ORIGINAL ARTICLE Downregulation of and Its Association With Cancer History and a Tendency Toward Poor Survival in Head and Neck Squamous Cell Carcinoma

Esra Gunduz, DDS, PhD; Mehmet Gunduz, MD, PhD; Levent Beder, MD, PhD; Hitoshi Nagatsuka, DDS, PhD; Kunihiro Fukushima, MD, PhD; Recep Sutcu, MD; Namik Delibas, MD; Noboru Yamanaka, MD, PhD; Kenji Shimizu, PhD; Noriyuki Nagai, DDS, PhD

Objective: To examine the role of TESTIN as a candi- ables to mRNA expression status of TESTIN revealed a date tumor suppressor in head and neck carcino- significant difference in terms of cancer history (P=.03). genesis. Moreover, a higher smoking ratio and a family cancer history were also associated with downregulation of Design: Mutation and messenger RNA (mRNA) expres- TESTIN, although the difference was not statistically sig- sion analyses. nificant (P=.43 and P=.16, respectively). Kaplan-Meier survival analysis demonstrated a worse survival rate Setting: Academic research. among the patients with low TESTIN expression com- pared with the patients with normal-high TESTIN ex- Patients: Paired normal and tumor samples were ob- pression. tained from 38 patients with primary head and neck squa- mous cell carcinoma. Conclusions: Our findings suggest that inactivation of Main Outcome Measures: Analysis and comparison TESTIN is involved in head and neck carcinogenesis of TESTIN gene mRNA expression and its relationship through its downregulation. Further studies in various to clinicopathologic variables. human cancer tissues using a large sample size and in vitro functional studies as well as clinical comparison Results: Mutation analysis showed a nucleotide and research studies would give us a better evaluation of change in 6 of the 38 tumor samples (16.0%). TESTIN’s role and its possible future application in mo- Semiquantitative mRNA expression analysis of TESTIN lecular diagnosis and treatment of different cancer types, revealed a decreased expression in approximately 50% including head and neck squamous cell carcinoma. of the tumors compared with their matched normal con- trols. Interestingly, comparison of clinicopathologic vari- Arch Otolaryngol Head Neck Surg. 2009;135(3):254-260

EAD AND NECK SQUAMOUS mutational inactivation allows cells to dis- Author Affiliations: cell carcinoma (HNSCC) play 1 or more phenotypes of neoplastic Departments of Oral Pathology is one of the most fre- growth. Loss of heterozygosity (LOH) is and Medicine (Drs E. Gunduz, quent cancers that lead to considered an indication of a TSG pres- M. Gunduz, Nagatsuka, and death, making it a major ence, whereby inactivation contributes to Nagai), Molecular Genetics health problem in the world. It includes oral, the development and/or progression of tu- (Drs E. Gunduz and Shimizu), H 2 and Otolaryngology–Head and oropharyngeal, nasopharyngeal, hypopha- mor. Detailed LOH analysis of polymor- Neck Surgery (Dr Fukushima), ryngeal, and laryngeal cancers and ac- phic loci distributed along a chromo- Graduate School of Medicine, counts for more than 644 000 new cases some can reveal a common minimal Dentistry, and Pharmaceutical worldwide each year, with a mortality rate deleted region where putative TSGs may Sciences, Okayama University, of 53% and a male predominance of 3:1.1 reside. Okayama, Japan; Department of Despite advanced technology in its detec- Previous cytogenetic studies as well as Otolaryngology–Head and Neck tion and treatment, HNSCC continues to microdeletion analysis have shown fre- Surgery, Wakayama Medical pose a great threat to human life. Recent ad- quent abnormalities of 7 in University, Wakayama, Japan vances in the technology and molecular bi- various cancer types, including HNSCC.3-10 (Drs M. Gunduz, Beder, and Yamanaka); and Department of ology of human cancer, including head and Moreover, microcell-mediated transfer of Medical Biochemistry, Suleyman neck carcinoma, have provided possible human into a murine squa- Demirel University, Faculty of novel diagnostic and prognostic markers. mous cell carcinoma cell line was found Medicine, Isparta, Turkey Tumor suppressor (TSGs) are de- to inhibit tumorigenicity of the cell line.11 (Drs Sutcu and Delibasi). fined as genetic elements whose loss or In another study, introduction of a single

