Soluble MICA and a MICA Variation as Possible Prognostic Biomarkers for HBV-Induced Hepatocellular Carcinoma
et al. (2012) Soluble MICA and a MICA Variation as Possible Prognostic Biomarkers for HBV-Induced
Hepatocellular Carcinoma. PLoS ONE 7(9): e44743. doi:10.1371/journal.pone.0044743
Soluble MICA and a MICA Variation as Possible Prognostic Biomarkers for HBV-Induced Hepatocellular Carcinoma
Vinod Kumar 0 1
Paulisally Hau Yi Lo 0 1
Hiromi Sawai 0 1
Naoya Kato 0 1
Atsushi Takahashi 0 1
Zhenzhong Deng 0 1
Yuji Urabe 0 1
Hamdi Mbarek 0 1
Katsushi Tokunaga 0 1
Yasuhito Tanaka 0 1
Masaya Sugiyama 0 1
Masashi Mizokami 0 1
Ryosuke Muroyama 0 1
Ryosuke Tateishi 0 1
Masao Omata 0 1
Kazuhiko Koike 0 1
Chizu Tanikawa 0 1
Naoyuki Kamatani 0 1
Michiaki Kubo 0 1
Yusuke Nakamura 0 1
Koichi Matsuda 0 1
Erica Villa, University of Modena & Reggio Emilia, Italy
0 Funding: This work was conducted as a part of the BioBank Japan Project that was supported by the Ministry of Education , Culture, Sports , Science and Technology of the Japanese government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
1 1 Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 2 Center for Genomic Medicine, The Institute of Physical and Chemical Research (RIKEN), Kanagawa, Japan, 3 Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan, 4 Unit of Disease Control Genome Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 5 Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Aichi, Japan, 6 The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Chiba, Japan, 7 Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo , Tokyo , Japan
MHC class I polypeptide-related chain A (MICA) molecule is induced in response to viral infection and various types of stress. We recently reported that a single nucleotide polymorphism (SNP) rs2596542 located in the MICA promoter region was significantly associated with the risk for hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) and also with serum levels of soluble MICA (sMICA). In this study, we focused on the possible involvement of MICA in liver carcinogenesis related to hepatitis B virus (HBV) infection and examined correlation between the MICA polymorphism and the serum sMICA levels in HBV-induced HCC patients. The genetic association analysis revealed a nominal association with an SNP rs2596542; a G allele was considered to increase the risk of HBV-induced HCC (P = 0.029 with odds ratio of 1.19). We also found a significant elevation of sMICA in HBV-induced HCC cases. Moreover, a G allele of SNP rs2596542 was significantly associated with increased sMICA levels (P = 0.009). Interestingly, HCC patients with the high serum level of sMICA (.5 pg/ml) exhibited poorer prognosis than those with the low serum level of sMICA (#5 pg/ml) (P = 0.008). Thus, our results highlight the importance of MICA genetic variations and the significance of sMICA as a predictive biomarker for HBV-induced HCC.
Hepatocellular carcinoma (HCC) reveals a very high mortality
rate that is ranked the third among all cancers in the world .
HCC is known to develop in a multistep process which has been
related to various risk factors such as genetic factors, environment
toxins, alcohol and drug abuse, autoimmune disorders, elevated
hepatic iron levels, obesity, and hepatotropic viral infections .
Among them, chronic infection with hepatitis B virus (HBV) is one
of the major etiological factors for developing HCC with
considerable regional variations ranging from 20% of HCC cases
in Japan to 65% in China .
Interestingly, clinical outcome after the exposure to HBV
considerably varies between individuals. The great majority of
individuals infected with HBV spontaneously eliminate the viruses,
but a subset of patients show the persistent chronic hepatitis B
infection (CHB), and then progresses to liver cirrhosis and HCC
through a complex interplay between multiple genetic and
environmental factors . In this regard, genome wide association
studies (GWAS) using single nucleotide polymorphisms (SNPs)
have highlighted the importance of genetic factors in the
pathogenesis of various diseases including CHB as well as
HBVinduced HCC [5,6,7,8,9,10,11,12,13]. Recently, we identified a
genetic variant located at 4.7 kb upstream of the MHC class I
polypeptide-related chain A (MICA) gene to be strongly associated with
hepatitis C virus (HCV) -induced HCC development .
