human leukocyte antigen and killer immunoglobulin-like...
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Human Leukocyte Antigen and Killer Immunoglobulin-like receptor genes as outcome
predictors of hepatitis C virus-related hepatocellular carcinoma
Elisabetta Cariani1, Massimo Pilli2, Alessandro Zerbini3, Cristina Rota1, Andrea Olivani2, Paola
Zanelli4, Adele Zanetti4, Tommaso Trenti1, Carlo Ferrari2, Gabriele Missale2
1Clinical Pathology-Toxicology, Ospedale S. Agostino-Estense, Modena
2U.O. Infectious Diseases and Hepatology, 4U.O. Medical Genetics, Azienda Ospedaliero-
Universitaria di Parma
3Clinical Immunology, Allergy and Advanced Biotechnologies Unit, Department of Laboratory
Medicine, Azienda Ospedaliera ASMN, Istituto di Ricovero e Cura a Carattere Scientifico, Reggio
Emilia, Italy
Running title: KIR and HLA loci in HCV-related HCC
Keywords:
Natural Killer cells; Surgical resection; Radiofrequency thermal ablation; CD56; CD107a
Grant Support
Supported by a grant from Fondazione CARIPARMA (Parma, Italy); by the grant RBAP10TPXK
from Fondo per gli Investimenti della Ricerca di Base (Ministry of Education, University and
Research, Italy).
Corresponding Author
Gabriele Missale
U.O. Malattie Infettive ed Epatologia
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Azienda Ospedaliero-Universitaria di Parma
Tel +39 0521 702310 – Fax +39 0521 703857
e-mail: [email protected]
Disclosure of Potential Conflict of Interest
No potential conflicts of interest were disclosed
Electronic word count: 4699
Number of figures : 3
Number of tables: 3
Authors’ Contributions
Conception and design: E. Cariani, C. Ferrari, G. Missale
Development of methodology: E. Cariani, M. Pilli, G. Missale
Acquisition of data: M. Pilli, C. Rota, A. Olivani, P. Zanelli, A. Zanetti
Analysis and interpretation of data: E. Cariani, A. Zerbini, C. Ferrari, G. Missale
Writing, review, and/or revision of manuscript: E. Cariani, M. Pilli, A. Zerbini, C. Rota, A.
Olivani, P. Zanelli, A. Zanetti, T. Trenti, C. Ferrari, G. Missale
Administrative, technical or material support: M. Pilli, C. Rota
Study supervision: T. Trenti, C. Ferrari, G. Missale
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Translational relevance
Natural killer (NK) cells play a major role in anti-tumor immune response. The genotypes of NK
Killer Immunoglobulin-like receptors (KIRs) and of their HLA ligands are related to the
development of hepatocellular carcinoma (HCC) in patients with HCV infection. A better
understanding of the role played by the immunogenotypic background over HCC outcome might
represent a major breakthrough to improve the clinical management of HCC patients.
Our work shows that the immunogenetic profile is associated with NK-cell function and represents
a powerful outcome predictor in HCV-linked HCC. These results support a central role of NK cells
in the immune response against HCC with important implications in terms of development of
immunotherapeutic approaches and individualized monitoring schemes after treatment of early
HCC.
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Abstract
Purpose: We evaluated the impact of the Killer Immunoglobulin-like Receptors (KIRs) of natural
killer (NK) cells and of their Human Leukocyte Antigen (HLA) ligands over the clinical outcome of
HCV-related hepatocellular carcinoma (HCC) after curative treatment by either surgical resection
(SR) or radiofrequency thermal ablation (RTA).
Experimental Design: Sixty-one consecutive patients with HCV-related HCC underwent KIR
genotyping and HLA typing. A phenotypic/functional characterization of NK-cells was carried out
in patients with different KIR/KIR-ligand genotype.
Results: Activating KIR2DS5 was associated with significantly longer time to recurrence (TTR)
and overall survival (OS) (p<0.03 each). Homozygous HLA-C1 (p<0.02) and HLA-Bw4I80
(p<0.05) were expressed by patients with significantly better OS, while HLA-C2 (p<0.02) and
HLA-Bw4T80 (p<0.01) were associated with a worse OS. Multivariate analysis identified as
parameters independently related to TTR the type of treatment (SR vs RTA) (p<0.03) and HLA-C1
(p<0.03), whereas only KIR2DS5 was an independent predictor of longer OS (p<0.05). Compound
KIR2DL2-C1 and KIR3DS1-Bw4T80 genotypes were associated with better TTR (p<0.03) and
worse OS (p=0.02), respectively. A prevalent cytotoxic (CD56dim) NK phenotype was detected in
patients with both longer TTR and OS. Cytotoxic capacity measured by upregulation of CD107a
was significantly higher in subjects with HLA-C1 alone or combined with KIR2DL2/KIR2DL3.
