|Year : 2018 | Volume
| Issue : 4 | Page : 151-157
Prognostic significance of tetraspanin (CD81) expression in patients with acute myeloid leukemia
Ola A Elshoura1, Rasha A Elkholy1, Amal E Selim2, Nesreen M Sabry3
1 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt
3 Department of Clinical Oncology and Radiotherapy, Faculty of Medicine, Tanta University, Tanta, Egypt
|Date of Submission||09-Jun-2018|
|Date of Acceptance||20-Jul-2018|
|Date of Web Publication||10-Apr-2019|
Rasha A Elkholy
Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta
Source of Support: None, Conflict of Interest: None
Objective The aim was to determine the prognostic significance of CD81 expression in patients with acute myeloid leukemia (AML) and its effect on patients’ survival.
Patients and methods This study was carried out on 50 patients with newly diagnosed AML. Expression of CD81 was detected by flow cytometry, and then all the patients received chemotherapy and were observed for a period of 12 months regarding clinical and laboratory findings of remission and relapse.
Results Our results showed that positive expression of CD81 was detected in 30/50 (60%) patients. Clinical remission was achieved in six (12%) patients with positive expression of CD81, whereas it was achieved in 12 (24%) patients with negative CD81 expression. Relapse occurred in 19 (38%) patients with positive expression of CD81, whereas it occurred in six (12%) patients with negative CD81 expression. Death occurred in five (10%) patients with positive expression of CD81 and in two (4%) patients with negative expression of CD81.
Conclusion CD81 expression has a potential role as a prognostic marker as its expression was associated with patients with high-risk AML, with higher white blood cells, higher lactate dehydrogenase, and higher blast percentage in bone marrow, and M1 and M5 FAB subtypes. Decreased overall survival and disease-free survival in patients with CD81-positive expression than in patients with CD81-negative expression was also observed.
Keywords: acute myeloid leukemia, CD81, tetraspanin
|How to cite this article:|
Elshoura OA, Elkholy RA, Selim AE, Sabry NM. Prognostic significance of tetraspanin (CD81) expression in patients with acute myeloid leukemia. Egypt J Haematol 2018;43:151-7
|How to cite this URL:|
Elshoura OA, Elkholy RA, Selim AE, Sabry NM. Prognostic significance of tetraspanin (CD81) expression in patients with acute myeloid leukemia. Egypt J Haematol [serial online] 2018 [cited 2019 Oct 23];43:151-7. Available from: http://www.ehj.eg.net/text.asp?2018/43/4/151/255870
| Introduction|| |
Acute myeloid leukemia (AML) is a heterogenous group of malignant disorders characterized by expansion of immature myeloid precursors in bone marrow (BM) and peripheral blood. It results from acquiring genetic defects in either hematopoietic stem cells or progenitors cells, which lead to chromosomal rearrangement of multiple genes and finally malignant transformation .
It is the most common type of acute leukemia in adults, and without treatment, the condition is fatal within a short time, Although most young patients can achieve complete remission (CR) with chemotherapy, many of them relapse .
So identification of new prognostic markers is highly important, especially those potentially refining therapeutic options .
CD81 antigen, also named as tetraspanin antiproliferative antibody-1, is a cell surface transmembrane protein that belongs to the tetraspanin family (33 members in mammals) .
CD81 acts in the same way as other different tetraspanins and interacts with different partners in a cell type-dependent manner to regulate many processes, for example, CD81 interacts with CD19 to regulate B-cell signalling ,. CD81 interacts with CD3 and ICAM-1 to regulate the integrity of the immune synapse during T-cell activation . In connection with another tetraspanin CD9, CD81 plays a role in sperm–egg fusion, making it important for mammalian fertility . Moreover, CD81 is implicated in the life cycle of many human pathogens. It is involved in Plasmodium sporozoite and hepatitis C virus invasion of hepatocytes ,, and participates in the assembly and budding of human immunodeficiency virus and influenza A virus ,,.
