Long-term effect of photodynamic therapy on oral squamous cell carcinoma and epithelial dysplasia.

BACKGROUND: Oral squamous cell carcinoma (OSCC) treatment consists mainly of surgery, chemotherapy, and radiotherapy, alone or in combination. Epithelial dysplasia (ED) is also treated with surgery. However, these treatments can induce functional and/or aesthetic disturbances. Photodynamic therapy (PDT) can preserve organs. Although short-term studies have shown good progress, long-term evaluations have not yet been conducted. This study aimed to clarify the long-term effects of PDT on OSCC and ED. METHODS: Patients who underwent PDT with the first (porfimer sodium) or second generation photosensitizers (talaporfin sodium) for early OSCC (T1 and T2) and ED were included in this study. The long-term prognosis was assessed. RESULTS: Twenty-three patients were included. Complete response (CR) was observed in 19 patients (82.6%) and partial response (PR) in 4 patients (17.4%) 4 weeks after PDT. Regarding long-term progress, local region recurrence occurred in 11 of 19 CR cases (57.9%), and the term of recurrence was 27.4 ± 30.4 months. Surgical resection was performed in all local recurrence and PR cases, and 3 patients died of the underlying disease. CONCLUSIONS: PDT provides a good outcome in the short term, but its long-term effects are limited.

The effect of photodynamic therapy with talaporfin sodium, a second-generation photosensitizer, on oral squamous cell carcinoma: A series of eight cases.

OBJECTIVE: To assess the effect of photodynamic therapy (PDT) with talaporfin sodium, a second-generation photosensitizer, on oral squamous cell carcinoma (SCC). METHODS: Eight patients who were diagnosed with oral SCC without any metastasis and underwent talaporfin sodium-mediated PDT (t-PDT) were included in this study. Biopsies were performed 4-6 weeks after t-PDT. The clinical response was evaluated using Response Evaluation Criteria in Solid Tumors. RESULTS: Complete response (CR) was achieved in six of eight cases, and two cases showed partial response (PR) as a clinical outcome of t-PDT. Recurrence occurred in one of the CR cases 9 months after irradiation. The patient underwent tumor resection and no recurrence was found after surgery. The two cases with PR died from the cancer despite additional PDT. CONCLUSION: t-PDT is an effective treatment strategy for oral SCC. Talaporfin sodium has an advantage with regard to early elimination from the body compared with porfimer sodium.

Prognostic Prediction of Oral Squamous Cell Carcinoma by E-Cadherin and N-Cadherin Expression in Overall Cells in Tumor Nests or Tumor Cells at the Invasive Front.

Epithelial-mesenchymal transition (EMT) is a significant process in the invasion and metastasis of cancers including oral squamous cell carcinoma (OSCC), and the cadherin switch has been identified as one of the hallmarks of EMT. The aim of the present study was to evaluate the significance of the cadherin switch in the prognosis of OSCC and generate a model for prognostic predictions. Seventy-six biopsy and/or initial surgical specimens from OSCC patients were immunohistochemically analyzed for the expression of E-cadherin and N-cadherin in either overall OSCC cells in tumor nests or in OSCC cells at the invasive front. Among 76 OSCC cases, overall OSCC cells in tumor nests were negative for the expression of E-cadherin in 10 cases and positive for that of N-cadherin in 53 cases. Among 10 cases negative for the expression of E-cadherin, 4 cases were positive for that of N-cadherin. In OSCC cells at the invasive front, the expression of E-cadherin was negative in 62 cases, while that of N-cadherin was positive in 39 cases. Among 62 cases negative for the expression of E-cadherin, 33 cases were positive for that of N-cadherin. A logistic regression analysis showed that a model using the evaluation of N-cadherin expression in overall OSCC cells in tumor nests with a cut-off point of 70 years old was the best fit model. These results suggest that N-cadherin has significant value in prognostic predictions for OSCC patients.

Low-risk population among patients with tumor-node-metastasis stage III/IV oral squamous cell carcinoma.