MICA is highly expressed on viral-infected cells or cancer cells,
and acts as ligand for NKG2D to activate antitumor effects of
Natural killer (NK) cells and CD8+ T cells [15,16]. Our previous
results indicated that a G allele of SNP rs2596542 was significantly
associated with the lower cancer risk and the higher level of soluble
MICA (sMICA) in the serum of HCV-induced HCC patients,
demonstrating the possible role of MICA as a tumor suppressor.
However, elevation of serum sMICA was shown to be associated
with poor prognosis in various cancer patients [17,18,19,20].
Matrix metalloproteinases (MMPs) can cleave MICA at a
transmembrane domain  and release sMICA proteins from
cells. Since sMICA was shown to inhibit the antitumor effects of
NK cells and CD8+ T cells by reduction of their affinity to binding
to target cells [22,23], the effect of MICA in cancer cells would be
modulated by the expression of MMPs. To elucidate the role of
MICA in HBV-induced hepatocellular carcinogenesis, we here
report analysis of the MICA polymorphism and serum sMICA
level in HBV-induced HCC cases.
Materials and Methods
The demographic details of study participants are summarized
in Table 1. A total of 181 HCC cases, 597 CHB patients, and
4,549 non-HBV controls were obtained from BioBank Japan that
was initiated in 2003 with the funding from the Ministry of
Education, Culture, Sports, Science and Technology, Japan .
In the Biobank Japan Project, DNA and serum of patients with 47
diseases were collected through collaborating network of 66
hospitals throughout Japan. List of participating hospitals is shown
in the following website (http://biobankjp.org/plan/
member_hospital.html). A total of 226 HCC cases, 102 CHB
patients, and 174 healthy controls were additionally obtained from
the University of Tokyo. The diagnosis of chronic hepatitis B was
conducted on the basis of HBsAg-seropositivity and elevated
serum aminotransferase levels for more than six months according
to the guideline for diagnosis and treatment of chronic hepatitis
(The Japan Society of Hepatology, http://www.jsh.or.jp/
medical/gudelines/index.html). Control Japanese DNA samples
(n = 934) were obtained from Osaka-Midosuji Rotary Club,
Osaka, Japan. All HCC patients were histopathologically
diagnosed. Overall survival was defined as the time from blood
sampling for sMICA test to the date of death due to HCC. Patients
who were alive on the date of last follow-up were censored on that
date. All participants provided written informed consent. This
research project was approved by the ethics committee of the
University of Tokyo and the ethics committee of RIKEN. All
clinical assessments and specimen collections were conducted
according to Declaration of Helsinki principles.
Genotyping platforms used in this study were shown in Table 1.
We genotyped 181 HCC cases and 5,483 non-HBV control
samples using either Illumina Human Hap610-Quad or Human
Hap550v3. The other samples were genotyped at SNP rs2596542
MICA variable number tandem repeat (VNTR) locus
Genotyping of the MICA VNTR locus in 176 HBV-induced
HCC samples was performed using the primers reported
previously by the method recommended by Applied Biosystems
(Foster City, CA) . Briefly, the 59 end of forward primer was
labeled with 6-FAM, and reverse primer was modified with
GTGTCTT non-random sequence at the 59 end to promote Plus
A addition. The PCR products were mixed with Hi-Di
Formamide and GeneScan-600 LIZ size standard, and separated
by GeneScan system on a 37306l DNA analyzer (Applied
Biosystems, Foster City, CA). GeneMapper software (Applied
Biosystems, Foster City, CA) was employed to assign the repeat
fragment size (Figure S1).
Quantification of soluble MICA
We obtained serum samples of 111 HBV-positive HCC
samples, 129 HCV-positive HCC samples, and 60 non-HBV
controls from Biobank Japan. Soluble MICA levels were measured
by sandwich enzyme-linked immunosorbent assay, as described in
the manufacturers instructions (R&D Systems, Minneapolis, MN).
The association between an SNP rs2596542 and HBV-induced
HCC was tested by Cochran-Armitage trend test. The Odds ratios
were calculated by considering a major allele as a reference.