Furthermore, many studies showed that tetraspanins are involved in many stages of carcinogenesis as well as in angiogenesis and metastasis .
Specifically, CD81 may be of great value in hematopoiesis as it allows hematopoietic stem cells to re-enter quiescence , and its expression on plasma cells in multiple myeloma was associated with decreased patients survival .
The present study aimed to determine the prognostic significance of CD81 expression in patients with AML and its effect on patients’ survival.
| Patients and methods|| |
The current study was carried out from May 2016 to May 2018 on 50 patients with newly diagnosed AML referred to Hematology/Oncology Unit, Internal Medicine Department and Clinical Oncology and Radiotherapy Department. This study was carried out according to the declaration of Helsinki. After research ethical committee approval, an informed written consent was taken from all patients. Newly diagnosed patients with AML were included in the study, and any patient with malignant diseases other than AML or patients with AML who received treatment were excluded from the study. These cases were diagnosed on the basis of clinical presentation, complete blood picture, BM examination, morphological and cytochemical smears as well as immunophenotyping.
Patients were subjected to full history taking, thorough clinical examination, abdominal ultrasonography, and laboratory investigations, including the following.
- Complete blood picture was done on an ERMA PCE-210N cell counter (Tokyo, Japan) with examination of Giemsa-stained smears.
- BM aspiration was done with examination of Giemsa-stained smears and cytochemical-stained smears.
- Immunophenotyping for AML diagnosis was performed using the following monoclonal antibodies :
- Myeloid lineage markers: CD13 PE, CD33 PE,CD117 PE, cyt anti MPO PE, glycophorin A PE, CD71 FITC, and CD61 FITC.
- Monocytic lineage markers: CD14 PE and CD64 FITC.
- B-lymphoid lineage markers: CD10 PE, CD19 PE, anti-TDT FITC, and cyt CD79a PE.
- T-lymphoid lineage markers: CD2 FITC, CD7 FITC, and cyt CD3 APC.
- Nonspecific lineage markers: CD45 Per CP, HLA-DR FITC, and CD34 PE.
- These markers were supplied by Becton Dickinson (Mountain View, California, USA). Positive or Negative results of the target population are defined according to cutoff, which is 20% for all markers, except for CD34 and cytoplasmic markers, for which, it is 10% .
- Erythrocyte sedimentation rate (ESR).
- Lactate dehydrogenase (LDH) determination.
Flow cytometric analysis of CD81 APC on BM samples was done for all patients using BD kits. Cutoff for CD81 was 20% .
Samples were analyzed by four-color flow cytometry using the Becton Dickinson (BD) FACS Calibur instrument (Becton Dickinson, San Diego, California, USA). For each analysis, 10 000 events were acquired and analyzed using the Cell Quest software (Becton Dickinson, version 3, verify software House Topsham). An acquisition gate was done based on CD45 and SS to gate on blast cells. Isotype antibody was used to prevent nonspecific binding.
Follow-up of the patients
After being fully investigated, all the patients received chemotherapy and were observed for 12 months regarding clinical and laboratory findings of remission and relapse, taking care to estimate the date of first CR, date of relapse, and date of death or last seen alive.
Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. (IBM Corp., Armonk, New York, USA). Comparisons between groups for categorical variables were assessed using χ2-test. Independent t-test was used to compare two groups for quantitative variables. Kaplan–Meier survival curve was used, and Cox regression was done for the significant relation with disease-free survival (DFS) and overall survival (OS). Spearman coefficient was used to correlate between quantitative variables. Significance of the obtained results was judged at the P value less than or equal to 0.05 level .
| Results|| |
This study was conducted on 50 patients with newly diagnosed AML. Their ages ranged from 18 to 73 years, with a mean value of 46.26±16.59 years. There were 28 (56%) males and 22 (44%) females, with a male to female ratio of 1.2 : 1. A total of 35 (70%) patients presented with hepatosplenomegaly, 13 (26%) with lymphadenopathy, 11 (22%) with fever, 34 (68%) purpura and ecchymosis, 18 (36%) with bleeding per gum, and 40 (80%) with pallor. Patients were classified according to French–American–British (FAB) classification as follow: one (2%) patient was M0, 10 (20%) patients were M1, 13 (26%) were M2, four (8%) were M3, two (4%) were M3V, nine (18%) were M4, 10 (20%) were M5, and one (2%) was M7. Laboratory data of the studied group are provided in [Table 1].
