TCR Repertoire as a Novel Indicator for Immune Monitoring and Prognosis Assessment of Patients With Cervical Cancer.

There is increasing evidence that deep sequencing-based T cell repertoire can sever as a biomarker of immune response in cancer patients; however, the characteristics of T cell repertoire including diversity and similarity, as well as its prognostic significance in patients with cervical cancer (CC) remain unknown. In this study, we applied a high throughput T cell receptor (TCR) sequencing method to characterize the T cell repertoires of peripheral blood samples from 25 CC patients, 30 cervical intraepithelial neoplasia (CIN) patients and 20 healthy women for understanding the immune alterations during the cervix carcinogenesis. In addition, we also explored the signatures of TCR repertoires in the cervical tumor tissues and paired sentinel lymph nodes from 16 CC patients and their potential value in predicting the prognosis of patients. Our results revealed that the diversity of circulating TCR repertoire gradually decreased during the cervix carcinogenesis and progression, but the circulating TCR repertoires in CC patients were more similar to CIN patients than healthy women. Interestingly, several clonotypes uniquely detected in CC patients tended to share similar CDR3 motifs, which differed from those observed in CIN patients. In addition, the TCR repertoire diversity in sentinel lymphatic nodes from CC patients was higher than in tumor tissues. More importantly, less clonotypes in TCR repertoire of sentinel lymphatic node was associated with the poor prognosis of the patients. Overall, our findings suggested that TCR repertoire might be a potential indicator of immune monitoring and a biomarker for predicting the prognosis of CC patients. Although functional studies of T cell populations are clearly required, this study have expanded our understanding of T cell immunity during the development of CC and provided an experimental basis for further studies on its pathogenesis and immunotherapy.

Establishing a Th17 based mouse model for preclinical assessment of the toxicity of candidate microbicides.

BACKGROUND: To effectively block the invasion of human immunodeficiency virus (HIV)-1 on mucosal surface, vaginal anti-HIV-1 microbicides should avoid inflammatory responses and disruption of mucosa integrity because these will facilitate transepithelial viral penetration and replication. However, existing models fail to predict and evaluate vaginal mucosal toxicity induced by microbicides, and most importantly, they are unable to identify subtle or subclinical inflammatory reactions. This study was designed to develop a cost-effective in vivo model to evaluate microbicide safety in a preclinical study which can recapitulate the mucosal topical reaction. METHODS: A murine model was employed with nonoxynol-9 (N-9) as the topical stimulant within the vagina. Different concentrations of N-9 (1%, 3%, and 4%) were topically applied to the vagina for five consecutive days. A panel of inflammatory cytokines including interleukine-2 (IL-2), IL-4, IL-6, IL-17A, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and immuno-regulatory IL-10 were assayed in vaginal lavage. Cytokines were quantified by using cytometric bead array (CBA) and reverse transcript (RT) real-time PCR. Histopathological evaluation of vaginal tissues was conducted on hematoxylin-eosin stained slides and scored with a semi-quantitative system according to the severity of epithelial disruption, leucocyte infiltration, edema, and vascular injection. The association between the cytokines and histopathological scores was assessed by linear regression analysis. RESULTS: All three concentrations of N-9 induced inflammatory cytokine production. The 4% N-9 application resulted in a consistent production of cytokines in a time-dependent manner. The cytokines reached peak expression on day three with the exception of IL-4 which reached its peak on day one. Histopathological examination of 4% N-9 treated cervicovaginal tissues on day three showed intensive damage in four mice (sores: 10 - 13) and moderate damage in one mouse (score: 8), which were significantly associated with both inflammatory cytokines IL-17A and IL-6 and anti-inflammatory cytokines IL-4 and IL-10. Interestingly, IL-17A showed significant positive association with inflammatory cytokine TNF-α (r = 0.739; P < 0.05), anti-inflammatory cytokines IL-10 (r = 0.804; P < 0.01) and IL-4 (r = 0.668; P < 0.05). CONCLUSIONS: Our data demonstrate that a panel of cytokines (IL-17A, IL-6, IL-4 and IL-10) could be used as surrogate biomarkers to predict the histopathological damage. Th17 may play a central role in orchestrating inflammatory cytokine responses. This Th17 based mouse model is cost-effective and suitable to assess the toxicity of candidate microbicides in preclinical studies.

