CME/SAM. Acute Myeloid Leukemia With Monosomal Karyotype. Morphologic, Immunophenotypic, and Molecular Findings

Transcription

1 AJCP / Original Article Acute Myeloid Leukemia With Monosomal Karyotype Morphologic, Immunophenotypic, and Molecular Findings Olga K. Weinberg, MD, 1 Robert S. Ohgami, MD, PhD, 2 Lisa Ma, 2 Katie Seo, 2 Li Ren, 2 Jason R. Gotlib, MD, MS, 2 Mahesh Seetharam, MD, 3 Athena Cherry, PhD, 2 and Daniel A. Arber,MD 2 CME/SAM From the 1 Boston Children s Hospital, Boston, MA; 2 Stanford University Medical Center, Stanford, CA; and 3 Arizona Oncology, Scottsdale, AZ. Key Words: Acute myeloid leukemia; Monosomal karyotype; Myelodysplasia Am J Clin Pathol August 214;142: DOI: 1.139/AJCPMLO84JDNVLNK ABSTRACT Objectives: Acute myeloid leukemia (AML) with monosomal karyotype (MK) recently has been reported to be associated with worse outcome than the traditional complex karyotype. Methods: In this retrospective study of 111 patients with AML, we identified 14 patients with MK (13% of all patients with AML) using the definition proposed by Breems et al. Results: Five (36%) of these 14 patients had a loss of a single chromosome in the presence of other structural abnormalities, and nine (64%) had a loss of two or more autosomal chromosomes. Patients with presented at an older age, with lower bone marrow blasts, and their blasts less frequently expressed CD34. Most patients with had morphologic multilineage dysplasia and were predominantly subclassified as having AML with myelodysplasia-related changes (AML-MRC). Molecular analysis showed a significant absence of NPM1 and FLT3 in patients with. Conclusions: Outcome data showed that patients with had significantly worse overall survival, diseasefree survival, and complete response compared with the rest of the patients with AML as well as within the AML-MRC group. Upon completion of this activity you will be able to: define complex karyotype and monosomal karyotype in acute myeloid leukemia (AML). provide correlation between complex karyotype and overall survival in AML. describe the relationship between complex karyotype and morphologic findings in AML. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 274. Exam is located at Acute myeloid leukemia (AML) is a heterogeneous disease with diverse clinical features, prognosis, and pathogenesis. Cytogenetic analysis is well accepted as one of the most important prognostic factors in AML. Using pretreatment cytogenetic analysis, risk stratification systems divide patients with AML into favorable, intermediate, and unfavorable risk groups. 1,2 Complex karyotype has been strongly associated with unfavorable outcome in multiple studies. 1,2 In the past, different definitions for complex karyotype have been used, which include three, four, or five unrelated cytogenetic abnormalities. 3-5 Most recently, Breems et al 6 proposed the concept of monosomal karyotype (MK), defined by the presence of at least two separate autosomal monosomies or one monosomy plus one or more structural abnormalities. Using this definition, different studies have confirmed that 19 Am J Clin Pathol 214;142: DOI: 1.139/AJCPMLO84JDNVLNK

2 AJCP / Original Article comprises a very poor prognostic subgroup of patients with AML. 6 In a study of 733 patients with AML with cytogenetic abnormalities, MK was shown to be a stronger prognostic predictor of poor outcome compared with the traditionally defined complex karyotype. 6 Patients with MK had a 4-year overall survival of 4% compared with 21% in patients with other unfavorable karyotypes but without MK. Other studies have also confirmed that MK is an independent predictor of very poor prognosis in the elderly. 7 In fact, this remains true even in a cohort of patients with only unfavorable cytogenetics. 