Simultaneous VENTANA IHC and RT-PCR testing of ALK status in Chinese non-small cell lung cancer patients and response to crizotinib.

BACKGROUND: ALK rearrangement-advanced NSCLC patients respond to crizotinib. ALK rearrangement is currently determined with RT-PCR. VENTANA IHC is a standard method to identify ALK protein overexpression in NSCLC; however, VENTANA IHC has rarely been used to determine the response to crizotinib in Chinese patients with NSCLC and ALK overexpression. To better clarify the clinical implication of VENTANA IHC to detect ALK rearrangements, we conducted this study to analyze VENTANA IHC and RT-PCR in a large cohort of Chinese patients with NSCLC undergoing screening for ALK rearrangements. METHODS: A total of 1720 patients with NSCLC who had ALK rearrangements detected by VENTANA IHC and/or RT-PCR were included in this analysis. We compared the efficacy and survival of ALK-positive patients detected by VENTANA IHC and RT-PCR. We used NGS to identify patients in whom the two methods were inconsistent. RESULTS: Among 1720 patients, 187 (10.87%) were shown to be ALK-positive by VENTANA IHC and/or RT-PCR, and 66 received crizotinib treatment. We identified 10.27% (172/1674) of patients as ALK-positive by the VENTANA IHC method, and 12.73% (41/322) of patients had ALK rearrangements by the RT-PCR method. Twenty-nine of 276 (10.51%) ALK-positive patients were simultaneously analyzed using VENTANA IHC and RT-PCR. The overall response rates were 65.90% (29/44) by VENTANA IHC and 55.88% (19/34) by RT-PCR. The disease control rates were 86.36% (38/44) by VENTANA IHC and 76.47% (26/34) by RT-PCR. In contrast, the median progression-free survival for VENTANA IHC and RT-PCR was 8.5 and 9.2 months, respectively. The VENTANA IHC and RT-PCR results obtained for 6 of 17 ALK-positive patients were inconsistent based on NGS; specifically, 4 patients had EML4-ALK fusions, 2 patients had non EML4-ALK fusions, 1 patient had a KCL1-ALK fusion, and one patient had a FBXO36-ALK fusion. CONCLUSIONS: VENTANA IHC is a reliable and rapid screening tool used in routine pathologic laboratories for the identification of suitable candidates for ALK-targeted therapy. VENTANA IHC has moderate sensitivity and a slightly higher association with response to therapy with ALK inhibitors, and some VENTANA IHC-positive, but RT-PCR-negative cases may benefit from crizotinib.

TP53 mutations predict for poor survival in ALK rearrangement lung adenocarcinoma patients treated with crizotinib.

BACKGROUND: Advanced non-small cell lung cancer (NSCLC) patients who harbor anaplastic lymphoma kinase (ALK) rearrangement are sensitive to an ALK inhibitor (crizotinib), but not all ALK-positive patients benefit equally from crizotinib treatment. We analyze the impact of TP53 mutations on response to crizotinib in patients with ALK rearrangement NSCLC. METHODS: Sixty-six ALK rearrangement NSCLC patients receiving crizotinib were analyzed. 21 cases were detected successfully by the next generation sequencing validation FFPE before crizotinib. TP53 mutations were evaluated in 8 patients in relation to disease control rate (DCR), objective response rate (ORR), progression-free survival (PFS) and overall survival (OS). RESULTS: TP53 mutations were observed in 2 (25.00%), 1 (12.50%), 1 (12.50%) and 4 (50.00%) patients in exons 5, 6, 7 and 8, respectively. The majority of patients were male (75.00%, 6/8), less than 65 years old (62.50%, 5/8) and never smokers (75.00%, 6/8). ORR and DCR for crizotinib in the entire case series were 61.90% and 71.43%, respectively. Statistically significant difference was observed in terms of PFS and OS between TP53 gene wild group and mutation group patients (P=0.038, P=0.021, respectively). CONCLUSIONS: TP53 mutations reduce responsiveness to crizotinib and worsen prognosis in ALK rearrangement NSCLC patients.

Comparison of the c-MET gene amplification between primary tumor and metastatic lymph nodes in non-small cell lung cancer.

BACKGROUND: c-MET has recently been identified as a promising novel target in non-small cell lung cancer (NSCLC). We detected the consistency of c-MET gene amplification in metastatic lymph nodes and tumor tissues of NSCLC patients and discuss the clinical application value of c-MET gene amplification in metastatic lymph nodes. METHODS: Real-time fluorescent quantitative PCR was used to test tumor tissues in 368 NSCLC patients and 178 paired metastatic lymph node samples. The amplification consistency in metastatic lymph nodes and tissue samples were compared and the correlation between c-MET gene amplification and the clinical characteristics of patients was analyzed. RESULTS: The c-MET gene amplification rate was 8.97% (33/368) in tumor tissues. Of the 178 paired cases, c-MET gene amplification was positive in 7.95% (15/178) of cancerous tissues and 18.54% (33/178) of metastatic lymph nodes. c-MET gene amplification was detected more frequently in metastatic lymph nodes than in primary cancerous tissue. When metastatic lymph nodes were used as surrogate samples of primary cancerous tissues, the sensitivity was 86.67% (13/15) and the specificity was 87.69% (143/163). CONCLUSIONS: Screening for c-MET gene amplification in lymph node metastases could determine which patients are eligible for tyrosine kinase inhibitor therapy. Lymph node metastasis can predict c-MET gene amplification in a primary tumor and guide the clinical use of c-MET gene targeted drugs.

