Microsatellite instability mutator phenotype in hepatocellular carcinoma in non-alcoholic and non-virally infected normal livers

Franck Chiappini1,4,5, Marine Gross-Goupil1,4, Raphaël Saffroy1,4, Daniel Azoulay2,4, Jean-François Emile3,4, Luc-Antoine Veillhan2, Valérie Delvart2, Stephan Chevalier5, Henri Bismuth2, Brigitte Debuire1,4 and Antoinette Lemoine1,4,6

1 Service de Biochimie et Biologie Moléculaire, 2 Centre Hépato-biliaire and 3 Anatomie Pathologique, Hôpital Paul Brousse, Assistance Publique–Hôpitaux de Paris and 4 INSERM U268, IFR 89, Faculté de Médecine Paris-Sud, Université Paris XI, 14 Avenue Paul Vaillant Couturier, 94804 Villejuif Cedex, France and 5 Pfizer, Centre de Recherche, 37401 Amboise, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Microsatellite instability (MSI) seems to be a rare event in hepatocarcinogenesis and might actually be associated with the progression of hepatocellular carcinoma (HCC) in which the liver is often the site of chronic hepatitis or cirrhosis. The aim of this work was to define the MSI phenotype in HCC affecting exclusively normal livers to avoid slippage errors due to cirrhosis. One hundred and sixty-four patients with HCC affecting non-cirrhotic livers were operated on in our hospital between 1984 and 2001. We analyzed 37 patients selected for low alcohol consumption and the absence of HBV or HCV infection. All the livers were histologically normal. MSI was analyzed according to the criteria defined during the conference consensus workshop for colorectal cancer. High MSI (MSI-H > 30%) was found in 6 (16%) and low MSI (MSI-L < 30%) in 10 (27%) of the 37 HCCs. None of the 10 microsatellite markers tested were altered in the remaining 21 tumors (57%). Immunohistochemistry showed that normal amounts of hMLH1 and hMSH2 were present both in MSI-H and in MSI-L HCCs. MSI-H was significantly associated with more aggressive histological tumor features and a shorter median delay before recurrence. Thus, we have found a small subgroup of HCC tumors which can be considered as a new clinical/histological entity.

Abbreviations: HNPCC, hereditary non-polyposis colorectal cancer; MSI, microsatellite instability DNA mismatch repair; MSI-H, high MSI; MSI-L, low MSI; MSS, MMR microsatellite stable


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disorder that accounts for ~1–3% of all colorectal cancers that exhibit ubiquitous microsatellite instability (MSI) (1). HNPCC is caused by a germline mutation in one of the genes responsible for DNA mismatch repair (MMR). More than 90% of the mutations detected in HNPCC kindreds are located in the MSH2 or MLH1 genes (2,3). A deficiency in this system, which repairs the short DNA heteroduplex loops and nucleotide mismatches that occur during DNA synthesis, changes the length of nucleotide repeat sequences resulting in MSI.

Due to the heterogeneous nature of the mutations in the MMR genes and the time and cost of direct sequencing, an initial screen is usually performed to identify individuals who may have HNPCC. A panel of experts (4) recommended the use of a reference panel of five loci (two mononucleotide repeats and three dinucleotide repeats) for the identification of high MSI (MSI-H) and low MSI (MSI-L) tumors. MSI has been reported in other familial or sporadic cancers, such as endometrial (5), esophageal (6), gastric (7) and pancreatic (8) cancers.

It is still unclear whether some HCCs have MMR errors. Microsatellite alterations have been widely and frequently reported by different teams, including ours in liver cirrhosis, mainly when cirrhosis is associated with HBV infection (913). However, the frequency of MSI in HCC tumors exhibits large variations. More than 10% (10–43%) of HCCs in European, American, Japanese and black Southern African patients have been reported to exhibit MSI at one or more microsatellite markers (1020), whereas this phenotype has rarely been observed in other European, American, Japanese, Taiwanese, Korean and Chinese series of HCC tumors (2130). Moreover, analysis of the instability of BAT26, a monomorphic microsatellite marker that reliably predicts MSI-H, has been shown to rarely be altered (10,14,22,24,28). Possible explanations for this are differences in etiology, the fact that different approaches were used to define the presence of this microsatellite mutator phenotype, differences in the type and number of markers analyzed and differences in the cut-off criteria. Moreover, most of the studies used DNA extracted from the surrounding liver tissues as reference DNA, although several teams (1013), including ours (9), have detected insertion/deletion mutations in cirrhotic tissues.

