- Email: [email protected]
Stromal myofibroblasts in squamous cell carcinoma of the tongue in young patients – a multicenter collaborative study
Vol. 118 No. 4 October 2014
Stromal myofibroblasts in squamous cell carcinoma of the tongue in young patients e a multicenter collaborative study Felipe Paiva Fonseca, DDS, MSc,a Ricardo Della Coletta, DDS, PhD,a Marina Barbosa Azevedo, DDS Student,a Ana Carolina Prado Ribeiro, DDS, PhD,b Ana Maria Pires Soubhia, DDS, PhD,c Glauco Issamu Miyahara, DDS, PhD,c Roman Carlos, DDS,d Paula Farthing, DDS, PhD,e Keith D. Hunter, DDS, PhD,e Paul M. Speight, DDS, PhD,e Pablo Agustin Vargas, DDS, PhD, FRCPath,a Oslei Paes Almeida, DDS, PhD,a Marcio Ajudarte Lopes, DDS, PhD,a and Alan Roger Santos-Silva, DDS, PhDa University of Campinas (UNICAMP), Piracicaba, Brazil; Instituto do Cancer do Estado de São Paulo (ICESP), São Paulo, Brazil; Univ Estadual Paulista (UNESP), Araçatuba, Brazil; Centro Clinico de Cabeza y Cuello, Guatemala City, Guatemala; University of Sheffield, Sheffield, UK.
Objective. The purpose of this study was to evaluate the presence of myofibroblasts, frequently associated with a more aggressive neoplastic behavior, in oral tongue squamous cell carcinoma (TSCC) of young patients and to compare with the distribution observed in older patients. Study Design. Tumor samples from 29 patients younger than 40 years old affected by TSCC were retrieved and investigated for the presence of stromal myofibroblasts by immunohistochemical reactions against a smooth muscle actin, and the results obtained were compared to TSCC cases affecting older patients. Results. No positive reaction could be found in the stromal areas devoid of neoplastic tissue, whereas myofibroblasts were present in 58.6% of the lesions in young patients and in 75.9% of the older ones. No significant difference was found when comparing the invasive front and the overall stroma of both groups, and no correlation could be obtained with stromal a smooth muscle actin expression, higher tumor grades or clinical stage (P > .05). Conclusion. There was no significant difference between the presence of stromal myofibroblasts of TSCC affecting young and old individuals. (Oral Surg Oral Med Oral Pathol Oral Radiol 2014;118:483-489)
Oral cancer is a health problem worldwide, accounting for approximately 25 to 30 % of all malignant tumors in developing countries and ranking among the top three types of cancer in areas of the Asiatic continent, mostly because of the increased use of products containing tobacco.1-4 Oral squamous cell carcinoma (OSCC) is the most common subtype of oral cancer, responsible for 95% of oral malignant tumors.5,6 Although it is frequently diagnosed in the adult population in the fifth decade of life, OSCC incidence in young patients (age <40) has been shown to be increasing in different parts of the globe, but the exact mechanisms and the new possible etiologic factors involved in its onset in young people remain obscure.7 Moreover, the biological behavior of OSCC affecting young patients has been a The authors state that they have no potential conflict of interest that could bias the results obtained in the present study. a Department of Oral Diagnosis, University of Campinas (UNICAMP), Piracicaba Dental School, Piracicaba, Brazil. b Service of Dental Oncology, Instituto do Cancer do Estado de São Paulo (ICESP), Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil. c Department of Pathology and Clinical Propedeutic, Univ Estadual Paulista (UNESP), Araçatuba Dental School, Araçatuba, Brazil. d Centro Clinico de Cabeza y Cuello, Guatemala City, Guatemala. e Academic Unit of Oral and Maxillofacial Pathology, The University of Sheffield, School of Clinical Dentistry, Sheffield, UK. Received for publication Feb 12, 2014; returned for revision Jul 11, 2014; accepted for publication Jul 21, 2014. Ó 2014 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2014.07.012
matter of controversy, since some authors have reported a more aggressive clinical course compared to lesions in older individuals,8-11 whereas others found no significant difference.12-14 More recent studies concluded that young patients with tongue SCC represent a distinct clinical and biological entity because of an increased number of nonsmoking and nondrinking patients among this particular group of oral cancer patients, and a decreased ratio of male-tofemale individuals.15,16 In addition, increased genomic instability and other underlying genetic features that distinguish lesions in young persons from older patients with tongue SCC have been recently reported.17,18 Myofibroblasts were first identified in granulation tissue and wounds.1,19,20 They exhibit an intermediary phenotype between fibroblasts and smooth muscle cells, being characterized by the expression of a smooth muscle actin (a-SMA).5 In the last few years, myofibroblasts have been found to be a major component in the stroma of different human malignancies and are now believed to play an important role in the epithelial/
Statement of Clinical Relevance No significant difference exist between the stromal myofibroblastic component of tongue squamous cell carcinoma affecting young and old individuals and myofibroblasts may not be responsible for any different behavior that may exist between both groups. 483
ORAL AND MAXILLOFACIAL PATHOLOGY 484 Fonseca et al.
OOOO October 2014
Fig. 1. Histopathological distribution of overall stroma and invasive front area of TSCC used in the present study. A and B, Microscopic image under low magnification illustrating the invasive tumor front (bottom) and stromal (top) region of the tumor (H&E; 100). C, Higher magnification of the overall stromal component of the tumors (H&E; 200). D, Higher magnification of the invasive front of the tumor (H&E; 200; TSCC, tongue squamous cell carcinoma).
stromal signaling that would lead to malignant transformation, growth, and progression of epithelial tumors.1,19 It has been shown that through an exchange of cytokines, extracellular matrix proteins, and enzymes between cancer and stromal cells, myofibroblasts would also promote neoplastic invasion and migration, thereby being implicated in the aggressive potential of numerous epithelial malignancies.21-23 Recent studies have shown that myofibroblasts stimulate OSCC growth, proliferation, and invasiveness, and that malignant cells may directly induce myofibroblast transdifferentiation, illustrating the molecular cross talk between both components in the oral carcinogenesis process.19,23-25 Hence, because of the apparent increased aggressiveness of OSCC in young patients suggested in the literature and the recently described association of myofibroblasts with the progression of numerous human cancers, the authors in this study evaluated the presence of myofibroblasts in the stroma of SCC affecting the lateral border of the oral/ mobile tongue of patients younger than 40 years old, with the purpose of testing the hypothesis that such a stromal component would be differently distributed between both groups due to their speculated different biological behavior.
