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Head and neck squamous cell carcinoma: mismatch repair immunohistochemistry and promoter hypermethylation of hMLH1 gene
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American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 528 – 536 www.elsevier.com/locate/amjoto
Head and neck squamous cell carcinoma: mismatch repair immunohistochemistry and promoter hypermethylation of hMLH1 gene Heba Mohamed Tawfik, MDa , Nehad M.R. Abd El-Maqsoud, MDa , Balegh H.A. Abdel Hak, MDb,⁎, Yasser M. El-Sherbiny, MDc a
Department of Pathology, Faculty of Medicine Minia University, Minia, Egypt b ENT Department, Faculty of Medicine, El Minia University, Minia, Egypt c Department of Clinical Pathology, Faculty of Medicine, El Minia University, Minia, Egypt Received 19 August 2010
Abstract
Squamous cell carcinomas of the head and neck are the sixth most frequently occurring cancers and the seventh leading cause of cancer-related deaths worldwide. Epigenetic alteration, using promoter hypermethylation of hMLH1 gene, is important for the development of head and neck squamous cell carcinoma (HNSCC). Aim of this Work: The aim of the present study is to analyze the relationship between protein expression and promoter hypermethylation of the hMLH1 gene in HNSCC and correlating inactivation of this gene with clinical parameters. Materials and Methods: Paired normal and tumor specimens from 49 patients with HNSCC were collected from Otolaryngology Department, Minia University Hospital, from 2006 to 2009. We analyzed hMLH1 protein expression and promoter hypermethylation by immunohistochemical and methylation-specific polymerase chain reaction (MSP). Results: Decreased hMLH1 protein expression and hMLH1 promoter hypermethylation were shown in 15 (30.6%) and 14 (28.6%) cases, respectively. Eleven cases showed dysplasia and or carcinoma in situ in the surface squamous epithelia, and all were positively stained for the hMLH1 protein. hMLH1 promoter hypermethylation was detected in 10 (20.4%) cases of normal-appearing squamous mucosa adjacent to invasive carcinoma. Thirteen (86.7%) of 15 cases that were negative for the hMLH1 protein showed promoter hypermethylation, whereas 33 (97%) of 34 cases positive for the protein were negative of promoter methylation. Promoter hypermethylation was detected in 1 (7.1%) case in which invasive tumor cells were moderately positive for the hMLH1 protein. No significant correlation was observed between hMLH1 protein expression or hMLH1 promoter hypermethylation and any of clinicopathologic parameters. Conclusions: hMLH1 gene may be detected early in head and neck squamous carcinogenesis. Promoter hypermethylation is an important mechanism for hMLH1 gene inactivation in HNSCC. © 2011 Elsevier Inc. All rights reserved.
1. Introduction In Egypt, head and neck cancer represents 17% of all malignant tumors. The median age of patients is 50 years, HMT and NMR are equally contributed in performing the immunohistochemical (IHC) and molecular parts of the research. ⁎ Corresponding author. ENT Department, Faculty of Medicine, El Minia University, Minia 61111, Egypt. Tel.: +20113791900, 0020145099994 (mobile). E-mail address: [email protected] (B.H.A.A. Hak). 0196-0709/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.amjoto.2010.11.005
with male predominance of 3:1 [1]. Squamous cell carcinomas of the head and neck (SCCHN), including those of the oral cavity, pharynx, and larynx, are the sixth most frequently occurring cancers and the seventh leading cause of cancer-related deaths worldwide [2,3], with tobacco habits, alcohol consumption, and human papillomavirus infection being the major risk factors for this type of cancer [4,5]. Epigenetic alterations together with accumulation of genetic aberrations have been established in SCCHN. Promoter CpG island hypermethylation may represent an
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attractive alternative mechanism for inactivation of the hMLH1 gene in head and neck squamous cell carcinoma (HNSCC). It was reported that methylation of CpG-rich islands is an important epigenetic mechanism by which multiple genes are inactivated [6,7]. Increasing evidence indicates that promoter CpG island hypermethylation is associated with transcriptional silencing of genes and is stably inherited through mitosis in cancer [8]. Thus, aberrant promoter hypermethylation is proved to be an important mechanism that can be used as a marker for SCCHN. Furthermore, because this epigenetic mechanism is potentially reversible, it can be targeted for therapeutic intervention [7,9]. It has been shown that loss of activity of some key genes may occur through epigenetic means [10]. Loss of gene function by transcriptional silencing of selected genes may play a crucial role in the development and progression of sporadic human tumors [11]. DNA repair genes are also inactivated by the process of promoter hypermethylation during carcinogenesis. The mismatch DNA repair gene human MutL homologue 1 (hMLH1) [12–17] is inactivated by promoter hypermethylation in numerous human tumors [16,18]. The epigenetic inactivation via promoter hypermethylation of hMLH1 gene 19 was mapped to a chromosome region 3p21.3-23, has been linked to increased microsatellite instability in sporadic colorectal cancers and hereditary nonpolyposis colorectal cancer [15]. Promoter hypermethylation of the hMLH1 gene has also been reported in certain SCCHN. The inactivation of the hMLH1 gene by this mechanism has been implicated in SCCHN progression but not tumor initiation [16]. Promoter hypermethylation of the hMLH1 gene has been demonstrated in multiple tumor types, including HNSCC [16,19,20], lung cancer [21], gastric [22], colorectal [23], esophageal carcinoma [24], and ovarian cancers [25]. The present study aims to analyze the relationship between protein expression and promoter hypermethylation of the hMLH1 gene in HNSCC and correlating inactivation of this gene with clinical parameters.
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Table 1 Clinicopathological features of the 49 examined head and neck cancer cases Clinicopathologic features Age (y) b50 ≥50 Sex Male Female Tobacco habit Yes No Tumor site Oral cavity Pharynx Larynx Tumor stage T1 T2 T3 T4 N stage N0 N1 N2 N3 Clinical stage I II III IV
No. of patients
%
15 34
30.6 69.4
45 4
91.8 8.2
35 14
71.4 28.6
19 4 26
38.8 8.1 53.1
8 12 9 20
16.3 24.5 18.4 40.8
17 7 16 9
34.7 14.3 32.7 18.4
8 8 9 24
16.3 16.3 18.4 49
T, tumor; N, lymph node. Clinical stage was determined by combining T, N, and M (distant metastasis) stages.
