Clinical Cancer Research Prevention Award AACR Membership
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nomoto, S.
Right arrow Articles by Sidransky, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nomoto, S.
Right arrow Articles by Sidransky, D.
Clinical Cancer Research Vol. 8, 481-487, February 2002
© 2002 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Mitochondrial D-loop Mutations As Clonal Markers in Multicentric Hepatocellular Carcinoma and Plasma1

Shuji Nomoto, Katsuya Yamashita, Katsumi Koshikawa, Akimasa Nakao and David Sidransky2

Department of Otolaryngology–Head and Neck Surgery, Head and Neck Cancer Research Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196 [S. N., D. S.], and Department of Surgery II, Nagoya University School of Medicine, Nagoya, Japan 466–8550 [K. Y., K. K., A. N.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Hepatocellular carcinoma (HCC) is a highly malignant tumor prone to multicentric occurrence. Differentiation between a true relapse of HCC and a second primary tumor is of clinical importance. We sought to identify mitochondrial mutations in HCC and test their use as clonal markers in this disease.

Experimental Design: Primary HCC tissue samples were obtained from 19 patients and analyzed for mutations within the mitochondrial displacement loop (D-loop). The discovered mutations were used to determine tumor clonality and provided the basis for detection of tumor DNA in corresponding plasma samples.

Results: Thirteen of 19 HCC cases (68%) were identified as having D-loop mitochondrial DNA (mtDNA) mutations in at least one tumor. In 3 of these 13 cases, the same mutation was observed in multiple tumors, indicating monoclonal origin. Remarkably, in 8 of 13 mutated cases, we detected deletion/insertion mutations in the C-tract, a recently reported hotspot and potential replication start site of the closed, circular mitochondrial genome. In addition, we detected mutant mtDNA in 8 of 10 tested paired plasma DNA samples using a highly sensitivity molecular assay.

Conclusions: mtDNA mutations within the D-loop control region are a frequent event in HCC, providing a molecular tool for the determination of clonality. In addition, detection of tumor-specific mtDNA mutations in plasma DNA needs to be explored further for monitoring patients with primary HCC.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HCC3 is one of the most common malignancies worldwide and is generally associated with a poor prognosis (1) . The disease is highly prevalent in Asia but relatively rare in developed countries, although the incidence is increasing in both the United States and Japan (2) . Many environmental factors, including aflatoxins, alcoholism, and hemochromatosis, as well as a susceptible genetic background, have been implicated in the development and progression of HCC (3) . However, chronic HBV and HCV infection are by far the best documented factors associated with the progression of chronic hepatitis to cirrhosis and eventually to HCC (4) .

Although other therapeutic approaches have been introduced to treat HCC (5) , surgery remains the treatment of choice. The progression and outcome of truly relapsed HCC compared with a second primary tumor are distinct (6) , and thus, clonal analysis of the initial and recurrent HCC is of clinical significance. Although several studies have addressed HCC clonal determination (7, 8, 9) , reported molecular methods are still of limited use clinically or are technically challenging.

Recently, we and others (10 , 11) identified a high frequency of missense and frameshift mutations in mtDNA from primary human neoplasms. These mutations were most frequently found in the noncoding D-loop region (11) , especially in a polymorphic cytosine mononucleotide repeat between nucleotides 303 and 315. Although occasional defects of complex III (ubiquinone-cytochrome c-oxidoreductase) and IV (cytochrome c-oxidase) within the respiratory chain have been detected in both normal and cirrhotic livers, (12) the existence of mtDNA alterations in HCC was not generally recognized. More recently, one group reported frequent mtDNA mutations in HCC, as well as in noncancerous tissue (13) .

We report frequent mutations in the control region of the mitochondrial genome in tissue samples from HCC. The high frequency of mutations within the control region of the mtDNA provided a tool to determine the clonal origin of multiple HCCs in individual patients. In addition, mutant mtDNA was detected in matched plasma samples using sensitive techniques and might one day prove useful for management of this disease.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Clinical HCC Samples.
HCC and adjacent nontumorous liver tissues from 19 patients were surgically resected in Nagoya University Hospital between 1989 and 2000. Thirteen of 19 patients had synchronous multiple HCC, which were resected during their first operation. In the other 6 patients, hepatic tumors reappeared between 10 months and 5 years after the first curative operation and were resected by a surgical approach. Serological markers for HBV and HCV infections and {alpha}-fetoprotein levels were recorded.