METHODS Tissue samples The study sample consisted of formalin-fixed, paraffinembedded TSCCs (squamous cell carcinoma affecting the lateral border of the oral/mobile tongue) retrieved from four different oral pathology centers at the University of Campinas (Piracicaba Dental School, Brazil),
Universidade Estadual Paulista (Araçatuba Dental School, Brazil), Centro Clínico de Cabeza y Cuello (Guatemala) and the University of Sheffield (School of Clinical Dentistry, UK) in a 20-year period from 1988 to 2008. Those patients previously treated by surgery, radiotherapy, or chemotherapy, and those cases with insufficient tissue for analysis and a lack of clinical information, were excluded. The international approach was undertaken because of the low incidence of TSCC in young patients and to enable a sufficient number of tongue lesions to be collected. Demographic data (age and gender), social habits (tobacco and alcohol consumption), and stage of disease at diagnosis, according to the 2002 Union for International Cancer Control (UICC) TNM classification of malignant tumors, were collected from the patients’ clinical charts. Original H&E-stained slides were reviewed by two oral pathologists, and tumors were classified as well-, moderately, or poorly differentiated. The results were compared with those of a control group of patients older than 40 years selected from the archives of Piracicaba Dental School and matched by tumor site, histopathologic grade, and clinical stage of disease at diagnosis. This study was approved by the Ethics Committee for Human Studies, Piracicaba Dental School, and the South Sheffield Research Ethics Committee. Immunohistochemistry Immunohistochemical analyses were performed using the streptavidin-biotin peroxidase complex method as previously described.26 Briefly, the reactions were conducted on 3 mm sections of the original
OOOO Volume 118, Number 4
ORIGINAL ARTICLE Fonseca et al. 485
Table I. Clinical features of the young and old patients studied in the current investigation Young patients (age <40 y)
Old patients (age 40 y)
No.
Stage
Sex
Age
Tobacco
Alcohol
Stage
Sex
Age
Tobacco
Alcohol
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV
F F M M M M M M M M F M M F F M F F M M M M M M M F F M M
36 21 28 39 34 31 37 37 35 36 40 26 25 33 35 36 31 29 40 31 36 38 38 40 16 36 36 26 29
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV
F F F F F F M M F F M M M F M F F F F F F F F F F F F F F
53 71 69 71 71 57 57 62 41 74 66 58 77 67 53 52 67 57 56 68 58 67 54 74 56 50 60 73 57
þ þ þ þ þ þ þ þ þ NS þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ NS þ þ þ þ þ þ þ þ þ þ þ þ þ
NS, not specified; (þ), positive; (-), negative; F, female; M, male.
formalin-fixed, paraffin-embedded tissues, which were dewaxed with xylene and then hydrated in an ethanol series. Antigen retrieval was carried out, and endogenous peroxidase activity was blocked using 10% hydrogen peroxide in five baths, each of 5 minutes. After washing in PBS buffer (pH 7.4), slides were incubated overnight with the primary antibody: Monoclonal mouse anti-aSMA, diluted 1:200 (Dako Corp., Carpenteria, CA). All slides were subsequently exposed to avidin-biotin complex and horseradish peroxidase reagents (LSAB Kit -DakoCytomation, Dako, Glostrup, Denmark) and diaminobenzidin tetrahydrochloride (DAB, Sigma, St. Louis, MO), and subsequently counterstained with Carazzi’s hematoxylin. Normal vessels were used as internal positive control, whereas the negative control was obtained by omitting the primary specific antibody. The presence of a-SMA positive cells was classified as negative (0) (0% to 5% of positivity in the area analyzed), weak (1) (6% to 50% of positivity in the area analyzed) or strong (2) (>50% of positivity in the area analyzed) in two different areas of each case: The stroma within the tumor, considered as the overall area for the purposes of this study, and the tumor front, which represented the band of tissue between the invasive
tumor front and adjacent normal connective tissue (Figure 1), in a blinded analysis performed by three of the authors.23 Statistical analysis Correlations between the presence of myofibroblasts and clinicopathological parameters were performed by Fisher’s exact test at 5% significance GraphPad Prism version 5.0 (San Diego, CA).
RESULTS There were 29 young patients with TSCC (Table I). The mean age of these patients at the time of the biopsy was 32.93 years (range 16-40), with a male preponderance (male-to-female ratio of 2.2:1). A proportion of 51.7% of the patients were smokers and 62.1% were alcohol users. The matched group of 29 older patients presented a mean age of 61.93 years (range 41-77) and had a female preponderance with a male-to-female ratio of 1:3.2. Most of the older patients reported both tobacco (82.8%) and alcohol consumption (69.1%). Both groups were also matched according to UICC clinical staging, comprising 14 patients classified as stage I/II
ORAL AND MAXILLOFACIAL PATHOLOGY 486 Fonseca et al.
OOOO October 2014
Table II. Intensity of aSMA staining in each case according to histopathological grade of tumors Young patients (age <40 y) No.
Histologic grade
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Well Well Well Well Well Well Well Well Well Well Well Well Poor Poor Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate
Old patients (age 40 y)
Front
Overall Stroma
0 1 1 0 1 0 1 0 0 0 0 0 2 1 2 0 0 0 0 1 2 0 2 0 2 1 2 0 2
0 1 1 0 2 0 1 1 0 0 0 0 2 2 0 0 0 1 1 1 2 0 2 0 2 1 2 1 2
Histologic grade Well Well Well Well Well Well Well Well Well Well Well Well Poor Poor Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate
Front
Overall Stroma
1 0 1 0 0 1 1 1 2 2 2 1 1 1 2 2 0 0 2 0 2 0 0 0 0 0 1 2 1
1 2 1 0 0 2 2 2 1 2 1 1 2 0 1 2 0 1 0 0 0 0 0 0 1 1 1 2 1
0, negative; 1, weak; 2, strong.
and 15 patients in stages III/IV in each group, and according to histopathologic grade (12 well-, 15 moderately, and 2 poorly differentiated cases; Table II). Immunohistochemical positivity for aSMA was seen in 67.2% (n ¼ 39) of the tumors studied. It was noted that in the stromal area devoid of neoplastic tissue, no positive reaction could be found. With regard to TSCC cases affecting young patients, aSMA positivity was found in 58.6% of the cases (n ¼ 17). Among these, there was a strong reaction in the advancing front area in 6 cases, a weak reaction in another 7 cases, and a negative reaction in the remaining 4 cases, whereas reactivity was strong in the stromal compartment in 8 cases, but proved to be weak in the other 9 cases. In the lesions affecting older individuals, positivity was seen in 75.9% of the sample studied (n ¼ 22). Strong staining was present in the front compartment in 8 cases, weak staining in 10 cases, and negative staining in the remaining 4 cases. Stromal positivity was strong in 8 cases, weak in 11 cases, and negative in 3 cases. Comparing the expression of aSMA in young and adult patients in both front (Figure 2) and stromal (Figure 3) areas, no statistically significant difference could be obtained (P > .05).
No significant difference could be seen by comparing the presence of myofibroblasts between cases classified as stage I/II and stage III/IV (P > .05). Similarly, aSMA positivity did not significantly correlate with the histopathologic grade of the tumors (P > .05).
