The Egyptian Journal of Haematology

ORIGINAL ARTICLE
Year
: 2016  |  Volume : 41  |  Issue : 4  |  Page : 155--160

Novel immunophenotyping marker in acute myeloid leukemia: does it implicate prognosis?


Amira Y Abd El-Naby1, Amr Gawaly2, Shereen Awni3,  
1 Lecturer in Clinical Pathology Department, Tanta University, Tanta, Egypt
2 Assistant Professor in Internal Medicine Department, Tanta University, Tanta, Egypt
3 Lecturer in Internal Medicine Department, Tanta University, Tanta, Egypt

Correspondence Address:
Amira Y Abd El-Naby
Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta
Egypt

Abstract

Introduction AML is a heterogeneous group of neoplasms that affect hematopoietic cells responsible for the production of myeloid lineages in bone marrow (BM). Novel immunophenotyping marker in AML (CD30) is a 120 kDa cell membrane glycoprotein that shares sequence homology with tumor necrosis factor (TNF) receptors; it is expressed by myeloblasts (CD34+ and/or CD117+) in a substantial number of cases of non Monocytic AML, either de novo or arising from MDS. CD30 expression is more common in AML patients with unfavorable chromosomal abnormality. It is suggested that CD30 could be a target for therapy by using anti-CD30 antibodies in a subset of patients with non Monocytic AML; also it might provide useful information for patient prognosis and stage of disease. Aim of work Is to assess novel immunophenotyping marker in AML (CD30) and cytogenetic abnormality and its correlation with prognosis. Materials and methods This prospective cohort study included 120 patients with newly diagnosed acute myeloid leukemia. Their ages ranged from (2 to 75) years with a mean of (40.15±23.55) years. Chromosomal analysis by karyotyping was done in all AML patients and Multicolor flow-cytometry immunophenotypic analysis was performed on bone marrow aspirates of the AML patients using fluroisothiocyanate (FITC) conjugated antibodies to assess CD30 expression on BM myeloblasts. Results The previous study showed that CD30 was expressed in all AML cases rather than M4 and M5 cases that showed negative expression. In addition, this study showed that there is more CD30 expression in myeloblasts with unfavorable chromosomal abnormalities. A significant association between platelets counts and CD30 expression was also observed. There was a higher degree of thrombocytopenia and a greater tendency to have higher leucocytic counts in patients having +ve CD30 expression than those with –ve CD30 expression. Conclusion The analysis of CD30 expression has a potential role to be used as a prognostic marker in AML.



How to cite this article:
Abd El-Naby AY, Gawaly A, Awni S. Novel immunophenotyping marker in acute myeloid leukemia: does it implicate prognosis?.Egypt J Haematol 2016;41:155-160


How to cite this URL:
Abd El-Naby AY, Gawaly A, Awni S. Novel immunophenotyping marker in acute myeloid leukemia: does it implicate prognosis?. Egypt J Haematol [serial online] 2016 [cited 2019 Dec 13 ];41:155-160
Available from: http://www.ehj.eg.net/text.asp?2016/41/4/155/198646


