- Email: [email protected]
Synchronous oral leiomyosarcoma and squamous cell carcinoma
Synchronous oral leiomyosarcoma and squamous cell carcinoma Pedro Diz Dios, MD, PhD,a José Cameselle Teijeiro, MD, PhD,b Francisco Barros Anguira, BS, PhD,c Crispian Scully, MD, PhD, MDS,d Emma Vázquez García, DDS, PhD,a and Abel GarcíaGarcía, MD, PhD,a Vigo, Spain, and London, England SANTIAGO DE COMPOSTELA UNIVERSITY AND UNIVERSITY OF LONDON
An unusual case of synchronous squamous cell carcinoma and leiomyosarcoma of the oral cavity is reported in a patient without any identified environmental risk or predisposing factors. The invasive squamous cell carcinoma involved the tongue, whereas the leiomyosarcoma was located in the soft palate. No immunostaining was found for human papillomavirus or Epstein-Barr virus, and in situ hybridization showed negativity for human papillomavirus DNA within the tumor cells. Alterations of bcl-2, c-erb-b2 and Rb oncoproteins were not found immunohistochemically. Overexpression of p53 was detected by immunohistochemistry in both tumors, but p53 gene mutations were not found by polymerase chain reaction. Neither loss of heterozygosity of p53 nor microsatellite instability was detected in this patient. The smooth muscle nature of the leiomyosarcoma was confirmed by immunohistochemical methods. To our knowledge, synchronous smooth muscle and epithelial oral tumors have not previously been reported. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2001;91:70-5)
Squamous cell carcinoma (SCC) is the most common epithelial malignant neoplasm of the oral cavity.1 Classically it has been related mainly to tobacco and alcohol abuse, although other factors have also been implicated.2 An etiologic role for human papillomavirus (HPV) has also been suggested.3 Leiomyosarcoma (LMS) is a malignant smooth muscle tumor, which is usually seen in the uterus and gastrointestinal tract4 but is rare in the oral cavity.5 Although the cause of LMS remains unknown, there have been reports associating it with trauma, estrogenic stimulation and ionizing irradiation,6 and Epstein-Barr virus (EBV).7 Multiple genetic alterations of oncogenes and tumor suppressor genes in the development of human tumors involve the tumor suppressor genes retinoblastoma (Rb) and p53,8 and alterations in expression of the antiapoptotic protein bcl-2 have also been detected.9 The purpose of this article is to describe a rare case of synchronous oral LMS and SCC. In addition to evaluating the clinicopathologic features of these lesions, extensive immunohistochemical studies, in situ hybridization, and molecular genetic studies were performed. Supported in part by a grant from the Ministerio de Sanidad y Consumo (F.I.S. project n° 97/0173), Spain. aSchool of Medicine and Dentistry, Santiago de Compostela University, Spain. bDepartment of Pathology, Hospital Clínico Universitario, Santiago de Compostela, Spain. cUnit of Molecular Medicine, Santiago de Compostela, Spain. dEastman Dental Institute, University of London, London, England. Received for publication Feb 14, 2000; returned for revision Mar 2, 2000; accepted for publication Aug 14, 2000. Copyright © 2001 by Mosby, Inc. 1079-2104/2001/$35.00 + 0 7/14/111093 doi:10.1067/moe.2001.111093
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Fig 1. Initial aspect of palatal lesion.
CASE REPORT A 67-year-old white man presented with white lesions in the mouth. The patient was a retired cabinetmaker, had stopped his cigarette habit 9 years previously (earlier he had an 8-pack-year smoking history), and was a nondrinker. He had 3 sisters, 2 of whom were alive and healthy and 1 who had died of a brain tumor. His medical history included chronic obstructive pulmonary disease, hypertension, intermittent claudication, and prostatic hyperplasia. He had no history of previous neoplasms or irradiation. His current medications included nifedipine and trifusal. An examination showed an adherent white plaque covering the soft palate, buccal mucosa, ventral surface of the tongue, and floor of the mouth. A painless reddish 15-mm diameter nodule was located on the soft palate (Fig 1). The lateral tongue surface had a cobblestone pattern with a small asymptomatic keratotic lesion (Fig 2). Biopsies of the lingual and palatal lesions were performed, revealing malignancy. Because of tumor infiltration of the surgical margins of the palatal lesion, the patient was recalled. After 5 weeks, the palatal lesion became sessile, and its
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Fig 2. Cobblestone pattern and keratotic lesion on tongue surface.
Fig 3. Palatal lesion after 5 weeks (volume had increased 5fold).
volume increased 5-fold (Fig 3). A surgical resection of the entire palatal lesion was then performed with the patient under general anesthesia. At that time, chest radiograph and laboratory tests, including hemogram, biochemical, syphilis, and immunological blood tests, showed normal results.
MATERIAL AND METHODS The biopsy specimens were fixed in buffered formalin and routinely embedded in paraffin. The 5-µm histologic sections were stained with hematoxylin-eosin, and immunohistochemical studies were performed on paraffin sections by using a panel of antibodies (Table I). The reactions were evaluated by the streptavidin-biotin complex technique (Strept ABComplex/HRP, Dako, Glostrup, Denmark). Nonimmune mouse and rabbit sera were substituted for the primary antibodies as negative controls. Appropriate positive controls were run concurrently for all antibodies tested.
