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Prognostic value of tissue polypeptide antigen in oral squamous cell carcinoma
Oral Oncology 47 (2011) 114–120
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Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Prognostic value of tissue polypeptide antigen in oral squamous cell carcinoma Sharada S. Sawant a,⇑, Devendra A. Chaukar b, Shreyas S. Joshi a, Prerana P. Dange a, Sadhana Kannan a, Shubhada Kane c, Anil K. D’Cruz b, Milind M. Vaidya a a
Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai, 410210 Maharashtra, India Oral Surgery, Head and Neck Unit, Tata Memorial Hospital, Parel, Mumbai 40012, India c Pathology Department, Tata Memorial Hospital, Parel, Mumbai 40012, India b
a r t i c l e
i n f o
Article history: Received 16 September 2010 Received in revised form 8 November 2010 Accepted 8 November 2010 Available online 3 December 2010 Keywords: Oral cancer Squamous cell carcinoma Cytokeratins Serum tumour markers Tissue polypeptide antigen Prognosis
s u m m a r y Human oral cancer has a high risk of locoregional relapse which is difficult to diagnose early due to lack of prognostic markers. We and others have shown aberrant expression of cytokeratin (CK) 8 and 18 in human oral cancer. Aberrant supra-basal expression of CK19 has been shown earlier. In this study, our aim was to develop a non-invasive test for prognostication of human oral cancer. Immunoradiometric assay (IRMA) was used to measure the circulating levels of TPA in the sera of 80 oral cancer patients before surgery and seven days after surgery. Elevated serum TPA levels were noted in the post surgery samples of 28 out of the 50 patients for whom clinical follow-up was available, as compared to their pre-surgery samples. TPA levels in the pre-surgery serum samples of patients were significantly higher than levels in the sera of healthy controls (p = 0.001). Elevated levels in patients correlated significantly with stage (p = 0.02), development of recurrence (p < 0.006), and impacted survival (p < 0.033). IHC analysis showed statistically significant correlation between expression of CK8, 18 and 19 in surrounding uninvolved tissues of the tumour and post surgery elevated serum TPA levels (p < 0.001). This suggests the possibility that CK fragments are released from the surrounding uninvolved tissues into the sera of patients after surgical removal of the tumour. Thus, our study indicates that TPA can be a useful tumour marker for the prediction of recurrence and poor prognosis in human oral cancer. Ó 2010 Elsevier Ltd. All rights reserved.
Introduction Human oral cancer is the sixth largest group of malignancies worldwide and the single largest malignancy in males in the Indian subcontinent.1,2 Major contributory factors for the high incidence of oral cancer are the habits of chewing tobacco and other allied products. Loco-regional recurrence and regional lymph node metastases are the major hurdles in the management of oral cancer. Early detection of disease progression remains a challenging task mainly due to the lack of adequate early prognostic markers. Primary oral carcinoma is usually superficial and easily detected. However, deeply invading tumours that recur after primary resections are usually detected only when they are large and no longer curable. The existing imaging modalities are not sensitive enough for the early diagnosis of tumour progression.3–6 CK belonging to the intermediate filament family of proteins are particularly useful tools for diagnosis in oncology. Due to the Abbreviations: CK, cytokeratins; TPA, tissue polypeptide antigen; H&N, head and neck; SCC, squamous cell carcinoma. ⇑ Corresponding author. Tel.: +91 22 27405134; fax: +91 22 27405085. E-mail address: [email protected] (S.S. Sawant). 1368-8375/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2010.11.005
enhanced proteolytic activity in malignant cells, CK fragments are released in circulation and can be quantified using various commercially available specific serological assays.7 In healthy individuals, the level of CK in the circulation is low and rises significantly in patients with carcinoma.8 The three most frequently used CK which are being evaluated as serum markers for their utility in clinical applications are tissue polypeptide antigen (TPA), tissue polypeptide specific antigen (TPS), and cytokeratin fragments 21-1 (Cyfra21-1). TPA assay is a broad spectrum assay which measures cytosolic fragments of CK8, 18, and 19. Correlation between TPA levels in the serum and disease progression has been shown earlier.9 TPA assay is being used for prognostication of lung and breast cancer.10–12 However, it has not been evaluated for the prognostication of human oral cancer. Earlier we and others have shown aberrant expression of simple epithelia specific CK8, 18 and 19 in precancerous lesions as well as squamous cell carcinoma (SCC) of the tongue and buccal mucosa (BM).13–15 In the present study, we have examined the serum levels of CK8, 18 and 19 using TPA assay in oral SCC patients before and 7 days after surgery and correlated it with the clinical parameters of the patients.
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Material and methods Patients Blood samples and tumour tissues along with their respective surrounding uninvolved areas were collected at the time of surgery from a total of 80 primary oral SCC of tongue (40 patients) and BM (40 patients) from the Tata Memorial Hospital (TMH), Mumbai, India. Blood samples were also collected 7 days after surgery. It was ensured that none of the patients had received any radiotherapy or chemotherapy before collection of the blood. Blood samples from 24 normal healthy, age and sex-matched individuals were also collected as a control group. Normal buccal mucosa tissues were obtained from 10 subjects during third molar tooth extraction from Ragas Dental College and Hospital, Chennai, India. This study was approved by the Human Ethics Committees of the respective Institutional Review Boards. Informed consent was obtained from the patients as well as healthy individuals. Clinical and histopathological records of the patients Clinico-pathological information like tumour site, size, histological grade, perineural invasion, perinodal extension, lymphovascular invasion, margins, bone, lymph node involvement, loco-regional recurrence, metastasis and survival was collected from the clinical and pathology records of TMH. The median time of clinical follow-up was 11.5 months. RIA for serum TPA
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80 whose clinical follow-up was obtained. SPSS software version 15 was used for statistical analysis. Differences with a probability value of <0.05 were considered statistically significant. Specificity, sensitivity, and positive and negative prediction values for TPA were determined. The cut-off values for serum TPA levels in patients were determined with reference to TPA values in healthy individuals using Kaplan Meier test. Pearson chi-square test was used for correlations between higher TPA levels in post surgery serum samples and clinical parameters of the patients. TPA levels in the post surgery samples and CK8, 18 and 19 localization in the surrounding un-involved areas of the tumour were correlated using Pearson Chi-Square Test.
