Impact of Residual Normal Metaphases in Core Binding Factor Acute Myeloid Leukemia

Abstract

Background

Karyotype allows for stratification of outcomes in acute myeloid leukemia (AML) patients. Previous data suggested the presence of residual normal cells improved prognosis in patients with monosomy 7. The Southwest Oncology Group (SWOG) reported the impact of residual normal metaphases in AML patients with monosomal karyotype (MK) and found a similar relationship. We determined the influence of residual normal metaphases in patients with core binding leukemia (CBF) AML.

Methods

The presence and total number of normal and abnormal metaphases was tallied for patients with CBF AML treated in 10 consecutive SWOG trials and used as a variable to determine the effect on complete remission, refractory disease, and overall survival (OS) rates.

Results

Among 113 CBF AML patients, median age of diagnosis was 45 years (range 18–77 years) and median OS was 4 years (CI- 2 years – not reached). Patients with inv(16) and no normal metaphases had improved OS compared to those with 1+ normal metaphases (p=0.00005), while no difference was noted for patients with t(8;21). Multivariate analysis demonstrated that having cells with a normal karyotype had a negative impact on survival (HR 2.11, 95% CI 1.09, 4.08, p-0.026). This shorter survival was a consequence of higher rate of refractory disease in older patients (OR 1.03, 95% CI 0.9998, 1.06, p- 0.05) and those with normal metaphases (HR 1.26, 95% CI 1.04, 1.51, p- 0.02).

Conclusion

In patients with CBF AML, presence of cells with normal metaphases and increasing age negatively impact the prognosis, especially in patients with inv(16).

INTRODUCTION

Cytogenetic evaluation is one of the most important prognostic tools in acute myeloid leukemia (AML) (1–4). In patients less than age 60, core binding factor (CBF) translocations are present in 15–20% of cases and predict for a favorable prognosis. (1–3) Previous observations have suggested variability in outcome in patients within this distinct cytogenetic group. For example, data from the Southwest Oncology Group (SWOG) and Cancer and Leukemia B Group (CALGB) demonstrated that patients with CBF AML who also have complex cytogenetics do considerably worse, while those with inv(16) accompanied by +22 appear to have a more favorable outcome. (5,6) Meanwhile, a refinement of the original Medical Research Council (MRC) dataset showed that the presence of additional cytogenetic abnormalities had no impact on survival of patients with t(8;21) and confirmed that patients with inv(16)/t(16;16) and additional karyotypic abnormalities, in particular +22, had a particularly favorable prognosis. (4) Estey et al have shown that patients with monosomy 7 but with some residual normal metaphases had better outcomes than patients with monosomy 7 but without normal metaphases (7) and similar findings have been recently demonstrated in AML patients with a monosomal karyotype (MK). (8) Here we investigated the impact of normal metaphases on CBF AML patients treated in SWOG clinical trials.

PATIENTS

We used data from 1,344 patients with previously untreated AML enrolled between 1986 and 2009 in one of ten successive SWOG clinical trials (S8600, S9031, S9034, S9126, S9333, S9500, S9617, S9918, S0106, and S0112).(9) Centrally reviewed and approved cytogenetic data from diagnosis was used for identification of patients with CBF AML. Informed consent was obtained from all subjects involved in this study and research was conducted in accordance with the Declaration of Helsinki. Induction therapies were grouped into those: (1) with “standard” doses of cytarabine (100 mg/m2 daily X 7 days), (2) with higher than “standard” doses of cytarabine (typically at least 1 g/m², per dose), (3) without cytarabine. For patients achieving a complete remission, consolidation therapy varied based on protocol design. Of the 82 patients who achieved CR, 71 received protocol consolidation therapy. Of these 71 patients, 57 received standard dose cytarabine during consolidation (53 received standard dose cytarabine during induction and 4 had no cytarabine during induction) and 14 received high-dose cyatarabine during induction (9 received standard dose during induction and 5 received high-dose during induction).

