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Aberrant Fragile Histidine Triad Gene Transcripts in Primary Hepatocellular Carcinoma and Liver Cirrhosis¹

Department of Internal Medicine and Gastroenterology [L. G., L. B., M. D. T., L. M., F. P., S. B., S. G., M. V.], Institute of Oncology "Felice Addarii" [P. C.], and Department of Surgery [A. M.], University of Bologna, 40138 Bologna, Italy

	ABSTRACT

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To determine whether transcriptional alterations of the fragile histidine triad (FHIT) gene play a role in the development and progression of human hepatocellular carcinoma (HCC) we used reverse transcription-PCR to examine mRNA FHIT expression in 28 paired samples of HCC (24 in cirrhotic and 4 in noncirrhotic livers) and matched noncancerous tissue and in 10 normal livers. We also assessed loss of heterozygosity of the polymorphic D3S1300 microsatellite marker in the intron between exons 5 and 6 of the FHIT gene. Abnormal FHIT transcripts were detected in 13 cases (46.4%): 10 in the cancerous tissue only, 1 with the same pattern in both cancerous and matched noncancerous tissue, and 2 in the noncancerous tissue only. The four HCCs that arose in noncirrhotic liver all showed abnormal FHIT transcripts. No alterations were found in normal livers. Sequence analysis of abnormally sized transcripts revealed that they were generated by the fusion of exons 3 or 4 with exons 8 or 9. Among the cancerous specimens, one case showed only an abnormal sized transcript derived from the fusion of exons 4 and 9 in the absence of any normal-sized transcript, and another case showed deletion of a sequence comprised between nucleotides -35 and 399 resulting in an exon 4–9 fusion not respecting the exons’ bounds. Loss of heterozygosity was found in two cases with abnormal FHIT transcripts and in only one case with normal transcript. Patients with aberrant FHIT transcripts showed a significantly higher relapse rate and shorter recurrence time (P = 0.001). This could be related to a primary genomic instability affecting particularly susceptible regions like FRA3B and could be associated with an increasing risk of recurrence without involving a causative role.

	INTRODUCTION

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Although development and progression of HCC³ seem to be the result of multiple structural and functional genomic changes, critical molecular steps have yet to be clearly defined (1) . Fragile sites are thought to be target areas of genetic lesions in human tumors, and there is evidence of viral integration at these sites (2 , 3) . HCC frequently develops in cirrhotic livers affected by chronic HBV or HCV infections. Evidence as to whether sustained viral replication represents one of the mechanisms that could alter the expression of genes located in fragile sites is contradictory (4) .

The most common fragile site in humans is FRA3B, which is aphidicolin-inducible and is located at 3p14.2, the region involved in the t(3;8) translocation associated with familial renal cell carcinoma (5) . The FHIT gene, located at 3p14.2 is composed of 10 exons covering 1.1 kb of mRNA spread out over 1 Mb of genomic DNA, including FRA3B and the t(3;8) (p14.2;q24.1) translocation site (6) . This genomic region shows homozygous deletions in several cancer-derived cell lines (7 , 8) . The open reading frame of the FHIT gene is located in exons 5 to 9, whereas the first four exons and exon 10 are not translated. The histidine triad (a signature domain for the HIT family of proteins) is located in exon 8. The FHIT protein is a typical diadenosine 5',5'''-P¹,P³ triphosphate hydrolase (EC 3.6.1.29) that produces ADP and AMP from the diadenosine substrate in vitro (9 , 10) . Its exact role in the process of tumorigenesis remains unknown.

Aberrant transcripts of the FHIT gene were first described in about 50% of esophageal, stomach, and colon carcinomas (6) and later were found in 80% of small cell lung cancer, 40% of non-small cell lung cancer (11) , 30% of primary breast cancer (12) , 57% of Merkel cell carcinomas (13) , and other human malignancies. Deletion of 3p has been demonstrated in limited percentages of surgical HCCs, although few detailed reports are available (14) .