DISCUSSION OSCC is regarded as a disease of the elderly, affecting patients over 45 years of age; however, an increased number of young patients diagnosed with this condition has been reported in recent decades.2,27 Although the incidence of OSCC affecting young adults usually ranges from 2% to 6% in most specialized services worldwide,7,12,14,28 a higher incidence has been recently described in parts of the USA,29 western Europe,12 Latin America,6,7 and Asia.15 Related to this variability in the epidemiology of OSCC affecting young patients are uncertainties related to their biological and clinical behavior. Cases in young patients have been considered to represent a more aggressive disease than those diagnosed in older patients, and may also not be associated with tobacco use and alcohol intake, suggesting that alternative etiologic factors may be involved with its onset.7,13,30 However, recent
OOOO Volume 118, Number 4
ORIGINAL ARTICLE Fonseca et al. 487
Fig. 2. aSMA immunohistochemical expression in the front area of TSCC affecting patients older and younger than 40 years of age. A, Strong reactivity for aSMA in the invasive front areas of the tumor (streptavidin-biotin; 50). B, Higher magnification detailing the strong positivity of aSMA in the invasive front region (streptavidin-biotin; 100). C, Negative/weak reactivity for aSMA in the invasive front areas of the tumor (streptavidin-biotin; 50). D, Higher magnification detailing the negative/weak positivity of aSMA in the invasive front region (streptavidin-biotin; 100; TSCC, tongue squamous cell carcinoma; aSMA, a smooth muscle actin).
Fig. 3. aSMA immunohistochemical expression in the stromal area of TSCC affecting patients older and younger than 40 years of age. A, Strong reactivity for aSMA in the overall stromal region of the tumor (streptavidin-biotin; 50). B, Higher magnification detailing the strong positivity of aSMA in the neoplastic stroma (streptavidin-biotin; 100). C, Negative/weak reactivity for aSMA in the overall stromal areas of the tumor (streptavidin-biotin; 50). D, Higher magnification detailing the negative/weak positivity of aSMA in the stromal region (streptavidin-biotin; 100; TSCC, tongue squamous cell carcinoma; aSMA, a smooth muscle actin).
investigations have shown no significant differences in the prognosis, survival, and treatment response of young patients afflicted by OSCC compared with older ones, and molecular evaluation of the lesions has failed to show significant differences, again suggesting that no relevant difference exists.10,27,31,32
Although activation of the host stroma has been considered an important early step for tumorigenesis in different human cancers,20 studies investigating the stromal constituents of OSCC affecting young individuals and their relevance for the biological behavior of these tumors are very scarce, and specific studies of
ORAL AND MAXILLOFACIAL PATHOLOGY 488 Fonseca et al.
the presence of myofibroblasts, have never been conducted. Therefore, in the present manuscript, the authors evaluated the presence of myofibroblasts in the invasive front area of the neoplastic islands and in the overall stromal compartment of TSCC of young patients with the purpose of better understanding the biological features of this uncommon clinical presentation of OSCC. The results obtained revealed that there was no statistically significant difference between the presence of myofibroblasts in young patients and in older individuals, either in the advancing front of the tumor or in the overall stroma of the neoplasms, that could suggest a more aggressive behavior. This was further supported by the lack of association between myofibroblasts and both the histopathologic grade of tumors and the clinical stage of patients. This is in accordance with other studies from our group, which failed to find differences in the histopathologic profile of OSCC from young and old patients.7,17 Determining the best cut-off value between young and old patients affected by OSCC has been a debatable field in literature. Although several authors claimed 45 years as the most appropriate age, providing larger samples of younger patients, most of the recent studies have used 40 years as the cut-off value, and therefore we opted for this value to better compare the obtained results with those recently described in literature.6-9 However, in a 2002 national-based study, Funk et al.33 described significant differences in the biological behavior of patients distributed in three different categories, those younger than 33 years, between 35 and 65 years, and older than 66 years. Although we observed a higher myofibroblast density in older patients (data not shown), once again no statistical significance could be reached by categorizing our patients in line with this classification. Several studies have shown that the microenvironment of neoplastic tissues plays an active role in tumor progression, and that to the conversion of normal epithelial tissue to dysplastic epithelium to carcinoma, the stroma would also change to an activated state where fibroblasts, one of the most important constituents of this compartment, are transdifferentiated to active myofibroblasts, representing a crucial event in human carcinogenesis.1,20,34 This change in the state of myofibroblasts seems to be mediated by growth factors and cytokines expressed by tumor cells, especially transforming growth factor beta (TGF-b), whereas myofibroblasts would in turn secrete numerous growth factors and inflammatory mediators that stimulate epithelial neoplastic cell proliferation.20,25,34 Hence, various studies have proved the importance of stromal elements in tumoral development, growth, invasion, and aggressiveness, illustrating myofibroblasts as cancer-induced host cells.35
OOOO October 2014
Our group and other authors previously found that myofibroblasts would also be present in the stroma of OSCC, significantly correlating to the rate of lymph node metastases and, consequently, to the final prognosis of affected patients.23 However, studies have failed to show any expression of aSMA in potentially malignant lesions of the oral cavity and in normal oral mucosa, highlighting that myofibroblasts appear to be associated with molecular cross talk between stromal elements and neoplastic epithelial cells.34-36 This is further supported in the present study by the lack of immunoreactivity in areas devoid of neoplastic islands. In conclusion, although further studies would be necessary for a better understanding of the biological features of OSCC affecting young patients and the role of myofibroblasts in its development and behavior, the results obtained in the current investigation suggest that no significant difference exists between the stromal myofibroblastic component of TSCC affecting young and old individuals. REFERENCES 1. Vered M, Allon I, Buchner A, Dayan D. Stromal myofibroblasts and malignant transformation in a 4 NQO rat tongue carcinogenesis model. Oral Oncol. 2007;43:999-1006. 2. Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer. Oral Oncol. 2009;45:309-316. 3. Coelho KR. Challenges of the oral cancer burden in India. J Cancer Epidemiol. 2012;2012:1-17. 4. Chaturvedi P, Vaishampayan SS, Nair S, et al. Oral squamous cell carcinoma arising in background of oral submucous fibrosis: A clinicopathologically distinct disease. Head Neck. 2013;35:14041409. 5. Vered M, Allon I, Buchner A, Dayan D. Stromal myofibroblasts accompany modifications in the epithelial phenotype of tongue dysplastic and malignant lesions. Cancer Microenviron. 2009;2: 49-57. 6. Pontes FSC, Carneiro JT, Fonseca FP, Silva TSP, Pontes HAR, Pinto DS. Squamous cell carcinoma of the tongue and floor of the mouth: Analysis of survival rate and independent prognostic factors in the Amazon region. J Craniofac Surg. 2011;22: 925-930. 7. Ribeiro ACP, Santos-Silva AR, Simonato LE, Salzedas LMP, Sundefeld MLMM, Soubhia AMP. Clinical and histopathological analysis of oral squamous cell carcinoma in young people. A descriptive study in Brazilians. Br J Oral and Maxillofacial Surgery. 2009;47:95-98. 8. Sarkaria JN, Harari PM. Oral tongue cancer in young adults less than 40 years of age: Rationale for aggressive therapy. Head Neck. 1994;16:107-111. 9. Vargas H, Pitman KT, Johnson JT, Galati LT. More aggressive behavior of squamous cell carcinoma of the anterior tongue in young women. Laryngoscope. 2000;110:1623-1626. 10. Siriwardena BSMS, Tilakaratne A, Amaratunga EAPD, et al. Analysis of histopathological and immunohistochemical differences of oral squamous cell carcinoma in young and old patients in Sri Lanka. J Oral Pathol Med. 2007;36:357-362. 11. Vered M, Dobriyan A, Dayan D, et al. Tumor-host histopathologic variables, stromal myofibroblasts and risk score, are significantly associated with recurrent disease in tongue cancer. Cancer Sci. 2010;101:274-280.
