndrome), 1 patient had relapsed before treatment, and the remaining 26 patients were newly diagnosed. Among them, 4 patients had favorable risk, 10 had intermediate risk and 21 had poor risk AML. The range and median of bone marrow blasts was 2.04\u0026ndash;94.6 and 26.98%, respectively. Blast data from 4 patients were missing. Regarding genetic mutations, there were 11 patients with the WT1 mutation, 4 with the NPM1 mutation, 5 with the RUNX1 mutation, 4 with the ASXL1 mutation, 4 with the TET mutation, 3 with the TP53 mutation, 2 with the BCOR mutation, 4 with the AML-ETO1 mutation and 3 with the NRAS mutation. The albumin level in the 35 patients fluctuated between 29 and 56 g/l, with a median albumin concentration of 37.6 g/l (normal albumin level, 35\u0026ndash;50 g/l). A total of 17 patients received 100 mg venetoclax once a day\u0026thinsp;+\u0026thinsp;5 ml posaconazole three times a day, and 18 patients took only venetoclax daily. The venetoclax concentration varied greatly, with the minimum and maximum concentrations at 250 and 5,370 ng/ml, respectively, and the median venetoclax concentration was 1,420 ng/ml. By the end of the follow-up, 3 patients relapsed after taking venetoclax. In total, 6 patients died: 1 from severe pneumonia, 2 from septic shock and 3 from AML. Baseline data and the grouping of patients are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, and Table I.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eAnalyses of the potential influential factors on blood venetoclax concentration\u003c/em\u003e. The present study assessed patient sex, age, albumin level and whether treatment was combined with posaconazole or not to explore their effects on venetoclax concentration. It was observed that age (P\u0026thinsp;=\u0026thinsp;0.689; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), albumin level (P\u0026thinsp;=\u0026thinsp;0.945; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and sex (P\u0026thinsp;=\u0026thinsp;0.771; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) were not significantly associated with blood venetoclax concentration. However, patients who received venetoclax and posaconazole had higher blood venetoclax concentrations than those who used venetoclax only (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The ranges in the venetoclax monotherapy group and venetoclax plus posaconazole group were 256-2,430 and 969-5,370 ng/ml, respectively. Furthermore, in the venetoclax monotherapy group, half of the patients (9/18) achieved a low concentration while the rest of the patients achieved high concentrations. In the venetoclax plus posaconazole group, the proportion of patients that achieved low concentrations, standard concentrations and high concentrations were 6% (1/17), 35% (6/17) and 59% (10/17), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eAnalyses of potential influential factors on BCL-2 expression.\u003c/em\u003e The present study revealed that BCL-2 expression in the 35 patients with AML at primary diagnosis was significantly correlated with bone marrow blast percentage (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, r\u0026thinsp;=\u0026thinsp;0.717; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). However, BCL-2 expression was not significantly correlated with age (P\u0026thinsp;=\u0026thinsp;0.943; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), sex (P\u0026thinsp;=\u0026thinsp;0.128; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG) and cytogenetic risk (P\u0026thinsp;=\u0026thinsp;0.170; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eIn all patients, a statistically significant difference was seen in BCL-2 expression between primary diagnosis and after using venetoclax within 28 days (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Statistically significant differences in BCL-2 expression before and after medication were also seen in the venetoclax monotherapy group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and in the venetoclax plus posaconazole group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Notably, significant differences in BCL-2 expression before and after medication were also detected in the low concentration group (P\u0026thinsp;=\u0026thinsp;0.002), the standard concentration group (P\u0026thinsp;=\u0026thinsp;0.031) and the high concentration group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003e \u003cem\u003eEfficacy analyses.\u003c/em\u003e Out of the 35 patients, 18 patients were in the low BCL-2 expression group and 17 patients were in the high BCL-2 expression group. A significant difference was observed in the number of patients with overall response (OR) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and in the number of patients that achieved CR (P\u0026thinsp;=\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) The proportion of patients with OR and CR in the low-expression and high-expression BCL-2 groups was 100% (18/18) vs. 41% (7/17), and 61% (11/18) vs. 12% (2/17), respectively. