ORIGINAL ARTICLE
Impact of HLA-G polymorphism on the outcome of allogeneic
hematopoietic stem cell transplantation for metastatic renal
cell carcinoma
R Crocchiolo1, O Ringden2, J-O Bay3, D Blaise4, B Omasic2, B Mazzi1, C Picard5, S Trinca1, L Barkholt2, J Peccatori1, S Gregori6, G Amodio6, K Fleischhauer1, F Ciceri1,7and M Bregni1,7on behalf of the STWP and CTIWP of EBMT
Renal cell carcinoma (RCC) is particularly sensitive to immune intervention. HLA-G, a non-classical HLA class I molecule with immunomodulatory properties, has been studied with regard to outcome after hematopoietic stem cell transplantation (HSCT), in particular the 14 bp insertion/deletion polymorphism in the 3′ untranslated region. Here we analyzed n = 56 patients affected by metastatic RCC who received an allogeneic HSCT between 1998 and 2006 in Milano, Marseille, Clermont-Ferrand and Stockholm. The 14 bp polymorphism was analyzed in correlation with overall survival (OS), PFS, acute and chronic GvHD. With a median follow-up of 13 years, a trend towards better outcome was observed when homozygosity for the 14bp-del allele was present: multivariate hazard ratio was 0.50 (95% confidence interval (CI): 0.23–1.13; P = 0.10) and 0.57 (95% CI: 0.26–1.26; P = 0.17) for OS and PFS, respectively, when 14bp-del/del was compared with 14bp-ins/X. Further exploratory analysis revealed a significant association between T/C at p3003 and improved OS (P = 0.05) and PFS (P = 0.006) compared with T/T. To our knowledge this is thefirst study on HLA-G and outcome after HSCT for a solid malignancy. After a coordinated multicenter study, we found that the more tolerogenic polymorphisms (14bp-del/del) is associated with better PFS and OS. Thefinding on p3003 deserves further investigation. Bone Marrow Transplantation (2018)53, 213–218; doi:10.1038/bmt.2017.243; published online 13 November 2017
INTRODUCTION
A graft-versus-tumor (GvT) effect has been postulated in leukemia1,2and other malignancies3after allogeneic hematopoie-tic stem cell transplantation (HSCT), to explain responses not attributable to chemotherapy. In solid tumors, a GvT effect has been consistently reported in renal cell carcinoma (RCC).4Efforts have been made to identify the targets of the GvT effect: among them, strong candidates are minor histocompatibility antigens (mHags),5 other putative tumor-associated antigens such as carbonic anhydrase IX,6 and a 10-amino-acid peptide antigen encoded from HERV-E endogenous retrovirus sequences.7 How-ever, no definitive evidence has been found. Meanwhile, clinical and laboratory parameters to identify patients that could benefit most from this complex procedure have been actively investigated.8,9
Human leukocyte antigen-G is a human non-classical HLA class I molecule expressed in immune privileged sites, which plays an essential role in promoting fetus–maternal tolerance.10–12 By alternative splicing, HLA-G generates seven different isoforms, four of which are membrane-bound (HLA-G1, G2, G3 and G4) and three are soluble (HLA-G5, G6 and G7). HLA-G modulates immune responses through several non-exclusive mechanisms: it inhibits cytolytic activities of both NK and CD8+cytotoxic T cells, and allo-specific T-cell proliferation.13
HLA-G expression is induced after allogeneic transplantation in vivo and expression of HLA-G by CD4+ T cells during mixed lymphocyte reaction inhibits allo-specific T-cell proliferation, suggesting that HLA-G plays a role in
modulating alloresponses.14–18 Human leukocyte antigen-G can also be expressed in pathological conditions, such as in flamma-tion, autoimmunity and cancer. Human leukocyte antigen-G expression has been identified in many solid tumors including colon cancer19and melanoma,20as well as in leukemia.21In these contexts, HLA-G acts as a negative regulator of the immune response through different mechanisms such as inhibition of angiogenesis, prevention of antigen recognition and T-cell migration, and suppression of T- and NK-cell cytotoxicity.