whereas 4 were female. The primary sites of the carcinomas were oral cavity (n = 19), pharynx (n = 4), and larynx (n = 26). Tumor sizes according to the TNM classification [26] were T1 (n = 8), T2 (n = 12), T3 (n = 9), and T4 (n = 20). Lymph node metastasis was present in 32 cases. Characteristics of the patients are presented in Table (1). 2.2. IHC staining
2. Materials and methods 2.1. Patients and tissue specimens Paired normal and tumor specimens from 49 patients with HNSCC were collected from 2006 to 2009 from freshly operated tumor and adjacent normal tissues from patients who underwent surgery before receiving any treatment from Otolaryngology Department, Minia University Hospital, Minia, Egypt. For all 49 cases, tissue sections were fixed in 10% formalin, processed, embedded in paraffin, sectioned at 5-μm thickness, and stained with hematoxylin and eosin for their diagnosis and to select the most representative sections for immunohistochemical (IHC) staining and DNA extraction. The mean (SD) age of the patients at surgery was 57.85 (8.5) years (range, 40–79 years), and 45 patients were male,
Formalin-fixed, paraffin-embedded tissue sections were cut at 5-μm for IHC staining by using a monoclonal antibody against hMLH1 protein (Clone ES05, Ready-to-Use; DAKO, Egypt). The standard avidin-biotin-peroxidase technique was used in this study. Antigen retrieval with microwave treatment was used in immunostaining. The normal squamous epithelia were used as internal positive controls. 2.3. Assessment of IHC staining The hMLH1 IHC staining results were assessed semiquantitatively as previously described [16]. The results are as follows: less than 10% positive cells being negative (−), 10% to 20% positive cells being mostly negative (+/−), 30% to 40% positive cells being weakly positive (+), 50% to 70% positive cells being moderately positive (++), 70% to 90%
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being strongly positive (+++), and more than 90% positive cells being very strongly positive (++++). 2.4. Tissue and DNA samples for molecular studies DNA samples were collected from 5 deparaffinized, 5μm-thick tissue sections from each tissue block. Blood DNA was used as a positive control, and water was served as a negative control. Sections were stained with hematoxylineosin to verify their diagnosis. DNA was isolated by phenol/ chloroform extraction after overnight incubation, with proteinase K at 37°C. DNA was extracted by the standard procedure of proteinase K digestion. 2.5. Bisulfite treatment DNA was extracted from paired tumor and nearby normal tissue of each patient. DNA from paraffin-embedded tissue was extracted as described previously. The genomic DNA previously isolated was precipitated with 250 mmol/L NaCl and isopropanol. Then the extracted DNA (0.25–1 μg) was treated with sodium bisulfite; because of the small amount of DNA isolated from the paraffin-embedded tissue samples, this step was performed according to a modified version of a previously described protocol [27]. MSP distinguishes unmethylated from hypermethylated alleles in a given gene based on sequence changes produced after bisulfite treatment of DNA, which converts unmethylated, but not methylated, cytosines to uracil, and subsequent PCR using primers specific to either methylated or unmethylated DNA [28,29]. 2.6. Promoter methylation assay for hMLH1 Amplification of promoter region of the hMLH1 gene is carried out in a Touchgene Gradient Thermal Cycler (Techne Inc, Princeton, NJ) in 50 µL PCR reaction mixture containing 2 µL of bisulfite-treated genomic DNA, dNTPs (each at 200 µmol/L), primers (50 pmol each per reaction), 2.5 mmol/ L MgCl2, and 1.25 units Hotstar Taq (Qiagen, Valencia, CA) in 1× PCR buffer. All reagents are supplied with the Qiagen Hotstar Taq Kit (Qiagen), except for the dNTP mix (Roche Molecular Biochemicals, Giza, Egypt). Primer sequences of hMLH1 for the unmethylated reaction are 5′-TTT TGA TGT AGA TGT TTT ATT AGG GTT GT-3′ (sense) and 5′-ACC ACC TCA TCA TAA CTA CCC ACA-3′ (antisense), and for the methylated reaction, they are 5′-ACG TAG ACG TTT TAT TAG GGT CGC-3′ (sense) and 5′-CCT CAT CGT AAC TAC CCG CG-3′ (antisense) as described previously [15]. The PCR conditions are similar, except for the annealing temperature for both methylated and unmethylated reactions: initial degeneration and hot start at 95°C for 15 minutes, then 40 cycles consisting of 30 seconds at 95°C, 30 seconds at 63°C (methylated reactions) or 60°C (unmethylated reactions), and 1 minute at 72°C, followed by a final 5-minute extension at 72°C. Positive and negative control DNA samples and control without DNA (dH2O) was included for each set of PCR reactions.
The bisulfite-modified human placental DNA (Sigma, Egyptial International Center for Import Cairo, Egypt) and CpGenome universal methylated human DNA (Intergen Co, New York, NY) served as negative and positive control, respectively. The PCR products were separated with 2.2% agarose gel electrophoresis and visualized with 0.1% ethidium bromide staining. 2.7. Statistical analysis Statistical analyses were performed using the SPSS version 17 for Windows (SPSS Inc, Chicago, IL) program package. The 2-sided χ2 test was used to compare categorical variables, if the sample size was large. Fischer exact test was used when the sample size was small; such test was used when comparing the associations of hMLH1 immunoreactivity with age and sex. P ≤ .05 was used as a significance criterion. 3. Results 3.1. IHC staining for the hMLH1 protein In all cases, normal squamous epithelium demonstrated very strong staining (++++) indicative of the presence of endogenous hMLH1 activity. The most actively proliferating cells (basal cells) in normal squamous epithelia were strongly positive for the hMLH1 protein in the nuclei, whereas the terminally differentiated superficial keratinocytes were weakly positive or negative for the protein. Eleven cases showed dysplasia of various degrees and or carcinoma in situ in the surface squamous epithelia. Results of hMLH1 protein expression was seen in less than 10% positive cells in 8 (16.3%) cases, between 10% and 20% positive cells being mostly negative (+/−) in 7 (14.3) cases, between 30% and 40% positive cells being weakly positive (+) in 4 (8.2%) cases, between 50% and 70% positive cells being moderately positive (++) in 4 (8.2%), between 70% and 90% being strongly positive (+++) in 12 (24.5%) cases, and more than 90% of tumor cells being very strongly positive (++++) in 14 (28.6%) cases (Fig. 1A–D). 3.2. Correlation between hMLH1 protein expression and clinicopathological parameters The correlation between hMLH1 protein and clinicopathological parameters is illustrated in Table (2). No significant association was found between hMLH1 protein expression and any of the clinicopathological parameters. 3.3. Promoter hypermethylation for hMLH1 We determined the expression of hMLH1 gene in normal tissues and in tumor tissues to confirm that observed differences in methylation patterns also reflected expression levels in the corresponding genes. Analysis for hMLH1 promoter hypermethylation was performed in all 49 cases of HNSCC. Aberrant hMLH1 promoter hypermethylation was seen in 14 (28.6%) cases,
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Fig. 1. (A) Immunohistochemical analysis of hMLH1 expression in cases of squamous cell hyperplasia (DAB chromogen, haematoxylin counterstain. ×400). (B) Immunohistochemical analysis of hMLH1 expression in cases of carcinoma in situ (DAB chromogen, haematoxylin counterstain. ×400). (C) Immunohistochemical analysis of hMLH1 expression in cases of invasive squamous cell carcinoma in which a majority of the invasive carcinoma cells are positive (Dab Chromogen, hematoxylin counterstain ×400). (D) Immunohistochemical analysis of hMLH1 expression in cases of invasive squamous cell carcinoma in which a majority of the invasive carcinoma cells are negative (DAB chromogen, haematoxylin counterstain. ×400).
and the remaining 35 cases (71.4%) showed no evidence of promoter hypermethylation. Positive and negative controls worked appropriately in each round of PCR reaction. Representative hMLH1 methylation-specific PCR is illustrated in Fig. 2. When analyses of normal-appearing squamous mucosa and adjacent invasive squamous cell carcinoma are simultaneously done, both invasive squamous cell carcinoma and corresponding normal-appearing squamous mucosa were positive for hMLH1 promoter hypermethylation in 10 (20.4%) cases. 3.4. Correlation between promoter hypermethylation status and clinicopathological parameters The correlation between hypermethylation pattern and clinicopathological parameters is illustrated in Table 3. No significant association was demonstrated when promoter
hypermethylation of hMLH1 genes was tested against history of tobacco, age, tumor site (oral cavity, pharynx, and larynx), T stage, M stage, or clinical stage of the tumor. 3.5. Correlation between hMLH1 protein expression and hMLH1promoter hypermethylation status hMLH1 gene promoter hypermethylation was highly significantly associated with decreased levels of protein expression (P b .001), indicating that the hMLH1 gene may be a prime target for epigenetic silencing in HNSCC. Thirteen (86.7%) of 15 cases that were negative for the hMLH1 protein showed promoter hypermethylation, whereas 33 (97%) of 34 cases positive for the protein were free of promoter methylation. Promoter hypermethylation was detected in 1 case (7.1%) in which invasive tumor cells were moderately positive for the hMLH1 protein (Table 4).
