Abstract
Objective
Previous studies have shown Snail expression integral to the epithelial-mesenchymal transition during tumor progression. However, its behavior in clinical head and neck squamous cell carcinomas (HNSCCs) is yet undefined. We therefore sought to (1) investigate clinical and histopathologic characteristics of Snail-positive HNSCC and (2) understand the link between Snail and other commonly used HNSCC tumor markers.
Study Design
A retrospective case-control study was conducted.
Setting
This study was conducted in a large-scale academic center.
Study Subjects
Of 51 consecutive HNSCC, 42 surgical resections were included.
Methods
Two separate pathologists performed standard histopathologic reviews along with immunohistochemistries (Snail, E-cadherin, p16, epidermal growth factor receptor [EGFR]) and in situ hybridization (human papilloma virus [HPV]). Medical review for all cases was performed.
Results
Twenty-two (52%) of 42 cases stained 4+ Snail (>75% staining). The remaining 20 cases were considered negative. Snail was strongly inversely related to E-cadherin expression ( ρ = −0.69, P < .001), but statistically independent from HPV, p16, or EGFR expression. Snail(+) tumors were equally represented from each anatomic subsite. Snail(+) tumors were strongly associated with poor differentiation ( P < .001) and basaloid classification ( P = .004). Snail(+) tumors were also strongly associated with lymphovascular invasion ( P = .02), but not perineural invasion. Ultimately, 11 (50%) of 22 of Snail(+) tumors demonstrated positive nodal metastasis and 11 (79%) of 14 node-positive cases were Snail(+) ( P = .02).
Conclusion
This pilot study provides promising evidence of Snail’ role as a molecular prognostic marker for HNSCC. Snail positivity is significantly predictive of poorly differentiated, lymphovascular invasive, as well as regionally metastatic tumors. Because Snail positivity appears independent of HPV, p16, and EGFR expression, Snail may prove to improve upon these markers’ predictive limitations.
1
Introduction
Patients with head and neck squamous cell carcinoma (HNSCC) are at considerable risk of mortality, with more than 300 000 deaths attributable to the disease worldwide each year . Ultimately, most HNSCC-related deaths are due to locoregional failures. In fact, cervical lymph node metastases reduce survival by 50%. Identification of molecular markers that can pinpoint patients with increased risk of regional spread will be of the utmost importance for effective management of HNSCC.
Inflammatory mediators are dysregulated in smokers and patients with tobacco-related malignancies such as HNSCC . A chronic increase in inflammatory mediators can lead to increased HNSCC promotion, invasion, and metastasis . Inflammatory cytokines and mediators found in the tumor microenvironment include prostaglandin E 2 (PGE 2 ) and interleukin-1 (IL-1). Interleukin-1 has been shown to induce activation of signal transduction pathways that regulate several early transcription factors involved in proinflammatory cytokine genes. Its subtype, IL-1 β , is not only integral for inflammatory signaling but has also been implicated in the progression of HNSCC. Increased secretion of IL-1 β has been shown within resistant or progressing oral tumors . Interleukin-1 β is one of the several cytokines known to potently up-regulate cyclooxygenase 2 (COX-2) expression in a variety of cells . Tumor COX-2 plays an important role in regulating diverse cellular functions within cancer cells . Specifically within HNSCC, COX-2 has been closely correlated with increased Snail expression and subsequent E-cadherin suppression . Snail transcription factor appears to be a key regulator due to its ability to bind to the E-cadherin promoter and block its subsequent expression . Fig. 1 presents a streamlined diagram of this pathway.
Loss of E-cadherin is frequently observed at sites of epithelial-mesenchymal transition during cancer development and progression and is closely correlated with poor prognosis . Although several E-cadherin transcriptional repressors have been characterized (ZEB1, Snail, E12/E47, Slug, Twist, and SIP-1) in HNSCC, local recurrence following treatment has been correlated specifically with Snail up-regulation .
Based on our previous in vitro and in vivo delineation of this pathway , the aim of this pilot research was to identify Snail as a clinical prognostic histopathologic marker within HNSCC. Currently, because Snail has yet to be characterized clinically in HNSCC, its predictive value along with its relationships to other histopathologic findings is completely unknown. In addition, although Snail’s direct cellular pathway has been described, we aim to define its connection with other unrelated HNSCC tumor markers. Based on initial study , we hypothesize that Snail will in fact show significant association with aggressive HNSCC features.
2
Methods
This study was approved by the University of California, Los Angeles Office of Protection of Research Subjects (institutional review board).