Statistical comparisons between genotypes and sMICA levels were
performed by Kruskal-Wallis test (if more than two classes for
comparison) or Wilcoxon rank test using R. Overall survival rate
of the patients was analyzed by Kaplan-Meier method in
combination with log-rank test with SPSS 20 software. The
period for the survival analysis was calculated from the date of
blood sampling to the recorded date of death or the last follow-up
date. Differences with a P value of ,0.05 were considered
Association of SNP rs2596542 with HBV-induced HCC
In order to examine the effect of rs2596542 genotypes on the
susceptibility to HBV-induced HCC, a total of 407 HCC cases
and 5,657 healthy controls were genotyped. The Cochran
Armitage trend test of the data revealed a nominal association
Chronic hepatitis B* BioBank Japan
Illumina Human Hap610-Quad
*Chronic hepatitis B patients without liver cirrhosis and liver cancer during enrollment.
**Healthy volunteers from Osaka Midosuji Rotary Club, Osaka, Japan.
between HBV-induced HCC and rs2596542 in which a risk allele
G was more frequent among HBV-induced HCC cases than an A
allele (P = 0.029, OR = 1.19, 95% CI: 1.021.4; Table 2). To
further investigate the effect of rs2596542 on the progression from
CHB to HBV-induced HCC, we genotyped a total of 699 CHB
cases without HCC. Although the progression risk from CHB to
HBV-induced HCC was not statistically significant with
rs2596542 (P = 0.197 by the Cochran Armitage trend test with
an allelic OR = 1.3 (0.941.36); Table 2), we found a similar trend
of association in which the frequency of a risk-allele G was higher
among HBV-induced HCC patients than that of CHB subjects.
Since we previously revealed that an A allele was associated with a
higher risk of HCV-induced HCC with OR of 1.36 , the
s2596542 alleles that increased the risk of HCC were opposite in
HBV-induced HCC and HCV-induced HCC.
Soluble MICA levels are associated with SNP rs2596542
We subsequently performed measurement of soluble MICA
(sMICA) in serum samples using the ELISA method in 176
HBVpositive HCC cases and 60 non-HBV controls. Nearly 30% of the
HBV-induced HCC cases revealed the serum sMICA level of
.5 pg/ml (defined as high) while the all control individuals except
one showed that of #5 pg/ml (defined as low) (P = 4.561026;
Figure 1A). Then, we examined correlation between SNP
rs2596542 genotypes and serum sMICA levels in HBV-positive
HCC cases. Interestingly, rs2596542 genotypes were significantly
associated with serum sMICA levels (P = 0.009; Figure 1B); 39% of
individuals with the GG genotype and 20% of those with the AG
genotype were classified as high for serum sMICA, but only 11%
of those with the AA genotype were classified as high (AA+AG vs
GG; P = 0.003) (Figure 1B). These findings were similar with our
previous reports in which a G allele was associated with higher
serum sMICA levels in HCV-induced HCC patients .
Negative association of variable number of tandem
repeat (VNTR) with sMICA level
The MICA gene harbors a VNTR locus in exon 5 that consists
of 4, 5, 6, or 9 repeats of GCT as well as a G nucleotide insertion
into a five-repeat allele (referred as A4, A5, A6, A9, and A5.1,
respectively). The insertion of G (A5.1) causes a premature
translation termination and results in loss of a transmembrane
domain, which may produce the shorter form of the MICA
protein that is likely be secreted into serum . However, the
association of this VNTR locus with serum sMICA level was
controversial among studies [14,26,27,28]. Therefore, we
examined the association between the VNTR locus and sMICA level in
HBV-induced HCC patients, and found no significant association
(Figure S1 and S2), concordant with our previous report for
HCVinduced HCC patients .