DNA Extraction.
All specimens were immediately fresh frozen after resection and stored at -80°C. Serial 10-µm sections were cut with a microtome. After staining with H&E for histological examination, the other sections were used for DNA extraction. With the exception of small areas comprised of fatty and necrotic cells, nearly all sections were comprised of >90% neoplastic cells. In 14 of 19 cases, we collected plasma samples before treatment, and these were stored at -80°C until extracted for DNA. Normal, tumor, and plasma DNA were prepared as described previously (14) .

Amplification and Purification of the Mitochondrial D-loop Fragment.
To avoid amplification of nuclear-encoded pseudogenes (15) , mtDNA fragments of >2.5 kb in length containing the D-loop region were amplified using the following primers F47: 5'-CGCACGGACTACAACCACGAC-3' (forward) and R15: 5'-TGTGGGGGGTGTCTTTGGGG-3' (reverse). In a PCR buffer containing 5% DMSO, 100 ng of genomic DNA were subjected to the step-down PCR protocol: initial denaturing step at 94°C for 5 min followed by 94°C for 45 s, 64°C for 1 min and 15 s, and 70°C for 3 min for two cycles; 94°C for 45 s, 61°C for 1 min and 15 s, and 70°C for 3 min for two cycles; 94°C for 45 s, 58°C for 1 min and 15 s, and 70°C for 3 min for 30 cycles; and a final extension at 70°C for 5 min. PCR products were gel purified using a Qiagen gel extraction Kit (Qiagen, Inc., Valencia, CA). In each experiment, DNA extracted from a mtDNA-negative cell line ({rho}0) was included as a negative control (16) .

Direct Sequencing of the D-loop Region of mtDNA.
Amplified fragments containing the mitochondrial D-loop region were sequenced using ({gamma}-33P) ATP 5' end-labeled sequencing primers and an AmpliCycle sequencing kit (Perkin-Elmer and Roche Molecular Systems, Inc., Brachburg, NJ) under the following cycle conditions: 95°C for 30 s, 52°C for 1 min, and 70°C for 1 min for 30 cycles. The sequence of the primers was, F44: 5'-GTCTTTAACTCCACCATTAG-3', F55: 5-ACCGTACATAGCACATTACA-3', F56: 5'-ATCACGATGGATCACAGGT-3', and F57: 5'-CGTTCAATATTACAGGCGAAC-3'. The sequenced products were analyzed on a denaturing 6% polyacrylamide gel.

PCR Fragment Length Detection Assay Surrounding the C-tract Region.
A simple PCR assay, based on the amplification of a 104-bp product that included the C-mononucleotide repeat, was performed for rapid detection of C-tract deletion/insertion mutations. In PCR buffer containing [32P] dCTP, 100 ng of genomic DNA were subjected to the following PCR protocol: initial denaturing at 94°C for 3 min followed by 94°C for 20 s, 55°C for 20 s, and 70°C for 15 s for 35 cycles and a final extension at 70°C for 5 min using primer sets of C-tract F: 5'-TCTGACCAGCCACTTTCCA-3 and C-tract R: 5'-GGGTTTGGCAGAGATGTGT-3'. PCR products were analyzed on a denatured 6% polyacrylamide gel and processed by autoradiography.

