[Clinical significance of serum squamous cell carcinoma antigen in patients with early cervical squamous cell carcinoma].

Objective: To explore the clinical significance of serum squamous cell carcinoma antigen (SCC-Ag) in early cervical squamous cell carcinoma. Methods: The clinicopathological data and follow-up information of 1435 patients with stage ⅠA2-ⅡA cervical squamous cell carcinoma were collected. The correlation between serum SCC-Ag level and clinicopathological feature and prognosis were analyzed. The best cut-off of serum SCC-Ag for predicting pelvic lymph node metastasis and survival of cervical squamous cell carcinoma patients were also identified. Results: The result of univariate analysis showed that The International Federation of Gynecology and Obstetrics (FIGO) staging, tumor size, depth of cervical stromal invasion, lymphovascular space involvement, pelvic lymph node metastasis, common iliac lymph node metastasis and para-aortic lymph node metastasis were significantly related with serum SCC-Ag level (all P<0.05). The result of multivariate logistic regression analysis showed that tumor size, depth of cervical stromal invasion, pelvic lymph node metastasis and common iliac lymph node metastasis were the independent risk factors of preoperative serum SCC-Ag>2.65 ng/ml (all P<0.001). Multivariate Cox regression analysis showed that lymphovascular space involvement, SCC-Ag>3.15 ng/ml, common iliac lymph node metastasis and tumor size >4 cm were the independent prognostic risk factors (all P<0.05). The univariate analysis showed that, the tumor size, FIGO stage, depth of cervical stromal invasion and SCC-Ag level were significantly related with the recurrence of 1 096 patients without postoperative high risk factors (all P<0.05). Multivariate logistic regression analysis showed that FIGO stage (OR=1.671) and SCC-Ag>2.65 ng/ml (OR=4.490) were the independent risk factors for recurrence (both P<0.05). The best cut off of SCC-Ag for predicting early postoperative cervical lymph node metastasis of cervical squamous cell carcinoma was 2.65 ng/ml, the sensitivity was 60.8%, the specificity was 71.8%. The best cut off of SCC-Ag for predicting prognosis of cervical squamous cell carcinoma was 3.15 ng/ml, the sensitivity was 53.5%, the specificity was 71.1%. Conclusions: Preoperative serum squamous cell carcinoma antigen is an independent prognostic risk factor of survival of patients with early cervical squamous cell carcinoma, and is significantly related with recurrence of patients without postoperative high-risk factors. It can be used as a reference factor for postoperative adjuvant radiotherapy.

Long noncoding RNA PCAT6 regulates cell growth and metastasis via Wnt/ß-catenin pathway and is a prognosis marker in cervical cancer.

OBJECTIVE: Previously, long noncoding RNAs (lncRNAs) have been reported to have critical regulatory roles in the progression of human cancers. LncRNA prostate cancer-associated transcript 6 (PCAT6) has been reported to act as an oncogene in several tumors. However, its expression and function in cervical cancer (CC) have not been investigated. In this study, we aim to reveal the functions of PCAT6 and the underlying mechanisms in CC. PATIENTS AND METHODS: We evaluated the expression levels of PCAT6 in CC tissues and cell lines using real-time PCR. The clinical data were interpreted by chi-square test, Kaplan-Meier survival analyses, univariate analysis, and multivariate analysis. The effect of PCAT6 on CC proliferation and metastasis was investigated by CCK-8 assay, EdU incorporation assay and transwell assay. The cell apoptosis was detected by apoptosis flow detection. RT-PCR and Western blotting were used to detect the expression levels of ß-catenin, cyclin D1 and c-myc. RESULTS: We found that PCAT6 expression was significantly up-regulated in human CC tissues and cell lines compared with their normal counterparts, and its high levels were associated with advanced FIGO stage, depth of cervical invasion and positively lymph node metastasis. Survival assays indicated that high PCAT6 expression had a negative influence on overall survival and disease-free survival. Moreover, multivariate analysis identified high PCAT6 expression as an unfavorable prognostic biomarker for CC patients. Functionally, knockdown of PCAT6 significantly suppressed CC cells proliferation, migration and invasion, and promoted apoptosis. Mechanistic investigation showed PCAT6 activates Wnt/ß-catenin signaling in CC cell lines by promoting the expression of ß-catenin, cyclin D1 and c-myc. CONCLUSIONS: Our results indicated that PCAT6 played oncogenic roles and can be used as a therapeutic target for treating human CC.