To determine whether the FHIT gene is disrupted and/or aberrantly transcribed in primary human HCC we examined mRNA by RT-PCR and LOH at 3p14.2 by PCR in 28 paired samples of HCC (from cirrhotic/noncirrhotic livers) and matched noncancerous tissue. We also investigated possible associations between FHIT abnormalities and some clinical variables (size of HCC, AFP, viral status, presence of capsule, presence of cirrhosis, and recurrence rate and time).

	PATIENTS AND METHODS

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Patients.
Neoplastic and matched noncancerous tissues from the same livers were obtained from 28 consecutive patients undergoing surgical resection for previously untreated HCC [21 men (mean age, 62.3 years; SD, 9.6 years); 7 women (mean age, 59.8 years; SD, 5.4 years)]. Patients’ characteristics (age, sex, viral status, and serum AFP determinations), size of the tumors, histological grade, presence of capsule, and cirrhotic changes in the surrounding liver are reported in Table 1 . In 24 cases, liver cirrhosis coexisted with HCC, whereas in the remaining 4 cases, the parenchyma surrounding the tumor was noncirrhotic.

After obtaining informed consent, 10 histopathologically normal liver specimens were obtained at surgery from selected sex- and age-matched patients undergoing cholecystectomy for cholelithiasis in the absence of clinical, biochemical, or virological signs of liver disease. The study protocol was in accordance with the 1994 Helsinki Declaration and was approved by the local ethical committee. After surgery, all of the patients received quarterly biochemical and ultrasonografic examination to detect any possible recurrence.

RNA Extraction and RT.
Total RNA was extracted from frozen tissue using Tri-Zol reagent (Life Technologies, Inc., Gaithersburg, MD ) according to the manufacturer’s instructions. cDNA was synthesized by RT from 1 µg of total RNA in a 20 µl final volume of 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, 200 µM dNTPs, 50 ng/µl oligo(dT), 300 ng/ml random hexamers, and 300 units of Superscript II (Life Technologies, Inc.). The reaction was conducted for 1 h at 37°C, and the enzyme was then inactivated by heating to 95°C for 5 min. Control reactions in which RT was omitted were included with all of the assays.

PCR Amplification and DNA Sequencing.
One µl of the RT reaction was used to amplify the FHIT cDNA as described by Ohta et al. (6) . To amplify FHIT exons 3–10, a nested PCR was carried out in 50 µl final volume with 30 ng of primers (5U2–3D2 and 5U1–3D1, according to Ohta and coworkers), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM each dNTP, and 0.5 units Taq Polymerase (Promega). The two consecutive rounds of amplification were performed using a Perkin-Elmer 9600 thermal cycler for 30 cycles at 94°C for 30 s, (denaturation), 60°C for 30 s (annealing), and 72°C for 60 s (elongation), followed by a final extension period of 6 min at 72°C.

The PCR products were resolved in 1.5% ethidium bromide agarose gel. Bands were excised from gels, and DNA was purified using a QIA QUICK gel extraction kit (QIAGEN, Hilden, Germany). PCR products excised from gels were directly sequenced using primers 5U1 and 3D1 with an automated Applied Biosystem Model 377 DNA sequencer (Perkin-Elmer, Foster City, CA). Each case was amplified at least twice, the second time with a single-step PCR using the inner primers 5U2 and 3D2, and only bands that were reproducibly amplified on different occasions were purified and sequenced. To check for the presence of mRNA and rule out any aspecific amplifications, 25 PCR cycles of -actin cDNA were performed after RT, both with and without the RT enzyme.