Cirrhosis is the most frequent type of liver disease underlying HCC. The inflammation and regeneration of liver tissues that occur in cirrhosis favor the development of genetic abnormalities. Thus, the MSI observed in HCCs on cirrhotic livers might be due to the clonal expansion of genetically modified cells that acquired a selective growth advantage following the accumulation of altered protooncogenes and tumor suppressor genes (3134).

HCC occurring in non-fibrotic livers in patients with no known etiological factors, such as high alcohol consumption or hepatotropic viral infection, are a rare, ill-defined subgroup of non-cirrhotic HCCs in which mechanisms of hepatocarcinogenesis remain unclear. These tumors may be an accurate model for studying hepatocarcinogenesis and to avoid the inflammatory/regenerative processes or virus integration in the genome that are associated with such slippage errors during DNA replication. Thus, our objective was to analyze the incidence of alterations in the panel of microsatellites defined by the experts (4) in 37 HCC tumors on normal livers selected among a series of 164 HCC tumors affecting histologically non-cirrhotic livers without HBV and/or HCV viral infection or high alcohol consumption.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients and samples
Between 1984 and 2001 a total of 629 HCC tumors were surgically resected in our institution, 164 of which affected non-cirrhotic livers. The histology was assessed just after surgery by a pathologist and then checked by a second pathologist. Livers showing signs of inflammation or mild (F1) fibrosis (n = 29) or steatosis (n = 14) were excluded from the study. The etiology was checked and patients with HBV (n = 18) or HCV infection (n = 5), who consumed >5 g alcohol/day (n = 15) or who had iron or copper loading disease (n = 4) were excluded. Thus, 79 HCCs (12.5%) were considered to have developed on ‘normal livers’. Reference DNA (from gall bladder or lymphocytes) and amplified DNA for all the microsatellite markers were available for only 37 patients. Thus, 37 patients (21 men and 16 women) operated on between 1987 and March 2001 and followed until December 2002 were included in the study. All patients underwent curative surgery involving either hepatectomy (n = 27) or orthotopic liver transplantation (n = 10).

Serological tests (e.g. HCV RNA and hepatitis B surface antigen) were routinely carried out before surgery using commercially available assays (Amplicor HCV Monitor; Roche Diagnostic Systems, Branchburg, PA). None of the patients had HCV or HBV or serum anti-HBc antibodies.

Paraffin-embedded (i) tumoral tissues containing >80% tumoral cells, (ii) distant paired non-tumoral liver tissue and (iii) non-hepatic tissue such as the gall bladder when available, were analyzed. When gall bladder tissue was not available (n = 20), a whole blood sample obtained at a distance from the site of surgery was analyzed. DNA was extracted from paraffin-embedded tissues or whole blood as described previously (9,14).

Molecular analysis for MSI status
Seven dinucleotide repeats (D2S123, D5S346, D17S250, D16S402, IFNA, D13S153 and D8S277) and three mononucleotide repeats in poly(A) tracts (BAT25, BAT26 and BAT40) were amplified by PCR. These markers included the five microsatellite markers (BAT25, BAT26, D5S346, D2S123 and D17S250) defined as the reference panel and two markers (BAT40 and D13S153) defined as alternative loci by ‘The International Workshop on Microsatellite Instability and RER Phenotypes in Cancer Detection and Familial Predisposition’ (4). The last three markers (D16S402, IFNA and D8S277) were used previously by us or other groups to detect MSI in HCC (1316,30). PCR was performed in a 25 µl volume containing 5 pmol each primer, 40 µM each dNTP, 10% formamide, standard PCR buffer containing 1.5 mM MgCl2 and 1 U Taq DNA polymerase (Qiagen, Illkirch, France) using a GeneAmp PCR system 2400 (Applied Biosystems, Courtaboeuf, France). The forward primers were labeled with FAM, HEX or TET fluorescent dye (Eurogentec, Seraing, Belgium). One microliter of DNA extracted from formalin-fixed, paraffin-embedded specimens of tumor tissue or 50 ng DNA extracted from paired peripheral blood samples obtained at a distance from the site of surgery was amplified. The amplification conditions consisted of 1 cycle of 2 min at 94°C, 35 cycles of 1 min at 92°C, 30 s at 50, 52 or 55°C, 45 s at 72°C and a final extension step of 7 min at 72°C. One microliter of the PCR product was diluted in 24 µl of formamide and separated by capillary electrophoresis using an ABI PRISM 310 sequencer (Applied Biosystems). Changes at microsatellite loci in DNA from tumor tissues were compared with those in non-hepatic DNA processed simultaneously. A tumor was defined as MSI-H when shifts occurred in at least 30% of assessable markers (4). Tumors in which <30% of assessable markers were altered were classified as MSI-L and were grouped together with microsatellite stable (MSS) tumors. When one of the normal alleles for a given marker was missing and no other fragments were present for that marker, the locus was scored as negative for MSI.