[The effects of VEGF-R inhibitor on podocytopathy of rats with type I diabetic nephropathy].

AIM: To explore the effect of VEGF inhibitor SU5416 on podocytopathy of rats with type I diabetic nephropathy. METHODS: Thirty male SD rats were randomly divided into three groups: normal control group(NC), diabetic nephropathy group(DN) and diabetic nephropathy treated with SU5416 group(SU5416). Rats with DN were induced by STZ. At the end of 8 weeks after SU5416 treatment, body weight (BW), kidney weight (KW), 24 h urine albuminuria excretion rate(24 h UAER), plasma glucose and creatinine were detected respectively. Renal morphology were stained with periodic acid-Schiff (PAS). And the expression of podocyte-specific genes nephrin and podocin were detected by immunofluorescence. The mRNA levels of genes and VEGF were assessed by real time-PCR respectively. RESULTS: Compared with NC group, DN rats'BW were decreased but the KW were increased, and the levels of blood glucose, creatinine, 24 h UAER and kidney cortex VEGF mRNA were significantly higher. The expression of nephrin and podocin were decreased(P<0.05), and GBM thickening and mesangial matrix expansion were developed. Treatment with SU5416 leads to a marked decrease of KW and the level of 24 h UAER. Concurrently, the expressions of nephrin and podocin were revert partly in response to SU5416(P<0.05), and pathological changes were successfully ameliorated. However, the KW, glucose, creatinine and the level of VEGF mRNA were not significantly affected by SU5416 treatment(P>0.05). CONCLUSION: VEGF-R inhibitor SU5416 can obviously ameliorate albuminuria and histologic changes, and restore the expression of podocyte-specific genes nephrin and podocin in DN rats, suggesting that VEGF-R inhibitor is beneficial for the repair of podocytes in DN, which might be an important adjunct for podocytopathy therapy.

[Smad7 instead of Smad6 blocks epithelial-mesenchymal transition induced by TGF-beta in human renal proximal tubule epithelial cells].

AIM: To investigate whether Smad6 and Smad7 can regulate TGF-beta-induced epithelial-mesenchymal transition in human renal proximal tubule epithelial cells. METHODS: Two recombinant adeno-associated viruses (AAV) expressing Smad6 and Smad7 genes were produced without helper virus and then they were delivered into human renal proximal tubule epithelial cells (HKCs). The cells were randomly divided into normal controls, TGF-beta1-treated group, Smad7-infected control, LacZ-infected control, TGF-beta1+Smad7 group or TGF-beta1+Smad6 group, and TGF-beta1 + LacZ group. 10 microg/L of TGF-beta1 was added into the cell culture at the time of 15 min, 30 min, 60 min, and 120 min and the third day. The levels of phospho-Smad2, alpha-smooth muscle actin (alpha-SMA) and E-cadherin proteins were measured by Western blot. The concentration of hydroxyproline excreted into the culture supernatant was determinated by ELISA. The morphological changes of the cells detected by inverted microscope. RESULTS: Compared with the cells in absence of TGF-beta1, the expression level of P-Smad2 in HKCs increased at 15 min, 30 min, 60 min, and 120 min with the TGF-beta1 stimulation. It reached peak at 30 min. TGF-beta1 treatment for 72 hours resulted in significant up-regulation of alpha-SMA protein expression and hydroxyproline secretion, but a marked decrease in E-cadherin expression in the culture of HKCs. 10 microg/L of TGF-beta1 treatment for 72 hours also resulted in marked alteration in cell morphology. The cells lost their regular cuboidal appearance, and become elongated and spindle shaped. In the Smad7-infected cells, the overexpression of Smad7 resulted in a marked decrease of alpha-SMA protein and hydroxyproline synthesis, but a increase of E-cadherin protein, as well as the retainment of epithelial phenotypic appearance. These effects were associated with the inhibition of TGF-induced Smad2 activation, whereas the overexpression of Smad6 had no effect on the TGF-beta-induced changes in HKCs. CONCLUSION: The overexpression of Smad7 instead of Smad6 can efficiently inhibit TGF-beta-induced epithelial-mesenchymal transition by blocking Smad2 activation in human renal proximal tubule epithelial cells.

[Construction and expression of adeno-associated virus vectors of Smad 6 and Smad 7 genes in human renal tubule epithelial cells].

AIM: To construct the adeno-associated virus(AAV) vectors of Smad 6 and Smad 7 genes and observe their expressions in human renal tubule epithelial cells. METHODS: The plasmids pcDNA3-Smad 6/flag and pcDNA3-Smad 7/flag were digested with BamH I and Xho I. Then the Smad 6/flag and Smad 7/flag gene fragments were cloned into plasmid pAAV-MCS, respectively to construct the recombinant pAAV-Smad 6/flag and pAAV-Smad 7/flag plasmids. The recombinant expression plasmid or pAAV-LacZ plasmid were co-transfected into the HEK 293 cells with pHelper and pAAV-RC by calcium-phosphate precipitation method. Recombinant AAV-2 viral particles were prepared from infected HEK293 cells and then were used to infect human renal tubule epithelial cells (HKCs), The expressions of Smad 6 and Smad 7 in HKCs were demonstrated by immunocytochemical staining. RESULTS: The recombinant AAV vectors of Smad 6 or Smad 7 genes were constructed and expressed in the HKCs successfully. CONCLUSION: These results indicate that AAV can deliver Smad 6 and Smad 7 genes to renal cells in-vitro, suggesting the recombinant AAV can be used for gene therapy of renal fibrosis.