Intermediate Cytogenetic Group Research Articles

Cytogenetic profile and risk of transformation to acute myeloid leukemia (AML) in Indonesian patients with myelodysplastic syndrome (MDS): a pilot study

Background Cytogenetics is a fundamental examination in the course and management of myelodysplastic syndrome (MDS) since it is widely used as a diagnostic and prognostic indicator for the disease. Some cytogenetic profiles are associated with a higher risk of acute myeloid leukemia (AML) transformation. This is the first study to evaluate the cytogenetic profile of Indonesian patients with MDS. Methods This prospective cohort study was conducted at the Cancer Center and several other referral hospitals. Patients with primary MDS aged >18 years were included in the study. Clinical examination, peripheral blood smear, and bone marrow aspiration were performed, followed by cytogenetic examination. The results were further categorized into revised international prognostic scoring system (IPSS-R) scores, and cytogenetic profiles were descriptively presented. Patients were followed up for one year to evaluate AML transformation. Results A total of 28 MDS patients, aged 66±12 years, were included in this study. The majority of the patients were male (n=17;60.7%), aged 65 years or above (n=19;67.9%), diagnosed with MDS-MLD (n=14;50%), and had an intermediate cytogenetic group (n=4;14.3%). The IPSS-R score was high in 6 (21.4%) patients and very high risk in 3 (10.7%) patients. During one-year follow-up, AML transformation occurred in 3 (10.7%) patients, and 10 (35.7%) patients ceased. Monosomy 7 was observed in 6 (21.4%) patients but in one metaphase each. Deletion of chromosome 5 (del(5)(q31)), del (16)(q21.1), and del (16)(q11.2) were found in a male patient with MDS-EB1. Conclusions Monosomy 7 and deletion of chromosome 5 have been identified in Indonesian patients with MDS. MDS-EB has the highest risk of AML transformation.

Prognostic relevance of combined IDH1 and NPM1 mutations in the intermediate cytogenetic de novo acute myeloid leukemia.

Despite the great advance in treatment, cytogenetically normal Acute myeloid leukemia (CN-AML) is still a challenging entity. The discovery of IDH1 mutation in AML together with the frequent co-mutations; NPM1 and FLT3-ITD throughs a new insight into the pathogenesis and outcome of CN-AML. Recently, there has been an increasing number of recurring mutations in other genes for which the forecasting effect is still required. Despite the large number of risk variables established, there are relatively few prognostic indicators that can help in treatment decisions in AML patients. This study aimed at recording the frequency of IDH1 and NPM1 mutations in newly diagnosed AML and, dual clinicopathological significance. IDH1 and NPM1 mutations were analyzed using High-Resolution Melting curve analysis PCR in 78 newly diagnosed AML patients; 30 pediatric and 48 adult AML patients. IDH1 mutation was detected in 6 out of the 48 adult AML cases (12.5%) and all of them had intermediate cytogenetic prognostic stratification. 5/6 mutant IDH1 patients showed NPM1 co-mutation (P-value= 0.008). Mutant IDH1 patients showed significant resistance to induction therapy (P-value <0.001) and even those who achieved complete remission were relapsed later. Within the intermediate cytogenetic group, the IDH1 mutated patients had short overall survival (HR 12.9, 95% CI (3.1- 53.45) and event-free survival (HR 15.7, 95% CI (2.99-82.72) and P-value <0.001). IDH1 mutation is closely linked to the intermediate cytogenetic stratified group and in particular old age patients and has a great impact on their survival.

Characteristics and Survival of 927 Moroccan Adults with Acute Myeloid Leukemia: Monocentric Experience

Acute myeloïd leukemia (AML) is the most frequent form of acute leukemia among adults and the most aggressive type of leukemia, which is associated with the lowest survival rate. Patients with AML are treated with intensive chemotherapy and many factors could influence the survival of these patients, such as age, cytogenetic abnormalities; white blood cell (WBC) counts. The aim of this work was to study the epidemiological and response profiles of AML adults patients in Morocco.Patients and Methods: A prospective, descriptive study conducted in the Hematology and Pediatric Oncology department, 20 August Hospital Casablanca, and concerned adult patients diagnosed with AML through a period of seven years (January 2011 to December 2017). Statistical analysis was performed using SPSS version 20. The overall survival and disease-free survival were evaluated by using the Kaplan–Meier method. Results: A total of 927 patients diagnosed with AML during the 7 year period. 466 (50.3%) were males and 461 (49.7%) were females. The median age of patients was 46years.The most represented age group was between 18 and 60 years old with a percentage of 83.2%.The FAB subtype M2 occurred most frequently (27%) followed by M1 (24.8%). The cytogenetic study showed that the majority of patients had a normal karyotype. The t (8; 21) was the most detected balanced translocation in our series and the intermediate cytogenetic group was the most represented group (65.4%). A total of 461 patients (53.54%) were treated according to the protocol AML11. The Disease-free survival (DFS) was significantly better for favorable cytogenetic group as compared to other cytogenetic groups (median survival of 41.58 months for the favorable group versus 29.07 months for the adverse group; p-value = 0.02).Conclusion: The age of AML patients was younger compared to other populations. The majority of patients had a normal karyotype and the commonest balanced translocation was the t (8; 21). Survival was higher in patients with good prognosis.

