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HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients
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HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients Ghada I. Mossallam a,⇑, Raafat Abdel Fattah b, Alaa El-Haddad c, Hossam K. Mahmoud b a
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Bone Marrow Transplantation Laboratory Unit, National Cancer Institute, Cairo University, Cairo, Egypt Medical Oncology Department, National Cancer Institute, Cairo University, Cairo, Egypt c Pediatric Oncology Department, National Cancer Institute, Cairo University, Cairo, Egypt b
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Article history: Received 20 June 2014 Accepted 15 December 2014 Available online xxxx
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Keywords: HLA-E Hematopoietic stem cell transplantation Graft versus leukemia (GVL) Relapse
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a b s t r a c t Human leukocyte antigen-E (HLA)-E in a non-classical major histocompatibility complex (MHC) class I (Ib) molecule. HLA-E-peptide complex acts as a ligand for natural killer (NK) cells and CD8+ T lymphocytes playing a dual role in natural and acquired immune responses. The difference in expression levels between HLA-E alleles was suggested to have impact on transplantation outcome. The aim of the study is to evaluate the clinical impact of HLA-E alleles on transplantation in a group of Egyptian patients. HLA-E genotyping was analyzed in eighty-eight recipients of stem cell transplantation using polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP). HLA-E*01:03 allele showed a trend toward lower cumulative incidence of relapse at 2 years compared to homozygous HLA-E*01:01 genotype (8% versus 21.5%, p = 0.09, HR: 0.30, CI: 0.91–1.69). HLA-E was the only factor showing near significant association with relapse incidence. HLA-E polymorphism did not affect the cumulative incidence of acute GVHD grades II–IV at 100 days, the 2-years cumulative incidence of extensive chronic GVHD, transplant related mortality (TRM) or overall survival (OS). Conclusion: the suggested association of HLA-E polymorphism with reduced risk of relapse needs verification in a larger cohort. However, its proposed role in GVL helps better understanding of alloreactivity of T cells and NK cells and their implication in immunotherapy post allogeneic hematopoietic stem cell transplantation. Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
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1. Introduction
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Allogeneic hematopoietic stem-cell transplantation (AHSCT) is a curative therapy for many hematologic malignancies. Post transplantation complications are mainly related to the development of graft versus host disease (GVHD), infection and relapse. Human leukocyte antigen-E (HLA-E) in a non-classical major histocompatibility complex (MHC) class I (Ib) molecule characterized by limited polymorphism and lower cell surface expression compared to classical Class Ia molecule [1,2]. It is an immunomodulatory molecule that can function as both immuno-tolerogenic and immuno-activating molecule and plays a dual role in natural and acquired immune responses [3,4]. The HLA-E-peptide complex can act as a ligand for the CD94/NKG2 receptors expressed on the surface of natural killer (NK) cells and represents a restriction element for TCR receptor of CD8+ T cells [5–7]. HLA-E molecule
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⇑ Corresponding author at: National Cancer Institute, Cairo University, Fom ElKhalig Square, Kasr El-Aini St., 11796 Cairo, Egypt. E-mail address: [email protected] (G.I. Mossallam).
binds and presents a restricted set of peptides derived from self or foreign proteins [8]. It binds nonamer peptides derived from signal sequences of other HLA class I molecules including HLA-A, -B,C, and -G [4,9]. Its surface expression is regulated by the availability of these peptides [10]. HLA-E is expressed on the extravillous trophoblasts and mediates maternal tolerance to semiallogeneic fetal graft [11]. HLA-E can also bind viruses [12,13], bacteria [14], tumors [4], stress-related peptides [7], and self-antigens [15]. Recognition by CD94/NKG2 is governed by the sequence of peptide bound to HLA-E [16]; CD94/NKG2A functions as an inhibitory receptor, while CD94/NKG2C functions as an activating receptor [17]. HLA-E mediates lysis of targets by CD8+ cytotoxic T cell (CTL) including NKCTL and regulates adaptive immune response through CD8+ suppressive T cells [5–7,18]. Two nonsynonymous functional alleles of HLA-E have been found, E*01:01 and E*01:03 [19]. An arginine at position 107 located within the a2 domain of the HLA-E heavy chain in HLAE107A (HLA-E*01:01) is replaced by a glycine in HLA-E107G (HLAE*01:03) [20]. The functional differences between the HLA-E alleles are related to the relative peptide affinity and cell surface
http://dx.doi.org/10.1016/j.humimm.2014.12.017 0198-8859/Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
Q1 Please cite this article in press as: Mossallam GI et al. HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.12.017
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expression, with the HLA-E*01:03 allele expressed at a higher levels than *01:01, due to its higher affinity to peptides and therefore higher surface stability [4,10]. HLA-E polymorphism has been associated with infection, cancer, recurrent spontaneous abortion, autoimmune diseases, and transplantation outcome [4,11,13–15,21]. HLA-E polymorphism was found to affect HSCT outcome; reports pointed to the association of HLA-E*01:03 genotype with protection from graft versus host disease and relapse as well as better survival, while HLAE*0101 homozygosity was associated with risk of infection post AHSCT [21–27]. The aim of the study is to evaluate the clinical impact of HLA-E alleles on transplantation in Egyptian patients after AHSCT from HLA-matched sibling donors.
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2. Patients and methods
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2.1. Patients
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The cohort consisted of 88 recipients transplanted from their HLA-identical siblings at Bone Marrow Transplantation (BMT) Unit, National Cancer Institute, Cairo University and at Nasser Institute between 2007 and 2010. Informed consent was obtained. They were 51 males and 37 females. Their median age was 30 years, range (2–48). They suffered from: acute myeloid leukemia (AML) 52/88(59.1%), chronic myeloid leukemia (CML) 21/88(23.9%) and myelodysplastic syndrome (MDS) 15/88(17%). Patients’ characteristics are mentioned in Table 1. Donor selection was done using serologic method (BIO-Rad) for HLA class I typing, while sequence specific oligonucleotide probe (SSOP) method (Inno-Lipa HLA-DRB1 plus) was used for class II typing. HLA-E genotyping was performed for recipients using polymerase chain reactionrestricted fragment length polymorphism.
