Microsatellite instability associated with primary head and neck cancers and secondary esophageal cancers

doi:10.1093/jjco/28.12.733

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Japanese Journal of Clinical Oncology Pages 733-739

Microsatellite Instability Associated with Primary Head and Neck Cancers and Secondary Esophageal Cancers
Introduction
Materials and Methods
   Cases
   DNA Extraction
   Microsatellite Assay
   Immunohistochemistry
   Analysis of Tobacco and Alcohol Involvement
Results
   Microsatellite Assay
   Immunohistochemistry
   Analysis of Tobacco and Alcohol Involvement
Discussion
Acknowledgments
References

Microsatellite Instability Associated with Primary Head and Neck Cancers and Secondary Esophageal Cancers

Hiroshi Kiriu^1,2, Hiroshi Yokozaki¹, Wataru Yasui¹, Katsuhide Ito² and Eiichi Tahara¹

¹First Department of Pathology and ²Department of Radiology, Hiroshima University School of Medicine, Hiroshima, Japan

Background: It is common that patients with head and neck cancers have secondary malignant neoplasm of esophageal cancer.
Methods: To know the genetic background of the development of these secondary cancers, we performed microsatellite assay at six loci and immunohistochemical analysis on head and neck cancers of eight patients with esophageal cancer and on those of 19 patients without esophageal cancer.
Results: Replication error (RER) at more than two loci was observed in two (25%) of eight double cancer patients, whereas it was not observed in the patients without the secondary cancer. Immunohistochemically, overexpression of cyclin D1 was detected in two (25%) of eight double cancer cases and in two (11%) of 19 non-double cancer cases, respectively, the incidence showing a higher tendency in the former.
Conclusions: The results suggest that microsatellite instability may be implicated in the development of head and neck double cancers and that RER (+) phenotype may serve as a biomarker to predict the development of secondary esophageal cancer in patients with head and neck cancer.

Key words: second malignant neoplasms - head and neck cancer - esophageal cancer - microsatellite instability - immunohistochemistry

Introduction

Second primary malignancies such as oral and esophageal cancer frequently develop in patients with head and neck cancers (1). We have also found that patients with head and neck cancers are at high risk of developing synchronous esophageal cancer and suggested that endoscopic screening is essential for these patients (2).

Recent studies on molecular biology have revealed that genetic instability is one of the most important predispositions for human multistep carcinogenesis (3,4). Replication errors (RERs) reflect genetically unstable status that may underlie carcinogenesis. RERs are frequently observed in human malignancies such as colorectal and gastric cancers (5-10) and there is a high frequency of RERs in patients with multiple primary cancer (11). Therefore, genetic instability is thought to increase the normal mutation rate and to cause multiple mutations in oncogenes and tumor suppresser genes (3).

On the other hand, head and neck cancer and esophageal cancer contain similar genetic abnormalities. For example, frequent loss of heterozygosity (LOH) has been detected on chromosomes 3p, 9p and 17p in head and neck cancer and esophageal cancer (12,13). In addition, gene amplification of the c-myc, hst-1, int-2, cyclin D and EGF receptor (EGFR) has been reported in both cancers (14-21).

In this study, we carried out microsatellite analysis with six loci for the detection of RERs and LOHs on head and neck cancer associated or not with esophageal cancer. We also conducted immunohistochemical analysis for the expression of EGFR, cyclin D and p27.

Materials and Methods

Cases

Between 1992 and 1996, eight patients with esophageal cancer who had head and neck cancer were found by routine digestive endoscopy with the Lugol dye method at the Department of Radiology, Hiroshima University School of Medicine. Six esophageal cancers were associated with head and neck cancer (Cases 1-6) synchronously. According to the time interval between two malignancies, synchronous cancers were defined as those diagnosed at the same time as or within a 6-month period of the identification of the primary lesion; metachronous cancers were defined as those occurring more than 6 months after such identification (22). A further patient was diagnosed as having esophageal cancer after 3 years of radiation therapy for tongue cancer (Case 7) and the final patient was diagnosed as having esophageal cancer after 5 years of radiation therapy for oropharyngeal cancer (Case 8). In these eight patients, esophageal cancer was defined as secondary cancer because it was discovered after the decision of first primary head and neck cancer. These eight patients who had secondary esophageal cancer synchronously or metachronously were designated as the double cancer group.

Nineteen patients with head and neck cancer without esophageal cancer (Cases 101-119) were analyzed as the non-double cancer group who were confirmed by routine digestive endoscopy with the Lugol dye method before or during the treatment and follow-up period. Normal gastric mucosa from each case obtained at the time of endoscopic examinations were used as normal control tissues.

All head and neck cancers and esophageal cancers in the double cancer group were squamous cell carcinoma. The tumor stages of head and neck cancer in each group were follows: double cancer group, (six cases at stage 2 and two cases at stage 4) and the non-double cancer group (seven cases at stage 1, three cases at stage 2, four cases at stage 3 and five cases at stage 4) (23).

DNA Extraction

DNA from formalin- and paraffin-embedded samples were extracted according to the method of Shibata et al. (24) with minor modifications. Tissues scraped from the paraffin sections were incubated at 55°C for 3 h in 50 µl of DNA extraction solution containing 400 µg of proteinase K. After the extraction, the solution was boiled for 5 min to inactivate proteinase K and used for polymerase chain reaction (PCR).

