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The chromosome 5q21 band minisatellite and head and neck cancer
Cancer Genetics and Cytogenetics 147 (2003) 87–88
Letter to the editor
The chromosome 5q21 band minisatellite and head and neck cancer Squamous cell carcinoma of the head and neck (HNSCC) is one of the most common cancers in Brazil. The most important prognostic factor for this carcinoma is related to complete surgical removal of the neoplasm [1]. When tumor is present at a margin of resection, the rate of local recurrence increases substantially and the survival rate decreases [2]. HNSCC patients often develop multiple premalignant lesions in their upper aerodigestive tract. This finding led to the field cancerization theory, which hypothesizes that the entire mucosa of the upper aerodigestive tract has an increased risk for the development of neoplastic lesions. The presence of multiple genetic abnormalities in histologically normal, tumor-adjacent mucosa from HNSCC patients supports this hypothesis [3]. Bugert et al. [4] describes a 33-base pair (bp) minisatellite repeat in the 5′-flanking region of the mutated in colon cancer (MCC) gene at chromosome band 5q21. The repetitive sequence was observed as unique in the human genome and was not expressed as part of a gene transcription unit. The authors [4] discuss the duplication of one allele in three renal tumors and the loss of one allele in three bladder tumors. Because loss of heterozygosity of the 5q21 region has been found in many types of cancer, such as those of the lung, liver, colorecum, esophagus, and oral area [5–9], the 33-bp minisatellite repeat may be analyzed for imbalance of these regions. To investigate loss of heterozygosity (LOH) on chromosome band 5q21 in HNSCC, we analyzed blood, tumors, and surgical margins from 64 patients by using polymerase chain reaction (PCR) and single-stranded conformation polymorphism (SSCP) techniques. The samples were obtained at the Sa˜o Jose´ do Rio Preto School of Medicine,
and at the Hospital Arnaldo Vieira de Carvalho, Sa˜o Paulo, Brazil. Sections of tumors used for the study were selected from areas with high proportions of malignant tissue. None of the patients had undergone treatment before surgery. Genomic DNA was extracted from all specimens by using proteinase K digestion. A nonradioactive PCR-SSCP analysis was used to detect the presence of polymorphisms in the minisatellite of chromosome band 5q21. Oligodeoxynucleotide primers and thermocycle PCR conditions were the same as described by Bugert et al. [4]. Amplified products were separated on polyacrylamide gels and visualized by silver staining according to standard protocols. Loss of heterozygosity was defined as a reduction in the signal of more than 80% in one of the alleles. Twenty-seven cases of primary HNSCC showed constitutive heterozygosity. There were four (28.5%) tumors with LOH (Table 1). In two cases, LOH were observed only in the tumors. One patient has been disease-free 2 years after surgery and another had a recurrence 1 year after surgery. The latter exhibited a band of 257 bp in blood, tumor, and marginal tissue, corresponding to a previously unreported fragment of six repeats. This fragment was also observed in blood samples from two sons and one daughter and might have originated by events such as slippage replication, saltatory replication, unequal crossing over, or gene conversion. In two cases, both tumor and surgical margins exhibited loss of the same allele: one patient died 1 month after surgery and the second one is alive and free of disease 3 years after surgery. Therefore, our data provide no evidence that LOH on chromosome band 5q21 may be involved in the progression of HNSCC. Our data do indicate that the tumor develops in a genetically altered field where loss of genes at region5q21 may be present [10].
