Expression of hypoxia-inducible factor-1α is significantly associated with the progression and prognosis of oral squamous cell carcinomas in Taiwan

Background:  Overexpression of hypoxia-inducible factor-1α (HIF-1α) has been found to be significantly associated with the tumor invasion, lymph node metastasis, clinical stage, and prognosis of a variety of human cancers.
Methods:  This study examined the expression of HIF-1α in 57 specimens of oral squamous cell carcinoma (OSCC), 41 specimens of oral epithelial dysplasia (OED, 12 mild, 17 moderate, and 12 severe OED cases), and 14 specimens of normal oral mucosa (NOM) by immunohistochemistry.
Results:  We found that the mean nuclear HIF-1α labeling indices (LIs) increased significantly from NOM (9 ± 6%) through mild OED (25 ± 18%), moderate OED (41 ± 27%), and severe OED (42 ± 22%) to OSCC samples (55 ± 23%, P  < 0.001). A significant correlation was found between the higher mean nuclear HIF-1α LI and OSCCs with larger tumor size ( P  < 0.001), regional lymph node metastasis ( P  < 0.001), or more advanced clinical stages ( P  < 0.001). Only larger tumor size ( P  = 0.002) and nuclear HIF-1α LI ≥ 60% ( P  = 0.048) were identified as independent unfavorable prognosis factor by multivariate analyses with Cox regression model. Kaplan–Meier curve showed that OSCC patients with a nuclear HIF-1α LI ≥ 60% had a significantly poorer cumulative survival than those with a nuclear HIF-1α LI < 60% (log-rank test, P  = 0.022).
Conclusions:  We conclude that the expression of HIF-1α is an early event in oral carcinogenesis. The nuclear HIF-1α LI in OSCC samples can predict the progression of OSCCs and the survival of OSCC patients.     

Expression of hepatocyte growth factor and c-met protein is significantly associated with the progression of oral squamous cell carcinoma in Taiwan

BACKGROUND: Hepatocyte growth factor (HGF) is a pleotropic growth factor that regulates cell proliferation, migration, survival, tumor angiogenesis, and tumor cell invasion and metastasis. Its diverse biological effects are mediated through its interaction with its receptor, c-met protein. METHODS: In this study, we examined the expression of HGF and c-met protein in 93 specimens of oral squamous cell carcinoma (OSCC), 10 specimens of oral epithelial dysplasia (OED), 14 specimens of oral epithelial hyperkeratosis (OEH), and 16 specimens of normal oral mucosa (NOM) by immunohistochemistry. The HGF and c-met labeling indices (LIs) in OSCC, OED, OEH, and NOM groups were calculated and compared between groups. The correlation between the expression of HGF or c-met in OSCCs and clinicopathological parameters, or survival of OSCC patients was analyzed statistically to investigate the possible influence of HGF or c-met on the progression and prognosis of OSCCs in Taiwan. RESULTS: Positive HGF or c-met staining was mainly cytoplasmic. The mean HGF LI increased significantly from NOM (3.1 +/- 5.1%) through OEH (32.5 +/- 19.8%) and OED (52.0 +/- 19.3%) to OSCC (71.9 +/- 28.6%; P = 0.000). The mean c-met LI also increased significantly from NOM (25.8 +/- 30.8%) and OEH (34.4 +/- 19.3%) through OED (53.0 +/- 20.0%) to OSCC (73.0 +/- 29.4%; P = 0.000). Statistical analysis showed that the c-met LI in either the tumor center or invasion front was significantly associated with T status, N status, and clinical staging of OSCC. However, only the HGF LI in the tumor invasion front was significantly correlated with N status and clinical staging of OSCC. CONCLUSION: Our results suggest that the expression of HGF and c-met protein is an early event in oral carcinogenesis in Taiwan. The HGF LI in the tumor invasion front and the c-met LI in either the tumor center or invasion front can predict the progression of OSCCs in Taiwan.     