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2.1.1. Conditioning regimens and GVHD prophylaxis Conditioning regimens included Flu/Bu: Fludarabine 30 mg/m2/ d IV daily for 4 days (D-10, D-9,D-4 and D-2) and Busulphan 4 mg/ kg p.o. daily for 4 days (D-8 to D-5); Flu/Alk: Fludarabine 30 mg/ m2/d IV daily for 4 days (D-7 to D-4) and Alkeran (Melphalan) 70 mg/m2/d IV for 2 days (D-3 and D-2); Bu/cy: Busulphan 4 mg/ kg p.o. daily for 4 days (D-7 to D-4) and Cyclophosphamide 60 mg/kg/d IV for 2 days (D-3 and D-2) given with 2-mercaptoethane sulfonate sodium (MESNA). GVHD prophylaxis was given by Cyclosporin-A (CSA) 3 mg/kg IV from day 1 to be changed to oral form (3 mg/kg) once the patient can swallow for 9–12 months post-transplant. GVHD prophylaxis included also short course IV methotrexate (15 mg/m2 day 1 and 10 mg/m2 days 3 and 6) with leucovorin rescue and mycophenolate mofetil (MMF) 1000 mg twice daily PO.
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2.2. HLA-E typing by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP)
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DNA was extracted from peripheral blood of recipients using QIAamp DNA Mini Kit (Qiagen, Germany), according to the manufacturer’s protocol. Detection of HLA-E polymorphism was performed using PCR-RFLP [28]. PCR reaction consisted of 25 ll, containing 100 ng DNA, 10 pmol of each primer: forward primer: 50 -GGCTGCGAGCTGGGGCCCGCC-30 , reverse primer: 50 -AGCCCTG TGGACCCTCTT-30 , 1X Go Taq buffer including 1.5 mM MgCl2, 1.25 U Go Taq DNA polymerase (Promega, Madison, USA) and 0.2 mM each dNTP (Thermo Scientific, Fermentas). The PCR cycling conditions consisted of initial denaturation at 94 °C for 5 min. This was followed by 35 cycles of 94 °C for 45 s, 61 °C for 45 s and 72 °C for 45 s then final extension step at 72 °C for 7 min. The resulted fragment by visualization on 2% gel was
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Table 1 Patient characteristics.
Age median (range) (y) Sex Male Female Disease at transplantation, n (%) Acute myeloid leukemia Myelodysplastic syndrome Chronic myeloid leukemia Stage of disease, n (%) Low, intermediate High Prior therapy, n (%) No Yes Gender combination (Recipient/Donor), n (%) M/M M/F F/F F/M Source of stem cells, n (%) Bone Marrow Peripheral blood Conditioning regimen, n (%) Myeloablative Busulfan/Cyclophosphamide Busulfan /Fludarabine Reduced intensity Fludarabine /Melphalan GVHD prophylaxis, n (%) Cyclosporine /Methotrexate Cyclosporine/MMF
Entire group n = 88
01:03 allele n = 54
01:01/01:01 n (34)
p-Value
30 (2–48)
31 (15–45)
29 (2–48)
0.21
53 (60.2) 35 (39.8)
34 (63) 20 (37)
19 (55.9) 15 (44.1)
0.66
52 (59.1) 15 (17) 21 (23.9)
30 (55.5) 9 (16.7) 15 (27.8)
22 (64.7) 6 (17.6) 6 (17.6)
0.55
79 (89.7) 9 (10.3)
47 (87) 7 (13)
32 (94.1) 2 (5.9)
0.45
34 (38.6) 54 (61.4)
23 (42.6) 31 (57.4)
11 (32.4) 23 (67.6)
0.46
29 22 13 24
(33) (25) (14.8) (27.2)
16 (29.7) 17 (31.6) 9 (16.4) 12 (22.3)
13 (38.2) 5 (14.7) 4 (11.7) 12 (35.4)
0.23
4 (4.5) 84 (95.5)
1 (1.9) 53 (98.1)
3 (8.8) 31 (91.2)
0.31
72 (81.8)
46 (85.2)
26 (76.4)
0.45
16 (18.2)
8 (14.8)
8 (23.6)
68 (77.2) 20 (22.8)
44 (81.5) 10 (18.5)
24 (70.6) 10 (29.4)
0.41
Low, intermediate: AML(CR1), CML (chronic phase) and MDS; High: AML (CR2) and CML (accelerated phase).
Q1 Please cite this article in press as: Mossallam GI et al. HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.12.017
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HLA-E*03:03/03:03 in 15/88 (17%). The allele frequency was 60.8% for HLA*01:01 allele and 39.2% for HLA*01:03 allele. The distribution of genotypes was in Hardy–Weinberg equilibrium (p > 0.05).
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280 bp. Allele determination was carried out by digestion of the amplified PCR products using the restriction enzyme HpaII (Thermo Scientific, Fermentas). The presence of HLA*01:03 allele was identified by the presence of a restriction site for the enzyme.
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2.3. Statistical methods
3.2. Association of HLA-E genotype with clinical outcome
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The cumulative incidence of aGVHD II–IV at 100 days did not show significant difference between recipients harboring HLAE*01:03 allele compared to those homozygous for HLA-E*01:01 genotype (p = 0.54, HR: 0.80, 95% CI: 0.52–2.80). Similarly, extensive cGVHD did not show significant difference in the cumulative incidence at 2 years in recipients with HLA-E*01:03 allele compared to those homozygous for HLA-E*01:01 genotype (p = 0.93, HR: 0.97, 95% CI: 0.49–2.16) (Table 2) (Figs. 1 and 2). The median duration of follow-up was 41 months, range (0.5–65). The cumulative incidence of relapse at 2 years was 9.5%. HLA-E*01:03 allele showed a trend toward lower cumulative
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Numerical data were expressed as median and range. Hardy– Weinberg equilibrium was tested using Pearson’s chi-squared test. Qualitative data were expressed as frequency and percentage. Chi-square test (Fisher’s exact test) was used to examine the relation between qualitative variables. Survival analysis was done using Kaplan–Meier method. Comparison between survival curves was done using log-rank test. Estimation of the hazard ratio was done using cox-regression model. Hazard ratio with its 95% confidence interval (CI) was used for risk assessment. Survival package was used to test the proportional hazards assumption. The assumption (proportionality) of cox proportional hazard models were met. Relapse incidence was estimated by the cumulative incidence method with death without relapse treated as a competing risk. Transplant related mortality (TRM) was calculated using cumulative incidence with relapse treated as a competing. A p value < 0.05 was considered significant.