Correlation Study on HLA-DR and CD117 (c-Kit) Expressions: Its Prognosis and Treatment Response in Acute Myeloid Leukemia Patients

IntroductionAcute myeloid leukemia (AML) is the most common type of acute leukemia in adults. HLA-DR and CD117 (c-Kit) are important diagnostic markers of AML. Our objective is to determine the prognostic significance of HLA-DR and CD117 expressions in newly diagnosed AML patients and determine the correlation between HLA-DR and CD117 expressions and other prognostic markers such as cytogenetic abnormalities, FLT3-ITD, response to treatment, and patient’s survival.MethodsThis study included 100 newly diagnosed AML patients. All patients were subjected to clinical, morphological, cytochemical, cytogenetic analysis, molecular genetic analysis to detect FLT3-ITD, and Flowcytometric detection of HLA-DR, CD117, and CD 34.ResultsThe results showed that HLA-DR expression was found in 75 patients (77.3%), while CD117 expression was found in 63 patients (64.9%). Patients with HLA-DR expression showed significantly higher mean Hb concentration, significantly higher platelet count, associated with AML-FAB subtypes (M0, M1, and M2), CD34 expression, and favorable cytogenetic group. M3 subtype was significantly associated with HLA-DR-ve. While patients with CD117 expression showed significantly lower platelets count. Double positive patients (HLA-DR+ve/CD117+ve) showed significant association with the intermediate cytogenetic group, while double-negative patients (HLA-DR-ve/CD117-ve) were associated with the favorable and intermediate cytogenetic group and either positive (HLA-DR+ve /CD117-ve or HLA-DR-ve/CD117+ve) associated with poor cytogenetic groups. FLT3-ITD expression had significantly worse overall survival.ConclusionThe current study suggested that the expression of CD117 and HLA-DR may be a prognostic marker in AML, as they are associated with M0, M1, and M2 FAB subtypes; moreover, patients with combined HLA-DR and CD117 positive expression are associated with CD34 expression and intermediate cytogenetic group.

Distribution of Pyrethroid Resistant Populations of Triatoma infestans in the Southern Cone of South America.

BackgroundA number of studies published during the last 15 years showed the occurrence of insecticide resistance in Triatoma infestans populations. The different toxicological profiles and mechanisms of resistance to insecticides is due to a genetic base and environmental factors, being the insecticide selective pressure the best studied among the last factors. The studies on insecticide resistance on T. infestans did not consider the effect of environmental factors that may influence the distribution of resistance to pyrethroid insecticides. To fill this knowledge gap, the present study aims at studying the association between the spatial distribution of pyrethroid resistant populations of T. infestans and environmental variables.Methodology/Principal FindingsA total of 24 articles reporting on studies that evaluated the susceptibility to pyrethroids of 222 field-collected T. infestans populations were compiled. The relationship between resistance occurrence (according to different criteria) with environmental variables was studied using a generalized linear model. The lethal dose that kills 50% of the evaluated population (LD50) showed a strong linear relationship with the corresponding resistance ratio (RR50). The statistical descriptive analysis of showed that the frequency distribution of the Log (LD50) is bimodal, suggesting the existence of two statistical groups. A significant model including 5 environmental variables shows the geographic distribution of high and low LD50 groups with a particular concentration of the highest LD50 populations over the region identified as the putative center of dispersion of T. infestans.Conclusions/SignificanceThe occurrence of these two groups concentrated over a particular region that coincides with the area where populations of the intermediate cytogenetic group were found might reflect the spatial heterogeneity of the genetic variability of T. infestans, that seems to be the cause of the insecticide resistance in the area, even on sylvatic populations of T. infestans, never before exposed to pyrethroid insecticides, representing natural and wild toxicological phenotypes. The strong linear relationship found between LD50 and RR50 suggest RR50 might not be the best indicator of insecticide resistance in triatomines.

