|Year : 2019 | Volume
| Issue : 1 | Page : 48-53
CD34 expression and FLT3 mutation are independent poor prognostic factors in normal karyotype acute myeloid leukemia
Mohamed A Elakkad, Ayman F Abdelhalim, Hossam E Salah, Ahmed Y Amer MSc
Haematology Unit, Internal Medicine Department, Zagazig University Hospitals, Zagazig, Egypt
|Date of Submission||16-Dec-2018|
|Date of Acceptance||31-Jan-2019|
|Date of Web Publication||27-Sep-2019|
Ahmed Y Amer
24 Furness Avenue, Blackpool FY3 7QQ, UK
Source of Support: None, Conflict of Interest: None
Background The prognostic impact of CD34 expression on malignant myeloid leukemia blast cells is still under debate. FLT3 mutation is a well-known poor prognostic factor in acute myeloid leukemia (AML). The aim of the study was to assess CD34 expression in this subgroup of AML patients to detect the association of CD34 expression with FLT3-internal tandem duplication (ITD) mutation and its impact on the outcome.
Patients and methods This prospective study, carried out at Zagazig University Hospitals, included 33 de-novo AML patients with a normal karyotype. CD 34 expression was detected by flow cytometry using FITC florescent monoclonal antibody and it was considered positive if a cutoff level of 10% expression was exceeded. FLT3-ITD mutation was detected by PCR.
Results CD34 was positive in 20 cases while 13 cases were negative. Twenty-one cases were FLT3-ITD wild type while 12 cases were mutated. FLT3 mutation was significantly linked to CD34 reactivity (P=0.048). CD34-positive cases were associated with a significantly less complete remission (CR) achievement rate (33 and 80% in CD34-positive and CD34 negative, respectively, P=0.03). Those with positive CD34 had significantly lower overall survival compared with CD34-negative cases. Multivariate cox regression survival analysis showed that positive CD34 was a predictor of poor survival and higher risk of mortality (HR=1.3 and P=0.027, confidence interval, 1.1–2.9).
Conclusion CD34 expression is a poor prognostic biomarker in normal karyotype AML. It is associated with and further worsens the poor prognosis in FLT3-mutated cases. Its role in different subgroups of AML layered by different genetic aberrations needs further study.
Keywords: acute myeloid leukemia, CD34, FLT3, normal cytogenetics, normal karyotype, prognostic
|How to cite this article:|
Elakkad MA, Abdelhalim AF, Salah HE, Amer AY. CD34 expression and FLT3 mutation are independent poor prognostic factors in normal karyotype acute myeloid leukemia. Egypt J Haematol 2019;44:48-53
|How to cite this URL:|
Elakkad MA, Abdelhalim AF, Salah HE, Amer AY. CD34 expression and FLT3 mutation are independent poor prognostic factors in normal karyotype acute myeloid leukemia. Egypt J Haematol [serial online] 2019 [cited 2019 Oct 23];44:48-53. Available from: http://www.ehj.eg.net/text.asp?2019/44/1/48/268009
| Introduction|| |
Acute myeloid leukemia (AML) is a malignancy of primitive blood-forming cells of the bone marrow characterized by defective proliferation and differentiation functions of hematopoietic precursor cells with subsequent accumulation of these cells and depression of normal hematopoiesis . It accounts for about 80% of acute leukemia in adults and 20% of acute leukemia in pediatrics .
The prognosis of AML patients varies greatly, ranging from short survival of few days to complete cure. The outcome can be in part predicted by age, performance status, and cytogenetics ,,.
CD34 is a surface marker that was described for the first time by Civin et al.  as a cell-surface glycoprotein. It has as a cell-to-cell adhesion promotor function . It may play a role as a mediator of attachment of bone marrow stem cells to bone marrow extracellular matrix or directly to stromal cells ,.
The prognostic role of CD34 was extensively studied in the late 1980s and 1990s. It has been identified as an adverse prognostic marker in many studies ,,,,. On the other hand, other studies contradicted these conclusions doubting any significant prognostic value of CD34 ,,,,,,. Owing to this controversy, CD34 expression was considered not to be of value as a prognostic marker by Kanda et al.  who conducted a meta-analysis of data of 2483 patients from 22 studies.