Results Serum TPA levels Cut-off value for serum TPA levels was 140.93 U/L (mean ±1 SD of normal). Values of sensitivity, specificity, and positive and negative prediction for TPA were 78%, 61%, 68% and 72%, respectively. TPA levels in the pre-surgery serum samples were significantly greater than the serum samples of healthy individuals by 82.07% (p = 0.001, t-test). The serum TPA concentrations obtained for the 80 patients were used to study the pre and post surgery TPA levels in tongue and BM. The pre-surgery mean serum TPA levels in BM samples were higher than tongue samples by 49.5%. The BM samples showed 19.46% decrease in TPA concentration after the surgery as compared to 14.35% in tongue samples (Fig. 1).
TPA levels were estimated using a TPA-M IRMA (two-site monoclonal/polyclonal immunoradiometric sandwich assay) kit from Prolifigen (DiaSorin – USA) according to the manufacturer’s instructions. Briefly, the serum sample was incubated with monoclonal antibody coated plastic beads in a tube. This was followed by addition of I125-labelled polyclonal antibody to TPA. The beads were washed with the washing buffer to remove unbound antibody. The antibody bound radioactivity was measured using Beta counter (Packard, USA). Standards with known concentrations were used for reference. Histopathology Five micrometer thick sections of formalin fixed and paraffin embedded tissues were stained with Haematoxylin and Eosin (H&E), to microscopically confirm that all the collected samples were SCC. Immunohistochemistry (IHC) IHC was performed on serial sections of both formalin fixed and cryostat sections using monoclonal antibodies directed against human CK8 (1:200, clone M20, Sigma, USA, and 1:100, NCL-CK8-TS1, Novocastra, UK), CK18 (1:200, clone CY-90, Sigma, USA, and 1:40, clone NCL-CK18, Novocastra, UK) and CK19 (1;100, clone NCLCK19, Novocastra, UK) as described earlier.16,17 Using an Avidin– Biotin–Peroxidase Complex kit (Vector Lab., USA). Stained slides were analysed under light microscope in a blind fashion, and the staining intensity was graded as negative ( ), weak (+), moderate (++) and intense (+++). Statistical analysis The statistical analyses of TPA levels before the surgery and seven days after surgery were performed on 50 patients out of
Figure 1 Graph showing mean ± SD serum TPA levels in pre and post surgery samples of tongue (A) and BM (B).
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CK8, 18 and 19 expression in tumour and surrounding uninvolved areas using immunohistochemistry IHC was carried out for the detection of localization of CK8, 18 and 19 expression in the tumour and its surrounding uninvolved areas, as well as in normal control tissues. None of the 10 normal BM tissues showed CK8 expression while mild or moderate supra-basal CK18 expression was seen in four and one tissues, respectively. CK19 expression was seen only in the basal layer of seven tissues (Fig. 2A D, G, and J). As these samples were collected from patients who had come for tooth extraction and did not have good oral hygiene, it is possible that these tissues were not fully normal. Thus aberrant expression of CK18 in these tissues could be indicative of initiation of abnormal cell differentiation. Results of IHC obtained using formalin fixed tissues were confirmed by cryostat sections. The surrounding uninvolved areas of 47/80 (58.75%) oral SCC samples showed concomitant CK8, 18 and 19 expression. CK8, 18 and 19 expression was seen in the stratified spinous layer in 58%,
61% and 35% of these samples, respectively, while it was seen in the basal layer in 9%, 4% and 40% of these samples, respectively. CK8 and 18 staining was seen in the cytoplasm of differentiated epithelium while CK19 staining was observed in the basal layer of the surrounding uninvolved areas with moderate intensity (Fig. 2A E, H, and K). CK8, 18 and 19 staining was predominantly seen at the invasive fronts of the higher grade tumours while CK19 staining was also observed in the outer layer of sub-epithelial tumour islands with high intensity (Fig. 2A F, I, and L). Thirty-two of these 47 samples also showed post surgery elevated serum TPA levels as compared to pre-surgery levels (p < 0.001, Chi-square test). Correlation between post surgery elevation in TPA levels and tumour recurrence/patient survival Serum TPA levels in 50 post surgery samples whose clinical follow-up could be obtained were further correlated with clinical parameters as described in Material and Methods. Twenty-eight
Figure 2A Representative images of Haematoxylin-Eosin and Immunohistochemical staining patterns of CK8, 18 and 19 expression in: normal oral tissues (A, D, G, and J), surrounding uninvolved tissues (B, E, H, and K) and oral SCC (C, F, I, and L).