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3. Results
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3.1. Frequency of HLA-E genotypes and alleles
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Analyzing HLA-E genotype, HLA-E*01:01/01:01 was detected in 34/88 (38.6%), HLA-E*01:01/01:03 in 39/88 (44.4%) and
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Table 2 Association of HLA-E genotype with transplantation outcome.
Acute GVHD Chronic GVHD Relapse Transplant related mortality Overall survival
p-Value
Hazards ratio
95% CI
0.54 0.93 0.09 0.53 0.47
0.80 0.97 0.30 0.54 0.78
0.58–2.80 0.49–2.16 0.91–1.69 0.31–2.88 0.40–1.52
Fig. 2. Cumulative incidence of cGVHD according to HLA-E genotype. No significant difference in the cumulative incidence of extensive cGVHD in recipients harboring HLA-E*01:03 allele compared to those homozygous for HLA-E*01:01 genotype (p = 0.93).
HLA-E*01:03 allele compared to homozygous HLA-E*01:01.
Fig. 1. Cumulative incidence of aGVHD according to HLA-E genotype. No significant difference in the cumulative incidence of aGVHD II-IV in recipients harboring HLAE*01:03 allele compared to those homozygous for HLA-E*01:01 genotype (p = 0.54).
Fig. 3. Cumulative incidence of relapse according to HLA-E genotype. Relapse incidence was estimated by the cumulative incidence method with death without relapse treated as a competing risk. HLA-E*01:03 allele showed a trend toward lower cumulative incidence of relapse compared to homozygous HLA-E*01:01 genotype (p = 0.09).
Q1 Please cite this article in press as: Mossallam GI et al. HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.12.017
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Table 3 Analysis of factors affecting relapse incidence.
HLA-E# Age (<30 y versus >30 y) Prior therapy (Yes versus No) Stage of disease (low, intermediate versus high) Disease at presentation (CR1, CML versus CR2, accelerated phase) Conditioning regimen (MA versus RIC) GVHD (Yes versus No)
p-Value
Hazards ratio
95% CI
0.09 0.39
0.30 1.81
0.91–1.69 0.45–7.26
0.28 0.45
0.42 2.22
0.49–11.38 0.27–18.34
0.58
0.75
0.07–18.67
0.75
1.28
0.26–6.19
0.29
0.48
0.52–8.40
#
HLA-E*01:03 allele versus homozygous HLA-E*01:01; y: year; CR: complete remission; MA: myeloablative; RIC: reduced intensity conditioning.
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incidence of relapse at 2 years compared to homozygous HLA-E*01:01 genotype (8% versus 21.5%, p = 0.09, HR: 0.30, CI: 0.91–1.69) (Fig. 3). Analyzing other factors affecting relapse, HLAE was the only factor showing near significant association with relapse incidence (Table 3). The cumulative overall survival (OS) at 2 years was 60%; no significant difference was observed between HLA-E*01:03 allele compared to homozygous HLA-E*01:01 genotype (60% versus 58%, p = 0.47, HR: 0.78, 95% CI: 0.40–1.52). The 2-years cumulative incidence of transplant related mortality (TRM) was 29.5%; the incidence was 31% versus 34% in HLA-E*01:03 allele compared to homozygous HLA-E*01:01 genotype, respectively (p = 0.53, HR = 0.54, 95% CI: 0.31–2.88) (Table 2). The causes of TRM were infection (n = 18), acute and chronic GVHD (n = 6), organ failure (n = 2). Infection as a cause of death occurred in 26.4% of HLAE*01:01 homozygous patients compared to 16.7% of those with HLA-E*01:03 allele, the difference, however, was not statistically significant (p = 0.26, HR: 1.80, 95% CI: 0.63–5.11).