8 The incidence and prognostic impact of MK had not been fully evaluated in the context of the 28 World Health Organization (WHO) classification of AML. Kayser et al 9 found that in their cohort, was significantly associated with myelodysplastic syndrome (MDS) related cytogenetic abnormalities and AML with recurrent genetic abnormalities, but the authors did not include morphology in this analysis. The 28 WHO approach classifies AML using genetic, immunophenotypic, biologic, morphologic, and clinical features. We have recently shown that the newly expanded AML with myelodysplasia-related changes (AML-MRC) group has a worse prognosis compared with AML not otherwise specified. The relationship of MK with AML-MRC has not been described, to our knowledge. Recently, efforts in the classification of AML have focused on identifying molecular markers with prognostic significance. Few studies have evaluated the correlation between these markers and MK. Of the molecular markers described in AML and investigated in MK, NPM1 and FLT3 were found at very low frequencies in. 9 CEBPA and WT1 mutational status in MK has not been reported. The goal of this study is to describe clinical, morphologic, cytogenetic, and molecular features of in a group of patients with AML. Materials and Methods Patients A total of 111 consecutive de novo patients with AML diagnosed at Stanford University Medical Center between May 25 and November 27 with adequate material for mutation analysis were studied as previously described. 1 All cases were diagnosed with bone marrow aspirates, blood smears, trephine biopsies, and/or flow cytometry according to 28 WHO criteria. 11 Clinical parameters, hemogram data, and flow cytometry results at the time of diagnosis were reviewed. Clinical follow-up information was obtained by retrospective review of the electronic charts. Cytogenetic risk group stratification was performed using Southwest Oncology Group criteria. 3 This study has been approved by Stanford University s Institutional Review Board. NPM1, FLT3, and CEBPA Mutational Analysis Genomic DNA was obtained from the Stanford Molecular Pathology Laboratory. The FLT3-ITD, FLT3-D835, and exon 12 NPM1 insertion mutations were detected by a multiplex polymerase chain reaction (PCR) followed by restriction enzyme digestion and capillary electrophoresis. The entire coding region of CEBPA was PCR amplified and sequenced. 1 WT1 Mutation Analysis Genomic DNA obtained from the Stanford Molecular Pathology Laboratory was added to a PCR mixture containing PCR buffer, MgCL₂, Q Solution, HotStarTaq DNA polymerase (all from Qiagen, Valencia, CA), dntp (Thermo Fisher Scientific, Waltham, MA), DNase and RNase-free H₂O, and forward and reverse primers. The cycles included 95 C for 15 minutes followed by 35 cycles at 94 C for 6 seconds, 59 C for 45 seconds, 72 C for 6 seconds, and then a final extension at 72 C for 1 minutes. PCR products were treated with USB ExoSAP-IT (Affymetrix, Sunnyvale, CA). The treated PCR products were then sequenced with the BigDye v.3.1 sequencing kit (Applied Biosystems, Foster City, CA) and forward or reverse primers. The sequencing reactions were then purified with Performa DTR gel filtration cartridges (EdgeBio, Gaithersburg, MD) and run on an ABI 31 sequencer and analyzed using ABI s Sequencing Analysis 5.1 software (Applied Biosystems). Statistical Analysis Overall survival (OS), disease-free survival (DFS), and complete response (CR) were defined as previously described. 12,13 These parameters were compared using Kaplan-Meier methods and the log-rank test. Univariate and multivariate Cox proportional hazards models were performed. Quantitative factors were treated as continuous variables in these regression models. Categorical variables were compared using the Fisher exact test. Results Overall AML Group In total, 111 patients, including 63 men and 48 women with a median age of 57 years (range, years), were studied. Stratification of patients into cytogenetic risk groups resulted in 15 patients with favorable, 72 with intermediate, and 24 with unfavorable cytogenetic risk status. Most patients received idarubicin and cytarabine or daunorubicin Am J Clin Pathol 214;142: DOI: 1.139/AJCPMLO84JDNVLNK 191