Full Text

 Introduction



Leukemia begins when white blood cells exposed to DNA loss or damage. Those errors are copied and passed on to subsequent generations of cells. The abnormal leukemic blast cells remain immature that never matures properly. They do not die off, but tend to multiply and accumulate within the body. They do not die off like normal cells, but tend to multiply and accumulate within the body [1], certain risk factors (e.g. age, genetics) are un-modifiable while there is controllable other factors (e.g. environmental, lifestyle-related variables) [2]. AML is heterogeneous disorder with clinical, pathological, chromosomal and molecular subtypes. Investigating novel cellular and molecular markers in such as CD30 that may affect prognosis and used as target therapy [3]. CD30 is a 120 kDa cell membrane glycoprotein that has sequence homology with tumor necrosis factor (TNF) receptors [4] which was known also as TNF receptor superfamily member 8 (TNFRSF8) following its molecular cloning in 1992 [5]. Its gene locus is on chromosome 1p36 [6]. The receptor is expressed on activated B- and T-cells, and intercedes in signal transductions involving nuclear factor-β (NF-K-β) transcription. CD30 is expressed on Reed-Sternberg cells of Hodgkin lymphoma, on a subset of non-Hodgkin lymphomas, including anaplastic large cell lymphoma (ALCL) [7], on embryonic carcinoma [8], a subset of diffuse large B-cell lymphomas and peripheral T-cell lymphomas, in aggressive forms of systemic mastocytosis [9] and in myeloid neoplasms [10]. CD30 expression is restricted to activated B- and T- lymphocytes [11] while some studies reported that cell surface CD30 expression in resting human peripheral blood CD8+ T cells producing Th2-type cytokines [12]. CD30 expression was detected in patients with acute myeloid leukemia (AML) or high-grade myelodysplastic [10]. The human CD30 comprises 595 amino acid residues. The extracellular and cytoplasmic domains of human CD30 consist of 362 and 188 residues, respectively. The extracellular part of the membrane-bound CD30 can be proteolytically cleaved by the action of a zinc-metalloprotease. This produces a soluble form of CD30 (sCD30) with a molecular mass of 85–90 kDa [13]. Shedding of CD30 is an active process of viable CD30 positive cells. The effect of CD30 signaling on tumor cells varies from enhanced proliferation and survival to induction of inhibiting growth and apoptosis [14],[15], 40% of AML patients with CD30 expression was shown to simultaneously express CD30v which is a truncated CD30 lacks trans-membrane and extracellular domains, presents mainly in the cytoplasm of alveolar macrophages. High CD30v expression was associated with increased blast cell, leucocytosis, and thrombocytopenia compared to those cases of AML with no expression of CD30 [16],[17]. It was shown that AML patients with a poor cytogenetic risk score and refractory to therapy exhibited greater CD30 expression compared with patients with de novo AML and with no treatment [10]. Brentuximab vedotin (Adcetris) is a CD30-directed antibody – drug conjugate comprising a chimeric IgG1 anti-CD30 antibody, an anti-microtubule molecule monomethylauristatin (MMAE), and a protease cleavable linker that covalently attaches MMAE to the IgG antibody. Upon binding to CD30-expressing cells, the Brentuximab – CD30 complex internalizes, and releases MMAE after proteolytic cleavage. Interruption of the microtubule assembly by MMAE culminates in cell cycle arrest and apoptosis [18],[19]. Brentuximabvedotin, also named as SGN-35 or ADCETRIS®, is an anti-CD30 chimeric antibody conjugated by a protease-cleavable linker to monomethylauristatin E, that disrupts microtubules Brentuximabvedotin induce durable responses in patients with refractory and relapsed Hodgkin lymphoma and anaplastic large cell lymphoma. Also the high risk AML patients with hopeless treatment has the opportunity that anti CD30 targeted therapy could be a solution. The drug recently approved by the United States Food and Drug Administration for patients with relapsed Hodgkin lymphoma and anaplastic large cell lymphoma [20].

 Material and methods



The present study include 120 newly diagnosed AML patients admitted to Hematology , Oncology , Pediatric and Internal Medicine Departments at Tanta University hospital from November 2012 to November 2014. There were 68 males (56.7%) and 52 females (43.3%). The ages of AML patients ranged from (20 to 75) years with a mean of (40.15±23.55) years. All studied cases were clinically examination stressed on pallor, purpra, hepatomegaly, splenomegaly and lymphadenopathy, any manifestations of infection. Laboratory investigations included Complete blood count, Bone marrow aspiration, Cytogenetic analysis, immunophenotyping and CD30 was measured by flow-cytometry.

Flow cytometric analysis for CD30

The acute leukemia panel as well as CD 30 was done using Beckton Dickinson FAC Scan flow cytometer (BD FACS). The leukemia panels included myeloid cell markers (CD13, 33, 117), lymphoid cell markers which were T cell markers (CD 2,7) and B cell markers (CD 10, 19), also Immature markers (MPO and CD34).

Automated cell quest software used for data acquisition and analysis, the instrument setted using calibrated beads provided by the manufacture. Isotopic quality control was used to avoid unspecific binding and autoflourescence.

10000 events were acquired. Light scatter histogram, forward light scatter versus log side scatter is used to delineate cell populations of interest (blasts) by bitmap drawing (gating). Gated fluorescence histograms are evaluated for positive cells by using cursor position from histograms for isotopic controls so that 98% of positives are defined. A case was defined as positive if >20% of the gated cells expressed the studied markers.