Conclusions: These results support a central role of NK-cells in the immune response against HCC,
providing a strong rationale for therapeutic strategies enhancing NK response and for individualized
post-treatment monitoring schemes.
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Introduction
Natural killer (NK) cells recognize target cells through interaction of surface inhibitory and
activating receptors with their ligands. NK-cells have been divided into two major populations, one
mainly cytotoxic in function (CD56dimCD16+) and the other mainly involved in cytokine secretion
(CD56bright, CD16dim or negative) (1).
NK cell receptors belong to two main families: the C-type lectins-like (NKG2) receptors and the
immunoglobulin (Ig)-like superfamily, including the killer cell Ig-like receptors (KIRs). KIR
genotypes can be grouped into haplotypes A and B, mainly including inhibitory (A) or activating
(B) KIRs, respectively (2). The B haplotypes contain variable numbers and combinations of KIR
genes.
The fine tuning of KIR-ligand interactions is the result of multi-level regulatory mechanisms
relying both on the quantity of KIR/HLA molecules expressed on NK and target cells, and on the
affinity of KIR-HLA interactions. Inhibitory KIR2DL1 receptor recognizes alleles of HLA-C with
lysine at position 80 (HLA-C2), whereas KIR2DL2 and KIR2DL3, segregating as alleles of a single
locus, specifically bind HLA-C alleles with asparagine at position 80 (HLA-C1) (3,4) with different
binding affinity (5): KIR2DL3/HLA-C1 interaction is thought to be weaker than both
KIR2DL2/HLA-C1 and KIR2DL1/HLA-C2. In addition KIR2DL2, and to a lesser extent
KIR2DL3, bind with low affinity HLA-C2 (5). KIR3DL1 recognizes the Bw4 motif of HLA-B
alleles (6), generating a stronger inhibitory signal when an isoleucine residue is present at position
80 (Bw4I80). Due to the relevant homology of activating KIR2DS2 and KIR3DS1 to inhibitory
KIR2DL2/DL3 and KIR3DL1, respectively, it has been speculated that they might share the same
ligands as the inhibitory KIR counterparts, but this assumption has not been demonstrated yet. By
contrast, KIR2DS1 has been shown to bind HLA-C2 as KIR2DL1, although with lower affinity (7).
NK-cells recognize and eliminate cells that fail to express self HLA molecules, such as virus-
infected and transformed cells. In hepatitis C virus (HCV) infection, impaired NK cell frequency
and function has been reported (8) and specific KIR/ligand genotypes have been implicated in the
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clinical evolution and therapeutic response: the KIR2DL3/HLA-C1 genotype has been associated
with the resolution of infection (9-11) as KIR3DS1 and HLA-Bw4, although with a weak protective
effect (9). By contrast, homozygous HLA-C2 (9) and KIR2DS3, in the presence of HLA-C2 (12)
were more frequent in patients with chronic hepatitis C compared to individuals with spontaneously
resolved infection. In patients with acute HCV infection, increased NK-cells cytotoxicity was
present in subjects expressing HLA-C1-specific KIR2DL2/3 and in particular in self-limited
infection (13). Other studies reported over-representation of homozygous KIR2DL3/HLA-C1 in
sustained responders (11,14) and of HLA-C2C2 in patients resistant to treatment (15).
Functional impairment of NK-cells has been observed in patients with hepatocellular carcinoma
(HCC) (16). The KIR3DS1/HLA-Bw4I80 genotype and HLA-C1 were interpreted as protective
against the development of HCV-related HCC (17), but no data are available about the potential
relationship between KIR/HLA genotypes and the prognosis of HCC after treatment. We have
evaluated the impact of the immunogenetic host background over the clinical outcome of HCV-
related HCC after curative treatment by either surgical resection (SR) or radiofrequency thermal
ablation (RTA).
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Materials and Methods
Patients
We evaluated 61 consecutive Caucasian HCC patients with HCV-related liver disease, that
underwent curative treatment by either SR or RTA at the University Hospital of Parma, Italy. HCC
diagnosis was made by ultrasonography (US) and computed tomography (CT) or magnetic
resonance imaging (MRI) in selected cases. The type of treatment was decided on the basis of liver
function (Child-Pugh score), comorbidities, age and location of HCC nodules. Patients with early
HCC were evaluated by a multidisciplinary group (interventional radiologist, hepatologist and
surgeon) in order to decide treatment allocation based on liver function, portal hypertension,
number, site and size of HCC lesions. Patients with one or two lesions less than 15 mm in diameter
were treated with percutaneus alcohol injection. All patients had not been previously treated for
HCC.
