Experimental and Molecular Pathology 96 (2014) 92–97
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Reduced FHIT expression is associated with tumor progression in sporadic colon adenocarcinoma Sanja Kapitanović a,⁎, Tamara Čačev a, Božo Lončar b, Tina Catela Ivković a, Šimun Križanac c, Krešimir Pavelić a a b c
Laboratory for Personalized Medicine, Ruđer Bošković Institute, Bijenička c. 54, 10000 Zagreb, Croatia Department of Surgery, Clinical Hospital Dubrava, Avenija Gojka Šuška 6, 10000 Zagreb, Croatia Department of Clinical Pathology, Clinical Hospital Dubrava, Avenija Gojka Šuška 6, 10000 Zagreb, Croatia
a r t i c l e
i n f o
Article history: Received 11 November 2013 Available online 25 December 2013 Keywords: FHIT Colon adenocarcinoma LOH mRNA Immunohistochemistry
a b s t r a c t Purpose: Tumor supressor gene FHIT was identified at chromosome 3p14.2 spanning the FRA3B fragile site and is very often inactivated in different types of cancer. The aim of this study was to examine the frequency of FHIT gene LOH as well as FHIT mRNA and protein expression in sporadic colon adenocarcinoma. Methods: The results of LOH, real-time qRT-PCR and imunohistochemical analyses were correlated with clinicopathological characteristics of patients and their tumors in order to evaluate the role of FHIT gene/protein in sporadic colon adenocarcinoma tumorigenesis. Results: One hundred and thirty one (96.3%) samples were informative for both markers and 33/131 (25.2%) demonstrated LOH. Expression of FHIT mRNA was significantly decreased in colon tumors relative to that in corresponding normal tissue (p = 7.2 × 10−6). Most of the samples (54.0%) were negative for FHIT protein, 26.4% adenocarcinomas showed a weak to moderate immunostaining and 19.6% adenocarcinomas showed strong FHIT immunostaining. No correlation was found between FHIT gene LOH status, mRNA expression or FHIT protein immunostaining and clinico-pathological characteristics. Expression of FHIT mRNA was significantly decreased in FHIT LOH positive tumors (p = 0.027). Patients with LOH negative tumors or FHIT protein positive tumors had longer survival but this findings were not statistically significant. Conclusions: Our overall results suggest that reduced expression of FHIT gene may be associated with the progression of these malignant tumors. © 2013 Elsevier Inc. All rights reserved.
Introduction Fragile histidine triad (FHIT) gene was isolated and identified by collaborating researchers in the USA and Japan in 1996 and it was shown to span the most common human fragile chromosomal site FRA 3B at 3p14.2 (Ohta et al., 1996; Sozzi et al., 1996). FHIT protein has diadenosine triphosphate hydrolase activity which is dependent upon the conserved histidine triad encoded by exon 8 (Barnes et al., 1996). The FHIT gene is very often inactivated in different types of cancer. In some tumors, particularly those associated with environmental carcinogens, alterations in the FHIT gene occur quite early in their development. In other cancers, FHIT inactivation seems to be a later event, possibly associated with progression to more aggressive neoplasias (Croce et al., 1999). The multiple genetic lesions within FHIT gene may be explained by the location of the gene within a fragile site highly susceptible to breakage induced by carcinogens (Wang and Perkins, 1984). Further studies of the FHIT gene demonstrated that its expression is altered in many human epithelial cancer cell lines including lung, bladder, breast, cervical carcinomas (Druck et al., 1997; Man et al., 1996; ⁎ Corresponding author. E-mail address: [email protected] (S. Kapitanović). 0014-4800/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexmp.2013.12.005
Negrini et al., 1996; Sozzi et al., 1996). Loss of heterozygosity (LOH) at the 3p14 locus has been frequently observed in squamous cell carcinomas of the head and neck, as well as, lung cancers, and is likely to be a crucial step leading to loss of function of the FHIT gene (Ahsee et al., 1994; Ishwad et al., 1996; Mao et al., 1996; Virgilio et al., 1996; Wu et al., 1994). In the study examining the role of FHIT gene in lung tumors LOH was observed in 80% of tumors from smokers in contrast to only 22% of tumors from nonsmokers indicating that FRA3B is a preferential target of tobacco smoke damage at the molecular level (Sozzi et al., 1996). In addition, other mechanisms of FHIT inactivation include aberrant transcription and promoter methylation, while point mutations are rare event in tumors (Campiglio et al., 1999; Virgilio et al., 1996). Abnormal FHIT transcripts, including deletions of exons and insertions, are found in high percentage of lung and breast cancers (Mao et al., 1996; Sozzi et al., 1997; Tokuchi et al., 1999; Wu et al., 1994) as well as in head and neck (Siprashvili et al., 1997) and thyroid cancer (McIver et al., 2000). Aberrant FHIT promoter methylation as a mechanism of loss of FHIT expression was frequently found in non-small cell lung cancer and breast cancer (Zöchbauer-Müller et al., 2001). Several studies have explored the FHIT gene status in sporadic colon cancer, mostly by LOH, immunohistochemistry and Western blot, as well as mRNA expression. However there is still no uniform conclusion
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about the role of the FHIT gene in colon cancer tumorigenesis (Cao et al., 2006; Hao et al., 2000; Thiagalingam et al., 1996; Wierzbicki et al., 2009; Yasugi et al., 2008). The aim of this study was to examine the frequency of FHIT gene LOH as well as FHIT protein and specific mRNA expression in sporadic colon adenocarcinoma. The results of molecular analyses were correlated with clinico-pathological characteristics of patients and their tumors in order to evaluate the role of this gene/protein in sporadic colon cancer tumorigenesis. Materials and methods Tumor and normal DNA Tumor and adjacent normal colon tissue from patients with sporadic colon adenocarcinoma used in our study was obtained from the Croatian Tumor Bank (Spaventi et al., 1994). The study included 136 patients (66 men and 70 women) with mean age of 65.2 years (age range between 34 and 95 years). All specimens were obtained during routine surgery performed in patients with colon carcinoma. Fresh samples of resected colon carcinoma were snap frozen in liquid nitrogen and stored in the Human Tumor Bank at −80 °C until needed. Before use in the study, each specimen was verified by a histopathologist. All specimens were examined by routine hematoxylin-eosin staining to determine the proportion of tumor cells in the sample (80%). Control normal DNA was extracted from histologically normal colon mucous adjacent (15 cm from the tumor) to the adenocarcinoma. Frozen tissue DNA extraction was performed using proteinase K digestion and phenol chloroform extraction. Written informed consent was obtained from all patients included in the study. The study was approved by the ethics committee of Clinical Hospital Dubrava, Zagreb and was performed in accordance with the ethical standards of Helsinki Declaration. Polymerase chain reaction (PCR) For LOH analysis at the FHIT gene locus, two sets of primers were used. Sequences of specific oligonucleotides used were: D3S1300F (5′AGC TCA CAT TCT AGT CAG CCT-3′) and D3S1300R (5′-GCC AAT TCC CCA GAT G-3′); D3S4103F (5′-TTC TAC TGC AAT CCA GCC TGG-3′) and D3S4103R (5′-GCC TTG GGT AGA TTT AT-3′). Genomic DNA (100 ng) was used as a template in a reaction volume of 25 μl containing 5 pmol of each primer, 50 μM of each dNTP, and 1 U Taq Gold DNA polymerase (Applied Biosystems, Foster City, California, USA). PCR reactions were carried out in an Applied Biosystems GeneAmp PCR System 2400 for 30 cycles. Annealing temperatures for each primer set were optimized in pilot studies before processing experimental samples.
