Low expression of fragile histidine triad gene correlates with high proliferation in head and neck squamous cell carcinoma

Oral Oncology 39 (2002) 56–63 www.elsevier.com/locate/oraloncology

Low expression of fragile histidine triad gene correlates with high proliferation in head and neck squamous cell carcinoma H. Minetaa,*, Ka. Miurab, S. Takebayashia, Ki. Misawaa, Y. Uedaa, I. Suzukia, M. Itoa, J. Wennerbergc a

Department of Otolaryngology Hamamatsu University School of Medicine, 1-20-1 Handayama, 431-3192 Hamamatsu, Japan b Division of Pathology, Hamamatsu University Hospital, 1-20-1 Hanadayama, 431-3192 Hamamatsu, Japan c Department of Otorhinolaryngology/Head and Neck Surgery, Lund University Hospital, S-221 85 Lund, Sweden Received 12 March 2002; accepted 15 April 2002

Abstract Frequent loss of heterozygosity in head and neck squamous cell carcinoma (HNSCC) has been found in several chromosomal regions such as 3p, 9p, 11q, 13q and 17p. Fragile histidine triad (FHIT) gene is located at 3p14.2 encompassing a common fragile site, and is identified as a tumor suppressor gene. We examined 57 patients with HNSCC using immunohistochemistry, western blot, and reverse transcriptase polymerase chain reaction. The association between FHIT expression and clinicopathologic characteristics including p53 and Ki-67 expressions was analyzed. Immunohistochemical analysis revealed 30 patients (53%) of low FHIT expression and 27 patients (47%) of high FHIT expression. Low FHIT expression significantly correlated with high Ki-67 expression, indicating that tumor cells with low FHIT expression can proliferate aggressively. No correlation was found between FHIT expression and clinical characteristics including age, gender, tumor size, lymph node status, stage grouping, histologic grade, p53 expression, and prognosis. FHIT alteration may play an important role in cancer development of HNSCC, however it did not contribute to the prognosis. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Fragile histidine triad (FHIT) gene; Head and neck cancer; Immunohistochemistry; Ki-67 expression

1. Introduction Improvement of molecular biology reveals that carcinogenesis is related with a series of genetic events based on the dysfunction of tumor suppressor genes, oncogenes or growth factors. Frequent loss of heterozygosity (LOH) in head and neck squamous cell carcinoma (HNSCC) has been found in several chromosomal regions such as 3p, 5q, 9p, 11q and 17p [1]. Rare (or hereditable) fragile sites are found on the chromosomes of less than 5% of the individuals, whereas common (or constitutive) fragile sites are found in all individuals. Common fragile sites predispose to DNA instability in cancer [2]. Fragile histidine triad (FHIT) gene is located at 3p14.2 encompassing a common fragile site (FRA3B), and is encoded by 10 exons in a 1.1 kb transcripts [3]. This gene is identified as a tumor suppressor gene, because overexpression of the wild-type FHIT gene inhibited tumor cell growth, and because tumor * Corresponding author. Tel.: +81-53-435-2252; fax: +81-53-4352253. E-mail address: [email protected] (H. Mineta).

growth was significantly suppressed by the direct injection of the FHIT expressing adenoviral vector [4]. Although FHIT protein hydrolyses diadenosine triphosphate to ADP and AMP [5], this activity does not attribute to tumor suppression [6]. Substrate binding or binding of FHIT to an interacting protein is more important in tumor suppression than hydrolytic activity [6]. It serves as intracellular and exrtracellular signaling molecules involving in cellular differentiation and apoptosis [7]. This protein expresses at low levels in most adult tissues [8], but is absent or undetectable in some solid tumors [9], including head and neck carcinoma [10–18]. Head and neck carcinoma accounts for up to 5% of all carcinomas [11]. It characterizes that the majority arise from the epithelium of aerodigestive tracts and are therefore squamous cell carcinoma and that patients with HNSCC and are often accompanied with the second primary cancer of the aerodigestive tracts. Carcinogenesis of HNSCC is related with chemical and physical exposures and viral infections. The fragile region of 3p14.2 is subject to the target of environmental carcinogens [19]. Although low FHIT expression

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H. Mineta et al. / Oral Oncology 39 (2002) 56–63

was found in HNSCC, it remains unclear whether FHIT expression correlated with clinicopathologic characteristics and prognosis. The purpose of this study is (1) to determine the incidence of FHIT alteration in HNSCC using reverse transcriptase polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry (IHC), and (2) to determine the association between FHIT expression and clinicopathologic characteristics including p53 expression, Ki-67 expression, and prognosis.