OOOO Volume 118, Number 4 12. Annertz1 K, Anderson H, Biörklund A, et al. Incidence and survival of squamous cell carcinoma of the tongue in Scandinavia, with special reference to young adults. Int J Cancer. 2002;101:95-99. 13. Sasaki T, Moles DR, Imai Y, Speight PM. Clinico-pathological features of squamous cell carcinoma of the oral cavity in patients <40 years of age. J Oral Pathol Med. 2005;34:129-133. 14. Udeabor SE, Rana M, Wegener G, Gellrich N, Eckardt AM. Squamous cell carcinoma of the oral cavity and the oropharynx in patients less than 40 years of age: A 20-year analysis. Head Neck Oncol. 2012;4:28-35. 15. Iamaroon A, Pattanaporn K, Pongsiriwet S, et al. Analysis of 587 cases of oral squamous cell carcinoma in northern Thailand with a focus on young people. Int J Oral Maxillofac Surg. 2004; 33:84-88. 16. Toner M, O’Regan EM. Head and neck squamous cell carcinoma in the young: A spectrum or a distinct group? Part 1. Head Neck Pathol. 2009;3:246-248. 17. Santos-Silva AR, Ribeiro ACP, Soubhia AMP, et al. High incidences of DNA ploidy abnormalities in tongue squamous cell carcinoma of young patients: An international collaborative study. Histopathology. 2011;58:1127-1135. 18. Toner M, O’Regan EM. Head and neck squamous cell carcinoma in the young: A spectrum or a distinct group? Part 2. Head Neck Pathol. 2009;3:249-251. 19. Sobral LM, Bufalino A, Lopes MA, Graner E, Salo T, Coletta RD. Myofibroblasts in the stroma of oral cancer promote tumorigenesis via secretion of Activin A. Oral Oncol. 2011;47: 840-846. 20. Thode C, Jørgensen1 TG, Dabelsteen E, Mackenzie I, Dabelsteen S. Significance of myofibroblasts in oral squamous cell carcinoma. J Oral Pathol Med. 2011;40:201-207. 21. Surowiak P, Murawa D, Materna V, et al. Occurrence of stromal myofibroblasts in the invasive ductal breast cancer tissue is an unfavorable prognostic factor. Anticancer Res. 2007;27: 2917-2924. 22. Tsujino T, Seshimo I, Yamamoto H, et al. Stromal myofibroblasts predict disease recurrence for colorectal cancer. Clin Cancer Res. 2007;3:2082-2090. 23. Kellermann MG, Sobral LM, Silva SD, et al. Myofibroblasts in the stroma of oral squamous cell carcinoma are associated with poor prognosis. Histopathology. 2007;51:849-853. 24. Kellermann MG, Sobral LM, Silva SD, et al. Mutual paracrine effects of oral squamous cell carcinoma cells and normal oral fibroblasts: Induction of fibroblast to myofibroblast transdifferentiation and modulation of tumor cell proliferation. Oral Oncol. 2008;44:509-517. 25. Marsh D, Suchak K, Moutasim KA, et al. Stromal features are predictive of disease mortality in oral cancer patients. J Pathol. 2011;223:470-481. 26. Andrade BAB, León JE, Carlos R, Delgado-Azañero W, Mosqueda-Taylor A, Almeida OP. Immunohistochemical
ORIGINAL ARTICLE Fonseca et al. 489
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
expression of p16, p21, p27 and cyclin D1 in oral nevi and melanoma. Head Neck Pathol. 2012;6:297-304. Warnakulasuriya S, Mak V, Möller H. Oral cancer survival in young people in South East England. Oral Oncol. 2007;43: 982-986. Llewellyn CD, Linklater K, Bell J, Johnson NW, Warnakulasuriya S. An analysis of risk factors for oral cancer in young people: A case-control study. Oral Oncol. 2004;40:204-213. Schantz SP, Yu GP. Head and neck cancer incidence trends in young Americans, 1973-1997, with a special analysis for tongue cancer. Arch Otolaryngol Head Neck Surg. 2002;128:268-274. Hirota SK, Braga FPF, Penha SS, Sugaya NN, Migliari DA. Risk factors for oral squamous cell carcinoma in young and older Brazilian patients: A comparative analysis. Med Oral Patol Oral Cir Bucal. 2008;13:E227-E231. Thomas L, Moore EJ, McGree ME, Olsen KD, Kasperbauer JL, Erickson LA, et al. Prognostic features, human papillomavirus status, and epidermal growth factor receptor expression in oral squamous cell carcinoma in young adults. Am J Otolaryngol Head Neck Med Surg. 2012;33:650-656. Benevenuto TG, Nonaka CFW, Pinto LP, Souza LB. Immunohistochemical comparative analysis of cell proliferation and angiogenic index in squamous cell carcinomas of the tongue between young and older patients. Appl Immunohistochem Mol Morphol. 2012;20:291-297. Funk GF, Karnell LH, Robinson RA, Zhen WK, Trask DK, Hoffman HT. Presentation, treatment, and outcome of oral cavity cancer: A national cancer data base report. Head Neck. 2002;24: 165-180. Barth PJ, Schweinsberg TS, Ramaswamy A, Moll R. CD34þ fibrocytes, a-smooth muscle antigen-positive myofibroblasts, and CD117 expression in the stroma of invasive squamous cell carcinomas of the oral cavity, pharynx, and larynx. Virchows Arch. 2004;444:231-234. Etemad-Moghadam S, Khalili M, Tirgary F, Alaeddini M. Evaluation of myofibroblasts in oral epithelial dysplasia and squamous cell carcinoma. J Oral Pathol Med. 2009;38:639-643. de-Assis EM, Pimenta LGGS, Costa-e-Silva E, Souza PEA, Horta MCR. Stromal myofibroblasts in oral leukoplakia and oral squamous cell carcinoma. Med Oral Patol Oral Cir Bucal. 2012;17:E733-e738.