The existence of a 14 bp insertion or deletion in the 3′ untranslated region (UTR) of exon 8 of HLA-G is known to affect the stability of HLA-G mRNA and consequently the expression of HLA-G.22,23 In line with this notion, the 14 bp insertion/deletion polymorphism of HLA-G proved to be associated with tolerance in different clinical conditions including autoimmunity and patholo-gical pregnancy. Importantly, the presence of 14 bp deletion has been found to be predictive for the incidence of GvHD after unrelated donors HSCT for beta-thalassemia,24,25suggesting a role for this polymorphism in the establishment of immunological tolerance also in the context of HSCT. Its importance after allografting for hematologic malignancies, as well as for solid tumors, has so far not been determined.
We therefore intended to assess whether the 14 bp polymorph-ism of HLA-G may be a predictive marker after allogeneic HSCT for metastatic renal cell cancer. Other polymorphisms occurring in the HLA-G sequence were also analyzed together with the entire 3′ 1
San Raffaele Hospital, Milano, Italy;2
Karolinska University Hospital, Stockholm, Sweden;3
CHU Clermont Ferrand, Clermont Ferrand, France;4
Institut Paoli-Calmettes, Aix-Marseille University, CNRS, INSERM, CRCM, Marseille, France;5
Aix Marseille Univ, CNRS, EFS, ADES, "Biologie des Groupes Sanguins", Marseille, France and6
San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milano, Italy. Correspondence: Dr R Crocchiolo, San Raffaele Hospital, Via Olgettina 60, 20132 Milano, Italy. E-mail: roberto.crocchiolo@gmail.com
7
These authors contributed equally to this work.
Received 23 July 2017; revised 29 August 2017; accepted 8 September 2017; published online 13 November 2017
UTR haplotypes as obtained from the combination of the single nucleotide polymorphisms (SNPs). Clinical parameters such as acute or chronic GvHD, overall survival (OS), PFS, transplant-related mortality (TRM) and rejection, have been analyzed.
PATIENTS AND METHODS
The study was conducted on behalf of the Solid Tumor and the Cellular Therapy & Immunobiology Working Parties of the European Society for Blood and Marrow Transplantation (EBMT) and included four participating centers: San Raffaele Hospital in Milano, Institut Paoli-Calmettes in Marseille, CHU Clermont Ferrand, Karolisnka University Hospital in Stockholm.
Patients affected by advanced stage RCC who received an allogeneic HSCT from an HLA-identical sibling or from an unrelated donor were included. The HSCTs were performed between 1995 and 2006 in the above cited centers, within a dedicated transplant program for patients affected by advanced-stage solid tumors treated with several therapeutic lines and who were supposed to benefit from the adoptive immunotherapy represented by HSCT, that is the GvT effect. The target dose for the infused stem cells was 4–8 × 106CD34+/kg of recipient and no ex-vivo graft manipulation was planned; therefore, the infused CD3+ were all those contained in the graft. The HLA-G 14 bp-ins/del polymorphism analysis was performed retrospectively from samples obtained by patients and/or their respective donors. Additionally, polymorphism analyses of the positions 3003, 3010, 3027, 3035, 3142, 3187, 3196, 3121 as well as the corresponding 3′UTR haplotypes (namely 1 to 7, 13) obtained from the combination of the SNPs26 were conducted in an attempt to provide exploratory data for further research. The study was approved by the IRB of each participating center and informed consent was obtained from the patients and the donors.
Sequence-based typing of the HLA-G 3′UTR
Human leukocyte antigen 3′UTR polymorphisms were determined on genomic DNA from peripheral blood by direct sequencing. Briefly, a 402 bp fragment encompassing bp +2885 to +3287 of the 3′UTR, was amplified using HLA-G_F: 5′-TCACCCCTCACTGTGACTGA-3′ and HLA-G_R: 5′-TTCTCATGTCTTCCATTTATTTTGTC-3′, as forward and reverse primers, respectively. Cycler conditions were: 95 °C for 15 min followed by 30 cycles at 93 °C for 1 min, 58 °C for 1 min and 72 °C for 1 min; the extension step was carried out at 72 °C for 10 min. The amplicon, encompassing the 14 bp
ins/del at position +2961 (classified as ins/ins, ins/del or del/del), and the seven SNPs described by Castelli et al.26namely at positions +3003, +3010, +3027, +3035, +3142, +3187, +3196 and +3121 was subjected to direct sequencing after purification by ExoSAP-IT (Affymetrix, Regensburg, Germany).