Table 1 Clinicopathologic characteristics of HNSCC patients with LOH at 5q21 Consumption
Tumor
Patient LOH(⫹)
Age
Sex
Tobacco
Alcohol
Site
Stagea
Status
LOH site
A B C D
72 48 85 70
Male Male Male Male
⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫺ ⫺
Larynx Glottis Larynx Tongue
IV III II II
Dead Alive, well Alive, well Recurrence
T/M T T/M T
Abbreviations: M, surgical margin; T, tumor. a UICC TNM staging system. 0165-4608/03/$ – see front matter 쑖 2003 Elsevier Inc. All rights reserved. doi: 10.1016/S0165-4608(03)00187-0
88
Letter to the editor / Cancer Genetics and Cytogenetics 147 (2003) 87–88
Table 2 Allele frequencies of the minisatellite repeat at chromosome band 5q21
Allele
No. of 32/33-bp Product repeats size (bp)
A1 A2 A3 A4
11 10 9 5
422 389 356 224
P.M. Cury E.M.G. Bertollo Faculdade de Medicina Av. Brig. Faria Lima, 5416 Sa˜o Jose´ do Rio Preto 15090, Brazil
Frequency Present study Bugert et al. (1998) 0.59 0.20 0.16 0.05
0.63 0.17 0.05 0.15
The screening of 102 European individuals by Bugert et al. [4] showed allele frequencies of 0.63, 0.17, 0.05, and 0.15 for alleles A1 (11 repeats), A2 (10 repeats), A3 (9 repeats), and A4 (5 repeats), respectively. Two alleles of different sizes were seen in 57 individuals, corresponding to a heterozygosity rate of 0.56. The present data, obtained from individuals living in Sa˜o Paulo, Brazil, showed deviation from Hardy-Weinberg equilibrium (X2 ⫽ 12.3), contrary to the results of Bugert et al. [4]. The allele frequencies in 64 patients are listed in Table 2. Thus, we suggest the following: 1) LOH of 5q21 is a frequent event in head and neck carcinomas; 2) loss of genes at 5q21 may be involved in the very early steps of head and neck tumorigenesis; 3) Hardy-Weinberg disequilibrium for the polymorphic minisatellite repeat at 5q21 may be attributed to the instability of the repeat; 4) the 257-bp fragment corresponds to a previously undescribed allele; and 5) larger studies are needed to determine whether the 33-bp minisatellite repeat may be considered as an informative marker for head and neck cancer. U.M. Mancini M.R.H. Este´cio Departamento de Biologia Instituto de Biociencias Letras e Ciencas Exatas Universidad Estadual Paulista R. Cristo´va˜o Colombo, 2265 Caixa Postal 136 Sa˜o Jose´ do Rio Preto 15054, Brazil J.F.F. Go´is E.E. Fukuyama P.J. Valentim Instituto do Caˆncer Arnaldo Vieira de Carvalho R. Dr. Cesario Motta Junior, 112 Sa˜o Paulo 01221, Brazil
E.H. Tajara Departamento de Biologia Instituto de Biociencias, Letras e Ciencas Exatas Universidad Estadual Paulista R. Cristo´va˜o Colombo, 2265 Caixa Postal 136 Sa˜o Jose´ do Rio Preto 15054, Brazil
References [1] Greenman J, Homer JJ, Stafford ND. Markers in cancer of the larynx and pharynx. Clin Otolaryngol 2000;25:9–18. [2] Brennan JA, Mao L, Hruban RH, Boyle JO, Eby YJ, Koch WM, Goodman SN, Sidransky D. Molecular assessment of histopathological staging in squamous-cell carcinoma of the head and neck. N Engl J Med 1995;16;332(7):429–35. [3] Thomson PJ. Field change and oral cancer: new evidence for widespread carcinogenesis? Int J Oral Maxillofac Surg 2002;31:262–6. [4] Bugert P, Kenck C, Kovacs G. A 33 pb. minisatellite repeat upstream of the “mutated in colon cancer” gene at chromosome 5q21. Electrophoresis 1998;19:1362–5. [5] Ashton-Rickardt PG, Whyllie AH, Bird CC, Dunlop MG, Steel CM, Morris RG, Piris J, Romanowski P, Wood R, White R, Nakamura Y. MCC, a candidate familial polyposis gene in 5q.21, shows frequent allele loss in colorectal and lung cancer. Oncogene 1991;6:1881–6. [6] Fujimori M, Tokino T, Hino O, Kitagawa T, Imamura T, Okamoto E, Mitsunobu M, Ishikawa T, Nakagama H, Harada H, Yagura M, Matsubara K, Nakamura Y. Allelotype study of primary hepatocellular carcinoma. Cancer Res 1991;51:89–93. [7] Boynton RF, Blount PL, Yin J, Brown VL, Huang Y, Tong Y, McDaniel T, Newkirk C, Resau JH, Raskind WH, Haggitt RC, Reid BJ, Meltzer SJ. Loss of heterozygosity involving the APC and MCC genetic loci occurs in the majority of human esophageal cancers. Proc Natl Acad Sci USA 1992;89:3385–8. [8] Hosoe S, Ueno K, Shigedo Y, Tachibana I, Osaki T, Kumagai T, Tanio Y, Kawase I, Nakamura Y, Kishimoto T. A frequent deletion of chromosome 5q21 in advanced small cell and non-small cell carcinoma of the lung. Cancer Res 1994;54:1787–90. [9] Mao EJ, Schwartz SM, Daling JR, Beckmann AM. Loss of heterozygosity at 5q21–22 (adenomatous polyposis coli gene region) in oral squamous cell carcinoma is common and correlated with advanced disease. J Oral Pathol Med 1998;27:297–302. [10] Braakhuis BJ, Tabor MP, Leemans CR, van der Waal I, Snow GB, Brakenhoff RH. Second primary tumors and field cancerization in oral and oropharyngeal cancer: molecular techniques provide new insights and definitions. Head Neck 2002;24:198–206.