Fig 4. Histopathologic study of palatal mass, consistent with leiomyosarcoma. A, Fascicular architecture, cigar-shaped nuclei, and numerous mitoses (hematoxylin-eosin, original magnification ×200). B, Actin immunopositivity (original magnification ×200).
DNA-DNA in situ hybridization for HPV nucleic acids with the Super Sensitive Detection System (BioGenex, San Ramon, Calif) with fluoresceinated probes derived from HPV type 6/11, 16/18, and 31/33 (BioGenex) were performed on paraffin sections. The latent membrane protein marker for EBV was analyzed by immunohistochemistry. The evaluation of particular p53 mutations after amplification by polymerase chain reaction of exons 4 to 8 was performed both in biopsy tissues by singlestrand conformation polymorphism (SSCP) and in blood by SSCP and cyclic sequencing.
RESULTS AND FOLLOW-UP Histolopathic examination of the excisional biopsy specimen of the palatal mass showed a predominantly fascicular growth pattern, with the tumor bundles intersecting each other at wide angles under the microscope. Nuclear atypia, numerous typical
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Fig 5. Verrucous hyperplasia (A) and SCC (B) of tongue (hematoxylin-eosin, original magnification ×200).
Table I. Antibodies used for immunohistochemical analysis Antigen Ks 8, 18 Ks 1, 2, 10, 11, 14, 15, 16, 19 Ks 1, 5, 10, 14 Vimentin Smooth muscle actin Muscle-specific actin Desmin CD34 Factor VIII S-100 protein Ki-67 p53 protein Bcl-2 protein C-erbB-2 Rb protein HPV EBV, LMP
Antibody (clone and source) M, CAM5.2 (Becton Dickinson, Erembodegem, Belgium) M, AE1/AE3 (Concepta, Biosystems, Barcelona, Spain) M, 34β E12 (Enzo, Farmingdale, NY) M, V9 (Concepta, Biosystems, Barcelona, Spain) M, 1A4 (BioGenex, San Ramon, Calif) M, HHF35 (Enzo) M, D33 (Dako, Glostrup, Denmark) M, BBEnd 10 (BioGenex) M, F8/86 (Dako) P (Dako) M, MIB-1 (Immunotech, Marseille, France) M, DO-7 (Dako) M, 124 (Dako) M, CB11 (Novocastra, Newcastle upon Tyne, UK) M, G3-245 (BioGenex) P (Dako) M, CS1-4 (Dako)
Dilution 1/5 1/50 1/10 1/500 1/100 1/40 1/100 1/40 1/40 1/1000 1/20 1/10 1/5 1/100 1/200 1/10,000 1/400
Antibody retrieval Protease, citrate/microwave Protease, citrate/microwave Protease, citrate/microwave Citrate/microwave None None None Citrate/microwave Protease Citrate/microwave Protease, citrate/microwave Citrate/microwave Overnight (4°C), citrate/microwave Citrate/microwave Citrate/microwave None Citrate/microwave
M, Monoclonal; P, polyclonal; Ks, keratins.
and atypical mitotic figures, and necrosis were found (Fig 4, A). On immunohistochemical investigation, the neoplastic cells showed reactivity for vimentin, actin (1A4 and HHF35) (Fig 4, B), and desmin (Table II). Nuclear positivity for MIB-1 was found in 83% of the neoplastic cells. However, immunostaining for cytokeratins, CD34, and factor VIII-related antigen
were negative. These findings were consistent with a diagnosis of LMS. The oral mucosal specimens were histologically normal or showed acanthosis (hyperplasia) with or without keratosis (Fig 5, A). Tissue from the tongue lesion was characterized by cellular atypia and loss of a normal maturation pattern with complete disorgani-
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Fig 6. Follow-up intraoral view, 2 years after surgical treatment.
zation of all layers and foci of stromal invasion; these findings are compatible with invasive SCC (Fig 5, B). The results of immunostaining for p53, bcl-2, C-erbb-2 and Rb oncoproteins are summarized in Table III. Diffuse nuclear staining for p53 was positive in both the LMS and SCC. No p53 mutations were found either in tumor or blood samples. Normal Rb protein expression was found in all tissues examined. HPV was not detectable either by immunohistochemistry or by in situ hybridization. The antibody directed against a latent membrane protein-1 (LMP) encoded by the BNLF1 gene of EBV showed no evidence of immunoreactivity. After surgical resection of both tumors, magnetic resonance imaging of the head and neck revealed no residual tumor. A thorough systemic work-up for metastatic disease (chest radiography, digestive tract endoscopy, and abdominal echography) failed to show any underlying occult malignancy. Subsequently, the patient has undergone periodic re-evaluation in the dentistry, otolaryngology, and internal medicine departments. Two new oral SCCs were diagnosed and surgically resected from the tongue after 2 and 19 months, respectively. For the treatment of underlying leukoplakia, systemic retinoids (isotretinoin) and carbon dioxide laser vaporization were performed without success (Fig 6).