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of these samples (69% tongue and 42% BM) showed elevated serum TPA levels post surgery. Ten out of 28 (35.7%) patients who showed increased post surgery serum TPA levels also showed recurrence. The average increase in post surgery TPA levels in these patients was 24.83% (p = 0.001, paired sample t-test) as compared to the pre-surgery levels. Post surgery serum TPA levels above the cutoff value correlated significantly with recurrence (p < 0.006, Log Rank Test) (Fig. 3A) and inversely with survival (p < 0.033, Log Rank Test) (Fig. 3B). Five of the 10 patients showing recurrence died before the last follow-up, their median follow-up period being
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9 months. Thus, recurrence and patient survival showed statistically significant correlation with elevated post surgery serum TPA levels. Correlation between post surgery serum TPA levels, CK localization in the surrounding uninvolved areas of the tumour and clinical parameters of the patients Of the 50 samples whose clinical correlation could be obtained, 28 samples showed elevated serum TPA levels. Twenty-three of these samples also demonstrated expression of CK8, 18 and 19 in their surrounding uninvolved areas. The staining intensity and layer-wise CK expression pattern is shown in Figs. 2B and 2C. These samples showed statistically significant correlation with tumour stage (p = 0.02, Chi-Square Test, Table 1). Ten patients who came up with recurrence and increased post surgery TPA levels also demonstrated CK8, 18 and 19 expression in surrounding un-involved areas (Fig. 4). Thus, in summary, our results show that post surgery increase in serum TPA levels correlates with tumour stage, recurrence and adversely affects survival in oral cancer patients. Further, increased post surgery serum TPA levels also correlate with CK8, 18 and 19 expression in surrounding uninvolved areas of the tumour. Discussion
Figure 2B CK8, 18 and 19 staining intensity in the surrounding uninvolved tissues.
Figure 2C Layer-wise expression of CK8, 18 and 19 in the surrounding uninvolved tissues.
Loco-regional recurrence and regional lymph node metastasis are major hurdles in the management of human oral cancer. There is a dire need for sensitive and specific prognostic markers that could predict disease recurrence and possibly help in controlling disease progression. Squamous cell carcinoma antigen (SCCAg), Carcinoembryonic antigen (CEA), Lipid associated sialic acid, SCC marker (TA-4), serum intercellular adhesion molecule-1 (S-ICAM1), Cyfra-21-1, TPS, etc., have been examined for their value in detecting head and neck cancers.10,18–25 However, due to their low sensitivity, these makers have not clinically proved to be useful in HNSCC. TPA measures soluble CK8, 18, and 19 fragments together in serum samples26 and is an example of a broad-spectrum cytokeratin assay demonstrating high sensitivity in cancer patients. CK are in routine use as diagnostic markers in various types of pathological conditions including cancer. Aberrant expression of CK8 and 18 in SCC has been widely reported irrespective of the site.13,27–32 Supra-basal expression of CK19 in precancerous lesions and SCC has been previously proposed as an early diagnostic
Figure 3 Graph showing correlation between post surgery TPA levels with (A) recurrence (p = 0.006) and (B) mortality (p = 0.033) using Kaplan–Meier analysis.
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Table 1 Statistical correlation between Clinico-pathological features of oral SCC patients and CK8, 18 and 19 expression in tumours, their respective surrounding uninvolved tissues and the post surgery elevated TPA levels. Variables
Age(Years) 50 n = 26 Sex Male n = 35 Female n = 15 Location Tongue n = 27 BM n = 23 Thickness 2 cm n = 8 Stage I n=3 II n = 9 III n = 6 IV n = 32 Size T1 n = 8 T2 n = 16 T3 n = 4 T4 n = 22 Node status N0 n = 21 N1 n = 10 N2b n = 16 N2c n = 3 Differentiation Well n = 3 Moderate n = 30 Poor n = 17
CK8, 18 and 19 expression together in SCC
CK8, 18 and 19 expression together in surrounding uninvolved areas
Negative
+
++
+++
Negative
0 2
3 4
16 14
5 6
1 1
2 0
4 4
20 9
9 2
0 2
3 5
16 12
1 1
2 6
0 2 0 0
p Values
++
+++
5 9
15 10
4 5
16 12
0.3
1 1
11 3
17 9
6 2
16 12
0.56
7 4
1 1
9 4
11 16
5 3
17 11
0.31
5 25
1 9
1 1
2 10
5 22
1 8
4 24
0.43
1 1 0 6
1 5 4 18
1 1 2 8
0 1 0 1
0 2 2 9
0 4 3 19
3 2 1 2
3 6 3 16
0.02
0 2 0 0
2 1 0 5
2 10 3 14
4 3 1 3
0 1 0 1
2 6 2 4
1 7 2 16
5 2 0 1
7 9 1 11
0.23
2 0 0 0
2 3 4 0
12 5 10 2
5 2 2 1
1 0 1 0
3 3 6 2
13 4 8 1
4 3 1 0
14 4 8 2
0.46
0 1 1
0 5 2
3 19 8
0 5 6
0 2 0
1 9 4
1 15 11
1 5 2
3 17 8
0.94
marker.28,33,34 In a recent follow-up study, CK18 has been proposed as prognostic marker for human oral cancer.15 These changes suggest their potential use as predictive markers for the prognosis of human oral cancer. Earlier reports from our laboratory have shown that CK8 and 18 are aberrantly expressed in a majority of the precancerous lesions and SCC of the oral cavity, and their expression in some way contributes to the malignant transformation of squamous epithelia.13,14,16,35 CK fragments in serum have been used to predict disease status in lieu of conventional methods. They have been used to predict overall disease status in various types of malignancies including
Figure 4 Post surgery elevated serum TPA levels along with CK8, 18 and 19 expression in surrounding uninvolved areas of samples showing recurrence.