Letter to the editor
The chromosome 5q21 band minisatellite and head and neck cancer Squamous cell carcinoma of the head and neck (HNSCC) is one of the most common cancers in Brazil. The most important prognostic factor for this carcinoma is related to complete surgical removal of the neoplasm [1]. When tumor is present at a margin of resection, the rate of local recurrence increases substantially and the survival rate decreases [2]. HNSCC patients often develop multiple premalignant lesions in their upper aerodigestive tract. This finding led to the field cancerization theory, which hypothesizes that the entire mucosa of the upper aerodigestive tract has an increased risk for the development of neoplastic lesions. The presence of multiple genetic abnormalities in histologically normal, tumor-adjacent mucosa from HNSCC patients supports this hypothesis [3]. Bugert et al. [4] describes a 33-base pair (bp) minisatellite repeat in the 5′-flanking region of the mutated in colon cancer (MCC) gene at chromosome band 5q21. The repetitive sequence was observed as unique in the human genome and was not expressed as part of a gene transcription unit. The authors [4] discuss the duplication of one allele in three renal tumors and the loss of one allele in three bladder tumors. Because loss of heterozygosity of the 5q21 region has been found in many types of cancer, such as those of the lung, liver, colorecum, esophagus, and oral area [5–9], the 33-bp minisatellite repeat may be analyzed for imbalance of these regions. To investigate loss of heterozygosity (LOH) on chromosome band 5q21 in HNSCC, we analyzed blood, tumors, and surgical margins from 64 patients by using polymerase chain reaction (PCR) and single-stranded conformation polymorphism (SSCP) techniques. The samples were obtained at the Sa˜o Jose´ do Rio Preto School of Medicine,
and at the Hospital Arnaldo Vieira de Carvalho, Sa˜o Paulo, Brazil. Sections of tumors used for the study were selected from areas with high proportions of malignant tissue. None of the patients had undergone treatment before surgery. Genomic DNA was extracted from all specimens by using proteinase K digestion. A nonradioactive PCR-SSCP analysis was used to detect the presence of polymorphisms in the minisatellite of chromosome band 5q21. Oligodeoxynucleotide primers and thermocycle PCR conditions were the same as described by Bugert et al. [4]. Amplified products were separated on polyacrylamide gels and visualized by silver staining according to standard protocols. Loss of heterozygosity was defined as a reduction in the signal of more than 80% in one of the alleles. Twenty-seven cases of primary HNSCC showed constitutive heterozygosity. There were four (28.5%) tumors with LOH (Table 1). In two cases, LOH were observed only in the tumors. One patient has been disease-free 2 years after surgery and another had a recurrence 1 year after surgery. The latter exhibited a band of 257 bp in blood, tumor, and marginal tissue, corresponding to a previously unreported fragment of six repeats. This fragment was also observed in blood samples from two sons and one daughter and might have originated by events such as slippage replication, saltatory replication, unequal crossing over, or gene conversion. In two cases, both tumor and surgical margins exhibited loss of the same allele: one patient died 1 month after surgery and the second one is alive and free of disease 3 years after surgery. Therefore, our data provide no evidence that LOH on chromosome band 5q21 may be involved in the progression of HNSCC. Our data do indicate that the tumor develops in a genetically altered field where loss of genes at region5q21 may be present [10].