A Clinical Measure of DNA Methylation Predicts Outcome in De Novo AML

Background: Despite advances, the majority of patients (pts) with acute myeloid leukemia (AML) will die of their disease. Current genetics-based risk classification schemes inadequately predict outcome. We sought to determine if a novel methylation-based biomarker could enhance risk stratification in AML.Methods: Using a novel methylation assay (xMELP) validated for the clinical laboratory, we performed DNA methylation assessment at 17 previously identified prognostic loci to calculate a summary methylation statistic (M-score, Wertheim et al. Clin Chem 2015) for 166 pts with de novo AML treated at the University of Pennsylvania (UPENN). Targeted next-generation sequencing (NGS) of 33 hematologic malignancy-associated genes was performed for a 136 pt subset. The association of M-score with other prognostic variables and outcome [complete remission (CR) and overall survival (OS)] was evaluated. Median follow-up was 68.1 mos (range 1.4-150.2) among 38 survivors and 10.5 mos (range 0.1-95.2) among those (n=128) deceased. The optimal M-score for identifying groups with differing OS in the total UPENN cohort defined a binary classifier. The classifier was validated in UPENN subgroups and in a separate ECOG ACRIN E1900 trial cohort.Results: The mean and median M-score for the UPENN cohort was 92.3 (95% confidence interval [CI], 87.4-97.2) and 91.4 (range, 30.8-97.3), respectively; the M-score was not significantly associated with age, gender, specimen type, blast percent, NPM1, or FLT3-ITD. Patients with favorable cytogenetics had a lower mean M-score than those with non-favorable cytogenetics; there was no difference between intermediate and unfavorable cytogenetic groups. Univariate analyses demonstrated that a 10-unit increase in M-score was associated with a 10% increase in the hazard of death (HR 1.1; P<.0001) and a 20% increase in the odds of failing to achieve CR (OR 1.2, P<.0001). In multivariable Cox analysis, higher M-score (HR 1.1, P=.011) and older age (P=.001) were significantly associated with increased hazard of death, while NPM1+/FLT3-ITD-status (P=.031) was associated with decreased hazard of death. In a multivariable logistic analysis, higher M-score (HR, 1.1, P=.034) and older age (P=.007) were associated with increased odds of failing to achieve CR, while favorable cytogenetics (P=.030) was associated with achievement of CR.Based on the maximization of the log-rank statistic, the optimal M-score cutpoint was 86, which defined a binary risk classifier (hazard of death for high vs low M-score: unadjusted HR 2.5, P<.0001; adjusted HR 1.9, P=.003). Median OS was 26.6 vs. 10.6 mos for low and high M-score groups (Figure). The CR rates for low and high M-score groups were 84% and 61%, respectively. The performance of the M-score classifier was confirmed in pts ≤ 60 yrs with intermediate cytogenetics (log-rank P=.001, OS in high vs low M-score groups: 36.4 vs 14.0 mos) and among pts who achieved CR with initial therapy (log-rank P<.00001, OS 43.9 vs 17.2 mos). The M-score classifier identified groups with different outcome regardless of allogeneic transplant.The M-score classifier was validated in the E1900 cohort (n=383). The mean and median M-scores were similar to the UPENN cohort and M-score was significantly associated with OS on multivariable analysis (P<0.0001). The M-score classifier identified E1900 subgroups with different OS (log-rank P<.00001; OS 29.5 vs 12.6 mos), a finding confirmed in the intermediate cytogenetics group, in those aged <50 and ≥50 yrs, and among pts assigned to both standard and high dose daunorubicin. Notably, high dose daunorubicin benefited patients with high M-scores (P=.001), but not those with low M-scores (P=.328).We also evaluated the prognostic value of the M-score in the context of an extended molecular profile. Random forest analysis of the UPENN cohort showed that M-score and age are the most robust predictors of OS, while a subset of recurrent mutations (FLT3-ITD, NPM1, IDH2, TET2, TP53, NRAS, CEBPA) also contribute to prognosis, but to a lesser degree.Conclusion: The M-score and associated classifier represent promising tools for clinical decision-making in AML and deserve further investigation. The prognostic value of the M-score may be superior to mutational analysis. Optimal methods for integration of M-score, clinical and genetic information are being defined. [Display omitted] DisclosuresLoren:Merck: Research Funding. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

An Integrated Genomic Classification That Includes Copy Number Alterations Is Highly Predictive of Post Induction Minimal Residual Disease (MRD) Status in Childhood Precursor B Lineage Acute Lymphoblastic Leukemia

IntroductionChildhood precursor B lineage ALL (B-ALL) is a genetically heterogeneous disease where the underlying genetics is an important determinant of outcome. Copy number alterations (CNA) have been described in B-ALL, which in conjunction with chromosomal abnormalities drive leukemogenesis. Some, especially IKZF1 deletions are prognostically relevant and influence disease outcome. However, there is no consensus on how these CNAs can be incorporated in a clinical setting. Recently, an integrated genomic classification has been proposed for ALL which includes CNA as well as cytogenetics based risk prediction. However, there have not been many studies which validated these suggestions or correlated them with immunophenotyping based MRD. Using end induction MRD as a surrogate marker of outcome we demonstrate that the integrated genomic profile is highly predictive of MRD clearance.Patients and Methods91 cases of childhood B-ALL (WHO 2008 criteria) were prospectively accrued over a 4 months. NCI risk was calculated as per standard recommendations. FISH detected recurrent cytogenetic abnormalities as well as iAMP(21); conventional karyotyping and flow cytometry determined the ploidy status. SALSA MLPA P335 was used to detect CNA in BALL following the manufacturers recommendations. Data was analyzed on the Coffalyzer software. Patients were divided into good and poor risk genetic abnormalities to stratify them according to the integrated genetic profile. The former included good risk cytogenetic (ETV6-RUNX1 or high hyperdiploidy) as well as good risk CNA profiles (no deletion of IKZF1, CDKN2A/B, PAR1, EBF1, ETV6 or RB1; isolated deletion of ETV6, PAX5 or BTG1 or ETV6 deletion with single deletion of BTG1, PAX5, or CDKN2A/B). Poor risk genetic abnormalities included high risk cytogenetic groups (BCR-ABL1, MLL rearranged, near haploidy, low hypodiploidy or iAMP21) as well as intermediate and poor risk CNA profiles (IKZF1/ PAR1/EBF1/RB1 deletion or any other CNA profiles) Cytogenetic abnormalities took precedence over CNA abnormalities as has been described (Moreman AV et al Blood 2014). MRD was detected using 9 colour flow cytometry (CD19, CD20, CD10, CD45, CD38, CD66c, CD123, CD34, CD58) on an end of induction bone marrow sample. In every case attempt was made to acquire 10,00,000 events. Syto 16 dye was used to correct the MRD value. Flow cytometry data was analyzed with Kaluza (v1.3). Two-tailed fishers exact test and chi squared test were applied for statistical analysis.ResultsMedian age was 5 years (range: 1-14), male predominant (58 males). Majority patients had good risk (50.5%) followed by intermediate (40.7%) and poor risk cytogenetics (8.8%). The frequencies of CNA were as follows; CDKN2A/B (23.1%), ETV6 (19.8%), IKZF (18.7%), PAX5 (14.3%), EBF1 (4.4%), BTG1 (4.4%), RB1 (3.3%). Using these data patients were classified into good risk (47.3%), intermediate (30.8%) and poor risk CNA profiles (22%). The cytogenetic and CNA risk profiles were compiled together into good risk genetic (74.7%) and poor risk (25.3%) profiles. MRD positivity (28.6%) ranged from 0.01% to 48.4% where as the rest were negative (71.4%). The CNA risk profiles showed a tendency for correlation with MRD status (p=0.08) whereas the integrated genetic profile showed a very high correlation with the MRD status (in which good risk patients were associated with MRD negative status) and NCI risk. In addition, the integrated genomic profile also predicted the MRD status in the intermediate cytogenetic group. (Table 1)ConclusionThis data seems to indicate that in addition to cytogenetics, CNA should be incorporated into routine clinical testing and risk algorithms for B-ALL. The integrated genomic classification is of prognostic relevance and offers an additional avenue for prognostication and risk adapted therapy. Table 1Correlation of immunophenotypic MRD, NCI Risk, Prednisolone Response and intermediate cytogenetics with integrated genetic profile.Variable TestedGood Genomic ProfilePoor Genomic ProfileStatistical SignificanceMRD StatusEnd Induction MRD Positive1214Significant (p=0.0003)End Induction MRD Negative569NCI RiskHigh NCI Risk1812Significant (p=0.03)Standard NCI Risk5011D+8 Prednisolone Response (n=83)Good Response5818Not Significant (p=1)Poor Response61Intermediate Cytogenetic RiskEnd Induction MRD Positive36Significant (p=0.05)End Induction MRD Negative197 DisclosuresNo relevant conflicts of interest to declare.