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City, USA) according to the manufacturer's protocols. Number of tumor samples analyzed was dependent upon sufficient RNA quality for real-time RT-PCR analysis. Real-time RT-PCR analysis of FHIT mRNA expression Real-time RT-PCR analysis for FHIT and RPLP0 (housekeeping gene) was performed using an ABI PRISM 7300 SDS (Applied Biosystems, Foster City, USA) and predeveloped TaqMan assay reagents Hs_00179987 (FHIT) and Hs99999902_m1 (RPLP0) (Applied Biosystems, Foster City, USA). PCR reaction was carried out according to the manufacturer's protocol. To compensate for inter-PCR variation normalization of the target gene (FHIT) with an endogenous control (RPLP0) was performed. Results are shown as ΔCt values which are the difference in cycle number required to raise the amount of PCR product above threshold. ΔCt is therefore inversely proportional to the amounts of mRNA (Kapitanović et al., 2004). Immunohistochemistry Immunohistochemistry was performed on formalin-fixed, paraffinembedded tissue. Expression of FHIT protein in 87 samples of sporadic colon adenocarcinoma was analyzed using primary rabbit polyclonal anti FHIT antibodies (Zymed Laboratories Inc, San Francisco, USA). Negative controls were performed by omission of the primary antibody. After deparaffinization in xylene, slides were rehydrated in ethanol and washed in phosphate-buffered saline (3 × 3 min). The endogenous peroxidase activity was quenched by 15 min incubation in methanol with 3% hydrogen peroxide (Sigma Chemical Co., Munich, Germany). Nonspecific binding was blocked by applying DAKO Protein Block SerumFree (DAKO, Glostrup, Denmark) in a humidity chamber for 10 min at room temperature. Slides were blotted, and the primary rabbit polyclonal antibody at concentration of 3 μg/ml was applied overnight at 4 °C. Slides were then washed three times in phosphate-buffered saline. DAKO EnVision™ + System, HRP (DAB) (DAKO) was used for visualization of positive reaction according to the manufacturer's instructions. The slides were counterstained with hematoxylin for 30 s, dehydrated and mounted in Canada balsam. Each slide was evaluated in the entire tumor area. The relative intensity of cell immunostaining was evaluated, semiquantitatively, so that no staining was denoted (0), weak/moderate staining (b 80% positive tumor cells) was denoted (1) and strong (≥80% positive tumor cells) was denoted (2). Statistical analysis
Polymorphic marker analysis was performed by non-denaturing polyacrylamide electrophoresis. For VNTR analysis, 5 μl of PCR product was mixed with 3 μl of loading buffer and loaded onto 1 mm thick, 35 × 30 cm, 10% nondenaturing polyacrylamide gel. Electrophoresis was performed in 1xTBE buffer for 16 h at 10 V/cm, at room temperature. The gels were silver stained. LOH was defined by visible change in allele:allele ratio in tumors compared with matching normal tissue. Allelic deletion of the FHIT gene was judged by positive LOH at any of the two sites.
Correlations between the FHIT LOH status and clinicopathological characteristics were explored from contingency tables. Tables were analyzed using the χ2 test or Fisher's exact test. Correlations were considered significant when the two tailed p-value was less than 0.05. Correlations of mRNA expression in normal and tumor tissue were analyzed with the Student's t-test. Correlations of mRNA expression and clinicopathological characteristics were analyzed with Student's t-test or with the analysis of variance where appropriate. A p-value less than 0.05 was considered statistically significant. All evaluations were performed using GraphPad Prism version 4.03 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. Survival analysis was performed using MedCalc statistical software (trial version 11.6.1). The probability of survival was calculated for the different subgroups by the Kaplan–Meier method.
RNA extraction and reverse transcription
Results
Total RNA was extracted from 50 pairs of resected colon carcinoma and corresponding normal tissue using Trizol reagent (Invitrogen, Carlsbad, USA) and ten micrograms of RNA were used for reverse transcription (High-Capacity cDNA Archive Kit, Applied Biosystems, Foster
LOH at the FHIT gene locus
Variable number of tandem repeats (VNTR) and LOH analysis
To analyze LOH at the FHIT gene we used two microsatellite markers within the FHIT gene, D3S1300 dinucleotide repeat and D3S4103
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trinucleotide repeat. At the D3S1300 locus 109 of 136 patients were informative (heterozygous) (80.1%) and 22 of the 109 (20.2%) demonstrated LOH. At the D3S4103 locus 109 of 136 patients were informative (heterozygous) (80.1%) and 22 of the 109 (20.2%) demonstrated LOH. One hundred and thirty one (96.3%) samples were informative for both markers and 33 of the 131 (25.2%) demonstrated LOH (Table 1). Fig. 1 represents examples of FHIT gene LOH at D3S4103 locus.
147 bp 123 bp
LOH at the FHIT gene locus and clinicopathological characteristics The results of LOH analysis were correlated to the clinicopathological characteristics of patients and their tumors (age, sex, tumor size, tumor grade and Dukes' stage of tumor) (Table 1). No correlation was found between the FHIT gene LOH and age or sex of patients (Table 1). In addition, no correlation was found between the FHIT gene LOH and tumor size (p = 0.765) as well as Dukes' stage and histological grade of tumor samples (p = 0.102 and p = 0.401, respectively) (Table 1). FHIT mRNA expression and clinicopathological characteristics Fifty samples of colon adenocarcinoma and corresponding normal tissue were examined for the FHIT mRNA expression. Expression of FHIT mRNA was decreased in colon tumors relative to that in corresponding normal tissue (p = 7.2 × 10− 6) (Fig. 2A). No statistically significant correlation was found between the FHIT mRNA expression and tumor size (p = 0.851) (Fig. 2B), histological grade (p = 0.173) (Fig. 2C) and Dukes' stage (p = 0.399) (Fig. 2D) of tumors. Expression of FHIT mRNA was statistically significant decreased in FHIT LOH positive tumors (p = 0.027) (Fig. 3). FHIT protein expression in colon adenocarcinoma Eighty seven samples of sporadic colon adenocarcinoma were examined for the presence of FHIT tumor suppressor protein. Forty seven (54.0%) samples were negative for FHIT protein (Fig. 4A). Twenty three (26.4.%) adenocarcinomas showed a weak to moderate immunostaining (1, positive b 80% of the tumor cells) (Fig. 4B) and seventeen (19.6%) adenocarcinomas showed strong FHIT immunostaining (2, positive ≥ 80% of the tumor cells) (Fig. 4C). The observed staining pattern was identical in all carcinomas and was represented by the cytoplasmic staining of tumor cells with negative surrounding normal tissue.