DISCUSSION To the best of our knowledge, this is the first case report of a synchronous SCC and LMS of the oral cavity. Spindle cell (sarcomatoid) carcinoma is a microscopic type of SCC that closely simulates sarcoma and might appear as an ulcerated and infiltrative mass or as a polypoid growth in the upper aerodigestive tract.2 In this patient’s palatal tumor, a blending between the LMS and SCC was not found. The palatal neoplasm had the histologic pattern and immunohistochemical profile of an LMS, and epithelial markers were negative.
Diz Dios et al 73
Primary intraoral LMS is exceptional because of the paucity of smooth muscle in that region. Izumi et al,5 in an extensive review, showed that only 60 cases had been published by 1995. Since then, 9 new cases have been described.10-17 Excluding tumors found in the maxillary sinus, lip, and perioral skin, the total number of cases of intraoral LMS reported is only 52. This tumor does not appear to affect a specific age group, but is more frequent in men than in women with a proportion of 2:1. The most frequently affected site is the jaws, whereas palatal involvement seems rare; only 4 cases in this area have been published.18 The LMS might be either painful or painless, and the development of a mass has been reported in all cases.18 The treatment of choice is surgical resection. The estimated survival rate is 70% at 2 years and 35% at 5 years.5 The p53 tumor suppressor gene is one of the most commonly implicated in tumorigenesis. About 34% to 79% of head and neck SCCs show an overexpression of p53 as detected by immunohistochemistry, and 45% of these tumors have p53 gene mutations.19 Some authors20 have correlated p53 overexpression with increased survival in patients with SCC of the tongue. However, in a recent study by Ma et al,21 61% of head and neck SCCs stained positive for p53, but this finding had no prognostic value. Multiple primary tumors are common in oral SCC, and the pattern of p53 expression is similar in these multiple tumors,22 which have been found to have a common clonal origin when analyzed karyotypically and by in situ hybridization by some investigators.23 Mutations of the p53 germ line are the molecular basis for most of the patients affected by Li-Fraumeni syndrome, and these individuals are prone to a variety of neoplasms, including bone and soft tissue sarcomas.24 In our case, both SCC and LMS overexpressed p53, but p53 gene mutations and loss of heterozygosity were not found. Concerning these results for p53, it is well recognized that overexpression of p53 does not always indicate a gene mutation.19 Yoo et al25 reported pRb abnormalities in 13% of head and neck tumors, most mutations correlating well with the absence of detectable pRb by immunohistochemical analysis. Loss of pRb expression has been related to recurrence rates and disease-free intervals in laryngeal carcinoma.26 On the other hand, somatic mutations of the Rb gene and altered patterns of pRb have been detected in sarcomas and have also been associated with aggressive behavior and poor clinical outcome.27,28 In our case, neither LMS nor SCC specimens showed altered pRb immunoexpression. The product of the bcl-2 oncogene is a membrane protein which, on upregulation, blocks apoptosis, providing a latency period for cells to become differentiated or tumorigenic. Bcl-2 protein overexpression
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Table II. Leiomyosarcoma, immunohistochemical profile Vimentin Smooth muscle actin (1A4) Muscle-specific actin (HHF35) Desmin CD34
+ + + + –
Cytokeratins (CAM5.2) Cytokeratins (AE1/AE3) Cytokeratins (34β 12) Factor VIII S 100
– – – – –
Table III. Oncoproteins, human papillomavirus, Epstein-Barr virus, and cellular proliferation (a) p 53 Bcl 2 Cerb B2 Rb HPV EBV MIB 1 (ki 67)
IHQ PCR/SSCP IHQ IHQ IHQ IHQ ISH IHQ IHQ
Leiomyosarcoma
SCC
Hyperplastic squamous mucosa
Normal mucosa
+ – – – + – – – 83%
+ ND – – + – – – 52%
– (b) ND – – + – – – (b) 30%
– (b) ND – – + – – – (b) 40%
IHQ, Immunohistochemistry; ISH, in situ hybridization; ND, not done; PCR, polymerase chain reaction; SSCP, single strand conformation polymorphism; (a), no mutations were found in germinal cell line (venous blood) by polymerase chain reaction; (b), nuclear immunostaining only in the basal layer.
has been detected in an early phase of lung SCC, and it has been suggested to be a significant prognostic factor.29 However, lack of any correlation between bcl2 overexpression and prognosis has been described in esophageal malignancies30 and in head and neck SCC.21 No overexpression of bcl-2 was found in the tumor samples of our patient. Overexpression of certain growth factor receptors has been reported both in human sarcomas31 and oropharyngeal carcinomas.32 Werkmeister et al33 have even suggested that erb-B amplifications in oral SCC play a significant role in disease-free survival. In this study, overexpression of c-erb-b2 was not observed. Reports of EBV in smooth muscle tumors in patients who are immunosuppressed suggests a possible etiologic role for the virus.7 However, in our case, the tumor was EBV-negative. Two early gene products of HPV 16, the E6 and E7 gene-encoded proteins, can bind with host cell regulatory proteins, including p53, hypophosphorylated pRb, and pRb-related proteins, thus inhibiting growth regulatory effects.34 HPV-DNA has been found in 20% to 30% of oral SCCs.35 However, in our study none of the samples contained HPV-DNA. After 3 years of follow-up, an asymptomatic enlarged cervical lymph node was detected. The patient was then referred to the otolaryngology department, and a supralaryngeal SCC was diagnosed (T2N1M0). The patient is currently receiving preoperative chemotherapy. As yet, there is no evidence of local recurrence or distant metastasis of LMS.