3 Weinberg et al / AML With Monosomal Karyotype and cytarabine as induction therapy and high-dose or standard-dose cytarabine for consolidation. The median OS was 171 days, and the median DFS was 82 days. CR was achieved in 64 (61%) of 15 patients. Frequency of MK and Relationship With Complex Karyotype Cytogenetic analysis was successful in all 111 patients with AML with 2 metaphases. A total of 14 (13%) patients with AML had MK as defined by Breems et al, 6 with either (1) loss of two or more distinct autosomal chromosomes or (2) one single autosomal chromosome in the presence of other structural abnormalities. Five (36%) of 14 patients had a loss of a single chromosome in the presence of other structural abnormalities, and the rest had a loss of two or more chromosomes. The most common chromosomes that were lost were chromosomes 7 and 17, which were present in 6 (43%) of all 14 patients with MK Table 1. Most of the patients with MK (13 of 14) had a complex karyotype (as defined by having three or more unrelated changes). Clinical Features and Course of Patients With Compared with other patients with AML, patients with MK were older at presentation (65 vs 54 years, P =.25), Table 1 Specific Chromosomes Lost in Cases of Acute Myeloid Leukemia With Monosomal Karyotype Chromosome No. (%) of Cases (n = 14) 7 6 (43) 17 6 (43) 18 5 (36) 5 4 (29) 21 4 (29) 2 3 (21) 3 2 (14) 12 2 (14) 22 2 (14) 4 1 (7) 9 1 (7) 13 1 (7) 19 1 (7) 2 1 (7) Table 2 Clinical and Pathologic Features of AML Cases and of patients older than 6 years, MK was frequent, seen in 11 (22%) of 49 patients. Review of hemogram data showed that patients with MK had a lower WBC count, while no difference in hemoglobin or hematocrit was found Table 2. Patients with MK had lower bone marrow blast counts, and their blasts more frequently lacked CD34 (Table 2). There was no association of MK with sex (P =.567). Using the 28 WHO classification, 13 (93%) of 14 patients with MK were classified as having AML-MRC (P =.3). Within this group, 3 (27%) of 11 patients had a reported history of MDS. Morphologic evaluation of the diagnostic bone marrow showed dysplasia in all three lineages in four (29%) patients and two lineages in nine (64%) patients of all MK cases. An example of and multilineage dysplasia is illustrated in Image 1A and Image 1B. One of the patients with MK had history of chemotherapy, which was considered therapy-related AML. Review of the prognostic molecular data in this group of patients with AML showed that patients with MK significantly lacked NPM1 and FLT3-ITD mutations with no differences in the distribution of D835, WT1, and CEBPA mutations Table 3, although the overall number of patients with CEBPA and WT1 mutations was small. Within the AML- MRC group, those with MK sought treatment at an older age (65 vs 6 years, P =.412) and with lower marrow blasts (35% vs 47%, P =.371), but no difference in presenting CBC was noted in this group. In univariate analysis, outcome data showed that patients with had worse OS (P =.1) and DFS (P =.7) Figure 1. Fewer patients with MK achieved complete remission (five of 14; 36% vs 61%; P =.211). When patients with MK were subdivided by number of chromosomes lost, no clinical difference was found between those who lost one chromosome or two or more (OS, P =.235). Within the AML-MRC group, patients with MK had shorter OS (P =.89), shorter DFS (P =.76), but no clinical difference in CR (P =.312) Figure 2. In multivariate analysis, cytogenetic risk group, MK, and age 6 years or older all correlated with decreased survival (all P <.1) Table 4. Characteristic Other AML P Value Age, mean (range), y 65 (48-76) 54 (17-82).25 Blast immunophenotype (CD34+), % Marrow blast, % Platelets, mean (range), k/ml 47 (14-181) 69 (11-27).51 WBC, mean (range), k/ml 7 ( ) 32 (.3-274).11 WHO AML-MRC, % AML, acute myeloid leukemia;, AML with monosomal karyotype; AML-MRC, AML with myelodysplasia-related changes; WHO, World Health Organization. 192 Am J Clin Pathol 214;142: DOI: 1.139/AJCPMLO84JDNVLNK

