Transtinib, a potent tyrosine kinase inhibitor inhibits L858R/T790M mutant NSCLC cell lines and xenografts.

Non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations initially respond well to the EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib. However, clinical efficacy is limited by the development of resistance. In most cases, this resistance is in the form of the T790M mutation. Here, we report the design, synthesis and biochemical evaluation of a novel series of irreversible EGFR tyrosine kinase inhibitors (EGFR-TKIs) that are derived from the anilinoquinazoline scaffold. Guided by molecular modeling, this series of analogs was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and to achieve high levels of anti-tumor activity in cell cultures and in xenografts. The most promising compound 13c ((E) -N - (4 - (4 - (3-fluorobenzyloxy) -3- chlorophenylamino) -7-ethoxyquinazolin-6-yl) -3- ((S) -pyrrolidin-2-yl)acrylamide, which we named Transtinib) displayed strong anti-proliferative activity against the H1975 and A431 cell lines with IC50 values of 34 nM and 62 nM, respectively. In xenograft models, Transtinib significantly decreases tumor size for a prolonged period of time. These results suggest that Transtinib is a potential cancer therapeutic drug lead for the inhibition of mutant EGFR to overcome the development of resistance.

Circulating tumor cells (CTCs) as a liquid biopsy material and drug target.

Circulating tumor cells (CTCs) have attracted interest as biomarkers of cancer metastases but only recently has a reliable method of CTC detection been developed. CTCs can be thought of as a liquid biopsy from the blood, and they can be used in pathological and molecular assays. CTCs may ideally replace metastatic tissue biopsies in the prediction and monitoring of therapeutic responses and tumor recurrence. CTCs can be used to guide therapeutic cancer management and serve as drug targets. For this reason, the potential of this technology and the limitations of currently available methods of CTC detection are addressed here. The clinical applications of CTCs include the prediction of cancer prognosis; selection and monitoring of therapeutic regimens; and drug target applications. The manner in which CTC molecular profiling can facilitate prognosis and the selection of cancer therapies.

25-(OH)VitD3, as a risk indicator in diagnosis of adenocarcinoma.

Vitamin D (VitD) comes from sunlight exposure and food intake. Apart from regulating calcium homeostasis and bone function, its levels also associate with the presence of development of adenocarcinoma. VitD can interact with VitD receptor (VDR), which heterodimerizes with retinoic X receptor (RXR) and then induces transcription of proteins that function in cell proliferation, differentiation, apoptosis, and angiogenesis. We reviewed and discussed the genes and their associated polymorphisms involved in the correlation between development of adenocarcinoma and VitD deficiency to highlight how VitD may be instrumental in cancerization. Furthermore, pilot epidemiological data show that the detection of 25-hydroxy-Vitamin D3 ((36.5±10.7 nmol/L, n=129) vs (81.4±19.8 nmol/L, n=81)) can be a promising approach in cancer diagnosis. In this review, we suggest that 25-hydroxy-Vitamin D3 can act as an indicator and/or risk assessment factor in early diagnosis, prognosis and treatment of adenocarcinoma.

[Screening for EGFR mutations in lung cancer by a novel real-time PCR with double-loop probe and Sanger DNA sequencing].