All patients undergoing liver resection were within Child-A score. HBsAg and anti-human
immunodeficiency virus were negative for all cases. All patients had the same postoperative follow-
up based on bidimensional and contrast-enhanced (CE) US every 4 months and dynamic CT or CE
MRI in any case of appearance of new liver nodules more than 1 cm in diameter or arterial
enhancement at the site of previous ablation. The clinico-pathological features of the patients are
shown in Table 1 .
The study was approved by the local ethical committee (Comitato Etico Indipendente of the
Azienda Ospedaliero-Universitaria of Parma, Italy). Patients gave written informed consent to
participate in the study.
KIR genotyping and HLA typing
DNA was extracted from frozen peripheral blood mononuclear cells (PBMC) derived from all
patients using the QIAamp DNA blood Mini Kit (Qiagen, Hilden, Germany). KIR genotypes were
determined by duplex real-time polymerase chain reaction (PCR) (18) on 5 ng DNA in 10 μl
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containing 1X SsoFastTM EvaGreen® Supermix (Bio-Rad Laboratories, Hercules, CA), 0.3 μM of
each KIR-specific primer, 0.1 μM of each internal control primer, in a Rotor Gene Q thermal cycler
(Qiagen) as follows: 2’ 98°C, 5” 98°C and 10” 62°C for 45 cycles. The PCR products were then
discriminated by melting (2’ 98°C, 10” 75°C, and ramping by 0.5°C/s up to 98°C). The KIR genes
analyzed are shown in Supplementary Figure S1.
HLA typing was performed in all patients by PCR Sequence Specific Priming on genomic DNA. In
all cases showing ambiguities for definition of the HLA-B or C supertypes C1, C2, Bw4, Bw6 and
Bw4 I80/T80 variants, high resolution typing was performed by PCR Sequence Specific
Oligonucleotide Probes (Proimmune Co, Oxford, UK).
Immunostaining of NK-cells
In 38 of 61 patients an aliquot of frozen PBMC obtained the same day or the day before treatment
was available for phenotypic analysis. PBMC were resuspended in RPMI 1640, and 8% human
serum. PBMC (3x105) were stained with CD3-PerCP (BD Biosciences-Pharmingen, San Jose, CA)
and CD56-APC (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were analyzed on FACSC-
CantoII flow cytometer (BD Biosciences, Franklin Lakes, NJ) by the FACSDiva Software.
Frequency of CD56dim and CD56bright NK-cells was defined for each patient evaluating different
fluorescence intensity of CD3-CD56+ cells.
Evaluation of cytotoxic function and interferon (IFN)-γ production in NK-cells
In 31 of 61 patients an aliquot of frozen PBMC obtained the same day or the day before treatment
was available for functional analysis. For the evaluation of the cytotoxic function, PBMC (1x106)
were incubated 16 hours at 37°C in the presence or absence of 1 ng/ml rhIL-15 (R&D Systems,
Abingdon, UK). PE/Cy5-5-conjugated CD107a (BD Biosciences) was then added with or without
K562 target cells at an E:T ratio of 5:1. After 1 h, 10 µg/ml of brefeldin A (Sigma-Aldrich, St.
Louis, MO) was added and cell were incubated further 3 h at 37°C, then harvested and stained for
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NK cell surface markers. Percentage of degranulating NK-cells was calculated by subtracting
CD107a staining after incubation without K562 to CD107a staining after incubation with K562
targets.
PBMC (1x106) were incubated for 14 h at 37°C with or without 10 ng/ml rhIL-12 and rhIL-18
(Sigma-Aldrich). Brefeldin A (Sigma-Aldrich) (10 µg/ml) was added for the last 3 h of incubation.
After staining with CD56-APC (Miltenyi Biotec) and CD3-APC/Cy7 (Biolegend, San Diego, CA),
cells were fixed and permeabilised using Fix and Perm medium A and B (Caltag Laboratories,
Burlingame, CA) according to the supplier’s instructions. Cells were stained with anti-IFN-γ
PerCP/Cy5-5 mAb (Biolegend). The proportion of IFN-γ producing cells was determined by
subtracting the percentage of IFN-γ+ cells in unstimulated samples from the one of IL-12/IL-18-
stimulated samples.