Oligonucleotide Mismatch Ligation Assay.
Because of the low concentration of DNA in the matched plasma samples, mtDNA mutations were not detected using sequence analysis alone, and a more sensitive oligonucleotide mismatch ligation assay was performed (17) . We applied this method to matched plasma DNA to 4 of 5 cases, in which we detected primary tumor missense mutations. In the cases with C-tract deletion/insertion mutations, we could not perform the assay because of a problem with oligo design. Fragments containing the mitochondrial D-loop region were PCR amplified and extracted using the method described previously. For each missense mutation, discriminating oligonucleotides that contained the mutated base at their 3' end were designed (position: 72, 5'-CTGGGGGGC-3' for patient 1, position: 16278, 5'-CTAGGATAC-3' for patient 14, position: 16390, 5'-CAGATAGGA-3' for patient 16, and position: 70, 5'-GTCTGGGGA-3' for patient 19). An immediately adjacent 3' oligonucleotide linker, along with the discriminating oligonucleotide (5'-ATGCACGCG-3' for patient 1, 5'-CAACAAACC-3' for patient 14, 5'-GTCCCTTGA-3' for patient 16, and 5'-GTATGCACG-3' for patient 19) was used as a substrate for the ligation reaction. In case 16, a blocking oligo (5'-TAGGGGTCCC-3') was used for decreasing the background. Discriminating oligonucleotides (250 ng) were mixed with the PCR amplified fragment and 40 ng of the {gamma}-[32P] end-labeled 3' oligonucleotide linker. The reactions were incubated at 37°C for 1 h in the presence of T4 DNA ligase (Life Technologies, Inc., Grand Island, NY), analyzed on denatured 12% polyacrylamide gels, and processed by autoradiography.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mutations in the D-loop Region of the Mitochondrial Genome.
The D-loop region of the mitochondrial genome was analyzed in all tumors from 19 patients with multiple HCCs and matching nontumorous tissue by direct sequencing. Representative results of the sequence analysis in normal tissue and paired tumor is shown in Fig. 1Citation . In case 1, all three tumors harbored a T to C transition. Detailed results of the mitochondrial sequence analysis are summarized in Table 1Citation .



View larger version (57K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Sequence detection of mutated mtDNA in samples obtained from multiple tumors. The mtDNA D-loop was analyzed by direct sequencing of the normal (noncancerous tissue of the liver), primary tumor (T1), second tumor (T2), and third tumor (T3), if available, from patients 1 (A), 13 (B), and 19 (C). Arrow, a single nucleotide change T > C at position 72 in all three tumors (A), whereas in cases 13 and 19, only the DNA from the primary tumor has a nucleotide change C > T at position 16209 (B) and G > A at position 70 (C), respectively.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Characteristics of 19 patients with multiple HCCa

 
Thirteen of 19 patients (68%) harbored a D-loop mtDNA mutation in at least one tumor. Because many mutations were in the D-loop, we also tested all samples with a simple PCR fragment length assay. This assay is based on the amplification of a 104-bp product that includes the C-mononucleotide repeat. Comparisons between direct sequence analysis and the PCR assay are shown in Fig. 2Citation . We observed C-tract deletion/insertion mutations in 8 of 19 cases (42%) and five other missense and deletion/insertion mutations in an additional 5 cases (26%). Eight cases (numbers 5, 7, 12–16, and 19) revealed mutations that were found in only one of the tumors tested (Fig. 2, a and b)Citation . One case (number 17) revealed the same mutation in two of three tumors, whereas all tumors from cases 1 and 10 shared identical mutations. Finally, in cases 2 and 4, each tumor tested harbored a different mutation. All of the somatic mutations detected were homoplasmic or near homoplasmic, confirming results from previous studies in other tumor types (10 , 11) .



View larger version (52K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Deletion/insertion mutations in a C mononucleotide repeat from the D-loop. In a, the DNA sequence shows a deletion/insertion mutation in only T1 of case 5 and in the primary tumor of cases 12 and 15. Arrows, the point of deletion/insertion mutation and the number of deleted nucleotides. b, a simple PCR fragment length assay based on amplification of a 104-bp product that includes the C-mononucleotide repeat corresponding to the sequence shown in a.

 
The results of our molecular analysis were compared with more standard clinical and pathological criteria in Table 1Citation ("Clinical Diagnosis"). These criteria are based on pathological findings, including tumor size, location of the lesion (e.g., right lobe or left lobe), and interval of occurrence. In some cases (e.g., cases 1 and 17), molecular analysis identified the presence of an identical mutation confirming the clinical diagnosis of metastases. In other cases (e.g., case 10), molecular analysis identified the presence of an identical mutation, whereas the clinical diagnosis was indeterminate. In some cases (e.g., case 4), distinct mutations confirmed the independent origin of the tumors by clinical analysis. In at least 1 case (case 2), independent mutations argued against the clinical diagnosis of metastases.

In addition to the somatic mutations shown above, we identified 38 polymorphisms, of which 5 are apparently newly described (Table 2)Citation . There was no significant association between HBV status, HCV status, age, or gender and the occurrence of mtDNA mutations (by Fisher’s exact test).