CXCR-4 knockdown by small interfering RNA inhibits cell proliferation and invasion of oral squamous cell carcinoma cells

Background:  Oral squamous cell carcinomas (OSCCs) are characterized by a high degree of local invasion and a high rate of metastases to cervical lymph nodes. Downregulation of CXCR-4 by siRNA inhibits invasion and growth of breast and colon cancer cells. However, there have been no reports on the downregulation of CXCR-4 by small interfering RNA (siRNA) in oral cancer cells.
Methods:  We generated two stable CXCR-4-knockdown clones (KBsi and KOSCC-25Bsi) from the KB and KOSCC-25B OSCC cell lines by lentiviral delivery. In vitro invasion and cell proliferation assays were used to investigate the effect of CXCR-4 downregulation on cell proliferation and invasiveness in KBsi and KOSCC-25Bsi. Immunohistochemistry was performed to evaluate the correlation between CXCR-4 expression and proliferation in 26 OSCC tissue samples.
Results:  CXCR4-knockdown OSCC cells showed reduced invasiveness. The invasiveness of KBsi decreased to 29.5% of the vector-infected controls, and KOSCC-25Bsi decreased to 38.1% of the control vector-infected cells ( P  <   0.05). The CXCR4-knockdown OSCC cells grew significantly slower than the vector-infected control cells. KBsi and KOSCC-25Bsi cells proliferated at 69.5% and 71.7%, respectively, of the rate of control vector-infected cells ( P  <   0.05). CXCR-4-positive group had significantly higher PCNA labeling index than CXCR-4-negative group in OSCC tissue samples.
Conclusion:  These results suggest that the downregulation of CXCR-4 induces anti-proliferative and anti-invasive effects in OSCC and that CXCR-4 might be a useful target molecule for the treatment of OSCC.     

Prognostic significance of Bcl-2 and Bax protein expression in the patients with oral squamous cell carcinoma

Background:  The aim of this study was to investigate the expression of the apoptosis-inhibitory Bcl-2 protein and the apoptosis-promoting Bax protein and to identify their association with the clinical parameters and prognosis of the patients with oral squamous cell carcinoma (OSCC).
Methods:  The expression of Bcl-2 and Bax proteins was evaluated by immunohistochemical staining in specimens from 110 patients with OSCC. Every section was scored according to both the percentage of positive staining tumor cells and the staining intensity. The Kaplan-Meier test and Cox proportional hazards regression analysis were performed to assess the correlation between the protein levels and the long survival rate of patients. The association between Bax, Bcl-2 immunoexpression and clinicopathologic variables was analyzed with the chi-square test and non-parametric analysis. The Bcl-2 and Bax immunoexpression in 20 oral mucosa samples were also investigated as normal control.
Results:  The results showed that the 5-year survival rate was significantly higher in the patients with the ratio of Bcl-2/Bax ≤ 1 than in those with Bcl-2/Bax > 1 (76.79 ± 6.69% vs. 59.26 ± 6.69%, P  = 0.0489). Bax immunoreactivity was significantly correlated with histological grading and lymph node metastasis. Univariate analysis indicated that the ratio of Bcl-2/Bax and lymph node metastasis were two independent factors related to the prognosis.
Conclusion:  The ratio of Bcl-2/Bax could be used as an effective biomarker to predict the prognosis of OSCC.     

Serum soluble CD44v6 levels in patients with oral and maxillofacial malignancy

Objective:  To determine the levels of serum sCD44v6 in patients with oral cancer and evaluate the value of serum sCD44v6 in adjuvant diagnosis, staging and monitoring treatment response in these patients.
Materials and Methods:  A total of 112 hospitalized patients with oral and maxillofacial malignancy and 28 healthy individuals were examined for serum sCD44v6 levels. Venous blood was collected from these patients and the healthy individuals. One week after treatment, venous blood was collected once again in 60 patients with oral and maxillofacial squamous cell carcinoma (OSCC).
Results:  The sCD44v6 concentration was not significantly different between patients with oral and maxillofacial malignancy and control group ( P  > 0.05). The levels of serum sCD44v6 in patients with OSCC and salivary carcinoma showed no difference with those in control group ( P  > 0.05). The sCD44v6 level in patients with stage III and IV disease was higher than that of patients with stage I and II and that of the control group, but the difference was not significant ( P  > 0.05).  Serum sCD44v6 levels in patients with OSCC after treatment became lower than that prevailed during pretreatment ( P  < 0.05).
Conclusion:  The possible roles of CD44v6 in the diagnosis of oral and maxillofacial malignancy deserve further elucidation and evaluation. Serum sCD44v6 may be a valuable marker in monitoring treatment response in patients with OSCC.     