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4. Discussion
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The HLA-E-peptide complex acts as a ligand for NK cells and cytotoxic T lymphocytes. The difference in expression levels between HLA-E alleles was suggested to have impact on transplantation outcome. In our cohort, a trend toward reduced cumulative incidence of relapse at 2 years was found with HLA-E*01:03 allele compared to HLA-E*01:01/01:01 genotype. Contrary to Weisdorf et al. [29] and Chen et al., [30], GVHD and disease stage did not affect risk of relapse after allogeneic transplantation. Our observation coincides with the recently reported data by Hosseini et al. [27], where an association was found between HLA-E*01:03 homozygosity and decreased incidence of relapse and better DFS. This finding was, however, not confirmed by other studies [22,23,25]. Graft versus leukemia (GVL) activity in preventing leukemia relapse is mediated by alloreactive donor CD8+ T cells and NK cells [4,31]. Our data suggest effective GVL effect in patients with HLA-E*01:03 allele. This effect is probably related to the higher affinity of HLA-E*01:03 allele to peptides and its higher cell surface expression compared to HLA-E*01:01/01:01. Although inhibition of immature NK after haploidentical transplantation through CD94/NKG2A recognition was observed by Nguyen et al. [32] and Haas et al. [33], however, did not find relationship between HLA-E genotype and NKG2A+KIR NK-cell response in unrelated transplant. Alternatively, activation may result from binding peptides other than HLA nonamers leading to competitive relief of CD94/NKG2A mediated inhibition [34]. Heat-shock proteins e.g. Hsp60 overexpressed on leukemic cells decreases inhibition via CD94/NKG2A [34,35]. On the other hand, binding
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of the activation receptors NKG2C rather than the NKG2A inhibitory receptors activates NK cells [17]. The role of NKG2C receptor post ASCT was recently supported by Foley et al. [36] who suggested that cytomegalovirus viremia-induced NKG2C may play role in eradication of residual leukemic cells besides controlling infectious disease. In a study conducted by Linn et al. [37], the NKG2C was upregulated exclusively in the ex vivo-expanded cytokine induced killer (CIK) (CD3+CD56+) cells that were cytolytic to susceptible AML targets. The role of CIK cells was supported in managing relapse post allogeneic HSCT with minimal GVHD [38–40]. CIK cells cytotoxicity could be improved after antibodydirected stimulation of NKG2C and CD94 [41].Other HLA-E-peptides are recognized by the CD8+ CTL mediating cytotoxicity and cytokine secretion, HLA-E may bind non-standard minor histocompatibility antigens (mHags) peptides that specifically mediate GVL [22]. Its independence of classic HLA class I in addition to its wide reactivity make it highly responsive to allogeneic stimulation [5]. Peptides with dual HLA-class I-A2 and HLA-E binding specificity were identified for the antioxidant enzyme peroxiredoxin expressed on stressed and malignant cells, contributing to T cell activation [42]. In line with the suggested defective presentation of HLA-E 0101 allele, Tamouza et al. [21,22] reported increased incidence of bacterial infection with HLA-E*0101 homozygous donor in URD transplant due to inefficient presentation of bacterial peptides. This association was, however, not found in our cohort may be due to the lower incidence of infection in related donor transplant. On the other hand, reports studying the effect of HLA-E polymorphism on aGVHD showed protective role for the HLA-E*01:03 genotype in related and unrelated transplants [22–24,26]. Tamouza et al. [22], claimed that competition of HLA-E*0103 with classic class I for mHag presentation to T cell protect from NK-mediated tissue damage. In our series, no association was observed between HLA-E polymorphism and the cumulative incidence of aGVHD in accordance with a recent study in HLA-matched unrelated donor (URD) transplant [25]. We did not also find association between HLA-E and the cumulative incidence of cGVHD in contrast to Hosseini et al. [26] who recorded reduced risk with HLA-E*01:03/01:03 genotype. The reduced post transplantation complications reported by Tamouza et al. [21,22] and Danzer et al. [23] with HLA-E*01:03 genotype was converted into reduced TRM and improved OS with HLA-E*01:03/01:03 genotype compared to HLA-E*01:01/01:01 genotype in related and unrelated HSCT. Similarly, Bogunia-Kubik et al. [43,44] and Hosseini et al. [26] found better overall survival with HLA-E*01:03/01:03 genotype compared to HLA-E*01:01/ 01:01 genotype. In our cohort, no association was observed between HLA-E polymorphism and the 2-years cumulative incidence of TRM or OS in accordance with Fürst et al. [25] in matched URD. The suggested association of HLA-E polymorphism with reduced risk of relapse needs verification in a larger cohort. However, its proposed role in GVL helps better understanding of alloreactivity of T cells and NK cells and their implication in immunotherapy post ASCT.
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Q1 Please cite this article in press as: Mossallam GI et al. HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.12.017
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[26] Hosseini E, Schwarer AP, Ghasemzadeh M. The impact of HLA-E polymorphisms in graft-versus-host disease following HLA-E matched allogeneic hematopoietic stem cell transplantation. Iran J Allergy Asthma Immunol 2012;11(1):15–21. [27] Hosseini E, Schwarer AP, Jalali A, Ghasemzadeh M. The impact of HLA-E polymorphisms on relapse following allogeneic hematopoietic stem cell transplantation. Leuk Res 2013;37(5):516–9. [28] Matte C, Lacaille J, Zijenah L, Ward B, Roger M. HLA-G and HLA-E polymorphisms in an indigenous African population. The ZVITAMBO study group. Hum Immunol 2000;61:1150–6. [29] Weisdorf D, Zhang MJ, Arora M, Horowitz MM, Rizzo JD, Eapen M. Graftversus-host disease induced graft-versus-leukemia effect: greater impact on relapse and disease-free survival after reduced intensity conditioning. Biol Blood Marrow Transplant 2012;18(11):1727–33. [30] Chen Y, Xu Y, Fu G, Liu Y, Peng J, Fu B, et al. Allogeneic hematopoietic stem cell transplantation for patients with acute leukemia. Chin J Cancer Res 2013;25(4):389–96. [31] Ruggeri L, Capanni M, Tosti A, Urbani E, Posati S, Aversa F, et al. Innate immunity against hematological malignancies. Cytotherapy 2002;4(4): 343–6. [32] Nguyen S, Beziat V, Dhedin N, Kuentz M, Vernant JP, Debre P, et al. HLA-E upregulation on IFN-gamma-activated AML blasts impairs CD94/NKG2Adependent NK cytolysis after haplo-mismatched hematopoietic SCT. Bone Marrow Transplant 2009;43(9):693–9. [33] Haas P, Loiseau P, Tamouza R, Cayuela JM, Moins-Teisserenc H, Busson M, et al. NK-cell education is shaped by donor HLA genotype after unrelated allogeneic hematopoietic stem cell transplantation. Blood 2011;117(3):1021–9. [34] Michaëlsson J, Teixeira de Matos C, Achour A, Lanier LL, Kärre K, Söderström K. A signal peptide derived from hsp60 binds HLA-E and interferes with CD94/ NKG2A recognition. J Exp Med 2002;196(11):1403–14. [35] Sedlackova L, Spacek M, Holler E, Imryskova Z, Hromadnikova I. Heat-shock protein expression in leukemia. Tumour Biol 2011;32(1):33–44. [36] Foley B, Cooley S, Verneris MR, Pitt M, Curtsinger J, Luo X, et al. Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated NKG2C+ natural killer cells with potent function. Blood 2012;119(11):2665–74. http://dx.doi.org/10.1182/blood-2011-10386995. Epub 2011 Dec 16. [37] Linn YC, Wang SM, Hui KM. Comparative gene expression profiling of cytokine-induced killer cells in response to acute myloid leukemic and acute lymphoblastic leukemic stimulators using oligonucleotide arrays. Exp Hematol 2005;33(6):671–81. [38] Laport GG, Sheehan K, Baker J, Armstrong R, Wong RM, Lowsky R, et al. Adoptive immunotherapy with cytokine-induced killer cells for patients with relapsed hematologic malignancies after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2011 Nov;17(11):1679–87. [39] Linn YC, Niam M, Chu S, Choong A, Yong HX, Heng KK, et al. The anti-tumour activity of allogeneic cytokine-induced killer cells in patients who relapse after allogeneic transplant for haematological malignancies. Bone Marrow Transplant 2012 Jul;47(7):957–66. [40] Nishimura R, Baker J, Beilhack A, Zeiser R, Olson JA, Sega EI, et al. In vivo trafficking and survival of cytokine-induced killer cells resulting in minimal GVHD with retention of antitumor activity. Blood 2008;112(6):2563–74. http://dx.doi.org/10.1182/blood-2007-06-092817. Epub 2008 Jun 18. [41] Sievers E, Neuberg J, Starkmuth M, Frank S, Strehl J, Schmidt-Wolf IG. Cytotoxicity of a CD94-enriched cell fraction from cytokine-induced killer (CIK) cells against tumor cells. Curr Trends Immun 2009;10:41–8. [42] Sensi M, Pietra G, Molla A, Nicolini G, Vegetti C, Bersani I, et al. Peptides with dual binding specificity for HLA-A2 and HLA-E are encoded by alternatively spliced isoforms of the antioxidant enzyme peroxiredoxin 5. Int Immunol 2009;21(3):257–68. [43] Bogunia-Kubik K, Jaskuła E, Polak M, et al. Non classical HLA-E and G gene polymorphisms affect the HSCT outcome. Bone Marrow Transplant 2010;45(Suppl. 2):S135 (abstract P533). [44] Bogunia-Kubik K, Polak M, Kos´cin´ska K, et al. Chromosome 6gene polymorphisms as the factors affecting the risk of HSCToutcome. Eur J Immunol 2009;39(Suppl. 1):S734 (abstract PD12/10).
Q1 Please cite this article in press as: Mossallam GI et al. HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.12.017
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HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients Ghada I. Mossallam a,⇑, Raafat Abdel Fattah b, Alaa El-Haddad c, Hossam K. Mahmoud b a
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Bone Marrow Transplantation Laboratory Unit, National Cancer Institute, Cairo University, Cairo, Egypt Medical Oncology Department, National Cancer Institute, Cairo University, Cairo, Egypt c Pediatric Oncology Department, National Cancer Institute, Cairo University, Cairo, Egypt b
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Article history: Received 20 June 2014 Accepted 15 December 2014 Available online xxxx
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Keywords: HLA-E Hematopoietic stem cell transplantation Graft versus leukemia (GVL) Relapse
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a b s t r a c t Human leukocyte antigen-E (HLA)-E in a non-classical major histocompatibility complex (MHC) class I (Ib) molecule. HLA-E-peptide complex acts as a ligand for natural killer (NK) cells and CD8+ T lymphocytes playing a dual role in natural and acquired immune responses. The difference in expression levels between HLA-E alleles was suggested to have impact on transplantation outcome. The aim of the study is to evaluate the clinical impact of HLA-E alleles on transplantation in a group of Egyptian patients. HLA-E genotyping was analyzed in eighty-eight recipients of stem cell transplantation using polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP). HLA-E*01:03 allele showed a trend toward lower cumulative incidence of relapse at 2 years compared to homozygous HLA-E*01:01 genotype (8% versus 21.5%, p = 0.09, HR: 0.30, CI: 0.91–1.69). HLA-E was the only factor showing near significant association with relapse incidence. HLA-E polymorphism did not affect the cumulative incidence of acute GVHD grades II–IV at 100 days, the 2-years cumulative incidence of extensive chronic GVHD, transplant related mortality (TRM) or overall survival (OS). Conclusion: the suggested association of HLA-E polymorphism with reduced risk of relapse needs verification in a larger cohort. However, its proposed role in GVL helps better understanding of alloreactivity of T cells and NK cells and their implication in immunotherapy post allogeneic hematopoietic stem cell transplantation. Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
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1. Introduction
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Allogeneic hematopoietic stem-cell transplantation (AHSCT) is a curative therapy for many hematologic malignancies. Post transplantation complications are mainly related to the development of graft versus host disease (GVHD), infection and relapse. Human leukocyte antigen-E (HLA-E) in a non-classical major histocompatibility complex (MHC) class I (Ib) molecule characterized by limited polymorphism and lower cell surface expression compared to classical Class Ia molecule [1,2]. It is an immunomodulatory molecule that can function as both immuno-tolerogenic and immuno-activating molecule and plays a dual role in natural and acquired immune responses [3,4]. The HLA-E-peptide complex can act as a ligand for the CD94/NKG2 receptors expressed on the surface of natural killer (NK) cells and represents a restriction element for TCR receptor of CD8+ T cells [5–7]. HLA-E molecule
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⇑ Corresponding author at: National Cancer Institute, Cairo University, Fom ElKhalig Square, Kasr El-Aini St., 11796 Cairo, Egypt. E-mail address: [email protected] (G.I. Mossallam).