Overexpression of p53 was the only “oncogenetic” alteration detected in the tumor specimens. History of heavy exposure to tobacco or alcohol, specific environmental risk factors, or special conditions, such as immunosuppression, were not identified. Moreover, smooth muscle tumors probably respond to carcinogenic agents in a different way than do tumors that act on the epithelium. We thank Gonzalo Ventoso Pereira for his technical assistance. REFERENCES 1. Weir JC, Davenport WD, Skinner RL. A diagnostic and epidemiologic survey of 15,783 oral lesions. J Am Dent Assoc 1987;115:439-42. 2. Rosai J. Oral cavity and oropharynx. In: Ackerman’s surgical pathology. 8th ed, vol 1. St Louis: Mosby-Year Book; 1996. p. 223-55. 3. Woods KV, Shillitoe EJ, Spitz MR, Schantz SP, Adler S. Analysis of human papillomavirus DNA in oral squamous cell carcinomas. J Oral Pathol Med 1993;22:101-8. 4. Enzinger FM, Weiss W. Soft tissue tumors. 2nd ed. St Louis: CV Mosby; 1988. 5. Izumi K, Maeda T, Cheng J, Saku T. Primary leiomyosarcoma of the maxilla with regional lymph node metastasis. Report of a case and review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;80:310-9. 6. Cook T, Fosko S. Unusual cutaneous malignancies. Semin Cutan Med Surg 1998;17:114-32. 7. Tulbach A, Al-Dayel F, Fawaz I, Rosai J. Epstein-Barr virusassociated leiomyosarcoma of the thyroid in a child with congenital immunodeficiency. A case report. Am J Surg Pathol 1999;23:473-6. 8. Bishop JM. Molecular themes in oncogenesis. Cell 1991;64:235-48. 9. Ravi D, Nalinakumari KR, Rajaram RS, Nair MK, Pillai MR.
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Expression of programmed cell death regulatory p53 and bcl-2 proteins in oral lesions. Cancer Lett 1996;105:139-46. Piatelli A, Artese L. Leiomyosarcoma of the tongue: a case report. J Oral Maxillofac Surg 1995;53:698-701. Le Toux G, Brette MD, Arnoux Y, Pasquiou, Besset G, Monteil JP. Localisation bucco-sinusienne d’un leiomyosarcome primitif. Ann Otolaryngol Chir Cervicofac 1995;112:244-7. Mizutani H, Tohnai I, Yambe M, Ueda M. Leiomyosarcoma of the maxillary gingiva: a case report. Nagoya J Med Sci 1995;58:165-70. Mesquita RA, Migliari DA, de Sousa SO, Alves MR. Leiomyosarcoma of the buccal mucosa: a case report. J Oral Maxillofac Surg 1998;56:504-7. Karlis V, Zaslow M, Minkowitz G, Glickman RS. Leiomyosarcoma of the mandible: report of a case and literature review. J Oral Maxillofac Surg 1996;54:1127-30. Laccourreye O, Cauchois R, Laccourreye L, Maurice D, Carnot F, Brasnu D. Primary leiomyosarcoma of the mandible. Am J Otolaryn 1996;17:415-9. Moussaoui O, Menard P. Leiomyosarcome de la gencive. A propos d’un cas. Revue de la literature. Rev Stomatol Chir Maxillofac 1998;99:132-7. Goldschmidt PR, Goldschmidt JD, Lieblich SE, Eisenberg E. Leiomyosarcoma presenting as a mandibular gingival swelling: a case report. J Periodontol 1999;70:84-9. Schenberg ME, Slootweg PJ, Koole R. Leiomyosarcomas of the oral cavity. Report of four cases and review of the literature. J Craniomaxillofac Surg 1993;21:342-7. Raybaud-Diogene H, Tetu B, Morency R, Fortin A, Monteil RA. p53 overexpression in head and neck squamous cell carcinoma: review of the literature. Eur J Cancer B Oral Oncol 1996;32B:14393. Sauter ER, Ridge JA, Gordon J, Eisenberg BL. P53 overexpression correlates with increased survival in patients with squamous carcinoma of the tongue base. Am J Surg 1992;164:651-3. Ma L, Ronai A, Riede UN, Köhler G. Expression of p53 and bcl2 in squamous cell carcinoma of the head and neck: an immunohistochemical study. Appl Immunohistochem 1998;6:77-83. Nakanishi Y, Noguchi M, Matsuno Y, Saikawa M, Mukai K, Shimosato Y, et al. p53 expression in multicentric squamous cell carcinoma and surrounding squamous epithelium of the upper aerodigestive tract. Immunohistochemical analysis of 95 lesions. Cancer 1995;75:1657-62. Worsham MJ, Wolman SR, Carey TE, Zarbo RJ, Benninger MS, Van Dyke DL. Common clonal origin of synchronous primary head and neck squamous cell carcinomas. Analysis by tumor karyotypes and fluorescence in situ hybridization. Hum Pathol 1995;26:251-61. Malkin D, Li FP, Strong LC, Fraumeni JF Jr, Nelson CE, Kim
