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ΔNp63α as early indicator of malignancy in surgical margins of an oral squamous cell carcinoma
Oral Oncology EXTRA (2005) 41, 129–131
http://intl.elsevierhealth.com/journal/ooex
CASE REPORT
DNp63a as early indicator of malignancy in surgical margins of an oral squamous cell carcinoma B. Mognetti a, E. Trione a, G. Corvetti b, E. Pomatto c, F. Di Carlo a, G.N. Berta a,*, V. Vercellino c a
Pharmacology Unit, Department of Clinical and Biological Science, University of Turin, Ospedale San Luigi, Reg. Gonzole 10, 10043 Orbassano (TO), Italy b Anatomy Unit, Department of Clinical and Biological Science, University of Turin, Italy c Unit of Oral and Maxillofacial Surgery, Department of Clinical and Biological Science, University of Turin, Italy Received 13 January 2005; accepted 11 February 2005
KEYWORDS
Summary Abnormalities in DNp63 expression have been regarded as early indicators of carcinogenesis of squamous origin, including oral carcinomas. In our hypothesis, the a isoform of DNp63 might represent an early marker for identifying neoplastic tissues left behind after surgical ablation of an oral carcinoma. To demonstrate this hypothesis, we are prospectively screening DNp63a overexpression in surgical margins. In one of our analysis we found a correlation between DNp63a overexpression and the presence of tumoral cells subsequently confirmed by histology. This observation supports our hypothesis while waiting for the main study to be completed. c 2005 Elsevier Ltd. All rights reserved.
Oral squamous cell carcinoma; RT-PCR; DNp63; Clean surgical margins
Introduction
Surgery remains the elective therapy for oral cancer. As important as the complete tumor ablation, the clearance of surgical margins at the primary site is widely thought to influence the subsequent * Corresponding author. Tel.: +39 0116705446; fax: +39 0119038639. E-mail address: [email protected] (G.N. Berta).
course of the disease in patients operated on for oral or oropharyngeal carcinoma. The 5-years tumor-specific survival of patients with a positive margin is poorer by 12% than for those patients with a negative margin, since 66% of patients with positive margins develop a primary site recurrence.1 McMahon et al. suggest to remove a 1-cm macroscopic margin2 but often a tissue undergoing a malignant transformation maintains normal anatomical features, and is therefore difficult to
1741-9409/$ - see front matter c 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ooe.2005.02.010
130 diagnose properly as tumoral at a gross morphological analysis. Hence, it is of paramount importance to find early diagnostic factors through which recognize a tissue undergoing tumoral transformation before any morphological changes occur. A good candidate to this goal might be DNp63a, an isoform of p63 (a member of the p53 gene family3) lacking the NH2-terminal transactivation domain. The DN isoforms block p53 activities by competing for DNA binding sites and therefore favor cell proliferation.4 Their expression has already been documented in oral5 as well as in other tumors6 of squamous origin. Currently, we are screening the morphologically normal clean surgical margins to detect any possible correlation between DNp63a expression and relapse. This prospectic work will require several years. Here we report a case we have recently identified and that we consider significant for supporting our hypothesis.
B. Mognetti et al.
Figure 1 Retrotranscription and PCR amplification of mRNA coding for DNp63a and GAPDH (internal control) in tumor, controlateral healthy mucosa and three different surgical margins after tumor ablation. One of the margins (no. 3) expresses as much DNp63a as the tumor tissue.
Case report A 68 year-old male patient was operated to surgically remove a lesion of the right floor of mouth previously diagnosed as squamous carcinoma of the oral mucosa. The patient never underwent chemotherapy or radiotherapy before surgery. The exsected clean surgical margins were routinely analyzed by the pathological service, whose first analysis considered the margins as clean. We snap-froze a tumor fragment, a portion of the ablated margins and a sample of healthy controlateral mucosa (considered as baseline control) and screened them for DNp63a messenger RNA (mRNA) expression by retrotranscription and polymerase chain reaction (RT-PCR). We found that DNp63a mRNA expression in one of the margins was 6.6 folds higher than the baseline level, and comparable to that measured in the tumor (Fig. 1). In this same margin, only a more accurate and time consuming histological analysis on seriated slides later recognized the presence of neoplastic cells of squamous origin (Fig. 2).
