| Background and AimsAcute promyelocytic leukemia (APL) is a distinct type of acute leukemia characterized by abnormal proliferation of promyelocytes, life threatening coagulopathy and the chromosome translocation t(15; 17)(q22; q21). Introduction of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has improved the prognosis of APL patients significantly.Prognosis of APL was affected by many factors. Early death, relapse and long-term complications decide overall survival (OS), disease free survival (DFS) and event free survival (EFS). Although administration of ATRA and arsenic agents revolutionizes the treatment of APL and makes it a kind of highly curable cancer, it is reported early death in APL remains high. Furthurmore some researches considered that improvement of induction treatment didn’t affect early death. McClellan believed that many serious patients died before recruitment of clinical trial and adequate induction therapy, and it was the reason that led to the underestimate of early death rate. And bleeding, leukocytosis and differentiation syndrome (DS) are still the main causes resulting in early death.ATRA, anthracycline-based chemotherapy and arsenic trioxide (ATO) are recommended by NCCN as the first line treatment for de novo APL. However, NCCN guildline doesn’t indicate when to use anthracycline-based chemotherapy during induction treatment for APL with different risk. This study tried to analyze the clinical features of early death occurred in different period with different risk, investigate the appropriate timing to initiate chemotherapy, compare the types of cytotoxic agents and assess the immune pattern associated with prognosis.1. Clinical features of early death occurred in different period with different risk in de novo APLIt is commonly believed that mortal hemorrhage is the main reason leading to early death in APL and delay of diagnosis and treatment should contribute to early death Further investigation on early death in APL may help to provide evidence for new treatment. This study try to compare early death occurred in different period with different risk.2. Investigation on timing of initiating chemotherapy in de novo APL with low/intermediate riskWBC counts usually rise after induction treatment of ATRA or ATO in de novo APL with low/intermediate APL. Then leukocytosis may increase the risk of differentiation syndrome (DS) and even lead to early death. However, too rapid administration of chemotherapy may make coagulopathy more serious. Thus it is necessary to discuss when to give chemotherapy to de novo APL patients with low/intermediate risk during induction treatment.3. Investigation on timing of initiating chemotherapy in de novo APL with high riskDe novo APL patients with high risk have high early death incidence at diagnosis because of leukocytosis and coagulopathy. Induction of ATRA may further increase WBC count, while anthracycline-based chemotherapy may make hemorrhage more serious and increase the risk of tumor lyses syndrome. However, NCCN guildline doesn’t give specific advice on when to use chemotherapy.4. Comparison of chemotherapy protocols during induction treatmentBased on clinical trial with large samples, such as AIDA, LPA and so on, NCCN recommends anthracycline based chemotherapy as the first line treatment. In China, homoharringtonine and hydroxyurea also are used widely during induction treatment in APL. In order to compare clinical effects and side effects, anthracyclined based chemotherapy, homoharringtonine and hyroxyurea are compared.5. Immunophenotypes and immune markers associated with prognosis in APLMost diagnostic tests for APL, including chromosome examination, polymerase chain reaction, and fluorescent in situ hybridization, are time-consuming. Flow cytometric immunophenotypic analysis has gained attention as an effective and rapid diagnostic tool for APL. It is well documented that CD2+, CD56+, and CD34+APL immunophenotypes are associated with lower overall survival (OS) rate, shorter remission, decreased incidence of remission, and increased incidence of early death, respectively. However, the relationship between these APL immunophenotypes and disease prognosis has not been fully explored. The present study investigated the efficacy of flow cytometric analysis for detecting CD2+, CD56+, and CD34+ APL immunophenotypes and other immune markers as diagnostic tools and predictors of early death and long-term prognosis in APL.Study populationA total of 212 patients with de novo APL, who were hospitalized at the Nanfang Hospital (Guangzhou, China) between January 2003 and December 2013, were retrospectively enrolled in this study. In this group,49 patients [34 males and 15 females with a median age of 32 years (range 15-84)] who died at diagnosis or soon after the onset of therapy were retrospectively enrolled in this study. The remaining 163 patients [91 males and 72 females with a median age of 31 years (range 15-54)] who achieved complete remission after induction treatment with ATRA, toxic agents and/or arsenic trioxide were included in the control group.163 patients accepted