Statistical analysis
Data on main pre-, peri- and post-transplant variables were collected from the participating centers and information were updated as of September 2016. Pre-transplant variables were: histology, disease sites at HSCT, stage, patient and donor age, gender and CMV serostatus, donor type (HLA-identical sibling, unrelated donor); peri-transplant: conditioning regimen, HSCT date; post-transplant: acute GvHD date and grading according to Glucksberg et al.,27chronic GvHD date and severity,28date of progression, date and status at last follow-up, cause of death. Estimates of OS and PFS were calculated using the Kaplan–Meier method. Rates of acute GvHD, chronic GvHD and TRM were provided. Owing to the results obtained in a preliminary analysis on the same population29 the HLA-G 14 bp polymorphisms‘ins/ins’ and ‘ins/del’ were grouped together and results compared with the one ‘del/del’, on a pre-specified basis, both for univariate and multivariate analyses. Because of the presence of multiple and heterogeneous confounding factors potentially affecting GvHD (that is, the duration of post-transplant immune suppression based on disease stage and/or center policies and/or protocols, previous therapies, donor type) or TRM (conditioning intensity, patient age and/or comorbidities), and in order to enhance the strength of results from a clinical perspective and in the light of potential future speculations, the multivariate models were built only for OS and PFS, after adjusting for the main above specified variables using Cox regression. Exploratory analysis conducted on SNPs at positions 3003, 3010, 3027, 3035, 3142, 3187, 3196 and 3121 included univariate comparisons with OS and PFS for each locus. The same analysis was performed using the 3′UTRs, after having pooled patients into a three-group (homozygous for the 3′UTR ‘2’, heterozygous for the 3′UTR ‘2’, else) and a four-group built on the association between the 14 bp ins/del and the +3142 G/C SNP (insG/insG, insG/delC, delC/delC, 3′UTR ‘3’/x) classifications, both based on the expected amount of HLA-G expression. Whenever a different polymorphism was disclosed between patient and donor, the one from the donor was used with the exception of those patients who suffered from graft rejection. No adjustment for multiple testing was made. P-value o0.05 was considered statistically significant. Analyses were performed using the SPSS software (v13.0 for Windows).
Table 1a. Main patient characteristics
Characteristic Total (N = 56)
Median age (range) 49 (23–66)
Male sex 38
ECOG performance status
0 41 1 10 2 5 Histology Clear cell 52 Non-clear cell 0 Missing 4 Site involvementa Nodes 31 Lung 42 Bone 4 Liver 10 Local relapse 10 CNS 3 Other 1
Previous cytokine treatment 54
aMultiple involvements.
Table 1b. Transplant and outcome characteristics
Characteristic Total (N = 56) Donor HLA-id sibling 48 Matched unrelated 8a Conditioning regimen Thio/Flu/Cy 25 Bu/Flu 10 Flu/Cy 15 Fludarabine 1 Flu/TBI 2 5 Engraftment 52 aGvHD 37 aGvHD G3-4 18 cGvHD 21 Extensive cGvHD 5 Day 100-TRM 9 (16%) 1-year OS 31(55%) cGvHD 21 Extensive cGvHD 5 Day 100-TRM 9 (16%) 1-year OS 31(55%)
Abbreviations: aGvHD= acute GvHD; cGvHD = chronic GvHD; TRM = transplant-related mortality; OS= overall survival.
a
All donors were 10/10-matched at allele typing.
RESULTS
A total of 56 patients were included in the present analysis, of which 51 were evaluable for the HLA-G 14 bp polymorphism. Since the results of the HLA-G 14 bp polymorphism were found to be identical for patient and donor in thefirst 27 pairs, the typing of the 24 additional transplants was performed on patients samples only, by the assumption that 14 bp polymorphism was identical in the presence of HLA compatibility.