+
Post surgery elevated TPA levels U/L
head and neck cancer.36–43 It has been shown that patients showing higher levels of circulating CK fragments especially TPA in lung cancer and Cyfra 21-1 in H&N cancer during the follow-up are at higher risk to develop loco-regional recurrence and metastases.38,44,45 However, to our knowledge, there is only one report available showing the status of circulating CK fragments (Cyfra 21-1 and TPS) in oral cancer.25 Hence, in this study, our aim was to evaluate the efficacy of TPA assay as a non-invasive tumour marker for the early prediction of progression of oral cancer. In the present study, TPA levels in SCC patients were significantly higher than those of healthy individuals indicating the prognostic value of TPA assay. The serum TPA levels correlated significantly with stage, recurrence and inversely with survival of the patients. Other studies have shown significantly elevated serum TPA levels in advanced disease and have correlated them with poor prognosis in genitourinary, lung and bladder cancer.41,46,47,48 Values of sensitivity and specificity for TPA in this study matched with some studies while they differed with studies from some other groups.46–50 This could be the outcome of variables such as type of malignancy, sample size, type of commercially available TPA detection kit, selected cut-off values, etc. We have compared TPA levels in the pre-surgery and post surgery serum samples of SCC of tongue and BM. Out of 50 samples, TPA levels remained high in 28 post surgery serum samples. Our study group consisted of selected patients who were surgically treated and had not undergone any pre or post surgical (till post surgery blood samples were collected) treatment. Here, although the follow-up period is short, response to treatment can be detected within seven days since the half-life of CK is less than 24 h.
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Various groups have reported correlation between elevated TPA levels and recurrence, although the time of follow-up differed in every case.25,44,51–54 Thus it is evident that there is inconsistency in the results showing higher TPA levels and time of survival. These differences could possibly be attributed to the fact that none of these studies have taken into account site specific CK expression. CK expression is site specific and different sites in the oral cavity differ in their CK expression pattern. Most of the earlier studies have not mentioned any specific sub-sites while reporting their results. Doweck et al.44 have suggested that before the marker is used routinely in patients with carcinoma of the H&N, further investigations in a particular sub-site of H&N cancer are necessary. In this context, it would be worth noting that the two prevalent sites of oral tumour formation in the Indian subcontinent are tongue and BM, the sites that have been investigated in this study. We have conducted IHC on surrounding areas of the tumour so as to understand the origin of circulating CK serum fragments after surgical excision of the tumour. Out of 28 post surgery samples showing elevated serum TPA, 23 samples showed expression of CK8, 18 and 19 in their respective surrounding uninvolved tissues. Importantly, immunostaining intensity in the tumour as well as surrounding uninvolved tissues was higher in the BM, as compared to the tongue. We have also recorded higher concentration of serum TPA levels in BM SCC as compared to tongue SCC. Earlier, Banal et al.38 have noted highest Cyfra 21-1 levels in tongue tumour samples and lowest in the tumours developing at other sites in the oral cavity. These observations indicate that CK expression and release pattern is based on the anatomical site of the oral cavity. The mechanism of release of soluble CK fragments into the circulation is not well understood. Different mechanisms have been proposed earlier, which include proteolytic degradation of CK in dying cells, abnormal mitosis, spillover of monomeric CK polypeptides from proliferating cells, apoptosis, etc.8,36,55,56 In one of the earlier studies the authors have proposed that CK serum fragments are released in the sera of patients from the surrounding uninvolved areas of the tumour.57 In the present study, we have shown aberrant expression of CK8, 18 and suprabasal expression of CK19 in surrounding uninvolved areas of the tumour. Thus this observation suggests that this surrounding population may be responsible for high TPA levels in sera of patients even after surgical removal of the tumour and warrants further investigations. To summarize, we have shown the presence of CK8, 18 and 19 fragments in the sera of oral cancer patients. We could correlate the higher TPA levels with tumour stage, recurrence and poor survival of the patients. We have also shown that the TPA levels remained high even after surgical removal of the tumour, which could be the result of presence of these proteins in the surrounding uninvolved areas. In conclusion, increased post surgery serum TPA levels could prove to be a useful marker of disease progression in human oral cancer, and TPA could be used as a useful non-invasive prognostic assay.
Conflicts of interest statement None declared.
Acknowledgements We acknowledge Tata Memorial Centre, IRG No. 371 for funding. We thank Professor Ranganathan from Ragas Dental College, Chennai for providing us normal human oral tissues and Ms.
