Prognostic Value of C-met Expression in Cholangiocarcinoma.

AIM: To explore the relationship of clinicopathological features and the proteins of C-met expression in the prognosis of cholangiocarcinoma. METHODS: Clinical data and the completed follow-up information of patients with cholangiocarcinoma who underwent cholangiocarcinoma operation from January 2004 to December 2010 were analyzed retrospectively. The relationship of clinicopathological features and C-met in the prognosis of the patients was analyzed. RESULTS: Patients with high expression of C-met had significantly shorter overall survival than those with low expression of C-met, the difference being statistically significant (P = .003). Patients with high C-met expression had significantly shorter disease-free survival time than those with low expression of C-met, the difference being statistically significant (P = .009). By COX multivariate analysis, high C-met expression in tumor tissues was an independent risk factor in predicting overall survival and disease-free survival for patients with cholangiocarcinoma (P = .038, .048, relative risk = 1.390, 1.427). CONCLUSION: Patients with high C-met expression in cancer tissues had shorter disease-free survival and overall survival. High expression of C-met is an independent risk factor for overall survival and disease-free survival.

Prognostic value of neutrophil distribution in cholangiocarcinoma.

AIM: To explore the relationship of clinicopathological features and the distribution of neutrophils in the tumor microenvironment with the prognosis of cholangiocarcinoma. METHODS: Two hundred and fifty-four formalin-fixed and paraffin embedded tissue blocks were analyzed, including tissues from cholangiocarcinoma (n = 254), and tumor adjacent tissues (n = 238). Tissue sections were stained for CD15 using immunohistochemical staining. CD15 expression was detected to identify the distribution of neutrophils in the local tumor microenvironment. The neutrophil density of the tumor tissues and the adjacent tumor tissues was detected to reflect their inflammatory status. Clinical data and follow-up information of cholangiocarcinoma patients who underwent surgery from January 2004 to December 2010 were analyzed retrospectively. The relationship between clinicopathological features and the distribution of neutrophils with prognosis of the patients were analyzed. RESULTS: The positive expression level of CD15 was only significantly related to the TNM stage. CD15 expression was higher in tumor tissues than in adjacent tissues (73.6% vs 54.6%), with significant differences. Patients with high expression of CD15 had significantly shorter overall survival (OS) than those with low expression of CD15 (median overall survival time 39.77 mo vs 16.87 mo, P = 0.008). Patients with high CD15 expression had significantly shorter disease free survival time (DFS) than those with low expression of CD15 (median DFS 38.27 mo vs 16.83 mo, P = 0.029). COX multivariate analysis indicated that high CD15 expression in tumor tissues was an independent risk factor for predicting OS for patients with cholangiocarcinoma [P = 0.012, relative risk (RR) = 1.601], but it was not an independent risk factor for predicting DFS (P = 0.073, RR = 1.462). CONCLUSION: Patients with high CD15 expression in cancer tissues had shorter DFS and OS. High expression of CD15 is an independent risk factor for OS.

[A standardized protocol for detection of ALK protein expression and gene fusion in lung adenocarcinoma cytologic specimens].