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Table 2 Correlation between hMLH1 protein expression and clinicopathological parameters Clinicopathological features
Age (y) b50 ≥50 Sex Male Female Tobacco habit Yes No Tumor site Oral cavity Pharynx Larynx Tumor stage T1 T2 T3 T4 N stage N0 N1 N2 N3 Clinical stage I II III IV
Negative (%)
hMLH1 protein expression
P
Mostly negative (%)
Weakly positive (%)
Moderately positive (%)
Strongly positive (%)
Very strongly positive (%)
4 (50) 4 (50)
3 (42.9) 4 (57.1)
0 (0) 4 (100)
2 (50) 2 (50)
2 (16.7) 10 (83.3)
4 (28.6) 10 (71.4)
.35⁎
7 (87.5) 1 (12.5)
7 (100) 0 (0)
4 (100) 0 (0)
4 (100) 0 (0)
10 (83.3) 2 (16.7)
13 (92.9) 1 (7.1)
.74⁎
6 (75) 2 (25)
3 (42.9) 4 (57.1)
2 (50) 2 (50)
3 (75) 1 (25)
11 (91.7 ) 1 (8.3)
10 (71.4) 4 (28.6)
.28⁎
3 (37.5) 1 (12.5) 4 (50)
4 (57.1) 0 (0) 3 (42.9)
2 (50) 0 (0) 2 (50)
1 (25) 0 (0) 3 (75)
5 (41.7) 2 (16.7) 5 (41.7)
4 (28.6) 1 (7.1) 9 (64.3)
.89
1 4 0 3
(12.5) (50) (0) (37.5)
2 (28.6) 2 (28.6) 1 (14.3) 2 (28.6)
1 (25) 0 (0) 1 (25) 2 (50)
0 (0) 3 (75) 0 (0) 1 (25)
2 (16.7) 0 (0) 2 (16.7) 8 (66.7)
2 (14.3) 3 (21.4) 5 (35.7) 4 (28.6)
.18
5 0 1 2
(62.5) (0) (12.5) (25)
3 (42.9) 2 (28.6) 1 (14.3) 1 (14.3)
1 (25) 1 (25) 2 (50) 0 (0)
2 (50) 1 (25) 1 (25) 0 (0)
2 (16.7) 0 (0) 7 (58.3) 3 (25)
4 (28.6) 3 (21.4) 4 (28.6) 3 (21.4)
.42
1 4 0 3
(12.5) (50) (0) (37.5)
2 (28.6) 1 (14.3) 2 (28.6) 2 (28.6)
1 (25) 0 (0) 1 (25) 2 (50)
0 (0) 2 (50) 1 (25) 1 (25)
2 (16.7) 0 (0) 0 (0) 10 (83.3)
2 (14.3) 1 (7.1) 5 (35.7) 6 (42.9)
.06
T, tumor; N, lymph node; Test of significance: χ2 test. P values less than .05 is considered significant. ⁎ Fisher exact test.
4. Discussion
Fig. 2. PCR based methylation analysis of hMLH1 gene. Aliquots (5µl) of PCR reaction products analyzed by 1% agarose electrophoresis. If No Methylation a PCR product was generated from the tumor while no PCR products were generated sized 115bp from normal tissue after methylation indicating an absence of hMLH1 gene methylation. 1st lane is ladder N) Normal tissue T) Tumour tissue.
Methylation is a very important epigenetic modification in humans, and changes in methylation patterns play an important role in tumorigenesis [30,31]. CpG islands are the region frequently targeted by hypermethylation events. These are GpC- and CpG-rich regions of approximately 1 kilobase that are usually associated with the promoter or 5′ end of genes. Abnormal methylation of these islands can efficiently repress transcription of the associated gene leading to mutations and deletions [31]. Promoter hypermethylation is a central mechanism for epigenetic inactivation of key genes in the development of SCCHN. DNA methylation is considered to be a reversible process; trial by intervention to revert the aberrant promoter methylation and thus prevent the inactivation of crucial tumorigenic genes is very promising in anticancer therapeutics [7,9]. Regarding hMLH1 protein expression, we found that hMLH1 was expressed in 69.4% of cases. In line with previous studies, that reported hMLH1 expression from 69% to 81.7% [16,19,20]; however, low incidence of hMLH1 protein expression 42% was reported [32]. We
H.M. Tawfik et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 528–536 Table 3 Correlation between hMLH1promoter hypermethylation status and clinicopathological parameters Clinicopathological features Age (y) b50 ≥50 Sex Male Female Tobacco habit Yes No Tumor site Oral cavity Pharynx Larynx Tumor stage T1 T2 T3 T4 N stage N0 N1 N2 N3 Clinical stage I II III IV
hMLH1 hypermethylation status Negative (%)
P
Positive (%)
8 (22.9) 27 (77.1)
7 (50) 7 (50)
.06⁎
32 (91.4) 3 (8.6)
13 (92.9) 1 (7.1)
.68⁎
26 (74.3) 9 (25.7)
9 (64.3) 5 (35.7)
.23⁎
12 (34.3) 3 (8.6) 20 (57.1)
7 (50) 1 (7.1) 6 (42.9)
.59
5 6 8 16
(14.3) (17.1) (22.9) (45.7)
3 (21.4) 6 (42.9) 1 (7.1) 4 (28.6)
.16
9 5 15 6
(25.7) (14.3) (42.9) (17.1)
8 (57.1) 2 (14.2) 1 (7.1) 3 (21.4)
.07
5 3 7 20
(14.3) (8.6) (20) (57.1)
3 (21.4) 5 (35.7) 2 (14.3) 4 (28.6)
.08
T, tumor; N, lymph node; Test of significance: χ2 test. P values less than .05 is considered significant. ⁎ Fisher exact test.
found no significant correlation between hMLH1 protein expression and any clinicopathologic parameters. Similar results were reported [20]. We also found that the most actively proliferating cells (basal cells) in normal squamous epithelia were strongly positive for the hMLH1 protein in the nuclei, whereas the
Table 4 Correlation between hMLH1 protein expression and hMLH1promoter hypermethylation status hMLH1 promoter hypermethylation status
hMLH1 protein
Negative Mostly negative Weakly positive Moderately positive Strongly positive Very strongly positive
Negative (%)
Positive (%)
1 (2.9) 1 (2.9) 4 (11.4) 3 (8.6) 12 (34.3) 14 (40)
7 (50) 6 (42.9) 0 (0) 1 (7.1) 0 (0) 0 (0)
P
b .001
Test of significance: χ2 test. P values less than .05 is considered significant.