View this table:
[in this window]
[in a new window]

  		Table 2 Summary of new polymorphisms found in HCC cases

 
Detection of Mutant mtDNA in Plasma Samples.
Plasma DNA was available preoperatively from 10 of 13 cases where a mtDNA mutation was detected in a primary HCC. We applied a more sensitive oligonucleotide mismatch ligation assay to detect missense mutations, and in 3 cases, we confirmed the mutant sequence in tumor DNA and documented a negative signal in the corresponding normal tissue. We then demonstrated that mutant mtDNA could be detected in the plasma DNA samples from all 3 of these cases (cases 1, 14, and 19; Fig. 3aCitation ). In the remaining case with a missense mutation (case 16), there were weak background bands in both normal and plasma DNA despite the use of a blocking oligo. We applied the simple PCR fragment length detection assay to identify mutant mtDNA in 6 cases with C-tract deletion/insertion mutations, and in 5 of these 6 cases, we detected the identical C-tract deletion/insertion mutations in matched plasma DNA (Fig. 3b)Citation . Overall, we detected mutant mtDNA in 8 of 10 plasma DNA samples. In all cases, the mtDNA mutant signal was much weaker than the tumor (range 1:200–1:500), reflecting significant dilution in or lack of mtDNA access to the plasma.



View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Detection of mutant mtDNA in plasma. In a, arrows identify mutated mitochondrial sequences (Cases 1, 14, and 19) by oligonucleotide mismatch ligation assay. Mutated mtDNA was found in T1 (Tumor 1), T2 (Tumor 2), and T3 (Tumor 3) of case 1, T2 of case 14, and T1 of case 19. Weaker and more diluted signals were observed in the plasma lanes (see "Materials and Methods"). In case 1, P1 (Plasma 1) and P2 (Plasma 2) indicate plasma samples obtained before first surgery (P1) and second (P2) one. b, simple PCR fragment length assay for a region of 104 bp containing the C-mononucteotide repeat. Arrows, a 1-bp deletion in T1 (Case 5), a 5-bp deletion in T1 (Case 12), and a 10-bp deletion in T1 (Case 15). The deleted signals were faintly detected in the plasma samples (1:200–500).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent advances in our understanding of the structure and function of mitochondria have led to the recognition that both inherited and acquired mitochondrial dysfunction may be responsible for diseases affecting the liver and other organ systems (18) . Previously, two types of liver disease with related mitochondrial alterations were reported (18) . Primary mitochondrial hepatopathies are conditions in which inherited defects in structure or function of the mitochondria are present, leading to hepatic failure or chronic liver dysfunction in affected children. Secondary mitochondrial hepatopathies are conditions in which the mitochondria are major targets during liver injury from another cause, such as metal overload, certain drugs and toxins, alcoholic liver injury, and conditions of oxidative stress. We found frequent mitochondrial mutations in HCC tissue. Mitochondria that undergo rapid replication in chronic hepatitis or in HCC may acquire and accumulate DNA damage more readily than those maintained under resting condition (13) . These somatic mitochondrial mutations in cirrhotic liver tissue or in HCC may be best classified with the secondary mitochondrial hepatopathies.

Recently, we and others (10 , 11) identified a high frequency of missense and frameshift mutations in the mtDNA from primary human neoplasms. In some cases, these mutations could lead to abnormal metabolic and apoptotic processes in neoplastic cells (e.g., many coding mutations were confined to respiratory complex I; Ref. 11 ). However, many identified somatic mutations are silent or occur in noncoding regions, and their significance is unclear (see further discussion below). The noncoding D-loop was found to be a mutational hot spot in bladder, lung, and head and neck neoplasms (11) . Mutations in this region may alter the function of the D-loop, as this represents a regulatory site for both replication and expression of the mitochondrial genome. We found acquired somatic mutations in 13 of 19 cases not present in the matched nontumorous lesions in the D-loop region. All of the somatic mutations detected were homoplasmic, confirming results from previous studies in which the mutated mtDNA apparently becomes dominant in tumor cells (10) .