Reprint requests: Dr. Alan Roger Santos-Silva, DDS, PhD Oral Diagnosis Department (Semiology) Piracicaba Dental School (UNICAMP) Av. Limeira, No. 901, 13.414-903 Piracicaba São Paulo Brazil [email protected]
Stromal myofibroblasts in squamous cell carcinoma of the tongue in young patients e a multicenter collaborative study Felipe Paiva Fonseca, DDS, MSc,a Ricardo Della Coletta, DDS, PhD,a Marina Barbosa Azevedo, DDS Student,a Ana Carolina Prado Ribeiro, DDS, PhD,b Ana Maria Pires Soubhia, DDS, PhD,c Glauco Issamu Miyahara, DDS, PhD,c Roman Carlos, DDS,d Paula Farthing, DDS, PhD,e Keith D. Hunter, DDS, PhD,e Paul M. Speight, DDS, PhD,e Pablo Agustin Vargas, DDS, PhD, FRCPath,a Oslei Paes Almeida, DDS, PhD,a Marcio Ajudarte Lopes, DDS, PhD,a and Alan Roger Santos-Silva, DDS, PhDa University of Campinas (UNICAMP), Piracicaba, Brazil; Instituto do Cancer do Estado de São Paulo (ICESP), São Paulo, Brazil; Univ Estadual Paulista (UNESP), Araçatuba, Brazil; Centro Clinico de Cabeza y Cuello, Guatemala City, Guatemala; University of Sheffield, Sheffield, UK.
Objective. The purpose of this study was to evaluate the presence of myofibroblasts, frequently associated with a more aggressive neoplastic behavior, in oral tongue squamous cell carcinoma (TSCC) of young patients and to compare with the distribution observed in older patients. Study Design. Tumor samples from 29 patients younger than 40 years old affected by TSCC were retrieved and investigated for the presence of stromal myofibroblasts by immunohistochemical reactions against a smooth muscle actin, and the results obtained were compared to TSCC cases affecting older patients. Results. No positive reaction could be found in the stromal areas devoid of neoplastic tissue, whereas myofibroblasts were present in 58.6% of the lesions in young patients and in 75.9% of the older ones. No significant difference was found when comparing the invasive front and the overall stroma of both groups, and no correlation could be obtained with stromal a smooth muscle actin expression, higher tumor grades or clinical stage (P > .05). Conclusion. There was no significant difference between the presence of stromal myofibroblasts of TSCC affecting young and old individuals. (Oral Surg Oral Med Oral Pathol Oral Radiol 2014;118:483-489)
Oral cancer is a health problem worldwide, accounting for approximately 25 to 30 % of all malignant tumors in developing countries and ranking among the top three types of cancer in areas of the Asiatic continent, mostly because of the increased use of products containing tobacco.1-4 Oral squamous cell carcinoma (OSCC) is the most common subtype of oral cancer, responsible for 95% of oral malignant tumors.5,6 Although it is frequently diagnosed in the adult population in the fifth decade of life, OSCC incidence in young patients (age <40) has been shown to be increasing in different parts of the globe, but the exact mechanisms and the new possible etiologic factors involved in its onset in young people remain obscure.7 Moreover, the biological behavior of OSCC affecting young patients has been a The authors state that they have no potential conflict of interest that could bias the results obtained in the present study. a Department of Oral Diagnosis, University of Campinas (UNICAMP), Piracicaba Dental School, Piracicaba, Brazil. b Service of Dental Oncology, Instituto do Cancer do Estado de São Paulo (ICESP), Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil. c Department of Pathology and Clinical Propedeutic, Univ Estadual Paulista (UNESP), Araçatuba Dental School, Araçatuba, Brazil. d Centro Clinico de Cabeza y Cuello, Guatemala City, Guatemala. e Academic Unit of Oral and Maxillofacial Pathology, The University of Sheffield, School of Clinical Dentistry, Sheffield, UK. Received for publication Feb 12, 2014; returned for revision Jul 11, 2014; accepted for publication Jul 21, 2014. Ó 2014 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2014.07.012
matter of controversy, since some authors have reported a more aggressive clinical course compared to lesions in older individuals,8-11 whereas others found no significant difference.12-14 More recent studies concluded that young patients with tongue SCC represent a distinct clinical and biological entity because of an increased number of nonsmoking and nondrinking patients among this particular group of oral cancer patients, and a decreased ratio of male-tofemale individuals.15,16 In addition, increased genomic instability and other underlying genetic features that distinguish lesions in young persons from older patients with tongue SCC have been recently reported.17,18 Myofibroblasts were first identified in granulation tissue and wounds.1,19,20 They exhibit an intermediary phenotype between fibroblasts and smooth muscle cells, being characterized by the expression of a smooth muscle actin (a-SMA).5 In the last few years, myofibroblasts have been found to be a major component in the stroma of different human malignancies and are now believed to play an important role in the epithelial/
Statement of Clinical Relevance No significant difference exist between the stromal myofibroblastic component of tongue squamous cell carcinoma affecting young and old individuals and myofibroblasts may not be responsible for any different behavior that may exist between both groups. 483
ORAL AND MAXILLOFACIAL PATHOLOGY 484 Fonseca et al.
OOOO October 2014
Fig. 1. Histopathological distribution of overall stroma and invasive front area of TSCC used in the present study. A and B, Microscopic image under low magnification illustrating the invasive tumor front (bottom) and stromal (top) region of the tumor (H&E; 100). C, Higher magnification of the overall stromal component of the tumors (H&E; 200). D, Higher magnification of the invasive front of the tumor (H&E; 200; TSCC, tongue squamous cell carcinoma).
stromal signaling that would lead to malignant transformation, growth, and progression of epithelial tumors.1,19 It has been shown that through an exchange of cytokines, extracellular matrix proteins, and enzymes between cancer and stromal cells, myofibroblasts would also promote neoplastic invasion and migration, thereby being implicated in the aggressive potential of numerous epithelial malignancies.21-23 Recent studies have shown that myofibroblasts stimulate OSCC growth, proliferation, and invasiveness, and that malignant cells may directly induce myofibroblast transdifferentiation, illustrating the molecular cross talk between both components in the oral carcinogenesis process.19,23-25 Hence, because of the apparent increased aggressiveness of OSCC in young patients suggested in the literature and the recently described association of myofibroblasts with the progression of numerous human cancers, the authors in this study evaluated the presence of myofibroblasts in the stroma of SCC affecting the lateral border of the oral/ mobile tongue of patients younger than 40 years old, with the purpose of testing the hypothesis that such a stromal component would be differently distributed between both groups due to their speculated different biological behavior.