binds and presents a restricted set of peptides derived from self or foreign proteins [8]. It binds nonamer peptides derived from signal sequences of other HLA class I molecules including HLA-A, -B,C, and -G [4,9]. Its surface expression is regulated by the availability of these peptides [10]. HLA-E is expressed on the extravillous trophoblasts and mediates maternal tolerance to semiallogeneic fetal graft [11]. HLA-E can also bind viruses [12,13], bacteria [14], tumors [4], stress-related peptides [7], and self-antigens [15]. Recognition by CD94/NKG2 is governed by the sequence of peptide bound to HLA-E [16]; CD94/NKG2A functions as an inhibitory receptor, while CD94/NKG2C functions as an activating receptor [17]. HLA-E mediates lysis of targets by CD8+ cytotoxic T cell (CTL) including NKCTL and regulates adaptive immune response through CD8+ suppressive T cells [5–7,18]. Two nonsynonymous functional alleles of HLA-E have been found, E*01:01 and E*01:03 [19]. An arginine at position 107 located within the a2 domain of the HLA-E heavy chain in HLAE107A (HLA-E*01:01) is replaced by a glycine in HLA-E107G (HLAE*01:03) [20]. The functional differences between the HLA-E alleles are related to the relative peptide affinity and cell surface
http://dx.doi.org/10.1016/j.humimm.2014.12.017 0198-8859/Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
Q1 Please cite this article in press as: Mossallam GI et al. HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.12.017
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expression, with the HLA-E*01:03 allele expressed at a higher levels than *01:01, due to its higher affinity to peptides and therefore higher surface stability [4,10]. HLA-E polymorphism has been associated with infection, cancer, recurrent spontaneous abortion, autoimmune diseases, and transplantation outcome [4,11,13–15,21]. HLA-E polymorphism was found to affect HSCT outcome; reports pointed to the association of HLA-E*01:03 genotype with protection from graft versus host disease and relapse as well as better survival, while HLAE*0101 homozygosity was associated with risk of infection post AHSCT [21–27]. The aim of the study is to evaluate the clinical impact of HLA-E alleles on transplantation in Egyptian patients after AHSCT from HLA-matched sibling donors.
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2. Patients and methods
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2.1. Patients
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The cohort consisted of 88 recipients transplanted from their HLA-identical siblings at Bone Marrow Transplantation (BMT) Unit, National Cancer Institute, Cairo University and at Nasser Institute between 2007 and 2010. Informed consent was obtained. They were 51 males and 37 females. Their median age was 30 years, range (2–48). They suffered from: acute myeloid leukemia (AML) 52/88(59.1%), chronic myeloid leukemia (CML) 21/88(23.9%) and myelodysplastic syndrome (MDS) 15/88(17%). Patients’ characteristics are mentioned in Table 1. Donor selection was done using serologic method (BIO-Rad) for HLA class I typing, while sequence specific oligonucleotide probe (SSOP) method (Inno-Lipa HLA-DRB1 plus) was used for class II typing. HLA-E genotyping was performed for recipients using polymerase chain reactionrestricted fragment length polymorphism.
80 81 82 83 84 85 86 87 88 89 90 91
96 97 98 99 100 101 102 103 104 105 106 107 108
2.1.1. Conditioning regimens and GVHD prophylaxis Conditioning regimens included Flu/Bu: Fludarabine 30 mg/m2/ d IV daily for 4 days (D-10, D-9,D-4 and D-2) and Busulphan 4 mg/ kg p.o. daily for 4 days (D-8 to D-5); Flu/Alk: Fludarabine 30 mg/ m2/d IV daily for 4 days (D-7 to D-4) and Alkeran (Melphalan) 70 mg/m2/d IV for 2 days (D-3 and D-2); Bu/cy: Busulphan 4 mg/ kg p.o. daily for 4 days (D-7 to D-4) and Cyclophosphamide 60 mg/kg/d IV for 2 days (D-3 and D-2) given with 2-mercaptoethane sulfonate sodium (MESNA). GVHD prophylaxis was given by Cyclosporin-A (CSA) 3 mg/kg IV from day 1 to be changed to oral form (3 mg/kg) once the patient can swallow for 9–12 months post-transplant. GVHD prophylaxis included also short course IV methotrexate (15 mg/m2 day 1 and 10 mg/m2 days 3 and 6) with leucovorin rescue and mycophenolate mofetil (MMF) 1000 mg twice daily PO.
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2.2. HLA-E typing by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP)
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DNA was extracted from peripheral blood of recipients using QIAamp DNA Mini Kit (Qiagen, Germany), according to the manufacturer’s protocol. Detection of HLA-E polymorphism was performed using PCR-RFLP [28]. PCR reaction consisted of 25 ll, containing 100 ng DNA, 10 pmol of each primer: forward primer: 50 -GGCTGCGAGCTGGGGCCCGCC-30 , reverse primer: 50 -AGCCCTG TGGACCCTCTT-30 , 1X Go Taq buffer including 1.5 mM MgCl2, 1.25 U Go Taq DNA polymerase (Promega, Madison, USA) and 0.2 mM each dNTP (Thermo Scientific, Fermentas). The PCR cycling conditions consisted of initial denaturation at 94 °C for 5 min. This was followed by 35 cycles of 94 °C for 45 s, 61 °C for 45 s and 72 °C for 45 s then final extension step at 72 °C for 7 min. The resulted fragment by visualization on 2% gel was
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Table 1 Patient characteristics.