Molecular diagnosis of sentinel lymph node metastases in cervical cancer using squamous cell carcinoma antigen.

PURPOSE: To clarify the prognostic value of molecular diagnosis of SLN metastases in cervical cancer using SCCA. EXPERIMENTAL DESIGN: All SLNs and primary tumors, part of non-SLNs, were harvested from 36 patients with cervical cancer. Expression levels of SCCA, cytokeratin 19 (CK19), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in 178 samples (29 primary tumors, 5 histologic positive nodes, 60 histologic negative SLNs, 69 non-SLNs, and 15 normal nodes) were assessed by quantitative reverse transcription-PCR assay. The quantitative value of SCCA or CK19 mRNA was described as each value relative to GAPDH mRNA. The cutoff value was set at the upper limit of the quantitative value of nodes from noncancer patients, and those above this value constituted the molecular metastasis group. RESULTS: The SCCA mRNA expression values were more than 1 x 10(3) in 28 primary tumors and all histologic positive nodes, and its expression levels in SLNs were higher than in non-SLNs. SLNs from patients with adverse prognostic features had higher SCCA mRNA expression levels. Four histologic negative SLNs were diagnosed molecular metastases based on SCCA mRNA. Two cases with histologically uninvolved pelvic nodes recurred. Survival analysis indicates that molecular lymphatic metastasis based on elevated SCCA mRNA level is the best predictor of recurrence. However, CK19 is not a suitable marker due to its low specificity and relative higher baseline expression in normal nodes. CONCLUSIONS: SCCA mRNA levels for molecular diagnosis of SLN metastases in cervical cancer more accurately identifies patients at risk for recurrence than the routine histology does.

Sentinel lymph node detection using methylene blue in patients with early stage cervical cancer.

OBJECTIVE: To evaluate the feasibility of sentinel lymph node (SLN) detection in patients with cervical cancer using the low-cost methylene blue dye and to optimize the application procedure. PATIENTS AND METHODS: Patients with stage Ib(1)-IIa cervical cancer and subjected to abdominal radical abdominal hysterectomy and pelvic lymphadenectomy were enrolled. Methylene blue, 2-4 ml, was injected into the cervical peritumoral area in 77 cases (4 ml patent blue in the other four cases) 10-360 min before the incision, and surgically removed lymph nodes were examined for the blue lymph nodes that were considered as SLNs. RESULTS: High SLN detection rate was successfully achieved when 4 ml of methylene blue was applied (93.9%, 46/49). Bilaterally SLN detection rate was significantly higher (78.1% vs. 47.1% P=0.027) in cases when the timing of application was more than 60 min before surgery than those with timing no more than 30 min. The blue color of methylene blue-stained SLNs sustained both in vivo and ex vivo, compared with the gradually faded blue color of patent blue that detected in 3 of 4 cases unilaterally. In the total of 112 dissected sides, the most common location of SLNs was the obturator basin (65.2%, 73/112), followed by external iliac area (30.4%, 34/112) and internal iliac area (26.8%, 30/112). Three patients who gave false negative results all had enlarged nodes. CONCLUSION: Methylene blue is an effective tracer to detect SLNs in patients with early stage cervical cancer. The ideal dose and timing of methylene blue application are 4 ml and 60-90 min prior surgery, respectively.

[Recurrence risk factors of platinum-sensitive epithelial ovarian cancer].