Table 1 Clinicopathological characteristics of 131 informative patients with colon adenocarcinoma stratified by FHIT LOH status. Characteristic
FHIT LOH Negative (%) 98 (74.8)
Age b70 years 59 (74.7) ≥70 years 36 (76.6) Sex Male 47 (73.4) Female 51 (76.1) Tumor size ≤5 cm 62 (77.5) N5 cm 28 (80.0) Histological grade (differentiation) Well 33 (91.7) Moderate 36 (73.5) Poor 13 (76.5) Dukes' stage A 25 (80.6) B 24 (66.7) C 44 (75.9)
p Positive (%) 33 (25.2) 20 (25.3) 11 (23.4)
0.810
17 (26.6) 16 (23.9)
0.724
18 (22.5) 7 (20.0)
0.765
3 (8.3) 13 (26.5) 4 (23.5)
0.102
6 (19.4) 12 (33.3) 14 (24.1)
0.401
Fig. 1. Loss of heterozygosity (LOH) at microsatellite FHIT gene locus D3S4103 in colon adenocarcinoma. M. DNA marker; N, Normal; T, Tumor; 1, 4 and 6 heterozygous (informative) with LOH; 2, 3 and 5 heterozygous without LOH.
FHIT protein expression and clinical parameters The results of immunohistochemical analysis of FHIT protein expression were correlated to the clinicopathological characteristics of patients and their tumors (age, sex, tumor size, tumor grade and Dukes' stage of tumor) (Table 2). No correlation was found between the FHIT protein positivity and the age of patients (p = 0.502) (Table 2). Most of the tumor samples strongly positive to FHIT protein were observed in female patients. There was statistically significant difference in FHIT immunostaining between male and female groups of patients (p = 0.043) (Table 2). No difference was observed between the FHIT protein expression and tumor size (p = .0.687) (Table 2). Most of the strongly positive FHIT tumors were in Dukes' A, and in the group of well differentiated tumors, but these finding were not statistically significant (p = 0.704 and p = 0.567, respectively) (Table 2). Survival analysis Survival analysis was performed to correlate the outcome of patients monitored during the 250 week period with the FHIT LOH status and FHIT protein expression. Survival analysis with regard to FHIT LOH status was performed in 60 patients who survived for more than four weeks after surgery. Fig. 5A shows the survival curves according to FHIT LOH status. The median survival time of the patients without FHIT LOH was 76.6 weeks, with 58.0% of them being alive at the end of the monitoring period. The median survival time of the patients with FHIT LOH was 39.6 weeks, with 50.0% of them being alive at the end of the monitoring period. Statistical analysis showed that there was no statistically significant difference (p = 0.500) between these two groups of patients according to FHIT LOH status. Survival analysis with regards to the FHIT protein expression was performed on 76 patients who survived for more than four weeks after surgery. Fig. 5B shows the survival curves according to FHIT protein positivity. The median survival time of the patients without FHIT protein expression was 72.2 weeks, with 51.1% of them being alive at the end of the monitoring period. The median survival time of the patients with FHIT protein expression was 104.1 weeks, with 69.6% of them being alive at the end of the monitoring period. Statistical analysis showed that patients with FHIT protein positive tumors had longer survival but the result was not statistically significant (p = 0.079). Discussion Colorectal cancer development and progression is a consequence of molecular genetic changes in many oncogenes and tumor suppressor genes (Vogelstein and Kinzler, 2004). FHIT gene, located at most common human fragile chromosomal site FRA (3p14.2) was first
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A FHIT mRNAexpression ( Δ Ct)
10
8
6
4 Median
2
25%-75%
FHIT mRNAexpression ( Δ Ct)
B
12
95
12
10
8
6
4 Median 25%-75%
2
Non-Outlier Range
Non-Outlier Range
0
0 N
≤ 5 cm
T
p=7.2x10-6
D
12
10
8
6
4 Median 25%-75%
2
FHIT mRNAexpression ( Δ Ct)
FHIT mRNAexpression ( Δ Ct)
C
> 5 cm
Tumor size p=0.851
12
10
8
6
4 Median
2
25%-75%
Non-Outlier Range
Non-Outlier Range
0
0 1
2
3
Histological grade (differentiation) p=0.173
A
B
C
Dukes' stage p=0.399
Fig. 2. FHIT gene mRNA expression. A. mRNA expression in normal vs. tumor tissue. mRNA expression with respect to tumor size (B), histological grade (C) and Dukes' stage (D).
identified as a candidate tumor suppressor gene in hereditary renal cell carcinoma. In addition, common deletions in the chromosomal region 3p12–3p14.2, were described in primary clear cell renal carcinoma (Lubinski et al., 1994). Since then FHIT gene loss has been detected
Fig. 3. FHIT mRNA expression with respect to FHIT gene LOH status.
in lung (Fong et al., 1997), gastric (Baffa et al., 1998), head and neck (Virgilio et al., 1996; Wu et al., 1994) and breast cancers (Campiglio et al., 1999; Man et al., 1996). Although the role of the FHIT gene in the development and progression of the colorectal cancer has been studied it is still poorly understood. The aim of this study was to examine the role of the FHIT gene in sporadic colon adenocarcinoma. For this purpose we have analyzed the loss of heterozygosity of the FHIT gene as well as its mRNA and protein expression by quantitative real-time PCR and immunohistochemistry. Several groups have examined the role of the FHIT gene in colorectal tumorigenesis with opposing conclusions. Wierzbicki et al. (2009) examined FHIT mRNA and protein expression in colorectal adenomas and carcinomas found the high FHIT mRNA expression in 60% of adenomas and 86% of CRC using the quantitative PCR analysis. In their study the immunohistochemical analysis showed comparable results. These findings are in agreement with the previously described results of Thiagalingam et al. (1996) who showed very high expression of the complete FHIT coding transcript in 29/31 cell lines derived from human colorectal cancers. The results of our study differ from those of Wierzbicki et al. (2009) as well as Thiagalingam et al. (1996). In our study expression of FHIT mRNA in colon adenocarcinoma tissue was significantly decreased relative to that in the corresponding normal tissue, but there was no correlation with tumor size, histological grade or Dukes' stage. In the
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S. Kapitanović et al. / Experimental and Molecular Pathology 96 (2014) 92–97 Table 2 Clinicopathological characteristics of 87 patients with colon adenocarcinoma stratified by FHIT protein staining. Characteristic
FHIT staining Negative (%) 47 (54.0)
Age b70 years 29 (50.0) ≥70 years 16 (64.0) Sex Male 24 (57.1) Female 22 (51.2) Tumor size ≤5 cm 29 (54.6) N5 cm 16 (53.3) Histological grade (Differentiation) Well 13 (44.8) Moderate 23 (57.5) Poor 10 (66.7) Dukes' stage A 7 (46.6) B 18 (48.6) C 21 (63.6) a b
p 1 (%)a 23 (26.4)
2 (%)b 17 (19.6)
16 (27.6) 5 (20.0)
13 (22.4) 4 (16.0)
0.502
14 (33.3) 8 (18.6)
4 (9.6) 13 (30.2)
0.043
12 (22.7) 9 (30.0)
12 (22.7) 5 (16.7)
0.687
8 (27.6) 11 (27.5) 3 (20.0)
8 (27.6) 6 (15.0) 2 (13.3)
0.567
4 (26.7) 11 (29.7) 7 (21.2)
4 (26.7) 8 (21.7) 5 (15.2)
0.704
1, positive b 80%. 2, positive ≥ 80%.