DH, et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 1990;250:1233-7. Yoo GH, Xu HJ, Brennan JA, Westra W, Hruban RH, Koch W, et al. Infrequent inactivation of the retinoblastoma gene despite frequent loss of chromosome 13q in head and neck squamous cell carcinoma. Cancer Res 1994;54:4603-6. Mizokami H, Sawatsubasi M, Tokunaga O, Shin T. Loss of retinoblastoma protein expression in laryngeal squamous cell carcinoma. Mod Pathol 1999;12:47-53. Cance WG, Brennan MR, Dudas ME, Huang CM, CordonCardo C. Altered expression of the retinoblastoma gene product in human sarcomas. N Engl J Med 1990;323:1457-61. Wunder JS, Czitrom AA, Kandel R, Andrulis IL. Analysis of alterations in the retinoblastoma gene and tumor grade in bone and soft tissue sarcomas. J Natl Cancer Inst 1991;83:194-7. Pezzella F, Turley H, Kuzu I, Tungekar MF, Dunnill MS, Pierce CB, et al. Bcl-2 protein in non-small-cell lung carcinoma. N Engl J Med 1993;329:690-4. Sarbia M, Bittinger F, Porschen R, Verreet P, Dutkowski P, Willers K, et al. Bcl-2 expression in squamous cell carcinomas of the oesophagus. Path Res Pract 1992;192:340-3. Garberoglio C, Cordon-Cardo C, Sorvillo J, Dudas M, Casper ES, Bestro J, et al. Expression of epidermal growth factor receptor and transforming growth factor alpha in human myogenic sarcomas. J Exp Clin Cancer Res 1992;11:83-7. Saranath D, Panchal RG, Nair R, Mehta AR, Sanghavi VD, Deo MD. Amplification and overexpression of epidermal growth factor receptor gene in human oropharyngeal cancer. Eur J Cancer B Oral Oncol 1992;28B:139-43. Werkmeister R, Brandt B, Joos U. The erb-B oncogenes as prognostic markers in oral squamous cell carcinomas. Am J Surg 1996;172:681-3. Shindoh M, Chiba I, Yasuda M, Saito T, Funaoka K, Kohgo T, et al. Detection of human papillomavirus DNA sequences in oral squamous cell carcinomas and their relation to p53 and proliferating cell nuclear antigen expression. Cancer 1995;76:1513-21. Chiba I, Shindoh M, Yasuda M, Yamazaki Y, Amemiya A, Sato Y, et al. Mutations in the p53 gene and human papillomavirus infection as significant prognostic factors in squamous cell carcinomas of the oral cavity. Oncogene 1996;12:1663-8.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21. 22.
23.
24.
25.
26. 27. 28. 29. 30. 31.
32.
33. 34.
35.
Reprint requests: Pedro Diz Dios, MD, PhD c/ Panamá 2; 2°Dcha 36203-Vigo Spain [email protected]
An unusual case of synchronous squamous cell carcinoma and leiomyosarcoma of the oral cavity is reported in a patient without any identified environmental risk or predisposing factors. The invasive squamous cell carcinoma involved the tongue, whereas the leiomyosarcoma was located in the soft palate. No immunostaining was found for human papillomavirus or Epstein-Barr virus, and in situ hybridization showed negativity for human papillomavirus DNA within the tumor cells. Alterations of bcl-2, c-erb-b2 and Rb oncoproteins were not found immunohistochemically. Overexpression of p53 was detected by immunohistochemistry in both tumors, but p53 gene mutations were not found by polymerase chain reaction. Neither loss of heterozygosity of p53 nor microsatellite instability was detected in this patient. The smooth muscle nature of the leiomyosarcoma was confirmed by immunohistochemical methods. To our knowledge, synchronous smooth muscle and epithelial oral tumors have not previously been reported. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2001;91:70-5)
Squamous cell carcinoma (SCC) is the most common epithelial malignant neoplasm of the oral cavity.1 Classically it has been related mainly to tobacco and alcohol abuse, although other factors have also been implicated.2 An etiologic role for human papillomavirus (HPV) has also been suggested.3 Leiomyosarcoma (LMS) is a malignant smooth muscle tumor, which is usually seen in the uterus and gastrointestinal tract4 but is rare in the oral cavity.5 Although the cause of LMS remains unknown, there have been reports associating it with trauma, estrogenic stimulation and ionizing irradiation,6 and Epstein-Barr virus (EBV).7 Multiple genetic alterations of oncogenes and tumor suppressor genes in the development of human tumors involve the tumor suppressor genes retinoblastoma (Rb) and p53,8 and alterations in expression of the antiapoptotic protein bcl-2 have also been detected.9 The purpose of this article is to describe a rare case of synchronous oral LMS and SCC. In addition to evaluating the clinicopathologic features of these lesions, extensive immunohistochemical studies, in situ hybridization, and molecular genetic studies were performed. Supported in part by a grant from the Ministerio de Sanidad y Consumo (F.I.S. project n° 97/0173), Spain. aSchool of Medicine and Dentistry, Santiago de Compostela University, Spain. bDepartment of Pathology, Hospital Clínico Universitario, Santiago de Compostela, Spain. cUnit of Molecular Medicine, Santiago de Compostela, Spain. dEastman Dental Institute, University of London, London, England. Received for publication Feb 14, 2000; returned for revision Mar 2, 2000; accepted for publication Aug 14, 2000. Copyright © 2001 by Mosby, Inc. 1079-2104/2001/$35.00 + 0 7/14/111093 doi:10.1067/moe.2001.111093
70
Fig 1. Initial aspect of palatal lesion.