Autologous Transplantation in 1st Cr Aml Patients with Purged or Unpurged Bone Marrow: Good Clinical Results in Favorable Cytogenetic Group and in those Infused with a Low Number of Marrow Cells

In a single institution, 31 patients affected with Acute Myeloid Leukaemia (AML) in 1st Complete Remission (CR) received autologous bone marrow transplantation (ABMT). Mafosfamide was employed in a non-randomized fashion to purge marrows, in 15 cases bone marrow cells were purged, while in 16 they were left unpurged. Dose of infused Total Nucleated Cells (TNC) was an important factor for myeloid engraftment (P=0.02). LFS was 58% in purged and 40% in unpurged groups (P=0.26). Patients having a good prognosis cariotype had a LFS of 100% while the group of all other patients had a LFS of 37.5%. Patients receiving a dose of TNC below to median had a LFS of 65% and those receiving a dose of TNC>median had a LFS of 28% (P=0.017). Purging significantly improved LFS in patients “not harbouring good cytogenetic abnormalities” (53% LFS in purged group versus 18% in unpurged group, P=0.05). In conclusion, ABMT is associated with excellent results in “good prognosis cytogenetic”. Purging may improve results in patients belonging to “intermediate cytogenetic group”. A high number of infused TNC produces a fast myeloid engraftment but a poor LFS.

Lack of CD34 Expression in Acute Myeloid Leukemia (AML) Is a Favorable Prognostic Factor in Patients Treated with Stem Cell Transplant Irrespective of the Cytogenetic Risk

Abstract 1894 Background:Most of the studies of prognostic factors in acute myeloid leukemia (AML) are focused on predicting outcome after standard chemotherapy treatment. The studies that looked at the importance of these prognostic variables in predicting outcome after hematopoietic stem cell transplantation (HSCT) are very limited. Considering post-HSCT prognostic factors in stratifying AML patients may add a new level of confidence in any risk stratification of these patients. Toward this goal, we investigated the clinical relevance of CD34 expression in the primary leukemic cells on the outcome of allogeneic HSCT. Methods:Data collected from immunophenotyping of 110 patients with AML who were treated with HSCT was reviewed and the expression of CD34 on the blast population as determined by flow cytometry was correlated with clinical behavior and outcome. All the patients were treated with allogeneic HSCT. The median age of these patients was 24 (range: 14–57) and included 88 patients in the intermediate cytogenetic group and 22 in the adverse cytogenetic group. Of the 110 patients 71, (64.5%) were treated with HSCT in first remission (CR1). Results:Twenty-eight (25%) of all AML patients studied did not express CD34 on the surface of the blasts at diagnosis. Patients with CD34 negative blasts had significantly longer overall survival (OS) (P=0.003) as well as longer event free survival (EFS) (P=0.01) when all patients were considered. In the subgroup of patients who received HSCT in CR1, OS and EFS were significantly longer (P=0.017 and P=0.027, respectively) in the CD34-negative patients (N=20). Furthermore, if we consider only patients in the intermediate cytogenetic group at diagnosis, patients with CD34-negative blasts had significantly longer OS (P=0.007) and EFS (P=0.026). Even in patients with adverse cytogenetic abnormalities, OS and EFS were also significantly longer in the CD34-negative patients (P=0.01 for both). This was true when all patients were considered. The same was true when only patients transplanted in CR1 (P=0.05 for EFS in intermediate cytogenetics and P=0.05 for EFS in patients with adverse cytogenetic) were evaluated. Multivariate analysis including CD34 expression and FLT3 mutation status was carried out on a subset of patients (N=63) and showed CD34 expression was an independent prognostic factor for survival and EFS, while FLT3 mutation status became no longer a predictor. Conclusion:Our data suggests that CD34 expression on the blast cells at the time of AML diagnosis may have an adverse prognostic impact even after allogeneic HSCT. Lack of CD34 expression is a powerful independent favorable prognostic factor for AML patients if these patients are treated with HSCT after induction chemotherapy, irrespective if they were in CR1 or in CR2, and irrespective of their cytogenetic risk at diagnosis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Expression of CD56 in Acute Myeloid Leukemia (AML) Is Associated with Poor Outcome When Patients Treated with Stem Cell Transplant in Second Remission but Not in the First Remission

