Development and validation of systems for genetic manipulation of the Old World tick-borne relapsing fever spirochete, Borrelia duttonii.

Relapsing fever (RF), a vector-borne disease caused by Borrelia spp., is characterized by recurring febrile episodes due to repeated bouts of bacteremia. RF spirochetes can be geographically and phylogenetically divided into two distinct groups; Old World RF Borrelia (found in Africa, Asia, and Europe) and New World RF Borrelia (found in the Americas). While RF is a rarely reported disease in the Americas, RF is prevalent in endemic parts of Africa. Despite phylogenetic differences between Old World and New World RF Borrelia and higher incidence of disease associated with Old World RF spirochete infection, genetic manipulation has only been described in New World RF bacteria. Herein, we report the generation of genetic tools for use in the Old World RF spirochete, Borrelia duttonii. We describe methods for transformation and establish shuttle vector- and integration-based approaches for genetic complementation, creating green fluorescent protein (gfp)-expressing B. duttonii strains as a proof of principle. Allelic exchange mutagenesis was also used to inactivate a homolog of the Borrelia burgdorferi p66 gene, which encodes an important virulence factor, in B. duttonii and demonstrate that this mutant was attenuated in a murine model of RF. Finally, the B. duttonii p66 mutant was complemented using shuttle vector- and cis integration-based approaches. As expected, complemented p66 mutant strains were fully infectious, confirming that P66 is required for optimal mammalian infection. The genetic tools and techniques reported herein represent an important advancement in the study of RF Borrelia that allows for future characterization of virulence determinants and colonization factors important for the enzootic cycle of Old World RF spirochetes.

Serum peptide expression and treatment responses in patients with advanced non-small-cell lung cancer.

Epidermal growth factor receptor (EGFR) mutation is an important predictor for response to personalized treatments of patients with advanced non-small-cell lung cancer (NSCLC). However its usage is limited due to the difficult of obtaining tissue specimens. A novel prediction system using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported to be a perspective tool in European countries to identify patients who are likely to benefit from EGFR tyrosine kinase inhibitor (TKI) treatment. In the present study, MALDI-TOF MS was used on pretreatment serum samples of patients with advanced non-small-cell lung cancer to discriminate the spectra between disease control and disease progression groups in one cohort of Chinese patients. The candidate features for classification were subsequently validated in a blinded fashion in another set of patients. The correlation between plasma EGFR mutation status and the intensities of representative spectra for classification was evaluated. A total of 103 patients that were treated with EGFR-TKIs were included. It was determined that 8 polypeptides peaks were significant different between the disease control and disease progression group. A total of 6 polypeptides were established in the classification algorithm. The sensitivity of the algorithm to predict treatment responses was 76.2% (16/21) and the specificity was 81.8% (18/22). The accuracy rate of the algorithm was 79.1% (34/43). A total of 3 polypeptides were significantly correlated with EGFR mutations (P=0.04, P=0.03 and P=0.04, respectively). The present study confirmed that MALDI-TOF MS analysis can be used to predict responses to EGFR-TKI treatment of the Asian population where the EGFR mutation status differs from the European population. Furthermore, the expression intensities of the three polypeptides in the classification model were associated with EGFR mutation.

Escherichia coli cultures maintain stable subpopulation structure during long-term evolution.

How genetic variation is generated and maintained remains a central question in evolutionary biology. When presented with a complex environment, microbes can take advantage of genetic variation to exploit new niches. Here we present a massively parallel experiment where WT and repair-deficient (∆mutL) Escherichia coli populations have evolved over 3 y in a spatially heterogeneous and nutritionally complex environment. Metagenomic sequencing revealed that these initially isogenic populations evolved and maintained stable subpopulation structure in just 10 mL of medium for up to 10,000 generations, consisting of up to five major haplotypes with many minor haplotypes. We characterized the genomic, transcriptomic, exometabolomic, and phenotypic differences between clonal isolates, revealing subpopulation structure driven primarily by spatial segregation followed by differential utilization of nutrients. In addition to genes regulating the import and catabolism of nutrients, major polymorphisms of note included insertion elements transposing into fimE (regulator of the type I fimbriae) and upstream of hns (global regulator of environmental-change and stress-response genes), both known to regulate biofilm formation. Interestingly, these genes have also been identified as critical to colonization in uropathogenic E. coli infections. Our findings illustrate the complexity that can arise and persist even in small cultures, raising the possibility that infections may often be promoted by an evolving and complex pathogen population.

Evolutionary determinants of genome-wide nucleotide composition.