A novel system auxiliary to the Union for International Cancer Control classification may allow the prognosis of patients with malignant tumors at similar stages to be predicted, as currently this is challenging. The present study generated a novel system to predict populations at low risk among patients with stage III/IV oral squamous cell carcinoma (OSCC). A total of 41 patients who were diagnosed at stages III/IV OSCC and underwent surgical tumor resection were analyzed. Band-like or follicular lymphocyte infiltration, intraepithelial micro-abscess formation and natural killer (NK) cell infiltration were histopathologically evaluated. Cox's proportional hazards regression model was used to identify prognostic factors, and a set of factors was selected from a combination of those prognostic factors to create a logic covariate model. A logic regression analysis for 41 patients with OSCC revealed that the presence of intraepithelial micro-abscesses and a lower density of NK cells were significantly associated with a favorable prognosis among patients with stage III/IV OSCC. These results suggested that the host innate immune responses, including neutrophil and NK cell infiltrations, are useful for prognostic prediction in patients with advanced malignant tumors.

Histopathological and immunohistochemical study in keratocystic odontogenic tumors: Predictive factors of recurrence.

The aim of the present study was to identify the most useful markers for predicting recurrence of keratocystic odontogenic tumors (KCOTs). A total of 65 tumor samples from 63 patients diagnosed with typical parakeratinized cysts and KCOTs between 1992 and 2014 were retrospectively studied. Clinical and histopathological data and treatment modality were reviewed. In addition, the expression profiles of Ki-67, cluster of differentiation (CD)34 and podoplanin were assessed using immunohistochemistry. The association between these factors and the rate of KCOT recurrence was evaluated. The presence of daughter cysts, epithelial islands and high Ki-67, CD34 and podoplanin expression levels were revealed to be associated with tumor recurrence. In particular, univariate analysis revealed that high CD34 expression levels were significantly associated with tumor recurrence (P=0.034), as was conservative surgical treatment (P=0.003). Multivariate analysis revealed that conservative treatment was the greatest independent risk factor for tumor recurrence (odds ratio=13.337; P=0.018). These results suggest that overexpression of CD34 may be a potent predictor of tumor recurrence and radical treatment of the teeth that are in contact with the tumors is recommended in order to prevent tumor recurrence.

Immunohistochemical study of vascular endothelial growth factor-C/vascular endothelial growth factor receptor-3 expression in oral tongue squamous cell carcinoma: Correlation with the induction of lymphangiogenesis.

The aim of the present study was to elucidate the associations between the expression of the vascular endothelial growth factor-C (VEGF-C)/VEGF receptor-3 (VEGFR-3) axis and lymphangiogenesis, regional lymph node metastasis and clinicopathological factors in oral tongue squamous cell carcinoma (OTSCC) using immunohistochemistry. The expression of VEGF-C, VEGFR-3 and podoplanin was immunohistochemically evaluated in specimens obtained from 65 patients with OTSCC (T1-2, N0) who had undergone radical surgery alone. The associations between the expression of VEGF-C, VEGFR-3 and podoplanin, and lymphangiogenesis, regional lymph node metastasis and clinocopathological factors were determined by immunohistochemical analysis. VEGF-C, VEGFR-3 and combined VEGF-C/VEGFR-3 expression was significantly higher in cases with regional recurrence compared with those without lymph node involvement (P<0.001). As regards lymphangiogenesis, a significant correlation was observed between podoplanin expression and VEGF-C, VEGFR-3 and combined VEGF-C/VEGFR-3 expression (P<0.001). Therefore, lymphangiogenesis in the peritumoral stroma was associated with lymph node metastasis. However, podoplanin expression did not exhibit a significant correlation with the progression of lymph node metastasis. The results of the present study suggest that the VEGF-C/VEGFR-3 axis may be associated with lymph node metastasis through lymphangiogenesis. Determining the VEGF-C/VEGFR-3 expression status may help predict which patients will develop regional recurrence and provide novel targets for therapies to suppress lymph node metastasis in the treatment of OTSCC.

Expression of the cancer stem cell markers CD44v6 and ABCG2 in tongue cancer: effect of neoadjuvant chemotherapy on local recurrence.