Table 1 Clinicopathologic characteristics of HNSCC patients with LOH at 5q21 Consumption
Tumor
Patient LOH(⫹)
Age
Sex
Tobacco
Alcohol
Site
Stagea
Status
LOH site
A B C D
72 48 85 70
Male Male Male Male
⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫺ ⫺
Larynx Glottis Larynx Tongue
IV III II II
Dead Alive, well Alive, well Recurrence
T/M T T/M T
Abbreviations: M, surgical margin; T, tumor. a UICC TNM staging system. 0165-4608/03/$ – see front matter 쑖 2003 Elsevier Inc. All rights reserved. doi: 10.1016/S0165-4608(03)00187-0
88
Letter to the editor / Cancer Genetics and Cytogenetics 147 (2003) 87–88
Table 2 Allele frequencies of the minisatellite repeat at chromosome band 5q21
Allele
No. of 32/33-bp Product repeats size (bp)
A1 A2 A3 A4
11 10 9 5
422 389 356 224
P.M. Cury E.M.G. Bertollo Faculdade de Medicina Av. Brig. Faria Lima, 5416 Sa˜o Jose´ do Rio Preto 15090, Brazil
Frequency Present study Bugert et al. (1998) 0.59 0.20 0.16 0.05
0.63 0.17 0.05 0.15
The screening of 102 European individuals by Bugert et al. [4] showed allele frequencies of 0.63, 0.17, 0.05, and 0.15 for alleles A1 (11 repeats), A2 (10 repeats), A3 (9 repeats), and A4 (5 repeats), respectively. Two alleles of different sizes were seen in 57 individuals, corresponding to a heterozygosity rate of 0.56. The present data, obtained from individuals living in Sa˜o Paulo, Brazil, showed deviation from Hardy-Weinberg equilibrium (X2 ⫽ 12.3), contrary to the results of Bugert et al. [4]. The allele frequencies in 64 patients are listed in Table 2. Thus, we suggest the following: 1) LOH of 5q21 is a frequent event in head and neck carcinomas; 2) loss of genes at 5q21 may be involved in the very early steps of head and neck tumorigenesis; 3) Hardy-Weinberg disequilibrium for the polymorphic minisatellite repeat at 5q21 may be attributed to the instability of the repeat; 4) the 257-bp fragment corresponds to a previously undescribed allele; and 5) larger studies are needed to determine whether the 33-bp minisatellite repeat may be considered as an informative marker for head and neck cancer. U.M. Mancini M.R.H. Este´cio Departamento de Biologia Instituto de Biociencias Letras e Ciencas Exatas Universidad Estadual Paulista R. Cristo´va˜o Colombo, 2265 Caixa Postal 136 Sa˜o Jose´ do Rio Preto 15054, Brazil J.F.F. Go´is E.E. Fukuyama P.J. Valentim Instituto do Caˆncer Arnaldo Vieira de Carvalho R. Dr. Cesario Motta Junior, 112 Sa˜o Paulo 01221, Brazil
E.H. Tajara Departamento de Biologia Instituto de Biociencias, Letras e Ciencas Exatas Universidad Estadual Paulista R. Cristo´va˜o Colombo, 2265 Caixa Postal 136 Sa˜o Jose´ do Rio Preto 15054, Brazil
References [1] Greenman J, Homer JJ, Stafford ND. Markers in cancer of the larynx and pharynx. Clin Otolaryngol 2000;25:9–18. [2] Brennan JA, Mao L, Hruban RH, Boyle JO, Eby YJ, Koch WM, Goodman SN, Sidransky D. Molecular assessment of histopathological staging in squamous-cell carcinoma of the head and neck. N Engl J Med 1995;16;332(7):429–35. [3] Thomson PJ. Field change and oral cancer: new evidence for widespread carcinogenesis? Int J Oral Maxillofac Surg 2002;31:262–6. [4] Bugert P, Kenck C, Kovacs G. A 33 pb. minisatellite repeat upstream of the “mutated in colon cancer” gene at chromosome 5q21. Electrophoresis 1998;19:1362–5. [5] Ashton-Rickardt PG, Whyllie AH, Bird CC, Dunlop MG, Steel CM, Morris RG, Piris J, Romanowski P, Wood R, White R, Nakamura Y. MCC, a candidate familial polyposis gene in 5q.21, shows frequent allele loss in colorectal and lung cancer. Oncogene 1991;6:1881–6. [6] Fujimori M, Tokino T, Hino O, Kitagawa T, Imamura T, Okamoto E, Mitsunobu M, Ishikawa T, Nakagama H, Harada H, Yagura M, Matsubara K, Nakamura Y. Allelotype study of primary hepatocellular carcinoma. Cancer Res 1991;51:89–93. [7] Boynton RF, Blount PL, Yin J, Brown VL, Huang Y, Tong Y, McDaniel T, Newkirk C, Resau JH, Raskind WH, Haggitt RC, Reid BJ, Meltzer SJ. Loss of heterozygosity involving the APC and MCC genetic loci occurs in the majority of human esophageal cancers. Proc Natl Acad Sci USA 1992;89:3385–8. [8] Hosoe S, Ueno K, Shigedo Y, Tachibana I, Osaki T, Kumagai T, Tanio Y, Kawase I, Nakamura Y, Kishimoto T. A frequent deletion of chromosome 5q21 in advanced small cell and non-small cell carcinoma of the lung. Cancer Res 1994;54:1787–90. [9] Mao EJ, Schwartz SM, Daling JR, Beckmann AM. Loss of heterozygosity at 5q21–22 (adenomatous polyposis coli gene region) in oral squamous cell carcinoma is common and correlated with advanced disease. J Oral Pathol Med 1998;27:297–302. [10] Braakhuis BJ, Tabor MP, Leemans CR, van der Waal I, Snow GB, Brakenhoff RH. Second primary tumors and field cancerization in oral and oropharyngeal cancer: molecular techniques provide new insights and definitions. Head Neck 2002;24:198–206.