Analysis of LOH.
A PCR-based approach was selected to assess allelic loss in cancerous tissue. DNA from 12 cases with normal transcripts and from 11 cases with aberrant transcripts was analyzed for LOH at locus 3p14.2 with 3S1300 (forward: AGC TCA CAT TCT AGT CAG CCT; reverse: GCC AAT TCC CCA GAT G) and D3S1234 (forward: CCT GTG AGA CAA AGC AAG AC; reverse: GAC ATT AGG CAC AGG GCT AA) microsatellite markers. After digestion with proteinase K, high molecular weight DNA was extracted by phenol-chloroform according to described protocols (16) . PCR was performed by a Perkin-Elmer 9600 thermal cycler using 100 ng of DNA in a 50 µl total volume and 100 ng of each primer, 0.5 units of Taq DNA polymerase (Promega) with 200 µM concentrations each of dATP, dGTP, and dTTP and 2.5 µM dCTP, in addition to [-³²P]dCTP at 1.0 mCi/reaction. Each PCR reaction was preceded by a denaturation step of 3' at 94°C and was then carried out for 19 cycles consisting of 20 s at 94°C, 20 s at 55°C, and 20 s at 72°C. The final extension step was lengthened to 6 min at 72°C. This procedure keeps the reaction in the linear part of the amplification curve so that differences in allelic band intensity reflect real differences in allelic ratio. Three µl of PCR reaction product were mixed with 3 µl of dimethyl formamide buffer and electrophoresed in a standard 6% polyacrylamide sequencing gel. The gel was fixed, dried, and processed for autoradiography on Kodak X-AR5 film. Allelic imbalance was assessed by comparing the intensities of the alleles in heterozygous cases of matched tumorous and nontumorous tissues both by visual inspection and by scanning densitometry of the autoradiograph (Gel Pro Analyzer, Immagini e Computer, Milan, Italy). In the normal heterozygous tissue, the intensity of the two allele bands is approximately equal. Allelic imbalance in tumor tissue was assessed when there was a >50% discrepancy in the allele ratios of the matched tumorous and nontumorous tissue DNA (in all of the cases, this was >80%). All of the cases were run twice to confirm the presence or absence of allelic imbalance.

Statistical Analysis.
Associations between the presence of aberrant FHIT transcripts and clinical variables were assessed by logistic regression (size and AFP) or Fisher’s exact test (presence of the capsule, viral status, and presence of cirrhosis; Ref. 17 ). Recurrence intervals were calculated from the date of surgery. Time-to-recurrence curves were computed using the Kaplan-Meier (18) product-limit method and compared by a log-rank test (19) . The median time to censoring was derived from a reversed Kaplan-Meier analysis (20) . Time-to-recurrence regressions were estimated using a proportional hazard model (21) . Analyses were performed using the statistical software StatView 5.0 (SAS Institute, Cary, NC).

	RESULTS

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Normal-sized and sequenced FHIT transcripts of 707 bp (6) were found in both cancerous and noncancerous tissues of all but one of the examined livers, as well as in the hepatic tissue of the 10 controls. Aberrant FHIT transcripts were detected in 13 cases (46.4%): 10 in the cancerous tissue only, 1 in both cancerous and matched noncancerous tissue, and 2 in the noncancerous tissue only. Thus, aberrant FHIT messengers were present in the cancerous tissue of 11 (39.3%) of 28 patients and in the hepatic tissue surrounding the tumors of 3 (10.3%) of 28 patients but were not present in control livers. No examined cases had a complete absence of FHIT transcripts. All but one of the patients with aberrant FHIT transcripts also presented normal FHIT mRNA whose relative abundance differed from patient to patient. One case showed an aberrant transcript only (Fig. 1 , Case 2).

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Observed splicing patterns of the FHIT gene. Numbers at the top of the figure represent the nucleotide corresponding to the bound () of exons E1-E10; the thicker top line is the open reading frame. Single cases are listed by the number at the left; Cases 1–10, aberrant transcripts in cancerous tissue; Case 11, aberrant transcripts detected in cirrhosis and HCC; Cases 12 and 13, aberrant transcripts detected in cirrhotic tissue only. Horizontal lines, retained exon sequences; two arrow heads, the interstitial deletions in Case 12.

View larger version (82K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Expression of the FHIT gene analyzed by RT-PCR and visualized by ethidium bromide-stained agarose gel electrophoresis. Tumor (Lane T) and matched nontumorous (Lane N) tissue from patients with HCC. Arrow (707 bp), normal transcript size; Lane M, 1 kb DNA ladder molecular weight marker (Life Technologies Inc); Lane B, RT-blank.