Cytogenetic analysis

Conventional cytogenetic analysis was performed on 48 and 72-hour’s cultures of BM aspirate specimens. At least 20 metaphases or all available metaphases were examined. Cytogenetic risk score was categorized as “good” “intermediate” or “poor” according to the revised UK Medical Research Council criteria for AML and International Prognostic Scoring System for AML so we simply grouped the cases as Unfavorable and Favorable prognosis. Analysis and Statistics of the present data was conducted, using the mean, standard deviation and chi-square test by SPSS V.20. (SPSS inc., Chicago, Illinois, USA).

 Result



The demographic data of the studied group was as follows: hemoglobin (Hb) concentration ranged from 4.2 to 13 g/dl with a mean value of 7.63±3.25; platelet counts ranged from 14 to 100×103/mm3 with a mean value of 51 766±23 447; total leukocytic counts ranged from 7 to 170×103/mm3 with a mean value of 52.303±45.677; PB blasts ranged from 10 to 80% with a mean value of 44.7±15.8; and BM blasts ranged from 34 to 94% with a mean value of 54.7±25.6 ([Table 1]).{Table 1}

As regards the CD30 expression, 20 of 30 AML patients showed CD30 expression (66.7%) and 10 cases were CD30-negative (33.3%) ([Figure 1]).{Figure 1}

On comparing the laboratory data of AML patients with positive CD30 expression and those with negative CD30 expression, there was a significance difference between CD30-positive and CD30-negative expression as regards total leukocytic counts and platelet count with no significance in Hb concentrations and the PB blasts count ([Table 2]).{Table 2}

As regards the French–American–British classification of the studied cases, the CD30-positive patients showed that 10 of them were of M2 subtype (50%) and 10 of them were of M3 subtype (50%). However, among the CD30-negative patients, one of them was of M2 subtype (10%), three were of M3 subtype (30%), two of them were of M4 subtype (20%), and four were of M5 subtype (40%); this was statistically significant ([Figure 2]).{Figure 2}

As regards the cytogenetic analysis of the studied AML patients, 15 of 120 patients (12.5%) were cytogenetically normal and 34 of 120 patients (28.3%) showed favorable cytogenetic abnormalities whereas 61 of 120 patients (45.8%) showed unfavorable cytogenetic abnormalities but the karyotyping failed in 10 patients (8.4%) ([Table 3]).{Table 3}

On follow-up of the CD30-positive cases after induction therapy, there was no significant difference in the mean value of total leukocytes count (TLC), Hb, platelets, and PB blasts before and after treatment. However, CD30-negative cases showed statistical significance after chemotherapy induction as regards the same parameters ([Table 4]).{Table 4}

On follow-up of the studied patients we found that, among patients with positive CD30, two patients showed remission followed by relapse (10%) within 1 month and 13 of them died (65%) during induction therapy. The remaining five patients (25%) showed no remission. However, among CD30-negative patients, four entered in complete remission (40%), one patient showed remission followed by relapse (10%) within 1 month, and four of them died (40%) during induction therapy. The remaining patient (10%) showed no remission ([Table 5]).{Table 5}

As regards survival, 52 of 80 CD30-positive patients (65%) died and 28 of 80 (35%) were alive until the end of the study. However, 16 of 40 (40%) CD30-negative patients died and 24 of 40 (60%) were alive until the end of the study ([Table 6]).{Table 6}

Kaplan–Meier curve used to show the overall survival of the studied AML patients ([Figure 3]).{Figure 3}

The purple curve (the positive group) is under the green one (the negative group); this signifies that there is a worse prognosis of AML patients with positive CD30 expression than those with no CD30 expression ([Figure 4] and [Figure 5]).{Figure 4} {Figure 5}

 Discussion



The previous study showed positive expression CD30 in 80/120 (66.7%) AML patients, while 40/120 (33.3%) were negative for CD30 expression. This was in agreement with Zheng et al. [10], who reported that CD30 is to be expressed on myeloblasts, but against Falini et al. [21] and RyouichiHorie et al. [22] who reported that Non-lymphohematopoietic tumors and AML usually lack the expression of the CD30. Zheng W et al. [23] founded that CD30 is expressed by myeloblasts in patients with AML or MDS. Because the study group was composed mostly of patients with high-risk AML or MDS who were option less for treatment, these data may increase the possibility that anti-CD30–targeted therapy could be a potential option for this patient group.