Soluble MICA levels are associated with survival of HCC
In order to evaluate the prognostic significance of serum sMICA
levels in HCC patients, we performed survival analysis of HCC
patients. A total of 111 HBV-infected HCC patients and 129
HCV-infected HCC patients were included in this analysis. The
mean survival period for HBV- and HCV-infected patients with
less than 5 pg/ml of serum sMICA were 67.1 months (95% CI:
61.173.1, n = 83), and 58.2 months (95% CI: 51.465.0, n = 85),
respectively. On the other hand, for patients with more than 5 pg/
ml of serum sMICA, the mean survival periods were 47.8 months
(95% CI: 34.830.9, n = 28) for HBV-induced HCC patients and
59.5 months (95% CI: 51.967.1, n = 44) for HCV-induced HCC
patients. The Kaplan-Maier analysis and log-rank test indicated
that among HBV-induced HCC subjects, the patients in the high
serum sMICA group showed a significantly shorter survival than
those in the low serum sMICA (P = 0.008; Figure 2). In addition,
we performed multi-variate analysis to test whether sMICA is an
independent prognostic factor by including age and gender as
covariates. The results revealed significant association of sMICA
levels with overall survival (P = 0.017) but not with age and gender
(Table S1).However, we found no association between the serum
sMICA level and the overall survival in the HCV-induced HCC
subjects (P = 0.414; Figure S3). Taken together, our findings imply
the distinct roles of the MICA variation and sMICA between
HBV- and HCV-induced hepatocellular carcinogenesis.
Vascular invasion in HBV-related HCC patients is
associated with soluble MICA levels
Since sMICA levels were associated with the overall survival of
HBV-related HCC patients, we tested whether sMICA levels
affect survival through modulating invasive properties of tumors or
size of the tumors. We tested the association between sMICA
levels and vascular invasion in 35 HBV-related HCC cases, among
whom 7 cases were positive and 21 cases were negative for
vascular invasion. We found significant association between
sMICA levels and vascular invasion (Figure 3; P = 0.014) in which
7 cases with positive vascular invasion showed high levels of
sMICA (mean = 54 pg/ml) than 21 cases without vascular
invasion (mean = 7.51 pg/ml). However, we found no association
between tumor size and sMICA levels (P = 0.56; data not shown).
These results suggest that sMICA may reduce the survival of
HBV-related HCC patients by affecting the invasive properties of
Several mechanisms such as HBV-genome integration into host
chromosomal DNA  and effects of viral proteins including
HBx  are shown to contribute to development and progression
of HCC, while the immune cells such as NK and T cells function
as key antiviral and antitumor effectors. MICA protein has been
HCC vs. Healthy control
Note: 407 HCC cases, 699 CHB subjects and 5,657 non-HBV controls were used in the analysis.
Chr.,chromosome; MAF, minor allele frequency; OR, odds ratio for minor allele; CI, confidence interval.
*Obtained by Armitage trend test.
Figure 1. Soluble MICA levels are associated with HBV-related HCC. (A) Correlation between soluble MICA levels and HBV-induced HCC
subjects. The y-axis displays the concentration of soluble MICA in pg/ml. The number of independent samples tested in each group is shown in the
xaxis. Each group is shown as a box plot and the mean values are shown in the x-axis. The difference between two groups is tested by Wilcoxon rank
test. The box plots are plotted using default settings in R. (B) Correlation between soluble MICA levels and rs2596542 genotype in HBV-positive HCC
subjects. The x-axis shows the genotypes at rs2596542 and y-axis display the concentration of soluble MICA in pg/ml. Each group is shown as a box
plot. P = 0.027 and 0.013 for AA vs. GG and AA vs. AG, respectively. The association between genotypes and sMICA levels was tested by Kruskal-wallis
test, whereas the difference in the sMICA levels between AA and GG is tested by Wilcoxon rank test. The box plots are plotted using default settings
considered as a stress marker of gastrointestinal epithelial cells
because of its induced expression by several external stimuli such
as heat, DNA damage, and viral infections [31,32,33,34]. Here,
we examined the association of rs2596542 and serum sMICA
levels with HBV-induced HCC. Like in HCV-induced HCC ,
our results from ELISA revealed a significantly higher proportion
of high serum sMICA cases (nearly 30%) in the HBV-induced
HCC group, compared to non-HBV individuals (1.7%).
Moreover, the serum sMICA level was significantly associated with
rs2596542, but not with the copy number differences of the
VNTR locus, as concordant with our previous report .