Immunochemistry of hMLH1 and hMSH2
Immunohistochemical analyses were performed on 4 mm thick sections of paraffin-embedded liver samples. The primary antibodies were mouse anti-MLH1 (G168-728; BD Pharmingen, San Diego, CA) and mouse anti-MSH2 (Ab-2; Oncogene Research Products, Boston, MA) monoclonal antibodies. The staining was amplified and revealed with a three step, avidin–biotin kit according to the manufacturer's instructions (DAB Detection Kit; Ventana Medical Systems Tucson, AZ).

Statistical analysis
Statistical analyses were performed using the Statview software system (Statview F-4.5). The {chi}2 test, Student's t-test and Fisher's exact test were used to determine whether there were any statistical differences in the clinical data between patients with MSI-H tumors and patients with MSI-L or MSS tumors. The probability of disease-free survival in the two groups was determined by use of the Kaplan–Meier technique and the significance of the difference was analyzed by the log rank test. A P value of <0.05 was considered to be statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Microsatellite alterations
We studied microsatellite instability in 37 hepatocellular carcinomas affecting normal livers by use of the panel of five microsatellite markers described by the consensus conference workshop in 1998 and five others that have already been used to detect MSI in HCC and other tumor types. A tumor was defined as MSI-H when shifts occurred in at least 30% of assessable markers (4). Tumors in which <30% of assessable markers were altered were classified as MSI-L. Tumors were considered to be MSS if none of the loci showed instability. The results are presented in Table I. Six of the 37 (16%) tumors were MSI-H, 10 (27%) were MSI-L and 21 (57%) were MSS. Alterations of at least one of the quasi-monomorphic mononucleotide markers (BAT25, BAT26 and BAT40) were observed in five tumors, two of which were considered as MSI-H tumors. Three independent assays gave reproducible nucleotide shifts on mononucleotide markers, however, all were smaller than 10 bp deletions (Figure 1). Two non-tumoral liver samples (patients 3 and 5) in which no histological alterations were observed presented an alteration of one microsatellite marker, a microsatellite also altered in the corresponding tumor.


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Table I. Characteristics of patients and tumors analyzed

 


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Fig. 1. Alterations of microsatellite markers in hepatocellular carcinoma. Example of patient 5.

 
hMLH1 and hMSH2 immunochemistry
All of the six MSI-H tumors and six MSI-L tumors stained positively for both hMLH1 and hMSH2 proteins (Figure 2). These proteins were present in a normal colon mucosa sample and absent from a HNPCC tumor used as controls.




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Fig. 2. Representative assessment of hMLH1 and hMSH2 expression in hepatocellular carcinomas. (A) hMLH1 expression in low grade HCC; (B) hMSH2 expression in high grade HCC.

 
Histological and clinical characteristics of patients according to MSI alterations
We compared the clinical and histological tumor characteristics of the patients with MSI-H tumors with those of patients with MSI-L or MSS tumors (Table II). No differences were observed in sex ratio or mean age. All of the patients with MSI-H tumors presented a unique nodule, whereas the mean number of HCC nodules was 3.0 ± 3.5 in the other group; the difference was not significant. The mean diameter of the largest nodule was significantly higher in patients with MSI-H tumors than in the other patients (14.0 ± 3 versus 10.0 ± 4.5 cm; P = 0.03). None of the patients with MSI-H tumors presented a normal capsule surrounding the tumor, whereas the capsule was present and normal in 14 of the 31 patients (45%) with MSI-L or MSS tumors (P < 0.05). No difference in the microvascular tumoral extension was observed between the two groups.


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Table II. Characteristics of patients and tumours according to h-MSI phenotype or not

 
Recurrence-free survival of patients according to MSI alterations
All the patients had undergone curative surgery. Figure 3 shows the Kaplan–Meier curves for disease-free survival rate for patients with MSI-H tumors and patients with MSI-L or MSS tumors. The survival rates were 67 and 80% at 12 months and 17 and 47% at 36 months in the MSI-H patients and in the patients without MSI-H, respectively (P = 0.02, log rank analysis). The median delay before recurrence was 12 months in the group of patients with MSI-H tumors, compared with 25 months in the other group.