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 copy of human chromosome 7 into a highly aggressive human prostate carcinoma cell line increased tumor la- Table 1. Clinicopathologic Characteristics of Patients tency by at least 2-fold.12 Similarly, insertion of an in- a tact human chromosome 7 into an immortalized hu- Characteristic Values man fibroblast cell line with LOH in the 7q31-32 region Tumor site suppressed immortality of the cells and restored their se- Oral cavity 17 (44.7) nescent ability.13 All these studies have provided strong Oropharynx 8 (21.1) Larynx 7 (18.4) evidence for the existence of a TSG(s) in chromosome Hypopharynx 1 (2.6) 7q31 region. Maxilla 5 (13.2) Based on these studies, we previously examined the Age, mean (SD), y 64.2 (9.3) chromosome region 7q22-31 using a set of highly poly- Sex morphic microsatellite markers to evaluate allele loss ra- Male 30 (78.9) tios and candidate TSGs in HNSCC.14 The study indi- Female 8 (21.1) cated 2 differentially deleted chromosomal areas at 7q31 T category T1 3 (7.9) around the markers D7S486 and D7S643. The presence T2 15 (39.4) of microdeletion in both loci but retention of the chro- T3 8 (21.1) mosomal region between these 2 peaks in some cases sug- T4 12 (31.6) gested that chromosome 7q31 included 2 different TSGs, N category at least in HNSCC. We have already shown that ING3 N0 19 (50.0) located at one of these regions (D7S643) has been in- N1 7 (18.4) 14 N2 12 (31.6) volved as a TSG in HNSCC. However, the role of the TNM stage other possible candidate gene in HNSCC remained un- I 1 (2.6) known. Therefore, in the current study, we focused on II 10 (26.3) the other highly deleted chromosomal area around the III 9 (23.7) marker D7S486, which is located in intron 6 of the IV 18 (47.4) b TESTIN gene, confirming its high ratio of deletion. In Differentiation Well 15 (40.6) fact, missense mutations and decreased expression of Moderate 17 (45.9) TESTIN were detected in various cancer cell lines, in- Poor 5 (13.5) cluding breast cancer, pancreatic cancer, and hemato- 15 logic malignant neoplasms. Moreover, adenoviral trans- a Values other than age are expressed as number (percentage). fection of TESTIN into breast and uterine cancer cell lines b One case with an unknown situation was not included. reduced tumor growth in mice.16 A recent study also dem- onstrated tumor suppressor function of TESTIN in a tion approved the study. The clinicopathologic characteris- knockout mouse model.17 Thus, we analyzed the expres- tics of the patients were shown in Table 1. sion level and mutation status of TESTIN to clarify its role in HNSCC. We also compared the messenger RNA ISOLATION, COMPLEMENTARY DNA RNA (mRNA) status of TESTIN with clinicopathologic PREPARATION, AND RT-PCR ANALYSIS variables. Total RNA samples were prepared using a modified acid METHODS guanidinium phenol chloroform method (ISOGEN; Nippon Gene Co Ltd, Tokyo, Japan). Total RNA was reverse- transcribed with a preamplification system (ReverTra Ace PATIENTS AND SAMPLES Kit; Toyobo Co Ltd, Osaka, Japan) starting with 2 µg of total RNA from each sample, according to the procedures pro- Paired normal and tumor samples were obtained from 38 vided by the supplier. TESTIN (GenBank NM_015641) mRNA patients with primary HNSCC between 1999 and 2005 at the expression in paired tumor and normal tissues was examined Department of Otolaryngology–Head and Neck Surgery, by duplex reverse transcriptase–polymerase chain reaction (RT- Okayama University Hospital, Okayama, Japan, after PCR). One microliter of each RT reaction was amplified in 50 informed consent was obtained from each patient. Normal µL of mixture containing 1.2mM magnesium chloride, 1X PCR control samples were obtained from grossly normal tissue as buffer, 200 µmol/L of each deoxynucleotide triphosphate, 20 far as possible from the tumor tissue. Although surgical mar- pmol of each primer, and1Uofrecombinant Thermus ther- gins were examined during surgery, we also confirmed the mophilus (rTth) DNA polymerase XL (Applied Biosystems, Fos- histopathologic appearance of the normal tissues by ter City, California). Thirty PCR cycles of TESTIN primers, RT-S hematoxylin-eosin staining. All tissues were frozen in liquid (5Ј- GAA TGA GAA GCT ATA CTG TGG C) and RT-AS (5Ј- nitrogen immediately after surgery and stored at −80°C until ATG GCT CGA TAC TTC TGG GTG), and 25 cycles of ␤- the extraction of RNA. The study included 30 men and 8 primers, S1 (5Ј-GGC CAA CCG CGA GAA GAT GAC) women (mean age, 64 years; age range, 40-81 years). The and AS1 (5Ј-GCT CGT AGC TCT TCT CCA GGG), were used histologic diagnosis in all cases was squamous cell carci- for amplification (the primers were designed with noma. None of the patients received preoperative chemo- GENETYX-MAC 10.1; Software Development Co Ltd, Tokyo, therapy or radiotherapy. The differentiation and diagnosis of Japan). An initial denaturation step at 94°C for 3 minutes was the tumor was based on the surgical pathology reports of the followed by 30 cycles of a denaturation step at 94°C for 30 sec- hospital. All clinical information was obtained from the onds, an annealing step at 60°C for 1 minute, and an exten- patient files, which included the initial diagnosis, treatment, sion step at 72°C for 1 minute. A final extension step at 72°C and follow-up data. The bioethics committee of the institu- for 7 minutes was added. ␤-Actin primers were added to each