533
terminally differentiated superficial keratinocytes were weakly positive or negative for the protein. The surface squamous epithelia showing dysplasia or carcinoma in situ were positively stained for the hMLH1 protein. Similar observation has been reported in other studies [16,19,20,33]. This is explained by the fact that the basal layers (basal keratinocytes) in the squamous mucosa consist of stem cell population that constantly divides to replace loss of cells from the mucosal surface. Therefore, basal keratin cytes normally contain high levels of endogenous hMLH1 protein [19]. In this study, 11 cases contained dysplastic and/or carcinoma in situ components, and all of them showed strong immunostaining for the hMLH1 protein. These findings indicate that the loss of hMLH1protein occurs in tumor progression rather than in tumor initiation in HNSCCs. Similar findings have been described in HNSCC tumors [16] and in sporadic gastric cancer [13]. Thus, it appears that during the process of tumor invasion, the loss of the hMLH1 protein or its function may be acquired but not at the preinvasive state in a variety of sporadic cancers and that this may be the result of accelerated mutation in other growth regulatory genes, such as tumor suppressor genes and/or proto-oncogenes [16]. In this study, a comprehensive analysis for hMLH1 promoter hypermethylation and protein expression levels was performed in 49 HNSCC cases; the results obtained were correlated with the clinicopathologic parameters. We found that hMLH1 promoter hypermethylation is significantly correlated with decreased hMLH1 protein expression (P b .001), supporting a possible relationship between promoter hypermethylation and hMLH1 gene inactivation in HNSCC. Similar results were reported [20]. Our data support the finding that promoter hypermethylation of hMLH1 gene is a frequent event in SCCHN. We found that promoter hypermethylation was detected in 28.6%. Consistent with previously published data, the rate of promoter hypermethylation range from 23% to 47% [20,34–36]. To our knowledge, no report on hMSH1 promoter hypermethylation in Egyptian patients with HNSCC was available before the current study. Similar to the current results, high frequency of promoter hypermethylation of hMLH1 in HNSCC tumors has been reported in Indian patients [36] and in the Turkish population [35]. High frequency of hMLH1 promoter hypermethylation also has been reported in the Polish population for laryngeal cancer [37] and in the Korean population for oral cancer [38]. In our study, we found that normal tissues adjacent to methylation-positive tumors also demonstrated promoter hypermethylation at a high frequency (20.4%). hMLH1 promoter hypermethylation was detected in 25% normalappearing squamous mucosa adjacent to invasive carcinoma [20,36]. This result indicated that hMLH1 promoter hypermethylation could occur early in the head and neck squamous carcinogenesis and, therefore, could be used as an epigenetic marker for early detection of head and neck
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cancer [20]. The study on promoter hypermethylation gives us an idea about silencing tumor suppressor genes, even in normal tissues. It has been concluded that methylation is an early event depending on the finding of histologically normal tissues adjacent to tumors and premalignant lesions that contain high levels of methylation of several genes [36,39,40]. We found that promoter hypermethylation of the hMLH1 gene was present in 13 (86.7%) of 15 cases in which the invasive carcinoma cells were negative or mostly negative for the protein by IHC staining. Our data support the role of promoter hypermethylation in causing the lack of hMLH1 protein expression in HNSCCs. In other studies, promoter hypermethylation of the hMLH1 gene was present in 12 (92%) of 13 cases and 7 of 8 cases (87.5%) in which the cells were mostly negative for the protein by IHC staining [16,19]. Although the possibility of mutation cannot be completely ruled out as the underlying cause for the lack of hMLH1 protein, particularly in 1 case that showed no evidence of promoter methylation [16], this possibility appeared to be small because somatic mutation in 1 of the Mismatch Repair Gene has only been detected in about 25% of human sporadic tumors that displayed increased Microsatelite instability [13]. These results, with those obtained from Liu et al [16,19], are consistent with the notion that promoter hypermethylation of the hMLH1 gene is primarily involved in the silencing of the gene in sporadic forms of human cancers [23,25,41]. Similar observation was noted in other tissues such as sporadic gastric [13], and endometrial carcinomas [42]. Paradoxically, we noticed that promoter hypermethylation was detected in 1 (7.1%) case in which invasive tumor cells were moderately positive for the hMLH1 protein. Similar observations were reported in HNSCC tumors where (23%) of invasive tumor cells were strongly positive for the hMLH1 protein showing promoter methylation [16] while in another study only 1 case 70% of the neoplastic cells were positive (++) for the hMLH1 protein [19]. In another study, it was found that promoter hypermethylation of the hMLH1 gene was detected in 5 (83.3%) of 6 cases with less than 50% hMLH1-positive tumor cells, in 9 (56.3%) of 16 cases with 50% to 69% hMLH1-positive tumor cells, in 10 (45.5%) of 22 cases with 70% to 89% hMLH1-positive tumors cells, and in 15 (19.7%) of 76 cases with greater than 90% hMLH1-positive tumor cells [20]. The explanation of this may be due to a small number of tumor cells that were negative for the hMLH1 protein, which may contain methylated CpG island in the hMLH1 promoter [19]. Alternatively, the methylation of the CpG sites being analyzed may be important but not sufficient for gene inactivation. Characterization of the CpG methylation status in a larger area of the hMLH1 promoter and its correlation with protein expression are needed to resolve these issues [16]. Our study of SCCHN patients showed no correlation between promoter hypermethylation of hMLH1 with tumor
site, nodal disease, or T stage. Promoter hypermethylation was also not associated with age, sex, or history of tobacco use [20,34–36], confirming that multiple genes are involved in the complex process of carcinogenesis. Furthermore, multiple mechanisms of genetic alteration may be responsible for initiation and development of SCCHN. The impact of inactivation via aberrant promoter hypermethylation of these genes cannot be ruled out based on these results; a study with greater numbers is needed [34]. We found no significant correlation between tobacco habit and promoter hypermethylation, despite of the fact that 9 cases (64.3%) were positive and 5 cases (35.7%) were negative. A positive significant correlation was reported [36]. In this study, we found that HNSCC tumors from stages I (21.4%) and II (35.7%) exhibited a high frequency of hypermethylation that remained constant in stages III and IV, and this observation suggests that hypermethylation is probably an early event, and this hypermethylated state is maintained during the tumor progression. Involvement of promoter hypermethylation in early stages of HNSCC development was supported by the observation that a premalignant condition also exhibited a high frequency of hypermethylation. It should be noted that hMLH1 proteins are implicated not only in postreplicative mismatch correction but also in cell killing by DNA-modifying agents. It has been reported that development of resistance to chemotherapeutic agents sometimes may be due to inactivation of hMLH1 by promoter hypermethylation [25,43]. This is again proved in other studies that found that the hMLH1 gene inactivation in association with promoter hypermethylation has been implicated in the development of resistance to chemotherapeutic agents, cisplatin, and/or carboplatin in human colon and ovarian cancers [25,44,45]. Thus, investigation of promoter hypermethylation and its relationship to defective hMLH1 gene function in HNSCC may provide invaluable information in relation to the development of tumor resistance to cisplatin/carboplatin, commonly used in the treatment of HNSCC and strategies designed for reversal of this process. It has been demonstrated both in vitro and in vivo that reexpression of the hMLH1 gene in ovarian cancer cell lines resistant to cisplatin by treatment with the demethylating agent, 5-azacytidine, results in sensitization of the resistant cells to cisplatin [25,46,47]. In summary, we have shown loss of hMLH1 protein expression in up to 30.6% of HNSCCs and that this loss of protein is largely due to the presence of promoter methylation in the hMLH1 gene. We have also demonstrated the presence of promoter methylation in 86.7% of HNSCCs that lost the hMLH1 protein expression. We thus propose that promoter hypermethylation may be an important mechanism for hMLH1 gene inactivation in a subset of HNSCCs. Studying the correlation of hMLH1 protein expression and/ or promoter hypermethylation with various clinical parameters, including response to chemotherapeutic agents in a large series of HNSCC patients, is recommended to
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determine the role of hMLH1 gene function in response of HNSCC to chemotherapeutic agents.