[Curative effect analysis of postoperative concurrent chemoradiotherapy on early-stage cervical cancer patients with intermediate-risk factors].

Objective: To compare the curative effect of postoperative concurrent chemoradiotherapy (CCRT) and radiation therapy (RT) alone on early-stage cervical cancer patients with intermediate-risk factors. Methods: Clinical data of patients with early stage (ⅠB-ⅡA) cervical cancer who underwent radical hysterectomy and pelvic lymphadenectomy in Zhejiang Cancer Hospital between January 2008 and December 2011 were retrospectively analyzed. Cervical squamous cell carcinoma patients with more than two intermediate-risk factors, including lymphatic vascular infiltration, depth of cervical stromal infiltration >1/2 cm or tumor size >4 cm, were screened. Among them, 80 patients were divided into the RT group, 137 patients were divided into the CCRT group. The survivals of these patients were analyzed. Results: At the end of the follow-up period, a total of 22 patients were dead, one patient was alive with neoplasm. There were 23 patients who occurred recurrence/ metastasis, 14 of them were in the RT group and 9 were in the CCRT group. The 5-year progression free survival (PFS) and 5-year overall survival (OS) rate of the entire cohort were 89.9% and 89.8%, respectively. The 5-year PFS rate of RT group was 82.4%, the 5-year PFS rate of CCRT group was 94.1%, and the difference between the two groups was statistically significant (P=0.013). The 5-year OS rate of RT group was 85.1%, the 5-year OS rate of CCRT group was 92.7%, and the difference between the two groups was statistically significant (P=0.049). Univariate analysis showed that therapeutic modality was associated with the 5-year DFS and OS of early-stage cervical cancer patients with intermediate-risk factors (P<0.05). The results of multivariate analysis showed that therapeutic modality was an independent prognostic factor of favorable PFS (RR=3.741, 95% CI: 1.506-9.289, P=0.004) and OS (RR=2.754, 95%CI=1.143-6.637, P=0.024). Neither of the two groups occurred severe anemia. However, the incidence of mild and moderate anemia in the CCRT group was higher than that of RT group (P<0.001). The incidence of leukocytopenia severer than grade Ⅲ in the CCRT group was significantly higher than that of RT group (P<0.001). However, the patients could recover quickly with the treatment of granulocyte colony-stimulating factor. Conclusion: Postoperative CCRT improves the survival of early stage cervical cancer patients with two or more intermediate-risk factors.

First Report of Ginger Rhizome Rot Caused by Fusarium oxysporum in China.