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 and for exon 5, EX5-S (5Ј- GAC TAG GTT GTT CTG GAT GGC TT) and EX5-AS (5Ј- TGT CAA CCC AAT TAA CAC AGA Centromere CAG). The PCR mixture contained 100 ng of genomic DNA, 1.2mM magnesium chloride, 1X PCR buffer, 200 mmol/L of each deoxynucleotide triphosphate, 20 pmol/L of each primer, and1UofrTth DNA polymerase XL (Applied Biosys- TESTIN D7S486 tems) in a 50-µL volume. Initial denaturation at 94°C for 3 118 MB minutes was followed by 30 cycles of a denaturation step at 94°C for 30 seconds, an annealing step at 56°C (exon 3) or 60°C (exons 4 and 5) for 1 minute, and an extension step at 72°C for 1 minute. A final extension step at 72°C for 7 min- utes was added. The resultant PCR products were purified using ExoSAP-IT reagent (USB Corp, Cleveland, Ohio) before sequence-specific PCR amplification. Purified PCR products were reamplified with a cycle sequencing kit (BigDye Termi- nator v1.1 Cycle Sequencing Kit; Applied Biosystems); then, ING3 they were ethanol precipitated and direct sequenced on an au- D7S643 122 MB tomated capillary sequencer using the primers described above (ABI 3130xl; Applied Biosystems).