Abstract 4880 Background:The CD56 is a neural cell adhesion molecule (N-CAM) expressed in natural killer cells. CD56 expression on the surface of leukemic cells in acute myeloid leukemia (AML) has been reported to correlate with higher rate of relapse and to be associated with poor outcome when patients are treated with conventional intensive chemotherapy. The role of allogeneic hematopoietic stem cell transplantation (HSCT) in improving outcome in this subgroup of AML patients is not known. We investigated whether the outcome of CD56-positive AML patients differ from average AML patients when treated with allogeneic HSCT after intensive chemotherapy and its prognostic relevance in patients transplanted in the first (CR1) or second complete remission (CR2). Methods:Immunophenotyping data of 106 patients with AML who were treated with allogeneic HSCT was reviewed. The expression of the CD56 on the blast population as determined by flow cytometry was correlated with clinical data and outcome. The median age of patients was 24 (range: 14–57) and included 72 patients in the intermediate cytogenetic group and 30 in the adverse cytogenetic group. Of the 106 patients, 71 (67%) were treated with HSCT in their first remission (CR1) and the remaining patients were treated in CR2. Results:Twenty nine (28%) of all studied patients expressed CD56 on the surface of the blasts at diagnosis. When we consider only patients treated with HSCT in CR1, 17 (24%) were CD56 positive. The overall survival (OS) and event free survival (EFS) for patients who were positive for CD56 were not significantly different from the rest of the patients in the CR1 group. In contrast, in the group of patients who were treated with HSCT after relapsing (CR2), patients with CD56 positivity had significantly shorter OS (P=0.03) and DFS (P=0.006). In this group, 11 of the 35 (31%) were CD56+ and their median survival was 8.85 months while the median has not been reached in the CD56 negative patients. Conclusion:Our data suggests that the expression of CD56 in AML is a significant poor prognostic factor if patients are treated with HSCT after relapsing in their second remission. Although this should be confirmed in larger studies, the CD56 expression in AML should be considered as an adverse risk factor and favors treatment with allogeneic HSCT in the first remission and not after relapse in second remission. AML patients with CD56 positive blasts have higher incidence of relapse after chemotherapy and they might lose their opportunity of good outcome if HSCT is delayed and performed only in the second remission. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

AML Patients In First CR with Intermediate Risk Cytogenetic and High Level of CD34+ Mobilization Have Better DFS After Allogeneic Transplantation.

AbstractAbstract 4563AML patients having a high apheretic yield in CD34+ cells during mobilization have a poor prognosis, independently from cytogenetic risk (Feller 2004, Keating 2004). It is however not known if prognostic value of PBSC mobilization is retained after different post remissional treatment and clinical usefullness of this prognostic information in dealing with intermediate cytogenetic group patients, the more represented AML subgroup in which post remissional therapy is still a controversial issue. METHODSA group of 64 AML adult patients in 1st CR was prospectively studied. PBSC mobilization was attempted following first consolidation course. Cytogenetic assessment at diagnosis was available in 95% of patients. Post remissional treatment was chosen based on cytogenetic risk, sibling donor availability and presence of others prognostic factors, MUD transplants were proposed in cytogenetic high risk patients or in selected young patients with intermediate cytogenetic, Autologous HSC Transplantation was proposed when allogeneic transplant was felt not indicated, no further therapy was administered in patients deemed ineligible for any kind of transplantation. Prognostic significance of CD34+ cells was evaluated measuring DFS in groups of patients identified from value of CD34+ peak in respect to 50th percentile (CD34 peak: 65×10e6/L) and 75th percentile (197×10e6/L). Those patients having a CD34+ cell peak below median value (65×10e6/L) were categorised as LOW MOBILIZER, those having a CD34+ peak between 65 and 197 ×10e6/L were categorised as GOOD MOBILIZER, and those having a CD34+ peak over 75th percentiles (197 ×10e6/L) were categorised as SUPERMOBILIZER. RESULTSIn our serie 40% of patients received an Allogeneic transplant after a myeloablative treatment, 49% an autologous transplant while 11% no further chemotherapy. Disease Free Survival (DFS) was 60%. CD34+ peak during mobilization, evaluated as a continuous variable, in a Cox regression model resulted important for DFS (hazard ratio:1.001; p=0.01). Importance of CD34+ peak was maintained also when the stratum of patients with intermediate cytogenetic risk was analysed (DFS: p=0.002). POOR MOBILIZER, GOOD MOBILIZER and SUPERMOBILIZER patients had statistically different DFS when cases were analysed as a whole (DFS: POOR M.: 70%; GOOD M. 50%; SUPER M: 30%; log rank: p=0.05); mobilizing efficiency was important for DFS also in intermediate cytogenetic group (DFS: POOR M. 70%; GOOD M. 40% DFS; SUPER M. 28%; log rank p= 0.02), importance was maintained also in patients treated by allogeneic transplantation or with autologous transplantation (fig.1).When allogeneic transplantation was compared to the group of patients receiving autologous transplantation or other post remissional strategies both in the cases as a whole and in the stratum of intermediate cytogenetic (table 1), the group of POOR mobilizer had after autologous transplants results non different from patients receiving allogeneic transplantation, however, the group of GOOD mobilizer patients had after allogeneic transplants a DFS significantly better compared to other treatment while SUPER mobilizer patients had poor results also when allogeneic transplantation was employed.In conclusion in AML in 1st CR, evaluation of CD34 peak during mobilization is a parameter providing prognostic informations useful also within intermediate cytogenetic group.INTERMEDIATE CYTOGENETIC RISK (n.42)DFS (All patients) (n.42)DFS (Autologous Tx Group) (n.16) or no Tx (n.6)Log rankDFS (Allogeneic Tx Group) (n.20)CD34+ LOW MOBILIZER70%65%NS80%CD34+ GOOD MOBILIZER40%0%p=0.0275%CD34+ SUPER MOBILIZER28%0%NS30%Log rankp=0.02p=0.005p=0.04 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Proteomics-based prediction of clinical response in acute myeloid leukemia