OBJECTIVE: To map the frequency and types of EGFR gene mutations present in lung cancer tissues. To evaluate the clinical applicability of a novel real-time double-loop probe PCR of which the ADx-EGFR kit is based, and to compare its performance with traditional Sanger DNA sequencing in the detection of somatic mutations of tumor genes. METHODS: A total of 208 formalin-fixed paraffin-embedded (FFPE) tumor samples were tested. Genomic DNA of the tissue samples was extracted and purified, and subjected to both traditional PCR amplification, Sanger sequencing of EGFR gene in exon 18, 19, 20, 21, and ADx's EGFR mutation detection kit. The mutation rates for EGFR gene in exon 18, 19, 20, 21, as well as the frequency of each mutation detected by the two methods, were analyzed. RESULTS: The traditional Sanger DNA sequencing technique was successfully performed in 196 out of 208 (94.2%) lung cancer samples, and 22 samples (11.2%) showed EGFR gene mutations. ADx-EGFR kit was successfully used in the lung cancers of all of the 208 cases (100.0%), and 40 samples (19.2%) showed mutations. In the lung cancer samples analyzed, mutations were mainly detected in the exon 19 and exon 21 L858R point mutation, i.e. 4.8% (10/208) and 11.6% (23/208) of total mutations, respectively, and the remaining mutations were rare. CONCLUSIONS: The success rate of ADx-EGFR real-time PCR for formalin-fixed and paraffin-embedded tissues samples is significantly higher than that of Sanger sequencing (P < 0.01). There are significant differences between the two methods. ADx-EGFR real-time PCR shows a much higher successful detection rate and mutation rate of lung cancer tissues compared with that of Sanger sequencing. As a result, the real-time PCR with ADx-EGFR kit is proved to have a good clinical applicability and a strong advantage over the traditional Sanger DNA sequencing. It is an effective and reliable tool for clinical screening of somatic gene mutations in tumors.

Association between class III ß-tubulin expression and response to paclitaxel/vinorebine-based chemotherapy for non-small cell lung cancer: a meta-analysis.

BACKGROUND: It has been proposed that the level of class III ß-tubulin gene expression can be used to predict clinical sensitivity to paclitaxel/vinorebine-based chemotherapy in non-small cell lung cancer (NSCLC) patients. However, whereas there are published reports supporting this association, there are also reports of studies that failed to find such an association. We conducted a meta-analysis of all relevant published data to provide a combined statistical assessment of the proposed association of expression variations of class III ß-tubulin with objective response and median survival in patients with NSCLC treated with paclitaxel/vinorebine-based chemotherapy. METHODS: We conducted the meta-analysis using data from ten studies, each of which evaluated the correlation between class III ß-tubulin expression levels and objective response in patients treated with paclitaxel/vinorebine-based chemotherapy for NSCLC patients. All eligible studies were searched by MEDLINE, EMBASE and CNKI databases. Overall odds ratios (ORs) of the objective response were calculated using the method of Mantel-Haenszel. The differences in objective responses between Caucasian and Asian patients treated with paclitaxel/vinorebine-based chemotherapy were compared. We also compared outcomes for patients treated with paclitaxel to those treated with vinorebine. RESULTS: There were a total of 552 patients in the ten studies that met our criteria for evaluation. High/positive expression of class III ß-tubulin was found in 279 patients (50.5%), and low/negative expression for this gene was found in 273 (49.5%) patients. The objective response rate for paclitaxel/vinorebine-based chemotherapy was significantly higher in patients with low/negative class III ß-tubulin expression (OR=0.28; 95% CI, 0.20-0.41; P<0.00001). Median survival time was longer for patients with low/negative expression of class III ß-tubulin compared with patients with high/positive expression (MR=1.40; CI, 0.89-0.90; P<0.00001). There was no significant difference in therapy between Caucasian and Asian patients treated with paclitaxel/vinorebine-based chemotherapy (Chi(2)=0.02, P=0.88). In our analysis, NSCLC patients treated with paclitaxel had more favorable clinical outcomes than those treated with vinorelbine (Chi(2)=3.69, P=0.05). CONCLUSIONS: By combining data from ten different studies, we found a correlation between low TUBB3 gene expression and favorable clinical outcome to anti-tubulin therapy. The correlation for the combined data was significantly stronger than it was for any of the individual studies. This result supports the usefulness of class III ß-tubulin mRNA level as a biomarker for sensitivity to paclitaxel/vinorebine-based chemotherapy in NSCLC patients.

[Synthesis and identification of artificial complete antigen 25-hydroxyvitamin D(3);].