Telomere STATISTICAL ANALYSIS

Figure 1. A relatively simple physical map of the chromosome 7q31 region. Pearson ␹2, Fisher exact, and t tests were used to evaluate The distances of the genes and markers are based on the latest mapping the correlation between TESTIN mRNA expression and the information from the National Center for Biotechnology Information (http: clinicopathologic characteristics of the patients. Survival //www.ncbi.nlm.nih.gov/genome/guide/human/) and the Genome Database curves were calculated according to Kaplan-Meier. The log- (http://www.gdb.org/) homepages. D7S643 is the center marker for the ING3 locus, and D7S486 is a specific marker for the TESTIN locus. MB indicates rank test was used to compare survival between low and mega–base pairs. normal-high expression of TESTIN. Overall survival in months was calculated for 5 years starting from the day after surgery. The duration of disease-free survival was deter- PCR tube at the end of the fifth cycle by holding the thermo- mined from the day after surgery to the initial recurrence of cycler at 94°C for awhile. Reproducibility was confirmed by pro- the surgically resected cancer, evaluated by clinical examina- cessing all samples 2 times. tion. All statistical analyses were performed with SPSS ver- sion 10 (SPSS Inc, Chicago, Illinois), and PϽ.05 was consid- QUANTIFICATION OF THE RT-PCR PRODUCTS ered significant.

The PCR products were separated through 2% agarose gel RESULTS and stained with ethidium bromide. As the sizes of the PCR products were 386 (bp) for ␤-actin and 446 bp for TESTIN, they were readily distinguishable. The intensity of MAPPING OF TESTIN GENE ethidium bromide staining of each band was measured by a CCD (charged-couple device) image sensor (Gel Print 2000/ The LOH at the chromosome 7q31 region has been re- VGA; Toyobo Co Ltd) and analyzed by a computer program ported for various cancers, including head and neck car- for band quantification (Quantity One; Toyobo Co Ltd). The cinoma. We recently redefined the map of the 7q31 re- value of tumor-specific TESTIN expression was determined gion according to the contiguous sequences and detected by calculating the ratio of the expression levels in the tumor 2 preferentially deleted areas around the markers D7S486 and in the matched normal sample, each of which was nor- 14 malized for the corresponding ␤-actin expression level (T, and D7S643. In that study, we documented ING3 as a TESTIN/␤-actin expression ratio in tumor sample; N, TSG at one of these frequently deleted loci, D7S643. The TESTIN/␤-actin expression ratio in matched normal sample; other marker, D7S486, is located in intron 6 of the and T/N ratio, the relative TESTIN expression in the tumor TESTIN gene. Figure 1 shows their relative locations sample compared with its matched normal sample after nor- at the chromosome 7q31 region. malization). Decreased and increased expression levels were classified as L and H when this ratio was less than 0.6 and mRNA EXPRESSION ANALYSIS OF TESTIN greater than 1.4, respectively, as reported previously.14 Class N (normal expression) represented the value of the ratio Because our previous data demonstrated frequent alle- between 0.6 and 1.4. lic loss at the TESTIN gene area (D7S486), we examined the mRNA expression status of TESTIN to see whether MUTATION ANALYSIS OF TESTIN it was inactivated with other mechanisms in HNSCC. In 38 available matched RNA samples, the expression lev- Each of the coding regions of exon 3 through 5 of the TESTIN els of TESTIN mRNA were compared by semiquantita- gene was amplified by PCR with intron-spanning primers de- ␤ signed using GENETYX-MAC 10.1 software (Software Devel- tive RT-PCR using -actin mRNA as a control. Seven- opment Co): for exon 3, EX3-S (5Ј- CGT GTT TTG TTT CCT teen of the 38 tumor tissue samples (45.0%) showed TCT TGC) and EX3-AS (5Ј- AGT AAT GAG AAC CCC GGA decreased expression of TESTIN mRNA compared with AG); for exon 4, EX4-S (5Ј- ATT GGC CTC CTT GTG GTT each paired normal tissue sample, while 12 samples CAG) and EX4-AS (5Ј- CAA ACA CGA TGA CCC TCT GTG); (31.0%) showed a similar level of expression in normal