Remarkably, eight of the mtDNA alterations were deletion/insertion mutations in a polymorphic mononucleotide repeat sequence (CCCCCCCTCCCCC) between nucleotides 303 and 315. We recently detected these alterations in 24% of all tumors tested (n = 161) and in every tumor type analyzed except ovary and prostate (19) . The biological reason for these alterations is unclear, but this region is the start site of replication of the closed, circular mitochondrial genome (20) . Replication of mtDNA begins with the synthesis of the heavy strand (H strand) using primer RNA, and the 3' termini of primer RNA were mapped to CSBs I-III (21 , 22) . We found frequent deletions or insertions only in the CSB II region in HCC samples but not in other CSBs, suggesting some functional relevance. Kang et al. (23) reported that the RNA-DNA hybrid at CSB II was more stable than those at the other CSBs in vivo. Interestingly, we did observe a 53- and 10-bp deletion in this area in two different primary HCCs from 1 case (case 2). The identification of larger deletions in this region serves as an impetus for further research on the mechanisms of late replication and processing of mtDNA in cancer.

Previous studies have used the patterns of HBV DNA integration in the cancer genome or selective chromosome inactivation for clonal analysis (7 , 8) . However, these methods can be applied only to HBV carriers or female patients, respectively. Because most patients with HCC are male and an increasing number of cases are associated with HCV, there is a need for clonal molecular markers. Recently, a comparative genomic hybridization study documented chromosomal aberration profile as a unique fingerprint in HCC (9) . Although comparative genomic hybridization profile comparisons can help establish clonality in HCC, this method is limited and requires special analysis systems. Tumor-specific molecular abnormalities have been investigated as a diagnostic tool in HCC. Frequent mutations of p53 were used for determination of clonality of multiple HCC lesions (24) . However, the frequency of p53 mutations in HCCs may be low because of complementary viral inactivation (25 , 26) , and as such, alterations of the p53 gene are generally found only in advanced stage (27) .

Clearly, tumors studied that have the identical mtDNA mutation are of the same origin. Cases 1, 10, tumor 2 of case 17, and those that display different mutations are likely to have arisen from separate independent foci (cases 2 and 4). If the primary tumor has a mutation and the second tumor reveals no mutation, they should be considered independent lesions (cases 12, 13, 15, 16, 19, and tumor 3 of case 17). It is difficult to determine clonality when the primary tumor does not reveal any type of mutation and the second tumor has a mutation, because mtDNA mutations may occur at any time during progression and may thus not always precede all waves of clonal outgrowth (cases 5 and 7). However, a tumor which has a different mutation compared with the primary lesion has at least had clonal divergence, although it may represent a metastasis from a clinical point of view.

In our study, the clinical diagnosis of metastasis or independent lesions was based on the pathological findings that include tumor size, location of the lesion, and interval of occurrence (28) , whereas in some cases, we could not determine their clonal relationship because we lacked more comprehensive markers. Although some discrepancies occurred between mtDNA mutation status and clinical diagnosis, mtDNA mutations can help establish clonal relationship and select an appropriate treatment strategy for new primary tumors or recurrent cancers. This molecular approach requires minimal DNA from each tumor and can be used to analyze routine needle biopsies.

We also demonstrated that mutant mtDNA could be detected in plasma samples from most patients with HCC. These results are probably because of the homoplasmic nature of the somatic mtDNA mutations within each cell, providing an unprecedented detection advantage because of the high copy number of mutant DNA. The low absolute abundance of mutant mtDNA detected in plasma suggests that mtDNA may not be readily introduced into or survive long in the circulation. This approach may prove useful as part of a panel of noninvasive DNA-based tests for the detection and monitoring of HCC after additional study.

We have shown that control region mtDNA mutations are a frequent event in HCC and that these mutations provide significant information in determining the clonality of multiple HCCs. These mutations may serve as sensitive markers to distinguish metastatic disease from the occurrence of multiple independent primary tumors.