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46. Buccheri G, Ferrigno D. Usefulness of tissue polypeptide antigen in staging, monitoring, and prognosis of lung cancer. Chest 1988;93(3):565–70. 47. Khanna OP, Wu B. Tissue polypeptide antigen (TPA) as a predictor for genitourinary cancers and their metastases. Urology 1987;30(2):06–110. 48. Maulard-Durdux C, Toubert ME, Hennequin C, Housset M. Serum tissue polypeptide antigen in bladder cancer as a tumor marker: a prospective study. J Clin Oncol 1997;15(12):3446–50. 49. Ecke ThorstenH, Schlechte HorstH, Schulze Guntram, Lenk SeverinV, Loening SA. Four Tumour Markers for Urinary Bladder Cancer - Tissue Polypeptide Antigen (TPA), HER-2/neu (ERB B2), Urokinase-type Plasminogen Activator Receptor (uPAR) and TP53 Mutation. Jpn J Clin Oncol 2000;30: 215–20. 50. Soletormos G, Nielsen D, Schioler V, Skovsgaard T, Dombernowsky P. Tumor markers cancer antigen 15.3, carcinoembryonic antigen, and tissue polypeptide antigen for monitoring metastatic breast cancer during first-line chemotherapy and follow-up. Clin Chem 1996;42:564–75. 51. Doweck I, Barak M, Greenberg E, Uri N, Kellner J, Lurie M, et al. Cyfra 21–1. A new potential tumor marker for squamous cell carcinoma of head and neck. Arch Otolaryngol Head Neck Surg 1995;121(2):177–81. 52. Ebert W, Dienemann H, Fateh-Moghadam A, Scheulen M, Konietzko N, Schleich T, et al. Cytokeratin 19 fragment CYFRA 21–1 compared with carcinoembryonic antigen, squamous cell carcinoma antigen and neuron-specific enolase in lung cancer. Results of an international multicentre study. Eur J Clin Chem Clin Biochem 1994;32(3):189–99. 53. van der Gaast A, Schoenmakers CH, Kok TC, Blijenberg BG, Cornillie F, Splinter TA. Evaluation of a new tumour marker in patients with non-small-cell lung cancer: Cyfra 21.1. Br J Cancer 1994;69(3):525–28. 54. Deng YF, Chen P, Lin YZ, Le JZ, Wu XL, Yu MQ, et al. Analytical and clinical evaluation of CYFRA 21-1 by electrochemiluminescent immunoassay in head and neck squamous cell carcinoma. J Laryngol Otol 2003;117(3):190–4. 55. Brabon AC, Williams JF, Cardiff RD. A monoclonal antibody to a human breast tumor protein released in response to estrogen. Cancer Res Jun 1984;44(6):2704–10. 56. Ku NO, Liao J, Omary MB. Apoptosis generates stable fragments of human type I keratins. J Biol Chem 1997;272(52):33197–203. 57. Kramer G, Erdal H, Mertens HJ, Nap M, Mauermann J, Steiner G, et al. Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18. Cancer Res 2004;64(5): 1751–6.
Contents lists available at ScienceDirect
Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Prognostic value of tissue polypeptide antigen in oral squamous cell carcinoma Sharada S. Sawant a,⇑, Devendra A. Chaukar b, Shreyas S. Joshi a, Prerana P. Dange a, Sadhana Kannan a, Shubhada Kane c, Anil K. D’Cruz b, Milind M. Vaidya a a
Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai, 410210 Maharashtra, India Oral Surgery, Head and Neck Unit, Tata Memorial Hospital, Parel, Mumbai 40012, India c Pathology Department, Tata Memorial Hospital, Parel, Mumbai 40012, India b
a r t i c l e
i n f o
Article history: Received 16 September 2010 Received in revised form 8 November 2010 Accepted 8 November 2010 Available online 3 December 2010 Keywords: Oral cancer Squamous cell carcinoma Cytokeratins Serum tumour markers Tissue polypeptide antigen Prognosis
s u m m a r y Human oral cancer has a high risk of locoregional relapse which is difficult to diagnose early due to lack of prognostic markers. We and others have shown aberrant expression of cytokeratin (CK) 8 and 18 in human oral cancer. Aberrant supra-basal expression of CK19 has been shown earlier. In this study, our aim was to develop a non-invasive test for prognostication of human oral cancer. Immunoradiometric assay (IRMA) was used to measure the circulating levels of TPA in the sera of 80 oral cancer patients before surgery and seven days after surgery. Elevated serum TPA levels were noted in the post surgery samples of 28 out of the 50 patients for whom clinical follow-up was available, as compared to their pre-surgery samples. TPA levels in the pre-surgery serum samples of patients were significantly higher than levels in the sera of healthy controls (p = 0.001). Elevated levels in patients correlated significantly with stage (p = 0.02), development of recurrence (p < 0.006), and impacted survival (p < 0.033). IHC analysis showed statistically significant correlation between expression of CK8, 18 and 19 in surrounding uninvolved tissues of the tumour and post surgery elevated serum TPA levels (p < 0.001). This suggests the possibility that CK fragments are released from the surrounding uninvolved tissues into the sera of patients after surgical removal of the tumour. Thus, our study indicates that TPA can be a useful tumour marker for the prediction of recurrence and poor prognosis in human oral cancer. Ó 2010 Elsevier Ltd. All rights reserved.
Introduction Human oral cancer is the sixth largest group of malignancies worldwide and the single largest malignancy in males in the Indian subcontinent.1,2 Major contributory factors for the high incidence of oral cancer are the habits of chewing tobacco and other allied products. Loco-regional recurrence and regional lymph node metastases are the major hurdles in the management of oral cancer. Early detection of disease progression remains a challenging task mainly due to the lack of adequate early prognostic markers. Primary oral carcinoma is usually superficial and easily detected. However, deeply invading tumours that recur after primary resections are usually detected only when they are large and no longer curable. The existing imaging modalities are not sensitive enough for the early diagnosis of tumour progression.3–6 CK belonging to the intermediate filament family of proteins are particularly useful tools for diagnosis in oncology. Due to the Abbreviations: CK, cytokeratins; TPA, tissue polypeptide antigen; H&N, head and neck; SCC, squamous cell carcinoma. ⇑ Corresponding author. Tel.: +91 22 27405134; fax: +91 22 27405085. E-mail address: [email protected] (S.S. Sawant). 1368-8375/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2010.11.005
enhanced proteolytic activity in malignant cells, CK fragments are released in circulation and can be quantified using various commercially available specific serological assays.7 In healthy individuals, the level of CK in the circulation is low and rises significantly in patients with carcinoma.8 The three most frequently used CK which are being evaluated as serum markers for their utility in clinical applications are tissue polypeptide antigen (TPA), tissue polypeptide specific antigen (TPS), and cytokeratin fragments 21-1 (Cyfra21-1). TPA assay is a broad spectrum assay which measures cytosolic fragments of CK8, 18, and 19. Correlation between TPA levels in the serum and disease progression has been shown earlier.9 TPA assay is being used for prognostication of lung and breast cancer.10–12 However, it has not been evaluated for the prognostication of human oral cancer. Earlier we and others have shown aberrant expression of simple epithelia specific CK8, 18 and 19 in precancerous lesions as well as squamous cell carcinoma (SCC) of the tongue and buccal mucosa (BM).13–15 In the present study, we have examined the serum levels of CK8, 18 and 19 using TPA assay in oral SCC patients before and 7 days after surgery and correlated it with the clinical parameters of the patients.