Age median (range) (y) Sex Male Female Disease at transplantation, n (%) Acute myeloid leukemia Myelodysplastic syndrome Chronic myeloid leukemia Stage of disease, n (%) Low, intermediate High Prior therapy, n (%) No Yes Gender combination (Recipient/Donor), n (%) M/M M/F F/F F/M Source of stem cells, n (%) Bone Marrow Peripheral blood Conditioning regimen, n (%) Myeloablative Busulfan/Cyclophosphamide Busulfan /Fludarabine Reduced intensity Fludarabine /Melphalan GVHD prophylaxis, n (%) Cyclosporine /Methotrexate Cyclosporine/MMF
Entire group n = 88
01:03 allele n = 54
01:01/01:01 n (34)
p-Value
30 (2–48)
31 (15–45)
29 (2–48)
0.21
53 (60.2) 35 (39.8)
34 (63) 20 (37)
19 (55.9) 15 (44.1)
0.66
52 (59.1) 15 (17) 21 (23.9)
30 (55.5) 9 (16.7) 15 (27.8)
22 (64.7) 6 (17.6) 6 (17.6)
0.55
79 (89.7) 9 (10.3)
47 (87) 7 (13)
32 (94.1) 2 (5.9)
0.45
34 (38.6) 54 (61.4)
23 (42.6) 31 (57.4)
11 (32.4) 23 (67.6)
0.46
29 22 13 24
(33) (25) (14.8) (27.2)
16 (29.7) 17 (31.6) 9 (16.4) 12 (22.3)
13 (38.2) 5 (14.7) 4 (11.7) 12 (35.4)
0.23
4 (4.5) 84 (95.5)
1 (1.9) 53 (98.1)
3 (8.8) 31 (91.2)
0.31
72 (81.8)
46 (85.2)
26 (76.4)
0.45
16 (18.2)
8 (14.8)
8 (23.6)
68 (77.2) 20 (22.8)
44 (81.5) 10 (18.5)
24 (70.6) 10 (29.4)
0.41
Low, intermediate: AML(CR1), CML (chronic phase) and MDS; High: AML (CR2) and CML (accelerated phase).
Q1 Please cite this article in press as: Mossallam GI et al. HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.12.017
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HLA-E*03:03/03:03 in 15/88 (17%). The allele frequency was 60.8% for HLA*01:01 allele and 39.2% for HLA*01:03 allele. The distribution of genotypes was in Hardy–Weinberg equilibrium (p > 0.05).
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280 bp. Allele determination was carried out by digestion of the amplified PCR products using the restriction enzyme HpaII (Thermo Scientific, Fermentas). The presence of HLA*01:03 allele was identified by the presence of a restriction site for the enzyme.
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2.3. Statistical methods
3.2. Association of HLA-E genotype with clinical outcome
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The cumulative incidence of aGVHD II–IV at 100 days did not show significant difference between recipients harboring HLAE*01:03 allele compared to those homozygous for HLA-E*01:01 genotype (p = 0.54, HR: 0.80, 95% CI: 0.52–2.80). Similarly, extensive cGVHD did not show significant difference in the cumulative incidence at 2 years in recipients with HLA-E*01:03 allele compared to those homozygous for HLA-E*01:01 genotype (p = 0.93, HR: 0.97, 95% CI: 0.49–2.16) (Table 2) (Figs. 1 and 2). The median duration of follow-up was 41 months, range (0.5–65). The cumulative incidence of relapse at 2 years was 9.5%. HLA-E*01:03 allele showed a trend toward lower cumulative
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Numerical data were expressed as median and range. Hardy– Weinberg equilibrium was tested using Pearson’s chi-squared test. Qualitative data were expressed as frequency and percentage. Chi-square test (Fisher’s exact test) was used to examine the relation between qualitative variables. Survival analysis was done using Kaplan–Meier method. Comparison between survival curves was done using log-rank test. Estimation of the hazard ratio was done using cox-regression model. Hazard ratio with its 95% confidence interval (CI) was used for risk assessment. Survival package was used to test the proportional hazards assumption. The assumption (proportionality) of cox proportional hazard models were met. Relapse incidence was estimated by the cumulative incidence method with death without relapse treated as a competing risk. Transplant related mortality (TRM) was calculated using cumulative incidence with relapse treated as a competing. A p value < 0.05 was considered significant.
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3. Results
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3.1. Frequency of HLA-E genotypes and alleles
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Analyzing HLA-E genotype, HLA-E*01:01/01:01 was detected in 34/88 (38.6%), HLA-E*01:01/01:03 in 39/88 (44.4%) and
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Table 2 Association of HLA-E genotype with transplantation outcome.
Acute GVHD Chronic GVHD Relapse Transplant related mortality Overall survival
p-Value
Hazards ratio
95% CI
0.54 0.93 0.09 0.53 0.47
0.80 0.97 0.30 0.54 0.78
0.58–2.80 0.49–2.16 0.91–1.69 0.31–2.88 0.40–1.52
Fig. 2. Cumulative incidence of cGVHD according to HLA-E genotype. No significant difference in the cumulative incidence of extensive cGVHD in recipients harboring HLA-E*01:03 allele compared to those homozygous for HLA-E*01:01 genotype (p = 0.93).
HLA-E*01:03 allele compared to homozygous HLA-E*01:01.
Fig. 1. Cumulative incidence of aGVHD according to HLA-E genotype. No significant difference in the cumulative incidence of aGVHD II-IV in recipients harboring HLAE*01:03 allele compared to those homozygous for HLA-E*01:01 genotype (p = 0.54).
Fig. 3. Cumulative incidence of relapse according to HLA-E genotype. Relapse incidence was estimated by the cumulative incidence method with death without relapse treated as a competing risk. HLA-E*01:03 allele showed a trend toward lower cumulative incidence of relapse compared to homozygous HLA-E*01:01 genotype (p = 0.09).
Q1 Please cite this article in press as: Mossallam GI et al. HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.12.017
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Table 3 Analysis of factors affecting relapse incidence.
HLA-E# Age (<30 y versus >30 y) Prior therapy (Yes versus No) Stage of disease (low, intermediate versus high) Disease at presentation (CR1, CML versus CR2, accelerated phase) Conditioning regimen (MA versus RIC) GVHD (Yes versus No)
p-Value
Hazards ratio
95% CI
0.09 0.39
0.30 1.81
0.91–1.69 0.45–7.26
0.28 0.45
0.42 2.22
0.49–11.38 0.27–18.34
0.58
0.75
0.07–18.67
0.75
1.28
0.26–6.19
0.29
0.48
0.52–8.40
#
HLA-E*01:03 allele versus homozygous HLA-E*01:01; y: year; CR: complete remission; MA: myeloablative; RIC: reduced intensity conditioning.