METHODS Tissue samples The study sample consisted of formalin-fixed, paraffinembedded TSCCs (squamous cell carcinoma affecting the lateral border of the oral/mobile tongue) retrieved from four different oral pathology centers at the University of Campinas (Piracicaba Dental School, Brazil),
Universidade Estadual Paulista (Araçatuba Dental School, Brazil), Centro Clínico de Cabeza y Cuello (Guatemala) and the University of Sheffield (School of Clinical Dentistry, UK) in a 20-year period from 1988 to 2008. Those patients previously treated by surgery, radiotherapy, or chemotherapy, and those cases with insufficient tissue for analysis and a lack of clinical information, were excluded. The international approach was undertaken because of the low incidence of TSCC in young patients and to enable a sufficient number of tongue lesions to be collected. Demographic data (age and gender), social habits (tobacco and alcohol consumption), and stage of disease at diagnosis, according to the 2002 Union for International Cancer Control (UICC) TNM classification of malignant tumors, were collected from the patients’ clinical charts. Original H&E-stained slides were reviewed by two oral pathologists, and tumors were classified as well-, moderately, or poorly differentiated. The results were compared with those of a control group of patients older than 40 years selected from the archives of Piracicaba Dental School and matched by tumor site, histopathologic grade, and clinical stage of disease at diagnosis. This study was approved by the Ethics Committee for Human Studies, Piracicaba Dental School, and the South Sheffield Research Ethics Committee. Immunohistochemistry Immunohistochemical analyses were performed using the streptavidin-biotin peroxidase complex method as previously described.26 Briefly, the reactions were conducted on 3 mm sections of the original
OOOO Volume 118, Number 4
ORIGINAL ARTICLE Fonseca et al. 485
Table I. Clinical features of the young and old patients studied in the current investigation Young patients (age <40 y)
Old patients (age 40 y)
No.
Stage
Sex
Age
Tobacco
Alcohol
Stage
Sex
Age
Tobacco
Alcohol
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV
F F M M M M M M M M F M M F F M F F M M M M M M M F F M M
36 21 28 39 34 31 37 37 35 36 40 26 25 33 35 36 31 29 40 31 36 38 38 40 16 36 36 26 29
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV III/IV
F F F F F F M M F F M M M F M F F F F F F F F F F F F F F
53 71 69 71 71 57 57 62 41 74 66 58 77 67 53 52 67 57 56 68 58 67 54 74 56 50 60 73 57
þ þ þ þ þ þ þ þ þ NS þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ NS þ þ þ þ þ þ þ þ þ þ þ þ þ
NS, not specified; (þ), positive; (-), negative; F, female; M, male.
formalin-fixed, paraffin-embedded tissues, which were dewaxed with xylene and then hydrated in an ethanol series. Antigen retrieval was carried out, and endogenous peroxidase activity was blocked using 10% hydrogen peroxide in five baths, each of 5 minutes. After washing in PBS buffer (pH 7.4), slides were incubated overnight with the primary antibody: Monoclonal mouse anti-aSMA, diluted 1:200 (Dako Corp., Carpenteria, CA). All slides were subsequently exposed to avidin-biotin complex and horseradish peroxidase reagents (LSAB Kit -DakoCytomation, Dako, Glostrup, Denmark) and diaminobenzidin tetrahydrochloride (DAB, Sigma, St. Louis, MO), and subsequently counterstained with Carazzi’s hematoxylin. Normal vessels were used as internal positive control, whereas the negative control was obtained by omitting the primary specific antibody. The presence of a-SMA positive cells was classified as negative (0) (0% to 5% of positivity in the area analyzed), weak (1) (6% to 50% of positivity in the area analyzed) or strong (2) (>50% of positivity in the area analyzed) in two different areas of each case: The stroma within the tumor, considered as the overall area for the purposes of this study, and the tumor front, which represented the band of tissue between the invasive
tumor front and adjacent normal connective tissue (Figure 1), in a blinded analysis performed by three of the authors.23 Statistical analysis Correlations between the presence of myofibroblasts and clinicopathological parameters were performed by Fisher’s exact test at 5% significance GraphPad Prism version 5.0 (San Diego, CA).
RESULTS There were 29 young patients with TSCC (Table I). The mean age of these patients at the time of the biopsy was 32.93 years (range 16-40), with a male preponderance (male-to-female ratio of 2.2:1). A proportion of 51.7% of the patients were smokers and 62.1% were alcohol users. The matched group of 29 older patients presented a mean age of 61.93 years (range 41-77) and had a female preponderance with a male-to-female ratio of 1:3.2. Most of the older patients reported both tobacco (82.8%) and alcohol consumption (69.1%). Both groups were also matched according to UICC clinical staging, comprising 14 patients classified as stage I/II
ORAL AND MAXILLOFACIAL PATHOLOGY 486 Fonseca et al.
OOOO October 2014
Table II. Intensity of aSMA staining in each case according to histopathological grade of tumors Young patients (age <40 y) No.
Histologic grade
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Well Well Well Well Well Well Well Well Well Well Well Well Poor Poor Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate
Old patients (age 40 y)
Front
Overall Stroma
0 1 1 0 1 0 1 0 0 0 0 0 2 1 2 0 0 0 0 1 2 0 2 0 2 1 2 0 2
0 1 1 0 2 0 1 1 0 0 0 0 2 2 0 0 0 1 1 1 2 0 2 0 2 1 2 1 2
Histologic grade Well Well Well Well Well Well Well Well Well Well Well Well Poor Poor Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate
Front
Overall Stroma
1 0 1 0 0 1 1 1 2 2 2 1 1 1 2 2 0 0 2 0 2 0 0 0 0 0 1 2 1
1 2 1 0 0 2 2 2 1 2 1 1 2 0 1 2 0 1 0 0 0 0 0 0 1 1 1 2 1
0, negative; 1, weak; 2, strong.
and 15 patients in stages III/IV in each group, and according to histopathologic grade (12 well-, 15 moderately, and 2 poorly differentiated cases; Table II). Immunohistochemical positivity for aSMA was seen in 67.2% (n ¼ 39) of the tumors studied. It was noted that in the stromal area devoid of neoplastic tissue, no positive reaction could be found. With regard to TSCC cases affecting young patients, aSMA positivity was found in 58.6% of the cases (n ¼ 17). Among these, there was a strong reaction in the advancing front area in 6 cases, a weak reaction in another 7 cases, and a negative reaction in the remaining 4 cases, whereas reactivity was strong in the stromal compartment in 8 cases, but proved to be weak in the other 9 cases. In the lesions affecting older individuals, positivity was seen in 75.9% of the sample studied (n ¼ 22). Strong staining was present in the front compartment in 8 cases, weak staining in 10 cases, and negative staining in the remaining 4 cases. Stromal positivity was strong in 8 cases, weak in 11 cases, and negative in 3 cases. Comparing the expression of aSMA in young and adult patients in both front (Figure 2) and stromal (Figure 3) areas, no statistically significant difference could be obtained (P > .05).
No significant difference could be seen by comparing the presence of myofibroblasts between cases classified as stage I/II and stage III/IV (P > .05). Similarly, aSMA positivity did not significantly correlate with the histopathologic grade of the tumors (P > .05).
