|Ahead of print publication
Clinical and hematological correlates of aberrant immunophenotypic profiles in adult and pediatric acute myeloid leukemia at presentation
Manupriya Sharma1, Neelam Varma2, Man Updesh Singh Sachdeva2, Parveen Bose2, Subhash Varma3
1 Department of Pathology, Dr. R. P. Government Medical College, Kangra, Himachal Pradesh, India
2 Departments of Hematology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
3 Departments of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India
Professor and Head, Department of Hematology, Post Graduate Institute of Medical Education and Research, Chandigarh - 160 012
Source of Support: None, Conflict of Interest: None
Background: Aberrant phenotypes in acute leukemia have been reported with varying frequencies in independent studies and their association with prognostic factors is still a matter of debate.
Aim: This study aims to identify the frequency of aberrant immunophenotypes in de novo acute myeloid leukemia (AML) and to evaluate their association with initial clinical and hematological features.
Materials and Methods: A total of 181 patients of de novo AML were included during the time (July 2010–June 2012). The immunophenotype of all cases of AML was studied by using flow cytometry.
Results: Aberrant lymphoid antigen expression was seen in 43.1% cases. Most frequent aberrant lymphoid antigen was CD7, seen in 26.5% cases. All French-American-British (FAB) subtypes except AML-M3 expressed aberrant lymphoid antigens. The expression was most common in AML-M4 in the current study. CD34 expression in AMLs was significantly associated with the expression of aberrant lymphoid antigens. Lymphoid antigen expression in adult AML was significantly associated with higher white blood cell (WBC) count (>50,000/mm3) and higher number of peripheral blasts (>70%).
Conclusion: In summary, CD7 is the most common aberrant lymphoid antigen expressed in AML. FAB subtype AML-M3 is usually not associated with aberrant lymphoid antigen expression. AML cases with CD34 positivity are more likely to express aberrant lymphoid markers. The current study also supports that aberrant lymphoid antigen expression in adult AML is associated with adverse presenting hematological features (WBC count >50,000/mm3, peripheral blasts >70%). Pediatric Ly + AML cases are not associated with adverse presenting clinical and biological features.
Keywords: Aberrant, acute leukemia, acute myeloid leukemia, immunophenotyping
|How to cite this URL:|
Sharma M, Varma N, Sachdeva MU, Bose P, Varma S. Clinical and hematological correlates of aberrant immunophenotypic profiles in adult and pediatric acute myeloid leukemia at presentation. J Can Res Ther [Epub ahead of print] [cited 2019 Nov 21]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=269749
| > Introduction|| |
Aberrant phenotypes in acute leukemia are characterized by variation in the patterns of antigen expression on neoplastic cells as compared to the process of normal hematopoietic maturation. In acute myeloid leukemia (AML), these aberrancies include cross-lineage expression (expression of lymphoid antigens in AML) or/and asynchronous antigen expression where early antigens are co-expressed with mature ones., Immunophenotyping is an indispensable tool for proper lineage assignment and identification of any aberrant phenotypes. It has been reported that aberrant phenotypes in AML occur with varying frequency (30%–88%) and there is still a controversy about its prognostic implication.,,,, This wide variation in incidence may be because of differences in flow cytometry (FCM) instruments, reagents, criteria for aberrancy, and variation in the binding patterns of monoclonal antibody clones. Although immunophenotyping is a valuable tool in acute leukemia diagnosis; many hospitals in India do not have the availability of flow cytometer. Consequently, those cases which do not reach referral tertiary care centers miss a proper lineage characterization and identification of any aberrant phenotypes. Identification of these aberrant phenotypes may be instrumental in making diagnosis, disease monitoring, and making specific treatment decisions. This particular study was planned for a span of 2 years to identify various immunophenotypic aberrancies in a large series of AML cases which were uniformly characterized using FCM. Initial clinical and hematological features were studied in relation to the expression of aberrant markers.
| > Materials and Method|| |
A prospective study was conducted in the department of hematology at a tertiary hospital for 2 years. All newly diagnosed cases of AML during this period were included in the study. All the patients underwent bone marrow examination for light microscopic evaluation and immunophenotyping on FCM. Written informed consents were obtained from all the patients. The study was approved by the Ethical Committee of the Institution.