Ginger (Zingiber officinale Roscoe) is an important commercial crop that is planted in 60,000 to 70,000 ha every year in Shandong Province, China. In 2010, rotted rhizomes of cultivar Laiwu Big Ginger were reported on 20 ha in Anqiu, Shandong Province, and yield losses of up to 70% were reported. The aboveground symptoms were the water-conducting portion of symptomatic rhizomes was discolored brown and had a black dry rot of the cortex tissues (3). Thirty symptomatic rhizomes were sampled from six fields in six farms. Komada's method (1) was used to isolate the pathogen. Ten pieces from each rhizome were washed with sterile distilled water and plated on Komada selective medium at 25°C. White fungal colonies turned orchid after 7 days of incubation. Two types of asexual spores were associated with the colonies: microconidia and macroconidia. The microconidia were the most abundantly produced spores and were oval, elliptical or kidney shaped, and produced on aerial mycelia. Macroconidia had three to five cells and gradually pointed or curved edges, varied in size from 3 to 5 × 19 to 36 µm. The rDNA of the internal transcribed spacer regions 1 and 2 and the 5.8S gene in five isolates were amplified using primers ITS1 and ITS4, and the nucleotide sequence was the same as isolate no. 3, which was deposited in GenBank (Accession No. KC594035). A BLAST search showed 99% identity with the strain Z9 of Fusarium oxysporum (EF611088). Pathogenicity tests of five isolates were carried out in a greenhouse and the pathogenicity test of isolate no. 3 was selected for the method description. Ten 1-month-old ginger plants (cv. Laiwu Big Ginger) were grown in plastic pots (diameter 20 cm) with sandy soil and inoculated. Ten plants were used as untreated controls. Isolate no. 3 was grown on casein hydrolysate medium (4) for 72 h and the spores were harvested in sterile distilled water. Aqueous spore suspensions of isolate no. 3 were adjusted with deionized water to 1 × 108 CFU/ml as the inoculum. The prepared inoculum was injected with a syringe into the soil around the rhizome of ginger plants. Inoculated plants were placed in the greenhouse at 24 to 26°C and assessed for rhizome rot on the 14th day after inoculation. Disease severity was recorded based on a scale in which - = no symptoms; 1 = small lesions on seedlings, no rot; 2 = seedling rot; and 3 = plant dead. Similar rhizome rot symptoms were observed after inoculation. The inoculated isolate was re-isolated from diseased rhizomes, confirming its pathogenicity. To our knowledge, this is the first report of rhizome rot of ginger caused by F. oxysporum in China. Rhizome rot of ginger caused by Fusarium spp. is well known in Asian countries such as India (2). References: (1) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (2) V. Shanmugam et al. Biol Control. 66:1, 2013. (3) E. E. Trujillo. Diseases of Ginger (Zingiber officinale) in Hawaii, Circular 62, Hawaii Agricultural Experiment Station, University of Hawaii, December, 1964. (4) G. E. Wessman. Appl. Microbiol. 13:426, 1965.

First Report of Leaf Spot Caused by Alternaria alternata on Marigold (Tagetes erecta) in Beijing, China.

Marigold (Tagetes erecta) is an important commercial crop and 200 ha are planted every year in the Beijing district of China. A leaf spot disease of T. erecta was observed during 2012 and 2013 in the Beijing district. The disease was widespread, with 60 to 75% of the fields affected. Leaves of the affected plants had small, brown, necrotic spots on most of the foliage. Yield losses of flowers of up to 20 to 30% were reported. The spots gradually enlarged, becoming irregular in shape, or remained circular, and with concentric rings or zones. In the later stages of infection, the spots coalesced, and the leaves withered, dried, and fell from the plants (4). A fungus was consistently isolated on potato dextrose agar (PDA) from the infected leaves of T. erecta. After 6 days of incubation at 26°C and a 12-h photoperiod, the fungus produced colonies that were flat, with a rough upper surface (2). The conidiophores were short. Conidia varied from 18 × 6 to 47 × 15 µm and were medium to dark brown or olive-brown in color, short beaked, borne in long chains, oval and bean shaped, with 1 to 5 transverse septa and 0 to 2 longitudinal septa. The rDNA of the internal transcribed spacer regions 1 and 2 and the 5.8S gene in seven isolates were amplified using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The nucleotide sequence was the same as isolate No. 7, which was deposited in GenBank (Accession No. KF307207). A BLAST search showed 97% identity with the strain Alternaria alternata GNU-F10 (KC752593). Seven isolates were also confirmed as A. alternata by PCR identification performed by specific primers (C_for/C_rev) of A. alternata (1). Seven isolates were grown on PDA for 2 weeks and the conidia harvested. A 5-µl drop of spore suspension (1 × 105 spores/ml) was placed on each leaflet of 140 detached, surface-sterilized T. erecta leaves. Twenty leaves were inoculated with sterile distilled water as a control. The leaves were incubated in a growth chamber at 80 to 90% relative humidity, 50 to 60 klx/m2 light intensity, and a 12-h photoperiod. After 6 days, leaf spots similar to the original developed at inoculation sites for all isolates and A. alternata was consistently re-isolated. The control leaves remained symptomless. The pathogenicity test was performed three times. Leaf spot of T. erecta caused by Alternaria spp. is well known in Asian countries such as Japan (3). To our knowledge, this is the first report of A. alternata on T. erecta in the Beijing district of China. References: (1) T. Gat. Plant Dis. 96:1513, 2012. (2) E. Mirkova. J. Phytopathol. 151:323, 2003. (3) K. Tomioka. J. Gen. Plant Pathol. 66:294, 2000. (4) T. Y. Zhang. Page 284 in: Flora Fungorum Sinicorum, Volume 16: Alternaria. Science Press, Beijing, 2003.