View larger version (50K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Sequences from matched normal and abnormal FHIT transcripts. Sequence analysis of FHIT transcripts in nontumorous and tumorous tissue from patients with HCC. A, Case 3, arrows, normal junctions between exons 3 and 4 (cirrhotic tissue, top) and abnormal fusion between exon 3 and 8 (HCC, bottom). B, Case 5, arrows, a normal junction between exon 8 and 9 (cirrhotic tissue, top) and abnormal fusion between exon 4 and 9 (HCC, bottom).

In 5 of 13 cases with aberrant transcripts and in 2 of 15 cases with normal transcripts, an alternative splicing of the FHIT gene was observed at the border between exons 9 and 10, with the deletion of an 11-bp sequence (nucleotides 450–460), outside the coding region, both in normal and tumor tissue, as reported previously (11) .

Point mutations were observed only in aberrant transcripts: (a) in two different cases at nucleotide 426 (CG transversion in one case, resulting in CGGGGG; and C deletion in another case); and (b) in one case at nucleotide 393 with the insertion of an adenine that resulted in a frameshift mutation. PCR products smaller than 707 bp were sometimes observed but were poorly reproducible, and the related DNA segments were not sequenced.

To assess possible associations between FHIT aberrant transcripts and allelic loss at 3p14.3, we chose D3S1300 (located in the intron between exon 5 and 6, which resulted in loss in all of the aberrant transcripts) and D3S1234, located between exons 8 and 9 of the FHIT gene. Among patients exhibiting abnormal FHIT transcripts, allelic loss at D3S1300 was found in two of seven heterozygous cases, whereas two cases could not be analyzed, and the remaining four cases were homozygous. One HCC with LOH had only abnormal FHIT transcripts (Table 1 , Case 2). Among cases with normal FHIT transcripts, eight cases retained heterozygosity, three cases became homozygous, and one case displayed LOH (three cases were not analyzed). Similar results were obtained with D3S1234, except for Case 5 (showing aberrant transcripts), which displayed LOH at D3S1300 and retained heterozygosity at D3S1234. Aberrant FHIT transcripts were detected in 5 of the 6 HBsAg-positive cases and in 8 of the 22 HBsAg-negative cases (borderline significance, see Table 2 ). Of all of the 28 livers with HCC, 4 turned out to be devoid of cirrhotic changes on histopathological examination. These four cases all presented aberrant FHIT transcripts only in the tumor. The presence of FHIT aberrations was not associated with the presence of tumor capsule, AFP, tumor size, or HCV infection (Table 2) .

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Kaplan-Meier estimated survival rates according to the presence of FHIT gene aberration. —-, cases presenting aberrant FHIT transcripts; - - - -, cases with normal FHIT transcripts; short vertical bar, censored observations.

	DISCUSSION

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One case presented only the abnormal transcript. The sequence analysis confirmed the loss of exons 5–8, without other sequence abnormalities. This patient was affected by multiple nodules of HCC in the left lobe, occurring in noncirrhotic liver without HBV or HCV infection. The histopathological examination of these three lesions showed a grade II (according to Edmondson and Steiner criteria) HCC of the trabecular type. The hystopathological characteristic, even if similar in the three lesions, are, however, insufficient to establish a metastatic or multifocal origin. Two of these lesions were analyzed, and both showed the same FHIT abnormality. Only normal FHIT messengers were present in the surrounding liver tissue.

In the present study, mRNA was extracted from tissue homogenates, and, therefore, it was not possible to assess whether the normal and abnormal messengers were produced by the same cells. Some reports, however, describe the simultaneous presence of normal and aberrant FHIT transcripts in monoclonal cell lines (6 , 12) . The coexistence of normal and aberrant transcripts in the same cell makes the biological significance of the aberrant transcripts unclear. Because a start codon is present on exon 9, transcripts lacking exons 5 and 6 might encode a 13-amino-acid peptide at the COOH-terminus of the FHIT protein. As yet, there is no direct evidence of this in HCC, although Kisielewski et al. (23) found, by Western blot analysis, a putative truncated pFHIT in cell lines and one primary tumor of the head and neck. Considering that the functional FHIT protein is a homodimer, it is conceivable that a defective peptide might dimerize and negatively affect the wild-type FHIT protein. This study addresses the evaluation of a possible role of aberrant FHIT transcripts as a putative prognostic marker rather than as a carcinogen.