The efficacy of neoadjuvant chemotherapy (NAC) is controversial, and no report supports NAC with a high evidence level. Recently, we showed that a deep surgical margin was resected very close to the tumor site in many NAC-treated oral squamous cell carcinoma patients, suggesting that NAC may lead to local recurrence and poor outcomes. The purpose of this study was to evaluate the effect of NAC on tumor local recurrence using cancer stem cell marker immunohistochemistry. We retrospectively analyzed 89 patients who underwent radical surgery for tongue cancer, and examined the effect of NAC on tumor local recurrence. Cancer stem cell marker (CD44v6 and ABCG2) expression was detected by immunohistochemistry. In our study, the local recurrence rate was 12.4%. CD44v6 and ABCG2 expression was significantly associated with regional lymph node metastasis, pattern of invasion, depth of invasion, perineural invasion and local recurrence, respectively. Tumor local recurrence was a significant independent predictive factor of the 5-year disease specific survival. CD44v6 or ABCG2 positivity in NAC-treated patients was significantly associated with tumor local recurrence. It was suggested that local recurrence in NAC-treated cases is associated with cancer stem-like cells. We propose that NAC leads to the selection and/or residue of more aggressive cancer stem-like cells.

Ameloblastic fibrodentinoma with a congenitally missing second premolar tooth: a case report.

Ameloblastic fibrodentinoma (AFD) is a rare benign mixed odontogenic tumor that occurs predominantly in children and young adults. AFD is usually associated with delayed eruption of the tooth and shows painless swelling. We present a case of AFD affecting an 8-year-old Japanese girl with a complaint of delayed eruption of her left mandibular second premolar tooth. Intraoral examination did not reveal any sign of pain or swelling in the left mandible. Panoramic radiography revealed a radiolucent and well-circumscribed lesion, which contained radiopaque material at the premolar lesion of the left mandible. The second deciduous molar tooth was unerupted and the second premolar tooth was congenitally lacking. Our provisional diagnoses were odontogenic tumor. The biopsy confirmed AFD. The treatment included total excision of the tumor and the second premolar tooth. No recurrence has been noted during the 2 years of follow-up.

Peripheral-type ameloblastic fibrodentinoma with features of so-called "immature dentinoma".

We report herein a rare case of peripheral-type ameloblastic fibrodentinoma (PAFD) with features of so-called "immature dentinoma" in the gingiva. The patient was a 51-year-old woman, who presented to our hospital complaining of mild swelling in the left upper buccal gingiva. Periapical radiography of the area did not reveal obvious bone resorption and the provisional clinical diagnosis was a benign tumor. The entire mass was excised under general anesthesia, and the pathological diagnosis was PAFD according to the classification by World Health Organization. More precisely, the detail of the histological examination revealed that our case was compatible with unusual type of ameloblastic fibrodentinoma, so-called "immature dentinoma". The postoperative course was satisfactory and the excised area has remained free of recurrence for 5 years.

Prognostic value of podoplanin expression in oral squamous cell carcinoma--a regression model auxiliary to UICC classification.

Podoplanin, a type I transmembrane glycoprotein with an effect of platelet aggregation, has been reported to be one of the possible prognostic factors of oral squamous cell carcinoma (OSCC). However, the biological significance of podoplanin is largely unclear. The aim of this study was to develop a practical model for the prediction of prognosis using the grade of podoplanin expression, and also to evaluate the biological function of podoplanin. Eighty-two specimens of patients with previously untreated OSCC, who underwent either biopsy or surgery, were histopathologically and immunohistochemically analyzed. These 82 cases were composed of 66 well-differentiated, 10 moderately differentiated and 6 poorly differentiated OSCC. Podoplanin was successfully immunostained in 78 specimens, and was detected in most cases, but the frequency of positive cells varied. The prognosis of patients with more than 50 % podoplanin-positive tumor cells was significantly poorer than that of the other patients. Multivariate hazards regression analysis suggested that a linear combination of covariates, OSCC patients with more or less than 50 % podoplanin expression, age of more or less than 70 years old, mode of invasion and T3, T4 or T2 versus T1 of the UICC T-stage classification was the most effective model for evaluating the prognosis of OSCC patients. Additionally, podoplanin expression had a significant relationship to UICC clinical stage and the expression of Ki-67. An effective regression model using podoplanin expression was developed for evaluating the prognosis of OSCC and the biological significance of podoplanin was suggested to be associated with the growth and/or progression of OSCC.

Treatment outcome of Photofrin-based photodynamic therapy for T1 and T2 oral squamous cell carcinoma and dysplasia.