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 and tumor tissue samples (Figure 2 and Table 2). In- whether this change was a simple polymorphism or a tu- creased expression of TESTIN was detected in 9 tumor mor-specific mutation, we analyzed corresponding nor- tissue samples (24.0%). mal DNA by direct sequencing. Our analysis detected the same substitutions in the normal tissues, suggesting that MUTATION ANALYSIS OF TESTIN the nucleotide and amino acid change in codon 221 of exon 4 was a polymorphism. A previous study demonstrated missense mutations in exons 3, 4, and 5 of TESTIN in 3 cell lines, including CEM THE RELATIONSHIP OF CLINICOPATHOLOGIC (leukemic cell line), MDAMB453 (breast cancer cell line), CHARACTERISTICS TO TESTIN 15 and CAOV3 (ovarian cancer cell line). Based on this in- mRNA EXPRESSION formation, we also examined the mutation status of ex- ons 3 through 5 of TESTIN in our 38 head and neck can- The relationship between the expression status of cer samples. The PCR amplification and subsequent direct TESTIN and the clinicopathologic characteristics of the sequencing of each sample demonstrated no substitu- patients is shown in Table 2. We compared these results tion in exons 3 and 5. However, we detected the same between the samples with and without decreased mRNA nucleotide change (codon 221 of TESTIN transcript 1 in expression of TESTIN. Seventeen of the 38 tumor samples exon 4, from GCC to GTC, with an amino acid sub- (45.0%) showed decreased TESTIN mRNA expression in stitution from alanine to valine) as reported in the tumor samples compared with their normal counter- MDAMB453 breast cancer cell line.15 Six (cases 5, 6, 13, parts. The mean age of the patients with and without de- 20, and 24 [heteromutant] and case 7 [homomutant]) creased TESTIN expression was similar (62.5 years vs 65.5 of 38 samples (16.0%) showed this nucleotide and amino years, respectively). A significant difference was de- acid change in our samples (Figure 3). Five of the 6 tected between TESTIN expression and sex. All tumor nucleotide changes were heterozygous, while only 1 samples but 1 (94.1%) with low TESTIN expression were sample displayed a homozygous substitution. To clarify from male patients, while 14 of 21 samples (66.7%) with normal or high TESTIN expression were from 20 34 4 1338 1 2 male patients (Table 3 [P Ͻ .05]). With regard to MN T NT NT NTNTNTNT alcohol consumption and smoking, there was no sig- TESTIN nificant difference between the decreased TESTIN Actin expression group and the normal-high TESTIN expression group. However, the cases with decreased TESTIN expression were associated with a higher rate Figure 2. TESTIN messenger RNA expression analysis in matched tumor and normal samples. Representative raw data from reverse of smoking compared with the cases with normal-high transcriptase–polymerase chain reaction analysis are shown. The upper band TESTIN expression (70.6% vs 42.1%, respectively; indicates the TESTIN message (446 base pairs [bp]); lower band, the ␤-actin Table 3). When the expression status of TESTIN was message (386 bp); N, nontumor tissue; T, tumor tissue; and M, size marker. The upper numbers represent the case numbers. Cases 20, 34, 4, 13, compared with the T stage, N stage, overall (TNM) and 38 demonstrate decreased expression of TESTIN, while cases 1 and 2 stage, and histologic differentiation, no significant demonstrate the normal and increased expressions of the gene, respectively. alterations were detected (Table 3).

Table 2. Quantification of TESTIN Messenger RNA Expression in Head and Neck Squamous Cell Carcinomaa

Case No. Ratio Class Case No. Ratio Class 1 1.16 N 20 0.15 L 2 2.08 H 21 0.44 L 3 0.54 L 22 1.27 N 4 0.52 L 23 0.38 L 5 1.84 H 24 0.89 N 6 0.57 L 25 14.00 H 7 0.54 L 26 2.60 H 8 3.42 L 27 1.35 N 9 3.25 H 28 0.98 N 10 2.60 H 29 0.60 L 11 1.20 N 30 0.54 L 12 24.80 H 31 0.75 N 13 0.45 L 32 0.90 N 14 3.39 H 33 0.56 L 15 0.60 L 34 0.30 L 16 0.46 L 35 0.50 L 17 1.38 N 36 3.67 H 18 1.29 N 37 0.72 N 19 1.35 N 38 0.48 L

a The ratio of the expression levels in the matched tumor and normal samples was calculated as described in the “Methods” section. Decreased and increased expression levels were classified as N (normal) when the value of the ratio was between 0.6 and 1.4 and as L (lower) and H (higher) when the ratio was less than 0.6 and greater than 1.4, respectively.