METHODS

Cytogenetic abnormalities were considered clonal if at least two metaphases had the same aberration in case of a structural abnormality or an extra chromosome or if at least 3 shared the same abnormality in case of a monosomy. The total number of normal and abnormal metaphases was tallied for each individual patient. Importantly, different culturing methods prior to G-banding metaphase preparation can result in variations in the proportion of normal versus abnormal cells. The central review process of the SWOG cytogenetics largely ensured proper culturing conditions for all patient samples in the study and hence the reliability of the comparison. No variability was noted in the distribution of normal metaphases in different studies. Complete remission (CR) was defined as bone marrow blasts < 5%; absence of blasts with Auer rods; absence of extramedullary disease; absolute neutrophil count > 1.0 × 10⁹/L (1000/μL); platelet count > 100 × 10⁹/L (100 000/μL) and independence of red cell transfusions. Complete remission with incomplete platelet recovery was not consistently captured in all studies and therefore not included in the analysis. Death during induction (early death, ED) was defined as death within 28 days of initiating therapy. Refractory disease (RD) was defined as patients alive past 28 days of initiating therapy who nonetheless never achieved CR or who relapsed within one year of achieving CR. Relapse-free survival (RFS) was calculated for patients who achieved CR from the date of CR to the date of the first of progression or death, and was censored at the data of last contact for patients last known to be alive without progression. Overall survival (OS) was calculated from the date from registration to the study until death from any cause and was censored at the date of last contact for patients last known to be alive. Fisher’s exact test was used to compare proportions. Survival curves were estimated using the Kaplan-Meier method and compared using the log-rank test. Association of relapse risk with the covariates age, gender, total white blood cell (WBC) and peripheral blast count at presentation and number of normal or abnormal cells was examined using proportional hazards regression models (for the outcome OS) and logistic regression models (for the outcomes CR, ED, and RD). The significance level for these analyses was ≤0.05.

RESULTS

Baseline characteristics

A total of 113 of the 1,344 patients (8%) were classified as CBF AML and had complete covariate information (41 CBF patients did not have complete covariate information and were excluded from further analysis). No differences in the rates of CR, ED, RD or OS were noted between the patients with complete covariates compared to those whose covariates were not available. Of these, 67 patients had inv(16) (59%) and 46 patients had t(8,21) (41%). The median age of 45 years with range 18–78; 80% received induction with standard dose cytarabine (SDAC), 13% with high dose cytarabine (HDAC), while the remaining 7% were treated with non-cytarabine (no-AC) containing regimens. The complete remission rate was similar in patients treated with on SDAC, HDAC and no-AC regimens (78%, 53% and 57%, respectively, p-value = 0.06). Similarly, no differences in induction mortality (ED) or RD were noted. An increasing number of normal metaphases was associated with decreased RFS in CBF patients (p=0.02), but there was no significant difference between RFS in inv(16) and t(8;21) when controlling for number of normal metaphases (p=0.57). The median survival for the entire cohort of 113 CBF AML patients was 4 years (95% confidence interval 2 years - ∞). No differences in OS at 4 years were noted in patients receiving cytarabine-containing regimens, but 4-year OS was lower in patients receiving no-AC induction (SDAC-57%, HADC- 53% and no-AC- 13%, p-value = 0.001).

Distribution of Metaphases in CBF AML

The presence of one or more normal metaphases was detected in 35% of CBF AML patients and ranged from 1of 20 cells to 20 of 22 cells. However, the proportion of patients with at least one or more normal metaphases was less in inv (16) than in t(8;21) (18/67 (27%) vs. 22/46 (48%) (p=0.03). The effect of having at least one normal metaphase differed in t(8;21) and inv (16). Thus among t(8,21) patients, the presence of 0 versus 1 or more normal metaphases had no significant impact on survival (unadjusted p= 0.2), while patients with inv(16) and one or more normal metaphases were noted to have a significantly worse survival than inv(16) patients with an absence of normal metaphases (see Figure 1, unadjusted p=0.00005). There was no evidence that either presence of one or more normal metaphases was associated with CR, ED, or RD in inv (16) or in t(8;21) (Table 1).

Figure 1.

Overall survival.

Table 1.

Incidence of CR, ED and RD among CBF AML patients according to presence or absence of normal metaphases.