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American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 528 – 536 www.elsevier.com/locate/amjoto
Head and neck squamous cell carcinoma: mismatch repair immunohistochemistry and promoter hypermethylation of hMLH1 gene Heba Mohamed Tawfik, MDa , Nehad M.R. Abd El-Maqsoud, MDa , Balegh H.A. Abdel Hak, MDb,⁎, Yasser M. El-Sherbiny, MDc a
Department of Pathology, Faculty of Medicine Minia University, Minia, Egypt b ENT Department, Faculty of Medicine, El Minia University, Minia, Egypt c Department of Clinical Pathology, Faculty of Medicine, El Minia University, Minia, Egypt Received 19 August 2010
Abstract
Squamous cell carcinomas of the head and neck are the sixth most frequently occurring cancers and the seventh leading cause of cancer-related deaths worldwide. Epigenetic alteration, using promoter hypermethylation of hMLH1 gene, is important for the development of head and neck squamous cell carcinoma (HNSCC). Aim of this Work: The aim of the present study is to analyze the relationship between protein expression and promoter hypermethylation of the hMLH1 gene in HNSCC and correlating inactivation of this gene with clinical parameters. Materials and Methods: Paired normal and tumor specimens from 49 patients with HNSCC were collected from Otolaryngology Department, Minia University Hospital, from 2006 to 2009. We analyzed hMLH1 protein expression and promoter hypermethylation by immunohistochemical and methylation-specific polymerase chain reaction (MSP). Results: Decreased hMLH1 protein expression and hMLH1 promoter hypermethylation were shown in 15 (30.6%) and 14 (28.6%) cases, respectively. Eleven cases showed dysplasia and or carcinoma in situ in the surface squamous epithelia, and all were positively stained for the hMLH1 protein. hMLH1 promoter hypermethylation was detected in 10 (20.4%) cases of normal-appearing squamous mucosa adjacent to invasive carcinoma. Thirteen (86.7%) of 15 cases that were negative for the hMLH1 protein showed promoter hypermethylation, whereas 33 (97%) of 34 cases positive for the protein were negative of promoter methylation. Promoter hypermethylation was detected in 1 (7.1%) case in which invasive tumor cells were moderately positive for the hMLH1 protein. No significant correlation was observed between hMLH1 protein expression or hMLH1 promoter hypermethylation and any of clinicopathologic parameters. Conclusions: hMLH1 gene may be detected early in head and neck squamous carcinogenesis. Promoter hypermethylation is an important mechanism for hMLH1 gene inactivation in HNSCC. © 2011 Elsevier Inc. All rights reserved.
1. Introduction In Egypt, head and neck cancer represents 17% of all malignant tumors. The median age of patients is 50 years, HMT and NMR are equally contributed in performing the immunohistochemical (IHC) and molecular parts of the research. ⁎ Corresponding author. ENT Department, Faculty of Medicine, El Minia University, Minia 61111, Egypt. Tel.: +20113791900, 0020145099994 (mobile). E-mail address: [email protected] (B.H.A.A. Hak). 0196-0709/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.amjoto.2010.11.005
with male predominance of 3:1 [1]. Squamous cell carcinomas of the head and neck (SCCHN), including those of the oral cavity, pharynx, and larynx, are the sixth most frequently occurring cancers and the seventh leading cause of cancer-related deaths worldwide [2,3], with tobacco habits, alcohol consumption, and human papillomavirus infection being the major risk factors for this type of cancer [4,5]. Epigenetic alterations together with accumulation of genetic aberrations have been established in SCCHN. Promoter CpG island hypermethylation may represent an
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attractive alternative mechanism for inactivation of the hMLH1 gene in head and neck squamous cell carcinoma (HNSCC). It was reported that methylation of CpG-rich islands is an important epigenetic mechanism by which multiple genes are inactivated [6,7]. Increasing evidence indicates that promoter CpG island hypermethylation is associated with transcriptional silencing of genes and is stably inherited through mitosis in cancer [8]. Thus, aberrant promoter hypermethylation is proved to be an important mechanism that can be used as a marker for SCCHN. Furthermore, because this epigenetic mechanism is potentially reversible, it can be targeted for therapeutic intervention [7,9]. It has been shown that loss of activity of some key genes may occur through epigenetic means [10]. Loss of gene function by transcriptional silencing of selected genes may play a crucial role in the development and progression of sporadic human tumors [11]. DNA repair genes are also inactivated by the process of promoter hypermethylation during carcinogenesis. The mismatch DNA repair gene human MutL homologue 1 (hMLH1) [12–17] is inactivated by promoter hypermethylation in numerous human tumors [16,18]. The epigenetic inactivation via promoter hypermethylation of hMLH1 gene 19 was mapped to a chromosome region 3p21.3-23, has been linked to increased microsatellite instability in sporadic colorectal cancers and hereditary nonpolyposis colorectal cancer [15]. Promoter hypermethylation of the hMLH1 gene has also been reported in certain SCCHN. The inactivation of the hMLH1 gene by this mechanism has been implicated in SCCHN progression but not tumor initiation [16]. Promoter hypermethylation of the hMLH1 gene has been demonstrated in multiple tumor types, including HNSCC [16,19,20], lung cancer [21], gastric [22], colorectal [23], esophageal carcinoma [24], and ovarian cancers [25]. The present study aims to analyze the relationship between protein expression and promoter hypermethylation of the hMLH1 gene in HNSCC and correlating inactivation of this gene with clinical parameters.
529
Table 1 Clinicopathological features of the 49 examined head and neck cancer cases Clinicopathologic features Age (y) b50 ≥50 Sex Male Female Tobacco habit Yes No Tumor site Oral cavity Pharynx Larynx Tumor stage T1 T2 T3 T4 N stage N0 N1 N2 N3 Clinical stage I II III IV
No. of patients
%
15 34
30.6 69.4
45 4
91.8 8.2
35 14
71.4 28.6
19 4 26
38.8 8.1 53.1
8 12 9 20
16.3 24.5 18.4 40.8
17 7 16 9
34.7 14.3 32.7 18.4
8 8 9 24
16.3 16.3 18.4 49
T, tumor; N, lymph node. Clinical stage was determined by combining T, N, and M (distant metastasis) stages.