In the current era of expanding understanding of cytogenetics and gene mutations involved in the pathogenesis of AML and its great impact on prognosis and management, CD34 has been reinvestigated to detect its significance in the light of these factors, for example FLT3, CEBPA, NPM1 mutation status, and different chromosomal aberrations ,,.
FMS-like tyrosine kinase 3 is a transmembrane tyrosine kinase receptor which stimulates cell proliferation upon activation. Mutations in the FLT3 gene, producing internal tandem duplications (FLT3-ITD) and activation of the FLT3 receptor tyrosine kinase, are common in AML, especially in patients with normal karyotypes. It has been associated with worse outcome in AML patients receiving intensive chemotherapy . FLT3-ITD mutational status is the main predictor of outcome in patients with intermediate-risk AML .
This study aimed to assess CD34 expression in AML patients with a normal karyotype together with FLT3-ITD to detect the association of CD34 expression and FLT3-ITD mutation and their impact on the outcome.
| Patients and methods|| |
This prospective study had been carried out at the Haematology Unit, Internal Medicine Department, Zagazig University Hospital and it included 33 de-novo AML patients with normal karyotypes. this study was approved by IRB, faculty of medicine zagazig university on 8/1/2017, IRB#: 3319/8-1-17. written consent was given by all participants. All adult normal karyotype AML patients of both sexes above 18 years of age with normal liver and renal functions and good PS were included. All patients had given their informed consent. All patients having severe cardiac, pulmonary, hepatic, renal, neurological, metabolic disease, or concomitant malignancies were excluded from the study. All patients were subjected to thorough history and physical examination, basic laboratory investigations including complete blood counts, liver and kidney functions, serum electrolytes, coagulation profile, hepatitis B surface antigen, hepatitis C antibody, HIV antibody, and bone marrow aspiration. Immunophenotyping of EDTA bone marrow sample was performed on Becton Dickinson BD (San Diego, CA, USA), FAC scan flow cytometer using acute leukemia panel (CD33, CD34, MPO, CD5, CD3, TDT, CD10, CD13, CD14, CD7, CD19, CD20, CD22, CD64, CD79a, HLA-DR) using a panel of fluorescein (FITC) and phycoerythrin conjugated MoAbs reactive with these antigens. Detection of CD34 expression by flow cytometry on malignant myeloid cells of bone marrow aspirate used FITC florescent monoclonal antibody and it was considered positive if a cutoff level of 10% expression was exceeded. Conventional cytogenetic study was done for all patients. PCR for FLT3 mutation was done for all patients.
All these patients were treated by induction 3 and 7 chemotherapy protocols, that is 3 days of adriamycin 25 mg/m2 and 7 days of continuous infusion of cytarabine 100 mg/m2. Bone marrow aspirate evaluation was carried out at day 14 of end of induction protocol. Response was assessed according to standardized international response criteria.
After achievement of remission, patients with FLT3 mutation were classified as of unfavorable risk and were considered for Haematopoietic stem cell transplantation (HSCT). Those with FLT3 wild type were challenged for consolidation of high-dose chemotherapy regimens such as HAM, that is high-dose Ara C 1.5 g/m2/12 h days 1–3, mitoxantrone 12 mg/m2 days 3–5 for four cycles, and then follow-up.
Patients were followed up 3 monthly after end of the treatment for a median follow-up period of 20 months. The follow up included physical examination and complete blood picture Bone marrow examination was done every 3–6 months unless a suspicious complete blood count result or clinical data warranted earlier assessment.
The collected data were computerized and statistically analyzed using SPSS program, version 24 (IBM, New York, USA). Data were tested for normal distribution using the Shapiro–Walk test. Qualitative data were represented as frequencies and relative percentages. χ2 test and Fisher’s exact were used to calculate the difference between qualitative variables as indicated. Qualitative data were compared by independent t test. All statistical comparisons were two tailed with a significance level of P value less than or equal to 0.05 indicating statistical significance. Kaplan–Meier method was used to estimate overall and disease-free survival and log-rank test compared survival curves (P value was considered significant at ≤0.05 levels).
| Results|| |
Our study included 33 de-novo AML patients. Their clinical and demographic characteristics are summarized in [Table 1].