Fig. 4. Immunohistochemical analysis of FHIT protein in colon adenocarcinoma. A. negative immunostaining; B. weak to moderate immunostaining (1, positive b 80% of the tumor cells); C. strong FHIT immunostaining (2, positive ≥ 80% of the tumor cells)(100×).
immunohistochemical analysis 53.4% of tumor samples were negative for FHIT protein. In only 23.3% of tumors a strong positive reaction to FHIT protein was detected and there was no correlation with clinicopathological characteristics of patients and their tumors. The difference between our study and findings of Wierzbicki et al. (2009) may be caused by the use of different methodologies, differences in the selection of primers, probes or antibodies for FHIT protein, as well as protocols. The study of Thiagalingam et al. (1996) was on the other hand performed on primary cell cultures from human colon cancers which can result in different findings when compared with the results obtained from fresh tumor tissues. However, our results are in agreement with the results of other groups. Hao et al. (2000) found reduced expression of FHIT protein in 38% colorectal tumors without metastases and in 62% tumors with
Fig. 5. Kaplan–Meier survival curves with respect to A. tumor LOH status and B. FHIT immunohistochemical staining.
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metastases. In our study 48.0% of non-metastatic and 63.6% of metastatic CRC were found to be negative for FHIT protein. In addition to these findings Hao et al. (2000) also described high expression of FHIT protein in colorectal adenomas, premalignant lesions leading to adenocarcinomas. Yasugi et al. (2008) also described significantly reduced or absent FHIT protein expression in colorectal carcinomas but in none of the analyzed adenomas. In the study of Cao et al. (2006) positive FHIT gene expression was found in 93.75% of normal colorectal tissue and decreased FHIT expression correlated significantly with the progression of colorectal carcinoma with 68.75% and 46.25%, FHIT positive adenomas and adenocarcinomas respectively. In their study down-regulation of FHIT protein expression was associated with down-regulation of Bax and up-regulation of Bcl-2 and Survivin, which resulted in the inhibition of apoptosis. Wierzbicki et al. described the highest occurrence of the FHIT gene LOH at the D3S1234 locus (32.5%) followed by the D3S1300 locus (24%) and the D3S1481 locus (11%) of informative cases. Our FHIT LOH analysis showed similar results. We analyzed FHIT LOH at two loci, and found that at D3S1300 locus LOH was present in 20.2% of informative cases, and same percentage of LOH was observed at D3S4103 locus. In our study, as well as, in study of Wierzbicki et al. (2009) FHIT mRNA levels were significantly lower in LOH positive cases. Overall informativity for both markers in our study was 80.1% and 25.2% tumors demonstrated LOH, no correlation with clinicopathological characteristics was observed. Patients with LOH negative tumors had longer survival but this finding was not statistically significant. When survival was examined in relation to FHIT protein status in tumor tissue patients, with FHIT protein positive tumors had longer survival but this difference was not statistically significant, as well. In conclusion, FHIT gene LOH and lower or absent expression of FHIT protein were present in significant proportion of colon adenocarcinomas included in our study. Expression of FHIT mRNA was significantly lower in tumors when compared to the expression in the corresponding normal tissue. Patients whose tumors were LOH positive as well as those with FHIT protein negative tumors had longer survival, and FHIT LOH significantly correlated with the reduced FHIT mRNA expression. These results suggest that reduced expression of FHIT gene may be associated with the progression of this malignant tumor, and further studies may be expected to add new insights into FHIT as a potential prognostic marker in colon adenocarcinoma. Conflict of interest statement The authors declare that there are no conflicts of interest. References Ahsee, K.W., Cooke, T.G., Pickford, I.R., Soutar, D., Balmain, A., 1994. An allelotype of squamous carcinoma of the head and neck using microsatellite markers. Cancer Res. 54, 1617–1621. Baffa, R., Veronese, M.L., Santoro, R., Mandes, B., Palazzo, J.P., Rugge, M., Santoro, E., Croce, C.M., Huebner, K., 1998. Loss of FHIT expression in gastric carcinoma. Cancer Res. 58, 4708–4714. Barnes, L.D., Garrison, P.N., Siprashvili, Z., Guranowski, A., Robinson, A.K., Ingram, S.W., Croce, C.M., Ohta, M., Huebner, K., 1996. FHIT, a putative tumor suppressor in humans, is a dinucleoside 5′,5″-P1, P3-triphosphate hydrolase. Biochemistry 35, 11529–11535. Campiglio, M., Pekarsky, Y., Menard, S., Tagliabue, E., Pilotti, S., Croce, C.M., 1999. FHIT loss of function in human primary breast cancer correlates with advanced stage of the disease. Cancer Res. 59, 3866–3869. Cao, J., Li, W., Xie, J., Du, H., Tang, W., Wang, H., Chen, X., Xiao, W., Li, Y., 2006. Downregulation of FHIT inhibits apoptosis of colorectal cancer: mechanism and clinical implication. Surg. Oncol. 15, 223–233. Croce, C.M., Sozzi, G., Huebner, K., 1999. Role of FHIT in human cancer. J. Clin. Oncol. 17, 1618–1624.