Response to chemotherapy is achieved in 60% to 70% of patients with acute myeloid leukemia. The ability to predict responders may help in stratifying patients and exploring different therapeutic approaches for nonresponders. Proteomics methods were used to search for predictive factors or combinations of factors. Peripheral blood plasma samples from 41 patients with confirmed acute myeloid leukemia with intermediate or poor cytogenetics were obtained prior to induction therapy for proteomic analysis. For each plasma sample, four fractions eluted from a strong anion column were applied to 3 different ProteinChip array surfaces and 12 surface-enhanced laser desorption/ionization spectra were generated. Peaks that correlated with response were identified, and decision trees incorporating these peaks along with various clinical and laboratory findings were constructed to predict response. Multiple decision trees were constructed. One peak, when combined with age, provided strong positive prediction of responders with 83% accuracy. A second tree, which combined one peak with both cytogenetics and the percent of monocytes in peripheral blood, detected responders with 95% accuracy. A third peak was adequate to predict responders in the intermediate cytogenetic group with 86% accuracy. Proteomic analysis should be further explored to define factors important in predicting clinical response in patients with acute myeloid leukemia.

Identification of Specific Genes Expression Signatures for CEBPa, NPM1 and FLT3 Genes Mutations in AML. A Study Based of the MILE Data Issued from 79 French Samples.

The MILE (Microarray innovations in Leukemia) project has recently reported on the efficiency and accuracy of micro-array use in the diagnosis and sub-classification of leukemia. The MILE algorithm classifier of AML recognized 6 subgroups according to major cytogenetic findings:t(15;17), t(8;21),inv(16), MLL rearrangement, complex karyotype and a last group including normal cytogenetics and other patients. In the latter group, beside age, leucocytosis and antecedent of previous hemopathy, molecular alterations, especially of CEBPa, NPM1 and FLT3 mutations have been shown to be frequent and to have a major prognosis value. In this work, we have attempted to enhance the MILE classification of AML by examining data for a gene signature specific of each of these mutations. The study was made using the data generated from HG-U133 plus 2.0 Affymetrix arrays from the 79 AML samples included from the French study centers of MILE phase 1 trial. Among these patients with AML, according to the gold standard classification used in the MILE project were 2 t(8;21) AML, 2 inv(16) AML and the others belonging to intermediate cytogenetic group class MILE 13 (mainly patients with normal karyotype). Into the cohort, 7 samples harbored CEBPa mutations, 33 samples have NPM1 mutation and 26 samples have FLT3-ITD. Data generated from GEP profiles were combined to these molecular data to obtain predictor of mutation status. Predictors were based on differential gene expression signature using SVM methods. Differentially expressed genes sets were obtained using volcano plot with p value of 0.05 and fold change of 2. The CEBPa predictor shows a sensitivity of 100% in the detection of CEBPa mutation with a specificity of 81%, only six sample were misclassified among them 3 were non informative and 3 were called as CEBPa mutated (2 t(8;21) samples and one AML with MLL alteration), the efficiency of the predictor was 83%. The NPM1 predictor shows a sensitivity of 87.9% in the detection of NPM1 mutation and a specificity of 75%, 7 samples were misclassified, among them 3 NPM1 mutated samples (2 were called as wt and one assigned as non informative) and at the other hand 4 NPM1 wt samples were called as mutated), efficiency of this predictor was 81%. To note using the set of genes used for this predictor, non supervised hierarchical tree clearly shows the dichotomy of NPM1 mutated samples into two sub-clusters according to the FLT3 status. Results were weaker for the FLT3-ITD predictor with a sensitivity of 57% and a specificity of 70% with 29 samples misclassified (9 miscalled and 20 without answer).These results shows that the use of the data generated by the MILE in the diagnosis of leukemia can be used in the detection of CEBPa, NPM1 and FLT3 mutations in AML that have a major value in the prognosis of patients. The use of this 3 new predictors, in second line after the use of MILE algorithm to discriminate AML belonging to the intermediate cytogenetic groups clearly improve the use of microarrays in the diagnosis of AML.