whereas 4 were female. The primary sites of the carcinomas were oral cavity (n = 19), pharynx (n = 4), and larynx (n = 26). Tumor sizes according to the TNM classification [26] were T1 (n = 8), T2 (n = 12), T3 (n = 9), and T4 (n = 20). Lymph node metastasis was present in 32 cases. Characteristics of the patients are presented in Table (1). 2.2. IHC staining
2. Materials and methods 2.1. Patients and tissue specimens Paired normal and tumor specimens from 49 patients with HNSCC were collected from 2006 to 2009 from freshly operated tumor and adjacent normal tissues from patients who underwent surgery before receiving any treatment from Otolaryngology Department, Minia University Hospital, Minia, Egypt. For all 49 cases, tissue sections were fixed in 10% formalin, processed, embedded in paraffin, sectioned at 5-μm thickness, and stained with hematoxylin and eosin for their diagnosis and to select the most representative sections for immunohistochemical (IHC) staining and DNA extraction. The mean (SD) age of the patients at surgery was 57.85 (8.5) years (range, 40–79 years), and 45 patients were male,
Formalin-fixed, paraffin-embedded tissue sections were cut at 5-μm for IHC staining by using a monoclonal antibody against hMLH1 protein (Clone ES05, Ready-to-Use; DAKO, Egypt). The standard avidin-biotin-peroxidase technique was used in this study. Antigen retrieval with microwave treatment was used in immunostaining. The normal squamous epithelia were used as internal positive controls. 2.3. Assessment of IHC staining The hMLH1 IHC staining results were assessed semiquantitatively as previously described [16]. The results are as follows: less than 10% positive cells being negative (−), 10% to 20% positive cells being mostly negative (+/−), 30% to 40% positive cells being weakly positive (+), 50% to 70% positive cells being moderately positive (++), 70% to 90%
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being strongly positive (+++), and more than 90% positive cells being very strongly positive (++++). 2.4. Tissue and DNA samples for molecular studies DNA samples were collected from 5 deparaffinized, 5μm-thick tissue sections from each tissue block. Blood DNA was used as a positive control, and water was served as a negative control. Sections were stained with hematoxylineosin to verify their diagnosis. DNA was isolated by phenol/ chloroform extraction after overnight incubation, with proteinase K at 37°C. DNA was extracted by the standard procedure of proteinase K digestion. 2.5. Bisulfite treatment DNA was extracted from paired tumor and nearby normal tissue of each patient. DNA from paraffin-embedded tissue was extracted as described previously. The genomic DNA previously isolated was precipitated with 250 mmol/L NaCl and isopropanol. Then the extracted DNA (0.25–1 μg) was treated with sodium bisulfite; because of the small amount of DNA isolated from the paraffin-embedded tissue samples, this step was performed according to a modified version of a previously described protocol [27]. MSP distinguishes unmethylated from hypermethylated alleles in a given gene based on sequence changes produced after bisulfite treatment of DNA, which converts unmethylated, but not methylated, cytosines to uracil, and subsequent PCR using primers specific to either methylated or unmethylated DNA [28,29]. 2.6. Promoter methylation assay for hMLH1 Amplification of promoter region of the hMLH1 gene is carried out in a Touchgene Gradient Thermal Cycler (Techne Inc, Princeton, NJ) in 50 µL PCR reaction mixture containing 2 µL of bisulfite-treated genomic DNA, dNTPs (each at 200 µmol/L), primers (50 pmol each per reaction), 2.5 mmol/ L MgCl2, and 1.25 units Hotstar Taq (Qiagen, Valencia, CA) in 1× PCR buffer. All reagents are supplied with the Qiagen Hotstar Taq Kit (Qiagen), except for the dNTP mix (Roche Molecular Biochemicals, Giza, Egypt). Primer sequences of hMLH1 for the unmethylated reaction are 5′-TTT TGA TGT AGA TGT TTT ATT AGG GTT GT-3′ (sense) and 5′-ACC ACC TCA TCA TAA CTA CCC ACA-3′ (antisense), and for the methylated reaction, they are 5′-ACG TAG ACG TTT TAT TAG GGT CGC-3′ (sense) and 5′-CCT CAT CGT AAC TAC CCG CG-3′ (antisense) as described previously [15]. The PCR conditions are similar, except for the annealing temperature for both methylated and unmethylated reactions: initial degeneration and hot start at 95°C for 15 minutes, then 40 cycles consisting of 30 seconds at 95°C, 30 seconds at 63°C (methylated reactions) or 60°C (unmethylated reactions), and 1 minute at 72°C, followed by a final 5-minute extension at 72°C. Positive and negative control DNA samples and control without DNA (dH2O) was included for each set of PCR reactions.
The bisulfite-modified human placental DNA (Sigma, Egyptial International Center for Import Cairo, Egypt) and CpGenome universal methylated human DNA (Intergen Co, New York, NY) served as negative and positive control, respectively. The PCR products were separated with 2.2% agarose gel electrophoresis and visualized with 0.1% ethidium bromide staining. 2.7. Statistical analysis Statistical analyses were performed using the SPSS version 17 for Windows (SPSS Inc, Chicago, IL) program package. The 2-sided χ2 test was used to compare categorical variables, if the sample size was large. Fischer exact test was used when the sample size was small; such test was used when comparing the associations of hMLH1 immunoreactivity with age and sex. P ≤ .05 was used as a significance criterion. 3. Results 3.1. IHC staining for the hMLH1 protein In all cases, normal squamous epithelium demonstrated very strong staining (++++) indicative of the presence of endogenous hMLH1 activity. The most actively proliferating cells (basal cells) in normal squamous epithelia were strongly positive for the hMLH1 protein in the nuclei, whereas the terminally differentiated superficial keratinocytes were weakly positive or negative for the protein. Eleven cases showed dysplasia of various degrees and or carcinoma in situ in the surface squamous epithelia. Results of hMLH1 protein expression was seen in less than 10% positive cells in 8 (16.3%) cases, between 10% and 20% positive cells being mostly negative (+/−) in 7 (14.3) cases, between 30% and 40% positive cells being weakly positive (+) in 4 (8.2%) cases, between 50% and 70% positive cells being moderately positive (++) in 4 (8.2%), between 70% and 90% being strongly positive (+++) in 12 (24.5%) cases, and more than 90% of tumor cells being very strongly positive (++++) in 14 (28.6%) cases (Fig. 1A–D). 3.2. Correlation between hMLH1 protein expression and clinicopathological parameters The correlation between hMLH1 protein and clinicopathological parameters is illustrated in Table (2). No significant association was found between hMLH1 protein expression and any of the clinicopathological parameters. 3.3. Promoter hypermethylation for hMLH1 We determined the expression of hMLH1 gene in normal tissues and in tumor tissues to confirm that observed differences in methylation patterns also reflected expression levels in the corresponding genes. Analysis for hMLH1 promoter hypermethylation was performed in all 49 cases of HNSCC. Aberrant hMLH1 promoter hypermethylation was seen in 14 (28.6%) cases,
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531
Fig. 1. (A) Immunohistochemical analysis of hMLH1 expression in cases of squamous cell hyperplasia (DAB chromogen, haematoxylin counterstain. ×400). (B) Immunohistochemical analysis of hMLH1 expression in cases of carcinoma in situ (DAB chromogen, haematoxylin counterstain. ×400). (C) Immunohistochemical analysis of hMLH1 expression in cases of invasive squamous cell carcinoma in which a majority of the invasive carcinoma cells are positive (Dab Chromogen, hematoxylin counterstain ×400). (D) Immunohistochemical analysis of hMLH1 expression in cases of invasive squamous cell carcinoma in which a majority of the invasive carcinoma cells are negative (DAB chromogen, haematoxylin counterstain. ×400).
and the remaining 35 cases (71.4%) showed no evidence of promoter hypermethylation. Positive and negative controls worked appropriately in each round of PCR reaction. Representative hMLH1 methylation-specific PCR is illustrated in Fig. 2. When analyses of normal-appearing squamous mucosa and adjacent invasive squamous cell carcinoma are simultaneously done, both invasive squamous cell carcinoma and corresponding normal-appearing squamous mucosa were positive for hMLH1 promoter hypermethylation in 10 (20.4%) cases. 3.4. Correlation between promoter hypermethylation status and clinicopathological parameters The correlation between hypermethylation pattern and clinicopathological parameters is illustrated in Table 3. No significant association was demonstrated when promoter
hypermethylation of hMLH1 genes was tested against history of tobacco, age, tumor site (oral cavity, pharynx, and larynx), T stage, M stage, or clinical stage of the tumor. 3.5. Correlation between hMLH1 protein expression and hMLH1promoter hypermethylation status hMLH1 gene promoter hypermethylation was highly significantly associated with decreased levels of protein expression (P b .001), indicating that the hMLH1 gene may be a prime target for epigenetic silencing in HNSCC. Thirteen (86.7%) of 15 cases that were negative for the hMLH1 protein showed promoter hypermethylation, whereas 33 (97%) of 34 cases positive for the protein were free of promoter methylation. Promoter hypermethylation was detected in 1 case (7.1%) in which invasive tumor cells were moderately positive for the hMLH1 protein (Table 4).