4 AJCP / Original Article A B Table 3 Frequency of Gene Mutations in AML With and Without a Monosomal Karyotype Characteristic NPM1 FLT3-ITD FLT3-D835 CEBPA WT1, No. Other AML, No. P value AML, acute myeloid leukemia;, AML with monosomal karyotype. Discussion It has long been accepted that AML with complex karyotype predicts an unfavorable prognosis. Recently, numerous studies have shown that MK is often associated with complex karyotype and predicts a very poor prognosis in AML. The overall frequency of MK in AML varies from 6% to 1% in previous studies but has been reported up to 2% in patients with AML older than 6 years.7 In our study, accounted for 13% of all patients with AML and 22% of all patients with AML 6 years and older. This difference in the reported rate of MK might be related to various techniques used in different laboratories. For instance, Haferlach et al13 used 24-color fluorescence in situ hybridization in addition to chromosome banding. In addition, the distribution of various types of patients with AML in each study is different. Compared with other patients with AML, patients with MK in our study also had lower counts and a higher frequency of previous MDS (27% of all patients). However, even when limiting analysis to only patients in the poor-prognosis AML-MRC group, patients with had lower counts. This confirms the poor prognosis of this group but suggests that most patients with MK may have had MDS, although only three (27%) patients had a reported history of MDS. In Table 4 Factors Identified as Significant for Overall Survival by Multivariate Analysis Variable P Value Overall survival Cytogenetic risk group.1 Monosomal karyotype.8 Age 6 y.3 Hazard Ratio (95% CI) 2.49 ( ) 2.41 ( ) 2.35 ( ) addition to presenting with low counts, patients with AMLMK had blasts with a lower rate of CD34 expression. Most of the previous studies on did not compare blast immunophenotype. A recent study suggested that CD11b was strongly associated with MK, but other antigens were not significantly expressed in comparison with other AML types.14 The authors found that in addition to MK and complex karyotype, patients with AML with CD11b were also older and had an inferior OS. While we did not assess for CD11b expression in these cases of AML, our results are nonetheless in concordance with those of Chen et al14; in our AML patient group, other antigens seen in the blast immunophenotype did not correlate with survival. 193 Am J Clin Pathol 214;142: DOI: 1.139/AJCPMLO84JDNVLNK Image 1 Multilineage dysplasia in acute myeloid leukemia with monosomal karyotype. Blasts are seen among dysplastic megakaryocytes (A) and hypogranular neutrophils and dysplastic erythroid cells (B). (Wright-Giemsa, 1,.)

5 Weinberg et al / AML With Monosomal Karyotype A 1. B , AML Length of Follow-up (d) 1,5 Recently, efforts in the classification of AML have focused on identifying molecular markers with prognostic significance. Few studies have evaluated the correlation between these markers and MK. Our analysis showed that of 14 patients with, only one demonstrated a FLT3-D835 mutation. FLT3-ITD, NPM1, CEBPA, and WT1 mutations were not seen in these patients with MK, although only NPM1 and FLT3-ITD reached clinical significance, likely due to a larger number of mutated cases in the series compared with the other gene mutations. Both CEBPA and AML Disease-Free Survival (d) 1, 1,2 Figure 1 Survival differences between patients with a monosomal karyotype (MK) and the rest of the patients with acute myeloid leukemia (AML). A, Overall survival difference between patients with AML and those with. P =.1. B, Disease-free survival. P =.7. A AML-MRC Length of Follow-up (d) AML-MRC Disease-Free Survival (d) Figure 2 Survival differences between patients with a monosomal karyotype (MK) and patients with acute myeloid leukemia (AML) with myelodysplasia-related changes (MRC). A, Overall survival difference between AML - MRC and AML- MK. P =.89. B, Disease-free survival. P =.76. B NPM1 mutations have been reported to be associated with more favorable prognosis in the absence of FLT3 mutation, which might explain their absence in this group. However, the presence of an FLT3 mutation is usually associated with worse survival in AML, which suggests that MK would provide additional information and might be defining a separate group of patients with a worse prognosis. In this study, similar to previous studies, was associated with worse OS, DFS, and CR. Furthermore, even when restricting the group to just AML-MRC, patients 194 Am J Clin Pathol 214;142: DOI: 1.139/AJCPMLO84JDNVLNK