One of the long-standing mysteries of evolutionary genomics is the source of the wide phylogenetic diversity in genome nucleotide composition (G + C versus A + T), which must be a consequence of interspecific differences in mutation bias, the efficiency of selection for different nucleotides or a combination of the two. We demonstrate that although genomic G + C composition is strongly driven by mutation bias, it is also substantially modified by direct selection and/or as a by-product of biased gene conversion. Moreover, G + C composition at fourfold redundant sites is consistently elevated above the neutral expectation-more so than for any other class of sites.

Fundamental effects of PD-1 antibody on the body: a brief report.

We report the case of a 61-year-old man with squamous cell carcinoma of the left lung and mediastinal lymph node metastases who received nivolumab and experienced a better antitumor effect than expected but died soon afterward. A clinical analysis showed that the grades of the treatment-related events did not cause death. The immunological impact of this treatment on his body, especially at his age and with concurrent infection, possibly augmented by the blockade of the PD-1/PD-L1 signaling pathway, is proposed to be one of the key factors for his death. Consequently, we suggest that the cellular immune status and the clinical characteristics of patients, especially the symptoms like concurrent infection, should be considered in the design of clinical protocol of such a kind of therapy.

Evolution and prognosis of breast osteosarcoma: A case report.

Breast osteosarcoma is a rare malignancy of unknown etiology, with no standard adjuvant treatment at present. The prognosis of the disease is poor, and it has a high propensity for recurrence and metastasis. The current report presents the case of a 52-year-old woman, in whom adenomyoepithelioma gradually developed into breast osteosarcoma following three separate surgeries. The patient survived for 41 months from the initial lesion occurrence and resection in the left breast; during this time, she underwent surgery and chemotherapy (liposomal doxorubicin and cisplatin) for the treatment of disease recurrence and lung metastasis, along with molecular-targeted therapy (sunitinib). However, the patient eventually succumbed to respiratory failure due to progressive disease. The present case underwent a clear pathological transformation process, and may provide a basis for an improved understanding of the clinical characteristics and treatment of breast osteosarcoma.

[Comparison of Efficacy and Safety of Different Therapeutic Regimens as 
Second-line Treatment for Small Cell Lung Cancer].

BACKGROUND AND OBJECTIVE: Small-cell lung cancer (SCLC) is an aggressive disease for which the mainstay of treatment is cytotoxic chemotherapy. Despite good initial responses most patients will relapse or progress after the first-line therapy. The evidence of a benefit from second-line chemotherapy is limited in patients with relapsed/advanced SCLC. Some drugs are recommended by guidelines, but more regimens are formulated based on experience in clinical. So we conducted this retrospective study in order to compare the efficacy and safety of different second-line treatment regimens. METHODS: We totally analyzed 309 patients received second-line treatment in our retrospective study. 157 patients received best supportive care (BSC), and the rest 152 patients received second-line chemotherapy. The Kaplan-Meier method survival curves and Log-rank test were used to analysis the differences among different groups. The endpoints were objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS). RESULTS: Patients administered second-line chemotherapy lived significantly longer, with a total OS from first-line therapy of 11.5 mo compared to 6.0 mo in patients with best supportive care alone (P<0.001), and the ORR, DCR, PFS and OS of the former (including the sensitive disease and resistance/refractory disease patients) were obviously better than that of the latter. The ORR and DCR of the patients who received second-line chemotherapy is 39.5% and 59.2%, respectively. The median PFS and OS from second-line chemotherapy were 3.3 mo and 5.3 mo. The patients who received second-line chemotherapy were divided by types of second-line regimens. The sensitive disease patients were from group A (VP-16-based rechallenge) and group B1 (CPT-11-based regimen). The ORR of the two groups were 48.6% and 35.3%, and the DCR were 68.6% and 58.8%, respectively. There was no statistically significant difference (P=0.264; P=0.400). The median PFS from second-line chemotherapy of the two groups were 4.0 mo and 3.0 mo, and the second-line median OS were 6.5 mo and 4.5 mo. There was no statistic difference (P=0.432; P=0.508). The resistance/refractory disease patients were divided into group B2 (CPT-11-based regimen), group C (PTX/DXL-based regimen) and group D (TPT-based regimen). There was no statistic difference in second-line ORR, DCR and median PFS among the three groups (P value is 0.521, 0.528 and 0.775, respectively); The median OS from second-line chemotherapy of the group D is longer than that of group B2 and group C, with statistical difference (P=0.043; P=0.030). The differences of grade III-IV hematologic toxicities among the four subgroups were not statistically different. The incidence of diarrhea in non-hematologic toxicities in patients who received irinotecan as second-line chemotherapy was higher than other three subgroups (P=0.029). CONCLUSIONS: Patients who progressed after the completion of first-line chemotherapy can gain survival benefit. The response and the PFS of the different second-line chemotherapies were similar. The patients who received the TPT-based regimen may gain longer overall survival than other resistance/refractory disease patients.
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[Sequential Treatment of Advanced Squamous Lung Cancer: 
First-line Gemcitabine +/- platinum Followed by Second-line Taxanes +/- platinum Versus Reverse Sequence].