   ACKNOWLEDGMENTS
 
We thank James Engles and Henning Usadel for helpful discussion. We also thank Robert Yochem and Robin Brewster for administrative support in preparing this study.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the Integrated Development of Novel Molecular Markers CA98-028 and a Virco, Inc. collaborative agreement. Under a licensing agreement between The Johns Hopkins University and Virco, Inc., Dr. Sidransky is entitled to a share of royalty received by the University on any future sales or products described in this article. The University and Dr. Sidransky own Virco, Inc. stock, which is subject to certain restrictions under University policy. Dr. Sidransky is a paid consultant to Virco, Inc. The terms of this arrangement are being managed by The Johns Hopkins University in accordance with its conflict of interest policies. Back

2 To whom requests for reprints should be addressed, at Department of Otolaryngology–Head and Neck Surgery, The Johns Hopkins University, 818 Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205-2196. Phone: (410) 502-5153; Fax: (410) 614-1411; E-mail: dsidrans{at}jhmi.edu Back

3 The abbreviations used are: HCC, hepatocellular carcinom; mtDNA, mitochondrial DNA; D-loop, displacement loop; HBV, hepatitis B virus; HCV, hepatitis C virus; CSB, conserved sequence block region. Back

Received 6/ 4/01; revised 10/24/01; accepted 10/29/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Parkin D. M., Stjernsward J., Muir C. S. Estimates of the worldwide frequency of twelve major cancers. Bull WHO, 62: 163-182, 1984.[Medline]
  2. El-Serag H. B., Mason A. C. Rising incidence of hepatocellular carcinoma in the United States. N. Engl. J. Med., 340: 745-750, 1999.[Abstract/Free Full Text]
  3. Chen C. J., Liang K. Y., Chang A. S., Chang Y. C., Lu S. N., Liaw Y. F., Chang W. Y., Sheen M. C., Lin T. M. Effects of hepatitis B virus, alcohol drinking, cigarette smoking, and familial tendency on hepatocellular carcinoma. Hepatology, 13: 398-406, 1991.[CrossRef][Medline]
  4. Chen P. J., Chen D. S. Hepatitis B virus infection and hepatocellular carcinoma: molecular genetics and clinical perspectives. Semin. Liver Dis., 19: 253-262, 1999.[Medline]
  5. Acunas B., Rozanes I. Hepatocellular carcinoma: treatment with transcatheter arterial chemoembolization. Eur. J. Radiol., 32: 86-89, 1999.[CrossRef][Medline]
  6. Okuda K., Tanaka M., Nakayama H., Saitsu H., Tanikawa K., Nakashima O., Kojiro M. Clinicopathologic comparison between resected hepatocellular carcinoma (HCC) and recurrent tumors: a special reference to multicentric carcinogenesis of HCC. Int. Hepatol. Commun., 1: 65-71, 1993.
  7. Yamamoto T., Kajino K., Kudo M., Sasaki Y., Arakawa Y., Hino O. Determination of the clonal origin of multiple human hepatocellular carcinomas by cloning and polymerase chain reaction of the integrated hepatitis B virus DNA. Hepatology, 29: 1446-1452, 1999.[CrossRef][Medline]
  8. Ochiai T., Urata Y., Yamano T., Yamagishi H., Ashihara T. Clonal expansion in evolution of chronic hepatitis to hepatocellular carcinoma as seen at an X-chromosome locus. Hepatology, 31: 615-621, 2000.[CrossRef][Medline]
  9. Chen Y. J., Yeh S. H., Chen J. T., Wu C. C., Hsu M. T., Tsai S. F., Chen P. J., Lin C. H. Chromosomal changes and clonality relationship between primary and recurrent hepatocellular carcinoma. Gastroenterology, 119: 431-440, 2000.[CrossRef][Medline]
  10. Polyak K., Li Y., Zhu H., Lengauer C., Willson J. K., Markowitz S. D., Trush M. A., Kinzler K. W., Vogelstein B. Somatic mutations of the mitochondrial genome in human colorectal tumours. Nat. Genet., 20: 291-293, 1998.[CrossRef][Medline]
  11. Fliss M. S., Usadel H., Caballero O. L., Wu L., Buta M. R., Eleff S. M., Jen J., Sidransky D. Facile detection of mitochondrial DNA mutations in tumors and bodily fluids. Science (Wash. DC), 287: 2017-2019, 2000.[Abstract/Free Full Text]