OBJECTIVE: The aim of this study was to establish a standardized protocol for detection of ALK protein expression and gene fusion in cytologic specimens. METHODS: Lung adenocarcinoma cytologic specimens were collected from seven hospitals in Beijing city. A detection protocol for ALK protein expression and gene fusion was designed according to the results of comparative experiment. Ventana immunohistochemical (IHC) ALK(D5F3) detecting ALK protein expression was performed in 203 prepared formalin-fixed paraffin-embedded (FFPE) cell blocks. ALK gene fusion in 98 EGFR gene wild type cytologic specimens and in 4 bronchoalveolar lavage fluid (BL) samples was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). ALK gene fusion in the Ventana IHC ALK (D5F3) positive samples was further tested by fluorescence in situ hybridization (FISH). Six patients with ALK IHC-positive result were followed up to analyze the responses of crizotinib therapy. Comparative experiments: (1) Comparison of the results of 4% neutral buffered formalin fixed for different time (24 h, 48 h, 72 h) on the Ventana IHC ALK (D5F3) staining was conducted in two cases of IHC ALK positive FFPE cell blocks; (2) Comparing qRT-PCR results for ALK fusion in samples from FFPE cell blocks and cytospin prepared slides in 10 cases of lung adenocarcinoma cytologic specimens. RESULTS: Among the specimens examined using the standardized protocol recommended by this study, 229 cases of cytologic specimens met the diagnostic criteria of lung adenocarcinoma. Among them, 207 cases obtained ALK gene test results (by at least one method), with an ALK test ratio of 90.4% (207/229). FFPE cell blocks were successfully prepared in 203 cases, Ventana IHC ALK (D5F3) were successfully performed in all the 203 FFPE cell blocks (100%), and the ALK protein positive detection rate was 10.3% (21/203). ALK fusion was tested in 98 FFPE cytologic samples of EGFR wild types by qRT-PCR, and 96 out of 98 (97.96%) cytologic samples were successfully performed.18 out of 19 IHC ALK-positive cases were verified to be of ALK fusion status by qRT-PCR. The concordance rate was 94.7% (Kappa=0.967, P<0.001) between Ventana IHC ALK (D5F3) and qRT-PCR, and the sensitivity of the Ventana IHC ALK (D5F3) assay compared with qRT-PCR was 100% and the specificity was 98.7%. FISH assay was used to verify the positive cases detected by Ventana IHC ALK (D5F3) staining. Two cases of low tumor cell content FFPE samples obtained indefinite results by FISH test. The six patients with positive ALK protein expression received crizotinib therapy, and 5 paitents got treated effectively. For two ALK IHC positive cases, which were 4% neutral buffered formalin fixed for 72 h, the result of Ventana IHC ALK (D5F3) staining became weakened obviously and uneven. In 10 cases of samples, total RNA was extracted from FFPE cytologic sections and cytospin prepared slides, and the results of qRT-PCR test and ALK gene fusion showed good concordance. CONCLUSIONS: The standardized protocol recommended in this study expands the detection types and quantity of cytologic specimens for ALK protein expression and gene fusion and increased the detection rate. Ventana IHC ALK (D5F3) is a reliable method for detecting ALK protein expression in FFPE cell blocks. The pathologic quality control procedure prior to Ventana IHC ALK (D5F3) is crucial for the accuracy of testing the ALK gene status. When FFPE cell blocks could not be prepared or prepared unsuccessfully from the cytologic specimens, qRT-PCR may be an alternative option for the detection of ALK gene fusion.

[A standard protocol for detection of EGFR mutations in cytologic specimens].

OBJECTIVE: The aim of this study was to establish a standard protocol for detection of EGFR mutations in cytologic specimens. METHODS: 287 cytologic samples were collected from the patients who were suspected of having lung cancer at six hospitals in Beijing. A detection protocol for EGFR mutations was designed. Two comparative experiments were carried out for the coincidence in EGFR mutation rates between direct sequencing (Seq) and amplification refractory mutation system (ARMS) methods, and between 40 matched cytologic samples with formaldehyde-fixed paraffin embedded (FFPE) cytologic blocks and cytospin slides. RESULTS: Tumor cells were found in 236 out of 287 cases (82.2%, 236/287) . Among them, there were 31 cases (13.1%, 31/236) of low tumor cell content samples and 205 cases (86.9%, 205/236) of high tumor cell content samples. 180 cases in the high tumor cell content samples (87.8%, 180/205) were diagnosed to be consistent with NSCLC. 25 out of 194 cases were ruled out or indefinite to be diagnosed as NSCLC by immunohistochemistry. By direct sequencing, the mutation rate of EGFR was 27.8% (50/180) in NSCLC samples and 28.2% (50/177) in adenocarcinoma samples (high tumor content samples) . By ARMS, the mutation rate of EGFR was 45.6% (82/180) in NSCLC samples and 46.3% (82/177) in adenocarcinoma samples (high tumor content samples). The EGFR mutation rate in low tumor content samples was 38.7% (12/31) , there was no significant difference in EGFR mutation rates between the groups of low tumor cell content samples and high tumor cell content samples (P = 0.12). The concordance rate of EGFR mutation rates was 100% between scraping tumor cells from slides samples and from FFEP blocks in the 40 matched samples. Forty-eight out of 180 definitive NSCLC patients received Gefitinib therapy. The FPS was 12 months in the gefitinib-treated ARMS⁺ group and 2 months in the ARMS⁻ group (P < 0.001), and the OS was 19 months in the gefitinib-treated ARMS⁺ group and 7 months in the ARMS⁻ group (P = 0.003), but no significant differences were found in the efficacy (PFS and OS) of Gefitinib between Seq⁺ and Seq⁻ groups (P = 0.227, P = 0.510, respectively), and Seq⁺/ARMS⁺ and Seq⁻/ARMS⁺ groups (P = 0.354, P = 0.334, respectively). CONCLUSIONS: The detection protocol for EGFR mutations in cytological specimens introduced in this study is tested to be reliable and feasible. Pathological evaluation and immunohistochemistry are important in the detection procedure of EGFR mutations in cytologic specimens. High sensitivity methods should be selected for detection of EGFR mutations in cytologic samples.