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Table 2 Correlation between hMLH1 protein expression and clinicopathological parameters Clinicopathological features
Age (y) b50 ≥50 Sex Male Female Tobacco habit Yes No Tumor site Oral cavity Pharynx Larynx Tumor stage T1 T2 T3 T4 N stage N0 N1 N2 N3 Clinical stage I II III IV
Negative (%)
hMLH1 protein expression
P
Mostly negative (%)
Weakly positive (%)
Moderately positive (%)
Strongly positive (%)
Very strongly positive (%)
4 (50) 4 (50)
3 (42.9) 4 (57.1)
0 (0) 4 (100)
2 (50) 2 (50)
2 (16.7) 10 (83.3)
4 (28.6) 10 (71.4)
.35⁎
7 (87.5) 1 (12.5)
7 (100) 0 (0)
4 (100) 0 (0)
4 (100) 0 (0)
10 (83.3) 2 (16.7)
13 (92.9) 1 (7.1)
.74⁎
6 (75) 2 (25)
3 (42.9) 4 (57.1)
2 (50) 2 (50)
3 (75) 1 (25)
11 (91.7 ) 1 (8.3)
10 (71.4) 4 (28.6)
.28⁎
3 (37.5) 1 (12.5) 4 (50)
4 (57.1) 0 (0) 3 (42.9)
2 (50) 0 (0) 2 (50)
1 (25) 0 (0) 3 (75)
5 (41.7) 2 (16.7) 5 (41.7)
4 (28.6) 1 (7.1) 9 (64.3)
.89
1 4 0 3
(12.5) (50) (0) (37.5)
2 (28.6) 2 (28.6) 1 (14.3) 2 (28.6)
1 (25) 0 (0) 1 (25) 2 (50)
0 (0) 3 (75) 0 (0) 1 (25)
2 (16.7) 0 (0) 2 (16.7) 8 (66.7)
2 (14.3) 3 (21.4) 5 (35.7) 4 (28.6)
.18
5 0 1 2
(62.5) (0) (12.5) (25)
3 (42.9) 2 (28.6) 1 (14.3) 1 (14.3)
1 (25) 1 (25) 2 (50) 0 (0)
2 (50) 1 (25) 1 (25) 0 (0)
2 (16.7) 0 (0) 7 (58.3) 3 (25)
4 (28.6) 3 (21.4) 4 (28.6) 3 (21.4)
.42
1 4 0 3
(12.5) (50) (0) (37.5)
2 (28.6) 1 (14.3) 2 (28.6) 2 (28.6)
1 (25) 0 (0) 1 (25) 2 (50)
0 (0) 2 (50) 1 (25) 1 (25)
2 (16.7) 0 (0) 0 (0) 10 (83.3)
2 (14.3) 1 (7.1) 5 (35.7) 6 (42.9)
.06
T, tumor; N, lymph node; Test of significance: χ2 test. P values less than .05 is considered significant. ⁎ Fisher exact test.
4. Discussion
Fig. 2. PCR based methylation analysis of hMLH1 gene. Aliquots (5µl) of PCR reaction products analyzed by 1% agarose electrophoresis. If No Methylation a PCR product was generated from the tumor while no PCR products were generated sized 115bp from normal tissue after methylation indicating an absence of hMLH1 gene methylation. 1st lane is ladder N) Normal tissue T) Tumour tissue.
Methylation is a very important epigenetic modification in humans, and changes in methylation patterns play an important role in tumorigenesis [30,31]. CpG islands are the region frequently targeted by hypermethylation events. These are GpC- and CpG-rich regions of approximately 1 kilobase that are usually associated with the promoter or 5′ end of genes. Abnormal methylation of these islands can efficiently repress transcription of the associated gene leading to mutations and deletions [31]. Promoter hypermethylation is a central mechanism for epigenetic inactivation of key genes in the development of SCCHN. DNA methylation is considered to be a reversible process; trial by intervention to revert the aberrant promoter methylation and thus prevent the inactivation of crucial tumorigenic genes is very promising in anticancer therapeutics [7,9]. Regarding hMLH1 protein expression, we found that hMLH1 was expressed in 69.4% of cases. In line with previous studies, that reported hMLH1 expression from 69% to 81.7% [16,19,20]; however, low incidence of hMLH1 protein expression 42% was reported [32]. We
H.M. Tawfik et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 528–536 Table 3 Correlation between hMLH1promoter hypermethylation status and clinicopathological parameters Clinicopathological features Age (y) b50 ≥50 Sex Male Female Tobacco habit Yes No Tumor site Oral cavity Pharynx Larynx Tumor stage T1 T2 T3 T4 N stage N0 N1 N2 N3 Clinical stage I II III IV
hMLH1 hypermethylation status Negative (%)
P
Positive (%)
8 (22.9) 27 (77.1)
7 (50) 7 (50)
.06⁎
32 (91.4) 3 (8.6)
13 (92.9) 1 (7.1)
.68⁎
26 (74.3) 9 (25.7)
9 (64.3) 5 (35.7)
.23⁎
12 (34.3) 3 (8.6) 20 (57.1)
7 (50) 1 (7.1) 6 (42.9)
.59
5 6 8 16
(14.3) (17.1) (22.9) (45.7)
3 (21.4) 6 (42.9) 1 (7.1) 4 (28.6)
.16
9 5 15 6
(25.7) (14.3) (42.9) (17.1)
8 (57.1) 2 (14.2) 1 (7.1) 3 (21.4)
.07
5 3 7 20
(14.3) (8.6) (20) (57.1)
3 (21.4) 5 (35.7) 2 (14.3) 4 (28.6)
.08
T, tumor; N, lymph node; Test of significance: χ2 test. P values less than .05 is considered significant. ⁎ Fisher exact test.
found no significant correlation between hMLH1 protein expression and any clinicopathologic parameters. Similar results were reported [20]. We also found that the most actively proliferating cells (basal cells) in normal squamous epithelia were strongly positive for the hMLH1 protein in the nuclei, whereas the
Table 4 Correlation between hMLH1 protein expression and hMLH1promoter hypermethylation status hMLH1 promoter hypermethylation status
hMLH1 protein
Negative Mostly negative Weakly positive Moderately positive Strongly positive Very strongly positive
Negative (%)
Positive (%)
1 (2.9) 1 (2.9) 4 (11.4) 3 (8.6) 12 (34.3) 14 (40)
7 (50) 6 (42.9) 0 (0) 1 (7.1) 0 (0) 0 (0)
P
b .001
Test of significance: χ2 test. P values less than .05 is considered significant.