BACKGROUND AND OBJECTIVE: Gemcitabine and taxanes are effective agents commonly used in advanced squamous lung cancer. The best treatment sequence, however, is unclear to our knowledge. So we conducted this retrospective study in order to compare the efficacy and toxicities of first-line Gemcitabine +/- platinum followed by second-line taxanes +/- platinum with the reverse sequence. METHODS: We totally analyzed 105 patients with stage IIIb-IV squamous lung cancer in our retrospective study. There were 49 patients receiving gemcitabine +/- platinum first-line followed by taxanes +/- platinum second-line (G-T group), and 56 patients receiving taxanes +/- platinum first-line followed by gemcitabine +/- platinum second-line (T-G group). The primary endpoint of the study was overall survival (OS), and the secondary endpoints included progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR) and toxicities. RESULTS: The median OS were 18.5 mo in G-T group and 19.0 mo in T-G group (P=0.520). The median PFS1 was 5.0 mo and 4.0 mo with first-line gemcitabine +/- platinum and taxanes +/- platinum, respectively (P=0.584). The median PFS2 was 2.7 mo and 2.5 mo with second-line gemcitabine +/- platinum and taxanes +/- platinum (P=0.432). The ORR1 of G-T group and T-G group were 36.73% and 33.92% (P=0.577), and DCR1 were 79.59% and 89.29% (P=0.186); the ORR2 of G-T group and T-G group were 4.08% and 5.36% (P=0.085), and DCR2 were 51.02% and 66.07%, respectively (P=0.118). Hematologic toxicities was more frequent in G-T group, the patients experienced more grade 3-4 lower hemoglobin (P=0.027) and thrombocytopenia (P=0.002). CONCLUSIONS: The efficacy of first line gemcitabine +/- platinum followed by second line taxanes +/- platinum and the reverse sequence was similar, and the toxicities was tolerable. Both sequential patterns were effective in advanced squamous lung cancer.
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Herpes B virus gD interaction with its human receptor--an in silico analysis approach.

BACKGROUND: The glycoprotein D (gD) is essential for Herpes B virus (BV) entry into mammalian cells. Nectin-1, an HSV-1 gD receptor, is found to be the receptor which mediated BV induced cell-cell fusion, while HVEM does not mediate fusion by BV glycoprotein. However, the specific sequence and structural requirements of the BV gD for the recognition of and binding to Nectin-1 are unknown. Moreover, the 3D structures of BV gD and the BV gD-receptor complex have not been determined. In this study, we propose a reliable model of the interaction of the BV gD with receptor using bioinformatics tools. RESULTS: The three-dimensional structures of two BV gD-receptor complexes were constructed using homology modelling and docking strategy. Based on the models of these complexes, the BV gD receptor interaction patterns were calculated. The results showed that the interface between the BV gD and nectin-1 molecule is not geometrically complementary. The computed molecular interactions indicated that two terminal extensions were the main region of BV gD that binds to nectin-1 and that hydrophobic contacts between the two molecules play key roles in their recognition and binding. The constructed BV gD-HVEM complex model showed that this complex had a lower shape complementarity value and a smaller interface area compared with the HSV-1 gD-HVEM complex, and the number of intermolecular interactions between BV gD-HVEM were fewer than that of HSV-1 gD-HVEM complex. These results could explain why HVEM does not function as a receptor for BV gD. CONCLUSION: In this study, we present structural model for the BV gD in a complex with its receptor. Some features predicted by this model can explain previously reported experimental data. This complex model may lead to a better understanding of the function of BV gD and its interaction with receptor and will improve our understanding of the activation of the BV fusion and entry process.

Clinical significance of serum BAP, TRACP 5b and ICTP as bone metabolic markers for bone metastasis screening in lung cancer patients.