CASE REPORT A 67-year-old white man presented with white lesions in the mouth. The patient was a retired cabinetmaker, had stopped his cigarette habit 9 years previously (earlier he had an 8-pack-year smoking history), and was a nondrinker. He had 3 sisters, 2 of whom were alive and healthy and 1 who had died of a brain tumor. His medical history included chronic obstructive pulmonary disease, hypertension, intermittent claudication, and prostatic hyperplasia. He had no history of previous neoplasms or irradiation. His current medications included nifedipine and trifusal. An examination showed an adherent white plaque covering the soft palate, buccal mucosa, ventral surface of the tongue, and floor of the mouth. A painless reddish 15-mm diameter nodule was located on the soft palate (Fig 1). The lateral tongue surface had a cobblestone pattern with a small asymptomatic keratotic lesion (Fig 2). Biopsies of the lingual and palatal lesions were performed, revealing malignancy. Because of tumor infiltration of the surgical margins of the palatal lesion, the patient was recalled. After 5 weeks, the palatal lesion became sessile, and its
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Diz Dios et al 71
Fig 2. Cobblestone pattern and keratotic lesion on tongue surface.
Fig 3. Palatal lesion after 5 weeks (volume had increased 5fold).
volume increased 5-fold (Fig 3). A surgical resection of the entire palatal lesion was then performed with the patient under general anesthesia. At that time, chest radiograph and laboratory tests, including hemogram, biochemical, syphilis, and immunological blood tests, showed normal results.
MATERIAL AND METHODS The biopsy specimens were fixed in buffered formalin and routinely embedded in paraffin. The 5-µm histologic sections were stained with hematoxylin-eosin, and immunohistochemical studies were performed on paraffin sections by using a panel of antibodies (Table I). The reactions were evaluated by the streptavidin-biotin complex technique (Strept ABComplex/HRP, Dako, Glostrup, Denmark). Nonimmune mouse and rabbit sera were substituted for the primary antibodies as negative controls. Appropriate positive controls were run concurrently for all antibodies tested.
Fig 4. Histopathologic study of palatal mass, consistent with leiomyosarcoma. A, Fascicular architecture, cigar-shaped nuclei, and numerous mitoses (hematoxylin-eosin, original magnification ×200). B, Actin immunopositivity (original magnification ×200).
DNA-DNA in situ hybridization for HPV nucleic acids with the Super Sensitive Detection System (BioGenex, San Ramon, Calif) with fluoresceinated probes derived from HPV type 6/11, 16/18, and 31/33 (BioGenex) were performed on paraffin sections. The latent membrane protein marker for EBV was analyzed by immunohistochemistry. The evaluation of particular p53 mutations after amplification by polymerase chain reaction of exons 4 to 8 was performed both in biopsy tissues by singlestrand conformation polymorphism (SSCP) and in blood by SSCP and cyclic sequencing.
RESULTS AND FOLLOW-UP Histolopathic examination of the excisional biopsy specimen of the palatal mass showed a predominantly fascicular growth pattern, with the tumor bundles intersecting each other at wide angles under the microscope. Nuclear atypia, numerous typical
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ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY January 2001
Fig 5. Verrucous hyperplasia (A) and SCC (B) of tongue (hematoxylin-eosin, original magnification ×200).
Table I. Antibodies used for immunohistochemical analysis Antigen Ks 8, 18 Ks 1, 2, 10, 11, 14, 15, 16, 19 Ks 1, 5, 10, 14 Vimentin Smooth muscle actin Muscle-specific actin Desmin CD34 Factor VIII S-100 protein Ki-67 p53 protein Bcl-2 protein C-erbB-2 Rb protein HPV EBV, LMP
Antibody (clone and source) M, CAM5.2 (Becton Dickinson, Erembodegem, Belgium) M, AE1/AE3 (Concepta, Biosystems, Barcelona, Spain) M, 34β E12 (Enzo, Farmingdale, NY) M, V9 (Concepta, Biosystems, Barcelona, Spain) M, 1A4 (BioGenex, San Ramon, Calif) M, HHF35 (Enzo) M, D33 (Dako, Glostrup, Denmark) M, BBEnd 10 (BioGenex) M, F8/86 (Dako) P (Dako) M, MIB-1 (Immunotech, Marseille, France) M, DO-7 (Dako) M, 124 (Dako) M, CB11 (Novocastra, Newcastle upon Tyne, UK) M, G3-245 (BioGenex) P (Dako) M, CS1-4 (Dako)
Dilution 1/5 1/50 1/10 1/500 1/100 1/40 1/100 1/40 1/40 1/1000 1/20 1/10 1/5 1/100 1/200 1/10,000 1/400
Antibody retrieval Protease, citrate/microwave Protease, citrate/microwave Protease, citrate/microwave Citrate/microwave None None None Citrate/microwave Protease Citrate/microwave Protease, citrate/microwave Citrate/microwave Overnight (4°C), citrate/microwave Citrate/microwave Citrate/microwave None Citrate/microwave
M, Monoclonal; P, polyclonal; Ks, keratins.