533
terminally differentiated superficial keratinocytes were weakly positive or negative for the protein. The surface squamous epithelia showing dysplasia or carcinoma in situ were positively stained for the hMLH1 protein. Similar observation has been reported in other studies [16,19,20,33]. This is explained by the fact that the basal layers (basal keratinocytes) in the squamous mucosa consist of stem cell population that constantly divides to replace loss of cells from the mucosal surface. Therefore, basal keratin cytes normally contain high levels of endogenous hMLH1 protein [19]. In this study, 11 cases contained dysplastic and/or carcinoma in situ components, and all of them showed strong immunostaining for the hMLH1 protein. These findings indicate that the loss of hMLH1protein occurs in tumor progression rather than in tumor initiation in HNSCCs. Similar findings have been described in HNSCC tumors [16] and in sporadic gastric cancer [13]. Thus, it appears that during the process of tumor invasion, the loss of the hMLH1 protein or its function may be acquired but not at the preinvasive state in a variety of sporadic cancers and that this may be the result of accelerated mutation in other growth regulatory genes, such as tumor suppressor genes and/or proto-oncogenes [16]. In this study, a comprehensive analysis for hMLH1 promoter hypermethylation and protein expression levels was performed in 49 HNSCC cases; the results obtained were correlated with the clinicopathologic parameters. We found that hMLH1 promoter hypermethylation is significantly correlated with decreased hMLH1 protein expression (P b .001), supporting a possible relationship between promoter hypermethylation and hMLH1 gene inactivation in HNSCC. Similar results were reported [20]. Our data support the finding that promoter hypermethylation of hMLH1 gene is a frequent event in SCCHN. We found that promoter hypermethylation was detected in 28.6%. Consistent with previously published data, the rate of promoter hypermethylation range from 23% to 47% [20,34–36]. To our knowledge, no report on hMSH1 promoter hypermethylation in Egyptian patients with HNSCC was available before the current study. Similar to the current results, high frequency of promoter hypermethylation of hMLH1 in HNSCC tumors has been reported in Indian patients [36] and in the Turkish population [35]. High frequency of hMLH1 promoter hypermethylation also has been reported in the Polish population for laryngeal cancer [37] and in the Korean population for oral cancer [38]. In our study, we found that normal tissues adjacent to methylation-positive tumors also demonstrated promoter hypermethylation at a high frequency (20.4%). hMLH1 promoter hypermethylation was detected in 25% normalappearing squamous mucosa adjacent to invasive carcinoma [20,36]. This result indicated that hMLH1 promoter hypermethylation could occur early in the head and neck squamous carcinogenesis and, therefore, could be used as an epigenetic marker for early detection of head and neck
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cancer [20]. The study on promoter hypermethylation gives us an idea about silencing tumor suppressor genes, even in normal tissues. It has been concluded that methylation is an early event depending on the finding of histologically normal tissues adjacent to tumors and premalignant lesions that contain high levels of methylation of several genes [36,39,40]. We found that promoter hypermethylation of the hMLH1 gene was present in 13 (86.7%) of 15 cases in which the invasive carcinoma cells were negative or mostly negative for the protein by IHC staining. Our data support the role of promoter hypermethylation in causing the lack of hMLH1 protein expression in HNSCCs. In other studies, promoter hypermethylation of the hMLH1 gene was present in 12 (92%) of 13 cases and 7 of 8 cases (87.5%) in which the cells were mostly negative for the protein by IHC staining [16,19]. Although the possibility of mutation cannot be completely ruled out as the underlying cause for the lack of hMLH1 protein, particularly in 1 case that showed no evidence of promoter methylation [16], this possibility appeared to be small because somatic mutation in 1 of the Mismatch Repair Gene has only been detected in about 25% of human sporadic tumors that displayed increased Microsatelite instability [13]. These results, with those obtained from Liu et al [16,19], are consistent with the notion that promoter hypermethylation of the hMLH1 gene is primarily involved in the silencing of the gene in sporadic forms of human cancers [23,25,41]. Similar observation was noted in other tissues such as sporadic gastric [13], and endometrial carcinomas [42]. Paradoxically, we noticed that promoter hypermethylation was detected in 1 (7.1%) case in which invasive tumor cells were moderately positive for the hMLH1 protein. Similar observations were reported in HNSCC tumors where (23%) of invasive tumor cells were strongly positive for the hMLH1 protein showing promoter methylation [16] while in another study only 1 case 70% of the neoplastic cells were positive (++) for the hMLH1 protein [19]. In another study, it was found that promoter hypermethylation of the hMLH1 gene was detected in 5 (83.3%) of 6 cases with less than 50% hMLH1-positive tumor cells, in 9 (56.3%) of 16 cases with 50% to 69% hMLH1-positive tumor cells, in 10 (45.5%) of 22 cases with 70% to 89% hMLH1-positive tumors cells, and in 15 (19.7%) of 76 cases with greater than 90% hMLH1-positive tumor cells [20]. The explanation of this may be due to a small number of tumor cells that were negative for the hMLH1 protein, which may contain methylated CpG island in the hMLH1 promoter [19]. Alternatively, the methylation of the CpG sites being analyzed may be important but not sufficient for gene inactivation. Characterization of the CpG methylation status in a larger area of the hMLH1 promoter and its correlation with protein expression are needed to resolve these issues [16]. Our study of SCCHN patients showed no correlation between promoter hypermethylation of hMLH1 with tumor
site, nodal disease, or T stage. Promoter hypermethylation was also not associated with age, sex, or history of tobacco use [20,34–36], confirming that multiple genes are involved in the complex process of carcinogenesis. Furthermore, multiple mechanisms of genetic alteration may be responsible for initiation and development of SCCHN. The impact of inactivation via aberrant promoter hypermethylation of these genes cannot be ruled out based on these results; a study with greater numbers is needed [34]. We found no significant correlation between tobacco habit and promoter hypermethylation, despite of the fact that 9 cases (64.3%) were positive and 5 cases (35.7%) were negative. A positive significant correlation was reported [36]. In this study, we found that HNSCC tumors from stages I (21.4%) and II (35.7%) exhibited a high frequency of hypermethylation that remained constant in stages III and IV, and this observation suggests that hypermethylation is probably an early event, and this hypermethylated state is maintained during the tumor progression. Involvement of promoter hypermethylation in early stages of HNSCC development was supported by the observation that a premalignant condition also exhibited a high frequency of hypermethylation. It should be noted that hMLH1 proteins are implicated not only in postreplicative mismatch correction but also in cell killing by DNA-modifying agents. It has been reported that development of resistance to chemotherapeutic agents sometimes may be due to inactivation of hMLH1 by promoter hypermethylation [25,43]. This is again proved in other studies that found that the hMLH1 gene inactivation in association with promoter hypermethylation has been implicated in the development of resistance to chemotherapeutic agents, cisplatin, and/or carboplatin in human colon and ovarian cancers [25,44,45]. Thus, investigation of promoter hypermethylation and its relationship to defective hMLH1 gene function in HNSCC may provide invaluable information in relation to the development of tumor resistance to cisplatin/carboplatin, commonly used in the treatment of HNSCC and strategies designed for reversal of this process. It has been demonstrated both in vitro and in vivo that reexpression of the hMLH1 gene in ovarian cancer cell lines resistant to cisplatin by treatment with the demethylating agent, 5-azacytidine, results in sensitization of the resistant cells to cisplatin [25,46,47]. In summary, we have shown loss of hMLH1 protein expression in up to 30.6% of HNSCCs and that this loss of protein is largely due to the presence of promoter methylation in the hMLH1 gene. We have also demonstrated the presence of promoter methylation in 86.7% of HNSCCs that lost the hMLH1 protein expression. We thus propose that promoter hypermethylation may be an important mechanism for hMLH1 gene inactivation in a subset of HNSCCs. Studying the correlation of hMLH1 protein expression and/ or promoter hypermethylation with various clinical parameters, including response to chemotherapeutic agents in a large series of HNSCC patients, is recommended to
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determine the role of hMLH1 gene function in response of HNSCC to chemotherapeutic agents.
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