2.1
Design
Because Snail has not been studied clinically in HNSCC, an exploratory study was designed from which future power calculations could be designed. We set out to include approximately 50 patients from which adequate pilot data could be gathered. Therefore, within a 12-month period, consecutive squamous cell carcinoma cases of a senior head and neck pathologist (C.L.) were reviewed. Tumors involving the oral cavity, oropharynx, or laryngeal (including supraglottis, glottal, and postcricoid) were included. This yielded 51 individual specimens. Five cases had very little tissue remaining to perform further staining, 2 cases had no cellblock, and 2 cases had inadequate staining (n = 42).
2.2
Immunohistochemistry
Tissue sections (4 μ m thick) were cut, deparaffinized in xylene, rehydrated in alcohols, and washed twice with water. Samples were then incubated in 0.01 mol/L citrate buffer (pH 6.0) for 25 minutes in a steamer to unmask antigens. Following cooling and rinsing with dH 2 O, samples were treated for 15 minutes with 3% H 2 O 2 diluted in methanol. Tissue sections were washed in dH 2 O and then in phosphate-buffered saline (PBS), then blocked with 10% normal horse serum for 30 minutes. Each tumor marker was stained on individual slides. For E-cadherin staining, the sections were incubated overnight at 4°C with 250 μ g/mL mouse antihuman E-cadherin diluted in normal horse serum (BD Transduction Biosciences, San Jose, CA). After extensive rinsing with PBS, samples were incubated for 40 minutes with 7.5 μ g/mL horse antimouse IgG-biotin (Vector Laboratories, Burlingame, CA) and rinsed with PBS. Samples were then incubated for 30 minutes at room temperature with the Vectastain ABC- kit (Vector Laboratories), followed by PBS washing, and then incubated with an alkaline phosphatase substrate kit (Vector Laboratories). Color development was followed under the microscope for 20 minutes. The color reaction was stopped by rinsing with dH 2 O. Samples were counterstained with hematoxylin. Lung and breast tumor specimens were used as a positive control for all immunohistochemistry staining. Negative controls included incubation with nonimmune pooled rabbit or goat IgG (rabbit IgG [Vector Laboratories] and goat IgG [Zymed; Invitrogen, Carlsbad, CA]) at the same concentration as the primary antibody. Goat antihuman Snail polyclonal IgG (1:50 dilution; Abcam, Cambridge, MA) was used for Snail immunohistochemistry.
p16 (prediluted mouse monoclonal; mtm laboratories, Westborough, MA) antigen retrieval involved heating at 95°C in 0.001 mol EDTA, pH 8.0, for 25 minutes in vegetable steamer, followed by a 15-minute cool down and rinse in 0.05 mol Tris-buffered saline with Tween 20. For EFGR (1:50 dilution of mouse monoclonal H11; Biocare Medical, Concord, CA), antigen retrieval involved enzymatic digestion with Proteinase K (DAKO, Carpenteria, CA) for 7 minutes followed by washing in Tris-buffered saline with Tween 20. Following 5-minute blocking with Ultra V Block (Lab Vision/ThermoScientific, Fremont, CA), primary antibodies were incubated for 30 minutes and coupled with Ultravision Value Polymer Detection System from LabVision/ThermoScientific with diaminobenzidine as chromogen. Slides were counterstained with hematoxylin (Harris). Immunostaining was performed on DAKO Autostainer.
All slides were reviewed by 2 of the investigators (M.F. and C.L.). Each slide was graded for percent cells positive for each stain and was subsequently scaled from (1+ to 4+). We examined the relationship between E-cadherin and Snail using the ordinal immunohistochemistry results (0+, 1+, etc) and found that they were significantly negatively correlated ( ρ = −0.69, P < .001). Subsequently, staining was dichotomized into positive (3+, 4+) and negative (1+, 2+) groups. The exception was Snail staining, which was dichotomized into positive (4+) and negative (1+, 2+, 3+) groups to increase test specificity.
2.3
In situ hybridization
Four-micrometer sections were baked for 15 minutes at 60°C and then labeled and programmed on Ventana XT autostainer to stain for human papilloma virus (HPV) High Risk (genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 66), DNA control probes using ISH IVIEW Blue Plus detection protocol. HPV staining was documented as either positive or negative.
2.4
Clinical Review
Only following the completion of histopathology, the 42 clinical charts were reviewed systematically. Pathologic TNM American joint committee on cancer (AJCC) staging was used for all primary tumor sites. Nodal staging was documented as “best of” from pathologic, radiologic, and then clinical.
2.5
Statistics
Descriptive statistics were derived for all analyzed variables. A P value less than .05 was required for significance. Cross-tabulation analyses were performed using Pearson χ 2 (for tables with values >5) and Fisher Exact (for tables with values <5) tests. Tests of mean were performed with unpaired t tests (SYSTAT 11, Chicago, IL).
2
Methods
This study was approved by the University of California, Los Angeles Office of Protection of Research Subjects (institutional review board).