In our series of HCCs, allelic loss at the FHIT gene was only occasionally found either in the presence or absence of aberrant transcripts. Indeed, only 2 HCCs with aberrant transcripts and 1 with wild-type transcripts showed LOH at the 3p14.2 band. A lack of association between LOH at this locus and the presence of splicing alterations in the FHIT gene is not an uncommon finding, and is well documented in breast cancer (12) . Therefore, LOH analysis with the D3S1300 and D3S1234 microsatellite markers might miss small alterations or deletions within the large 5th intron of this gene, which can affect the specificity of splicing. Because a polyclonal antibody recognizing the FHIT protein is now becoming available, it will be interesting to verify whether LOH or aberrant transcripts correspond to diminished or absent FHIT protein in single neoplastic cells.

As well as in the HCCs, we also observed aberrant FHIT transcripts in nontumorous cirrhotic liver tissue from three patients. In one case, liver tissue showed the same aberrant FHIT transcript of the matched HCC, whereas in the remaining two cases aberrant transcripts were found only in noncancerous tissue. One of the latter (Case 12 in Table 2 ) showed an exon-4-to-9 fusion that, to our knowledge, has not been previously reported in the literature. LOH analysis revealed homozygosity both at D3S1300 and D3S1234 markers. Because, in this case, LOH analysis showed a single band both in HCC and cirrhosis and because aberrant transcripts were detected in nonneoplastic tissue, we cannot exclude the possibility that both tissues lost the same allele. Because of the small amount of tissue in many cases of this retrospective series of HCCs, any further analysis on genomic DNA requiring relatively larger amounts of DNA was not performed.

Patients with aberrant FHIT transcripts in their liver showed a significantly higher recurrence rate and a significantly shorter disease-free time interval in comparison with patients displaying normal FHIT transcripts. All of the three patients with aberrant FHIT transcripts in liver cirrhosis experienced multiple early HCC recurrences at 8, 11, and 15 months after surgical resection. Liver cirrhosis is considered a precancerous condition, and FHIT abnormalities in the cirrhotic tissue could indicate generalized genomic instability favoring the emergence of neoplastic clones. Patients with recurrent HCCs include both a multicentric development and intrahepatic metastasis from primary nodules. The distinction between these two groups could suggest different possible roles of FHIT in hepatocarcinogenesis: a correlation with multicentric development would indicate a role in early carcinogenesis, whereas a correlation with intrahepatic metatstasis would indicate a role in malignant progression. Chen et al. (24) investigated, with the same technique used in this study, FHIT changes in a limited number of HCCs and identified aberrant FHIT transcripts in a high percentage (44.4%) of nontumorous liver parenchyma; they concluded that the presence of aberrant FHIT transcripts might not be related to liver carcinogenesis.

Experimental evidence concerning FHIT’s possible function as a tumor-suppressor gene are conflicting (22 , 25 , 26) . Panagopoulos et al. (25) found aberrant FHIT transcripts in nonneoplastic tissues and, thus, excluded a carcinogenic role. In our series, abnormal transcripts were found in a small fraction of nonneoplastic tissues (all with cirrhotic changes) and in none of the normal control livers. Thiagalingam et al. (26) found normal-sized RT-PCR products with a normal sequence in 29 of 31 colorectal cancers. They suggested that: (a) either FHIT is not involved or is functionally inactivated at the translational level in these kinds of tumors; (b) it is located adjacent to another target gene; or (c) its alterations can be related to the cancer-specific genetic instability of this region. Boldog et al. (27) recently reported a similarity in the repeat sequence composition between FRA3B and the Fragile X region and identified a FRA3B segment highly similar to a reported small poly-dispersed circular DNA, sequences of which are markedly elevated in damaged or unstable genomes. These authors suggested that a primary genomic instability affecting a particularly susceptible region like FRA3B could account for aberrant FHIT transcripts and allelic loss.

The present study suggests that the presence of aberrant FHIT transcripts could be a reliable marker of poor prognosis with a higher risk of early recurrence in patients with resected HCC. This in itself would not necessarily mean that the FHIT gene is directly related to hepatocarcinogenesis or tumor progression. Indeed, aberrant FHIT transcripts may merely subtend a high degree of genomic instability, which could account for a higher risk of neoplastic transformation.