BACKGROUND & OBJECTIVE: The prognosis of platinum-sensitive ovarian cancer patients was better than that of chemoresistant ones. However, platinum-sensitive ovarian cancer patients still have a high recurrence rate, which affects their prognosis. This study was designed to analyze clinical features and recurrence risk factors of platinum-sensitive epithelial ovarian cancer. METHODS: Factors that might relate to recurrence of 90 platinum-sensitive epithelial ovarian cancer patients, admitted in Cancer Center of Sun Yat-sen University from 1993 to 1999, with complete remission of more than 6 months, were assessed. Univariate analysis was performed using Chi(2) test; while multivariate analysis was carried out using Cox proportional hazard model. RESULTS: Among the 90 patients, 36(40.0%) relapsed with the median recurrence-free interval of 20 months. Pelvic cavity (18/36, 50.0%) was the most frequently involved. The 3-and 5-year survival rates of all patients were 79.6% and 69.5%; while those of the recurrent ones were 62.3% and 39.6%. Univariate analysis showed that the early FIGO stage group, mucinous type group, and no neoadjuvant chemotherapy group had lower recurrence rates than advanced FIGO stage group, non-mucinous type group, and neoadjuvant chemotherapy group, respectively (P=0.001, P=0.002, and P=0.025). Cox multivariate analysis showed that only FIGO stage was the independent risk factor of recurrence of ovarian cancer (risk ratio=1.771, P=0.003). There was no significant difference in recurrence rate between CBP and other postoperative chemotherapy regimen groups. More cycles of chemotherapy could not reduce the recurrence rate. CONCLUSION: Since FIGO stage is an independent recurrence risk factor of platinum-sensitive epithelial ovarian cancer patients, early diagnosis is the key point to decrease the recurrence rate.

[Expression of metastasis suppressor gene KAI1/CD82 in cervical squamous cell carcinoma and its clinical significance].

BACKGROUND & OBJECTIVE: Metastasis suppressor gene KAI1/CD82 plays an important role in infiltration and metastasis of several types of human cancers, while the researches on its relation with cervical carcinoma are far from adequate now. Therefore, immunohistochemical techniques were employed in this study to determine the expression of KAI1 gene, and explore its clinical significance in cervical squamous cell carcinoma. METHODS: SP immunohistochemistry was used to detect the expression of KAI1 gene in 99 specimens of cervical squamous cell carcinoma, 25 specimens of cervical intraepithelial neoplasm (CIN) II-III, and 18 specimens of normal cervix. Correlations of expression of KAI1 gene to clinicopathologic factors, and prognosis of cervical squamous cell carcinoma were statistically analyzed. RESULTS: The rates of negative, weak, moderate, and strong expression of KAI1 in cervical squamous cell carcinoma were 52.5% (52/99), 16.2% (16/99), 15.2% (15/99), and 16.2% (16/99), respectively, significantly lower than those in normal cervix, and CIN II-III (P=0.000). Expression of KAI1 has no correlation with FIGO stage, age, pelvic lymph node metastasis, tumor histological grade, depth of cervical infiltration, serum squamous cell carcinoma antigen (SCC) level, tumor size, and gross type of cervical lesion (P>0.05). Both univariate and multivariate analyses showed expression of KAI1 has no correlation with prognosis of cervical squamous cell carcinoma (P>0.05). CONCLUSION: KAI1 gene may be an early event in development of cervical cancer, and has no correlation with prognosis of cervical squamous cell carcinoma.

[Sentinel lymph node identification with methylene blue in cervical cancer].

BACKGROUND & OBJECTIVE: Study of sentinel lymph node(SLN) in cervical cancer has been initiated since recent years, and there are still a lot of unknown factors about SLN identification in cervical cancer. This study was to investigate influential factors of identifying SLN with methylene blue in cervical cancer. METHODS: For 41 patients with cervical cancer enrolled from Jun. 2002 to May 2003, 2-4 ml of methylene blue was injected into cervix at 4-6 sites around the tumor about 90-400 min before operation. The blue-dyed lymph node (BDLN) was considered as SLN. HE staining in step sections, and immunohistochemistry were applied to detect SLN. The influential factors of using methylene blue to detect SLN in cervical cancer were assessed based on the identification rate of SLN, and its false negative rate. RESULTS: SLNs were detected in 31 of 41(75.6%) patients with cervical cancer of stage Ib1-IIb. A total of 85 SLNs were identified,and most frequently located in obturator fossa. SLNs were successfully detected in 20 of 23 (87.0%) patients who had no preoperative radiotherapy or chemotherapy, while in only 11 of 18 (61.1%) patients who had preoperative radiotherapy and/or chemotherapy. SLNs were detected in only 17 of 27(63.0%) patients who were injected with 2-3 ml of methylene blue, while in all of 14 patients whose dose was 3.4-4 ml. Eight patients were confirmed of lymph node metastases by pathology. CONCLUSIONS: The dose of methylene blue recommended to detect SLN in cervical cancer is 3-4 ml. SLN varies in different sites, but most frequently located in obturator fossa.