Microsatellite Assay

We used oligonucleotide primer sets for the six microsatellite loci containing adenine mononucleotide repeats or cytosine-adenine dinucleotide repeats to observe RERs and LOHs; BAT25, BAT40, BATRII (adenine mononucleotide repeat), D3S1300, D9S171, TP53 (cytosine-adenine dinucleotide repeats). D3S1300 is a microsatellite marker located in the the FHIT gene (25-28). D9S171 is a microsatellite marker located near the p16 gene (29-31). TP53 is a microsatellite marker located in the p53 gene (13). PCR was performed according to Horii et al. (11) with some modifications. In brief, 15 µl volumes of reaction mixture, containing about 10-20 ng of DNA, 6.7 mM Tris-HCl (pH 8.8), 6.7 mM EDTA, 6.7 mM MgCl₂, 0.33 mM labelled primer with [[gamma]-³²P]ATP and 0.175 mM unlabelled primer, 1.5 mM dNTPs and 0.75 units of recombinant Taq DNA polymerase, was amplified for 40 cycles with the following regime: denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 30 s. PCR products were electrophoresed in 6% polyacrylamide-8 M urea-32% formamide gels and autoradiographed for 3-7 days on Fuji RX film.

Immunohistochemistry

Antibodies were used as follows: anti-p27 antibody (K25020, Transduction Laboratories, Lexington, KY), anti-EGFR antibody (EGFR133, Novocastra, Newcastle upon Tyne, UK) and anti-cyclin D1 antibody (Bcl-1, Medical Biological Laboratories, Nagoya, Japan). A modification of the immunoglobulin enzyme bridge technique (ABC method) was employed as described previously (32). Deparaffinized tissue sections were immersed in methanol containing 0.03% H₂O₂ for 30 min to block the endogenous peroxide activity. Microwave pretreatment in citrate buffer was used to retrieve the antigenicity. The sections were treated consecutively at room temperature with anti-p27 antibody (diluted 1:200), anti-cyclin D1 (diluted 1:100) or anti-EGFR (diluted 1:20) for 3 h, biotinylated anti-mouse (diluted 1:100; Vector Laboratories, Burlingame, CA) for 30 min and avidin DH-biotinylated horseradish peroxidase complex (Vectastain ABC kit, Vector) for 30 min. Peroxidase staining was performed for 10-15 min using a solution of 3,3[prime]-diaminobenzidene tetrahydrochloride in 50 mM Tris-HCl (pH 7.5) containing 0.001% H₂O₂. The sections were weakly counterstained with 0.1% hematoxylin or methylgreen. Immunoreactivity was graded as (-) to (+++) according to the number of cells stained and the intensity of the reaction in individual cells as follows: (-), almost no positive tumor cells; (+), 5-25% of tumor cells showed weak to moderate activity; (++), 25-50% of tumor cells showed intense immunoreactivity; (+++), >50% of tumor cells showed intense immunoreactivity.

Case	Age	Gender	Tumor	TNM	p27	EGFR	Cyclin D1
1	42	Male	Hypopharynx	T2N0M0	(+)	(+)	(+)
2	62	Male	Hypopharynx	T2N0M0	(++)	(-)	(++)
3	49	Male	Hypopharynx	T4N2M0	(-)	(++)	(-)
4	67	Male	Oropharynx	T2N0M0	(+)	(++)	(+)
5	77	Male	Oropharynx	T4N1M0	(+)	(+++)	(+)
6	69	Male	Tongue	T2N0M0	(+)	(+)	(+)
7	65	Male	Tongue	T2N0M0	(+)	(+)	(+)
8	68	Male	Oropharynx	T2N0M0	(++)	(+)	(++)
Incidence of overexpression (grade ++ and +++)						3/8 (38%)	2/8 (25%)
Incidence of reduced expression (grade - and +)					6/8 (75%)

Case	Age	Gender	Tumor	TNM	p27	EGFR	Cyclin D1
101	56	Male	Tongue	T1N0M0	(+)	(+)	(++)
102	68	Male	Tongue	T1N0M0	(+)	(+)	(+)
103	59	Male	Tongue	T1N0M0	(-)	(-)	(-)
104	71	Female	Tongue	T1N0M0	(-)	(++)	(+)
105	48	Female	Tongue	T1N0M0	(+)	(-)	(+)
106	53	Female	Tongue	T2N0M0	(++)	(-)	(-)
107	52	Male	Tongue	T2N0M0	(++)	(++)	(++)
108	67	Female	Tongue	T3N0M0	(-)	(+++)	(+)
109	68	Male	Tongue	T3N2M0	(-)	(+)	(+)
110	71	Male	Oropharynx	T1N0M0	(-)	(-)	(+)
111	79	Male	Oropharynx	T3N0M0	(++)	(-)	(-)
112	75	Male	Oropharynx	T2N2M0	(++)	(-)	(+)
113	67	Female	Upper Gum	T2N1M0	(-)	(++)	(+)
114	70	Male	Upper Gum	T4N2M0	(++)	(++)	(-)
115	67	Female	Oral Lip	T1N0M0	(+)	(+++)	(-)
116	53	Male	Oral Floor	T2N0M0	(+++)	(+++)	(+)
117	68	Male	Buccal Mucosa	T4N0M0	(-)	(-)	(-)
118	72	Male	Lower Gum	T4N1M0	(+)	(-)	(+)
119	68	Male	Hypopharynx	T3N1M0	(++)	(-)	(+)
Incidence of overexpression (grade ++ and +++)						7/19 (37%)	2/19 (11%)
Incidence of reduced expression (grade - and +)					12/19 (63%)

				J. S. Welsh, S. A. Thurman, and S. P. Howard Thymoma and Multiple Malignancies: A Case of Five Synchronous Neoplasms and Literature Review Clin. Med. Res., July 1, 2003; 1(3): 227 - 232. [Abstract] [Full Text] [PDF]