  12. Muller-Hocker J., Aust D., Rohrbach H., Napiwotzky J., Reith A., Link T. A., Seibel P., Holzel D., Kadenbach B. Defects of the respiratory chain in the normal human liver and in cirrhosis during aging. Hepatology, 26: 709-719, 1997.[CrossRef][Medline]
  13. Nishikawa M., Nishiguchi S., Shiomi S., Tamori A., Koh N., Takeda T., Kubo S., Hirohashi K., Kinoshita H., Sato E., Inoue M. Somatic mutation of mitochondrial DNA in cancerous and noncancerous liver tissue in individuals with hepatocellular carcinoma. Cancer Res., 61: 1843-1845, 2001.[Abstract/Free Full Text]
  14. Goelz S. E., Hamilton S. R., Vogelstein B. Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem. Biophys. Res. Commun., 130: 118-126, 1985.[CrossRef][Medline]
  15. Herrnstadt C., Clevenger W., Ghosh S. S., Anderson C., Fahy E., Miller S., Howell N., Davis R. E. A novel mitochondrial DNA-like sequence in the human nuclear genome. Genomics, 60: 67-77, 1999.[CrossRef][Medline]
  16. Trounce I., Schmiedel J., Yen H. C., Hosseini S., Brown M. D., Olson J. J., Wallace D. C. Cloning of neuronal mtDNA variants in cultured cells by synaptosome fusion with mtDNA-less cells. Nucleic Acids Res., 28: 2164-2170, 2000.[Abstract/Free Full Text]
  17. Jen J., Powell S. M., Papadopoulos N., Smith K. J., Hamilton S. R., Vogelstein B., Kinzler K. W. Molecular determinants of dysplasia in colorectal lesions. Cancer Res., 54: 5523-5526, 1994.[Abstract/Free Full Text]
  18. Treem W. R., Sokol R. J. Disorder of the mitochondria. Semin. Liver Dis., 18: 237-253, 1998.[Medline]
  19. Sanchez-Cespedes M., Parrella P., Nomoto S., Cohen D., Xiao Y., Esteller M., Jeronimo C., Nicol T., Koch W. M., Schoenberg M., Yang S. C., Fazio V. M., Giai M., Sidransky D. Identification of a mononucleotide repeat as a major target for mitochondrial DNA mutations in human tumors. Cancer Res., 61: 7015-7019, 2001.[Abstract/Free Full Text]
  20. Clayton D. Replication and transcription of vertebrate mitochondrial DNA. Annu. Rev. Cell Biol., 7: 453-478, 1991.[CrossRef]
  21. Chang D. D., Clayton D. A. Priming of human mitochondrial DNA replication occurs at the light-strand promoter. Proc. Natl. Acad. Sci. USA, 82: 351-355, 1985.[Abstract/Free Full Text]
  22. Chang D. D., Clayton D. A. A novel endoribonuclease cleaves at a priming site of mouse mitochondrial DNA replication. EMBO J., 6: 409-417, 1987.[Medline]
  23. Kang D., Miyako K., Kai Y., Irie T., Takeshige K. In vivo determination of replication origins of human mitochondrial DNA by ligation-mediated polymerase chain reaction. J. Biol. Chem., 272: 15275-15279, 1997.[Abstract/Free Full Text]
  24. Oda T., Tsuda H., Scarpa A., Sakamoto M., Hirohashi S. Mutation pattern of the p53 gene as a diagnostic marker for multiple hepatocellular carcinoma. Cancer Res., 52: 3674-3678, 1992.[Abstract/Free Full Text]
  25. Pontisso P., Belluco C., Bertorelle R., De Moliner L., Chieco-Bianchi L., Nitti D., Lise M., Alberti A. Hepatitis C virus infection associated with human hepatocellular carcinoma: lack of correlation with p53 abnormalities in Caucasian patients. Cancer (Phila.), 83: 1489-1494, 1998.[CrossRef][Medline]
  26. Shieh Y. S., Nguyen C., Vocal M. V., Chu H. W. Tumor-suppressor p53 gene in hepatitis C and B virus-associated human hepatocellular carcinoma. Int. J. Cancer, 54: 558-562, 1993.[Medline]
  27. Teramoto T., Satonaka K., Kitazawa S., Fujimori T., Hayashi K., Maeda S. p53 gene abnormalities are closely related to hepatoviral infections and occur at a late stage of hepatocarcinogenesis. Cancer Res., 54: 231-235, 1994.[Abstract/Free Full Text]
  28. Classification of primary liver cancer. First English Edition: Liver Cancer Study Group of Japan, Kanehara & Co., Ltd. Tokyo, Japan, Eizo Okamoto 1997.