anthracycline-based chemotherapy or other toxic agents such as Homoharringtonine and Hydroxyurea.96 were stratified as low/intermediate risk group [47 males and 49 females with a median age of 32 years (range 15-66)].73 were stratified as high risk group [54 males and 19 females with a median age of 32 years (range 15-67)]. Disease diagnosis was confirmed by bone marrow aspiration, chromosome karyotyping analysis, Fluorescence in Situ Hybridization analysis (FISH) and PCR tests. Demographics, clinical parameters and laboratory data at diagnosis were collected for analysis. Informed consent was obtained from all patients included in the study.Once the diagnosis of APL was suspected, ATRA (30mg/m2/d) was given as induction treatment as early as possible, until complete remission (CR) was achieved. Chemotherapy comprised treatments of idarubicin (8 mg/m2/day on days 1,3, and 5), daunorubicin (45 mg/m2/day on days 1,3, and 5), homoharringtonine (2 mg/m2/day on days 1-5), and/or cytarabine (100 mg/m2/days on days 1-7). Hydro xycarbamide was given after chemotherapy if WBC count was still high.36 patients simultaneously received arsenic trioxide (0.15 mg/kg/day for 14 days). Blood product support was applied to maintain the platelet≥30×109/L, hemoglobin≥70g/L, plasma fibrinogen≥1.5g/L.1. Analysis of early death in newly diagnosed acute promyelocytic leukemia patientsThe ED patients were divided into two groups:group 1 (death before treatment or within the first 3 days; more early death group; mED group; n=24), and group 2 (death during treatment at least 3 days after commencement; n=25). The patients were categorized according to Sanz’s risk stratification score:low risk, intermediate risk and high risk. Clinical characters were compared between group 1 and group 2, low/intermediate risk group and high risk group.2. Effects of different onsets of chemotherapy during induction treatment on new diagnosed APL with low/interme diate riskWBC counts in APL usually rise after induction treatment of ATRA. According to the WBC counts at the time chemotherapy was administrated,96 de novo APL patients with low/intermediate risk were divided into three groups. They were WBC≤4×109/L,4-15×109/L and ≥15×109/L groups. According to the time from initiation of ATRA to chemotherapy, the population was divided to two groups: chemotherapy was given within 3 days (within-3-days group) and at least 3 days after ATRA (3-days-after ATRA group).Clinical features, DS rate, infection, bleeding, bone marrow depression, CR rate, time needed to acquire CR, early death rate and other treatment effects were compared between the groups. Multivariate analysis also was used to find independent risk factors of events related to early death.3. Effects of different onsets of chemotherapy during induction treatment on new diagnosed APL with high risk73 APL patients with high risk were divided into three group:25 patients accepted ATRA and hydroxyurea or cytarabine at the same time (low-dose-chemotherapy group),35 patients accepted anthracycline based chemotherapy or homoharringtonine within the first 3 days after ATRA (early chemotherapy group),13 patients accepted ATRA as well as hydroxyurea (3g/d) or cytarabine (3g/d) and combined chemotherapy at least 3 days after ATRA (late chemotherapy group). Clinical features and clinical efficacy were compared.4. Comparison of chemotherapy protocoles during induction treatmentBefore October in 2009,71 new diagnosed APLpatients accepted chemotherapy of HA, DA or hydroxyurea/cytarabine (HA group, DA group, low dose chemotherapy group) during induction treatment. And the clinical efficacy was compared.54 patients accepted only idarubincin and 27 patients accepted DA chemotherapy between September in 2009 and December in 2013 also were compared.5. Immunophenotypes and immune markers associated with prognosis in APLA total of 132 patients with de novo APL were retrospectively analyzed. Immunophenotypes were determined by flow cytometry. Clinical features, immunophenotype, complete remission (CR), relapse, and five-year overall survival (OS) rate were assessed and subjected to multivariate analyses.Statistical analysesAll statistical analyses were performed using SPSS v.17.0 software (SPSS Inc., Chicago, USA). Clinical features are presented as percentages (%) for categorical variables and as mean values ± standard deviation (SD) for normally distributed continuous variables. The X2 test was used to analyze differences in the distribution of categorical variables between patient subsets. The t test or ANOVA analysis was used to detect differences in the distribution of continuous parametric variables. The Mann-Whitney test was used to analyze differences in the distribution of ranked variables. Multivariate analyses were performed using a binary logistic regression model. P values< 0.05 were considered statistically significant. A cutoff of> 10% was used to quantify the presence of a subpopulationof CD34+ and CD56+ cells, and a cutoff of> 20% was used for defining positivity for other antigens.Results1. Analysis of early death in newly diagnosed acute promyelocytic leukemia