CD34 was positive in 20 (61%) cases while 13 (39%) cases were negative. Twenty-one (66%) cases were FLT3-ITD wild type, while 12 (about 34%) cases were mutated. FLT3 mutation was significantly linked to CD34 reactivity (P=0.048) ([Table 2]). CD34-positive cases were associated with a significantly less complete remission (CR) achievement rate (33 and 80% in CD34 positive and CD34 negative, respectively, P=0.03). This was exactly the same with FLT3-ITD mutant cases (9 and 66% in FLT3 mutant and wild type, respectively, P=0.004).
|Table 2 Association between CD34 expression status with FLT-internal tandem duplication mutation|
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The impact of CD34 expression and FLT3-ITD mutation status on the overall survival and disease-free survival has been examined using the Kaplan–Meier log survival function. It showed that those with positive CD34 had significantly lower overall survival compared with CD34-negative cases (10.2 vs. 15.7 months with P=0.01). Combining CD34 expression and FLT3-ITD mutation status results showed that CD34 positivity further worsened the already worse prognosis of FLT3-ITD-mutated AML cases indicating that it is an independent risk factor in this group ([Figure 1] and [Figure 2]). The mean overall survival for each group is shown in [Table 3]. Multivariate cox regression survival analysis was used to assess if CD34 poor prognostic value on overall survival is independent of other prognostic factors in AML ([Table 4]). Positive CD34 was a predictor of poor survival and higher risk of mortality (HR=1.3 and P=0.027; confidence interval, 1.1–2.9).
|Figure 1 Correlation between overall survival and CD34 expression/FLT3-ITD mutation status. ITD, internal tandem duplications.|
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|Figure 2 Correlation between DFS survival and CD34 expression/FLT3-ITD mutation status. ITD, internal tandem duplications.|
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|Table 3 Correlation between overall survival and CD34 expression/FLT3-internal tandem duplication mutation status|
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|Table 4 Univariate and multivariate cox regression analyses of different prognostic factors for overall survival|
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| Discussion|| |
In our work, we defined CD34 positivity by a cutoff value of 10% of examined cellular population by flow cytometry. Most of previous studies used a 20% cutoff. However, Schuurhuis and colleagues have claimed that the true negative CD34 AML is only defined with a lower than 1% of CD34 expressing population. Using this cutoff for defining truly negative AML is associated with a significantly better outcome. They claim that excluding such cases from analysis leaves no prognostic value for CD34 .
Zeijlemaker and colleagues used a new definition, without using prior cutoff. This definition used the presence or absence of neoplastic CD34-positive cells, which appears to be a powerful predictor for event free survival (EFS) and overall survival (OS) in the entire group of AML patients. Therefore, this new definition not only explains conflicting results published in the past, but also indicates that this independent prognostic marker should be incorporated into AML risk stratification .
Twenty (61%) of the studied AML patients had a positive CD34 while only 13 (39%) cases were CD34 negative. According to Raspadori et al. , 47% of the AML patients were CD34 negative. Most of the studies that examined CD34 expression were near these but as this study examines CD34 in a different population (subgroup of AML cases), this can explain this difference.
In our study, we examined 33 patients of normal karyotype for FLT3-ITD mutation. Twenty-one cases of them were wild while 12 (about 34%) cases were mutated. This is very similar to what Ebrahim et al.  had found 35%. In our study 10/12 FLT3-mutated cases were CD34 positive while 10/21 cases of wild FLT3 were CD34 positive with a P value of 0.045. This is consistent with the findings of Ebrahim et al.  and Pollard et al. .
In our work, 15/33 (45%) patients achieved remission. Those with negative CD34 had significantly higher CR rate than CD34-positive cases (80 and 33%, respectively) with a P value of 0.03. These were close to the results from Geller et al.  (87 and 59%, respectively). However, according to Zeijlemaker et al.  this was 67 and 58%, respectively.