Discussion We show that the identification of DNp63a mRNA overexpression by RT-PCR in tissue surrounding a neoplastic lesion correlated with the presence of tumor cells (which could be a micrometastasis or a second primary tumor) as confirmed by histological analysis. This technique permits to analyze, by extracting and pooling mRNA, the whole tissue fragment, while, in order to individuate a small
Figure 2 Histological analysis of surgical margin no. 3. A nest of neoplastic cells of squamous origin is shown. H & E, bar = 40 lm.
neoplastic mass by histology, the section has to include it. This might be difficult when the lesion is composed by a few cells, and the histological analysis risks to neglect it. Therefore, we consider this test a good method to screen the clean surgical margins to support the classic histological analysis. We will follow up for long periods a large number of patients to verify if a correlation can be established between DNp63a positivity in left-in place tissues and relapse. If DNp63 overexpression, as it
DNp63a as early indicator of malignancy in surgical margins of an oral squamous cell carcinoma seems, occurs early in the development of squamous carcinomas,6 it may have important implications in early detection strategies and, therefore, on the poor prognosis characterizing this kind of cancer.
Acknowledgements This work was supported by CRT Foundation and by ENEL workers, Acqui Terme (Italy); BM is supported by a grant from Ricerca Scientifica Applicata, Regione Piemonte.
2.
3.
4.
5.
References 6. 1. Jones AS, Hanafi ZB, Nadapalan V, Roland NJ, Kinsella A, Helliwell TR. Do positive resection margins after ablative
131
surgery for head and neck cancer adversely affect prognosis? A study of 352 patients with recurrent carcinoma following radiotherapy treated by salvage surgery. Br J Cancer 1996; 74:128–32. McMahon J, O’Brien CJ, Pathak I, et al. Influence of condition of surgical margins on local recurrence and disease-specific survival in oral and oropharyngeal cancer. Br J Oral Maxillofac Surg 2003;41:224–31. Schmale H, Bamberger C. A novel protein with strong homology to the tumor suppressor p53. Oncogene 1997;15: 1363–7. Dietz S, Rother K, Bamberger C, Schmale H, Mossner J, Engeland K. Differential regulation of transcription and induction of programmed cell death by human p53-family members p63 and p73. FEBS Lett 2002;525:93–9. Chen YK, Hsue SS, Lin LM. Expression of p63 (TA and deltaN isoforms) in human primary well differentiated buccal carcinomas. Int J Oral Maxillofac Surg 2004;33:493–7. Massion PP, Taflan PM, Rahman SMJ, et al. Significance of p63 amplification and overexpression in lung cancer development and prognosis. Cancer Res 2003;63:7113–21.
http://intl.elsevierhealth.com/journal/ooex
CASE REPORT
DNp63a as early indicator of malignancy in surgical margins of an oral squamous cell carcinoma B. Mognetti a, E. Trione a, G. Corvetti b, E. Pomatto c, F. Di Carlo a, G.N. Berta a,*, V. Vercellino c a
Pharmacology Unit, Department of Clinical and Biological Science, University of Turin, Ospedale San Luigi, Reg. Gonzole 10, 10043 Orbassano (TO), Italy b Anatomy Unit, Department of Clinical and Biological Science, University of Turin, Italy c Unit of Oral and Maxillofacial Surgery, Department of Clinical and Biological Science, University of Turin, Italy Received 13 January 2005; accepted 11 February 2005
KEYWORDS
Summary Abnormalities in DNp63 expression have been regarded as early indicators of carcinogenesis of squamous origin, including oral carcinomas. In our hypothesis, the a isoform of DNp63 might represent an early marker for identifying neoplastic tissues left behind after surgical ablation of an oral carcinoma. To demonstrate this hypothesis, we are prospectively screening DNp63a overexpression in surgical margins. In one of our analysis we found a correlation between DNp63a overexpression and the presence of tumoral cells subsequently confirmed by histology. This observation supports our hypothesis while waiting for the main study to be completed. c 2005 Elsevier Ltd. All rights reserved.
Oral squamous cell carcinoma; RT-PCR; DNp63; Clean surgical margins
Introduction
Surgery remains the elective therapy for oral cancer. As important as the complete tumor ablation, the clearance of surgical margins at the primary site is widely thought to influence the subsequent * Corresponding author. Tel.: +39 0116705446; fax: +39 0119038639. E-mail address: [email protected] (G.N. Berta).