Fatty acid synthase expression in squamous cell carcinoma of the tongue: clinicopathological findings

Background:  Overexpression of fatty acid synthase (FAS), the cytosolic enzyme responsible for the conversion of dietary carbohydrates to fatty acids, has been reported in several human malignancies and pointed as a potential prognostic marker for some tumors. This study investigated whether FAS immunohistochemical expression is correlated with the clinicopathological characteristics of oral squamous cell carcinoma (OSCC).
Materials and methods:  The clinical features of 102 patients with OSCC of the tongue treated in a single institution were obtained from the medical records and all histopathological diagnoses were reviewed. The expression of FAS was determined by the standard immunoperoxidase technique in formalin-fixed and paraffin-embedded specimens and correlated with the clinicopathological characteristics of the tumors.
Results:  Eighty-one cases (79.41%) were positive for FAS. Microscopic characteristics such as histological grade ( P  < 0.05), lymphatic permeation ( P  < 0.001), perineural infiltration ( P  < 0.05), and nodal metastasis ( P  < 0.02) were associated with FAS status. A significantly lower survival probability for patients with advanced clinical stage (log-rank test, P  < 0.001), lymph nodes metastasis (log-rank test, P  < 0.001), presence of vascular permeation (log-rank test, P  = 0.05), and perineural invasion (log-rank test, P  = 0.01) was observed in the studied samples.
Conclusion:  The expression of FAS in OSCC of the tongue is associated with the microscopic characteristics that determine disease progression and prognosis.     

Apoptosis-associated markers and clinical outcome in human oral squamous cell carcinomas

Background:  Apoptosis is a genetically regulated cell death involved in the deletion of cells in normal or malignant tissues. Proteins of the Bcl-2 family play a key role in the control of apoptosis and carry out both pro-apoptotic and anti-apoptotic functions. The present study evaluated the prognostic value of Bcl-2 and Bax expression at the invasive front of oral squamous cell carcinoma (OSCC), taking clinicopathological findings into account.
Methods:  Fifty-six specimens of OSCC were randomly selected, and Bcl-2 and Bax expression was evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded pre-treated specimens at the invasive front of OSCC. Clinicopathological data were gathered and patient survival was analysed.
Results:  No significant relationship was found between Bcl-2 or Bax expression and clinical variables. Patients with Bcl-2 expression had a worse prognosis than those without Bcl-2 expression, but the difference did not reach statistical significance. Patients with Bax expression had a significantly better prognosis than those without Bax expression ( P  <   0.05). In univariate analyses, T category, mode of cancer invasion and Bax expression showed significant correlations. Multivariate analysis revealed that the mode of cancer invasion and Bax expression were significant and independent variables. Bax expression was found to be the strongest independent prognostic parameter. Patients with negative Bcl-2 expression and positive Bax expression had a significantly better prognosis ( P  <   0.005).
Conclusion:  We suggest that Bax expression at the invasive front of OSCC is a significant predictor of prognosis and that it is therefore important to investigate the expression of Bcl-2 and Bax in this disease.     

Shifts in cellular localization of moesin in normal oral epithelium, oral epithelial dysplasia, verrucous carcinoma and oral squamous cell carcinoma

BACKGROUND: Moesin, a member of ERM (ezrin/radixin/moesin) family, links actin filaments of cell surface structure to the cell membrane. The purpose of the study is to assess the shifts in cellular distribution of moesin in normal oral epithelium, oral epithelial dysplasia (OED), verrucous carcinoma (VC), and oral squamous cell carcinoma (OSCC). METHODS: The expression of moesin was evaluated immunohistochemically in paraffin-embedded tissues of 59 specimens of OSCC, 35 specimens of OED, 17 specimens of VC, and five specimens of normal oral epithelium. RESULTS: In the normal oral epithelia, all specimens showed a pattern of membranous expression against the anti-moesin antibody in the basal layer cells. In the OED specimens, moesin was dominantly expressed in the cell membrane except for the cornified layer. In VC and OSCC specimens, almost the whole of the carcinoma cells were stained with anti-moesin antibody. However, in OSCC samples, moesin was markedly expressed increasingly in the cytoplasm and decreasingly in the cell membrane, as compared with OED and VC. In addition, there was a significant correlation between the pattern of moesin expression and tumor differentiation in OSCC. CONCLUSIONS: Our results suggest that it is useful to detect the moesin expression as adjunct to screening mucosal lesions in the oral cavity.     