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Material and methods Patients Blood samples and tumour tissues along with their respective surrounding uninvolved areas were collected at the time of surgery from a total of 80 primary oral SCC of tongue (40 patients) and BM (40 patients) from the Tata Memorial Hospital (TMH), Mumbai, India. Blood samples were also collected 7 days after surgery. It was ensured that none of the patients had received any radiotherapy or chemotherapy before collection of the blood. Blood samples from 24 normal healthy, age and sex-matched individuals were also collected as a control group. Normal buccal mucosa tissues were obtained from 10 subjects during third molar tooth extraction from Ragas Dental College and Hospital, Chennai, India. This study was approved by the Human Ethics Committees of the respective Institutional Review Boards. Informed consent was obtained from the patients as well as healthy individuals. Clinical and histopathological records of the patients Clinico-pathological information like tumour site, size, histological grade, perineural invasion, perinodal extension, lymphovascular invasion, margins, bone, lymph node involvement, loco-regional recurrence, metastasis and survival was collected from the clinical and pathology records of TMH. The median time of clinical follow-up was 11.5 months. RIA for serum TPA
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80 whose clinical follow-up was obtained. SPSS software version 15 was used for statistical analysis. Differences with a probability value of <0.05 were considered statistically significant. Specificity, sensitivity, and positive and negative prediction values for TPA were determined. The cut-off values for serum TPA levels in patients were determined with reference to TPA values in healthy individuals using Kaplan Meier test. Pearson chi-square test was used for correlations between higher TPA levels in post surgery serum samples and clinical parameters of the patients. TPA levels in the post surgery samples and CK8, 18 and 19 localization in the surrounding un-involved areas of the tumour were correlated using Pearson Chi-Square Test.
Results Serum TPA levels Cut-off value for serum TPA levels was 140.93 U/L (mean ±1 SD of normal). Values of sensitivity, specificity, and positive and negative prediction for TPA were 78%, 61%, 68% and 72%, respectively. TPA levels in the pre-surgery serum samples were significantly greater than the serum samples of healthy individuals by 82.07% (p = 0.001, t-test). The serum TPA concentrations obtained for the 80 patients were used to study the pre and post surgery TPA levels in tongue and BM. The pre-surgery mean serum TPA levels in BM samples were higher than tongue samples by 49.5%. The BM samples showed 19.46% decrease in TPA concentration after the surgery as compared to 14.35% in tongue samples (Fig. 1).
TPA levels were estimated using a TPA-M IRMA (two-site monoclonal/polyclonal immunoradiometric sandwich assay) kit from Prolifigen (DiaSorin – USA) according to the manufacturer’s instructions. Briefly, the serum sample was incubated with monoclonal antibody coated plastic beads in a tube. This was followed by addition of I125-labelled polyclonal antibody to TPA. The beads were washed with the washing buffer to remove unbound antibody. The antibody bound radioactivity was measured using Beta counter (Packard, USA). Standards with known concentrations were used for reference. Histopathology Five micrometer thick sections of formalin fixed and paraffin embedded tissues were stained with Haematoxylin and Eosin (H&E), to microscopically confirm that all the collected samples were SCC. Immunohistochemistry (IHC) IHC was performed on serial sections of both formalin fixed and cryostat sections using monoclonal antibodies directed against human CK8 (1:200, clone M20, Sigma, USA, and 1:100, NCL-CK8-TS1, Novocastra, UK), CK18 (1:200, clone CY-90, Sigma, USA, and 1:40, clone NCL-CK18, Novocastra, UK) and CK19 (1;100, clone NCLCK19, Novocastra, UK) as described earlier.16,17 Using an Avidin– Biotin–Peroxidase Complex kit (Vector Lab., USA). Stained slides were analysed under light microscope in a blind fashion, and the staining intensity was graded as negative ( ), weak (+), moderate (++) and intense (+++). Statistical analysis The statistical analyses of TPA levels before the surgery and seven days after surgery were performed on 50 patients out of
Figure 1 Graph showing mean ± SD serum TPA levels in pre and post surgery samples of tongue (A) and BM (B).
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CK8, 18 and 19 expression in tumour and surrounding uninvolved areas using immunohistochemistry IHC was carried out for the detection of localization of CK8, 18 and 19 expression in the tumour and its surrounding uninvolved areas, as well as in normal control tissues. None of the 10 normal BM tissues showed CK8 expression while mild or moderate supra-basal CK18 expression was seen in four and one tissues, respectively. CK19 expression was seen only in the basal layer of seven tissues (Fig. 2A D, G, and J). As these samples were collected from patients who had come for tooth extraction and did not have good oral hygiene, it is possible that these tissues were not fully normal. Thus aberrant expression of CK18 in these tissues could be indicative of initiation of abnormal cell differentiation. Results of IHC obtained using formalin fixed tissues were confirmed by cryostat sections. The surrounding uninvolved areas of 47/80 (58.75%) oral SCC samples showed concomitant CK8, 18 and 19 expression. CK8, 18 and 19 expression was seen in the stratified spinous layer in 58%,
61% and 35% of these samples, respectively, while it was seen in the basal layer in 9%, 4% and 40% of these samples, respectively. CK8 and 18 staining was seen in the cytoplasm of differentiated epithelium while CK19 staining was observed in the basal layer of the surrounding uninvolved areas with moderate intensity (Fig. 2A E, H, and K). CK8, 18 and 19 staining was predominantly seen at the invasive fronts of the higher grade tumours while CK19 staining was also observed in the outer layer of sub-epithelial tumour islands with high intensity (Fig. 2A F, I, and L). Thirty-two of these 47 samples also showed post surgery elevated serum TPA levels as compared to pre-surgery levels (p < 0.001, Chi-square test). Correlation between post surgery elevation in TPA levels and tumour recurrence/patient survival Serum TPA levels in 50 post surgery samples whose clinical follow-up could be obtained were further correlated with clinical parameters as described in Material and Methods. Twenty-eight
Figure 2A Representative images of Haematoxylin-Eosin and Immunohistochemical staining patterns of CK8, 18 and 19 expression in: normal oral tissues (A, D, G, and J), surrounding uninvolved tissues (B, E, H, and K) and oral SCC (C, F, I, and L).
