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- μ m 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, 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 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

Formalin-fixed, paraffin-embedded tissue sections were cut at 5- μ m 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 . 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% 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, 5- μ m-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 hematoxylin-eosin 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 μ g) 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 . 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 .

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 MgCl ₂ , 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 .

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 (dH ₂ O) 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 χ ² 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.

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- μ m 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, 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 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

Formalin-fixed, paraffin-embedded tissue sections were cut at 5- μ m 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 . 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% 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, 5- μ m-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 hematoxylin-eosin 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 μ g) 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 . 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 .

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 MgCl ₂ , 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 .

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 (dH ₂ O) 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 χ ² 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.