AIM: To synthesize the 25-hydroxyvitamin D(3); artificial complete antigen and to prepare the specific antibody against 25-hydroxyvitamin D(3);. METHODS: The active group carboxyl was introduced into 25-hydroxyvitamin D(3); and formed 25-hydroxyvitamin D(3);-hemisuccinate which possessed the structure of the hapten by chemical modification. The EDC method was applied to conjugate 25-hydroxyvitamin D(3);-hemisuccinate to bovine serum albumin as an artificial immunogen. The coating antigen 25-hydroxyvitamin D(3);-hemisuccinate-OVA was obtained in the same way. Ultraviolet, SDS-PAGE and MALDI-TOF were used to identify 25-hydroxyvitamin D(3);-hemisuccinate-BSA. RESULTS: BALB/c mice were immunized with 25-hydroxyvitamin D(3);-hemisuccinate-BSA to generate the polyclonal antibody of the 25-hydroxyvitamin D(3); worth high titer and the immunogen, 25-hydroxyvitamin D(3);-hemisuccinate-BSA, was successfully prepared with coupling ratio (12±0.16):1(N=3) coupling. CONCLUSION: The high titer and good sensitivity of anti-25-hydroxyvitamin D(3); antibody are produced in sera immunized BALB/c mice, which made it possible to develop a clinical diagnostics for illness.

[Screening for K-ras mutations in colorectal and lung cancers by using a novel real-time PCR with ADx-K-ras kit and Sanger DNA sequencing].

OBJECTIVE: to map out the frequency and types of K-ras gene mutations present in colorectal and lung cancer patients; to evaluate the clinical applicability of a novel real-time double-loop probe PCR using the ADx-K-ras kit, and to compare its performance with the result by using traditional Sanger DNA sequencing in detection of somatic mutations of the tumor genes. METHODS: a total of 827 formalin-fixed paraffin-embedded (FFPE) blocks including 583 from the colorectal and 244 from the lung cancer patients were assayed. Genomic DNA of the sample tissues was extracted, purified and subjected to PCR amplification of K-ras gene codon 12 and 13 and DNA sequencing was carried on using both the traditional Sanger sequencing method and the ADx's K-ras mutation detection kit, respectively. The mutation rates for K-ras gene at codon 12 and 13, and the mutation frequencies detected by using both methods were analyzed. RESULTS: 533 out of 583 (91.4%) colorectal cancer samples and 144 out of 244 lung cancer samples (59.0%) were detected using the traditional Sanger DNA sequencing technique, and 583 out of 583 (100.0%) colorectal plus 244 out of 244(100.0%) lung cancers were detected, respectively by using the ADx-K-ras kit. Of the 583 colorectal cancer samples, 192 (32.9%) showed mutations by using the ADx-K-ras kit in comparing with a result of 160 samples (27.4%) with K-ras gene mutation by using the traditional Sanger DNA sequencing technique. Of the 244 lung cancer samples, 26 (10.7%) showed K-ras gene mutations by using ADx-K-ras kit, while in 144 samples detected by using the traditional Sanger DNA sequencing technique, only 12 samples (8.3%) showed K-ras gene mutations. In colorectal cancer analyzed, GGT→GAT at codon 12 was the most common event with 35.1% (66/188) mutations, followed by GGC→GAC at codon 13 with 26.6% (50/188) and GGT→GTT at codon 12 with 18.6% (35/188), while GGT→GCT at codon12 was the most rare with only 1.6% (3/188) of the total mutation cases. In patients with lung cancer analyzed, GGT→GTT at codon 12 was the most common mutation, accounting for 40.9% (9/22), and GGT→GCT at codon 12 the most rare with only about 4.5% (1/22) of the total mutation cases. CONCLUSIONS: K-ras gene mutations were present in colorectal cases, and significantly more frequent than that in lung cancer. There were significant statistical differences between the two methods. ADx-K-ras real-time PCR showed much higher successful detection rates and mutation ratios compared to Sanger sequencing. As a result, the real-time PCR with ADx-K-ras kit proves to have a good clinical applicability and a strong advantage over the traditional Sanger DNA sequencing. It is a effective and reliable tool for clinical screening of somatic gene mutations in tumors.