Regarding CD81 expression, CD81-positive expression was present in 30/50 (60%) patients of the studied group, and its level ranged from 22 to 96%. with mean value of 57.53±22.83, whereas CD81-negative expression was present in 20/50 (40%) patients of the studied group, and its level ranged from 0 to 14%, with mean value of 8.41±4.81. ([Figure 1] and [Figure 2]).
Positive expression of CD81 was detected in 30/50 patients. Of them, 17(56.66%) were males and 13 (43.33%) were females. The remaining 20 patients showed negative expression of CD81. Of them, 11 (55%) were males and nine (45%) were females. There was no statistically significant difference between CD81-positive group versus CD81-negative patient group regarding sex (P>0.05).
Regarding age and laboratory data of patients with AML with positive CD81 expression and those with negative CD81 expression, there was a statistical significance difference between the two groups regarding total leukocytic count, blast percentage in BM, and LDH, whereas there was no statistical significant difference regarding age, Hb concentrations, platelet count, PB blast percentage, and first hour ESR. Comparison between patients with CD81-positive AML group versus patients with CD81-negative AML group regarding age and laboratory data was provided in [Table 2].
|Table 2 Comparison between CD81-positive acute myeloid leukemia patient group versus CD81-negative acute myeloid leukemia patient group regarding age and laboratory data|
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Regarding FAB subtype, the frequency of CD81-positive expression was higher in M1 followed by M5, whereas the frequency of CD81-negative expression was higher in M2 followed by M4. The relation between FAB subtypes in studied patients with AML and CD81 expression is provided in [Table 3].
|Table 3 Relation between Frensh American British (FAB) subtypes in studied patients with acute myeloid leukemia and CD81 expression|
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A statistically significant positive correlation was detected between CD81 expression and white blood cells (WBCs), blast percentage in BM, and LDH, whereas a positive nonsignificant correlation was present between CD81 expression and age, first hour ESR, and blast percentage in PB.
A negative nonsignificant correlation was present between both Hb concentration and PLT count and CD81 expression. The correlation between CD81 expression with age and laboratory parameters is provided in [Table 4].
|Table 4 Correlation between CD81 expression with age and laboratory parameters|
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The 50 patients with newly diagnosed AML were treated and were followed up for 12 months. On follow-up of the studied patients we found that, among patients with positive CD81 expression, six (12%) patients showed CR until the end of the follow-up and five (10%) patients died during induction therapy. However, among CD81-negative expression patients, 12 (24%) patients entered in CR until the end of the follow-up and two (4%) patients died during induction therapy. The incidence of relapse was 25 (50%) patients, including 19 (38%) patients with positive CD81 expression and six (12%) patients with negative CD81 expression, with a statistically significant difference between the two groups. Distribution of the studied cases regarding survival is provided in [Table 5].
Patients with positive CD81 expression had statistical significantly shorter DFS and shorter OS compared with patients with negative CD81 expression (5.55 vs. 8.53 months and 6.90 vs. 9.65 months, respectively). Log-rank test for equality of survival distribution of both groups showed a highly significant difference (χ2=5.506, P<0.019, and χ2=5.990, P<0.014) ([Figure 3] and [Figure 4]).
|Figure 3 Kaplan–Meier survival curve for disease-free survival with CD81 expression.|
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|Figure 4 Kaplan–Meier survival curve for overall survival with CD81 expression.|
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| Discussion|| |
The current study was carried out on 50 newly diagnosed patients having AML. Their ages ranged from 18 to 73 years, with a mean value of 46.26±16.59. There were 28 (56%) male and 22 (44%) female patients, with male to female ratio of 1.2:1. This was in agreement with Ries Rhonda et al. , who reported that the incidence of AML is higher in males than females.