The clinical variables among patients and donors are shown in Table 1(a). The median year of HSCT was 2002 and the most used conditioning regimen was the association of thiotepa-fludarabine-ciclophosphamide (n = 25) followed by fludarabine-cyclophosphamide (n = 15). The remaining were busulfan-based regimens (n = 9),fludarabine-total body irradiation 2 Gy (n = 5) orfludarabine alone (n = 1). Donors were HLA-identical siblings in the majority of cases (n = 47, 85%) (Table 1 (b)). When analyzed with respect to the HLA-G 14 bp polymorphism, no statistically different distribution was observed among the conditioning regimens (P = 0.16) nor according to time to engraftment (P = 0.87).
As of September 2016, three patients are alive at +12 years, +13.5 years and +14 years after HSCT, having received further treatments due to progression after HSCT. The median OS and PFS of the entire cohort are 11 (range: 1–168+) and 6 (range: 1–102) months, respectively.
GvHD, transplant-related mortality, PFS and overall survival A total of 37 patients (67%) had any form of acute GvHD, in particular: n = 11 of grade 1 (20%), n = 14 of grade 2 (25%), n = 7 of grade 3 (13%) and n = 5 of grade 4 (9%). Chronic GvHD was observed in 21 patients (38%), of whomfive had extensive form (9%). No difference in GvHD occurrence was observed across the distinct HLA-G 14 bp polymorphism groups (P = 0.63). Ten patients (18%) died of an identifiable transplant-related cause in the absence of disease-related event: GvHD (n = 3), cardiac failure (n = 2), renal failure (n = 2), infectious pneumonia (n = 1), invasive fungal infection (n = 1), sudden death (n = 1).
All evaluable patients relapsed at a median of 186 days after transplant (range: 48–2312). As above specified, the median PFS and OS were six and 11 months after HSCT.
HLA-G polymorphism correlation analysis with PFS and OS Results of the HLA-G polymorphism analysis are shown in Table 2. The‘14bp-del/del’ genotype was detected in 13 out of the n = 51 evaluable patients. The HSCT outcomes of these patients were compared with those of the grouped‘14 ins/del’ plus ‘14 bp-ins/ins’ population (further referred to as ‘14 bp-ins/X’), both in Kaplan–Meier curves and Cox regression results. Five-year PFS probabilities of 14 bp-del/del vs 14 bp-ins/X were 8% (95% confidence interval (CI): 0–22) and 5% (95% CI: 0–12) respectively, with median estimates of 15 vs 6 months, P = 0.22 (Figure 1a). OS was 23% (95% CI: 0–46) and 10% (95% CI: 0–20) for the same groups, with median estimates of 29 vs 10 months respectively, P = 0.20 (Figure 1b). Taking into account the 14 bp-ins/X group as reference, the univariate hazard ratios (HRs) of 14 bp-del/del were 0.66 (95% CI: 0.34–1.28; P = 0.22) and 0.64 (95% CI: 0.32–1.28; P = 0.21) for PFS and OS, respectively. After adjustment, a trend towards better outcome for 14 bp-del/del patients was confirmed, with multivariate HRs of 0.57 (95% CI: 0.26–1.26; P = 0.17) and 0.50 (95% CI: 0.23–1.13; P = 0.10) for PFS and OS respectively. Patient gender (P = 0.01) and CMV serostatus (P = 0.04) were statistically significant in the PFS model whereas none of the other included variables was found to be significant in the OS model, including conditioning regimen and GvHD (P = 0.30 and P = 0.33 for conditioning regimen and GvHD with PFS in univariate respec-tively, P = 0.91 and P = 0.86 for conditioning regimen and GvHD
with OS in univariate). Correlation analyses for GvHD found only a trend towards more chronic GvHD among the patient–donor pairs who were homozygous for the 14 bp‘ins’: HR was 3.18 (95% CI: 0.83–12.26) for 14 bp-ins/ins vs 14 bp -del/X (P = 0.09 by logistic regression).