Potential cure of acute myeloid leukemia

Potential cure of acute myeloid leukemia (AML) is now a widely accepted idea, but it is uncertain whether there is heterogeneity in the failure rate in patients once they have been in complete remission (CR) for various periods of time. The long-term outcomes were analyzed in 1069 consecutive AML patients in first CR who were diagnosed and treated at the University of Texas M. D. Anderson Cancer Center between 1991 and 2003. RESULTS.: The failure rates as yearly risk of treatment failure were 69.1 in the first year, 37.7 in the second year, 17.0 in the third year, 7.6 in the fourth year, and 6.6 in the fifth year, suggesting that 3 years from the CR date is a convenient time to consider patients potentially cured. The effect of cytogenetics on relapse-free survival (RFS) remained constant throughout the first 3 years, whereas the effect of age increased with time. The probability of RFS for patients alive without disease recurrence at 3 years was 84.0% at 6 years. When the interaction between age and cytogenetics was examined for these patients, the outcomes of those with favorable cytogenetics were found to be excellent regardless of age. However, in the intermediate cytogenetic group, although patients aged <60 years had excellent outcomes, those aged > or =60 years were found to be at a substantial risk of disease recurrence even after 3 years of CR, with a 6-year RFS rate of 56.5%. There were only 6 patients with adverse cytogenetics in this cohort. The results of the current study demonstrate that the risk of treatment failure differs over time according to a combination of cytogenetics and age.

Prognostic Significance of Multidrug Resistance Proteins Expression in Acute Myeloid Leukemia.

Background: Conventional induction chemotherapy induces complete remission (CR) in 65–75% of adults with de novo acute myeloid leukemia (AML), and 15–20% of the patients have refractory disease. We investigated the prognostic significance of multidrug resistance proteins (P-glycoprotein (Pgp), BCRP, MRP1 and LRP) expression on AML blast cells before treatment.Methods: We included in analysis 30 patients (pts) with de novo AML. Expression of multidrug resistance proteins (MDR) was detected by indirect immunofluorescence technique and flow cytometry on bone marrow blast cells before chemotherapy. Expression of MDR proteins was considered as positive if at least 25% of the blast cells were stained by anti-MDR protein antibody. All pts received standard induction therapy (cytarabine, etoposide and idarubicin or daunorubicine).Results: Blast cells was defined as Pgp-positive in 64.3% of cases, BCRP+ in 42.9%, MRP1+ in 46.4%, and LRP+ in 64.3% of cases. After induction therapy 20 (66,7%) pts achieved CR and 10 pts (33.3%) were resistant. MDR proteins expression was observed more frequently in resistant group, then in a sensitive one (70% vs 61% for Pgp, 70% vs 33% for MRP1, 100% vs 44% for LRP, 80% vs 22% for BCRP, respectively), difference for LRP and BCRP was statistically significant (p=0.004). Blast cells of all resistant pts expressed 2–4 MDR proteins (all studied proteins − 40%, 3 of them − 40% and 2 proteins − 20%). In a group of pts archived CR the blast cells expressed 3 proteins only in 2 cases (10%), and the expression of all 4 proteins we observed only in 1 patient (5%) with very short CR duration (3 months). Other pts from this group express only one studied protein.According to chromosome analysis 18.2% of pts had favorable, 50% - intermediate and 31.8% unfavorable cytogenetic. Blast cells of all pts in cytogenetically unfavorable group expressed more then 1 protein, 3 or 4 MDR proteins expressed in 71,4% of cases. In favorable and intermediate cytogenetic groups blast cells expressed 1or 2 MDR proteins in 73,2% of cases, 3 or 4 proteins in 20%.Conclusions: The expression of MDR proteins in AML has a prognostic value with respect to CR achievement in pts receiving standard antracycline-Ara-C regimens. The detection of any single protein didn't have prognostic significance, only co-expression of 2 and more proteins predict unfavorable treatment outcome. We observed a correlation between the cytogenetic and the MDR phenotype.

Effect of Priming with Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) in Younger Adults with Newly Diagnosed Acute Myeloid Leukemia (AML): A Trial by the Acute Leukemia French Association (ALFA) Group.