patientsForty-nine ED patients diagnosed with APL were included in the analysis (23.11% of the entire cohort of APL patients). The ED group was divided into two groups:group 1 (death before treatment or within the first 3 days; more early death group; mED group; n=24), and group 2 (death during treatment at least 3 days after commencement; n=25).12/24 patients who died before treatment or within the first 3 days did not receive any induction treatment. In the ED group 30 patients were stratified as high-risk at diagnosis.12.2% of the patients were □ 55 years of age (n=6). An ECOG score of 3 to 4 was observed in 95.9% of the total ED patients (n=47).Main differences between group 1 and group 2 were as follow:WBC count was observed higher (53.88±64.49 vs 24.53±29.59×109/L, P=0.012); 3-4 grade bleeding frequence was greater (91.7%vs60.90%, P=0.005); median time from the onset of symptoms to the initial hospitalization (median7vs10days, Z=186.50, P=0.022), median time from diagnosis to death (median 1 vs13days, Z=17.060, P=0.000) and median time from the onset of symptoms to death (median 9vs24days, Z=67.50, P=0.000) were significantly shorter in groupl than group 2.Compared with CR group, creatine level was higer (P=0.000,0.002), LDH level was higher (P=0.000,0.001), PT prolongation was more frequent (P=0.000,0.004), median ISTH score was greater (P=0.000,0.000),3-4 grade bleeding was greater (P=0.000,0.003) in these two ED groups than CR group. Significant differences between group 1 and CR group include proportion of high risk cases, WBC count at diagnosis, PLT count at diagnosis, APTT prolongation and DIC incidence, all of which are more serious in group 1.19/134 (14.2%) low/intermediate risk patients with APL suffered ED patients compared to 30/78 high-risk patients (38.5%).The LDH level was higher in the high-risk ED group (P=0.002). PT prolongation however was more frequent in the early death patients of the high-risk group (56.7%; P=0.014). Similarly, the incidence of grade 3-4 bleeding was higher in the high-risk group (83.3%; P=0.049). No delay in diagnosis was observed for the ED patients in the high-risk group. In contrast, both the time from the onset of symptoms to the initial hospitalization and from the onset of symptoms to death was shorter for the ED patients in the high-risk group than for the patients in the low/intermediate risk group (8d vs 10d, X2=184.50, P=0.037; 11d vs 24d, X2=145.50, P=0.004).2. Effects of different onsets of chemotherapy during induction treatment on new diagnosed APL with low/interme diate riskAccording to the WBC counts at the time chemotherapy was administrated,96 de novo APL patients with low/intermediate risk were divided into three groups. They were WBC≤4×109/L,4～15×109/L and ≥15×109/L groups. DS incidences in these three groups were 0%,11.10% and 40.00% respectively (x2=186.50, P=0.000). Once WBC count increased as high as more than 15×109/L, DS incidence was as high as 40%. As for 3-4 grade bone marrow depression, there was no significant difference between these three groups. But infection rates were different. In WBC≤4×109/L group,3-4 grade infection rate was 71.4%, significantly higher than other two groups (33.30%,43.30%, x2=8.440, P=0.015). Rate of 3-4 grade bleeding in WBC>15×109/L group was most serious. They were 9.5%,29.30% and 42.90%.Clinical effects were compared between these three groups. CR rate was the highest in WBC 4～15×109/L group (100%) and the lowest in WBC≥15×109/L group (73.7%). The difference was significant (x2=13.740, P=0.000). The early death rates were 4.8%,0.0% and 26.70% respectively, which are significantly different (X2=15.739, P=0.000).DS incidence,3-4 grade bone marrow depression,3-4 grade infection, CR rate, early death rate were not significantly different between within-3-days group and 3 -days-after ATRA group. However, bleeding events related to chemotherapy occurred more in 3-days-after ATRA group (33.3% vs 7.4%, X2=6.773, P=0.009). And time to achieve CR was longer in 3-days-after ATRA group (30.77±11.60 days vs 37.64+10.00 days, t=-1.924, P=0.004).Binary logistic regression analysis showed WBC count at the time chemotherapy was given was the independent risk factor of DS and early death. However, time from initiation of ATRA to chemotherapy, WBC count at diagnosis, PLT count at diagnosis were not risk factors of DS, early death and failure of remission.3. Effects of different onsets of chemotherapy during induction treatment on new diagnosed APL with high riskDS incidence,3-4 grade bone marrow depression and bleeding related to chemotherapy were not significantly different among low-dose-chemotherapy group, early chemotherapy group and late chemotherapy group.3-4 grade infection rates were 13.00%,57.10% and 69.20%, which are significant different (X2=14.655, P=0.001). Time to achieve CR was 29.27±6.78,28.54±8.08 and 50.00±21.75 days in these three groups (F=11.851, P=0.000), which was longert in late chemotherapy group than other two groups (P=0.000,0.000). CR rate were 40.0%,68.6% and 84.6% (X2=8.605, P=0.014), which was the lower in low-dose-chemotherapy group than