Statistical analysis
The differences between groups of continuous variables were analyzed by t-test for unpaired data.
Categorical variables were compared by the Chi-square test or Fisher exact test, as appropriate.
Survival curves were estimated by the Kaplan-Meier method and compared by log-rank test. For
continuous variables, ROC curve analysis was used to define threshold levels discriminating
subgroups of patients with survival/time to recurrence higher or lower than median values, for
subsequent analysis by the log-rank test. Cox proportional hazards regression model was performed
for multivariate survival analysis. Only variables available for all patients and presenting p<0.1 in
the univariate analysis (Table 2) were included in the multivariate model. A p value <0.05 (two-
tailed) was considered significant.
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Results
Clinical outcome
The study was performed on 61 consecutive Caucasian patients with diagnosis of HCV-related
HCC allocated to treatment by either SR or RTA. (Table 1). The median overall survival (OS) was
43 months and the median time to the first HCC recurrence (TTR) was 17 months. By dividing
patients according to the treatment received, age was significantly higher and liver function worse
in RTA-treated patients, while HCC nodules were on average larger in patients treated by SR.
Subjects that performed SR showed a significantly better OS and TTR compared to patients treated
by RTA (Tables 1 and 2). Female sex was associated with significantly shorter OS, and Child score
with worse TTR; age, number and size of HCC nodules did not affect survival (Table 2). From
these results, survival differences related to treatment seem to be most likely due to the treatment
allocation criteria, since RTA-treated patients were significantly older and had a worse Child score.
Moreover, 3 patients who underwent surgical resection during the same period, died within 2
months from surgery. These patients were not included in the study because the effect of HLA and
KIR associations could not be evaluated.
Individual KIRs and HLA expression and their association with disease progression and overall
survival
KIRs frequencies identified in our patients cohort were comparable to the ones previously reported
in Caucasian patients from Northern Italy (19,20), without any significant difference in the
prevalence of each reported KIR (Supplementary Figure S1).
The 15 patients carrying the KIR2DS5 gene, that is present in a fraction of the KIR B haplotypes,
showed significantly longer TTR and OS (Figure 1). KIR2DS5 is an activating receptor in linkage
disequilibrium with KIR2DS1, that also showed a trend towards a protective effect against
recurrence (Table 2). No other analyzed KIRs were significantly correlated to clinical outcome.
Homozygous HLA-C1 and HLA-Bw4, I80 variant, were associated with significantly better OS,
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while HLA-C2 and HLA-Bw4T80 were associated with a worse OS (Figure 1 and Table 2).
Multivariate analysis identified as independent parameters associated with outcome the type of
treatment (SR vs RTA) and HLA-C1 for TTR, and only KIR2DS5 for OS (Table 3). By analyzing
the effect of HLA-C1 copy number on survival, we detected that HLA-C1 homozygous patients
showed significantly longer OS compared to HLA-C1 heterozygous patients, and both longer OS
and TTR compared to those homozygous for HLA-C2 (Figure 2A).
Effect of the compound KIR-HLA genotype on disease progression and overall survival
The inhibitory receptors KIR2DL2 and KIR2DL3 share the same ligand HLA-C1. Activating
KIR2DS2 is in strong linkage disequilibrium and highly homologous to KIR2DL2, however
experimental evidence of that KIR2DS2 binds HLA-C1 is not available at present. KIR2DL1 and
KIR2DS1 bind HLA-C2, while KIR3DL1 binds HLA-Bw4, with higher and lower affinity for I80
and T80 variants, respectively (21). Since KIR3DS1 and KIR3DL1 segregate as alleles of the same
locus and show 97% similarity in their extracellular domains, they may share the same HLA ligand.
For the remaining KIRs genotyped in this study, the binding HLA molecule is not known.
Since almost all our patients were positive for both KIR2DL1 and KIR3DL1 (Supplementary
Figure S1), the effect of compound KIR-HLA genotype on disease progression was not evaluated
for these molecules,. Altogether, combined survival analysis was performed for KIR2DL2/2DL3-
C1, KIR2DS1-C2, KIR2DS2-C1 and KIR3DS1-Bw4 (I80 and T80). Significant associations were
found for KIR2DL2-C1 and KIR2DS2-C1 with a better TTR and for KIR3DS1-Bw4T80 with a
worse OS (Figure 2B and Table 2).