Anti-tumor efficacy of Cloretazine (VNP40101M) alone and in combination with fludarabine in murine tumor and human xenograft tumor models.

Cloretazine (VNP40101M), a new sulfonylhydrazine alkylating agent, has demonstrated broad-spectrum anti-tumor activity in preclinical studies. In this study, Cloretazine was evaluated both as a monotherapy and in combination with fludarabine in murine tumor and human tumor xenograft models. Cloretazine significantly inhibited the growth of subcutaneously implanted tumors, including B16F10 murine melanoma in C57BL/6 mice, and H460 human lung carcinoma and WiDr human colon carcinoma in athymic nude CD1 mice. The inhibition of tumor growth by Cloretazine was dose dependent, increasing from 42.2 to 87% as the dose escalated from 100 to 150 mg/kg. Cloretazine showed equivalent efficacy but lower toxicity compared to cyclophosphamide in these models. The combination therapy, consisting of a single dose of 10 mg/kg Cloretazine plus five doses of 70 mg/kg fludarabine, given every other day intraperitoneally, significantly increased the long-term survival of BDF1 mice bearing the L1210 murine leukemia. On Day 65 post-tumor implantation, the combination therapy yielded a 90% survival rate compared to 40% for Cloretazine alone and 0% for fludarabine alone.

Tumor-targeted Salmonella expressing cytosine deaminase as an anticancer agent.

The study was designed to evaluate whether TAPET-CD, an attenuated strain of Salmonella typhimurium expressing Escherichia coli cytosine deaminase (CD), was capable of converting nontoxic 5-fluorocytosine (5-FC) to the active antitumor agent 5-fluorouracil (5-FU). The antitumor effect of TAPET-CD plus 5-FC against subcutaneously implanted colon tumors was also evaluated. TAPET-CD was given to tumor-bearing mice by a single bolus intravenous administration followed with 5-FC by intraperitoneal administration. TAPET-CD accumulated in tumors at levels 1000-fold higher than that in normal tissues and high levels of 5-FU were detected in tumors in mice treated with both TAPET-CD and 5-FC. No 5-FU could be detected in normal tissues. Inhibition of tumor growth was observed in mice treated with either TAPET-CD alone or TAPET-CD in combination with 5-FC (TAPET-CD/5-FC), but not with 5-FC alone. TAPET-CD/5-FC inhibited tumor growth by 88%-96%, compared to TAPET-CD alone, which inhibited tumor growth by 38%-79%. These data suggest that tumor-targeting Salmonella could be used to deliver prodrug-converting enzyme selectively to tumors and produced anti-tumor effects when the corresponding prodrug was also given.

Live bacteria as anticancer agents and tumor-selective protein delivery vectors.

The development of novel cancer therapies that are selective for cancer cells with limited toxicity to normal tissues is a challenge for oncology researchers. Microorganisms, such as viruses with selectivity for tumor cells or tumor micro-environments, have been investigated as potential arsenals for decades. Genetically-modified, non-pathogenic bacteria have begun to emerge as potential antitumor agents, either to provide direct tumoricidal effects or to deliver tumoricidal molecules. Attenuated Salmonella, Clostridium and Bifidobacterium are capable of multiplying selectively in tumors and inhibiting their growth, representing a new approach for cancer treatment. Because of their selectivity for tumor tissues, these bacteria would also be ideal vectors for delivering therapeutic proteins to tumors. VNP20009, an attenuated strain of Salmonella typhimurium, and its derivative, TAPET-CD, which expresses an Escherichia coli cytosine deaminase (CD), are particularly promising, and are currently undergoing phase I clinical trials in cancer patients.