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) There was no significant difference in the number of patients who achieved CRi (P\u0026thinsp;=\u0026thinsp;0.658) and the number of patients who achieved PR (P\u0026thinsp;=\u0026thinsp;1) between the two different BCL-2 expression groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThere was no significant difference in the number of patients who achieved CRi (P\u0026thinsp;=\u0026thinsp;0.177), PR (P\u0026thinsp;=\u0026thinsp;0.177) or OR(P\u0026thinsp;=\u0026thinsp;0.711) between the venetoclax monotherapy group and the venetoclax plus posaconazole group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). However, there was a significant difference in the number of patients who achieved CR (P\u0026thinsp;=\u0026thinsp;0.002) between these two groups. In the venetoclax monotherapy group and venetoclax plus posaconazole group, 2 and 11 patients achieved CR, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn the low blood venetoclax concentration group, the numbers of patients with OR, and those that achieved CR, CRi and PR were 6, 1, 3 and 2 patients, respectively. In the standard concentration group, the number of patients were 5, 4, 1 and 0, respectively. Furthermore, in the high blood venetoclax concentration group the numbers were 14, 8, 2 and 4 patients, respectively. There was no significant difference in the number of patients in the three different venetoclax concentration groups (low venetoclax concentration vs. standard venetoclax concentration vs. high venetoclax concentration) that were OR(P\u0026thinsp;=\u0026thinsp;0.680), achieved CR (P\u0026thinsp;=\u0026thinsp;0.068), achieved CRi (P\u0026thinsp;=\u0026thinsp;1) or achieved PR (P\u0026thinsp;=\u0026thinsp;0.696) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cem\u003eSafety analyses\u003c/em\u003e. Regarding adverse events, similar results were recorded between the venetoclax monotherapy group and the venetoclax plus posaconazole group, as follows:\u003c/p\u003e \u003cp\u003eGroup 1 (without agranulocytosis or fever), P\u0026thinsp;=\u0026thinsp;0.603; group 2 (fever with agranulocytosis), P\u0026thinsp;=\u0026thinsp;0.489; group 3 (only agranulocytosis), P\u0026thinsp;=\u0026thinsp;0.711 ; group 4 (only fever), P\u0026thinsp;=\u0026thinsp;0.264; 0\u0026ndash;2 grade leukopenia, P\u0026thinsp;=\u0026thinsp;0.489; 3\u0026ndash;4 grade leukopenia, P\u0026thinsp;=\u0026thinsp;0.489; 0\u0026ndash;2 grade anemia, P\u0026thinsp;=\u0026thinsp;1; 3\u0026ndash;4 grade anemia, P\u0026thinsp;=\u0026thinsp;1; 0\u0026ndash;2 grade thrombocytopenia, P\u0026thinsp;=\u0026thinsp;1; 3\u0026ndash;4 grade thrombocytopenia, P\u0026thinsp;=\u0026thinsp;1; alanine aminotransferase (ALT) increase, P\u0026thinsp;=\u0026thinsp;1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, C, E, G and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNo significant difference was observed in the number of adverse-exposure reactions between the patients within different venetoclax concentration groups (low concentration vs. standard concentration vs. high concentration), as follows: Group 1 (without agranulocytosis or fever), P\u0026thinsp;=\u0026thinsp;0.472; group 2 (fever with agranulocytosis), P\u0026thinsp;=\u0026thinsp;0.510; group 3 (only agranulocytosis), P\u0026thinsp;=\u0026thinsp;0.886; group 4 (only fever), P\u0026thinsp;=\u0026thinsp;0.071; 0\u0026ndash;2 grade leukopenia, P\u0026thinsp;=\u0026thinsp;0.343; 3\u0026ndash;4 grade leukopenia, P\u0026thinsp;=\u0026thinsp;0.343; 0\u0026ndash;2 grade anemia, P\u0026thinsp;=\u0026thinsp;0.260; 3\u0026ndash;4 grade anemia, P\u0026thinsp;=\u0026thinsp;0.260; 0\u0026ndash;2 grade thrombocytopenia, P\u0026thinsp;=\u0026thinsp;0.307; 3\u0026ndash;4 grade thrombocytopenia, P\u0026thinsp;=\u0026thinsp;0.307; ALT increase, P\u0026thinsp;=\u0026thinsp;0.208 (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, D, F, H and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Throughout the study, no renal function impairments and tumor lysis syndrome were observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eEffect of BCL-2 expression and venetoclax concentration on prognosis.\u003c/em\u003e OS of the patients was not significantly associated with initial BCL-2 expression (high expression vs. low expression, P\u0026thinsp;=\u0026thinsp;0.11; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In addition, no significant differences in OS were determined among the three venetoclax concentration groups (low concentration vs. standard venetoclax, P\u0026thinsp;=\u0026thinsp;0.91; low concentration vs. high concentration, P\u0026thinsp;=\u0026thinsp;0.32; standard venetoclax vs. high concentration, P\u0026thinsp;=\u0026thinsp;0.40; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), or between the venetoclax monotherapy group and the venetoclax plus posaconazole group (P\u0026thinsp;=\u0026thinsp;0.50; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding PFS, patients with low initial BCL-2 expression had a better trend than those in the high BCL-2 expression group (P\u0026thinsp;=\u0026thinsp;0.04; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). There were no obvious significant differences in PFS among the different venetoclax concentration groups and between the two treatments groups, respectively (low venetoclax concentration vs. standard concentration, P\u0026thinsp;=\u0026thinsp;0.48; high concentration vs. standard concentration, P\u0026thinsp;=\u0026thinsp;0.71; low concentration vs. high concentration, P\u0026thinsp;=\u0026thinsp;0.06; venetoclax monotherapy vs. venetoclax plus posaconazole, P\u0026thinsp;=\u0026thinsp;0.38). However, the PFS trend in the high concentration group was higher than that in the low concentration group, but this was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and F).