2. Patients methods 2.1. Patients We analyzed 57 patients with HNSCC without distant metastasis. All patients were treated at the Department of Otolaryngology, Hamamatsu University School of Medicine, Hamamatsu, Japan. They were followed until December 2001, with the follow up period varying from 13 to 67 months. The clinical information including age, gender, tumor size, lymph node status, stage grouping, histologic grade and outcome was obtained from the clinical records. Primary tumor size, lymph node status and stage grouping were classified according to the 1997 UICC criteria [20]. Two pieces of specimens were collected from the same patients at the surgery. Two pieces of specimens were collected from the same patients at the surgery—one from the tumor tissue and another from the adjacent normal tissue. Access to human tissue were complied with the guidelines of the Ethics Committee of Hamamatsu University School of Medicine—one from the tumor tissue and another from the adjacent normal tissue. 2.2. Immunohistochemistry (IHC) Sections of 5mm paraffin-embedded tissue were dewaxed with xylene, hydrated through graded alcohol, and rehydrated in water. They were microwaved in a citrated buffer three times for 5 min, and endogenous peroxidase activity was blocked using 0.5% hydrogen peroxide in methanol for 30 min. A gout serum (20%) was applied to the sections for 10 min as a blocking reagent to reduce nonspecific binding. The primary antibodies of anti-FHIT (1:200 dilution, IBL, Fujioka, Japan), anti-p53 (1:1000 dilution, DAKO, Copenhagen, Denmark) and anti-Ki-67 (1:100 dilution, Immunotech, Marsseille, France) were used. Sections were incubated at 4
C overnight. They were incubated with the biotinylated anti-rabbit immunoglobulin donky antibody (1:1000 dilution, Nichirei, Tokyo, Japan) for anti-FHIT polyclonal antibody, and with the biotinylated antimouse immunoglobulin rabbit antibody (1:1000 dilution, Nichirei) for anti-p53 and anti-Ki-67 monoclonal antibodies for 30 min. They were incubated with strep-

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tavidin peroxidase reagents (Strept-ABComplex, DAKO) for 30 min, followed by treatment with diaminobenzidine solution (Nichirei) for 5 min for FHIT and p53 immunostainings and with 3-amino-9-ethylcarbazole solution (Nichirei) for Ki-67 immunostaining, and counterstained with hematoxylin. 2.3. Immunohistochemical evaluation Immunohistochemical evaluation was performed by a pathologist (Ka. M) in a blind test without knowledge of the clinical parameters and outcome. A tumor was considered high FHIT expression if the expression was noted in 10% or more tumor cells, while a tumor was considered low FHIT expression if the expression was noted in fewer than 10% of the tumor cells with reserved high reactivity in admixed non-tumor cells. p53 and Ki-67 expressions were considered high expression if the expressions were noted in 50 and 25% or more tumor cells, respectively, while they were considered low expression if the expressions were noted in fewer than 50 and 25% of the tumor cells. 2.4. Western blot The protein extracts of 30mg in a lysis buffer containing 100 mM Tris (pH 7.4), 0.15 M NaCl, and 1% SDS were electrophoresed on a 12.5% SDS/polyacrylamide gel, and were transferred to a Hybond-PVDF membrane (Amersham, Buckinghamshire, England). Blots were incubated in a blocking buffer (Dulbecco’s PBS buffer containing 3% bovin serum albumin and 3% skim milk) for 60 min at room temperature, then they were immunoblotted with the anti-human FHIT rabbit polyclonal antibody (IBL) at a dilution of 1:1000 at 4
C overnight. After rinsing the blots with PBS, they were incubated with horseradish peroxidase cojugated anti-rabbit immunoglobulin (Amersham) at a dilution of 1:1000 for 1 h at room temperature. After washing the blots, they were developed with a detection kit (ECL, Amersham). The optimized bands were analyzed by comparison with the density of the bands from the adjacent normal tissue. Actin (Anti-actin antibody: I-19, 1:1000 dilution, Santa Cruz Biotechnology Inc., Santa Cruz, CA) levels were analyzed as controls for protein loading. 2.5. RT-PCR RT-PCR analysis was performed in 30 patients. Total RNA was extracted using an ISOGEN kit (Nippon Gene, Toyama, Japan) and RT-PCR was carried out using a RT-PCR high kit (TOYOBO, Osaka, Japan) according to the manufacturer’s protocols. Reverse transcription reaction was incubated at 42
C for 20 min and 99
C for 5 min. Oligonucleotides used in PCR amplification of FHIT transcripts were as follows [3]:

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H. Mineta et al. / Oral Oncology 39 (2002) 56–63

P1 (sense strand in exon1 and 2): 50 -ATCCTGGAAGCTTTGAAGCTCA-30 , P2 (antisense strand in exon 10): 50 -TCACTGGTTGAATACAGGA-30 , P3 (sense strand in exon3): 50 -TCCGTAGTGCTATCTACATCC-30 , P4 (antisense strand in exon 10): 50 -CATGCTGATTCAGTTCCTCTTG-30 . PCR amplification was carried out in a final volume of 25 ml containing 0.1mM of each P1 and P2 primer, 0.125 mM of each dNTP, 1reaction buffer (Boehringer, Mannheim, Germany), 1.25 units Taq Polymerase (Boehringer), and 1ml of cDNA [21]. The PCR consisted of an initial denaturation step at 95
C for 5 min, fol-

lowed by 25 cycles of 50 s at 95
C, 50 s at 58
C, and 70 s at 72
. The nested PCR amplification was performed with primers P3 and P4, and 1 ml of the first PCR product for 30 cycles under the same condition as the first PCR. The nested PCR products were subjected to 6% polyacrylamide gel, and were visualized by ethidium bromide staining. The density of band for tumor tissue were compared with that for adjacent normal tissue. G3PDH levels were analyzed as controls. 2.6. Statistical analysis The association between discrete variables and FHIT expression by IHC was tested by the Fisher’s exact probability test or the Mann–Whitney’s U test. Relapse

Fig. 1. Fragile histidine triad (FHIT), Ki-67, and p53 immunostaining of two patients with maxillary carcinoma (A–C) and with tongue carcinoma (D–F). (A) FHIT immunostaining showed that tumor cells stained negative, while lymphocytes around tumour tissue and vascular endothelial cell were stained positive (Original magnification 40). (B) Almost tumor cells were stained negative for Ki-67 (40). (C) Approximately 70% of tumor cells were stained positive for p53. (40). (D) Most tumor cells were stained positive for FHIT (100). (E) Approimately 38% of tumor cells were stained positive for Ki-67 (100). (F) Approximately 75% of tumor cells were stained positive for p53 (100).

H. Mineta et al. / Oral Oncology 39 (2002) 56–63

free survival (RFS) curves were constructed using the method of Kaplan–Meier and tested by the logrank test. A significant difference was identified when the probability was less than 0.05.

3. Results We analyzed 57 patients with HNSCC. The patients’ age ranged from 21 to 90 years with a mean of 62 years. Positive cells for p53 or Ki-67 were stained dark brown or red on the nucleus. Positive cells for FHIT were stained dark brown on the cytoplasm. IHC revealed 30 patients (53%) of low FHIT expression and 27 patients (47%) of high FHIT expression (Fig. 1). The frequency of the FHIT expression does not significantly depend on the primary sites (Table 1). Table 2 showed a correlation between FHIT expression and clinical characteristics. No correlation was found between FHIT expression and clinical characteristics including age, gender, tumor size, lymph node status, stage grouping, and histologic grade. The analysis of p53 expression demonstrated 28 patients (50%) of low p53 expression and 28 patients (50%) of high p53 expression, which was not significant. The analysis of Ki-67 expression demonstrated 33 patients (62%) of low Ki-67 expression and 20 patients (38%) of high Ki-67 expression, and it also showed 16 (59%) of 27 patients with low FHIT expression showed high Ki-67 expression, while 22 (85%) of 26 patients with high FHIT expression showed low Ki-67 expression. The frequency of high Ki-67 expression in the patients with low FHIT expression was significantly higher than that in the patients with high FHIT expression, suggesting that tumor cells with low FHIT expression correlated with high proliferation. A Kaplan–Meier curve demonstrated that the 5-year RFS rate of the patients with low FHIT expression (52%) was not significantly different from that of the patients with high FHIT expression (44%). RT-PCR analysis revealed 17 patients (50%) with FHIT alterations including three patients (18%) with aberrant transcripts, two patients (12%) with decreased Table 1 The primary sites and fragile histidine triad (FHIT) expression

Oral cavity Oropharynx Hypopharynx Larynx Paranasal sinus

Total (n=57)

Low FHIT expressiona (n=30)

High FHIT expressiona (n=27)

20 5 5 15 12

8 3 4 8 7

12 2 1 7 5

(40%) (60%) (80%) (53%) (58%)

(60%) (40%) (20%) (47%) (42%)

a The FHIT immunohistochemical evaluation was performed according to our criteria: low expression ( <10% tumor cell cytoplasm positive) and high expression (510%).