[Clinical analysis of 8 cases of vulvar Paget's disease].

BACKGROUND & OBJECTIVE: Vulvar Paget's disease is a rare disease. It has a relatively high misdiagnosis rate and there are still controversies regarding its treatment. The objective of this study was to investigate its clinical features and summarize the experience of the management of this disease in order to gain a better acknowledge of this rare disease and improve its cure rate. METHODS: The clinical records of 8 cases of vulvar Paget's disease admitted in Cancer Center, Sun Yet-sen University from January 1964 to December 2001 were analyzed. RESULTS: The average age of 8 cases was 66.5 years, and the mean time from the onset of the disease to diagnosis was 5 years. Pathologically, intraepithelial Paget's disease was the commonest (5/8), followed by invasive Paget's disease (2/8) and Paget's disease with underlying adenocarcinoma (1/8). Eight cases underwent altogether 10 times of surgery, and radical vulvectomy was the most frequently used procedure (6/10). One case of Paget's disease with underlying adenocarcinoma died 21 months after surgery and 1 case of intraepithelial Paget's disease died of other etiology, 5 cases achieved long-term disease free survival, 1 case developed recurrence. CONCLUSION: Surgery is the first choice for the patients of vulvar Paget's disease. For those patients whose lesions are extensive,or old aged,or not suitable for long-term follow-up, a radical vulvectomy is more preferable and a lower recurrence rate is expected.

[Expression of p21(WAF1) and its relationship with p53 and PCNA protein in epithelial ovarian cancer].

BACKGROUND & OBJECTIVE: Recent researches manifested that down-regulation of p21(WAF1) had relationship with carcinogenesis and development in various tumors, but its association with epithelial ovarian cancer (EOC) was not clear. This study was designed to investigate the role of p21(WAF1) in the tumorigenesis and development of EOC and its relationship with p53 and proliferating cell nuclear antigen (PCNA) protein. METHODS: Fifty-five EOC tissues, 32 benign ovarian tumor tissues, and 30 normal ovarian tissues were collected. Reverse transcription polymerase chain reaction (RT-PCR) was applied to determine the p21(WAF1)mRNA expression. Immunohistochemistry was applied to examine the protein expression of p21(WAF1), p53, and PCNA. The relationship between the expression of these markers and the clinicopathological characteristics, prognosis of the patients was analyzed. RESULTS: The positive rates of p21(WAF1)mRNA in EOC, benign ovarian tumor, and normal ovary were 40%, 56.25%, and 73.33%, respectively (P=0.012). The positive rates of p21(WAF1) protein were 36.36%, 56.25%, and 80%, respectively (P=0.001). The positive expression rates of p21(WAF1)mRNA and its protein in EOC were lower than those of the other two groups, while the positive expression rates of p53 and PCNA protein in EOC were higher than those of the other two groups (P< 0.05). Expression of p21(WAF1)mRNA had positive relation to its protein, negative relation to PCNA protein, no relation to p53 protein, while expression of p21(WAF1) protein had negative relation to p53 and PCNA protein in EOC. Low-expression of p21(WAF1) protein was associated with advanced FIGO stage (P=0.032), but not with age, histological type, pathological grade, and remnant tumor (P >0.05). There was no relationship between p21(WAF1)mRNA and former parameters (P >0.05). Univariate analysis showed that the patients with low-expression of p21(WAF1)mRNA and p21(WAF1) protein had poor prognosis (P< 0.05). CONCLUSION: p21(WAF1) is down-regulated in EOC. p21(WAF1) might be able to be used as a marker to predict the prognosis of patients with EOC.