Bone marrow examination
Bone marrow examination was done from the posterior superior iliac spine using Jamshidi bone marrow aspiration needle (No. 16 for adults and No. 18 for pediatric patients). Approximately 1 ml of bone marrow aspirate was taken separately for making smears and in ethylenediaminetetraacetic acid (EDTA) vials for FCM. Bone marrow biopsy was taken in all cases with Jamshidi trephine biopsy needle (No. 11 for adults and No. 13 for pediatric cases). It was fixed in 10% buffered formalin. Smears were examined using May–Grunwald Giemsa and special stains using myeloperoxidase (MPO) and Periodic acid–Schiff. All cases were evaluated morphologically based on the FAB criteria.
Approximately 1 ml of bone marrow aspirate was collected in EDTA vials. Samples were analyzed within 24 h of collection. Immunophenotypic analysis was performed in all cases, and it was based on a pretitrated four-color combination cocktail comprising of fluorescein isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC) conjugated monoclonal antibodies (Moab), i.e., CD19-FITC, CD10-PE, CD20-APC, CD22-PE, CD1a-PE, CD2-FITC, CD3-PE, CD4-PE, CD8-FITC, CD5-PE, CD7-FITC, CD13-PE, CD33-APC, CD117-APC, CD14-FITC, CD11b-PE, CD34-PE, human leukocyte antigen-DR-FITC on the surface of leukemic cells and intracytoplasmic Igμ chain-PE/FITC, cytoplasmic CD3-FITC/APC, CD79a-PE, MPO-FITC antigens and nuclear TdT-APC, along with CD45PerCP for gating of blasts. All antibodies were procured from BD Biosciences, California, USA. Briefly, 50 μL of blood sample was taken in each test tube and processed with 2 ml of red blood cell lysing solution. The sample was washed twice with phosphate buffered saline (PBS) and re-suspended in PBS and counts were adjusted to approximately 1 × 106 cells per tube. Pretitrated cocktails of Moab were added and incubated in dark for 20–30 min at room temperature. The cells were then again washed with PBS to remove any unbound Moab, and the cell button was resuspended in 500 μL of PBS. The nuclear and cytoplasmic antigens were processed using permeabilization with “BD FACS Permeabilizing solution 2” for 10 min. Flow cytometric analysis was performed on flow cytometer (BD FACS caliber/BD FACS Canto II) and analyzed with cell quest/FACS Diva, respectively. Results were obtained by gating the blast cells with side scatter analysis versus CD45PerCP gating. FCM data were analyzed based on dot plots. Negative controls were simultaneously run in every case. For surface and intracytoplasmic antigens, marker positivity was considered when more than 20% of blast cells were positive with the exception of anti-MPO, which was reported as positive at a cutoff of 10%. Besides isotype control for assessing nonspecific binding in MPO staining, lymphocyte population in each sample was assessed as the internal negative control for MPO.
| > Results|| |
A total of 181 newly diagnosed cases of AML were included in the study. The patient age range was wide (range, 1–81 years; median 30 years) with male/female ratio (M: F) of 1.42:1. Among all AML cases, 135 cases were adults (range, 17–81 years; median 40 years) with M: F ratio of 1.1:1. Forty-six cases were in the pediatric age group with M: F ratio of 1.23:1.
Expression of aberrant phenotype
Overall, 78 (43.1%) AML cases showed aberrant lymphoid antigens. Aberrancy for T-lineage associated markers (CD7/CD4/CD8) was most common, comprising 73% (57/78) of total aberrancies. Aberrancy for B-lineage associated markers (CD19/CD10) constituted 17.9% (14/78), followed by both B and T-cell aberrancy of 8.9% (7/78). Among all antigens, the most common aberrant lymphoid antigen was CD7 (26.5% of cases), followed by CD19 (11% of cases). The expression of CD7 was more common in childhood AML (28.2%) as compared to adult AML cases (25.9%) though the difference was not statistically significant. CD19 expression was more common in adult AML cases (12.6%) than childhood AML cases (6.5%). The detailed expression of aberrant B and T-lymphoid antigens is described in [Table 1].
|Table 1: Lymphoid antigen expression in 181 (46 pediatric and 135 adult) cases of acute myeloid leukemia|
Click here to view
Correlation of aberrant phenotype with French–American–British subtypes
All FAB subtypes except AML-M3 showed expression of aberrant phenotypes. Overall, the most common FAB subtype with aberrant lymphoid phenotype was AML-M4, present in 76% (16/21) cases. The aberrant phenotypic expression was next seen in AML-M0 and AML-M1 subtypes, constituting 62.5% each (10/16 and 5/8 cases, respectively). The association between aberrant lymphoid antigen expression and FAB subtype is shown in [Table 2] and [Table 3]. CD7 expression was seen in all FAB subtypes except AML-M3 and AML-M6. CD7 expression was prominently seen in AML-M0 and AML-M7 (70%, 7/10 cases). CD19 expression was most frequently seen in AML-M5 (50%, 8/16 cases) followed by AML-M2 (25%, 5/20 cases).