NK-cell phenotype and function
First we evaluated whether effector NK-cell phenotype (CD56dim) could be associated with a better
clinical outcome. Cut-off values of CD56dim% able to discriminate patients with TTR or OS higher
than median values (17 and 43 months, respectively) were identified by ROC curve analysis. Indeed
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patients with a frequency of NK cells with a CD56dim phenotype higher than the above-mentioned
cut-offs showed significantly longer TTR and OS (Figure 3 upper left panels). We then compared
NK-cell function of patients with specific HLA, KIR and HLA-KIR genotypes showing a
significant effect on survival analysis. The distribution of HLA and KIR genotypes in the subgroup
of patients with samples available for functional analysis was comparable to the one of the whole
cohort (data not shown). Cytotoxic capacity, measured by upregulation of CD107a, was
significantly higher in subjects with HLA-C1 alone or associated with KIR2DL2/KIR2DL3 (Figure
3 upper right panel and lower panels). No significant differences could be found for IFN-γ
production (data not shown).
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Discussion
KIR receptors and their HLA ligands have been implicated as risk factors for several human tumors,
chronic inflammatory diseases, and viral infections (22). A recent genome-wide association study
(23) identified an association between risk of HCC development in patients with chronic hepatitis
and a single nucleotide polymorphism in the gene encoding for MHC-associated chain A (MICA),
the ligand of NKG2D, an activating NK receptor (24). This observation strongly supports a relevant
role for NK-cells in HCC pathogenesis.
In the present study we evaluated the impact of the immunogenetic profile on the clinical outcome
of HCC patients undergoing curative treatment by either SR or RTA. Although both these treatment
strategies are able to eradicate neoplastic nodule(s), the relapse rate is high since, in addition to the
intrahepatic dissemination of primary tumor, de novo HCC may develop in the pre-tumorous
environment of liver cirrhosis. The direct clinical consequences of the underlying liver disease have
also a major impact on OS after treatment. Since the biological characteristics of the tumor and the
host immune response are both involved in HCC progression (25-27), it is likely that the
immunogenetic background determining the NK-cell response may play a role on the risk of relapse
and ultimately on the survival of HCC patients.
A significant association was observed between KIR2DS5, an activating receptor part of haplotype
B, and longer OS and TTR, but the implications of our results could not be further investigated since
the ligand of KIR2DS5 has not been identified yet. Whether KIR2DS5 is a marker for a haplotype
involved in HCC outcome, or whether NK-cells bearing KIR2DS5 play a direct role in the clinical
outcome due to specific functional characteristics remains to be determined. A survival advantage of
patients homozygous for HLA-C1 (HLA-C1C1) and therefore lacking HLA-C2 was also shown,
together with an association of HLA-Bw4 variants I80 and T80 with longer or shorter OS,
respectively. Homozygous HLA-C1, as well as HLA-Bw4I80, has already been reported as
protective against development of HCV-related HCC (17). Our results further support the role of
these genotypes in human HCC, showing their association with survival after curative treatment.
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Multivariate survival analysis identified as independent parameters associated with outcome the
type of treatment and HLA-C1 for TTR, and only KIR2DS5 for OS. These observations suggest
that the therapeutic approach is a major determinant for disease recurrence, together with the HLA-
C genotype, but does not represent an independent risk factor for OS, where the immunogenetic
profile appears to be the only independent predictive variable.
Previous studies mainly analyzed the implications of the co-expression of inhibitory KIRs and their
HLA ligands, that leads to the maturation of fully competent (“licensed”) NK-cells both in mice and
in humans (28,29). The opposite situation may occur when activating KIRs are co-expressed with
their ligands, as it was shown for KIR2DS1 that recognizes HLA-C2 (30). Indeed the compound
KIR2DS1/HLA-C2 immunogenetic profile would lead to reduced functional competence of NK-
cells. In HCC patients we observed that the concurrent presence of KIR2DL2 and HLA-C1 was
associated with longer TTR, possibly due to the protective effect of NK-cells that had reached full
functional competence upon maturation. Consistent with this view, HLA-Bw4I80 (a high-affinity
ligand of inhibitory KIR3DL1, expressed by 60/61 patients) was associated with longer survival,
whereas the opposite was observed in patients with HLA-Bw4T80, a low affinity ligand of KIR3DL1
(21,31). In addition, the combined presence of KIR3DS1 and HLA-Bw4T80 was associated with
reduced OS. Previous reports suggested a protective effect of HLA-Bw4I80 in association with
activating KIR3DS1 towards the development of HCV-linked HCC (17), the progression of acquired
immunodeficiency syndrome (32) and, with weak effect, the development of chronic HCV infection
(9). Although the binding of KIR3DS1 to HLA-Bw4 has not been convincingly demonstrated so far,
these results suggest that a less effective interaction of KIR3DS1 and HLA-Bw4T80 in HCC patients
might result in reduced NK function and impaired survival. The significance of the association
between KIR2DS2/HLA-C1 compound genotype and longer TTR is uncertain: to date, no
experimental evidence of HLA-C1 binding by KIR2DS2 is available. Therefore, it cannot be ruled
out that this association only derives from the strict linkage disequilibrium between KIR2DL2 and
KIR2DS2.