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Sozzi, G., Sard, L., De Gregorio, L., Marchetti, A., Musso, K., Buttitta, F., Tornielli, S., Pellegrini, S., Veronese, M.L., Manenti, G., Incarbone, M., Chella, A., Angeletti, C.A., Pastorino, U., Huebner, K., Bevilaqua, G., Pilotti, S., Croce, C.M., Pierotti, M.A., 1997. Association between cigarette smoking and FHIT gene alterations in lung cancer. Cancer Res. 57, 2121–2123. Spaventi, R., Pečur, L., Pavelić, K., Pavelić, Z.P., Spaventi, Š., Stambrook, P.J., 1994. Human tumour bank in Croatia: a possible model for a small bank as a part of future European tumour bank network. Eur. J. Cancer 30A, 419. Thiagalingam, S., Lisitsyn, N.A., Hamaguchi, M., Wigler, M.H., Willson, J.K., Markowitz, S.D., Leach, F.S., Kinzler, K.W., Vogelstein, B., 1996. Evaluation of the FHIT gene in colorectal cancers. Cancer Res. 56, 2936–2939. Tokuchi, Y., Kobayashi, Y., Hayashi, S., Hayashi, M., Tanimoto, K., Hashimoto, T., Nishida, K., Ishikawa, Y., Nakagawa, K., Satoh, Y., Yamamoto, M., Tsuchiya, E., 1999. Abnormal FHIT transcripts found in both lung cancer and normal lung tissue. Genes Chromosomes Cancer 24, 105–111. Virgilio, L., Shuster, M., Gollin, S.M., Veronese, M.L., Ohta, M., Huebner, K., Croce, C.M., 1996. FHIT gene alterations in head and neck squamous cell carcinomas. Proc. Natl. Acad. Sci. U. S. A. 93, 9770–9775. Vogelstein, B., Kinzler, K.W., 2004. Cancer genes and the pathways they control. Nat. Med. 10, 789–799. Wang, N., Perkins, K.L., 1984. Involvement of band 3p14 in t (3;8) hereditary renal carcinoma. Cancer Genet. Cytogenet. 11, 479–481. Wierzbicki, P.M., Adrych, K., Kartanowicz, D., Dobrowolski, S., Stanislawowski, M., Chybicki, J., Godlewski, J., Korybalski, B., Smoczynski, M., Kmiec, Z., 2009. Fragile histidine triad (FHIT) gene is overexpressed in colorectal cancer. J. Physiol. 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Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Reduced FHIT expression is associated with tumor progression in sporadic colon adenocarcinoma Sanja Kapitanović a,⁎, Tamara Čačev a, Božo Lončar b, Tina Catela Ivković a, Šimun Križanac c, Krešimir Pavelić a a b c
Laboratory for Personalized Medicine, Ruđer Bošković Institute, Bijenička c. 54, 10000 Zagreb, Croatia Department of Surgery, Clinical Hospital Dubrava, Avenija Gojka Šuška 6, 10000 Zagreb, Croatia Department of Clinical Pathology, Clinical Hospital Dubrava, Avenija Gojka Šuška 6, 10000 Zagreb, Croatia
a r t i c l e
i n f o
Article history: Received 11 November 2013 Available online 25 December 2013 Keywords: FHIT Colon adenocarcinoma LOH mRNA Immunohistochemistry
a b s t r a c t Purpose: Tumor supressor gene FHIT was identified at chromosome 3p14.2 spanning the FRA3B fragile site and is very often inactivated in different types of cancer. The aim of this study was to examine the frequency of FHIT gene LOH as well as FHIT mRNA and protein expression in sporadic colon adenocarcinoma. Methods: The results of LOH, real-time qRT-PCR and imunohistochemical analyses were correlated with clinicopathological characteristics of patients and their tumors in order to evaluate the role of FHIT gene/protein in sporadic colon adenocarcinoma tumorigenesis. Results: One hundred and thirty one (96.3%) samples were informative for both markers and 33/131 (25.2%) demonstrated LOH. Expression of FHIT mRNA was significantly decreased in colon tumors relative to that in corresponding normal tissue (p = 7.2 × 10−6). Most of the samples (54.0%) were negative for FHIT protein, 26.4% adenocarcinomas showed a weak to moderate immunostaining and 19.6% adenocarcinomas showed strong FHIT immunostaining. No correlation was found between FHIT gene LOH status, mRNA expression or FHIT protein immunostaining and clinico-pathological characteristics. Expression of FHIT mRNA was significantly decreased in FHIT LOH positive tumors (p = 0.027). Patients with LOH negative tumors or FHIT protein positive tumors had longer survival but this findings were not statistically significant. Conclusions: Our overall results suggest that reduced expression of FHIT gene may be associated with the progression of these malignant tumors. © 2013 Elsevier Inc. All rights reserved.
Introduction Fragile histidine triad (FHIT) gene was isolated and identified by collaborating researchers in the USA and Japan in 1996 and it was shown to span the most common human fragile chromosomal site FRA 3B at 3p14.2 (Ohta et al., 1996; Sozzi et al., 1996). FHIT protein has diadenosine triphosphate hydrolase activity which is dependent upon the conserved histidine triad encoded by exon 8 (Barnes et al., 1996). The FHIT gene is very often inactivated in different types of cancer. In some tumors, particularly those associated with environmental carcinogens, alterations in the FHIT gene occur quite early in their development. In other cancers, FHIT inactivation seems to be a later event, possibly associated with progression to more aggressive neoplasias (Croce et al., 1999). The multiple genetic lesions within FHIT gene may be explained by the location of the gene within a fragile site highly susceptible to breakage induced by carcinogens (Wang and Perkins, 1984). Further studies of the FHIT gene demonstrated that its expression is altered in many human epithelial cancer cell lines including lung, bladder, breast, cervical carcinomas (Druck et al., 1997; Man et al., 1996; ⁎ Corresponding author. E-mail address: [email protected] (S. Kapitanović). 0014-4800/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexmp.2013.12.005
Negrini et al., 1996; Sozzi et al., 1996). Loss of heterozygosity (LOH) at the 3p14 locus has been frequently observed in squamous cell carcinomas of the head and neck, as well as, lung cancers, and is likely to be a crucial step leading to loss of function of the FHIT gene (Ahsee et al., 1994; Ishwad et al., 1996; Mao et al., 1996; Virgilio et al., 1996; Wu et al., 1994). In the study examining the role of FHIT gene in lung tumors LOH was observed in 80% of tumors from smokers in contrast to only 22% of tumors from nonsmokers indicating that FRA3B is a preferential target of tobacco smoke damage at the molecular level (Sozzi et al., 1996). In addition, other mechanisms of FHIT inactivation include aberrant transcription and promoter methylation, while point mutations are rare event in tumors (Campiglio et al., 1999; Virgilio et al., 1996). Abnormal FHIT transcripts, including deletions of exons and insertions, are found in high percentage of lung and breast cancers (Mao et al., 1996; Sozzi et al., 1997; Tokuchi et al., 1999; Wu et al., 1994) as well as in head and neck (Siprashvili et al., 1997) and thyroid cancer (McIver et al., 2000). Aberrant FHIT promoter methylation as a mechanism of loss of FHIT expression was frequently found in non-small cell lung cancer and breast cancer (Zöchbauer-Müller et al., 2001). Several studies have explored the FHIT gene status in sporadic colon cancer, mostly by LOH, immunohistochemistry and Western blot, as well as mRNA expression. However there is still no uniform conclusion
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about the role of the FHIT gene in colon cancer tumorigenesis (Cao et al., 2006; Hao et al., 2000; Thiagalingam et al., 1996; Wierzbicki et al., 2009; Yasugi et al., 2008). The aim of this study was to examine the frequency of FHIT gene LOH as well as FHIT protein and specific mRNA expression in sporadic colon adenocarcinoma. The results of molecular analyses were correlated with clinico-pathological characteristics of patients and their tumors in order to evaluate the role of this gene/protein in sporadic colon cancer tumorigenesis. Materials and methods Tumor and normal DNA Tumor and adjacent normal colon tissue from patients with sporadic colon adenocarcinoma used in our study was obtained from the Croatian Tumor Bank (Spaventi et al., 1994). The study included 136 patients (66 men and 70 women) with mean age of 65.2 years (age range between 34 and 95 years). All specimens were obtained during routine surgery performed in patients with colon carcinoma. Fresh samples of resected colon carcinoma were snap frozen in liquid nitrogen and stored in the Human Tumor Bank at −80 °C until needed. Before use in the study, each specimen was verified by a histopathologist. All specimens were examined by routine hematoxylin-eosin staining to determine the proportion of tumor cells in the sample (80%). Control normal DNA was extracted from histologically normal colon mucous adjacent (15 cm from the tumor) to the adenocarcinoma. Frozen tissue DNA extraction was performed using proteinase K digestion and phenol chloroform extraction. Written informed consent was obtained from all patients included in the study. The study was approved by the ethics committee of Clinical Hospital Dubrava, Zagreb and was performed in accordance with the ethical standards of Helsinki Declaration. Polymerase chain reaction (PCR) For LOH analysis at the FHIT gene locus, two sets of primers were used. Sequences of specific oligonucleotides used were: D3S1300F (5′AGC TCA CAT TCT AGT CAG CCT-3′) and D3S1300R (5′-GCC AAT TCC CCA GAT G-3′); D3S4103F (5′-TTC TAC TGC AAT CCA GCC TGG-3′) and D3S4103R (5′-GCC TTG GGT AGA TTT AT-3′). Genomic DNA (100 ng) was used as a template in a reaction volume of 25 μl containing 5 pmol of each primer, 50 μM of each dNTP, and 1 U Taq Gold DNA polymerase (Applied Biosystems, Foster City, California, USA). PCR reactions were carried out in an Applied Biosystems GeneAmp PCR System 2400 for 30 cycles. Annealing temperatures for each primer set were optimized in pilot studies before processing experimental samples.