Several studies have already indicated the roles of sMICA as
prognostic markers for different types of malignant diseases
[17,18,19,20]. Therefore, it is of medical importance to test
whether serum sMICA levels can be used as a prognostic marker
for patients with HCC. To our best knowledge, this is the first
study to demonstrate the prognostic potential of sMICA for
HBVpositive HCC patients; we found 19.3 months of improvement in
survival among patients carrying less than 5 pg/ml of serum
sMICA, compared to those having more than 5 pg/ml.
On the contrary, we found no significant correlation between
sMICA levels and the prognosis of HCV-induced HCC cases.
These opposite effects of MICA variation could be explained by the
following mechanism. The individuals who carry the G allele
would express high levels of membrane-bound MICA upon HCV
infection and thus lead to the activation of immune cells against
virus infected cells. On one hand, HBV infection results in
increased expression of membrane-bound MICA as well as MMPs
through viral protein HBx , which would result in the
elevation of sMICA and the reduction of membrane-bound
MICA. Since sMICA could block CD8+T cells, NK-CTL, and
NK cells, higher sMICA would cause the inactivation of immune
surveillance system against HBV infected cells. In other words,
HBV may use this strategy to evade immune response and hence,
higher levels of sMICA could be associated with lower survival rate
among HBV-associated HCC. On the other hand, since HCV is
not known to induce the cleavage of membrane bound MICA,
individuals with low level membrane bound MICA expression
(carriers of rs2596542-allele A) could be inherently susceptible for
HCV-induced HCC. Thus, HBx-medicated induction of MMPs
could partially explain the intriguing contradictory effect of MICA
between HBV-induced HCC and HCV-induced HCC. Since we
observed significant correlation of sMICA levels with vascular
invasion, it may be the case that high levels of sMICA cause poor
prognosis of HBV-related HCC cases by making tumors more
aggressive and invasive. However it is important in future to
determine the ratio of membrane-bound MICA to sMICA in case
of HCV- and HBV-related HCC.
Interestingly, the immune therapy against melanoma patients
induced the production of auto-antibodies against MICA .
Anti-MICA antibodies would exert antitumor effects through
antibody-dependent cellular cytotoxicity against cells expressing
membrane-bound MICA and/or activation of NK cells by
inhibiting the sMICA-NKG2D interaction. However, further
studies are necessary, using well-defined HBV-related HCC
cohort, to investigate whether sMICA levels could be included
as an additional factor to predict the survival rate among
HBVrelated HCC subjects. Taken together, our results indicate the
potential of MICA variant and sMICA as prognostic biomarkers.
Thus, MICA could be a useful therapeutic target for
Figure S1 MICA repeat genotyping using
capillarybased method. The alleles are annotated using GeneMapper
software based on the size of the PCR product (185 bp = A4 allele,
188 bp = A5, 189 bp = A5.1, 191 bp = A6 and 200 bp = A9). The
inset at the base of each peak shows the size of the PCR product
with corresponding allele call by the software. The figure display
all observed heterozygotes at A5.1 allele.
Figure S3 Kaplan-Meier curves of the patients with
HCV-induced HCC. The patients were divided into two groups
according to their sMICA concentration (,5 pg/ml or .5 pg/
ml). Statistical difference was analyzed by log-rank test. The y-axis
shows the cumulative survival probability and x-axis display the
months of the patients` survival after blood sampling.
We would like to thank all the patients and the members of the Rotary
Club of Osaka-Midosuji District 2660 Rotary International in Japan, who
donated their DNA for this work. We also thank Ayako Matsui and Hiroe
Tagaya (the University of Tokyo), and the technical staff of the Laboratory
for Genotyping Development, Center for Genomic Medicine, RIKEN for
their technical support.
Conceived and designed the experiments: VK KM YN. Performed the
experiments: VK PHL YU HM ZD. Analyzed the data: VK PHL CT RM.
Contributed reagents/materials/analysis tools: YN NK AT MK HS KT
YT MS MM RT MO KK NK. Wrote the paper: VK PHL KM YN.
1. Kew MC ( 2010 ) Epidemiology of chronic hepatitis B virus infection, hepatocellular carcinoma, and hepatitis B virus-induced hepatocellular carcinoma . Pathol Biol (Paris) 58: 273 - 277 .