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Activating KIR–HLA genotypes appear to increase the risk of developing some virus-related tumors,
such as cervical cancer due to papilloma virus infection and nasopharyngeal carcinoma associated
with Epstein Barr virus infection (33,34). An increased risk of developing HCC was also reported in
patients with chronic HBV infection carrying HLA-C1C1, HLA-Bw4I80 and 22 bp-deleted form of
KIR2DS4 (KIR2DS4/1D) (35), thus suggesting that increased NK cell function might represent a risk
factor for HBV-related HCC. The apparent discrepancy between these observations and those
reported in HCV-linked HCC (17) might derive from differences in the specific immunopathogenic
mechanisms. Since NK-cells exert both antiviral and anti-tumor functions, an enhanced effector
phenotype might be protective against tumor development altogether increasing the pro-
inflammatory anti-viral environment and the consequent tissue damage, both favouring a pre-
neoplastic condition. However, in our study the tumor had already developed and
immunosurveillance by NK-cells would represent an immunological mechanism affecting clinical
outcome.
The impact of the immunogenetic background was further analyzed by phenotypic and functional
characterization of NK-cells. Higher percentage of CD56dim NK cells, indicative of prevalent effector
cytotoxic phenotype, was associated with both longer TTR and OS, and NK-cells from patients
carrying HLA-C1 had significantly higher cytotoxic activity. In addition, the combined presence of
KIR2DL2/KIR2DL3 and HLA-C1 was associated with increased cytotoxic function, further
supporting the relevance of NK-cell licensing for the achievement of full functional competence. The
same association was reported in patients with spontaneously resolving acute HCV infection (13).
This is to our knowledge the first study addressing the relevance of KIR genotype on the prognosis of
HCV-related HCC after curative treatment. Results suggest a central role of NK-cells in the immune
response against HCC, thus supporting the rationale for developing immunomodulatory strategies
aimed at the enhancement of NK response (36) especially in patients showing a favorable
immunogenotypic background. In addition, NK cell phenotype and function may provide information
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useful to predict outcome and tailor follow-up strategies after HCC treatment. Thus our results, if
confirmed on larger series of patients, will be relevant for the implementation of individualized post-
treatment monitoring schemes and novel therapeutic strategies in patients with HCC.
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Figure Legends
Figure 1.
Individual KIRs and HLA molecules associated with better TTR and/or OS.
Only KIR and HLA ligands significantly associated with TTR and/or OS are shown. p values were
determined by the log-rank test.
Of 61 patients, 15 were carriers of the KIR2DS5 gene and 19 were homozygous for HLA-C1.
KIR2DS5 gene carriers presented better TTR and OS. Homozygous expression of HLA-C1 was
associated with better OS. Among 48 patients positive for HLA-Bw4, at least one copy of the I80 or
T80 variants was present in 31 and 23 patients, respectively. The I80 variant (either one or two
copies) was associated with better OS, while opposite behavior was shown by the T80 variant.
Figure 2. Effect of HLA-C1 and compound KIR/HLA genotypes on TTR and OS.
A: Homozygous HLA-C1 (HLA-C1C1; 2 copies, detected in 19 patients) was associated with
significantly longer OS compared to heterozygous HLA-C1 (HLA-C1C2; 1 copy, detected in 29
patients) and with significantly longer TTR and OS compared to homozygous HLA-C2 (HLA-
C2C2; 0 copies, detected in 13 patients). Survival curves are referred to each of the 3 possible
genotypes (HLA-C1 homozygous, heterozygous or null), while bar panels show the progressive
effect of HLA-C1 copy number on median TTR and OS. B: Effect of the compound KIR2DL2-
KIR2DS2/HLA-C1 and KIR3DS1/HLA-Bw4T80 genotypes on TTR and OS, respectively.