Survival analysis for our patients showed that those with positive CD34 had significantly lower overall survival compared with CD34-negative cases (10.2 vs. 15.7 months with P=0.01). Same results were identified for disease-free survival (5.9 vs. 12.3 months with P=0.002). These results are consistent with the data from many studies ,,,,. However, it contradicts results from other studies ,,,,,,. Reasons for these discrepancies include specimen analyzed (bone marrow or peripheral blood), erythrocyte-lysed whole blood versus gradient density mononuclear cell fractions, use of cryopreserved versus fresh samples, detection systems employed (flow cytometry, immunofluorescence microscope, immune-enzymatic technique), use of different CD34 antibodies recognizing distinct CD34 epitopes (classes I, II, III), degree of intensity for CD34 antigen, cutoff levels for the discrimination of positive and negative cases (5–20%, percentage of leukemic cells present in the sample examined), patients analyzed [de-novo or secondary AML; childhood or adult acute lymphoblastic leukaemia (ALL)], biologic characteristics of acute leukemic cells (chromosome or gene abnormalities), and lastly the type of chemotherapy regimen used .
To examine the effect of CD34 expression on the outcome in FLT3-mutated versus wild cases, we compared the overall and disease-free survival of four groups based on both CD34 expression and FLT3 mutation status. Those with wild-type FLT3 and negative CD34 (11 cases) had the best outcome with 16.9 months overall survival and 11.8 months of disease-free survival (P=0.0005), while those with CD34-positive expression and mutated FLT3 (10 cases) the mean overall survival was 5.4 months and the disease-free survival was only 1.7 months. These results typically match the results of Ebrahim et al.  as well as the results from Schuurhuis et al. .
Finally, multivariate cox regression model examined the impact of CD34 on overall survival with other prognostic factors to test its independent prognostic effect. It showed quite clearly that CD34 positivity is associated with higher mortality rates and poorer outcome independent of all other risk factors (HR=1.3 and P=0.027; confidence interval, 1.1–2.9). This is consistent with what Ebrahim et al.  has proved in their study.
| Conclusion|| |
Positive CD34 is a marker for less CR achievement. CD34 is associated with FLT3-ITD mutation and is an independent factor for worse outcome in this patient group.
More studies are strongly needed to investigate the function of CD34 in AML. As the role of gene mutations and chromosomal aberrations in subclassifying AML is expanding, extensive studies of these AML subtypes based on their cytogenetics and role of CD34 and other immunophenotypes in identifying these subgroups and their prognostication is highly recommended.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin
Appelbaum FR, Gundacker H, Head DR, Slovak ML, Willman CL, Godwin JE et al.
Age and acute myeloid leukemia. Blood
Juliusson G, Antunovic P, Derolf A, Lehmann S, Mollgard L, Stockelberg D et al.
Age and acute myeloid leukemia: real world data on decision to treat and outcomes from the Swedish Acute Leukemia Registry. Blood
Schlenk RF, Benner A, Krauter J, Büchner T, Sauerland C, Ehninger G et al.
Individual patient data-based meta-analysis of patients aged 16 to 60 years with core binding factor acute myeloid leukemia: a survey of the German Acute Myeloid Leukemia Intergroup. J Clin Oncol
Byrd JC, Mrózek K, Dodge RK, Carroll AJ, Edwards CG, Arthur DC 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 (CALGB 8461): presented in PA. Blood
Civin CI, Strauss LC, Brovall C, Fackler MJ, Schwartz JF, Shaper JH. Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J Immunol
Sidney LE, Branch MJ, Dunphy SE, Dua HS, Hopkinson A. Concise review: evidence for CD34 as a common marker for diverse progenitors. Stem Cells
Nielsen JS, McNagny KM. CD34 is a key regulator of hematopoietic stem cell trafficking to bone marrow and mast cell progenitor trafficking in the periphery. Microcirculation
Healy L, May G, Gale K, Grosveld F, Greaves M, Enver T. The stem cell antigen CD34 functions as a regulator of hemopoietic cell adhesion. Proc Natl Acad Sci U S A
Raspadori D, Lauria F, Ventura MA, Rondelli D, Visani G, de Vivo A et al.
Incidence and prognostic relevance of CD34 expression in acute myeloblastic leukemia: analysis of 141 cases. Leuk Res
Myint H, Lucie NP. The prognostic significance of the CD34 antigen in acute myeloid leukaemia. Leuk Lymphoma
Lee EJ, Yang J, Leavitt RD, Testa JR, Civin CI, Forrest A et al.
The significance of CD34 and TdT determinations in patients with untreated de novo acute myeloid leukemia. Leukemia
Campos L, Guyotat D, Archimbaud E, Devaux Y, Treille D, Larese A et al.