CD81-positive expression was present in 30/50 (60%) patients of the studied group, and its level ranged from 22 to 96%, with mean value 57.53±22.83, whereas CD81-negative expression was present in 20 (40%) patients of the studied group, and its level ranged from 0 to 14%, with mean value of 8.41±4.81. This was in agreement with Boyer et al. , who stated that positive expression of CD81 was present in 69% of patients with AML.
However, in a study by Guihard et al. , the frequency of positive CD81 expression was 35% among patients with AML.
No significant difference was noted between age of patients who were positive for CD81 expression and those who were negative for the expression of CD81. This finding was in concordance with a study done by Guihard et al. , who reported nonsignificant difference between CD81-positive group and CD81-negative group regarding age. However, this was in contrast with Boyer et al., who stated that patients with AML with CD81-positive blast cells were of higher age.
Although male predominance was observed in the present study (male to female ratio 1.2 : 1), nonsignificant association was detected between sex and positive CD81 expression; this was in concordance with a study done by Boyer et al. .
With respect to laboratory findings, we found a statistical significant difference between CD81 +ve group and CD81-negative group regarding initial total WBCs count, LDH, and BM blast percentage. These results are in agreement with Boyer et al., who reported that the statistical difference was significant between the two groups and leucocytosis.
On the contrary, no statistical significant difference between CD81-positive group and CD81-negative group was present regarding Hb concentrations, platelets count, the PB blast percentage, and first hour ESR. These results are in agreement with those reported by Boyer et al. .
In the present work, regarding correlation study, a statistical significant positive correlation was detected between WBCs, blast percentage in BM, and LDH and CD81 expression, whereas a positive nonsignificant correlation was present between age, blast percentage in P.B, and first hour ESR and CD81 expression. A negative nonsignificant correlation was present between both Hb concentration and PLTs count and CD81 expression. No other studies or parameters had any correlation with CD81 expression.
Regarding FAB subtypes and the positive expression of CD81, the frequency was higher in M1 followed by M5, whereas the frequency of CD81-negative expression was higher in M2 followed by M4. These results are in agreement with Boyer et al. , who stated that the CD81 expression in the AML-blast population was more frequent in M1 compared with other FAB subtypes.
In the present study, regarding response of patients to chemotherapy only, six (12%) patients with CD81+ve expression achieved CR till the end of the study, whereas 12 (24%) patients with CD81-ve expression achieved remission till the end of the study, and the difference between them was statistically significant. The number of patients who relapsed after remission or did not achieve remission was higher in CD81-positive group in relation to CD81-negative group, with 19 (38%) and six (12%) patients respectively, and the difference between them was statistically significant. Overall, five (10%) patients with CD81-positive expression died, whereas two (4%) patients with CD81-positive expression died during induction therapy, with no statistical difference between the two groups. These results could be explained by the finding of Gonzales et al. , who proved that blast cells expressing CD81 were 30 to 50% more resistant to chemotherapy, and overexpression of CD81 increased AML cell adhesion, migration and blast homing and engraftment efficiency.
Patients with positive CD81 expression had statistical significantly shorter DFS and shorter OS compared with patients with negative CD81 expression (5.55 vs.8.53 months and 6.90 vs. 9.65 months, respectively). These results were in agreement with those reported by Guihard et al., Boyer et al. , and Gonzales et al. , who stated that CD81 expression had a negative effect on patient survival.
| Conclusion|| |
CD81 expression has a potential role as a prognostic marker, as its expression was associated with patients with high-risk AML having higher WBCs, higher LDH, higher blast percentage in BM, and M1 and M5 FAB subtypes; moreover, patients with CD81-positive expression have higher incidence of relapse and decreased OS and DFS than patients with CD81-negative expression.