Metallothionein and p-Akt proteins in oral dysplasia and in oral squamous cell carcinoma: an immunohistochemical study

Background:  Oral leukoplakia (OL) is the main potentially malignant lesion of the oral cavity, and oral squamous cell carcinoma (OSCC) accounts for more than 95% of all malignant neoplasms in the oral cavity. Therefore, the aim of this study was to verify the immunoexpression of p-Akt and Metallothionein (MT) proteins in dysplasic and neoplasic oral lesions.
Methods:  Immunohistochemical studies were carried out on 10 normal epithelium, 30 OL and 15 OSCC paraffin-embedded samples. Immunoperoxidase reaction for p-Akt and MT proteins was applied on the specimens, and the positivity of the reactions was calculated for 1000 epithelial cells.
Results:  Using the ANOVA and the Tukey's post hoc statistical analyses, it was observed a significant difference in the immunoexpression for p-Akt and MT when the OSCC samples were compared with normal and dysplasic epithelial groups. In addition, the Pearson's correlation test showed a significant correlation between the proteins' expression.
Conclusion:  Based on the data obtained, p-Akt and MT activation may play an important role in the conversion of a potentially malignant oral lesion to a malignant carcinoma since its earlier stages.     

Expression of CXCR4 in oral squamous cell carcinoma: correlations with clinicopathology and pivotal role of proliferation

J Oral Pathol Med (2010) 39 : 63–68
Background:  The chemokine SDF-1 and its receptor CXCR4 play active role in the metastasis and proliferation of several malignancies.
Methods:  In this study, we used an immunohistochemical technique in 91 specimens of oral squamous cell carcinoma (SCC), and flow cytometry technique in oral SCC cell line, and then evaluated the role of proliferation of CXCR4 using MTT assay in oral SCC cell line.
Results:  The expression of CXCR4 in 91 specimens of oral SCC was 62.6% and in oral SCC cell line was 68.6%. There was a significant association between the expression of CXCR4 and lymph node metastasis ( P  = 0.012), tumor size ( P  = 0.01), UICC stage ( P  = 0.016), tumor histology grade ( P  < 0.001). SDF-1 stimulated proliferation of oral SCC cell and CXCR4 neutralization by monoclonal antibodies decreased proliferation.
Conclusions:  Our results suggest that CXCR4 might be a novel biomarker to evaluate the biological behavior of oral SCC. CXCR4 inhibitors or antagonists might be potential anticancer agents to suppress tumor proliferation.     

The close association between dental and periodontal treatments and oral ulcer course in behcet's disease: a prospective clinical study

Objective:  The aim of the study was to evaluate the influence of dental and periodontal treatments to the course of oral ulcers in patients with Behcet's disease (BD).
Methods:  Fifty-eight consecutive BD patients with oral ulcers were studied. Twenty-nine patients were in the intervention group (F/M: 15/14, mean age: 39.6 ± 6.9 years) and 29 (F/M: 15/14, 39.4 ± 10.6 years) were followed with a conventional treatment approach. In addition to oral hygiene education, dental and periodontal treatments were carried out in the intervention group, whereas the control group was only given oral hygiene education. Patients were evaluated in the pre-treatment observation period (1 month), treatment period (1 month) and 6 months after treatment.
Results:  An increase in the number of new oral ulcers (4.1 ± 3.5) was observed within 2 days during the treatment compared with 3–30 days during treatment month (2.3 ± 1.2) ( P  = 0.002). However, 6 months after the treatment, the number of oral ulcers (1.9 ± 1.5) was significantly lower compared with the pre-treatment observation (4.8 ± 3.2) ( P  = 0.000) and treatment periods (6.4 ± 2.3) in the intervention group ( P  = 0.05), whereas a similar oral ulcer presence was observed in the control group (2.8 ± 2.4, 3.7 ± 2.3 and 4.8 ± 4.3, respectively) ( P  > 0.05). Dental and periodontal indices were also better in the intervention group during the 6-month follow-up.
Conclusion:  Our results suggest that, in BD patients, dental and periodontal therapies could be associated with a flare-up of oral ulcers in the short term, but may decrease their number in longer follow-up. They also lead to a better oral health.     