Sensitization of leukemic cells with GM-CSF may enhance the cytotoxicity of chemotherapy in younger adults with newly diagnosed AML. In a multicenter randomized trial, 259 patients (median age, 27 years; 15–50) were randomized at baseline to receive a timed sequential induction regimen (DNR 80mg/m2/d d1-3, AraC 500mg/m2/d d1-3 and 500mg/m2/12h d8-10, Mitox 12mg/m2/d d8-9) either without (135 patients) or with GM-CSF (124 patients) administered i.v. at 5μg/kg/d from day 1 to day 10. Patients reaching CR (eventually after salvage therapy combining AraC 3g/m2/12h d1,3,5,7 and Amsa 100mg/m2/d d1-3) and not eligible for allogeneic transplantation in CR1 were then randomized to compare the ALFA-9000 consolidation (Mitox 12mg/m2/d d1-3, VP16 200mg/m2/d d8-10, AraC 500mg/m2/d d1-3,8-10) (Castaigne et al., Blood 2004) to the CALGB postremission chemotherapy, which includes 4 cycles of HD-AraC (3g/m2/12h d1,3,5) followed by 4 additional DNR/AraC courses (Mayer et al, N Engl J Med 1994). Patients initially randomized in the GM-CSF arm received GM-CSF concurrently with chemotherapy during all cycles of consolidation therapy. The effects of GM-CSF on EFS were assessed in the entire cohort and according to distinct prognostic subgroups. After induction therapy, the proportions of CR were not significantly different in the two groups (91% with GM-CSF vs 87% without). After a median follow-up of 3 years, patients receiving GM-CSF had a higher rate of EFS than those who did not receive GM-CSF (3-year EFS, 42% vs 34%, p = 0.06). GM-CSF did not significantly improve OS. Patients with intermediate risk cytogenetics benefited from GM-CSF therapy (3-year EFS, 50% vs 35%, p = 0.05), while GM-CSF did not improve the outcome in the subgroups with favorable (p = 0.8) or unfavorable cytogenetics (p = 0.3). The benefit of GM-CSF in patients with intermediate karyotype was in part related to a lower rate of relapse (29% vs 47% at 3 years, p = 0.05) and a lower TRM (19% vs 23% at 3 years). Patients with abnormal intermediate karyotype benefited more of GM-CSF therapy (3-year EFS, 55% vs 19%, p = 0.03) than those with normal karyotype (3-year EFS, 47% vs 42%, p = 0.4). However, GM-CSF did not significantly improve OS in both groups. The effect of GM-CSF in the intermediate cytogenetic group was increased when considering only patients following the second randomization (consolidation chemotherapy ± GM-CSF) (3-year EFS, 49% vs 31%, p = 0.03; 3-year OS, 65% vs 43%, p = 0.05). GM-CSF also influenced favorably the intermediate group when considering a prognostic classification based on cytogenetics and the number of chemotherapy courses required for achieving CR (Wheatley et al., Br J Haematol 1999) (3-year EFS, 50% vs 34%, p = 0.04; 3-year OS, 63% vs 44%, p = 0.04).We confirmed here that sensitization of leukemic cells with hematopoietic growth factors is a clinically means of enhancing the efficacy of chemotherapy in younger adult patients with intermediate risk AML.

Acute Erythroid Leukemia (AEL): A Clinical, Morphological, Cytogenetic and Follow-Up Study of 69 Patients.

AEL is a uncommon type of acute leukemia. The subjects in this retrospective study were 69 patients diagnosed as having AEL to assess the incidence of different subtypes, detect prognostic factors, and compare there profile with others AML. 63 patients (82,5%) were diagnosed of erythroleukemia (EL) and 7 (17,5%) were pure erythroid leukemia according to WHO classification. 63,6% of EL presented erythroid maturation (immature/mature erythroid ratio <25%), and 36,4% >25%. Three of seven pure erythroid leukemia showed immature erythroid precursors identified by ultrastructural methods, and four had erythroid cells at all maturation stage. 49 (71%) were de novo AEL, only 3 pure erythroid leukemia, 9 (13%) were therapy related, in nine (13%) was preceded by a MDS, and 2 suffer a BC-CML, all of this later, pure erythroid leukemia. The three cases of pure erythroid proliferations with only immature precursors were two Down's syndrome, both 2 years old, and one BC-CML. The chromosomal abnormalities were classified by SWOG and MRC. BC-CML were excluded from survival analyses. A p value <0.01 was considered significant. There were 38 males and 31 females. The median age was 62 years. The medians hemoglobin 80 g/l; white cell 3,8x109/L; absolute neutrophil 1,0x109/L and platelet 36,0x109/L. Multilineage dysplasia were present in 74,5% (41/55). The overall incidence of chromosomal abnormalities was 79,7%(55/69). 35(50,7%) patients had complex karyotype, 26/49(53,1%) in de novo acute erythroid leukemia. Single abnormalities include 4 monosomy 7, 2 del(5q), two +8, two + 21. 39/64 patients (60,9%) received intensive chemotherapy (9 were consolidated with SCT) and 19/39 (48,7%) achieved CR. Remission rate was 83,3% for normal karyotype and 85,7% and 62,5% for intermediate cytogenetic groups according SWOG (p=0,003) and MCR(p=0,037). The median OS was 4 months with a projected actuarial DFS at 12 months 32,5%. Patients under 40 years (p=0,0001) with normal karyotype (p=0.0003) or intermediate SWOG (p=0.0002) or MRC (p=0.0019) citogenetic groups, who received intensive chemotherapy (p<0.0001) had a longer OS, and younger patients (p=0.0056) with normal karyotype (p=0.0038), or intermediate SWOG (p=0.0006) or MRC (p=0.039) citogenetic groups, consolidated with SCT (p=0.0023) showed the longest DFS. Multivariate analysis showed that only karyotype, is a powerful prognostic indicator both for OS and DFS, besides treatment for OS and age for DFS. The higher risk of relapse is for ≥65 years old patients (OR=2,84) with unknown (OR=7,86) or unfavourable (OR=4,29) SWOG groups and the higher risk of death is for unfavourable (OR=4,33) SWOG groups and those treat with supportive care (OR =3,31). When AEL were compared with a consecutive series of 339 de novo AML, 109 of them ≥ 65 years, and 68 therapy related AML, EL excluded, these series are similar for age and sex but AEL present more frequent multilineage dysplasia (p<0,001), and SWOG or MRC unfavourable citogenetic groups (p<0,001), beside the low remission rate, high risk of relapse and death, was similar at elderly patients AML and therapy related AML.