The exploratory analyses of the polymorphisms at the eight distinct positions (p) 3003, 3010, 3027, 3035, 3142, 3187, 3196 were conducted on n = 52 evaluable patients. Those on position 3121 were not run because the genotype was T/T in all the pairs tested (see Table 2). No significant association with PFS or OS was found, with the exception of the T/C polymorphism at p3003 that was associated with better PFS (P = 0.006) and OS (P = 0.05) with respect to T/T (Figures 2a and b). Of note, 7 out of the 10 patients with the ‘favorable’ T/C polymorphism at p3003 had the 14
bp-Table 2. Distribution of polymorphisms among the evaluable patients 14 bp n. of pts % of evaluable pts 3UTR n. of pts % of evaluable ptsa del/del 13 25 1/1 4 8 ins/del 26 51 1/2 6 12 ins/ins 12 24 1/3 2 4 missing 5 1/4 5 10 P3003 1/5 3 6 T/C 10 19 1/6 1 2 T/T 42 81 1/7 5 10 missing 4 2/2 6 12 P3010 2/3 1 2 C/C 19 37 2/4 2 4 C/G 24 46 2/5 1 2 G/G 9 17 2/6 4 8 missing 4 2/7 3 6 P3027 3/3 1 2 A/A 2 4 3/4 2 4 C/A 10 19 3/5 1 2 C/C 40 77 3/7 1 2 missing 3 4/7 1 2 P3035 7/7 2 4 C/C 34 65 2/13 1 2 C/T 16 31 missing 3 T/T 2 4 missing 4 P3142 C/C 10 19 C/G 26 50 G/G 16 31 missing 4 P3187 A/A 26 50 A/G 22 42 G/G 4 8 missing 4 P3196 C/C 28 54 C/G 18 35 G/G 6 12 missing 4 P3121 T/T 52 100 missing 4
Abbreviation: UTR= untranslated region.aTotal may exceed 100% due to rounding.
del/del polymorphism, thus we cannot exclude an indirect effect played at least in part by 14 bp; nevertheless, after combining the two variables in a bi-variate model only the p3003 retained statistical significance, with P = 0.02 for PFS (P = 0.18 for OS). No additional meaningful information were provided by the correla-tion analyses using the 3′UTR haplotypes (data not shown). DISCUSSION
The prognosis of metastatic RCC has considerably improved over the last decade. A growing understanding of the underlying biology of RCC has identified critical pathways in tumorigenesis, and their inhibition as a promising therapeutic strategy in RCC. Several agents, including tyrosine kinase inhibitors, mammalian target of rapamycin inhibitors and most recently immune checkpoint inhibitors, have demonstrated antitumor activity in randomized trials. Their introduction into clinical practice has favorably changed the prognosis of RCC from 6–18 to 24–30 month-overall survival.30 Clear-cell RCC may be susceptible to immune therapy: interleukin-2, especially at high doses, and/or interferon-alpha have induced responses and occasionally long-term PFS in 4–15% of patients in previous studies.31 In 2000, Childs and coworkers at NIH published thefirst series of patients with renal cancer treated with a non-myeloablative allogeneic HSCT: they observed a 53% response rate, including complete
responses, in a cytokine-refractory series.4 After this first report, several patient series with different conditioning regimens and prophylaxis against GvHD have been published.32Most of them are small (less than 10 patients), and eight report a GvT effect, albeit with highly variable response rates (8–57%). The largest series of allograft in RCC reported so far comprised 124 patients, and included the prospective study of the French ITAC group and the EBMT STWP experience.9 All of these patients received a peripheral-blood allogeneic graft after various,fludarabine-based, non-myeloablative conditioning. Only three patients (2.4%) rejected the stem cell graft. Transplant-related mortality was 16% at one year, most often associated with acute GvHD. Complete (n = 4) or partial (n = 24) responses were observed at median 150 (range 42–600) days post-transplant. The regression of metastases was associated with time from diagnosis to allogeneic transplant, mismatched donor and acute GvHD of grades II to IV. Factors associated with survival included chronic GvHD, donor lymphocyte infusions, o3 metastatic sites, and a Karnofsky score 470. Patients with chronic GvHD who had received donor lymphocyte infusions (DLI; n = 17) had a 2-year survival of 70%. In a long-term follow-up of our series at San Raffaele Hospital, we found that 20% patients were alive at a median time of 65 months after allografting.33
In this series of metastatic RCC patients treated at four transplant centers from Italy, Sweden and France on behalf of
1.0 0.8 0.6 0.4 0.2 0.0 0 24 48 72 96 120 Months after HSCT P robability P robability Months after HSCT 0 24 48 72 96 120 144 168 192 1.0 0.8 0.6 0.4 0.2 0.0 Overall survival Progression-free survival 14bp-del/del 14bp-del/del
14bp-ins/del + ins/ins 14bp-ins/del + ins/ins
a b
Figure 1. Kaplan–Meier estimates of transplant outcome according to the 14-bp polymorphism: n = 13 patients homozygous for the HLA-G del/del polymorphism compared to n= 38 patients ins/ins and ins/del grouped together (referred to as ‘ins/X’ in the text). (a) PFS and (b) OS.