Some studies showed that CD30 was negatively expressed in all M4 and M5 cases. This result comes in agreement with Zheng et al. [10], who reported that CD30 is often absent in monocytic blasts or background maturing and/or mature monocytes. This is in contrast to CD30 ligand that is preferentially expressed in AML with monocytic differentiation. As regards total leucocytic counts of the positive group, it ranged from 20.3–170×103/mm3 with a mean value of (59.4±49.9)×103/mm3 while in the negative group TLC ranged from 7–50.5×103/mm3 with a mean value of (24.4±15.49)×103/mm3. This showed the existence of positive associations between CD30 expression and the high leukocyte counts, this is equal with O’Donnell et al. [3], who stated that TLC greater than 20×103/mm3 is associated with CD30 expression.

As regards platelets count of the positive CD30 group, it ranged from (14–90)×103/mm3 with a mean value of (45.8±22.2×103/mm3) while in the negative CD30 group it ranged from 31–100×103/mm3 with a mean value of (63.73±22.32×103/mm3). This showed significant association between platelets counts and CD30 expression, as there was a higher degree of thrombocytopenia with patients having +ve CD30 expression than those with –ve CD30 expression, this is in agreement with http://www.ncbi.nlm.nih.gov/pubmed/?term=Emadi%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22966972, and file:///D:\amira\papers%20amira\22Karp%20JE%22Karp%20JE" 22Karp%20JE"Karp [4], who reported that expression of CD30 and CD30-ligand (CD30L) of 40% patients with acute myeloid leukemia (AML), is associated with increased leukocyte counts and decreased platelet count as compared to those cases of AML with no expression of CD30.

In our study there were 93.5% of unfavorable cytogenetic abnormalities AML patients showed CD30 +ve while only 6.5% showed CD30-ve and this is in agreement with Rezaei et al. [24], who reported that Patients with AML with a poor cytogenetic risk score, may be refractory to therapy, also exhibited greater CD30 expression.

In our study, follow up of the CD30 positive cases before and after induction therapy showed that they poorly responded to treatment as regards the CBC and Bone marrow blasts counts. The mean value of TLC before induction therapy was 59.4±49.9×103 and after therapy became 41.6±22.5×103. The mean value of HB before therapy was 7.88±3.19 gm/dl and after therapy became 8.85±2.79 gm/dl. The mean value of platelets count was 45.8±22.16×103 and after therapy became 54.77±19.1×103. The mean value of PB blasts before therapy was 48.2±22.2% and after therapy became 40.21±7.36%. The mean value of BM blasts before therapy was 65.25±19.72% and after therapy became 59.3±6.24%. On other hand the negative CD30 group, they showed a better response to conventional therapy in which, the mean value of TLC before induction therapy was 24.4±15.4×103 and after therapy became 54.71±14.2%. The mean value of HB before therapy was 8.6±2.1 gm/dl and after therapy became 9.3±3.3 gm/dl. The mean value of platelets count was 63.73±22.3×103 and after therapy became 150.6±24.2×103. The mean value of PB blasts before therapy was 43±20.5% and after therapy became 0.01±0.0%. The mean value of BM blasts before therapy was 65.5±18% and after therapy became 2.14±0.2%.