BACKGROUND: Photodynamic therapy (PDT) is a minimally invasive treatment modality for early and superficial malignancy or premalignancy in the head and neck regions. However, few studies have examined the use of Photofrin-mediated PDT to manage early carcinoma and dysplasia in the oral cavity. METHODS: Between January 2004 and November 2008, 25 T1 to T2 patients with N0 oral squamous cell carcinoma and mucosal dysplasia in the oral cavity were treated by Porfimer sodium (Photofrin(®))-mediated PDT at Nagasaki University Hospital. Clinical responses were evaluated according to the guidelines of the Response Evaluation Criteria in Solid Tumors (RECIST). After the PDT and a 2-year follow-up period, disease specific survival rates were then calculated. RESULTS: A total of 30 regions in 25 patients (18 with squamous cell carcinoma and 7 with epithelial dysplasia with hyperkeratosis in the oral cavity) were treated by PDT. Complete response was achieved in 24 of the 25 patients (96%), with a partial response found in the remaining patient. For the three patients who exhibited recurrence at 4, 5, and 15 months after PDT, salvage surgery or a second PDT was performed. Of these three patients, one died due to another disease, while one died due to local lymphatic metastasis that occurred during the follow-up period. Overall, the disease specific survival rate was 95.8%. Treatment-related edema and pain emerged within 24h after irradiation. Pain control using non-steroid anti-inflammatory drugs and opiates was required for 3-4 weeks in all patients. Complete healing was attained at 4-6 weeks after the treatment. No persistent problems related to functional or esthetic outcomes were noted.

Predictors of locoregional recurrence in T1-2N0 tongue cancer patients.

Locoregional recurrence of oral tongue squamous cell carcinoma (OTSCC) has been considered a poor prognostic entity in terms of survival rate. The purpose of this study was to evaluate the incidence of locoregional recurrence and to identify significant risk factors for locoregional recurrence in early-stage OTSCC. We retrospectively reviewed the records of 58 patients who underwent radical surgery for T1-2N0 OTSCC. The local recurrence and regional recurrence rates were 10.3 % (6/58 patients) and 15.5 % (9/58 patients) in this study, respectively. The survival rate of patients with local recurrence was 66.7 %, which was significantly lower than that (96.2 %) of patients without local recurrence, whereas the survival rates of patients with or without regional recurrence were not significantly difference. Pattern of invasion (POI), neoadjuvant chemotherapy (NAC) and the status of the surgical margin were identified as factors influencing local recurrence. In particular, the status of the deep surgical margin was a high potential independent risk factor. The deep surgical margin was resected closely in many NAC-treated cases, suggesting that NAC may lead to local recurrence and a poor outcome. No efficacy of NAC was observed, suggesting that the standard treatment for early OTSCC is surgery alone.

The observational study of delayed wound healing after tooth extraction in patients receiving oral bisphosphonate therapy.

INTRODUCTION: In this study, we investigated whether such a discontinuation of oral bisphosphonate (BP) for 3 months might influence the incidence of BP-related osteonecrosis of the jaw (BRONJ) and wound healing after tooth extraction in patients receiving oral BP therapy. MATERIAL AND METHODS: There were a total of 434 teeth in 201 patients (18 males and 183 females). The patients were divided into two groups depending on whether or not they underwent a 3-month discontinuation of BP therapy (BP- and BP+) before tooth extraction. In this observational study investigated delayed wound healing after tooth extraction in patients receiving oral BP therapy. RESULTS: In all cases of the BP- group, there were no BRONJ although there was delayed wound healing in two cases. However, in one case of the BP+ group, oral BP was continued because it was deemed high risk to discontinue treatment by the patient's physician. In this case, an intraoral fistula was still present with bone exposure at 120 weeks after extraction (BRONJ stage 1). CONCLUSION: This study supports the idea of a drug holiday and encourages further clinical research on this topic of tooth extraction in patients receiving oral BP therapy.

Prediction of outcome of patients with oral squamous cell carcinoma using vascular invasion and the strongly positive expression of vascular endothelial growth factors.