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 A Case 15. Normal: GCC/GCC (alanine/alanine) Table 3. Relationship Between TESTIN Messenger RNA Expression and Clinicopathologic Characteristics T T G T C C T C C A T G G C C C C C A C T G C T G C T 170 180 190 TESTIN Messenger RNA Expression

Normal- Lowa Higha P Characteristic (n = 17) (n = 21) Value Sexb Female 1 (5.9) 7 (33.3) .05 Male 16 (94.1) 14 (66.7) Age, mean (SD), yc 62.5 (9.5) 65.5 (9.2) .32 Smokingc,d Yes 12 (70.6) 8 (42.1) .43 No 5 (29.4) 11 (57.9) Alcohol consumptiond,e Yes 9 (52.9) 8 (44.4) .61 Case 5. Tumor, heteromutant: GCC/GTC (alanine/valine) B No 8 (47.1) 10 (55.6) a T T G T C C T C C A T G N C C C C C A C T G C T G C T TNM stage Early stage (I-II) 4 (23.5) 7 (33.3) .72 170 180 190 Late stage (III-IV) 13 (76.5) 14 (66.7) T categoryd,f Early T (T1-T2) 8 (47.0) 10 (47.6) .97 Late T (T3-T4) 9 (53.0) 11 (52.4) N categoryd N(0) 7 (41.1) 12 (57.1) .33 N(ϩ) 10 (58.9) 9 (42.9) Differentiation,d,e Well 8 (45.1) 7 (35.0) .46 Moderate-poor 9 (52.9) 13 (65.0) Personal cancer historyb,e Absent 10 (62.5) 18 (94.7) .03 Present 6 (37.5) 1 (5.3) Family cancer historyd,f C Case 7. Tumor, homomutant: GTC/GTC (valine/valine) Absent 8 (47.1) 14 (70.0) .16 T T G T C C T C C A T G A C C C C C A C T G C T G C T Present 9 (52.9) 6 (30.0) 160170 180 a Values other than age are expressed as number (percentage). b Fisher exact test. c t Test. d Pearson ␹2 test. e Cases with unknown status regarding smoking (n = 2), alcohol consumption (n = 3), differentiation (n = 1), and personal (n = 3) and family (n = 1) cancer history were not included for evaluation. f According to the International Union Against Cancer 1997 TNM classification system.

expression had a history of chondrosarcoma, gastric cancer, tongue cancer, non-Hodgkin lymphoma, tongue Figure 3. Representative examples of samples with normal and mutant cancer, and colon carcinoma, respectively. On the other TESTIN sequences. Codon 221 of TESTIN transcript 1 in exon 4, from GCC hand, although a tendency of a higher ratio of family to GTC, with an amino acid substitution from alanine to valine. The sequences are read in an antisense direction. cancer history was detected in the cases with decreased TESTIN expression, no statistically significant change was shown (Table 3). An important and potentially significant finding was revealed when the expression status of TESTIN was com- mRNA EXPRESSION OF TESTIN AND pared with cancer history: 10 of 16 cases (62.5%) with SURVIVAL ANALYSIS low TESTIN mRNA expression had no cancer history, while all but 1 of the cases (94.7%) with normal-high When we examined the relationship between survival and TESTIN expression had no cancer history (P Ͻ .03, the expression status of TESTIN, no statistically signifi- Table 3). In other words, 6 of the 16 cases (37.5%) with cant difference was detected in either overall or disease- decreased TESTIN expression were associated with a can- free survival (Figure 4A and B). In terms of disease- cer history, while only 1 of the 19 cases (5.3%) with nor- free survival, the rates among all cases were similar, mal-high TESTIN expression had a cancer history. One although the cases with low TESTIN expression had a case (No. 12) with high TESTIN mRNA expression had lower ratio of survival (Figure 4A, P=.28). As shown in a history of mucoepidermoid carcinoma, while 6 cases Figure 4A, the mean (SD) disease-free survival in the low (Nos. 3, 4, 6, 20, 29, and 34) with low TESTIN mRNA expression group was 31 (7) months (95% confidence