DISCUSSION OSCC is regarded as a disease of the elderly, affecting patients over 45 years of age; however, an increased number of young patients diagnosed with this condition has been reported in recent decades.2,27 Although the incidence of OSCC affecting young adults usually ranges from 2% to 6% in most specialized services worldwide,7,12,14,28 a higher incidence has been recently described in parts of the USA,29 western Europe,12 Latin America,6,7 and Asia.15 Related to this variability in the epidemiology of OSCC affecting young patients are uncertainties related to their biological and clinical behavior. Cases in young patients have been considered to represent a more aggressive disease than those diagnosed in older patients, and may also not be associated with tobacco use and alcohol intake, suggesting that alternative etiologic factors may be involved with its onset.7,13,30 However, recent
OOOO Volume 118, Number 4
ORIGINAL ARTICLE Fonseca et al. 487
Fig. 2. aSMA immunohistochemical expression in the front area of TSCC affecting patients older and younger than 40 years of age. A, Strong reactivity for aSMA in the invasive front areas of the tumor (streptavidin-biotin; 50). B, Higher magnification detailing the strong positivity of aSMA in the invasive front region (streptavidin-biotin; 100). C, Negative/weak reactivity for aSMA in the invasive front areas of the tumor (streptavidin-biotin; 50). D, Higher magnification detailing the negative/weak positivity of aSMA in the invasive front region (streptavidin-biotin; 100; TSCC, tongue squamous cell carcinoma; aSMA, a smooth muscle actin).
Fig. 3. aSMA immunohistochemical expression in the stromal area of TSCC affecting patients older and younger than 40 years of age. A, Strong reactivity for aSMA in the overall stromal region of the tumor (streptavidin-biotin; 50). B, Higher magnification detailing the strong positivity of aSMA in the neoplastic stroma (streptavidin-biotin; 100). C, Negative/weak reactivity for aSMA in the overall stromal areas of the tumor (streptavidin-biotin; 50). D, Higher magnification detailing the negative/weak positivity of aSMA in the stromal region (streptavidin-biotin; 100; TSCC, tongue squamous cell carcinoma; aSMA, a smooth muscle actin).
investigations have shown no significant differences in the prognosis, survival, and treatment response of young patients afflicted by OSCC compared with older ones, and molecular evaluation of the lesions has failed to show significant differences, again suggesting that no relevant difference exists.10,27,31,32
Although activation of the host stroma has been considered an important early step for tumorigenesis in different human cancers,20 studies investigating the stromal constituents of OSCC affecting young individuals and their relevance for the biological behavior of these tumors are very scarce, and specific studies of
ORAL AND MAXILLOFACIAL PATHOLOGY 488 Fonseca et al.
the presence of myofibroblasts, have never been conducted. Therefore, in the present manuscript, the authors evaluated the presence of myofibroblasts in the invasive front area of the neoplastic islands and in the overall stromal compartment of TSCC of young patients with the purpose of better understanding the biological features of this uncommon clinical presentation of OSCC. The results obtained revealed that there was no statistically significant difference between the presence of myofibroblasts in young patients and in older individuals, either in the advancing front of the tumor or in the overall stroma of the neoplasms, that could suggest a more aggressive behavior. This was further supported by the lack of association between myofibroblasts and both the histopathologic grade of tumors and the clinical stage of patients. This is in accordance with other studies from our group, which failed to find differences in the histopathologic profile of OSCC from young and old patients.7,17 Determining the best cut-off value between young and old patients affected by OSCC has been a debatable field in literature. Although several authors claimed 45 years as the most appropriate age, providing larger samples of younger patients, most of the recent studies have used 40 years as the cut-off value, and therefore we opted for this value to better compare the obtained results with those recently described in literature.6-9 However, in a 2002 national-based study, Funk et al.33 described significant differences in the biological behavior of patients distributed in three different categories, those younger than 33 years, between 35 and 65 years, and older than 66 years. Although we observed a higher myofibroblast density in older patients (data not shown), once again no statistical significance could be reached by categorizing our patients in line with this classification. Several studies have shown that the microenvironment of neoplastic tissues plays an active role in tumor progression, and that to the conversion of normal epithelial tissue to dysplastic epithelium to carcinoma, the stroma would also change to an activated state where fibroblasts, one of the most important constituents of this compartment, are transdifferentiated to active myofibroblasts, representing a crucial event in human carcinogenesis.1,20,34 This change in the state of myofibroblasts seems to be mediated by growth factors and cytokines expressed by tumor cells, especially transforming growth factor beta (TGF-b), whereas myofibroblasts would in turn secrete numerous growth factors and inflammatory mediators that stimulate epithelial neoplastic cell proliferation.20,25,34 Hence, various studies have proved the importance of stromal elements in tumoral development, growth, invasion, and aggressiveness, illustrating myofibroblasts as cancer-induced host cells.35
OOOO October 2014
Our group and other authors previously found that myofibroblasts would also be present in the stroma of OSCC, significantly correlating to the rate of lymph node metastases and, consequently, to the final prognosis of affected patients.23 However, studies have failed to show any expression of aSMA in potentially malignant lesions of the oral cavity and in normal oral mucosa, highlighting that myofibroblasts appear to be associated with molecular cross talk between stromal elements and neoplastic epithelial cells.34-36 This is further supported in the present study by the lack of immunoreactivity in areas devoid of neoplastic islands. In conclusion, although further studies would be necessary for a better understanding of the biological features of OSCC affecting young patients and the role of myofibroblasts in its development and behavior, the results obtained in the current investigation suggest that no significant difference exists between the stromal myofibroblastic component of TSCC affecting young and old individuals. REFERENCES 1. Vered M, Allon I, Buchner A, Dayan D. Stromal myofibroblasts and malignant transformation in a 4 NQO rat tongue carcinogenesis model. Oral Oncol. 2007;43:999-1006. 2. Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer. Oral Oncol. 2009;45:309-316. 3. Coelho KR. Challenges of the oral cancer burden in India. J Cancer Epidemiol. 2012;2012:1-17. 4. Chaturvedi P, Vaishampayan SS, Nair S, et al. Oral squamous cell carcinoma arising in background of oral submucous fibrosis: A clinicopathologically distinct disease. Head Neck. 2013;35:14041409. 5. Vered M, Allon I, Buchner A, Dayan D. Stromal myofibroblasts accompany modifications in the epithelial phenotype of tongue dysplastic and malignant lesions. Cancer Microenviron. 2009;2: 49-57. 6. Pontes FSC, Carneiro JT, Fonseca FP, Silva TSP, Pontes HAR, Pinto DS. Squamous cell carcinoma of the tongue and floor of the mouth: Analysis of survival rate and independent prognostic factors in the Amazon region. J Craniofac Surg. 2011;22: 925-930. 7. Ribeiro ACP, Santos-Silva AR, Simonato LE, Salzedas LMP, Sundefeld MLMM, Soubhia AMP. Clinical and histopathological analysis of oral squamous cell carcinoma in young people. A descriptive study in Brazilians. Br J Oral and Maxillofacial Surgery. 2009;47:95-98. 8. Sarkaria JN, Harari PM. Oral tongue cancer in young adults less than 40 years of age: Rationale for aggressive therapy. Head Neck. 1994;16:107-111. 9. Vargas H, Pitman KT, Johnson JT, Galati LT. More aggressive behavior of squamous cell carcinoma of the anterior tongue in young women. Laryngoscope. 2000;110:1623-1626. 10. Siriwardena BSMS, Tilakaratne A, Amaratunga EAPD, et al. Analysis of histopathological and immunohistochemical differences of oral squamous cell carcinoma in young and old patients in Sri Lanka. J Oral Pathol Med. 2007;36:357-362. 11. Vered M, Dobriyan A, Dayan D, et al. Tumor-host histopathologic variables, stromal myofibroblasts and risk score, are significantly associated with recurrent disease in tongue cancer. Cancer Sci. 2010;101:274-280.