and atypical mitotic figures, and necrosis were found (Fig 4, A). On immunohistochemical investigation, the neoplastic cells showed reactivity for vimentin, actin (1A4 and HHF35) (Fig 4, B), and desmin (Table II). Nuclear positivity for MIB-1 was found in 83% of the neoplastic cells. However, immunostaining for cytokeratins, CD34, and factor VIII-related antigen
were negative. These findings were consistent with a diagnosis of LMS. The oral mucosal specimens were histologically normal or showed acanthosis (hyperplasia) with or without keratosis (Fig 5, A). Tissue from the tongue lesion was characterized by cellular atypia and loss of a normal maturation pattern with complete disorgani-
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Fig 6. Follow-up intraoral view, 2 years after surgical treatment.
zation of all layers and foci of stromal invasion; these findings are compatible with invasive SCC (Fig 5, B). The results of immunostaining for p53, bcl-2, C-erbb-2 and Rb oncoproteins are summarized in Table III. Diffuse nuclear staining for p53 was positive in both the LMS and SCC. No p53 mutations were found either in tumor or blood samples. Normal Rb protein expression was found in all tissues examined. HPV was not detectable either by immunohistochemistry or by in situ hybridization. The antibody directed against a latent membrane protein-1 (LMP) encoded by the BNLF1 gene of EBV showed no evidence of immunoreactivity. After surgical resection of both tumors, magnetic resonance imaging of the head and neck revealed no residual tumor. A thorough systemic work-up for metastatic disease (chest radiography, digestive tract endoscopy, and abdominal echography) failed to show any underlying occult malignancy. Subsequently, the patient has undergone periodic re-evaluation in the dentistry, otolaryngology, and internal medicine departments. Two new oral SCCs were diagnosed and surgically resected from the tongue after 2 and 19 months, respectively. For the treatment of underlying leukoplakia, systemic retinoids (isotretinoin) and carbon dioxide laser vaporization were performed without success (Fig 6).
DISCUSSION To the best of our knowledge, this is the first case report of a synchronous SCC and LMS of the oral cavity. Spindle cell (sarcomatoid) carcinoma is a microscopic type of SCC that closely simulates sarcoma and might appear as an ulcerated and infiltrative mass or as a polypoid growth in the upper aerodigestive tract.2 In this patient’s palatal tumor, a blending between the LMS and SCC was not found. The palatal neoplasm had the histologic pattern and immunohistochemical profile of an LMS, and epithelial markers were negative.
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Primary intraoral LMS is exceptional because of the paucity of smooth muscle in that region. Izumi et al,5 in an extensive review, showed that only 60 cases had been published by 1995. Since then, 9 new cases have been described.10-17 Excluding tumors found in the maxillary sinus, lip, and perioral skin, the total number of cases of intraoral LMS reported is only 52. This tumor does not appear to affect a specific age group, but is more frequent in men than in women with a proportion of 2:1. The most frequently affected site is the jaws, whereas palatal involvement seems rare; only 4 cases in this area have been published.18 The LMS might be either painful or painless, and the development of a mass has been reported in all cases.18 The treatment of choice is surgical resection. The estimated survival rate is 70% at 2 years and 35% at 5 years.5 The p53 tumor suppressor gene is one of the most commonly implicated in tumorigenesis. About 34% to 79% of head and neck SCCs show an overexpression of p53 as detected by immunohistochemistry, and 45% of these tumors have p53 gene mutations.19 Some authors20 have correlated p53 overexpression with increased survival in patients with SCC of the tongue. However, in a recent study by Ma et al,21 61% of head and neck SCCs stained positive for p53, but this finding had no prognostic value. Multiple primary tumors are common in oral SCC, and the pattern of p53 expression is similar in these multiple tumors,22 which have been found to have a common clonal origin when analyzed karyotypically and by in situ hybridization by some investigators.23 Mutations of the p53 germ line are the molecular basis for most of the patients affected by Li-Fraumeni syndrome, and these individuals are prone to a variety of neoplasms, including bone and soft tissue sarcomas.24 In our case, both SCC and LMS overexpressed p53, but p53 gene mutations and loss of heterozygosity were not found. Concerning these results for p53, it is well recognized that overexpression of p53 does not always indicate a gene mutation.19 Yoo et al25 reported pRb abnormalities in 13% of head and neck tumors, most mutations correlating well with the absence of detectable pRb by immunohistochemical analysis. Loss of pRb expression has been related to recurrence rates and disease-free intervals in laryngeal carcinoma.26 On the other hand, somatic mutations of the Rb gene and altered patterns of pRb have been detected in sarcomas and have also been associated with aggressive behavior and poor clinical outcome.27,28 In our case, neither LMS nor SCC specimens showed altered pRb immunoexpression. The product of the bcl-2 oncogene is a membrane protein which, on upregulation, blocks apoptosis, providing a latency period for cells to become differentiated or tumorigenic. Bcl-2 protein overexpression