KIR2DL2 was detected in 34 patients, 2 of whom homozygous, and 26 also positive for HLA-C1
(homozygous or heterozygous); KIR2DS2 was detected in 33 patients, 25 also positive for HLA-C1
(homozygous or heterozygous); KIR3DS1 (homozygous or heterozygous) was detected in 18
patients, 9 of whom also positive for HLA-Bw4T80 (all heterozygous, either Bw4T80/BwI80 or
Bw4T80/Bw6).
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18
Figure 3. Analysis of NK-cell phenotype and cytotoxicity.
Upper panel: left, Kaplan-Meier curves of TTR and OS of HCC patients with different %CD56dim
NK-cells. The cut-off value between high or low CD56dim% was determined by ROC curve
analysis. The area under the ROC curve (AUC) for TTR was 0.73 (0.56-0.90), and a cut-off value
of 88.95% CD56dim showed 75% sensitivity and 76.2% specificity. The AUC for OS was 0.77
(0.66-0.96), with 72.2% sensitivity and 78.6% specificity for a cut-off of 89.25% CD56dim; right,
cytotoxic potential measured by upregulation of CD107a in CD3-CD56+ cells upon stimulation
(mean±standard error) in subjects with or without HLA-C1/C2 or combined KIR2DL2/DL3-C1.
Lower panel: Flow cytometry analysis of NK cells cytotoxic potential is shown for a representative
patient (RTA14), positive for HLA-C1C2 and HLA-Bw4(T80)/Bw6. The KIR genotype of patient
RTA14 is shown in Supplementary Figure S1. The two plots on the left show gating of NK cells
after overnight PBMCs activation with IL-15, while the remaining plots show staining for CD107a
on gated NK-cells (CD3-CD56+) after incubation with or without K562 target cells.
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1
Total
Surgical
resection
(SR)
Radiofrequency
Thermal Ablation
(RTA)
p value
(SR vs RTA)
Patients #
61
28
33
NA
Gender (M/F)
33/28 19/9 14/19 NS
Mean age at diagnosis ±
SD (years)
70.2±7.99 67.9±8.97 72.15±6.57 0.037
Child A/B
55/6 28/0 27/6 0.027
Median nodules # (range)
1 (1-5) 1 (1-5) 1 (1-3) NS
Mean nodules size ± SD
(mm)
42.3±23.6 58±25.7 29.9±11.7 <0.0001
Median TTR (months)
17
34
9
0.0002
Median OS (months)
43 84 41 0.05
Table 1. Clinical characteristics of patients.
SR: surgical resection; RTA: radiofrequency thermal ablation; NA: not applicable; NS: not
significant; M: males; F: females; SD: standard deviation.
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2
TTR
OS
p value
HR (95% CI)
p value
HR (95% CI) Gender (M/F)
0.15
0.65 (0.36-1.17)
0.014
0.43 (0.22-0.84)
Age at diagnosis* 0.077 1.70 (0.94-3.05) 0.064 1.86 (0.96-3.61) Child (A vs B) 0.048 0.28 (0.08-0.99) 0.38 0.51 (0.11-2.31)
Treatment (SR vs RTA) <0.001 0.496 (0.246-1.00) 0.0493 0.312 (1.00-4.06)
KIR2DS5 0.024 0.5 (0.27-0.91) 0.029 0.47 (0.24-0.93) KIR2DS1
0.06
0.57 (0.32-1.02)
0.11
0.58 (0.29-1.14)
HLA-Bw4I80
0.72
0.90 (0.51-1.59)
0.0499
0.43 (0.18-1)
HLA-Bw4T80
0.57
1.20 (0.64-2.23)
0.008
2.93 (1.33-6.43)
HLA-C1
0.057
0.44 (0.19-1.03)
0.13
0.50 (0.2-1.24)
HLA-C2
0.093
1.65 (0.92-2.98)
0.011
2.36 (1.22-4.54)
KIR2DL2+/C1+ vs C1-
0.027
0.29 (0.09-0.86)
0.56
0.72 (0.24-2.19)
KIR2DL3+/C1+ vs C1-
0.073
0.42 (0.16-1.08)
0.310
0.61 (0.23-1.59)
KIR2DS2+/C1+ vs C1- KIR3DS1+/I80+ vs I80-
0.026
0.91
0.28 (0.09-0.86)
1.08 (0.27-4.36)
0.5897
0.079
0.7371 (0.24-2.23)
0.17 (0.02-1.23)
KIR3DS1+/T80+ vsT80-
0.81
1.17 (0.32-4.30)
0.020
5.64 (1.32-24.16)
Table 2. TTR and OS of HCC patients according to clinical characteristics, KIRs, KIR ligands, and
compound KIR-HLA genotypes. Only parameters presenting p<0.1 in at least one analysis (TTR
and/or OS) are shown.