The authors thank the patients and the nurses of Internal Medicine, Clinical Oncology and Radiotherapy and Clinical Pathology Departments for their assistance in conducting the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Estey E. Acute myeloid leukemia:update on diagnosis, risk stratification and management. Am J Hematol
Tangen JM, Floisand Y, Foss-Abrahamsen J. Survival in adults with acute myelogenous leukemia. Tidsskr Nor Laegeforen
Boyer T, Guihard S, Roumier C, Peyrouze P, Gonzales F, Berthon C et al.
Tetraspanin CD81 is an adverse prognostic marker in acute myeloid leukemia. Oncotarget
Oren R, Takahashi S, Doss C, Levy R, Levy S. TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins. Mol Cell Biol
Mattila PK, Feest C, Depoil D, Treanor B, Montaner B, Otipoby KL et al.
The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling. Immunity
Maecker HT, Levy S. Normal lymphocyte development but delayed humoral immune response in CD81-null mice. J Exp Med
Rocha-Perugini V, Zamai M, González-Granado JM, Barreiro O, Tejera E, Yañez-Mó M et al.
CD81 controls sustained T cell activation signaling and defines the maturation stages of cognate immunological synapses. Mol Cel Biol
Rubinstein E, Ziyyat A, Prenant M, Wrobel E, Wolf JP, Levy S et al.
Reduced fertility of female mice lacking CD81. Develop Biol
Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R et al.
Binding of hepatitis C virus to CD81. Science
Silvie O, Rubinstein E, Franetich JF, Prenant M, Belnoue E, Rénia L et al.
Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity. Nat Med
Monk PN, Partridge LJ. Tetraspanins: gateways for infection. Infect Disord Drug Targets
Nydegger S, Khurana S, Krementsov DN, Foti M, Thali M. Mapping of tetraspanin-enriched microdomains that can function as gateways for HIV-1. J Cell Biol
He J, Sun E, Bujny MV, Kim D, Davidson MW, Zhuang X. Dual function of CD81 in influenza virus uncoating and budding. PLoS Pathogens
Thali M. Tetraspanin functions during HIV-1 and influenza virus replication. Biochem Soc Trans
Hemler ME. Tetraspanin proteins promote multiple cancer stages. Nat Rev Cancer
Lin KK, Rossi L, Boles NC, Hall BE, George TC, Goodell MA. CD81 is essential for the re-entry of hematopoietic stem cells to quiescence following stress-induced proliferation via deactivation of the Akt pathway. PLoS Biol
Paiva B, Gutierrez NC, Chen X, Vidriales MB, Montalban MA, Rosinol L et al.
Clinical significance of CD81 expression by clonal plasma cells in high-risk smoldering and symptomatic multiple myeloma patients. Leukemia
Matutes E, Morillo R, Cotovsky D. Immunophenotypic analysis of acute myeloid leukemia. In: Lewis S, Bain B, Bates I, editos. Practical hematology
10th ed. England: Harcout Publishers Limited; 2006. 335.
Dawson BD, Trapp RG. Basic & clinical biostatistics
. 3rd ed. New York, NY: Medical Publication Division, Lange Medical Book/ McGraw-Hill: 2001. 161–218
Ries Rhonda E, Daniel C, Kunter G, Kasai Y, Miner T. Distinct pattern of mutations occurring in de novo AML versus AML arising in the setting of severe congenital neutropenia. Blood
Guihard S, Boyer T, Roumier C, Peyrouze P, Gonzales F, Preudhomme C et al.
CD81 as new potential marker in acute myeloid leukemia. Hematologica
Gonzales F, Boyer T, Peyrouze P, Guihard S, Roumier C, Berthon C, Preudhomme C, Cheok M. Targeting aberrant expression of CD81 impacts cell adhesion and migration, drug resistance and prognosis of acute myeloid leukemia. Blood
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]