course of the disease in patients operated on for oral or oropharyngeal carcinoma. The 5-years tumor-specific survival of patients with a positive margin is poorer by 12% than for those patients with a negative margin, since 66% of patients with positive margins develop a primary site recurrence.1 McMahon et al. suggest to remove a 1-cm macroscopic margin2 but often a tissue undergoing a malignant transformation maintains normal anatomical features, and is therefore difficult to
1741-9409/$ - see front matter c 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ooe.2005.02.010
130 diagnose properly as tumoral at a gross morphological analysis. Hence, it is of paramount importance to find early diagnostic factors through which recognize a tissue undergoing tumoral transformation before any morphological changes occur. A good candidate to this goal might be DNp63a, an isoform of p63 (a member of the p53 gene family3) lacking the NH2-terminal transactivation domain. The DN isoforms block p53 activities by competing for DNA binding sites and therefore favor cell proliferation.4 Their expression has already been documented in oral5 as well as in other tumors6 of squamous origin. Currently, we are screening the morphologically normal clean surgical margins to detect any possible correlation between DNp63a expression and relapse. This prospectic work will require several years. Here we report a case we have recently identified and that we consider significant for supporting our hypothesis.
B. Mognetti et al.
Figure 1 Retrotranscription and PCR amplification of mRNA coding for DNp63a and GAPDH (internal control) in tumor, controlateral healthy mucosa and three different surgical margins after tumor ablation. One of the margins (no. 3) expresses as much DNp63a as the tumor tissue.
Case report A 68 year-old male patient was operated to surgically remove a lesion of the right floor of mouth previously diagnosed as squamous carcinoma of the oral mucosa. The patient never underwent chemotherapy or radiotherapy before surgery. The exsected clean surgical margins were routinely analyzed by the pathological service, whose first analysis considered the margins as clean. We snap-froze a tumor fragment, a portion of the ablated margins and a sample of healthy controlateral mucosa (considered as baseline control) and screened them for DNp63a messenger RNA (mRNA) expression by retrotranscription and polymerase chain reaction (RT-PCR). We found that DNp63a mRNA expression in one of the margins was 6.6 folds higher than the baseline level, and comparable to that measured in the tumor (Fig. 1). In this same margin, only a more accurate and time consuming histological analysis on seriated slides later recognized the presence of neoplastic cells of squamous origin (Fig. 2).
Discussion We show that the identification of DNp63a mRNA overexpression by RT-PCR in tissue surrounding a neoplastic lesion correlated with the presence of tumor cells (which could be a micrometastasis or a second primary tumor) as confirmed by histological analysis. This technique permits to analyze, by extracting and pooling mRNA, the whole tissue fragment, while, in order to individuate a small
Figure 2 Histological analysis of surgical margin no. 3. A nest of neoplastic cells of squamous origin is shown. H & E, bar = 40 lm.
neoplastic mass by histology, the section has to include it. This might be difficult when the lesion is composed by a few cells, and the histological analysis risks to neglect it. Therefore, we consider this test a good method to screen the clean surgical margins to support the classic histological analysis. We will follow up for long periods a large number of patients to verify if a correlation can be established between DNp63a positivity in left-in place tissues and relapse. If DNp63 overexpression, as it
DNp63a as early indicator of malignancy in surgical margins of an oral squamous cell carcinoma seems, occurs early in the development of squamous carcinomas,6 it may have important implications in early detection strategies and, therefore, on the poor prognosis characterizing this kind of cancer.
Acknowledgements This work was supported by CRT Foundation and by ENEL workers, Acqui Terme (Italy); BM is supported by a grant from Ricerca Scientifica Applicata, Regione Piemonte.
2.
3.
4.
5.
References 6. 1. Jones AS, Hanafi ZB, Nadapalan V, Roland NJ, Kinsella A, Helliwell TR. Do positive resection margins after ablative
131
surgery for head and neck cancer adversely affect prognosis? A study of 352 patients with recurrent carcinoma following radiotherapy treated by salvage surgery. Br J Cancer 1996; 74:128–32. McMahon J, O’Brien CJ, Pathak I, et al. Influence of condition of surgical margins on local recurrence and disease-specific survival in oral and oropharyngeal cancer. Br J Oral Maxillofac Surg 2003;41:224–31. Schmale H, Bamberger C. A novel protein with strong homology to the tumor suppressor p53. Oncogene 1997;15: 1363–7. Dietz S, Rother K, Bamberger C, Schmale H, Mossner J, Engeland K. Differential regulation of transcription and induction of programmed cell death by human p53-family members p63 and p73. FEBS Lett 2002;525:93–9. Chen YK, Hsue SS, Lin LM. Expression of p63 (TA and deltaN isoforms) in human primary well differentiated buccal carcinomas. Int J Oral Maxillofac Surg 2004;33:493–7. Massion PP, Taflan PM, Rahman SMJ, et al. Significance of p63 amplification and overexpression in lung cancer development and prognosis. Cancer Res 2003;63:7113–21.