Progressive increase of human papillomavirus carriage rates in potentially malignant and malignant oral disorders with increasing malignant potential

Introduction:  We investigated the potential role of human papillomaviruses (HPVs) in potentially malignant oral disorders, oral leukoplakia (OL) and oral lichen planus (OLP), and in oral squamous cell cancer (OSCC) in an Eastern Hungarian population with a high incidence of OSCC.
Methods:  Excised tumor samples (65 OSCC patients) and exfoliated cells from potentially malignant lesions (from 44 and 119 patients with OL and OLP, respectively) as well as from healthy controls (72 individuals) were analysed. OLPs were classified based on clinical appearance, 61 patients had erosive–atrophic lesions (associated with higher malignancy risk, EA-OLP) and 58 had non-erosive non-atrophic lesions (with lower risk of becoming malignant, non-EA-OLP), respectively. Exfoliated cells collected from apparently healthy mucosa accompanied each lesion sample. HPV was detected by MY/GP polymerase chain reaction (PCR) and genotyped by restriction analysis of amplimers. Copy numbers in lesions were determined using real-time PCR. Prevalence rates, copy number distributions, and association with risk factors and diseases were analysed using chi-square test, t -test, and logistic regression, respectively.
Results:  We detected HPVs significantly more frequently in lesions than in controls ( P  ≤ 0.001 in all comparisons). HPV prevalence increased gradually with increasing severity of lesions (32.8, 40.9, and 47.7% in OLP, OL, and OSCC, respectively). Copy number distribution patterns roughly corresponded to prevalence rates, but OLP and OL were comparable. HPV prevalence differed significantly between EA-OLP and non-EA-OLP groups (42.6 vs. 22.4%); EA-OLP group showed a prevalence similar to that found in OL.
Conclusion:  HPVs may be involved in the development or progression of not only OSCC but also of potentially malignant oral lesions.     

Oral distribution of Candida species and presence of oral lesions in Brazilian leprosy patients under multidrug therapy

Objective:  The aim of this study was to evaluate the prevalence of Candida spp. and presence of oral lesions in Brazilian leprosy patients under multidrug therapy (MDT).
Methods:  Thirty-eight individuals (18 males and 20 females, median age 53 years) clinically and microbiologically diagnosed as leprosy (lepromatous variant), and under MDT for at least 45 days were studied. The control group constituted by 38 healthy individuals (median age 53.5), matched to the test group in relation to age, gender and oral conditions. Oral rinses were collected and the Candida identification was performed by phenotypic tests. The existence of Candida dubliniensis among the isolates was analyzed using a validated multiplex PCR assay. Twenty-nine leprosy patients were examined intra-orally for the presence of lesions. Data were analyzed by z- and Mann–Whitney tests (α = 5%).
Results:  Yeast carriage rate between leprosy patients (65.8%) and controls (47.4%) was similar ( P  = 0.099), and no significant difference between yeast counts was observed ( P  = 0.1004). Candida albicans was the most frequently isolated species in both groups. In the leprosy group, Candida tropicalis and Candida parapsilosis were also identified. In the control group, we additionally identified Candida tropicalis , Candida glabrata and Candida kefyr. Candida dubliniensis was not detected. No leprosy-related oral lesion was registered.
Conclusion:  Within the limits of the study, we concluded that Brazilian leprosy patients under MDT showed similar levels of carriage and Candida species distribution in relation to the controls.     

Use of Patient Self-Report Oral Health Outcome Measures in Assessment of Dental Treatment Outcomes

Objective: To assess the sensitivity of a newly developed brief measure of oral health-related quality of life (OQOL). Methods: Self-assessed oral health and OQOL were measured in three groups of patients who had presented for either prophylaxis ( n =  32), endodontic care ( n =  15), or for a denture ( n =  16) in a dental school setting before and after treatment. Main outcome measures included the single-item self-report of oral health (OH-1) and the 6- and 12-item versions of a new OQOL instrument. General linear modeling was used to compute means of self-reported oral health by treatment group. Results: Of the 63 patients who completed the baseline questionnaire, 44 (70 percent) returned questionnaires after treatment. The sample averaged 43  ±  15 years, 48 percent male and 55 percent with some college education. Ethnic representation included 35 percent White, 33 percent Black, and 32 percent other – mostly Latino. The mean self-reported number of teeth was 20.6. In terms of sensitivity, significant differences were observed between the treatment groups on the items assessing being upset ( P <  0.05), feeling depressed ( P <  0.05), and uncomfortable about the appearance of teeth or dentures ( P <  0.05). However, magnitude of change, as measured by an effect size, was characterized as minimal to small in the recall and endodontic groups and borderline moderate in the denture group. Conclusion: The measure was sensitive to differences within groups, with a small to borderline magnitude of change.     