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transcripts, and 12 patients (70%) without transcripts, and the remaining 17 patients (50%) with normal transcripts out of 34 patients studied (Table 3, Fig. 2). Western blot analysis showed 21 patients (37%) of low FHIT expression and 36 patients (63%) of high FHIT expression, and it also showed 16 (76%) of 21 patients with low FHIT expression in western blot revealed low FHIT expression in IHC (Table 3, Fig. 3). The result of IHC was significantly associated with those of RT-PCR and western blot analyses, respectively (P=0.007 and P=0.016).

4. Discussion We set out to determine whether FHIT expression correlated with clinical characteristics in HNSCC. We detected low FHIT expression in 30 (53%) of 57 patients using IHC, and it correlated with high Ki-67 expression. Immunohistochemical analysis showed a simple and reliable method to detect FHIT alterations in tumor cells. Our results of RT-PCR and western blot analyses were significantly related with that of IHC. Sozzi et al. also revealed a good correlation between IHC and southern, RT-PCR and western blot analyses [22]. Because FHIT alteration mostly causes deletion at exon 5 instead of point mutation, this gene was often analyzed by LOH, RT-PCR, IHC and cDNA sequencing methods. However, RT-PCR may depend on PCR conditions, especially, nested PCR which may overexpress the shorter PCR products may be attributable to the result of RT-PCR [23]. Normal cell contamination is also attributable to the results of RT-PCR and western blot . Although IHC depends on its techniques, stain interpretation, and cut off point [24], it can analyze a lot of specimens at a short period. Using both IHC and RT-PCR analyses can guarantee FHIT genetic alterations. FHIT alteration plays a role in carcinogenesis of HNSCC. We found 53% of low FHIT expression in HNSCC. Low FHIT expression was found in 3–61% of HNSCC using clinical specimens [10–18]. Low level (3%) depends on the PCR condition of a single set of 40 PCR cycles [12]. Fragile sites are susceptible to carcinogen-induced alterations, and the disruption of FRA3B fragile sites will cause FHIT deletions [25]. HNSCC is associated with environmental carcinogens including tobacco smoking, alcohol, viral infection of human papilloma virus (HPV) or Epstein–Barr virus. HPV associated cervical carcinoma [26] or lung carcinoma with smokers [27,28] were significantly associated with low FHIT expression, and also a significant correlation between heavy smoking and alcohol use and FHIT inactivation was shown in esophageal cancer [29].

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FHIT altered tumor cells implied high proliferation, and they might contribute to tumor growth. Our results revealed that FHIT expression did not correlated with clinicopathologic characteristics except for Ki-67 expression. We could find the significant correlation between FHIT expression and Ki-67 expression according to the cut off point of 25% of Ki-67 expression, although we could not find it according to our previous criteria of 10%. It suggests that tumor cells with low FHIT expression proliferate aggressively. However, FHIT expression did not contribute to the prognosis. The 5-year RFS rate of patients with low FHIT expression was not significantly different from that of patients

with high FHIT expression. Ki-67 is known to be the most indicative marker of cell proliferation, and its overexpression predicts a considerable risk of aggressive clinical behavior. The significant correlation between low FHIT expression and tumor aggressiveness was found in endometrial [30] and urinary tract [19] carcinomas. They were all adenocarcinomas. The common fragile site may be more sensitive to environmental mutagen in adenocarcinoma than squamous cell carcinoma [2]. Some reports showed that low FHIT expression was significantly associated with poor prognosis [28,30,31]. Our data showed that FHIT alteration indicated the high proliferation of tumor resulted in

Table 2 The fragile histidine triad (FHIT) protein expression and clinicopathologic characteristics Total (n=57)

Low FHIT expressiona (n=30)

High FHIT expressiona (n=27)

P-value

61 (21–90) 44:13

61 (37–81)) 23: 7

60 (21–90) 21: 6

0.875b 0.921b 0.140d

5 21 13 18

1 (20%) 10 (48%) 10 (77%) 9 (50%)

4 (80%) 11 (52%) 3 (23%) 9 (50%)

Lymph node statusc 0 1 2 3

24 12 17 4

12 (50%) 8 (67%) 10 (59%) 0 ( 0%)

12 (50%) 4 (33%) 7 (41%) 4 (100%)

Stagec I II III IV

3 11 10 33

1 (33%) 5 (45%) 9 (90%) 15 (45%)

2 (67%) 6 (55%) 1 (10%) 18 (55%)

Age, mean(range) Gender (male:female) Tumor sizec 1 2 3 4

0.126d

0.296e

0.543d

Histologic grade Poorly Moderately Well

7 17 33

5 (71%) 9 (53%) 16 (48%)