Vascular invasion and lymph node metastasis have been used as histopathological prognosticators of cancers including oral squamous cell carcinoma (OSCC). In addition to metastatic potential via blood vessels, tumor-induced angiogenesis might also be associated with prognosis. However, the efficacy of combined evaluation of vascular invasion and angiogenesis-associated molecules for the prognosis of OSCC remains obscure. This is also the case in lymph node metastasis and lymphovasculogenesis-associated molecules. The aim of this study was to examine factors related to prognosis to improve the accuracy of prognostic prediction of OSCC using vasculogenesis-associated markers. Ninety specimens of patients from 1991 to 2002 with previously untreated OSCC, who underwent either biopsy or surgery, were histopathologically and immunohistochemically analyzed using antibodies for vascular endothelial growth factor (VEGF)-A, VEGF-C, cyclooxygenase (COX)-2 and Midkine. The ninety cases were composed of 72 well-differentiated, 12 moderately differentiated and 6 poorly differentiated OSCC. Efficient models of prognostic prediction were evaluated by extensive statistical analyses. The presence of vascular invasion or lymph node metastasis was confirmed to be significantly associated with poor prognosis in the univariate analysis. Multivariate logic regression analysis suggested that patients with the strongly positive expression of either VEGF-A or VEGF-C had a significant association with poor prognosis even in patients without vascular invasion and in early-stage patients. Neither COX-2 nor Midkine contributed to predict the prognosis of the patients. The strongly positive expression of VEGF-A or VEGF-C was suggested to reinforce the histopathological diagnosis of vascular invasion and improve the accuracy and efficacy of prognostic prediction of OSCC.

Predictive factor for photodynamic therapy effects on oral squamous cell carcinoma and oral epithelial dysplasia.

OBJECTIVE: The aim of this study was to investigate the correlation between the immunohistochemical expression of proliferating cell nuclear antigen (PCNA), factor VIII, and CD34 (markers of endothelial cells), and vascular endothelial growth factor (VEGF) and the recurrence of oral squamous cell carcinoma (OSCC) and oral epithelial dysplasia (OED) subjected to photodynamic therapy (PDT). DESIGN: Twenty-one biopsy specimens (14 cases of OSCC and 7 cases of OED) before PDT were immunohistochemically investigated in terms of their expressions of PCNA, factor VIII, CD34 and VEGF. The percentages of the total sample area that were immunopositive for factor VIII (percentage factor VIII immunopositive area: PFIA) CD34 (PCIA) and VEGF (PVIA) were calculated using computer-assisted image analysis for quantitative assessment of endothelial cells or VEGF expression in the lesions. The PCNA labelling index (LI) was evaluated as a proliferation marker. RESULTS: Five cases of OSCC and one case of OED recurred 4 to 30 months after PDT. We found that the average PVIA was 14.5% in the no-recurrence group and 1.7% in the recurrence group. The difference between these values was statistically significant (P=0.0483). On the other hand, the average PCNA LI was 30.3% in the no-recurrence group and 24.3% in the recurrence group; the average PFIA was 3.7% in the no-recurrence group and 1.6% in the recurrence group; and the average PCIA was 2.0% in the no-recurrence group and 1.4% in the recurrence group. There were no significant differences between the two groups for any of these markers (P=0.3379, P=0.1195, P=0.4835, respectively). CONCLUSIONS: These results provide clinical data indicating that VEGF expression may be a useful predictive marker for the effects of PDT in OSCC and OED.

Engineering bone formation from human dental pulp- and periodontal ligament-derived cells.

A robust method for inducing bone formation from cultured dental mesenchymal cells has not been established. In this study, a method for generating bone tissue in vivo from cultured human dental pulp- and periodontal ligament-derived cells (DPCs and PDLCs, respectively) was designed using exogenous bone morphogenetic protein 2 (BMP2). DPCs and PDLCs showed enhanced alkaline phosphatase (ALP) activity and calcified nodule formation in medium containing dexamethasone, ß-glycerophosphate, and ascorbic acid (osteogenic medium). However, the addition of recombinant human bone morphogenetic protein 2 (rhBMP2) to osteogenic medium remarkably increased ALP activity and in vitro calcification above the increases observed with osteogenic medium alone. rhBMP2 also significantly upregulated the expression of osteocalcin, osteopontin, and dentin matrix protein 1 mRNA in both cell types cultured in osteogenic medium. Finally, we detected prominent bone-like tissue formation in vivo when cells had been exposed to rhBMP2 in osteogenic medium. In contrast, treatments with osteogenic medium or rhBMP2 alone could not induce abundant mineralized tissue formation. We propose here that treatment with rhBMP2 in osteogenic medium can make dental mesenchymal tissues a highly useful source of cells for bone tissue engineering. In addition, both DPCs and PDLCs showed similar and remarkable osteo-inducibility.