2. Sherman M ( 2010 ) Hepatocellular carcinoma: epidemiology , surveillance, and diagnosis. Semin Liver Dis 30 : 3 - 16 .
3. Perz J , Armstrong G , Farrington L , Hutin Y , Bell B ( 2006 ) The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide . J Hepatol 45 : 529 - 538 .
4. Chen CJ , Chen DS ( 2002 ) Interaction of hepatitis B virus, chemical carcinogen, and genetic susceptibility: multistage hepatocarcinogenesis with multifactorial etiology . Hepatology 36 : 1046 - 1049 .
5. Cui R , Okada Y , Jang SG , Ku JL , Park JG , et al. ( 2011 ) Common variant in 6q26-q27 is associated with distal colon cancer in an Asian population . Gut 60 : 799 - 805 .
6. Kumar V , Matsuo K , Takahashi A , Hosono N , Tsunoda T , et al. ( 2011 ) Common variants on 14q32 and 13q12 are associated with DLBCL susceptibility . J Hum Genet England . pp. 436 - 439 .
7. Cui R , Kamatani Y , Takahashi A , Usami M , Hosono N , et al. ( 2009 ) Functional variants in ADH1B and ALDH2 coupled with alcohol and smoking synergistically enhance esophageal cancer risk . Gastroenterology 137 : 1768 - 1775 .
8. Urabe Y , Tanikawa C , Takahashi A , Okada Y , Morizono T , et al. ( 2012 ) A genome-wide association study of nephrolithiasis in the Japanese population identifies novel susceptible loci at 5q35 .3, 7p14.3 and 13q14.1. PLOS Genet 8 ( 3 ): e1002541 .
9. Tanikawa C , Urabe Y , Matsuo K , Kubo M , Takahashi A , et al. ( 2012 ) A genome-wide association study identifies two susceptibility loci for duodenal ulcer in the Japanese population . Nat Genet 44 ( 4 ): 430 - 434 .
10. Hata J , Matsuda K , Ninomiya T , Yonemoto K , Matsushita T , et al. ( 2007 ) Functional SNP in an Sp1-binding site of AGTRL1 gene is associated with susceptibility to brain infarction . Hum Mol Genet 16 : 630 - 639 .
11. Kamatani Y , Wattanapokayakit S , Ochi H , Kawaguchi T , Takahashi A , et al. ( 2009 ) A genome-wide association study identifies variants in the HLA-DP locus associated with chronic hepatitis B in Asians . Nat Genet 41 : 591 - 595 .
12. Mbarek H , Ochi H , Urabe Y , Kumar V , Kubo M , et al. ( 2011 ) A genome-wide association study of chronic hepatitis B identified novel risk locus in a Japanese population . Human Molecular Genetics 20 : 3884 - 3892 .
13. Zhang H , Zhai Y , Hu Z , Wu C , Qian J , et al. ( 2010 ) Genome-wide association study identifies 1p36.22 as a new susceptibility locus for hepatocellular carcinoma in chronic hepatitis B virus carriers . Nat Genet 42 : 755 - 758 .
14. Kumar V , Kato N , Urabe Y , Takahashi A , Muroyama R , et al. ( 2011 ) Genomewide association study identifies a susceptibility locus for HCV-induced hepatocellular carcinoma . Nature genetics 43 : 455 - 458 .
15. Jinushi M , Takehara T , Tatsumi T , Kanto T , Groh V , et al. ( 2003 ) Expression and role of MICA and MICB in human hepatocellular carcinomas and their regulation by retinoic acid . Int J Cancer 104 : 354 - 361 .
16. Bauer S , Groh V , Wu J , Steinle A , Phillips JH , et al. ( 1999 ) Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA . Science 285 : 727 - 729 .
17. Holdenrieder S , Stieber P , Peterfi A , Nagel D , Steinle A , et al. ( 2006 ) Soluble MICA in malignant diseases . Int J Cancer 118 : 684 - 687 .
18. Nu ckel H , Switala M , Sellmann L , Horn PA , Durig J , et al. ( 2010 ) The prognostic significance of soluble NKG2D ligands in B-cell chronic lymphocytic leukemia . Leukemia 24 : 1152 - 1159 .