2.1
Design
Because Snail has not been studied clinically in HNSCC, an exploratory study was designed from which future power calculations could be designed. We set out to include approximately 50 patients from which adequate pilot data could be gathered. Therefore, within a 12-month period, consecutive squamous cell carcinoma cases of a senior head and neck pathologist (C.L.) were reviewed. Tumors involving the oral cavity, oropharynx, or laryngeal (including supraglottis, glottal, and postcricoid) were included. This yielded 51 individual specimens. Five cases had very little tissue remaining to perform further staining, 2 cases had no cellblock, and 2 cases had inadequate staining (n = 42).
2.2
Immunohistochemistry
Tissue sections (4 μ m thick) were cut, deparaffinized in xylene, rehydrated in alcohols, and washed twice with water. Samples were then incubated in 0.01 mol/L citrate buffer (pH 6.0) for 25 minutes in a steamer to unmask antigens. Following cooling and rinsing with dH 2 O, samples were treated for 15 minutes with 3% H 2 O 2 diluted in methanol. Tissue sections were washed in dH 2 O and then in phosphate-buffered saline (PBS), then blocked with 10% normal horse serum for 30 minutes. Each tumor marker was stained on individual slides. For E-cadherin staining, the sections were incubated overnight at 4°C with 250 μ g/mL mouse antihuman E-cadherin diluted in normal horse serum (BD Transduction Biosciences, San Jose, CA). After extensive rinsing with PBS, samples were incubated for 40 minutes with 7.5 μ g/mL horse antimouse IgG-biotin (Vector Laboratories, Burlingame, CA) and rinsed with PBS. Samples were then incubated for 30 minutes at room temperature with the Vectastain ABC- kit (Vector Laboratories), followed by PBS washing, and then incubated with an alkaline phosphatase substrate kit (Vector Laboratories). Color development was followed under the microscope for 20 minutes. The color reaction was stopped by rinsing with dH 2 O. Samples were counterstained with hematoxylin. Lung and breast tumor specimens were used as a positive control for all immunohistochemistry staining. Negative controls included incubation with nonimmune pooled rabbit or goat IgG (rabbit IgG [Vector Laboratories] and goat IgG [Zymed; Invitrogen, Carlsbad, CA]) at the same concentration as the primary antibody. Goat antihuman Snail polyclonal IgG (1:50 dilution; Abcam, Cambridge, MA) was used for Snail immunohistochemistry.
p16 (prediluted mouse monoclonal; mtm laboratories, Westborough, MA) antigen retrieval involved heating at 95°C in 0.001 mol EDTA, pH 8.0, for 25 minutes in vegetable steamer, followed by a 15-minute cool down and rinse in 0.05 mol Tris-buffered saline with Tween 20. For EFGR (1:50 dilution of mouse monoclonal H11; Biocare Medical, Concord, CA), antigen retrieval involved enzymatic digestion with Proteinase K (DAKO, Carpenteria, CA) for 7 minutes followed by washing in Tris-buffered saline with Tween 20. Following 5-minute blocking with Ultra V Block (Lab Vision/ThermoScientific, Fremont, CA), primary antibodies were incubated for 30 minutes and coupled with Ultravision Value Polymer Detection System from LabVision/ThermoScientific with diaminobenzidine as chromogen. Slides were counterstained with hematoxylin (Harris). Immunostaining was performed on DAKO Autostainer.
All slides were reviewed by 2 of the investigators (M.F. and C.L.). Each slide was graded for percent cells positive for each stain and was subsequently scaled from (1+ to 4+). We examined the relationship between E-cadherin and Snail using the ordinal immunohistochemistry results (0+, 1+, etc) and found that they were significantly negatively correlated ( ρ = −0.69, P < .001). Subsequently, staining was dichotomized into positive (3+, 4+) and negative (1+, 2+) groups. The exception was Snail staining, which was dichotomized into positive (4+) and negative (1+, 2+, 3+) groups to increase test specificity.
2.3
In situ hybridization
Four-micrometer sections were baked for 15 minutes at 60°C and then labeled and programmed on Ventana XT autostainer to stain for human papilloma virus (HPV) High Risk (genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 66), DNA control probes using ISH IVIEW Blue Plus detection protocol. HPV staining was documented as either positive or negative.
2.4
Clinical Review
Only following the completion of histopathology, the 42 clinical charts were reviewed systematically. Pathologic TNM American joint committee on cancer (AJCC) staging was used for all primary tumor sites. Nodal staging was documented as “best of” from pathologic, radiologic, and then clinical.
2.5
Statistics
Descriptive statistics were derived for all analyzed variables. A P value less than .05 was required for significance. Cross-tabulation analyses were performed using Pearson χ 2 (for tables with values >5) and Fisher Exact (for tables with values <5) tests. Tests of mean were performed with unpaired t tests (SYSTAT 11, Chicago, IL).
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