in late chemotherapy group (P=0.009). Early death rates were 56.0%,25.7% and 15.4% (X2=8.439, P=0.015), which was the higher in low-dose-chemotherapy group than other two groups (P=0.017,0.006).4. Comparison of chemotherapy protocoles during induction treatmentDS rates were 22.7%,25.8% and 38.9% in HA, DA and low dose chemotherapy group respectively. Rates of 3-4 grade infection were 40.9%,45.2% and 16.7% in these three groups. And rates of 3-4 grade bleeding were 6.7%,7.1% and 0.0%. Not only DS,3-4 grade infection but also 3-4 grade bleeding rates were not significantly different. However,3-4 grade bone marrow depression were statistically different in these three groups (90.5%,90.3%,29.4%, X2=14.655, P=0.000), which was lower in low dose chemotherapy group than other two groups (P=0.000,0.000). As for clinical effects, CR rates (86.4%,90.30%,88.9%), time needed to achieve CR (38,35, 31 days), early death incidence (9.1%,3.2%,5.6%) during induction treatment were all not statistically different.Comparisons the groups of DA and Ida showed DS rates (37% vs 24.1%),3-4 grade bone marrow depression (77.8% vs 90.7%),3-4 grade infection (40.7%vs 53.7),3-4 grade bleeding (8.3% vs 4.3%), CR rates (70.4% vs 85.2%), early death incidence (29.6% vs 14.8%) and time needed to achieve CR (28.5 vs 30 days) were not significant different.5. Immunophenotypes and immune markers associated with prognosis in APLCD64 expression is common in APL (78.4%), but is highly variable. Data for CD9 was collected for 87 patients, of which 96.6% (84 patients) were CD9+. Forty-two of the patients with CD9+ expressed bright CD9, CD2, and CD56. Data for CD2 and CD56 were collected for 101 and 113 patients, respectively. Of which,12 (11.9%) and 4 patients (9.3%) were CD2+ and CD56+, respectively.12 cases were CD2 positive in 101 new diagnosed APL. WBC counts before treatment in the CD2+ APL group were significantly higher than in the CD2- APL group ([15.06 ± 22.49] x109/l vs. [34.97 ± 57.6] x109/l; t=-2.263, P=0.028). More patients in the CD2+ APL group expressed CD34 than patients in the CD2- APL group (13.74% vs.3.63%; x2=-2.055, P=0.006).There is no differences in CD56 expression between these two groups. Patients with CD2+ APL had a significantly higher early death rate (50% vs.15.7%, x2=5.741, P=0.016), lower incidence of CR (50% vs.91.1%, X2=5.741, P=0.042), and lower five-year OS rate (41.7% vs.74.2%, X2=5.346, P=0.018) than patients with CD2- APL. However, five year-relapse rates between these two groups were similar.Multivariate analyses revealed that WBC count before administration of anthracycline-based chemotherapy was an independent risk factor for the occurrence of differentiation syndrome (DS) (P=0.006, OR=1.022,95% confidence interval [CI] = 1.006-1.038). WBC count before anthracyc line-based chemotherapy also influenced the occurrence of early death (P=0.004, OR=1.026,95% CI=1.008-1.045) and remission failure (P=0.002, OR=1.028,95%CI=1.010-1.046). Analysis revealed that CD2+, CD34+, and CD56+ immunophenotypes were not independent risk factors for DS, remission failure, five-year survival, and five year-relapse.Conclusions1. Leukocytosis and rapid progression are main different characteristics between de novo APLpatients who died before treatment or within the first 3 days and those died at least 3 days after commencement. However, common characteristics between these two groups include leukocytosis, more frequence of PT prolongation, high ISTH score and more serious bleeding than those who achieved CR.LDH level, PT prolongation and incidence of grades 3 or 4 bleeding were higher in high-risk group than ED and low/intermediate risk groups. We found high WBC in APL patients impairs blood coagulation and contributes to ED. Current therapeutic strategies to reduce incidence of ED in these cases are not adequate and need attention.2. For low/intermediate risk APL, when combined chemotherapy is given as WBC count is between 4×109/L and 15×109/L, the patients may be benefitted the most from induction treatment. However it is worth being discussed whether chemotherapy should be administrated for those whose WBC count remains low.3. Chemotherapy is recommended to be administrated within the first 3 days after ATRA treatment for high-risk APL, in order to control leukocytosis, reduce the risk of intracranial hemorrhage and the incidence of early death.4. For those new diagnosed APL patients who cannot tolerate standard dose chemotherapy, Hydroxyurea may be a good choice. HA protocol has the similar long term and short term effects as other anthracycline based chemotherapy. HA chemotherapy is worth being recommended as an effective and economic treatment.5. Both CD64 and CD9 expression may help to diagnose APL rapidly. When compared with patients with CD2-APL, patients with CD2+ APL had a significantly higher incidence of early death and lower five-year OS rate. Multivariate analysis has demonstrated that WBC count before treatment is the only known independent risk factor that predicts prognosis for APL in this study population. |
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