1.0 0.8 0.6 0.4 0.2 0.0 0 24 48 72 96 120 0 24 48 72 96 120 144 168 192 1.0 0.8 0.6 0.4 0.2 0.0
Progression-free survival Overall survival
Months after HSCT Months after HSCT P robability P robability a b
Figure 2. Kaplan–Meier estimates of transplant outcome according to the HLA-G polymorphism at position 3003T/C (n = 10; continuous line) compared to T/T (n= 42; dotted line). (a) PFS. Median estimates were 16 (range: 2–102) and 5 (range: 1–69) months respectively, P = 0.006; (b) OS. Median estimates were 29 (range: 5–168) and 9 (range: 1–162) months respectively, P = 0.05.
the Solid Tumor and the Cellular Therapy & Immunobiology Working Parties of the EBMT, we report an improved PFS and overall survival after HSCT in patients homozygous for the 14-bp deletion polymorphism of HLA-G gene compared with ins/X genotypes. Although not statistically significant, the observed difference in outcomes such as PFS and OS may be the result of the more tolerogenic setting when del/del polymorphism is present compared with the ins/ins and ins/del pooled together. To our knowledge this is the first observation in the setting of allogeneic transplant for a solid tumor, suggesting a role of HLA-G polymorphism in this setting.
Previous studies reported an association between HLA-G 14-bp polymorphism and GvHD in HSCT for beta-thalassemia24,25 but also for other hematological diseases,34,35 confirming, with only one exception,35that the risk of GvHD is higher in the presence of homozygosity for the 14-bp deletion. The observed association did not translate into survival differences in the reported series for thalassemia, possibly due to the lack of GvT effect needed in this situation, and the distinct conditioning regimens as well as GvHD prophylaxis protocols and donor types that might explain the conflicting results observed in the series with various hematolo-gical conditions,34,35though the so-called‘14-bp paradox’ has also been evoked.36
Thefinding of a significant correlation between T/C polymorph-ism at p3003 and better PFS and OS is worthy of note; although adjustment for multiple comparisons has not been made in the exploratory analyses, this strong association deserves to be further studied in the future, either to confirm and to investigate the rationale behind the results.
Our results show no apparent effect of the 14-bp polymorphism on acute GvHD, and this might be due to the underlying disease and transplant protocols, aimed at maximizing the GvT through the modulation of GvHD prophylaxis, thus probably overcoming the impact of HLA-G in this specific situation. Indeed, RCC patients represented a population at high risk of early progression within the immune suppression window after transplant, due to the presence of active disease at the time of allograft; and this is a different picture compared with both thalassemia and acute leukemia.24,25,34,35
We acknowledge some limitations of the study, in particular its retrospective nature, being a registry-based analysis, and the limited number of evaluable patients, preventing for the achieve-ment of more solid statistical conclusions. We cannot exclude that the analysis of a larger series may have led to statistically significant results, due to higher patient numbers and more comprehensive multivariate analysis.
Nevertheless, this report represents the first study in this specific setting, originating from a joint effort of four experienced transplant centers across three European countries and on behalf of EBMT. Although allogeneic HSCT is rarely administered to RCC patients nowadays due to significant changes in therapy with the advent of new molecules, this procedure represents a platform for immunotherapy in which HLA-G appears to play a significant role. Our results might have implications for new protocols of immune checkpoint blockade treatment of RCC patients, in which exploration of the role of HLA-G is warranted. Finally, the present results confirm the role of immunotherapy in metastatic RCC and might open new perspectives in a field where great successes have been achieved in the last years but there is still room for improvement.
CONFLICT OF INTEREST
RC is employee of MolMed S.p.A. at the time of submission. The remaining authors declare no conflict of interest
ACKNOWLEDGEMENTS
We are grateful to the Solid Tumor Working Party of the EBMT forfinancial support to the study.
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