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City, USA) according to the manufacturer's protocols. Number of tumor samples analyzed was dependent upon sufficient RNA quality for real-time RT-PCR analysis. Real-time RT-PCR analysis of FHIT mRNA expression Real-time RT-PCR analysis for FHIT and RPLP0 (housekeeping gene) was performed using an ABI PRISM 7300 SDS (Applied Biosystems, Foster City, USA) and predeveloped TaqMan assay reagents Hs_00179987 (FHIT) and Hs99999902_m1 (RPLP0) (Applied Biosystems, Foster City, USA). PCR reaction was carried out according to the manufacturer's protocol. To compensate for inter-PCR variation normalization of the target gene (FHIT) with an endogenous control (RPLP0) was performed. Results are shown as ΔCt values which are the difference in cycle number required to raise the amount of PCR product above threshold. ΔCt is therefore inversely proportional to the amounts of mRNA (Kapitanović et al., 2004). Immunohistochemistry Immunohistochemistry was performed on formalin-fixed, paraffinembedded tissue. Expression of FHIT protein in 87 samples of sporadic colon adenocarcinoma was analyzed using primary rabbit polyclonal anti FHIT antibodies (Zymed Laboratories Inc, San Francisco, USA). Negative controls were performed by omission of the primary antibody. After deparaffinization in xylene, slides were rehydrated in ethanol and washed in phosphate-buffered saline (3 × 3 min). The endogenous peroxidase activity was quenched by 15 min incubation in methanol with 3% hydrogen peroxide (Sigma Chemical Co., Munich, Germany). Nonspecific binding was blocked by applying DAKO Protein Block SerumFree (DAKO, Glostrup, Denmark) in a humidity chamber for 10 min at room temperature. Slides were blotted, and the primary rabbit polyclonal antibody at concentration of 3 μg/ml was applied overnight at 4 °C. Slides were then washed three times in phosphate-buffered saline. DAKO EnVision™ + System, HRP (DAB) (DAKO) was used for visualization of positive reaction according to the manufacturer's instructions. The slides were counterstained with hematoxylin for 30 s, dehydrated and mounted in Canada balsam. Each slide was evaluated in the entire tumor area. The relative intensity of cell immunostaining was evaluated, semiquantitatively, so that no staining was denoted (0), weak/moderate staining (b 80% positive tumor cells) was denoted (1) and strong (≥80% positive tumor cells) was denoted (2). Statistical analysis
Polymorphic marker analysis was performed by non-denaturing polyacrylamide electrophoresis. For VNTR analysis, 5 μl of PCR product was mixed with 3 μl of loading buffer and loaded onto 1 mm thick, 35 × 30 cm, 10% nondenaturing polyacrylamide gel. Electrophoresis was performed in 1xTBE buffer for 16 h at 10 V/cm, at room temperature. The gels were silver stained. LOH was defined by visible change in allele:allele ratio in tumors compared with matching normal tissue. Allelic deletion of the FHIT gene was judged by positive LOH at any of the two sites.
Correlations between the FHIT LOH status and clinicopathological characteristics were explored from contingency tables. Tables were analyzed using the χ2 test or Fisher's exact test. Correlations were considered significant when the two tailed p-value was less than 0.05. Correlations of mRNA expression in normal and tumor tissue were analyzed with the Student's t-test. Correlations of mRNA expression and clinicopathological characteristics were analyzed with Student's t-test or with the analysis of variance where appropriate. A p-value less than 0.05 was considered statistically significant. All evaluations were performed using GraphPad Prism version 4.03 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. Survival analysis was performed using MedCalc statistical software (trial version 11.6.1). The probability of survival was calculated for the different subgroups by the Kaplan–Meier method.
RNA extraction and reverse transcription
Results
Total RNA was extracted from 50 pairs of resected colon carcinoma and corresponding normal tissue using Trizol reagent (Invitrogen, Carlsbad, USA) and ten micrograms of RNA were used for reverse transcription (High-Capacity cDNA Archive Kit, Applied Biosystems, Foster
LOH at the FHIT gene locus
Variable number of tandem repeats (VNTR) and LOH analysis
To analyze LOH at the FHIT gene we used two microsatellite markers within the FHIT gene, D3S1300 dinucleotide repeat and D3S4103
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trinucleotide repeat. At the D3S1300 locus 109 of 136 patients were informative (heterozygous) (80.1%) and 22 of the 109 (20.2%) demonstrated LOH. At the D3S4103 locus 109 of 136 patients were informative (heterozygous) (80.1%) and 22 of the 109 (20.2%) demonstrated LOH. One hundred and thirty one (96.3%) samples were informative for both markers and 33 of the 131 (25.2%) demonstrated LOH (Table 1). Fig. 1 represents examples of FHIT gene LOH at D3S4103 locus.