|Table 2: Frequency of lymphoid marker expression in different French-American-British subtypes|
Click here to view
|Table 3: Distribution of various lymphoid antigens among different FAB subtypes|
Click here to view
Relation of aberrant phenotype with adverse prognostic factors
Based on the positivity of aberrant lymphoid antigens (Ly+), AML cases were stratified into two groups: Ly + AML and Ly − AML. The expression of aberrant lymphoid antigens in both adult and pediatric AML cases was compared with known prognostic factors. The clinical and biological characteristics of Ly + AML and Ly − AML are summarized in [Table 4]. No difference was found between Ly + AML and Ly − AML group with regard to age, gender, median hemoglobin, median white blood cell (WBC) count, median platelet count, and clinical features at presentation such as lymphadenopathy, hepatomegaly, and splenomegaly. On analyzing the adult and pediatric groups separately, CD34 expression was significantly more in adult Ly + AML group than adult Ly − AML group (P = 0.001). Higher WBC count (>50,000/mm3) was seen more in adult Ly + AML group than adult Ly − AML group (P = 0.050). Higher peripheral blasts% (>70%) was seen in adult Ly + AML than adult Ly − AML (P = 0.039). No difference was seen between pediatric Ly + AML cases and Ly − AML cases with respect to CD34 expression, WBC count (>50,000/mm3), and peripheral blasts (>70%).
|Table 4: Lymphoid antigen expression in adult and pediatric acute myeloid leukemia cases in relation to clinical and biological features|
Click here to view
| > Discussion|| |
In the present study of 181 newly diagnosed cases of AML, aberrant expression of lymphoid antigens was seen in 43.1% of AML cases. The degree of lymphoid antigen expression in AML has varied between 30%–88% in various reports.,,,, This wide variation in the incidence of aberrant phenotypes can be attributed to factors like differences in the criteria used for aberrancy, the cutoff value for confirming the positivity of a particular antigen, number of antigens studied, sample size and differences in the instruments, reagents, and monoclonal antibody clones used for FCM. In the current study, a cutoff value of ≥20% of blast cells positivity was used to characterize the presence of a particular antigen, and a uniform FCM method was used to characterize all acute leukemia cases. The highest frequency of 88% of aberrant phenotypes in AML is reported by Bahia et al. This particular study also included the asynchronous expression of antigens as the criteria for aberrancy. However, excluding the asynchronous expression of antigens, aberrant lymphoid antigens were seen in 34% of AML cases.
CD7 was the most frequently expressed lymphoid antigen, seen in 26.5% (48/181) cases in our study. The aberrant expression of CD7 was more frequently seen in pediatric AMLs (28.2%) in comparison to adult-AMLs (25.9%). Second-most common aberrantly expressed lymphoid antigen was CD19 in 11% cases, followed by CD4 in 8.8% cases, CD10 in 2.8% cases, and CD8 in 0.6% cases. Majority of studies have identified CD7 as the most frequent lymphoid antigen, as seen in 20.5% cases by Zheng et al., 25.7% cases by Bahia et al., 37% by Legrand et al.,, There are few studies where other lymphoid markers were found to be expressed more frequently than CD7. Bhushan et al. found CD19 as the most common lymphoid antigen (32%). CD7 was seen in 15% of cases in this study. Reading et al. found CD4 as the most common lymphoid antigen (61%), followed by CD7 (24%). Khalidi et al. found CD20 as the most common lymphoid antigen (17%). It is believed that CD7 is expressed early in hematopoietic ontogeny and is usually co-expressed with early antigens. Supporting this view, we found CD34 expression in 75% of CD7-positive AML cases. Few studies have reported an inferior outcome in AML cases showing CD7 expression.,,, Co-expression (CD34 + CD7 + AML) is reported to be associated with multiple drug resistant proteins and a worse prognosis.