Histopathological change of oral malignant tumour and epithelial dysplasia subjected to photodynamic therapy.

OBJECTIVES: The purpose of this study is to analyze the morphological change of cell nuclei and the change of proliferating activity of oral malignancy and epithelial dysplasia between before and after photodynamic therapy in order to predict recurrence. MATERIAL AND METHODS: We experienced 14 cases of oral squamous cell carcinoma, one case of verrucous carcinoma and seven cases of epithelial dysplasia treated by photodynamic therapy (PDT). The mean nuclear area (NA) and coefficient of variation of the nuclear area (NACV) of 100 nuclei per slide were calculated using computer-assisted image analysis in hematoxylin and eosin stained biopsy specimens before and after PDT. Additionally, proliferating cell nuclear antigen (PCNA) immunohistochemistry was carried out in each specimen. RESULTS: The mean NA after PDT was significantly lower than that before PDT in the nonrecurrent group. However, there was no significant difference in mean NA before and after PDT in the recurrent group. There were no significance differences in NACV before and after PDT in either the nonrecurrent or recurrent group. Furthermore, the PCNA labelling indices of the specimens after PDT was significantly lower than that before PDT in both the nonrecurrent and the recurrent group. CONCLUSIONS: Mean nuclear area in the biopsy specimen after photodynamic therapy is likely to be a predictive marker for the recurrence of oral squamous cell carcinoma or epithelial dysplasia subjected to photodynamic therapy, while coefficient of variation of the nuclear area and proliferating cell nuclear antigen labelling indices are less helpful in predicting the recurrence of such lesions.

Hypoxia-inducible factor 1 alpha in oral squamous cell carcinoma and its relation to prognosis.

The aim of this study was to investigate the correlation between the expression of hypoxia-inducible factor 1 alpha (HIF-1 alpha) and proliferative activity in tumor cells, lymph node metastasis, as well as prognosis in patients with oral squamous cell carcinoma (OSCC). Fifty-seven biopsy specimens of OSCC were investigated for the expression of HIF-1 alpha and proliferating cell nuclear antigen (PCNA) by immunohistochemistry. None of the patients had received any prior treatments. The percentage of HIF-1 alpha immunopositive area (PHIA) was calculated using computer-assisted image analysis for quantitative assessment of HIF-1 alpha expression. The PCNA labeling index (LI) was evaluated as a proliferation marker. We found that the mean PHIA in all stages was 12.1% in the poor prognosis patients, and it was 6.4% in the good prognosis patients. There was a significant difference of PHIA between poor prognosis and good prognosis patients (P=0.0065). Furthermore, the mean PHIA in the patients who had no metastatic lymph nodes was 7.5%, while it was 11.7% in the patients who had metastatic lymph nodes. There was also a significant difference of PHIA between patients who had no metastatic lymph nodes and those who had metastatic lymph nodes (P=0.0487). On the other hand, significant correlation between PHIA and PCNA LI was not observed. These results provide the clinical data indicating that HIF-1 alpha may play an important role in lymph node metastasis and prognosis in patients with OSCC.

Intraosseous carcinoma of the mandible composed of cells expressing myoepithelial cell-associated antigens.

Carcinomas are rarely observed centrally in mandibular and maxillary bone. Etiology of intraosseous squamous cell carcinoma is thought to involve derivation from remnants of the odontogenic epithelium. Mucoepidermoid carcinoma is also rarely observed centrally in the mandibular and maxillary bone. Previous analyses raised the possibility that the tumor arose from entrapped salivary elements or from metaplastic odontogenic epithelia. Herein is described an intramandibular carcinoma with characteristic various morphologies. The tumor was composed of epidermoid cells, clear cells, mucous cells and intermediate cells, which were surrounded by cellular-rich collagenous stroma. The epidermoid cells, clear cells and mucous cells formed solid sheets, and the clear cells and intermediate cells formed trabecular structures. Sparse cell regions were partly accompanied by clear cells and mucous cells. Immunohistochemistry was positive for myoepithelial markers in the trabecular portion of the tumor. These findings suggest that this is a unique case of intramandibular mucoepidermoid carcinoma that includes tumor cells expressing myoepithelial cell-associated antigens.