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 interval [CI], 19-44 months), while that in the normal- high expression group was 40 (5) months (95% CI, 30-50 A months), with no significant difference between the 1.0

groups (P=.28, log-rank test). However, interestingly, a 0.9 shorter survival was shown in the patients with de- P = .28 creased TESTIN mRNA expression compared with the 0.8 0.7 patients without such alteration in terms of overall sur- Normal-high expression vival, almost reaching statistical significance (Figure 4B, 0.6 P=.09). As shown in Figure 4B, the overall survival in the low expression group was 42 (6) months (95% CI, 0.5 0.4 31-53 months) and that in the normal-high expression Low expression

group was 52 (4) months (95% CI, 44-59 months), and Disease-Free Survival 0.3 the log-rank test showed that patients with low TESTIN 0.2 expression had an almost significantly shorter overall sur- vival than those with normal-high expression (P=.09). 0.1 Overall, while the 5-year survival ratio of the cases with decreased TESTIN mRNA expression was approxi- 0 10 20 30 40 50 60 Time, mo mately 50%, it was nearly 80% among the cases without decreased TESTIN mRNA expression (Figure 4B). B 1.0 High-normal expression 0.9 COMMENT 0.8 Our previous study demonstrated frequent allelic loss at 0.7 Low expression the marker D7S486 location, which is an intragenic 0.6 14 marker and confirms the deletion of the TESTIN gene. 0.5 The appearance of 2 different peaks of allelic loss sug- P = .09 gested that TESTIN is likely to be another TSG involved 0.4 in HNSCC other than ING3, which was identified by our Overall Survival Rate 0.3 group as a TSG from the 7q31 area. In fact, an increas- 0.2 ing number of studies support a tumor suppression role for TESTIN. The detection of tumor-specific mutations 0.1