Aberrant lymphoid phenotypes in AML were seen in all FAB subtypes except AML-M3. The expression was most common in AML-M4 in 76% of cases, followed by AML-M0 and AML-M1 in 62.5% cases each and AML-M2 in 41.6% cases. Similar to our results, the aberrant lymphoid marker was not a common finding in AML-M3 in many other studies.,,,, However, there is still no consensus on the most common FAB subtype associated with lymphoid antigen expression. Our study finds AML-M4 to be the most common FAB subtype. Bhushan et al. reported AML-M5 to be the most common FAB subtype associated with lymphoid antigen expression and AML-M2 was the most common FAB subtype with Bahia et al., In the present study, CD7 was seen in all FAB subtypes except AML-M3 and AML-M6. Similarly, Bahia et al. found CD7 in all FAB subtypes except AML-M3 and AML-M6. Thalhammer-Scherrer et al. did not find CD7 positive cases with AML-M3, AML-M6, and AML-M7 morphology. Bahia et al. and Zheng et al. found thatCD19 expression was highest in AML-M2., We could not find an association between CD19 expression and AML-M2. In our study, CD19 expression was most frequently noted in AML-M5.
A number of clinical and biological features are known to predict the prognosis in AML. Association between aberrant lymphoid antigens and prognostic factors is still debatable. Some studies have reported AML with aberrant phenotypes to be associated with poor prognosis,,, while other studies reported favorable prognosis and few other studies report no prognostic value. In our study, we compared Ly + AML and Ly − AML patients with adverse prognostic factors. No significant association was seen between the aberrant phenotypes and adverse prognostic factors in children. In adults, the expression of CD34 was significantly more in Ly + AML patients than Ly − AML cases (P = 0.001). However, contrary to our observations, Kawai S et al. found CD34 to be more associated with pediatric Ly + AML cases. Bhushan et al. did not find any association between CD34 expression and aberrant phenotypes. The expression of CD34 on leukemic blasts might be attributed to a developmental arrest at an immature stage or an aberrant continuous spectrum of expression. CD34 expression in AML is regarded as a poor prognostic factor. Leukocytosis (>50,000/mm3) is regarded as a negative prognostic factor in AML. In our study, Ly + AML adult patients were more associated with leukocytosis (WBC count >50,000/mm3; P = 0.050) than Ly − AML patients. Peripheral blasts (>70%) were significantly more in Ly + AML patients (P = 0.039). The current study definitely highlights an association between aberrant lymphoid antigens expression in adult AML cases and adverse presenting hematological factors. Nevertheless, this study had its limitations, the biggest being that it could not take into account the follow-up data of the patients to document correlation between immune-phenotype of leukemic cells and treatment response since that might have more categorically established overall prognostic significance of expression of lymphoid markers in AML. In fact, the aim of the current study was mainly to generate data from Indian population regarding the expression of aberrant markers in AML cases.
| > Conclusion|| |
In summary, we conclude that in our series, 43.1% of AML cases showed aberrant lymphoid antigens. CD7 was the most common aberrant lymphoid antigen expressed in AML. FAB subtype AML-M3 is not associated with the expression of aberrant lymphoid antigens. Childhood Ly + AML cases are not associated with adverse presenting clinical and hematological factors. Adult AML cases with higher WBC count (>50,000/mm3), higher blast count% (>70%), and expression of CD34 antigen are more likely to express aberrant lymphoid antigens. The identification of these aberrant phenotypes may help in making a proper diagnosis, monitoring minimal residual disease, and making alternate treatment decisions. Longer period studies evaluating the treatment responses in these patients are needed to definitely comment on the prognostic significance of expression of aberrant markers in AML cases.
| > References|| |
Ball ED, Davis RB, Griffin JD, Mayer RJ, Davey FR, Arthur DC, et al.
Prognostic value of lymphocyte surface markers in acute myeloid leukemia. Blood 1991;77:2242-50.
Reading CL, Estey EH, Huh YO, Claxton DF, Sanchez G, Terstappen LW, et al.
Expression of unusual immunophenotype combinations in acute myelogenous leukemia. Blood 1993;81:3083-90.
Bhushan B, Chauhan PS, Saluja S, Verma S, Mishra AK, Siddiqui S, et al.
Aberrant phenotypes in childhood and adult acute leukemia and its association with adverse prognostic factors and clinical outcome. Clin Exp Med 2010;10:33-40.
Macedo A, Orfão A, Vidriales MB, López-Berges MC, Valverde B, González M, et al.
Characterization of aberrant phenotypes in acute myeloblastic leukemia. Ann Hematol 1995;70:189-94.
Khalidi HS, Medeiros LJ, Chang KL, Brynes RK, Slovak ML, Arber DA, et al.
