Specific immunohistochemical detection of the BRAF V600E mutation in primary and metastatic papillary thyroid carcinoma.

PURPOSE: To evaluate the clinical value of immunohistochemistry (IHC) using anti-BRAF V600E antibody (clone VE1) for specific detection of the BRAF V600E mutant protein in formalin-fixed paraffin-embedded papillary thyroid carcinoma (PTC) specimens. METHODS: A total of 118 PTC cases and 116 control cases processed between January 2008 and June 2010 were selected and created tissue microarrays (TMAs) for the study. BRAF V600E (VE1) IHC was performed on tissue sections from PTC cases to determine mutation status. Molecular tests (Sanger sequencing/ARMS) were used to confirm the BRAF V600E gene mutation in primary PTC. RESULTS: A uniformly cytoplasmic staining throughout the tumors was observed in IHC-positive cases. BRAF V600E was detected in 68.6% (81/118) of PTC samples by IHC and in 61.9% (73/118) by Sanger sequencing/ARMS. The overall concordance between IHC and Sanger sequencing/ARMS was 93.2% (110/118). The sensitivity and specificity of the BRAF V600E IHC was 100% (73/73) and 82.2% (37/45), respectively. Positive and negative predictive values were 90.1% (73/81) and 100% (37/37), respectively. Expression of the BRAF V600E mutant protein was detected in all of 59 cases of primary carcinoma and corresponding metastatic carcinoma in lymph nodes. The concordance between IHC staining in primary and metastatic PTC was 100% (59/59). CONCLUSION: IHC using VE1 antibody for detection of the BRAF V600E mutant protein expression in PTC showed high sensitivity and acceptable specificity, which are critical for diagnostic purposes. IHC staining for BRAF V600E showed uniformly cytoplasmic expression in both primary tumor and metastatic nodes. Therefore, IHC has high practical value for the detection of the BRAF V600E mutation in metastatic and primary PTC.

Expression and function of CXCL12/CXCR4/CXCR7 in thyroid cancer.

The contribution of CXCL12/CXCR4/CXCR7 axis to cancer progression has been increasingly recognized. However, its role in thyroid cancer development remains unclear. The present study aimed to examine the expression and function of CXCL12 and its receptors in thyroid cancer. The expression of CXCL12/CXCR4/CXCR7 in human tissue specimens of papillary, follicular, medullary, and anaplastic thyroid carcinoma, follicular adenoma, Hashimoto's thyroiditis and nodular goiter were examined by immunohistochemistry using a tissue microarray. CXCR4 and CXCR7 were over-expressed in human thyroid cancer cells K1 by transduction of recombinant lentivirus. The effect of overexpression of CXCR4 and CXCR7 on K1 cell proliferation and invasion and the molecular mechanism underlying the effect were investigated. CXCL12 was exclusively expressed in papillary thyroid carcinoma tissue but absent in other types of thyroid malignancies and benign lesions. CXCR7 was widely expressed in the endothelial cells of all types of malignancy but only occasionally detected in benign lesions. CXCR4 was expressed in 62.5% of papillary thyroid carcinoma tissue specimens and in 30-40% of other types of malignancy, and it was either absent or weakly expressed in benign lesions. CXCL12 stimulated the invasion and migration of K1 cells overexpressing CXCR4, but did not affect K1 cells overexpressing CXCR7. K1 cell proliferation was not affected by overexpression of CXCR4 or CXCR7. Overexpression of CXCR4 in K1 cells significantly increased AKT and ERK phosphorylation and markedly induced the expression and activity of matrix metalloproteinase-2 (MMP­2). Thus, CXCL12 may be an effective diagnostic marker for papillary thyroid carcinoma, and CXCL12/CXCR4/CXCR7 axis may contribute to thyroid cancer development by regulating cancer cell migration and invasion via AKT and ERK signaling and MMP-2 activation.

[Characteristics of aerobic short-cut nitrification granular sludge].

The physical properties and microbiological characteristics of aerobic short-cut granular sludge, which was cultivated in a lab-scale aerated upflow sludge bed (AUSB) reactor, were investigated. When the short-cut nitrification process was performed stably, the ratios of VSS/ SS of short-cut granules were kept at about 80%, the amount of granules with diameter larger than 1.0 mm was about 70% of the total, and the wet density of granules with diameter larger than 0.8mm was about 1022 kg/m3. The fluorescence in situ hybridization (FISH) results indicated the ammonia oxidation bacteria (AOB) were mainly located in the surface layer of the granules, and the nitrite oxidation bacteria (NOB) were in the inner layer. The results of most probable number (MPN) showed that, as the short-cut process was operated stably, the amount of AOB was much more than that of NOB, and sometimes the AOB amount was even 10 thousands times more than that of NOB. All these results indicated that, using the seed granules or their debris as the support media, AOB and NOB were attached and grown on the surface of the media, and finally the aerobic short-cut nitrification granules were formed.

[Community structure of nitrification bacteria in aerobic short-cut nitrification granule].

By using a lab-scale aerated upflow sludge bed reactor, the inoculated anaerobic granule was cultivated to aerobic nitrification granule, and then converted to short-cut nitrification granule with the short-cut nitrification efficiencies above 90%. Appling real-time quantity PCR, and florescent in situ hybridization techniques, the ecological community structure of nitrification bacteria in aerobic granules were studied. The results show that there existed a layered structure in the aerobic granule, the ammonia oxidizing bacteria (AOB) was mainly located in the surface area of the granule, and the nitrite oxidizing bacteria (NOB) was mainly located in the inner area of the granule, was just adjacent to the AOB. There was no active bacteria in the inner core area. The amount of AOB in the granules increased, as the ammonia loading rate of the reactor was increased gradually. The percentage of AOB in the total amount of Eubacteria in the granule was 0.45%, 5.20%, 15.37%, 48.55% respectively, as the ammonia loading rate of the reactor were 0, 0.4, 1.0 and 2.2 kg/(m3 x d) respectively, and the nitrosofication efficiency were 0%, 35%, 50%, 99% relatively.

[Diversity and quantity of different microorganisms in methanogenic granular sludge].

PCR-DGGE, FISH and RTQ-PCR techniques were used to study the microbial community structure and quantity of different microorganisms in the methanogenic granule in different phases from a lab-scale anaerobic reactor. The results indicated that as the organic loading rate of the reactor increasing, the archaea community changed more significantly than bacterial community. Most bacteria were located in the out layer of granule, while most archaea were located in the inner layer. The quantity of archaea was a little less than bacteria, and the quantity of methanosaeta increased significantly.