In conclusion, our study demonstrates that FHIT transcripts are altered in a percentage of cases of small resectable HCCs and in a minority of cases of liver cirrhosis, and that their presence can be associated with earlier tumor recurrence. The presence of altered FHIT transcripts may, thus, represent a negative prognostic factor even if the low rate of LOH makes the role of FHIT in HCC development unlikely.

	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 (60%) by a grant from Ministero Università e Ricerca Scientifica e Tecnologica.

² To whom requests for reprints should be addressed, at Dipartimento di Medicina Interna e Gastroenterologia, Università di Bologna, Divisione di Medicina Interna, via Albertoni, 15, 40138 Bologna, Italy. Phone: 39-51-6362260; Fax: 39-51-6362260; E-mail: Bolondi{at}almadns.unibo.it

³ The abbreviations used are: HCC, human hepatocellular carcinoma; FHIT, fragile histidine triad; RT, reverse transcription; AFP, -feto-protein; LOH, loss of heterozygosity; HBV, hepatitis B virus; HCV, hepatitis C virus

Received 5/ 3/99; revised 8/11/99; accepted 8/11/99.

	REFERENCES

Top ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Farber E. The step-by-step development of epithelial cancer: from phenotype to genotype. Adv. Cancer Res., 70: 21-48, 1996.[Medline]
2. Popescu N. C., Zimonjic D., Di Paolo J. A. Viral integrations, fragile sites and protooncogenes in human neoplasia. Hum. Genet., 84: 383-386, 1989.
3. Wilke M. C., Hall B. K., Hoge A., Paradee W., Smith D. I., Glover T. W. FRA3B extends over a broad region and contains a spontaneous HPV integration site: direct evidence for the coincidence of viral integration sites and fragile sites. Hum. Mol. Genet., 5: 187-195, 1996.[Abstract/Free Full Text]
4. Sutherland G. R. Fragile sites and cancer breakpoints: the pessimistic view. Cancer Genet. Cytogenet., 31: 5-7, 1988.[Medline]
5. Cohen A. J., Li F. P., Berg S., Marchetto D. J., Tsai S., Jacobs S. C., Brown R. S. Hereditary renal-cell carcinoma associated with a chromosomal translocation. N. Engl. J. Med., 301: 592-595, 1979.[Medline]
6. Ohta M., Inoue H., Cotticelli M. G., Kastury K., Baffa R., Palazzo J., Siprashvili Z., Mori M., McCue P., Druck T., Croce C. M., Huebner K. The human FHIT gene, spanning the chromosome 3p14.2 fragile site and renal carcinoma associated translocation breakpoint, is abnormal in digestive tract cancers. Cell, 84: 587-597, 1996.[Medline]
7. Lisitsyn N. A., Lisitsina N. M., Dalbagni G., Barker P., Sanchez C. A., Gnarra J., Linehan W. M., Reid B. J., Wigler M. H. Comparative genome analysis of tumors: detection of DNA losses and amplification. Proc. Natl. Acad. Sci. USA, 92: 946-951, 1995.
8. Kastury K., Baffa R., Druck T., Ohta M., Cotticelli M. G., Inoue H., Negrini M., Rugge M., Huang D., Croce C. M., Palazzo J., Huebner K. Potential gastrointestinal tumor suppressor locus at 3p14.2 FRA3B site identified by homozygous deletions in tumor cell lines. Cancer Res., 56: 978-983, 1996.[Abstract/Free Full Text]
9. Barnes L. D., Garrison P. N., Siprashvili Z., Guranowski A., Robinson A. K., Ingrma S. W., Croce C. M., Ohta M., Huebner K. Fhit, a putative tumor suppressor in humans, is a dinucleoside 5', 5'''-P1, P3-triphosphate hydrolases. Biochemistry, 35: 11529-11535, 1996.[Medline]