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of these samples (69% tongue and 42% BM) showed elevated serum TPA levels post surgery. Ten out of 28 (35.7%) patients who showed increased post surgery serum TPA levels also showed recurrence. The average increase in post surgery TPA levels in these patients was 24.83% (p = 0.001, paired sample t-test) as compared to the pre-surgery levels. Post surgery serum TPA levels above the cutoff value correlated significantly with recurrence (p < 0.006, Log Rank Test) (Fig. 3A) and inversely with survival (p < 0.033, Log Rank Test) (Fig. 3B). Five of the 10 patients showing recurrence died before the last follow-up, their median follow-up period being
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9 months. Thus, recurrence and patient survival showed statistically significant correlation with elevated post surgery serum TPA levels. Correlation between post surgery serum TPA levels, CK localization in the surrounding uninvolved areas of the tumour and clinical parameters of the patients Of the 50 samples whose clinical correlation could be obtained, 28 samples showed elevated serum TPA levels. Twenty-three of these samples also demonstrated expression of CK8, 18 and 19 in their surrounding uninvolved areas. The staining intensity and layer-wise CK expression pattern is shown in Figs. 2B and 2C. These samples showed statistically significant correlation with tumour stage (p = 0.02, Chi-Square Test, Table 1). Ten patients who came up with recurrence and increased post surgery TPA levels also demonstrated CK8, 18 and 19 expression in surrounding un-involved areas (Fig. 4). Thus, in summary, our results show that post surgery increase in serum TPA levels correlates with tumour stage, recurrence and adversely affects survival in oral cancer patients. Further, increased post surgery serum TPA levels also correlate with CK8, 18 and 19 expression in surrounding uninvolved areas of the tumour. Discussion
Figure 2B CK8, 18 and 19 staining intensity in the surrounding uninvolved tissues.
Figure 2C Layer-wise expression of CK8, 18 and 19 in the surrounding uninvolved tissues.
Loco-regional recurrence and regional lymph node metastasis are major hurdles in the management of human oral cancer. There is a dire need for sensitive and specific prognostic markers that could predict disease recurrence and possibly help in controlling disease progression. Squamous cell carcinoma antigen (SCCAg), Carcinoembryonic antigen (CEA), Lipid associated sialic acid, SCC marker (TA-4), serum intercellular adhesion molecule-1 (S-ICAM1), Cyfra-21-1, TPS, etc., have been examined for their value in detecting head and neck cancers.10,18–25 However, due to their low sensitivity, these makers have not clinically proved to be useful in HNSCC. TPA measures soluble CK8, 18, and 19 fragments together in serum samples26 and is an example of a broad-spectrum cytokeratin assay demonstrating high sensitivity in cancer patients. CK are in routine use as diagnostic markers in various types of pathological conditions including cancer. Aberrant expression of CK8 and 18 in SCC has been widely reported irrespective of the site.13,27–32 Supra-basal expression of CK19 in precancerous lesions and SCC has been previously proposed as an early diagnostic
Figure 3 Graph showing correlation between post surgery TPA levels with (A) recurrence (p = 0.006) and (B) mortality (p = 0.033) using Kaplan–Meier analysis.
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Table 1 Statistical correlation between Clinico-pathological features of oral SCC patients and CK8, 18 and 19 expression in tumours, their respective surrounding uninvolved tissues and the post surgery elevated TPA levels. Variables
Age(Years) 50 n = 26 Sex Male n = 35 Female n = 15 Location Tongue n = 27 BM n = 23 Thickness 2 cm n = 8 Stage I n=3 II n = 9 III n = 6 IV n = 32 Size T1 n = 8 T2 n = 16 T3 n = 4 T4 n = 22 Node status N0 n = 21 N1 n = 10 N2b n = 16 N2c n = 3 Differentiation Well n = 3 Moderate n = 30 Poor n = 17
CK8, 18 and 19 expression together in SCC
CK8, 18 and 19 expression together in surrounding uninvolved areas
Negative
+
++
+++
Negative
0 2
3 4
16 14
5 6
1 1
2 0
4 4
20 9
9 2
0 2
3 5
16 12
1 1
2 6
0 2 0 0
p Values
++
+++
5 9
15 10
4 5
16 12
0.3
1 1
11 3
17 9
6 2
16 12
0.56
7 4
1 1
9 4
11 16
5 3
17 11
0.31
5 25
1 9
1 1
2 10
5 22
1 8
4 24
0.43
1 1 0 6
1 5 4 18
1 1 2 8
0 1 0 1
0 2 2 9
0 4 3 19
3 2 1 2
3 6 3 16
0.02
0 2 0 0
2 1 0 5
2 10 3 14
4 3 1 3
0 1 0 1
2 6 2 4
1 7 2 16
5 2 0 1
7 9 1 11
0.23
2 0 0 0
2 3 4 0
12 5 10 2
5 2 2 1
1 0 1 0
3 3 6 2
13 4 8 1
4 3 1 0
14 4 8 2
0.46
0 1 1
0 5 2
3 19 8
0 5 6
0 2 0
1 9 4
1 15 11
1 5 2
3 17 8
0.94
marker.28,33,34 In a recent follow-up study, CK18 has been proposed as prognostic marker for human oral cancer.15 These changes suggest their potential use as predictive markers for the prognosis of human oral cancer. Earlier reports from our laboratory have shown that CK8 and 18 are aberrantly expressed in a majority of the precancerous lesions and SCC of the oral cavity, and their expression in some way contributes to the malignant transformation of squamous epithelia.13,14,16,35 CK fragments in serum have been used to predict disease status in lieu of conventional methods. They have been used to predict overall disease status in various types of malignancies including
Figure 4 Post surgery elevated serum TPA levels along with CK8, 18 and 19 expression in surrounding uninvolved areas of samples showing recurrence.