\u003c/p\u003e \u003cp\u003eThere were no significant differences in EFS between patients in the high and low initial BCL-2 expression groups (P\u0026thinsp;=\u0026thinsp;0.79; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Furthermore, the differences in EFS were not significant between the standard concentration and low concentration (P\u0026thinsp;=\u0026thinsp;0.75), and standard concentration and high concentration (P\u0026thinsp;=\u0026thinsp;0.10) groups. However, EFS was significantly different between the high concentration and low concentration groups (P\u0026thinsp;=\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). There was no evident difference in EFS between patients treated with venetoclax and those treated with venetoclax plus posaconazole (P\u0026thinsp;=\u0026thinsp;0.31; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eI).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have suggested that upregulation of BCL-2 is association with the occurrence and development of AML (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). BCL-2 can accelerate the progression of AML, and high BCL-2 expression has been detected in both relapsed AML cells and treatment-resistant AML cells (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). The present study revealed that BCL-2 expression among individuals was highly variable, which is consistent with other previous research (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe current study demonstrated that BCL-2 expression was correlated with the proportion of bone marrow blasts in patients with AML at initial diagnosis. It has previously been reported that, compared with in healthy individuals, the expression of BCL-2 is upregulated in patients newly diagnosed with AML; however, BCL-2 expression was not shown to be related to the proportion of bone marrow blasts, which differs from the present conclusion (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Based on the present results, irrespective of the different treatment groups or different venetoclax concentration groups, there were significant differences in the expression of BCL-2 before and after medication. This suggests that venetoclax is a factor that may reduce BCL-2 expression in patients with AML. Through analysis of the low and high BCL-2 expression groups, it was revealed that BCL-2 expression levels at initial onset may have a negative impact on whether patients with AML exhibit CR after treatment, which may be related to the upregulation of BCL-2 leading to relapse and venetoclax drug resistance (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince most patients who responded to the treatment achieved CR, the significant difference in the number of patients achieving OR in the low BCL-2 expression group and the high BCL-2 expression group may be related to the fact that the majority of patients that achieved OR in the two groups had achieved CR. That is, the significant difference in the number of patients achieving OR in different BCL-2 expression groups may be caused by the significant difference in the number of patients achieving CR. Furthermore, it was revealed that patients with low BCL-2 expression had better PFS than those who had high BCL-2 expression; however, there was no significant difference in OS and EFS between the two groups. Although the present findings are similar to those of previous studies (\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), some other studies that have questioned whether BCL-2 upregulation can really affect the prognosis of patients with AML (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Notably, a previous study did confirm the hypothesis that BCL-2 upregulation could lead to relapse and drug resistance in patients with AML (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e); thus, BCL-2 may affect the prognosis of patients with AML in this manner. The present study indicated that venetoclax may improve treatment efficacy and prognosis of patients with AML by reducing BCL-2 expression.\u003c/p\u003e \u003cp\u003eAccording to a previous study and clinical practice, posaconazole can be used for antifungal prophylaxis in patients with AML (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Furthermore, it has been reported to significantly increase blood venetoclax concentration; therefore, the venetoclax dose can be reduced by \u0026ge;\u0026thinsp;75% due to posaconazole (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In addition, this previous study revealed that, relative to 400 mg venetoclax alone, coadministration of 100 mg venetoclax with posaconazole (300 mg) can significantly increase mean venetoclax peak concentration and area under the concentration time curve of 0\u0026ndash;24 h. The results of the present study revealed that, compared with in the 400 mg venetoclax dose group, the combined venetoclax and posaconazole group appeared to have a higher concentration of venetoclax in the blood. Almost all of the patients who were treated with venetoclax and posaconazole met or even exceeded the standard concentration. Notably, venetoclax concentrations in the combined venetoclax and posaconazole group were higher than those in the venetoclax monotherapy group. This is consistent with the conclusions from a previous study, thus suggesting that posaconazole can significantly increase blood venetoclax concentrations (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). One of the concerns of the present study was that some of the patients (7/18) who received 400 mg venetoclax at the beginning had to decrease their venetoclax dose due to other reasons (such as severe agranulocytosis, anemia and thrombocytopenia) during treatment. However, the blood venetoclax concentrations of the patients fluctuated