First Report in China of Soft Rot of Ginger Caused by Pythium aphanidermatum.

Ginger (Zingiber officinale Roscoe) is an important commercial crop planted on more than 13,000 ha annually in Anqiu city, Shandong Province, China. From 2010 to 2011, the incidence of Pythium soft rot disease on cv. Laiwu Big Ginger reached 40 to 75% in Anqiu and yield losses of up to 60% were observed. The disease symptoms included brown spots on ginger rhizomes followed by soft rot, stems and leaves above ground becoming withered and yellow, and water soaking on the collar region. The soft rot did not produce offensive odors, which is different from bacterial rots (2). Forty symptomatic rhizomes were sampled from eight farms. Martin's method (1) was used to isolate the pathogen. Ten pieces from each rhizome were washed with sterile distilled water for 30 s and plated on Martin's selective medium at 26°C in a chamber without light. Colonies grew with cottony aerial mycelium. Main hyphae were 5.7 to 9.6 µm wide. Globose sporangia consisting of terminal complexes of swollen hyphal branches were 11.4 to 18.3 µm wide. The average diameter of zoospores was 9.2 µm. The oogonia were globose and smooth, with a diameter of 21 to 33 µm. The sequences of the rRNA gene internal transcribed spacer (ITS) regions 1 and 2 and the 5.8S gene of five isolates were amplified using primers ITS1 and ITS4 (4), and the nucleotide sequence was the same as isolate No. 2, which was deposited in GenBank (Accession No. KC594034). A BLAST search showed 99% identity with Pythium aphanidermatum strain 11-R-8 (Accession No. JQ898455.1). Pathogenicity tests of five isolates were carried out in a greenhouse. Sixty plants (cv. Laiwu Big Ginger) were grown for 30 days in plastic pots (diameter 20 cm) in sandy soil (pH 5.48) and inoculated. Ten plants were used as untreated controls. Five isolates were grown on Martin's liquid medium for 72 h and the spores were harvested in sterile distilled water. Aqueous spore suspensions of the five isolates were adjusted with deionized water to 1 × 108 CFU/ml and injected with a syringe into the soil around the rhizome of the plants. Plants were then placed in the greenhouse at 24 to 26°C and assessed for rhizome rot on the 14th day after inoculation. The inoculated isolates were recovered from the diseased rhizomes, confirming their pathogenicity. To our knowledge, this is the first report of ginger Pythium soft rot caused by P. aphanidermatum in China. Ginger Pythium soft rot caused by P. myriotylum is reported in Taiwan (3). References: (1) F. N. Martin. Page 39 in: The Genus Pythium. American Phytopathological Society, St. Paul, MN, 1992. (2) E. E. Trujillo. Diseases of Ginger (Zingiber officinale) in Hawaii, Circular 62, Hawaii Agricultural Experiment Station, University of Hawaii, December 1964. (3) P. H. Wang. Lett. Appl. Microbiol. 36:116, 2003. (4) T. J. White. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.