147 bp 123 bp
LOH at the FHIT gene locus and clinicopathological characteristics The results of LOH analysis were correlated to the clinicopathological characteristics of patients and their tumors (age, sex, tumor size, tumor grade and Dukes' stage of tumor) (Table 1). No correlation was found between the FHIT gene LOH and age or sex of patients (Table 1). In addition, no correlation was found between the FHIT gene LOH and tumor size (p = 0.765) as well as Dukes' stage and histological grade of tumor samples (p = 0.102 and p = 0.401, respectively) (Table 1). FHIT mRNA expression and clinicopathological characteristics Fifty samples of colon adenocarcinoma and corresponding normal tissue were examined for the FHIT mRNA expression. Expression of FHIT mRNA was decreased in colon tumors relative to that in corresponding normal tissue (p = 7.2 × 10− 6) (Fig. 2A). No statistically significant correlation was found between the FHIT mRNA expression and tumor size (p = 0.851) (Fig. 2B), histological grade (p = 0.173) (Fig. 2C) and Dukes' stage (p = 0.399) (Fig. 2D) of tumors. Expression of FHIT mRNA was statistically significant decreased in FHIT LOH positive tumors (p = 0.027) (Fig. 3). FHIT protein expression in colon adenocarcinoma Eighty seven samples of sporadic colon adenocarcinoma were examined for the presence of FHIT tumor suppressor protein. Forty seven (54.0%) samples were negative for FHIT protein (Fig. 4A). Twenty three (26.4.%) adenocarcinomas showed a weak to moderate immunostaining (1, positive b 80% of the tumor cells) (Fig. 4B) and seventeen (19.6%) adenocarcinomas showed strong FHIT immunostaining (2, positive ≥ 80% of the tumor cells) (Fig. 4C). The observed staining pattern was identical in all carcinomas and was represented by the cytoplasmic staining of tumor cells with negative surrounding normal tissue.
Table 1 Clinicopathological characteristics of 131 informative patients with colon adenocarcinoma stratified by FHIT LOH status. Characteristic
FHIT LOH Negative (%) 98 (74.8)
Age b70 years 59 (74.7) ≥70 years 36 (76.6) Sex Male 47 (73.4) Female 51 (76.1) Tumor size ≤5 cm 62 (77.5) N5 cm 28 (80.0) Histological grade (differentiation) Well 33 (91.7) Moderate 36 (73.5) Poor 13 (76.5) Dukes' stage A 25 (80.6) B 24 (66.7) C 44 (75.9)
p Positive (%) 33 (25.2) 20 (25.3) 11 (23.4)
0.810
17 (26.6) 16 (23.9)
0.724
18 (22.5) 7 (20.0)
0.765
3 (8.3) 13 (26.5) 4 (23.5)
0.102
6 (19.4) 12 (33.3) 14 (24.1)
0.401
Fig. 1. Loss of heterozygosity (LOH) at microsatellite FHIT gene locus D3S4103 in colon adenocarcinoma. M. DNA marker; N, Normal; T, Tumor; 1, 4 and 6 heterozygous (informative) with LOH; 2, 3 and 5 heterozygous without LOH.
FHIT protein expression and clinical parameters The results of immunohistochemical analysis of FHIT protein expression were correlated to the clinicopathological characteristics of patients and their tumors (age, sex, tumor size, tumor grade and Dukes' stage of tumor) (Table 2). No correlation was found between the FHIT protein positivity and the age of patients (p = 0.502) (Table 2). Most of the tumor samples strongly positive to FHIT protein were observed in female patients. There was statistically significant difference in FHIT immunostaining between male and female groups of patients (p = 0.043) (Table 2). No difference was observed between the FHIT protein expression and tumor size (p = .0.687) (Table 2). Most of the strongly positive FHIT tumors were in Dukes' A, and in the group of well differentiated tumors, but these finding were not statistically significant (p = 0.704 and p = 0.567, respectively) (Table 2). Survival analysis Survival analysis was performed to correlate the outcome of patients monitored during the 250 week period with the FHIT LOH status and FHIT protein expression. Survival analysis with regard to FHIT LOH status was performed in 60 patients who survived for more than four weeks after surgery. Fig. 5A shows the survival curves according to FHIT LOH status. The median survival time of the patients without FHIT LOH was 76.6 weeks, with 58.0% of them being alive at the end of the monitoring period. The median survival time of the patients with FHIT LOH was 39.6 weeks, with 50.0% of them being alive at the end of the monitoring period. Statistical analysis showed that there was no statistically significant difference (p = 0.500) between these two groups of patients according to FHIT LOH status. Survival analysis with regards to the FHIT protein expression was performed on 76 patients who survived for more than four weeks after surgery. Fig. 5B shows the survival curves according to FHIT protein positivity. The median survival time of the patients without FHIT protein expression was 72.2 weeks, with 51.1% of them being alive at the end of the monitoring period. The median survival time of the patients with FHIT protein expression was 104.1 weeks, with 69.6% of them being alive at the end of the monitoring period. Statistical analysis showed that patients with FHIT protein positive tumors had longer survival but the result was not statistically significant (p = 0.079). Discussion Colorectal cancer development and progression is a consequence of molecular genetic changes in many oncogenes and tumor suppressor genes (Vogelstein and Kinzler, 2004). FHIT gene, located at most common human fragile chromosomal site FRA (3p14.2) was first
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A FHIT mRNAexpression ( Δ Ct)
10
8
6
4 Median
2
25%-75%
FHIT mRNAexpression ( Δ Ct)
B
12
95
12
10
8
6
4 Median 25%-75%
2
Non-Outlier Range
Non-Outlier Range
0
0 N
≤ 5 cm
T
p=7.2x10-6
D
12
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4 Median 25%-75%
2
FHIT mRNAexpression ( Δ Ct)
FHIT mRNAexpression ( Δ Ct)
C
> 5 cm
Tumor size p=0.851
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8
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4 Median
2
25%-75%
Non-Outlier Range
Non-Outlier Range
0
0 1
2
3
Histological grade (differentiation) p=0.173
A
B
C
Dukes' stage p=0.399
Fig. 2. FHIT gene mRNA expression. A. mRNA expression in normal vs. tumor tissue. mRNA expression with respect to tumor size (B), histological grade (C) and Dukes' stage (D).
identified as a candidate tumor suppressor gene in hereditary renal cell carcinoma. In addition, common deletions in the chromosomal region 3p12–3p14.2, were described in primary clear cell renal carcinoma (Lubinski et al., 1994). Since then FHIT gene loss has been detected
Fig. 3. FHIT mRNA expression with respect to FHIT gene LOH status.