10. Lima C. D., Klein M. G., Hendrickson W. A. Structure-based analysis of catalysis and substrate definition in the HIT protein family. Science (Washington DC), 278: 286-290, 1997.[Abstract/Free Full Text]
11. Sozzi G., Veronese M. L., Negrini M., Baffa R., Cotticelli M. G., Inoue H., Tornielli S., Pillotti S., DeGregorio L., Pastorino V., Pierotti M. A., Ohta M., Huebner K., Croce C. M. The FHIT gene at 3p14.2 is abnormal in lung cancer. Cell, 85: 17-26, 1996.[Medline]
12. Negrini M., Monaco C., Vorechovsky I., Ohta M., Druck T., Baffa R., Huebner K., Croce C. M. The FHIT gene at 3p14.2 is abnormal in breast carcinomas. Cancer Res., 56: 3173-3179, 1996.[Abstract/Free Full Text]
13. Sozzi G., Alder H., Tornielli S., Corletto V., Baffa R., Veronese M. L., Negrini M., Pilotti S., Pierotti M. A., Huebner K., Croce C. M. Aberrant FHIT transcripts in Merkel cell carcinomas. Cancer Res., 56: 2472-2474, 1996.[Abstract/Free Full Text]
14. Fujimori M., Tokino T., Hino O., Kitagawa T., Imamura T., Okamoto E., Mitsunobu M., Ishikawa T., Nakagama H., Harada H., Yagura M., Matsubara K., Nakamura Y. Allelotype study of primary hepatocellular carcinoma. Cancer Res., 51: 89-93, 1991.[Abstract/Free Full Text]
15. Edmondson H. A., Steiner P. E. Primary carcinoma of the liver. A study of 100 cases among 48,900 necropsies. Cancer (Phila.), 7: 462-503, 1954.[Medline]
16. Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual, Ed. 2, 9.16–9.23 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989.
17. Matthews D. E., Farewell V. T. Using and Understanding Medical Statistics Ed. 3 Basel Karger 1996.
18. Kaplan E. L., Meier P. Nonparametric estimation for incomplete observations. J. Am. Stat. Assoc., 53: 457-481, 1958.
19. Lee E. T. Statistical Methods for Survival Data Analysis John Wiley New York 1992.
20. Altman D. G., De Stavola B. L., Love S. B., Stepniewska K. A. Review of survival analyses published in cancer journals. Br. J, Cancer, 72: 511-518, 1995.[Medline]
21. Cox D. R. Regression models and life tables (with discussion). J. R. Stat. Soc., 34: 87-220, 1972.
22. Sozzi G., Huebner K., Croce C. M. FHIT in human cancer. Adv. Cancer Res., 74: 141-166, 1998.[Medline]
23. Kisielewski A. E., Xiao G. H., Liu S. C., Klein-Szanto A. J., Novara M., Sina J., Bleicher K., Yeung R. S., Goodrow T. L. Analysis of the FHIT gene and its product in squamous cell carcinomas of the head and neck. Oncogene, 17: 83-91, 1998.[Medline]
24. Chen Y. J., Chen P. H., Chang J. G. Aberrant FHIT transcripts in hepatocellular carcinomas. Br. J. Cancer., 77: 417-420, 1998.[Medline]
25. Panagopoulos I., Thelin S., Mertens F., Mitelman F., Aman P. Variable FHIT transcripts in non-neoplastic tissues. Genes, Chromosomes, Cancer, 19: 215-219, 1997.[Medline]
26. Thiangalingam S., Lisitsyn N. A., Hamaguchi M., Wigler M. H., Willson J. K. V., Markowitz S. D., Leach F. S., Kinzler K. W., Vogelstein B. Evaluation of the FHIT gene in colorectal cancers. Cancer Res., 56: 2936-2939, 1996.[Abstract/Free Full Text]
27. Boldog F., Gemmill R. M., West J., Robinson M., Robinson L., Li E., Roche J., Todd S., Waggoner B., Lundstrom R., Jacobson R., Jacobson J., Mullokandov M. R., Klinger H., Drabkin H. A. Chromosome 3p14 homozygous deletions and sequence analysis of FRA3B. Hum. Mol. Genet., 6: 193-203, 1997.[Abstract/Free Full Text]

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