This article has been cited by other articles:


Home page
 J. Mol. Diagn.Home page
S. Zhou, K. Kassauei, D. J. Cutler, G. C. Kennedy, D. Sidransky, A. Maitra, and J. Califano
An Oligonucleotide Microarray for High-Throughput Sequencing of the Mitochondrial Genome
J. Mol. Diagn., September 1, 2006; 8(4): 476 - 482.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
M. Goggins
Molecular Markers of Early Pancreatic Cancer
J. Clin. Oncol., July 10, 2005; 23(20): 4524 - 4531.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
J. P. Jakupciak, W. Wang, M. E. Markowitz, D. Ally, M. Coble, S. Srivastava, A. Maitra, P. E. Barker, D. Sidransky, and C. D. O'Connell
Mitochondrial DNA as a Cancer Biomarker
J. Mol. Diagn., May 1, 2005; 7(2): 258 - 267.
[Abstract] [Full Text] [PDF]

Home page
 Cold Spring Harb Symp Quant BiolHome page
D.C. WALLACE
Mitochondria and Cancer: Warburg Addressed
Cold Spring Harb Symp Quant Biol, January 1, 2005; 70(0): 363 - 374.
[Abstract] [PDF]

Home page
 Genome ResHome page
A. Maitra, Y. Cohen, S. E.D. Gillespie, E. Mambo, N. Fukushima, M. O. Hoque, N. Shah, M. Goggins, J. Califano, D. Sidransky, et al.
The Human MitoChip: A High-Throughput Sequencing Microarray for Mitochondrial Mutation Detection
Genome Res., May 1, 2004; 14(5): 812 - 819.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
L-J C Wong, D-J Tan, R-K Bai, K-T Yeh, and J Chang
Molecular alterations in mitochondrial DNA of hepatocellular carcinomas: is there a correlation with clinicopathological profile?
J. Med. Genet., May 1, 2004; 41(5): e65 - e65.
[Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. Tang, S. Baez, M. Pruyas, A. Diaz, A. Calvo, E. Riquelme, and I. I. Wistuba
Mitochondrial DNA Mutation at the D310 (Displacement Loop) Mononucleotide Sequence in the Pathogenesis of Gallbladder Carcinoma
Clin. Cancer Res., February 1, 2004; 10(3): 1041 - 1046.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
N. Y.L. Lam, T. H. Rainer, R. W.K. Chiu, G. M. Joynt, and Y.M. D. Lo
Plasma Mitochondrial DNA Concentrations after Trauma
Clin. Chem., January 1, 2004; 50(1): 213 - 216.
[Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. Suzuki, S. Toyooka, K. Miyajima, T. Iizasa, T. Fujisawa, N. B. Bekele, and A. F. Gazdar
Alterations in the Mitochondrial Displacement Loop in Lung Cancers
Clin. Cancer Res., November 15, 2003; 9(15): 5636 - 5641.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
R. W.K. Chiu, L. Y.S. Chan, N. Y.L. Lam, N. B.Y. Tsui, E. K.O. Ng, T. H. Rainer, and Y.M. D. Lo
Quantitative Analysis of Circulating Mitochondrial DNA in Plasma
Clin. Chem., May 1, 2003; 49(5): 719 - 726.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
I. H. N. Wong, J. Zhang, P. B. S. Lai, W. Y. Lau, and Y. M. Dennis Lo
Quantitative Analysis of Tumor-derived Methylated p16INK4a Sequences in Plasma, Serum, and Blood Cells of Hepatocellular Carcinoma Patients
Clin. Cancer Res., March 1, 2003; 9(3): 1047 - 1052.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
O. Okochi, K. Hibi, T. Uemura, S. Inoue, S. Takeda, T. Kaneko, and A. Nakao
Detection of Mitochondrial DNA Alterations in the Serum of Hepatocellular Carcinoma Patients
Clin. Cancer Res., September 1, 2002; 8(9): 2875 - 2878.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nomoto, S.
Right arrow Articles by Sidransky, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nomoto, S.
Right arrow Articles by Sidransky, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online