greatly in the two groups with different administration regimens, which was consistent with the conclusions from a previous study (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). It may be hypothesized that this, on the one hand, is due to the differences in bioavailability of venetoclax among different individuals (based on factors such as ethnicity, liver condition and dietary habits), and on the other hand, this could be related to the high heterogeneity in posaconazole concentrations among patients (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Notably, the CR rate in the present study (11%) was better than previously reported in patients with AML given only venetoclax (6%) (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). It may be hypothesized that the reason for this difference could be that the subjects included in previous study were almost all patients with relapsed refractory AML with a poor prognosis. In particular, different outcomes were identified in different medication groups. In the present study, it appears that in the venetoclax monotherapy group and the venetoclax plus posaconazole group, there was a significant difference in the number of patients achieving CR. Since a higher proportion of patients achieved CR in the venetoclax plus posaconazole group, this may be related to the higher venetoclax concentration in this group.\u003c/p\u003e \u003cp\u003eNotably, there were no obvious differences in the occurrence of adverse events between the venetoclax monotherapy group and the venetoclax plus posaconazole group. The present study compared the prognosis of the two groups and revealed that, although there was no significant difference in OS, PFS and EFS between the two groups, the EFS trend of the venetoclax monotherapy group was higher than that of the venetoclax plus posaconazole group. This may be because of the fact that a larger proportion of patients met or exceeded the standard venetoclax concentration in blood in the combination group. Even though there was no significant difference in the number and proportion of adverse events between the two groups, a higher venetoclax concentration may lead to an earlier onset of adverse reactions. Since only one study (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) has explored the effect of two drug administration regimens on the treatment efficacy and prognosis of patients with AML, and the sample size of this study was small, this finding should be further explored in a study with an expanded sample size.\u003c/p\u003e \u003cp\u003eThe present results are similar to the results of a previous study on the effect of venetoclax concentration with different combination regimens on the treatment efficacy in patients with AML (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e); there were no significant differences in the number of patients who responded to treatment, achieved CR, achieved CRi or achieved PR among the low concentration, standard concentration and high concentration groups in the present study. As for the occurrence of adverse events, there was no apparent difference among the different concentration groups, which is also similar to the results from recent literature (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). When comparing the three different venetoclax concentrations, no effect of venetoclax concentration on the OS or PFS of the 35 patients with AML was determined, which is consistent with a previous study (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). In addition, the EFS of the low concentration group was markedly longer than that of the high concentration group. It could be hypothesized that the increase in blood venetoclax concentration may aggravate myelosuppression, leading to increased and premature adverse reactions. However, the difference in EFS between the low concentration group and the standard concentration group was not significant; therefore, the standard venetoclax concentration may have similar safety to the low concentration. Based on the results regarding the occurrence of adverse reactions and changes in treatment strategies, we believe that in the real world, because of the occurrence of adverse reactions, not all patients are suitable for the standard venetoclax combined with low-dose azacytidine regimen recommended by the guidelines. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In a number of cases, venetoclax and posaconazole were not initiated at the same time, which can also be used as a less toxic option than traditional \u0026ldquo;3\u0026thinsp;+\u0026thinsp;7\u0026rdquo; chemotherapy regimen. However, the selection of administration order, dose, interval time and timing of administration are all issues that need to be further solved. As a treatment option that may be less toxic than traditional \u0026ldquo;3\u0026thinsp;+\u0026thinsp;7\u0026rdquo; chemotherapy regimen, its efficacy, association with the occurrence of adverse reactions and the prognosis of patients should be further compared with those of recommended or traditional therapies in future studies. In addition, the number of patients in the present study was small and the conclusions drawn need to be further validated in a larger population. Furthermore, the results may be biased due to the small sample size, thus a subgroup analysis of patients receiving AZA/AZA\u0026thinsp;+\u0026thinsp;cytarabine was not conducted in this study, which is a limitation of the present study that needs to be addressed in a larger sample in the future.