in lung (Fong et al., 1997), gastric (Baffa et al., 1998), head and neck (Virgilio et al., 1996; Wu et al., 1994) and breast cancers (Campiglio et al., 1999; Man et al., 1996). Although the role of the FHIT gene in the development and progression of the colorectal cancer has been studied it is still poorly understood. The aim of this study was to examine the role of the FHIT gene in sporadic colon adenocarcinoma. For this purpose we have analyzed the loss of heterozygosity of the FHIT gene as well as its mRNA and protein expression by quantitative real-time PCR and immunohistochemistry. Several groups have examined the role of the FHIT gene in colorectal tumorigenesis with opposing conclusions. Wierzbicki et al. (2009) examined FHIT mRNA and protein expression in colorectal adenomas and carcinomas found the high FHIT mRNA expression in 60% of adenomas and 86% of CRC using the quantitative PCR analysis. In their study the immunohistochemical analysis showed comparable results. These findings are in agreement with the previously described results of Thiagalingam et al. (1996) who showed very high expression of the complete FHIT coding transcript in 29/31 cell lines derived from human colorectal cancers. The results of our study differ from those of Wierzbicki et al. (2009) as well as Thiagalingam et al. (1996). In our study expression of FHIT mRNA in colon adenocarcinoma tissue was significantly decreased relative to that in the corresponding normal tissue, but there was no correlation with tumor size, histological grade or Dukes' stage. In the
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S. Kapitanović et al. / Experimental and Molecular Pathology 96 (2014) 92–97 Table 2 Clinicopathological characteristics of 87 patients with colon adenocarcinoma stratified by FHIT protein staining. Characteristic
FHIT staining Negative (%) 47 (54.0)
Age b70 years 29 (50.0) ≥70 years 16 (64.0) Sex Male 24 (57.1) Female 22 (51.2) Tumor size ≤5 cm 29 (54.6) N5 cm 16 (53.3) Histological grade (Differentiation) Well 13 (44.8) Moderate 23 (57.5) Poor 10 (66.7) Dukes' stage A 7 (46.6) B 18 (48.6) C 21 (63.6) a b
p 1 (%)a 23 (26.4)
2 (%)b 17 (19.6)
16 (27.6) 5 (20.0)
13 (22.4) 4 (16.0)
0.502
14 (33.3) 8 (18.6)
4 (9.6) 13 (30.2)
0.043
12 (22.7) 9 (30.0)
12 (22.7) 5 (16.7)
0.687
8 (27.6) 11 (27.5) 3 (20.0)
8 (27.6) 6 (15.0) 2 (13.3)
0.567
4 (26.7) 11 (29.7) 7 (21.2)
4 (26.7) 8 (21.7) 5 (15.2)
0.704
1, positive b 80%. 2, positive ≥ 80%.
Fig. 4. Immunohistochemical analysis of FHIT protein in colon adenocarcinoma. A. negative immunostaining; B. weak to moderate immunostaining (1, positive b 80% of the tumor cells); C. strong FHIT immunostaining (2, positive ≥ 80% of the tumor cells)(100×).
immunohistochemical analysis 53.4% of tumor samples were negative for FHIT protein. In only 23.3% of tumors a strong positive reaction to FHIT protein was detected and there was no correlation with clinicopathological characteristics of patients and their tumors. The difference between our study and findings of Wierzbicki et al. (2009) may be caused by the use of different methodologies, differences in the selection of primers, probes or antibodies for FHIT protein, as well as protocols. The study of Thiagalingam et al. (1996) was on the other hand performed on primary cell cultures from human colon cancers which can result in different findings when compared with the results obtained from fresh tumor tissues. However, our results are in agreement with the results of other groups. Hao et al. (2000) found reduced expression of FHIT protein in 38% colorectal tumors without metastases and in 62% tumors with
Fig. 5. Kaplan–Meier survival curves with respect to A. tumor LOH status and B. FHIT immunohistochemical staining.
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metastases. In our study 48.0% of non-metastatic and 63.6% of metastatic CRC were found to be negative for FHIT protein. In addition to these findings Hao et al. (2000) also described high expression of FHIT protein in colorectal adenomas, premalignant lesions leading to adenocarcinomas. Yasugi et al. (2008) also described significantly reduced or absent FHIT protein expression in colorectal carcinomas but in none of the analyzed adenomas. In the study of Cao et al. (2006) positive FHIT gene expression was found in 93.75% of normal colorectal tissue and decreased FHIT expression correlated significantly with the progression of colorectal carcinoma with 68.75% and 46.25%, FHIT positive adenomas and adenocarcinomas respectively. In their study down-regulation of FHIT protein expression was associated with down-regulation of Bax and up-regulation of Bcl-2 and Survivin, which resulted in the inhibition of apoptosis. Wierzbicki et al. described the highest occurrence of the FHIT gene LOH at the D3S1234 locus (32.5%) followed by the D3S1300 locus (24%) and the D3S1481 locus (11%) of informative cases. Our FHIT LOH analysis showed similar results. We analyzed FHIT LOH at two loci, and found that at D3S1300 locus LOH was present in 20.2% of informative cases, and same percentage of LOH was observed at D3S4103 locus. In our study, as well as, in study of Wierzbicki et al. (2009) FHIT mRNA levels were significantly lower in LOH positive cases. Overall informativity for both markers in our study was 80.1% and 25.2% tumors demonstrated LOH, no correlation with clinicopathological characteristics was observed. Patients with LOH negative tumors had longer survival but this finding was not statistically significant. When survival was examined in relation to FHIT protein status in tumor tissue patients, with FHIT protein positive tumors had longer survival but this difference was not statistically significant, as well. In conclusion, FHIT gene LOH and lower or absent expression of FHIT protein were present in significant proportion of colon adenocarcinomas included in our study. Expression of FHIT mRNA was significantly lower in tumors when compared to the expression in the corresponding normal tissue. Patients whose tumors were LOH positive as well as those with FHIT protein negative tumors had longer survival, and FHIT LOH significantly correlated with the reduced FHIT mRNA expression. These results suggest that reduced expression of FHIT gene may be associated with the progression of this malignant tumor, and further studies may be expected to add new insights into FHIT as a potential prognostic marker in colon adenocarcinoma. Conflict of interest statement The authors declare that there are no conflicts of interest. References Ahsee, K.W., Cooke, T.G., Pickford, I.R., Soutar, D., Balmain, A., 1994. An allelotype of squamous carcinoma of the head and neck using microsatellite markers. Cancer Res. 54, 1617–1621. Baffa, R., Veronese, M.L., Santoro, R., Mandes, B., Palazzo, J.P., Rugge, M., Santoro, E., Croce, C.M., Huebner, K., 1998. Loss of FHIT expression in gastric carcinoma. Cancer Res. 58, 4708–4714. Barnes, L.D., Garrison, P.N., Siprashvili, Z., Guranowski, A., Robinson, A.K., Ingram, S.W., Croce, C.M., Ohta, M., Huebner, K., 1996. FHIT, a putative tumor suppressor in humans, is a dinucleoside 5′,5″-P1, P3-triphosphate hydrolase. Biochemistry 35, 11529–11535. Campiglio, M., Pekarsky, Y., Menard, S., Tagliabue, E., Pilotti, S., Croce, C.M., 1999. FHIT loss of function in human primary breast cancer correlates with advanced stage of the disease. Cancer Res. 59, 3866–3869. Cao, J., Li, W., Xie, J., Du, H., Tang, W., Wang, H., Chen, X., Xiao, W., Li, Y., 2006. Downregulation of FHIT inhibits apoptosis of colorectal cancer: mechanism and clinical implication. Surg. Oncol. 15, 223–233. Croce, C.M., Sozzi, G., Huebner, K., 1999. Role of FHIT in human cancer. J. Clin. Oncol. 17, 1618–1624.
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