6 AJCP / Original Article with MK had significantly worse OS and DFS. However, in our group, there are too few patients without an associated complex karyotype, and therefore in our analysis, there is a strong association between MK and complex karyotype, and the two are not independent. This might be related to the small number of patients with MK in this study, but the frequency of MK in our patient group is similar to what is found in other studies. Perrot et al 8 found that MK retained independent significance only in OS and CR. In summary, MK appears to define a poor-prognosis group that is more frequent in older patients with AML. It is most commonly associated with a complex karyotype and other, previously recognized unfavorable cytogenetic abnormalities. In this study, it is not independent of complex karyotype, but further investigations are needed to evaluate this point. Address reprint requests to Dr Weinberg: Dept of Pathology, Children s Hospital Boston, 3 Longwood Ave, Boston, MA 2115; Olga.Weinberg@childrens.harvard.edu. References 1. Mrozek K, Heerema NA, Bloomfield CD. Cytogenetics in acute leukemia. Blood Rev. 24;18: Mrozek K, Bloomfield CD. Chromosome aberrations, gene mutations and expression changes and prognosis in adult acute myeloid leukemia. Hematology Am Soc Hemat Educ Program. 26;1: Slovak ML, Kopecky KJ, Cassileth PA, et al. Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/Eastern Cooperative Oncology Group Study. Blood. 2;96: Visani G, Bernasconi P, Boni M, et al. The prognostic value of cytogenetics is reinforced by the kind of induction/ consolidation therapy in influencing the outcome of acute myeloid leukemia: analysis of 848 patients. Leukemia. 21;15: Byrd JC, Mrozek K, Dodhe RK, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALBG 8461). Blood. 22;1: Breems DA, Van Putten WL, De Greef GE,et al. Monosomal karyotype in acute myeloid leukemia: a better indicator of poor prognosis than a complex karyotype. J Clin Oncol. 28;26: Medeiros BC, Othus M, Fang M, et al. Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia: the Southwest Oncology Group (SWOG) experience. Blood. 21;116: Perrot A, Luquet I, Pigneux A, et al; Groupe Ouest-Est des Leucémies Aiguës et Maladies du Sang. Dismal prognostic value of monosomal karyotype in elderly patients with acute myeloid leukemia: a GOELAMS study of 186 patients with unfavorable cytogenetic abnormalities. Blood. 211;118: Kayser S, Zucknick M, Döhner K, et al. Monosomal karyotype in adult acute myeloid leukemia: prognostic impact and outcome after different treatment strategies. Blood. 212;119: Ohgami RS, Ma L, Ren L, et al. DNA methylation analysis of ALOX12 and GSTM1 in acute myeloid leukaemia identifies prognostically significant groups. Br J Haematol. 212;159: Swerdlow SH, Campo E, Harris NL, et al, eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France; IARC; Weinberg OK, Seetharam M, Ren L, et al. Clinical characterization of acute myeloid leukemia with myelodysplasia-related changes as defined by the 28 WHO classification system. Blood. 29;113: Haferlach C, Alpermann T, Schnittger S, et al. Prognostic value of monosomal karyotype in comparison to complex aberrant karyotype in acute myeloid leukemia: a study on 824 cases with aberrant karyotype. Blood. 212;119: Chen MH, Atenafu E, Craddock KJ, et al. CD11b expression correlates with monosomal karyotype and predicts an extremely poor prognosis in cytogenetically unfavorable acute myeloid leukemia. Leuk Res. 213;37: Am J Clin Pathol 214;142: DOI: 1.139/AJCPMLO84JDNVLNK 195