19. Tamaki S , Sanefuzi N , Kawakami M , Aoki K , Imai Y , et al. ( 2008 ) Association between soluble MICA levels and disease stage IV oral squamous cell carcinoma in Japanese patients . Hum Immunol 69 : 88 - 93 .
20. Li K , Mandai M , Hamanishi J , Matsumura N , Suzuki A , et al. ( 2009 ) Clinical significance of the NKG2D ligands, MICA/B and ULBP2 in ovarian cancer: high expression of ULBP2 is an indicator of poor prognosis . Cancer Immunol Immunother 58 : 641 - 652 .
21. Salih H , Rammensee H , Steinle A ( 2002 ) Cutting edge: down-regulation of MICA on human tumors by proteolytic shedding . J Immunol 169 : 4098 - 4102 .
22. Groh V , Wu J , Yee C , Spies T ( 2002 ) Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation . Nature 419 : 734 - 738 .
23. Jinushi M , Takehara T , Tatsumi T , Hiramatsu N , Sakamori R , et al. ( 2005 ) Impairment of natural killer cell and dendritic cell functions by the soluble form of MHC class I-related chain A in advanced human hepatocellular carcinomas . J Hepatol 43 : 1013 - 1020 .
24. Nakamura Y ( 2007 ) The BioBank Japan Project . Clin Adv Hematol Oncol 5 : 696 - 697 .
25. Ota M , Katsuyama Y , Mizuki N , Ando H , Furihata K , et al. ( 1997 ) Trinucleotide repeat polymorphism within exon 5 of the MICA gene (MHC class I chain-related gene A): allele frequency data in the nine population groups Japanese , Northern Han , Hui, Uygur, Kazakhstan, Iranian, Saudi Arabian , Greek and Italian. Tissue Antigens 49 : 448 - 454 .
26. Tamaki S , Sanefuzi N , Ohgi K , Imai Y , Kawakami M , et al. ( 2007 ) An association between the MICA-A5.1 allele and an increased susceptibility to oral squamous cell carcinoma in Japanese patients . J Oral Pathol Med 36 : 351 - 356 .
27. Tamaki S , Kawakami M , Yamanaka Y , Shimomura H , Imai Y , et al. ( 2009 ) Relationship between soluble MICA and the MICA A5.1 homozygous genotype in patients with oral squamous cell carcinoma . Clin Immunol 130 : 331 - 337 .
28. Lu M, Xia B , Ge L , Li Y , Zhao J , et al. ( 2009 ) Role of major histocompatibility complex class I-related molecules A*A5.1 allele in ulcerative colitis in Chinese patients . Immunology 128 : e230 - 236 .
29. Bonilla Guerrero R , Roberts LR ( 2005 ) The role of hepatitis B virus integrations in the pathogenesis of human hepatocellular carcinoma . J Hepatol 42 : 760 - 777 .
30. Bouchard MJ , Schneider RJ ( 2004 ) The enigmatic X gene of hepatitis B virus . J Virol 78 : 12725 - 12734 .
31. Groh V , Bahram S , Bauer S , Herman A , Beauchamp M , et al. ( 1996 ) Cell stressregulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium . Proc Natl Acad Sci U S A 93 : 12445 - 12450 .
32. Groh V , Steinle A , Bauer S , Spies T ( 1998 ) Recognition of stress-induced MHC molecules by intestinal epithelial gammadelta T cells . Science 279 : 1737 - 1740 .
33. Groh V , Rhinehart R , Randolph-Habecker J , Topp M , Riddell S , et al. ( 2001 ) Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virus-infected cells . Nat Immunol 2 : 255 - 260 .
34. Gasser S , Orsulic S , Brown EJ , Raulet DH ( 2005 ) The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor . Nature 436 : 1186 - 1190 .
35. Lara-Pezzi E , Gomez-Gaviro MV , Galvez BG , Mira E , Iniguez MA , et al. ( 2002 ) The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression . J Clin Invest 110 : 1831 - 1838 .
36. Jinushi M , Hodi F , Dranoff G ( 2006 ) Therapy-induced antibodies to MHC class I chain-related protein A antagonize immune suppression and stimulate antitumor cytotoxicity . Proc Natl Acad Sci U S A 103 : 9190 - 9195 .