	0 normal metaphases (N=73)			1 or more normal metaphases (N=40)
	t(8;21) (N=24)	Inv(16) (N=49)	All	t(8;21) (N=22)	inv(16) (N=18)	All
CR	75%	75%	75%	76%	61%	69%
ED	8%	2%	4%	0%	11%	5%
RD	57%	49%	52%	55%	69%	61%

CR- Complete remission, ED- Early death, Resistant- Resistant disease.

Regression Analysis of Number of Normal Metaphases

Given the results in Figure 1, we used regression models to investigate the association between number of normal cells and outcomes. Among t(8;21) patients, there was no association between number of normal cells and OS (hazard ratio [HR] 1.01, 95% CI 0.93, 1.11, p = 0.69). However, among inv (16) patients, increased numbers of normal cells was associated with decreased OS (HR = 1.15, 95% CI 1.07, 1.24, p = 0.0003). There were no significant associations between number of normal metaphases and CR or ED, either in all CBF patients or in the subsets of t(8;21) and inv (16) patients. Among all CBF patients, increasing number of normal of normal metaphases was associated with increased risk RD (odds ratio = 1.26, 95% CI 1.04, 1.51, p = 0.02) when controlling for number of abnormal metaphases, age, gender, WBC, and blasts (Table 2). There was no evidence that the association between number of normal metaphases and RD differed between t(8;21) and inv (16) patients when controlling for number of abnormal metaphases, age, gender, WBC, and blasts (p=0.49).

Table 2.

Multivariate logistic model for number of normal and abnormal cells and refractory disease rate.

Covariate	OR	95% CI	P-value
1 or more normal metaphases	1.26	(1.04, 1.51)	0.02
1 or more abnormal metaphases	1.13	(0.98, 1.29)	0.08
Age	1.03	(0.9998, 1.06)	0.05
Gender (ref=female)	1.76	(0.72, 4.26)	0.21
WBC	0.99	(0.98, 1.01)	0.45
Peripheral blasts	1.02	(0.997, 1.05)	0.08

WBC- white blood cell count, OR- Odds ratio, CI- Confidence interval

DISCUSSION

The provocative results presented in this analysis confirm previous reports demonstrating significant differences between subgroups of patients with CBF AML (t(8;21) AML versus inv(16) AML). Specifically, in this study we found that inv(16) karyotypes are less likely to have any normal metaphases. Also, our results demonstrate that unlike AML patients with adverse risk cytogenetics (i.e. monosomy 7 and MK) (7,8), an increasing number of cells with normal metaphases increases the risk of relapse and negatively impacts survival in patients with inv (16) CBF AML: at least one normal metaphase had a significant impact on 5-year survival (60% vs 14%, p = 0.00005). These findings are likely a result of a higher rate of refractory disease (Table 2, p = 0.02) as the presence of one or more normal metaphases and age were the only independent variables associated with refractory disease in the logistic regression model.

Dose escalation of cytarabine in patients with CBF karyotypes significantly increases the duration of remission and likely cure rates.(10) Retrospective data has shown that patients with CBF karyotype treated with HDAC have superior 5-year relapse-free survivals compared to those patients with normal karyotpye(10) but the expected 5-year OS rate for CBF AML patients is still only 50%.(5,6) Therefore, a significant proportion of CBF AML patients have a high risk of relapse and identifying pre-treatment predictors of relapse could improve outcomes in these patients. Previously, presence of +22 in patients with inv(16) and mutations in the c-Kit receptor have been associated with better and worse outcomes, respectively. (11) Presence of at least one normal metaphase may be an indicator of patients with higher relapse risk.

Reasons for this differential response and outcome are uncertain, however, the presence of normal metaphases, which appear to be intrinsically less sensitive to higher doses of cytarabine than leukemia blasts with CBF karyotpyes, could identify patients with favorable risk AML that have higher risk refractory disease and consequently higher risk of death. Conversely, in patients with monosomy 7 or MK AML, presence of normal metaphases improved outcomes. As AML cells with complex and unfavorable karyotypes are clinically highly resistant to cytarabine, the presence of normal metaphases may identify patients with distinct leukemic clones that are more sensitive to conventional treatment.

In summary, data presented here potentially identifies another subgroup of patients with CBF AML associated with refractory disease and poor outcomes. These surprising and provocative results in CBF AML undoubtedly should be confirmed independently by other groups.