\u003c/p\u003e \u003cp\u003eIn conclusion, primary BCL-2 expression may be associated with the proportion of bone marrow blasts, but not with the cytogenetic risk stratification of AML. BCL-2 expression in samples from patients with AML was revealed to be significantly different before and after receiving venetoclax, regardless of blood venetoclax concentration and treatment group. Compared with in the high BCL-2 expression group, patients with AML and low BCL-2 expression had a better CR rate, ORR and PFS. Venetoclax concentration varied greatly among patients and posaconazole was revealed to be a strong factor in increasing blood venetoclax concentration. Blood venetoclax concentration also affected the EFS of patients with AML, but it was not found to have a significant effect on treatment efficacy. Notably, BCL-2 expression, venetoclax concentration and posaconazole were shown to have no obvious effects on the OS of patients with AML.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Basic and Applied Basic Research Fundation of Guangdong Province (grant no. 2023A1515010249) and the President Foundation of ZhuJiang Hospital, Southern Medical University (grant no. yzjj2022ms2). Thanks to all the organizations and groups that funded this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Guangdong Provincial Basic and Applied Basic Research Fund of China (grant no. 2023A1515010249) and the President Foundation of ZhuJiang Hospital, Southern Medical University (grant no. yzjj2022ms2) Yuxian Huang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuxian Huang conceived and designed the experiments. Mengqi Guo, Yingzhi He and Jingwen Du collected data. Mengqi Guo, Dezhi Qiu and Yinjie Qin analyzed data. Mengqi Guo, Yingzhi He and Jingwen Du wrote the paper. Mengqi Guo responsible for data visualization and use of related softwares. Mengqi Guo and Yuxian Huang confirm the authenticity of all the raw data. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the patients for data collection and use. The present study was approved by the Ethics Committee of Zhujiang Hospital of Southern Medical University and is in accordance with the guidelines of The Declaration of Helsinki (approval no. 2023-KY-086-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors disclosed no relevant relationships.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGoel H, Rahul E, Gupta I et al (2021) Molecular and genomic landscapes in secondary \u0026amp; therapy related acute myeloid leukemia[J]. Am J Blood Res 11(5):472\u0026ndash;497\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeinberg OK, Porwit A, Orazi A et al (2023) The International Consensus Classification of acute myeloid leukemia[J]. 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Blood Adv 4(15):3528\u0026ndash;3549\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"BCL-2 expression, posaconazole, venetoclax concentration, acute myeloid leukemia","lastPublishedDoi":"10.21203/rs.3.rs-4188474/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4188474/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBCL-2 was the first gene identified to have antiapoptotic effects and venetoclax is an oral selective BCL-2 inhibitor, which has great potential in the treatment of patients with acute myeloid leukemia (AML) who are not candidates for intensive therapy. Notably, posaconazole, an oral antifungal drug, is also a strong factor that can affect blood venetoclax concentrations. To the best of our knowledge, the relationship between BCL-2 expression, posaconazole and venetoclax, as well as the influence of them on treatment efficacy and the prognosis of patients with AML, has not been reported. Therefore, in the present study, the relationship between BCL-2 expression and blood venetoclax concentration was analyzed in 35 patients with AML. BCL-2 mRNA expression levels were examined by reverse transcription quantitative PCR. Blood venetoclax concentrations were measured using high-performance liquid chromatography-tandem mass spectrometry. The results revealed that among patients with AML, those with lower primary BCL-2 expression had a higher complete remission (CR) rate (P = 0.005), overall response rate (P \u0026lt; 0.0001) and progression-free survival time (P = 0.04). Posaconazole was revealed to be a strong factor that was able to increase blood venetoclax concentration (P \u0026lt; 0.001) and CR rate in the venetoclax plus posaconazole group compared with that in the venetoclax monotherapy group (P = 0.002); however, no significant difference was identified in the occurrence of adverse reactions between these groups. Among low and high blood venetoclax concentration groups, the event-free survival of the former group was significantly higher (P = 0.013). In conclusion, the results of the present study could be used to guide clinical practice in the treatment of AML.\u003c/p\u003e","manuscriptTitle":"Effect of oral posaconazole on venetoclax plasma concentration and efficacy in patients with acute myeloid leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-04 19:30:19","doi":"10.21203/rs.3.rs-4188474/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be81d66d-fb2e-40a7-8640-da65a288f608","owner":[],"postedDate":"April 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-28T02:07:05+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-04 19:30:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4188474","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4188474","identity":"rs-4188474","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}