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Fig. 3. Kaplan–Meier curves for metastasis-free survival for the two groups of patients are shown: patients with MSI-H and patients without MSI-H.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We analyzed the incidence of alterations of a panel of microsatellites defined by the Amsterdam consensus conference workshop for colorectal cancer in a series of HCC affecting histologically normal livers (4). Sixteen percent of tumors were classified as MSI-H (>30%) and all of them stained positively for both hMLH1 and hMSH2. Patients with MSI-H tumors presented more aggressive histological features and a worse prognosis than patients with MSI-L or MSS.

HCC is usually preceded by cirrhosis, and the resulting genetic alterations are still poorly understood. Genetic studies on compensated cirrhotic or end-stage cirrhotic patients have revealed a number of molecular abnormalities in the cirrhotic parenchyma. A number of different genetic alterations have been observed, the most common of which is loss of heterozygosity (1115,23,2730,3437). LOH at some crucial loci may play a significant role in the initiation, progression or metastatic spread of HCC, suggesting that tumor or metastasis suppressor genes in the vicinity of the altered microsatellite loci have been lost or altered. As LOH of tumor suppressor genes is an early feature of different precancerous lesions in other organs such as the prostate, the colon epithelium and Barett's mucosa, cirrhotic tissue can be considered to be a favorable tissue for premalignant and malignant lesions. MSI has also been described in cirrhosis, mainly when infection by HBV was observed (913). Recently, rheumatoid arthritis synovium, another inflammatory tissue, has been shown to exhibit abundant MSI (38) with high MSH3 and low MSH6 in western blot analysis. However, in cirrhosis it was not possible to distinguish between MSI due to errors in MMR genes and the occasional slippage errors that occur during DNA replication, indicative of a clonal population and not reflecting significant genome instability.

To avoid the influence of inflammatory/regenerative processes in fibrosis or cirrhosis that could provoke such slippage errors during DNA replication, we analyzed the incidence of MSI in non-fibrotic HCC. HCCs affecting non-fibrotic livers considered to be ‘normal’ are rare. Most ‘non-cirrhotic’ livers present signs of fibrosis or steatosis. This has been reported in some cases of HBV and HCV infection (3948). Thus, we excluded all livers with fibrosis or steatosis and all patients with HBV or HCV infection from our study, even if the liver tissue was histologically normal. We also excluded patients with a high alcohol intake. All livers from patients were examined by two independent pathologists who confirmed the absence of fibrosis and steatosis. The characteristics of the population corresponded to those previously published (3948), with a non-predominance of males, a younger age and a large mean tumor size compared with the series of HCCs that developed on cirrhotic livers. Thus, HCC on ‘healthy’ liver probably represents a good model to explore genetic abnormalities involved in hepatocarcinogenesis.

We chose to study the five microsatellites recommended following the consensus conference in Amsterdam (4) and five others that have already been used to detect MSI in HCC and other tumor types. We analyzed our results in exactly the same way as described by the experts. No previous studies on HCC were performed using the Amsterdam criteria and this panel of microsatellite markers. DNA was extracted from paraffin-embedded tissues containing >80% tumor cells. It has been claimed that fixation and paraffin embedding generate artefactual genomic instability, but similar results have been observed with tumors that had been frozen and with tumors that had been fixed and embedded in paraffin (9,17).

According to the Amsterdam criteria, 6 of 37 tumors (16%) exhibited MSI-H (i.e. alterations in >30% of the loci analyzed). This subgroup of MSI-H HCCs represents 2% of all HCCs removed in our institution between 1984 and 2001. Interestingly, the histological characteristics and the prognosis of the group of patients with MSI-H tumors were worse than those of the group of patients with MSI-L or MSS tumors. Patients with MSI-H tumors tended to present a unique and large nodule without capsule, corresponding to a more aggressive tumor. These results are different from those observed in other digestive tumors such as HNPCC, for which MSI is an indicator of good prognosis (1,49). This suggests that genetic or environmental factors different from those involved in HNPCC tumors inducing MSI can induce such aggressive tumors.