Surface marker expression in adult acute myeloid leukaemia: correlations with initial characteristics, morphology and response to therapy. Br J Haematol
Borowitz MJ, Gockerman JP, Moore JO, Civin CI, Page SO, Robertson J et al.
Clinicopathologic and cytogentic features of CD34 (My 10)-positive acute nonlymphocytic leukemia. Am J Clin Pathol
Selleri C, Notaro R, Catalano L, Fontana R, Del Vecchio L, Rotoli B. Prognostic irrelevance of CD34 in acute myeloid leukaemia. Br J Haematol
Ciolli S, Leoni F, Caporale R, Pascarella A, Salti F, Rossi-Ferrini P. CD34 expression fails to predict the outcome in adult acute myeloid leukemia. Haematologica
Bradstock K, Matthews J, Benson E, Page F, Bishop J. Prognostic value of immunophenotyping in acute myeloid leukemia. Australian Leukaemia Study Group. Blood
Lamy T, Goasguen JE, Mordelet E, Grulois I, Dauriac C, Drenou B et al.
P-glycoprotein (P-170) and CD34 expression in adult acute myeloid leukemia (AML). Leukemia
Arslan O, Akan H, Beksac M, Ozcan M, Koc H, Ilhan O et al.
Lack of prognostic value of CD34 in adult AML. Leuk Lymphoma
Fruchart C, Lenormand B, Bastard C, Boulet D, Lesesve JF, Callat MP et al.
Correlation between CD34 expression and chromosomal abnormalities but not clinical outcome in acute myeloid leukemia. Am J Hematol
Kyoda K, Nakamura S, Hattori N, Takeshima M, Nakamura K, Kaya H et al.
Lack of prognostic significance of CD34 expression in adult AML when FAB M0 and M3 are excluded . Am J Hematol
Kanda Y, Hamaki T, Yamamoto R, Chizuka A, Suguro M, Matsuyama T et al.
The clinical significance of CD34 expression in response to therapy of patients with acute myeloid leukemia: an overview of 2483 patients from 22 studies. Cancer
Juncà J, Garcia O, Garcia-caro M, Vila J, Zamora L, Rodríguez-hernández I et al.
CD34 expression and the outcome of nucleophosmin 1-mutated acute myeloid leukemia. Ann Hematol
Dang H, Chen Y, Kamel-Reid S, Brandwein J, Chang H. CD34 expression predicts an adverse outcome in patients with NPM1-positive acute myeloid leukemia. Hum Pathol
Ebrahim EK, Assem MM, Amin AI, Kamel MM, El Meligui YM, Metwally AM. FLT3 internal tandem duplication mutation, cMPL and CD34 expressions predict low survival in acute myeloid leukemia patients. Ann Clin Lab Sci
Schmid C, Labopin M, Socié G, Daguindau E, Volin L, Huynh A et al.
Outcome of patients with distinct molecular genotypes and cytogenetically normal AML after allogeneic transplantation. Blood
Patel JP, Gönen M, Figueroa ME, Fernandez H, Sun Z, Racevskis J et al.
Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med
Schuurhuis GJ, Kelder A, Terwijn M, Rutten AP, Smit L, Zweegman S et al.
The prognostic value of CD34 expression in acute myeloid leukemia. A mystery solved. Blood
Zeijlemaker W, Kelder A, Wouters R, Valk PJM, Witte BI, Cloos J et al.
Absence of leukaemic CD34+cells in acute myeloid leukaemia is of high prognostic value: a longstanding controversy deciphered. Br J Haematol
Pollard JA, Alonzo TA, Gerbing RB, Woods WG, Lange BJ, Sweetser DA et al.
FLT3 internal tandem duplication in CD34+/CD33- precursors predicts poor outcome in acute myeloid leukemia. Blood
Geller RB, Zahurak M, Hurwitz CA, Burke PJ, Karp JE, Piantadosi S et al.
Prognostic importance of immunophenotyping in adults with acute myelocytic leukaemia: the significance of the stem-cell glycoprotein CD34 (My10). Br J Haematol
Basso G, Lanza F, Orfao A, Moretti S, Castoldi G. Clinical and biological significance of CD34 expression in acute leukemia. J Biol Regul Homeost Agents
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]