Transplantation of endothelial progenitor cells in treating rats with IgA nephropathy.

BACKGROUND: Therapeutic options in IgAN are still limited. The aim of this study is to explore the feasibility of using endothelial progenitor cell to treat IgAN in rat model. METHODS: Rat bone marrow mononuclear cells (BM-MNCs) obtained with density gradient centrifugation were cultured in vitro, and induced into endothelial progenitor cells (EPCs). EPCs were identified by surface marker CD34, CD133 and VEGFR2 (FLK-1) and by Dil-Ac-LDL/FITC-UEA-1 double staining. EPCs were labeled with PKH26 prior to transplantation. Rat model of IgAN was established by oral administration of bovine serum albumin together with lipopolysaccharide via the caudal vein and subcutaneous injection of CCL4. Kidney paraffin sections were stained by H&E and PAS. Immunofluorescence was used to assess IgA deposition in the glomeruli. Peritubular capillary (PTC) density was determined by CD31 staining. Monocyte chemoattrant protein-1 (MCP-1), hypoxia-inducible factor-1α (HIF-1α) and CD105 were also measured by immunohistochemistry, western blotting and real-time fluorescent quantitative PCR. RESULTS: The transplanted BM-EPCs were successfully located in IgAN rat kidney. After transplantation, Urinary red blood cell, urine protein, BUN, Scr and IgA serum level were significantly decreased, so were the areas of glomerular extracellular matrix and the IgA deposition in the glomeruli. In addition, PTC density was elevated. And the expression levels of HIF-1α and MCP-1 were significantly down-regulated, while the expression of CD105 was up-regulated. All these changes were not observed in control groups. CONCLUSION: The BM-EPCs transplantation significantly decreases the expansion of glomerular extracellular matrix and the deposition of IgA in the glomeruli; lowers the expression of inflammatory factors; increases PTC density; improves ischemic-induced renal tissue hypoxia, all of which improves the renal function and slows the progress of IgA nephropathy.

A preliminary report on adjuvant analgesic efficacy of HANS in opioid tolerant patients with cancer pain.

OBJECTIVE: To observe the adjuvant analgesic efficacy of Han's Acupoint Nerve Stimulator (HANS) in opioid tolerant patients with cancer pain. METHODS: A prospective non-controlled study was conducted. Opioid tolerant patients with cancer pain were enrolled and treated with both routinely analgesics and adjuvant HANS (2/100 Hz for 30 min/d, 5 d on and 2 d off for two weeks). Cancer pain, quality of life (QOL), anxiety and depression were assessed before enrollment and on d 8 and d 15 with the BPI-C, EORTC QLQ-C30, and self-rating anxiety scale (SAS)/self-rating depression scale (SDS), respectively; the therapeutic frequency of breakthrough pain (BP) and daily opioid dose were also recorded. RESULTS: Totally 47 patients meeting the inclusion criteria participated in this study; 43 patients completed the two-week treatment and assessment. The mean scores of patient's "worst" and "least" pain intensity assessed with BPI-C decreased significantly on d 8 and d 15; the therapeutic frequency of BP also significantly decreased; but the average daily dose of opioids did not change significantly. For the nine symptoms in EORTC QLQ-C30 assessment, the mean scores of pain, fatigue, constipation and insomnia were significantly lower on d 8 and d 15 compared with baseline; the mean scores of the overall health status, nausea/vomiting and the incidence rates of both anxiety and depression also decreased significantly on d 15. CONCLUSIONS: To opioid tolerant patients with cancer pain, adjuvant treatment with HANS could improve pain release and patients' QOL by decreasing the severity of pain, fatigue, constipation, insomnia and other concomitant symptoms; it could also decrease the incidence rates of anxiety and depression.