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Table II. Leiomyosarcoma, immunohistochemical profile Vimentin Smooth muscle actin (1A4) Muscle-specific actin (HHF35) Desmin CD34
+ + + + –
Cytokeratins (CAM5.2) Cytokeratins (AE1/AE3) Cytokeratins (34β 12) Factor VIII S 100
– – – – –
Table III. Oncoproteins, human papillomavirus, Epstein-Barr virus, and cellular proliferation (a) p 53 Bcl 2 Cerb B2 Rb HPV EBV MIB 1 (ki 67)
IHQ PCR/SSCP IHQ IHQ IHQ IHQ ISH IHQ IHQ
Leiomyosarcoma
SCC
Hyperplastic squamous mucosa
Normal mucosa
+ – – – + – – – 83%
+ ND – – + – – – 52%
– (b) ND – – + – – – (b) 30%
– (b) ND – – + – – – (b) 40%
IHQ, Immunohistochemistry; ISH, in situ hybridization; ND, not done; PCR, polymerase chain reaction; SSCP, single strand conformation polymorphism; (a), no mutations were found in germinal cell line (venous blood) by polymerase chain reaction; (b), nuclear immunostaining only in the basal layer.
has been detected in an early phase of lung SCC, and it has been suggested to be a significant prognostic factor.29 However, lack of any correlation between bcl2 overexpression and prognosis has been described in esophageal malignancies30 and in head and neck SCC.21 No overexpression of bcl-2 was found in the tumor samples of our patient. Overexpression of certain growth factor receptors has been reported both in human sarcomas31 and oropharyngeal carcinomas.32 Werkmeister et al33 have even suggested that erb-B amplifications in oral SCC play a significant role in disease-free survival. In this study, overexpression of c-erb-b2 was not observed. Reports of EBV in smooth muscle tumors in patients who are immunosuppressed suggests a possible etiologic role for the virus.7 However, in our case, the tumor was EBV-negative. Two early gene products of HPV 16, the E6 and E7 gene-encoded proteins, can bind with host cell regulatory proteins, including p53, hypophosphorylated pRb, and pRb-related proteins, thus inhibiting growth regulatory effects.34 HPV-DNA has been found in 20% to 30% of oral SCCs.35 However, in our study none of the samples contained HPV-DNA. After 3 years of follow-up, an asymptomatic enlarged cervical lymph node was detected. The patient was then referred to the otolaryngology department, and a supralaryngeal SCC was diagnosed (T2N1M0). The patient is currently receiving preoperative chemotherapy. As yet, there is no evidence of local recurrence or distant metastasis of LMS.
Overexpression of p53 was the only “oncogenetic” alteration detected in the tumor specimens. History of heavy exposure to tobacco or alcohol, specific environmental risk factors, or special conditions, such as immunosuppression, were not identified. Moreover, smooth muscle tumors probably respond to carcinogenic agents in a different way than do tumors that act on the epithelium. We thank Gonzalo Ventoso Pereira for his technical assistance. REFERENCES 1. Weir JC, Davenport WD, Skinner RL. A diagnostic and epidemiologic survey of 15,783 oral lesions. J Am Dent Assoc 1987;115:439-42. 2. Rosai J. Oral cavity and oropharynx. In: Ackerman’s surgical pathology. 8th ed, vol 1. St Louis: Mosby-Year Book; 1996. p. 223-55. 3. Woods KV, Shillitoe EJ, Spitz MR, Schantz SP, Adler S. Analysis of human papillomavirus DNA in oral squamous cell carcinomas. J Oral Pathol Med 1993;22:101-8. 4. Enzinger FM, Weiss W. Soft tissue tumors. 2nd ed. St Louis: CV Mosby; 1988. 5. Izumi K, Maeda T, Cheng J, Saku T. Primary leiomyosarcoma of the maxilla with regional lymph node metastasis. Report of a case and review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;80:310-9. 6. Cook T, Fosko S. Unusual cutaneous malignancies. Semin Cutan Med Surg 1998;17:114-32. 7. Tulbach A, Al-Dayel F, Fawaz I, Rosai J. Epstein-Barr virusassociated leiomyosarcoma of the thyroid in a child with congenital immunodeficiency. A case report. Am J Surg Pathol 1999;23:473-6. 8. Bishop JM. Molecular themes in oncogenesis. Cell 1991;64:235-48. 9. Ravi D, Nalinakumari KR, Rajaram RS, Nair MK, Pillai MR.
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Reprint requests: Pedro Diz Dios, MD, PhD c/ Panamá 2; 2°Dcha 36203-Vigo Spain [email protected]