* Age ≥ vs < median (71 years); HR: hazard ratio; CI: confidence interval.
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3
TTR
OS
p value HR (95% CI) p value HR (95% CI)
Gender (M vs F) NA NA 0.342 0.70 (0.33-1.46)
Age at diagnosis
( ≥71 vs <71 years) 0.106 1.67 (0.89-3.12) 0.114 1.85 (0.86-3.96)
Child (A vs B) 0.106 0.43 (0.160-1.19) NA NA
Treatment (SR vs RTA) 0.021 0.43 (0.21-0.88) 0.992 0.99 (0.42-2.34)
KIR2DS5 (pos vs neg) 0.664 0.74 (0.19-2.91) 0.047 0.408 (0.17-0.99)
KIR2DS1 (pos vs neg) 0.698 0.78 (0.22-2.79) NA NA
HLA-C1 (pos vs neg) 0.023 0.40 (0.18-0.88) NA NA
HLA-C2 (pos vs neg) 0.961 1.02 (0.50-2.09) 0.067 2.17 (0.99-4.98)
Table 3. Multivariate analysis of TTR and OS.
NA: not applicable (p>0.10 in the univariate analysis).
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urvi
val
60
80
100KIR2DS5-KIR2DS5+
p=0.0293
surv
ival
60
80
100 KIR2DS5-KIR2DS5+
p=0.0244
Fig. 1
Perc
ent s
u
0 50 100 1500
20
40
Perc
ent s
0 50 100 1500
20
40
OS months0 50 100 150
al 80
100 HLA-C1C1HLA-C1C2/HLA-C2C2
TTR months0 50 100 150
Perc
ent s
urvi
va
20
40
60
80p=0.0105
100 HLA-Bw4 I80- 100 HLA-Bw4 T80-
OS months0 50 100 150
0
cent
sur
viva
l
40
60
80
100HLA-Bw4 I80+
p=0.0499
cent
sur
viva
l
40
60
80
100HLA-Bw4 T80+
p=0.0075
OS months
Perc
0 50 100 1500
20
OS months
Perc
0 50 100 1500
20
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R-13-0986
surv
ival
60
80
100HLA-C2C2HLA-C1C2HLA-C1C1
urvi
val
60
80
100
HLA-C2C2HLA-C1C2HLA-C1C1
p=0.034
Fig. 2
Perc
ent
0 50 100 1500
20
40 p=0.122
Perc
ent s
u
0 50 100 1500
20
40
60
HLA-C1C1
TTR months
HLA-C1C1
p=0.022
OS months
0 50 100
150
HLA-C2C2
HLA-C1C2
p=0.037
0 50 100
150
HLA-C2C2
HLA-C1C2
p=0.018
TTR monthsmedian (range)
OS monthsmedian (range)
100 KIR2DL2+/C1- KIR3DS1+/Bw4T80-
A
100 KIR2DS2+/C1-
cent
sur
viva
l
40
60
80
100KIR2DL2+/C1+
p=0.0265
ent s
urvi
val
40
60
80
100 KIR3DS1+/Bw4T80+
p=0.0197
cent
sur
viva
l
40
60
80
100 S /CKIR2DS2+/C1+
p=0.0262
TTR months
Perc
0 50 100 1500
20
OS months
Perc
0 50 100 1500
20
BTTR months
Perc
0 50 100 1500
20
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TTR months
Perc
en
t su
rviv
al
0 50 100 150 0
20
40
60
80
100
p=0.0002
CD56DIM low
CD56DIM high
OS months
Perc
en
t su
rviv
al
0 50 100 150 0
20
40
60
80
100
p=0.0175
CD56DIM low
CD56DIM high
% C
D5
6/C
D1
07
a+
C1-
C1+ C
2-C2+
KIR
2DL2+
/C1-
KIR
2DL2+
/C1+
KIR
2DL3+
/C1-
KIR
2DL3+
/C1+
0
10
20
30
40
50
p=0.035 p=0.0075 p=0.0086
FSC CD3 CD56
SSC
CD
56
CD
10
7a
CD
10
7a
CD56
Fig. 3
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Published OnlineFirst August 12, 2013.Clin Cancer Res Elisabetta Cariani, Massimo Pilli, Alessandro Zerbini, et al. virus-related hepatocellular carcinomareceptor genes as outcome predictors of hepatitis C Human Leukocyte Antigen and Killer Immunoglobulin-like
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