OOOO Volume 118, Number 4 12. Annertz1 K, Anderson H, Biörklund A, et al. Incidence and survival of squamous cell carcinoma of the tongue in Scandinavia, with special reference to young adults. Int J Cancer. 2002;101:95-99. 13. Sasaki T, Moles DR, Imai Y, Speight PM. Clinico-pathological features of squamous cell carcinoma of the oral cavity in patients <40 years of age. J Oral Pathol Med. 2005;34:129-133. 14. Udeabor SE, Rana M, Wegener G, Gellrich N, Eckardt AM. Squamous cell carcinoma of the oral cavity and the oropharynx in patients less than 40 years of age: A 20-year analysis. Head Neck Oncol. 2012;4:28-35. 15. Iamaroon A, Pattanaporn K, Pongsiriwet S, et al. Analysis of 587 cases of oral squamous cell carcinoma in northern Thailand with a focus on young people. Int J Oral Maxillofac Surg. 2004; 33:84-88. 16. Toner M, O’Regan EM. Head and neck squamous cell carcinoma in the young: A spectrum or a distinct group? Part 1. Head Neck Pathol. 2009;3:246-248. 17. Santos-Silva AR, Ribeiro ACP, Soubhia AMP, et al. High incidences of DNA ploidy abnormalities in tongue squamous cell carcinoma of young patients: An international collaborative study. Histopathology. 2011;58:1127-1135. 18. Toner M, O’Regan EM. Head and neck squamous cell carcinoma in the young: A spectrum or a distinct group? Part 2. Head Neck Pathol. 2009;3:249-251. 19. Sobral LM, Bufalino A, Lopes MA, Graner E, Salo T, Coletta RD. Myofibroblasts in the stroma of oral cancer promote tumorigenesis via secretion of Activin A. Oral Oncol. 2011;47: 840-846. 20. Thode C, Jørgensen1 TG, Dabelsteen E, Mackenzie I, Dabelsteen S. Significance of myofibroblasts in oral squamous cell carcinoma. J Oral Pathol Med. 2011;40:201-207. 21. Surowiak P, Murawa D, Materna V, et al. Occurrence of stromal myofibroblasts in the invasive ductal breast cancer tissue is an unfavorable prognostic factor. Anticancer Res. 2007;27: 2917-2924. 22. Tsujino T, Seshimo I, Yamamoto H, et al. Stromal myofibroblasts predict disease recurrence for colorectal cancer. Clin Cancer Res. 2007;3:2082-2090. 23. Kellermann MG, Sobral LM, Silva SD, et al. Myofibroblasts in the stroma of oral squamous cell carcinoma are associated with poor prognosis. Histopathology. 2007;51:849-853. 24. Kellermann MG, Sobral LM, Silva SD, et al. Mutual paracrine effects of oral squamous cell carcinoma cells and normal oral fibroblasts: Induction of fibroblast to myofibroblast transdifferentiation and modulation of tumor cell proliferation. Oral Oncol. 2008;44:509-517. 25. Marsh D, Suchak K, Moutasim KA, et al. Stromal features are predictive of disease mortality in oral cancer patients. J Pathol. 2011;223:470-481. 26. Andrade BAB, León JE, Carlos R, Delgado-Azañero W, Mosqueda-Taylor A, Almeida OP. Immunohistochemical
ORIGINAL ARTICLE Fonseca et al. 489
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
expression of p16, p21, p27 and cyclin D1 in oral nevi and melanoma. Head Neck Pathol. 2012;6:297-304. Warnakulasuriya S, Mak V, Möller H. Oral cancer survival in young people in South East England. Oral Oncol. 2007;43: 982-986. Llewellyn CD, Linklater K, Bell J, Johnson NW, Warnakulasuriya S. An analysis of risk factors for oral cancer in young people: A case-control study. Oral Oncol. 2004;40:204-213. Schantz SP, Yu GP. Head and neck cancer incidence trends in young Americans, 1973-1997, with a special analysis for tongue cancer. Arch Otolaryngol Head Neck Surg. 2002;128:268-274. Hirota SK, Braga FPF, Penha SS, Sugaya NN, Migliari DA. Risk factors for oral squamous cell carcinoma in young and older Brazilian patients: A comparative analysis. Med Oral Patol Oral Cir Bucal. 2008;13:E227-E231. Thomas L, Moore EJ, McGree ME, Olsen KD, Kasperbauer JL, Erickson LA, et al. Prognostic features, human papillomavirus status, and epidermal growth factor receptor expression in oral squamous cell carcinoma in young adults. Am J Otolaryngol Head Neck Med Surg. 2012;33:650-656. Benevenuto TG, Nonaka CFW, Pinto LP, Souza LB. Immunohistochemical comparative analysis of cell proliferation and angiogenic index in squamous cell carcinomas of the tongue between young and older patients. Appl Immunohistochem Mol Morphol. 2012;20:291-297. Funk GF, Karnell LH, Robinson RA, Zhen WK, Trask DK, Hoffman HT. Presentation, treatment, and outcome of oral cavity cancer: A national cancer data base report. Head Neck. 2002;24: 165-180. Barth PJ, Schweinsberg TS, Ramaswamy A, Moll R. CD34þ fibrocytes, a-smooth muscle antigen-positive myofibroblasts, and CD117 expression in the stroma of invasive squamous cell carcinomas of the oral cavity, pharynx, and larynx. Virchows Arch. 2004;444:231-234. Etemad-Moghadam S, Khalili M, Tirgary F, Alaeddini M. Evaluation of myofibroblasts in oral epithelial dysplasia and squamous cell carcinoma. J Oral Pathol Med. 2009;38:639-643. de-Assis EM, Pimenta LGGS, Costa-e-Silva E, Souza PEA, Horta MCR. Stromal myofibroblasts in oral leukoplakia and oral squamous cell carcinoma. Med Oral Patol Oral Cir Bucal. 2012;17:E733-e738.
Reprint requests: Dr. Alan Roger Santos-Silva, DDS, PhD Oral Diagnosis Department (Semiology) Piracicaba Dental School (UNICAMP) Av. Limeira, No. 901, 13.414-903 Piracicaba São Paulo Brazil [email protected]