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incidence of relapse at 2 years compared to homozygous HLA-E*01:01 genotype (8% versus 21.5%, p = 0.09, HR: 0.30, CI: 0.91–1.69) (Fig. 3). Analyzing other factors affecting relapse, HLAE was the only factor showing near significant association with relapse incidence (Table 3). The cumulative overall survival (OS) at 2 years was 60%; no significant difference was observed between HLA-E*01:03 allele compared to homozygous HLA-E*01:01 genotype (60% versus 58%, p = 0.47, HR: 0.78, 95% CI: 0.40–1.52). The 2-years cumulative incidence of transplant related mortality (TRM) was 29.5%; the incidence was 31% versus 34% in HLA-E*01:03 allele compared to homozygous HLA-E*01:01 genotype, respectively (p = 0.53, HR = 0.54, 95% CI: 0.31–2.88) (Table 2). The causes of TRM were infection (n = 18), acute and chronic GVHD (n = 6), organ failure (n = 2). Infection as a cause of death occurred in 26.4% of HLAE*01:01 homozygous patients compared to 16.7% of those with HLA-E*01:03 allele, the difference, however, was not statistically significant (p = 0.26, HR: 1.80, 95% CI: 0.63–5.11).
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4. Discussion
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The HLA-E-peptide complex acts as a ligand for NK cells and cytotoxic T lymphocytes. The difference in expression levels between HLA-E alleles was suggested to have impact on transplantation outcome. In our cohort, a trend toward reduced cumulative incidence of relapse at 2 years was found with HLA-E*01:03 allele compared to HLA-E*01:01/01:01 genotype. Contrary to Weisdorf et al. [29] and Chen et al., [30], GVHD and disease stage did not affect risk of relapse after allogeneic transplantation. Our observation coincides with the recently reported data by Hosseini et al. [27], where an association was found between HLA-E*01:03 homozygosity and decreased incidence of relapse and better DFS. This finding was, however, not confirmed by other studies [22,23,25]. Graft versus leukemia (GVL) activity in preventing leukemia relapse is mediated by alloreactive donor CD8+ T cells and NK cells [4,31]. Our data suggest effective GVL effect in patients with HLA-E*01:03 allele. This effect is probably related to the higher affinity of HLA-E*01:03 allele to peptides and its higher cell surface expression compared to HLA-E*01:01/01:01. Although inhibition of immature NK after haploidentical transplantation through CD94/NKG2A recognition was observed by Nguyen et al. [32] and Haas et al. [33], however, did not find relationship between HLA-E genotype and NKG2A+KIR NK-cell response in unrelated transplant. Alternatively, activation may result from binding peptides other than HLA nonamers leading to competitive relief of CD94/NKG2A mediated inhibition [34]. Heat-shock proteins e.g. Hsp60 overexpressed on leukemic cells decreases inhibition via CD94/NKG2A [34,35]. On the other hand, binding
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of the activation receptors NKG2C rather than the NKG2A inhibitory receptors activates NK cells [17]. The role of NKG2C receptor post ASCT was recently supported by Foley et al. [36] who suggested that cytomegalovirus viremia-induced NKG2C may play role in eradication of residual leukemic cells besides controlling infectious disease. In a study conducted by Linn et al. [37], the NKG2C was upregulated exclusively in the ex vivo-expanded cytokine induced killer (CIK) (CD3+CD56+) cells that were cytolytic to susceptible AML targets. The role of CIK cells was supported in managing relapse post allogeneic HSCT with minimal GVHD [38–40]. CIK cells cytotoxicity could be improved after antibodydirected stimulation of NKG2C and CD94 [41].Other HLA-E-peptides are recognized by the CD8+ CTL mediating cytotoxicity and cytokine secretion, HLA-E may bind non-standard minor histocompatibility antigens (mHags) peptides that specifically mediate GVL [22]. Its independence of classic HLA class I in addition to its wide reactivity make it highly responsive to allogeneic stimulation [5]. Peptides with dual HLA-class I-A2 and HLA-E binding specificity were identified for the antioxidant enzyme peroxiredoxin expressed on stressed and malignant cells, contributing to T cell activation [42]. In line with the suggested defective presentation of HLA-E 0101 allele, Tamouza et al. [21,22] reported increased incidence of bacterial infection with HLA-E*0101 homozygous donor in URD transplant due to inefficient presentation of bacterial peptides. This association was, however, not found in our cohort may be due to the lower incidence of infection in related donor transplant. On the other hand, reports studying the effect of HLA-E polymorphism on aGVHD showed protective role for the HLA-E*01:03 genotype in related and unrelated transplants [22–24,26]. Tamouza et al. [22], claimed that competition of HLA-E*0103 with classic class I for mHag presentation to T cell protect from NK-mediated tissue damage. In our series, no association was observed between HLA-E polymorphism and the cumulative incidence of aGVHD in accordance with a recent study in HLA-matched unrelated donor (URD) transplant [25]. We did not also find association between HLA-E and the cumulative incidence of cGVHD in contrast to Hosseini et al. [26] who recorded reduced risk with HLA-E*01:03/01:03 genotype. The reduced post transplantation complications reported by Tamouza et al. [21,22] and Danzer et al. [23] with HLA-E*01:03 genotype was converted into reduced TRM and improved OS with HLA-E*01:03/01:03 genotype compared to HLA-E*01:01/01:01 genotype in related and unrelated HSCT. Similarly, Bogunia-Kubik et al. [43,44] and Hosseini et al. [26] found better overall survival with HLA-E*01:03/01:03 genotype compared to HLA-E*01:01/ 01:01 genotype. In our cohort, no association was observed between HLA-E polymorphism and the 2-years cumulative incidence of TRM or OS in accordance with Fürst et al. [25] in matched URD. The suggested association of HLA-E polymorphism with reduced risk of relapse needs verification in a larger cohort. However, its proposed role in GVL helps better understanding of alloreactivity of T cells and NK cells and their implication in immunotherapy post ASCT.
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Q1 Please cite this article in press as: Mossallam GI et al. HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.12.017
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Q1 Please cite this article in press as: Mossallam GI et al. HLA-E polymorphism and clinical outcome after allogeneic hematopoietic stem cell transplantation in Egyptian patients. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.12.017
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