 Conclusion



CD30 expression has a potential role as a prognostic marker as CD30 positive expression is increased in high risk AML patients with unfavorable chromosomal abnormalities with higher leucocytosis than CD30 negative AML patients.Also CD30 could be used for therapy by using anti-CD30 antibodies in non Monocytic AML patients who failed to respond to conventional therapy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Pieters R, Carroll WL. Biology and treatment of acute lymphoblastic leukemia. Pediatr Clin North Am 2008; 55:1–20.
2Campana D, Pui CH. Childhood leukemia. In: Abeloff MD, Armitage JO, Niederhuber JE, Kastan MB, McKena WG, editors. Clinical oncology. 4th ed. Philadelphia, PA: Elsevier Churchill Livingstone; 2008.
3O’Donnell MR, Abboud CN, Altman J, Appelbaum FR, Arber DA, Attar E et al. Acute myeloid leukemia. J Natl Compr Canc Netw 2012; 10:984–1021.
4Emadi A, Karp JE. CD30 in myeloid malignancies: hitting the bull’s-eye with an available dart. Leuk Lymphoma 2013; 54:679–680.
5Fonatsch C, Latza U, Dürkop H, Rieder H, Stein H. Assignment of the human CD30 (Ki-1) gene to 1p36. Genomics 1992; 14:825–826.
6Haase D, Fonatsch C, Freund M. Karyotype instability in myelodysplastic syndromes − a specific step in pathogenesis preceding clonal chromosome anomalies. Leuk Lymphoma 1992; 8:221–228.
7Emadi A, Karp JE. CD30 in myeloid malignancies: hitting the bull’s-eye with an available dart. Leuk Lymphoma 2013; 54:679–680.
8Gerli R, Muscat C, Bistoni O, Falini B, Tomassini C, Agea E et al. High levels of the soluble form of CD30 molecule in rheumatoid arthritis (RA) are expression of CD30+ T cell involvement in the inflamed joints. Clin Exp Immunol 1995; 102:547–550.
9Valent P, Jäger E, Mitterbauer-Hohendanner G, Müllauer L, Schwarzinger I, Sperr WR et al. Idiopathic bone marrow dysplasia of unknown significance (IDUS): definition, pathogenesis, follow up, and prognosis. Am J Cancer Res 2011; 1:531–541.
10Zheng W, Medeiros LJ, Young KH, Goswami M, Powers L, Kantarjian HH et al. CD30 expression in acute lymphoblastic leukemia as assessed by flow cytometry analysis. Leuk Lymphoma 2014; 55:624–627.
11Al-Shamkhani A. The role of CD30 in the pathogenesis of haematopoietic malignancies. Curr Opin Pharmacol 2004; 4:355–359.
12Gattei V, Degan M, Gloghini A, de Iuliis A, Improta S, Rossi FM et al. CD30 ligand is frequently expressed in human hematopoietic malignancies of myeloid and lymphoid origin. Blood 1997; 89:2048–2059.
13Hansen HP, Kisseleva T, Kobarg J, Horn-Lohrens O, Havsteen B, Lemke H. A zinc metalloproteinase is responsible for the release of CD30 on human tumor cell lines. Int J Cancer 1995; 63:750–756.
14Rezaei N, Haji-Molla-Hoseini M, Aghamohammadi A, Pourfathollah AA, Moghtadaie M, Pourpak Z. Increased serum levels of soluble CD30 in patients with common variable immunodeficiency and its clinical implications. J Clin Immunol 2008; 28:78–84.
15Eliopoulos AG, Wang CC, Dumitru CD, Tsichlis PN. Tpl2 transduces CD40 and TNF signals that activate ERK and regulates IgE induction by CD40. EMBO J 2003; 22:3855–3864.
16Masuya M, Kita K, Shimizu N, Ohishi K, Katayama N, Sekine T et al. Biologic characteristics of acute leukemia after myelodysplastic syndrome. Blood 1993; 81:3388–3394.
17Fathi AT, Preffer FI, Sadrzadeh H, Ballen KK, Amrein PC, Attar EC et al. CD30 expression in acute myeloid leukemia is associated with FLT3-internal tandem duplication mutation and leukocytosis. Leuk Lymphoma 2013; 54:860–863.
18Essa EA, El Halim SM, Abo-Elenin A, El Bendary A, Abdou SH, Farag W. Study of gene expression of CD30 variant (CD30v) and CD30 ligand (CD30L) in acute leukemia. Egypt J Immunol 2007; 14:11–20.
19Rassidakis GZ, Thomaides A, Atwell C, Ford R, Jones D, Claret FX, Medeiros LJ JunB expression is a common feature of CD30+ lymphomas and lymphomatoid papulosis. Mod Pathol 2005; 18:1365–1370.
20Rothe A, Sasse S, Goergen H, Eichenauer DA, Lohri A, Jager U et al. Brentuximab vedotin for relapsed or refractory CD30-positive hematologic malignancies: the GHSG experience. Blood 2012; 120:1470–1472.
21Falini B, Pileri S, Pizzolo G, Dürkop H, Flenghi L, Stirpe F et al. CD30 (Ki-1) molecule: a new cytokine receptor of the tumor necrosis factor receptor superfamily as a tool for diagnosis and immunotherapy Blood 1995; 85:1–14.
22Horie R, Watanabe T. CD30: expression and function in health and disease. Semin Immunol 1998; 10:457–470.
23Zheng W, Medeiros LJ, Hu Y, Powers L, Cortes JE, Ravandi-Kashani F et al. CD30 expression in high-risk acute myeloid leukemia and myelodysplastic syndromes. Clin Lymphoma Myeloma Leuk 2013; 13:307–314.
24Rezaei A, Adib M, Mokarian F, Tebianian M, Nassiri R. Leukemia markers expression of peripheral blood vs bone marrow blasts using flow cytometry. Med Sci Monit 2003; 9:CR359–CR362.