MSI characterizing the mutator phenotype described in HNPCC tumors is considered to be the consequence of exonic mutation or promoter methylation of MMR genes, rendering them dysfunctional. However, other factors, such as hypoxic stress due to histone deacetylation (50), reactive oxygen species (38) or radioactive compounds (51), have also been described as inducing MSI via MMR gene dysregulation. Several studies have reported variable MSI incidence in HCC (1030). However, the role of MMR gene inactivation in HCC is still controversial. MacDonald et al. (15) observed LOH at hMSH2 and/or hMLH1 gene loci associated with MSI and Yano et al. (18) observed mutations in the hMSH2 gene in 7 of 42 HCCs. Hypermethylation of the hMLH1 promoter was recently reported (52,53). Conversely, neither hMLH1 hypermethylation nor mutation was detected in a series of 55 japanese HCC (54) and Wang et al. (22) showed that the expression of hMLH1 and hMSH2 was normal in 36 HCC tumors, none of which exhibited BAT26 alterations. Moreover, other genes with mononucleotide repeats in the coding region, such as TGFBRII, IGFIIR and the proapoptotic Bax, as well as MMR genes such as MSH6 and MSH3, were not reported to be targets for frameshift mutations, even in HCC tumors with a MSI phenotype (2528). In our group of patients, immunochemistry showed that all tumors expressed both hMLH1 and hMSH2, the two genes most frequently mutated in other MSI+ tumors, suggesting the absence of either inactivating mutations in hMLH1 or hMSH2 or promoter hypermethylation of hMLH1.

MSI-H has been described in a substantial proportion of non-HNPCC tumors (4). To date, only a few reports have allowed the assessment of the etiology of MSI in non-HNPCC cancers (5463). In prostate (58,59,63) and bladder (61) tumors, MSI and disruption of the MMR system was clearly shown. In other cases, such as head and neck cancers (57), no MMR gene involvement could be demonstrated. However, the type of altered repeat seems to be different in non-HNPCC compared with HNPCC tumors. Thus, whereas in colorectal cancers an alteration in the mononucleotide repeat marker BAT26 alone seems sufficient for MSI-H determination (4,64), this seems not to be the case for cancers not belonging to the HNPCC spectrum (60,65). This observation also seems to be valid for our group of HCCs. Although in our study only reproducible and significant allele size variations have been considered for assessment of mononucleotide repeat alterations, we observed that variations are less extensive than generally observed in MSI-H colorectal tumors. With a model based on the use of a transgene with 26 A residues transfected into a cell line, it was shown that the length of the poly(A) repeat mutation was proportional to the number of divisions over longer time intervals (66). The extensive poly(A) deletions observed in MSI-H colorectal carcinoma likely reflect hundreds of divisions since loss of MMR. Mutations were also described as proportional to the number of divisions for dinucleotide repeats (67). Thus, the shorter length poly(A) repeats observed in our group of HCCs could reflect differences in the kinetics of the MMR defect along hepatocarcinogenesis or more subtle alterations in the MMR system, compared with traditional HNPCC tumors. The pathophysiological mechanisms involved in these patients were certainly different from the classical genetic factors involved in tumors belonging to the HNPCC spectrum. In addition, some carcinogens were also described as inducing MSI (68,69).

Thus, we do not yet fully understand the genetic mechanism of this instability. More aggressive histological features, absence of defective expression of hMLH1 and hMSH2 genes and altered microsatellite profiles suggest a mechanism distinct from those involved in HNPCC tumors. Thus this instability may be attributed to genetic alterations in other MMR genes not yet discovered or to more subtle alterations in the MMR system, such as an imbalance in the relative amounts of MMR proteins, perhaps implicating MMR disturbance by environmental factors, as recently described (38).

In conclusion, we have shown the presence of ‘true’ MSI-H defined according to the criteria of the consensus conference workshop (4) in 16% of HCC tumors from non-alcoholic and non-virally infected normal livers that cannot be attributed to slippage errors due to cirrhosis. This small group of HCCs, representing 2% of the HCCs removed in our institution, which is below the percentage of MSI reported for HNPCC tumors, can be considered as a new clinical/histological entity.


    Notes
 
6 To whom correspondence should be addressed. Email: antoinette.lemoine{at}pbr.ap-hop-paris.fr Back


    Acknowledgments
 
This work was supported by the Fondation de l'Avenir, Fondation Adrienne et Pierre Sommer and ICIG (Institut du Cancer et d'Immunogénétique, Villejuif).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 19, 2003; revised October 1, 2003; accepted November 17, 2003.





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