E‐cadherin downregulation and Twist overexpression since early stages of oral carcinogenesis

There is some evidence of Twist participation in oral carcinogenesis; however, little is known about its interaction with E‐cadherin in oral squamous cell carcinoma (OSCC) development. This experimental study included an immunohistochemical analysis of Twist and E‐cadherin proteins in paraffin‐embedded specimens of oral leukoplakia (OL), OSCC, and normal oral mucosa. In addition, it was also performed a Western blot and double‐immunofluorescence analysis of Twist and E‐cadherin expression in OSCC cell lines. Significant differences in Twist and E‐cadherin immunoexpression were observed between normal oral mucosa and OL, with an inverse relation since the earliest stages of oral dysplasia (r = ?0,512; P < 0.001). Western blot and double‐immunofluorescence analysis showed differences in Twist and E‐cadherin expression among human oral keratinocytes and OSCC cell lines suggesting that downregulation of E‐cadherin occurs in a dependent manner of Twist in OSCC. Our results showed a possible value of Twist and E‐cadherin in the prediction of risk of oral epithelium malignant transformation.     

A composite index for determining the impact of oral ulcer activity in Behcet's disease and recurrent aphthous stomatitis

Background:  Although number, frequency and healing time of oral ulcers and pain are generally used for clinical practice and studies in Behcet's disease (BD) and recurrent aphthous stomatitis (RAS), no standardized activity index is currently present to monitor clinical manifestations associated with oral ulcers. The aim of this study was to develop a standardized composite index (CI) to assess oral ulcer activity in BD and RAS.
Methods:  In this cross-sectional study, 121 patients with BD and 45 patients with RAS were included. Sixty-five percentage of BD and 68.9% of RAS patients were in active stage during the previous 3 months. The developed CI included the presence of oral ulcers, ulcer-related pain and functional status and was evaluated in patients with both active and inactive disease for content validity.
Results:  Composite index score was observed to be higher in active patients with RAS (6.94 ± 2.19) compared with active BD patients (6.01 ± 2.04) ( P  = 0.04). The number of oral ulcers and healing time of oral ulcers were significantly higher in RAS compared with BD ( P  = 0.018, P  = 0.001 respectively). CI score correlated with the number of oral ulcers in both BD and RAS ( P  = 0.000, P  = 0.002 respectively). CI score was '0' for inactive patients without oral ulcer in BD and RAS.
Conclusions:  The presented CI as an oral ulcer activity index seems to be a reliable and suitable tool for evaluating the clinical impact and disease-specific problems in BD and RAS.     

口腔黏膜癌前病变和口腔鳞癌组织Fas/FasL的表达及其意义

目的：研究调亡相关基因Fas／FasL在正常口腔黏膜、上皮异常增生和口腔鳞癌组织中的表达及意义。方法：应用免疫组织化学方法检测10例正常口腔黏膜、26例上皮异常增生、38例口腔鳞癌组织及肿瘤浸润淋巴细胞（TIL）中Fas／FasL的表达。结果：Fas在正常口腔黏膜中广泛表达;上皮异常增生和鳞癌组织表达明显下调（P〈0．05）;Fas表达与口腔鳞癌分化程度有关;FasL在正常口腔黏膜不表达;上皮异常增生和鳞癌组织表达明显上调（P〈0．05）;FasL表达与口腔鳞癌分化程度无关（P〉0．05）;TIL细胞Fas、FasL阳性表达率为81．6％和84．2％。结论：Fas表达与口腔黏膜上皮细胞的自然分化成熟、衰老及口腔鳞癌的形成和肿瘤的恶性度有关;FasL的表达上调可能是口腔鳞癌组织免疫反攻击的体现;Fas、FasL可作为监测口腔上皮癌变的标记物。