+
Post surgery elevated TPA levels U/L
head and neck cancer.36–43 It has been shown that patients showing higher levels of circulating CK fragments especially TPA in lung cancer and Cyfra 21-1 in H&N cancer during the follow-up are at higher risk to develop loco-regional recurrence and metastases.38,44,45 However, to our knowledge, there is only one report available showing the status of circulating CK fragments (Cyfra 21-1 and TPS) in oral cancer.25 Hence, in this study, our aim was to evaluate the efficacy of TPA assay as a non-invasive tumour marker for the early prediction of progression of oral cancer. In the present study, TPA levels in SCC patients were significantly higher than those of healthy individuals indicating the prognostic value of TPA assay. The serum TPA levels correlated significantly with stage, recurrence and inversely with survival of the patients. Other studies have shown significantly elevated serum TPA levels in advanced disease and have correlated them with poor prognosis in genitourinary, lung and bladder cancer.41,46,47,48 Values of sensitivity and specificity for TPA in this study matched with some studies while they differed with studies from some other groups.46–50 This could be the outcome of variables such as type of malignancy, sample size, type of commercially available TPA detection kit, selected cut-off values, etc. We have compared TPA levels in the pre-surgery and post surgery serum samples of SCC of tongue and BM. Out of 50 samples, TPA levels remained high in 28 post surgery serum samples. Our study group consisted of selected patients who were surgically treated and had not undergone any pre or post surgical (till post surgery blood samples were collected) treatment. Here, although the follow-up period is short, response to treatment can be detected within seven days since the half-life of CK is less than 24 h.
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Various groups have reported correlation between elevated TPA levels and recurrence, although the time of follow-up differed in every case.25,44,51–54 Thus it is evident that there is inconsistency in the results showing higher TPA levels and time of survival. These differences could possibly be attributed to the fact that none of these studies have taken into account site specific CK expression. CK expression is site specific and different sites in the oral cavity differ in their CK expression pattern. Most of the earlier studies have not mentioned any specific sub-sites while reporting their results. Doweck et al.44 have suggested that before the marker is used routinely in patients with carcinoma of the H&N, further investigations in a particular sub-site of H&N cancer are necessary. In this context, it would be worth noting that the two prevalent sites of oral tumour formation in the Indian subcontinent are tongue and BM, the sites that have been investigated in this study. We have conducted IHC on surrounding areas of the tumour so as to understand the origin of circulating CK serum fragments after surgical excision of the tumour. Out of 28 post surgery samples showing elevated serum TPA, 23 samples showed expression of CK8, 18 and 19 in their respective surrounding uninvolved tissues. Importantly, immunostaining intensity in the tumour as well as surrounding uninvolved tissues was higher in the BM, as compared to the tongue. We have also recorded higher concentration of serum TPA levels in BM SCC as compared to tongue SCC. Earlier, Banal et al.38 have noted highest Cyfra 21-1 levels in tongue tumour samples and lowest in the tumours developing at other sites in the oral cavity. These observations indicate that CK expression and release pattern is based on the anatomical site of the oral cavity. The mechanism of release of soluble CK fragments into the circulation is not well understood. Different mechanisms have been proposed earlier, which include proteolytic degradation of CK in dying cells, abnormal mitosis, spillover of monomeric CK polypeptides from proliferating cells, apoptosis, etc.8,36,55,56 In one of the earlier studies the authors have proposed that CK serum fragments are released in the sera of patients from the surrounding uninvolved areas of the tumour.57 In the present study, we have shown aberrant expression of CK8, 18 and suprabasal expression of CK19 in surrounding uninvolved areas of the tumour. Thus this observation suggests that this surrounding population may be responsible for high TPA levels in sera of patients even after surgical removal of the tumour and warrants further investigations. To summarize, we have shown the presence of CK8, 18 and 19 fragments in the sera of oral cancer patients. We could correlate the higher TPA levels with tumour stage, recurrence and poor survival of the patients. We have also shown that the TPA levels remained high even after surgical removal of the tumour, which could be the result of presence of these proteins in the surrounding uninvolved areas. In conclusion, increased post surgery serum TPA levels could prove to be a useful marker of disease progression in human oral cancer, and TPA could be used as a useful non-invasive prognostic assay.
Conflicts of interest statement None declared.
Acknowledgements We acknowledge Tata Memorial Centre, IRG No. 371 for funding. We thank Professor Ranganathan from Ragas Dental College, Chennai for providing us normal human oral tissues and Ms.
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