The immunophenotype of adult acute myeloid leukemia: High frequency of lymphoid antigen expression and comparison of immunophenotype, French-American-British classification, and karyotypic abnormalities. Am J Clin Pathol 1998;109:211-20.
Bahia DM, Yamamoto M, Chauffaille Mde L, Kimura EY, Bordin JO, Filgueiras MA, et al.
Aberrant phenotypes in acute myeloid leukemia: A high frequency and its clinical significance. Haematologica 2001;86:801-6.
El-Sissy AH, El-Mashari MA, Bassuni WY, El-Swaayed AF. Aberrant lymphoid antigen expression in acute myeloid leukemia in Saudi Arabia. J Egypt Natl Canc Inst 2006;18:244-9.
Ossenkoppele GJ, van de Loosdrecht AA, Schuurhuis GJ. Review of the relevance of aberrant antigen expression by flow cytometry in myeloid neoplasms. Br J Haematol 2011;153:421-36.
Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al.
The morphological classification of acute lymphoblastic leukaemia: Concordance among observers and clinical correlations. Br J Haematol 1981;47:553-61.
Zheng J, Wang X, Hu Y, Yang J, Liu J, He Y, et al.
Acorrelation study of immunophenotypic, cytogenetic, and clinical features of 180 AML patients in China. Cytometry B Clin Cytom 2008;74:25-9.
Legrand O, Perrot JY, Baudard M, Cordier A, Lautier R, Simonin G, et al.
The immunophenotype of 177 adults with acute myeloid leukemia: Proposal of a prognostic score. Blood 2000;96:870-7.
Chang H, Yeung J, Brandwein J, Yi QL. CD7 expression predicts poor disease free survival and post-remission survival in patients with acute myeloid leukemia and normal karyotype. Leuk Res 2007;31:157-62.
Ferrara F, Finizio O, Rosa CD, Mele G, Mettivier V, Rametta V, et al.
Acute myeloid leukemia expressing T-cell antigens: Clinico-hematological report on six cases. Leuk Lymphoma 1990;3:217-22.
Bradstock K, Matthews J, Benson E, Page F, Bishop J. Prognostic value of immunophenotyping in acute myeloid leukemia. Australian leukaemia study group. Blood 1994;84:1220-5.
Kita K, Miwa H, Nakase K, Kawakami K, Kobayashi T, Shirakawa S, et al.
Clinical importance of CD7 expression in acute myelocytic leukemia. The Japan cooperative group of leukemia/Lymphoma. Blood 1993;81:2399-405.
Chen SW, Li CF, Chuang SS, Tzeng CC, Hsieh YC, Lee PS, et al.
Aberrant co-expression of CD19 and CD56 as surrogate markers of acute myeloid leukemias with t(8;21) in Taiwan. Int J Lab Hematol 2008;30:133-8.
Jha R, Grover G, Bose P. Lymphoid associated antigen expression in new cases of acute myeloid leukemia. J Pathol Nepal 2013;3:487-90.
Thalhammer-Scherrer R, Mitterbauer G, Simonitsch I, Jaeger U, Lechner K, Schneider B, et al.
The immunophenotype of 325 adult acute leukemias: Relationship to morphologic and molecular classification and proposal for a minimal screening program highly predictive for lineage discrimination. Am J Clin Pathol 2002;117:380-9.
Smith LJ, Curtis JE, Messner HA, Senn JS, Furthmayr H, McCulloch EA, et al.
Lineage infidelity in acute leukemia. Blood 1983;61:1138-45.
Cross AH, Goorha RM, Nuss R, Behm FG, Murphy SB, Kalwinsky DK, et al.
Acute myeloid leukemia with T-lymphoid features: A distinct biologic and clinical entity. Blood 1988;72:579-87.
Drexler HG, Thiel E, Ludwig WD. Acute myeloid leukemias expressing lymphoid-associated antigens: Diagnostic incidence and prognostic significance. Leukemia 1993;7:489-98.
Kawai S, Zha Z, Yamamoto Y, Shimizu H, Fujimoto T. Clinical significance of childhood acute myeloid leukemias expressing lymphoid-associated antigens. Pediatr Hematol Oncol 1995;12:463-9.
Taussig DC, Vargaftig J, Miraki-Moud F, Griessinger E, Sharrock K, Luke T, et al.
Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34(-) fraction. Blood 2010;115:1976-84.
[Table 1], [Table 2], [Table 3], [Table 4]