as well as decreased mRNA expression in human tumor 0 10 20 30 40 50 60 samples and in vitro functional analysis all suggested that Time, mo TESTIN is a TSG in various cancers.15-19 20 Knudson’s definition of a classic TSG requires inac- Figure 4. Disease-free (A) and overall (B) survival rates in patients with head tivation of both alleles of a candidate gene in tumors. In- and neck squamous cell carcinoma and decreased or normal-high TESTIN activation of these classic TSGs occurs through the de- expression. Kaplan-Meier survival curves for the total number of cases are letion of one of its alleles and mutation in its other allele. stratified by TESTIN messenger RNA expression. The cases were divided into a low expression group and a normal-high expression group as described in We previously found that one of the TESTIN alleles the “Methods” section. Statistical significance was defined as PϽ.05. A, The (D7S486) was deleted in our cases.14 Therefore, in mean (SD) disease-free survival was 31 (7) months in the low expression the present study, we examined the inactivation status group (95% confidence interval [CI], 19-44 months) and 40 (5) months in the normal-high expression group (95% CI, 30-50 months) (P=.28, log-rank of TESTIN’s second allele in HNSCC. Mutation analysis test). B, The mean (SD) overall survival was 42 (6) months in the low revealed nucleotide and amino acid substitutions in exon expression group (95% CI, 31-53 months) and 52 (4) months in the 4 in a considerable number of the cancer cases. Exis- normal-high expression group (95% CI, 44-59 months) (P=.09, log-rank tence of this possible mutation was previously reported test). in a breast cancer cell line.15 However, analysis of corre- sponding normal samples for the tumor cases with this moter occurs, the result is a decrease in substitution showed the same alteration, suggesting that mRNA expression and then possibly a decrease in the pro- it was not a tumor-specific somatic change but a poly- tein product and deficient function of TSG, resulting in morphism, which can still influence TESTIN func- cancer development. To identify the possibility of such tion and be important during carcinogenesis. To clarify a result, we analyzed the mRNA expression status of the importance of this polymorphic change, in vitro func- TESTIN in matched normal-tumor cases and showed tional studies of the preparation of the expression vec- downregulation of TESTIN in approximately 50% the head tors for each variant should be performed. and cancer cases compared with their corresponding nor- Recently, a new class of TSG with haploid insuffi- mal controls. This finding suggested that inactivation of ciency, in which one of alleles is lost and the other allele TESTIN as a class 2 TSG is likely to occur, especially in is haploinsufficient, has been described. These hemizy- head and neck cancer. gous TSGs showed a tumor-prone phenotype when chal- Comparison of the patients’ clinicopathologic vari- lenged with carcinogens.21,22 Moreover, meth- ables with their TESTIN mRNA expression level dem- ylation has also been attributed to the inactivation onstrated interesting findings, the most remarkable of mechanism of TSG.23 Whether haploinsufficiency or pro- which was a significant association with a history of can-

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 cer. Clearly, the patients with a low TESTIN expression Japan Science and Technology Agency (Dr M. Gunduz); level had a higher rate of cancer history. Although it was by Sumitomo Trust Haraguchi Memorial Cancer Research not statistically significant (P=.43), a higher rate of smok- Promotion (Dr M. Gunduz); and by a research grant from ing was also shown in the patients with low TESTIN ex- AstraZeneca (Dr M. Gunduz). pression level. Therefore, a higher rate of cancer history Additional Contributions: Surgeons from the Depart- could be related with early smoking in those cases. How- ment of Otolaryngology–Head and Neck Surgery, ever, a tendency of a higher rate of family cancer history Okayama University, generously provided fresh samples was also shown in the patients with low TESTIN expres- for this experiment. sion, although it was far from reaching statistical signifi- cance. Nevertheless, the higher ratio of a personal as well REFERENCES as a family cancer history and the finding of a polymor- phism with unknown significance in a considerable num- 1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer ber of the patients suggest that TESTIN inactivation or a J Clin. 2005;55(2):74-108. decrease in its activity could be strongly related to the 2. Croce CM. Genetic approaches to the study of the molecular basis of human cancer. Cancer Res. 1991;51(18)(suppl):5015s-5018s. development of head and neck cancer. On the other hand, 3. Cowan JM, Beckett MA, Ahmed-Swan S, Weichselbaum RR. Cytogenetic evi- survival analysis demonstrated a possible prognostic value dence of the multistep origin of head and neck squamous cell carcinomas. J Natl of TESTIN in HNSCC. An almost significant difference Cancer Inst. 1992;84(10):793-797. was revealed between low and normal-high TESTIN ex- 4. Edelson MI, Scherer SW, Tsui LC, et al. Identification of a 1300 kilobase deletion pression in terms of overall survival. Although TESTIN unit on chromosome 7q31.3 in invasive epithelial ovarian carcinomas. Oncogene. 1997;14(24):2979-2984. expression alone is not enough evidence for a strong 5. Lin JC, Scherer SW, Tougas L, et al. Detailed deletion mapping with a refined prognostic association, evaluation of the expression sta- physical map of 7q31 localizes a putative tumor suppressor gene for breast can- tus of other TSGs in the together with cer in the region of MET. Oncogene. 1996;13(9):2001-2008. TESTIN expression may provide important information, 6. 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