2 (29%) 8 (47%) 17 (52%)

Ki-67 expressionf Low High

33 20

11 (33%) 16 (80%)

22 (67%) 4 (20%)

p53 expressiong Low High

28 28

16 (57%) 13 (46%)

12 (43%) 15 (54%)

5-Year relapse free survival (Kaplan–Meier,%)

49

52

44

0.001b

0.422b

0.401h

a The FHIT immunohistochemical evaluation was performed according to our criteria: low expression ( <10% tumor cell cytoplasm positive) and high expression (510%). b A P value was identified by the Fisher’s exact probability test. c Tumor size, lymph node status, and stage grouping were classified according to the 1997 International Union Against Cancer criteria [20]. d A P value was identified by the Mann–Whitney’s U test. e A P value between the two groups of stage I, II and stage III, IV was identified by the Fisher’s exact probability test. f Ki-67 immunohistochemical evaluation was performed according to our criteria: low expression ( <25% tumor cell nuclei positive) and high expression (525%). g p53 Immunohistochemical evaluation was performed according to our criteria: low expression ( <50% tumor cell nuclei positive) and high expression (550%). h A P value was identified by the log-rank test.

H. Mineta et al. / Oral Oncology 39 (2002) 56–63 Table 3 The relationship between the result of immunohistochemistry and those of western blot and RT-PCR of FHIT Total (n=57)

Immunohistochemistry

P value

Low expressiona (n=30)

High expressiona (n=27)

RT-PCR (n=34) Abberent 17 Normal 17

12 (17%) 5 (29%)

5 (29%) 12 (71%)

0.016b

Western blot (n=57)c Low expression 21 High expression 36

16 (76%) 14 (39%)

5 (24%) 22 (61%)

0.007b

a The FHIT immunohistochemical evaluation was performed according to our criteria: low expression ( <10% tumor cell cytoplasm positive) and high expression (510%). b A P value was identified by the Fisher’s exact probability test. c The western blot evaluation of FHIT was performed according to our criteria: low expression (the density of tumor
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aggressive behavior, however it did not contribute to the prognosis. FHIT disruption is independent of p53 abnormality. We did not find the significant association between FHIT and p53 alterations. A concomitant association between FHIT and p53 abnormalities was found in smoke-related lung carcinoma [32,33] and estrogenindependent endometrial carcinoma [30]. We supported that genetic instability leading to the inactivation of FHIT gene was not induced by p53 mutations which caused the dysfunction of DNA mismatch repair and genome stabilization [32], and that both FHIT and p53 genes played a specific role as a sensor of carcinogenic damage through both different pathways [33]. Effects on cell growth inhibition and apoptosis induced by FHIT seem different from those of p53 and RB. Further study’s needed for analysis in a large number of patients with HNSCC, and for analysis regulating FHIT expression. In conclusion we found 53% of low FHIT expression in HNSCC, and we found the significant correlation between FHIT expression and Ki-67 expression. FHIT

Fig. 2. (A) RT-PCR analysis showed fragile histidine triad (FHIT) expression of four paired specimens of head and neck squamous cell carcinoma (T) and adjacent normal tissue (N) (Lanes 1 and 2 from the patient with laryngeal carcinoma, Lanes 3 and 4 from the patient with hypopharyngeal carcinoma, Lanes 5 and 6 from the patient with tongue carcinoma, and Lanes 7 and 8 from the patient with maxillary carcinoma). Lanes 3 and 5 demonstrated the smaller abberant bands. (B) cDNA sequencing analysis of the abberant band (**) of lane 5 showed the deletion from exon 5 to exon 8. Abberant PCR product was cut from gel. DNA was purified using a Quiamp II kit (Qiagen, Tokyo, Japan) and then directly sequenced by thermal cycling with fluorescent dye terminators using P3 and P4 primers. Reactions were analysed on an automated sequencer (model ABI 373A, Applied Biosystems, Foster City, CA).

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Fig. 3. Western blot analysis showed fragile histidine triad (FHIT) expression of three paired specimens of head and neck squamous cell carcinoma (T: Lane 1, 3, and 5) and adjacent normal tissue (N: Lane 2, 4, and 6). Lanes 1 and 2 from the patient with hypopharyngeal carcinoma showed that tumor expressed FHIT less than normal tissue. Lanes 3 and 4 from the patient with maxillary carcinoma showed that both tumor and normal tissue expressed FHIT. Lanes 5 and 6 from the patient with tongue carcinoma showed that only normal tissue, but not tumor expressed FHIT.

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