BACKGROUND: We investigated the clinical significance of serum bone-specific alkaline phosphatase (BAP), tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) and type I collagen carboxyterminal telopeptide (ICTP) as bone metabolic markers for bone metastasis (BM) screening in lung cancer patients. METHODS: Newly diagnosed advanced lung cancer patients with (N = 130) and without (N = 135) BM were enrolled in the study. Serum BAP, TRACP 5b and ICTP were measured before the treatment. RESULTS: BAP, TRACP 5b and ICTP values were higher in patients with BM compared with patients without BM (all P < 0.0001). Area under ROC curve (AUC) of BAP, TRACP 5b and ICTP was 0.760, 0.753 and 0.835 (all P < 0.0001), respectively. The cut-off values for BAP, TRACP 5b and ICTP were 21.8 µg/l, 7.8 U/l and 8.8 µg/l, respectively. When TRACP 5b and ICTP were combined, AUC was elevated to 0.895 (P < 0.0001), and the cut-off values were TRACP 5b 7.6 U/l and ICTP 8.4 µg/l. CONCLUSIONS: We conclude that serum BAP, TRACP 5b and ICTP may serve as useful tools for BM screening in lung cancer patients.

[How to make the choice in the retreatment of EGFR-TKI for advanced NSCLC patients who benefited from prior gefitinib therapy: the original drug or switching to a second EGFR-TKI?].

BACKGROUND AND OBJECTIVE: For advanced non-small cell lung cancer (NSCLC) patients who benefited from prior epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy, the choice of a second TKI therapy has gradually become a new strategy for the treatment. Some investigators recommend that the second therapy should be continued with the original TKI; however, other investigators recommend the administration of another TKI. The aim of this study is to explore which choice is more reasonable. METHODS: In retrospect, patients with advanced NSCLC or with postoperative relapse of advanced NSCLC achieved complete response (CR), partial response (PR) or stable disease (SD) in prior Gefitinib therapy, progression free survival (PFS) ≥3 months. They received repeated Gefitinib or Erlotinib at an interval of at least one month. The analysis was carried out with respect to efficacy and optimal population of the two groups. RESULTS: A total of 61 patients were enrolled into the study, 30 in Gefitinib group and 31 in Erlotinib group. Baseline characteristics of the two groups were comparable. In the comparison between patients treated with Gefitinib and with Erlotinib, no statistical differences were seen for response rate (10% vs 22.6%, P=0.300,6), disease control rate (60% vs 74.2%, P=0.237,8), median PFS (3.0 vs 3.5 months, P=0.494,5), or median OS (8.3 vs 8.5 months, P=0.140,8). Multivariate analysis showed that in the initial dose of Gefitinib, PFS≥6 months (HR=0.317, 95%CI: 0.102-0.984, P=0.046,9). With an interval ≥3 months (HR=0.224, 95%CI: 0.071-0.713, P=0.011,3) between two doses of TKI, the risk of disease progression was reduced; but if with an interval ≥3 months (HR=0.262, 95%CI: 0.097-0.705, P=0.008,0), the risk of death was reduced. CONCLUSIONS: Advanced NSCLC patients who benefited from prior Gefitinib therapy can benefit again either with the original drug Gefitinib or the alternative drug Erlotinib when a second TKI therapy is resumed. Such benefit is related to PFS of initial TKI therapy and time interval between two doses of TKI.

[Preliminary study of MALDI-TOF mass spectrometry-based screening of patients with the NSCLC serum-specific peptides].

BACKGROUND AND OBJECTIVE: The improved survival of patients with lung cancer depends on early diagnosis of lung cancer. However, the traditional diagnostic techniques have several limitations. Mass spectrometry (MS) has been applied as a core technology for cancer diagnosis in preliminary proteomic studies. The aim of this study is to explore the differences in the serum peptide levels of patients with non-small cell lung cancer (NSCLC) and healthy individuals using matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-MS. A NSCLC serum classification model was then established. METHODS: One hundred and thirty three cases of patients with NSCLC serum specimens and 132 cases of healthy human serum specimens were randomly divided into two groups in accordance with the ratio of three to one without age and gender differences. The training group was used to establish the classification model, this group included serum samples from 100 NSCLC cases and 100 healthy individuals. The test group for validating the proposed model was composed of the remaining serum samples from 33 NSCLC cases and 32 healthy individuals. Peptides were extracted from the samples using magnetic beads--immobilized metal affinity capture--copper, and their mass spectra were obtained using an automated MALDI-TOF-MS system. The MS data from the training group was analyzed using the ClinproToolTM software to identify the individual peptide fragments and establish the classification model. The sensitivity and specificity of the model were verified by blind testing with the test group. RESULTS: Among the 131 different peptide peaks, ranging from m/z 1,000 Da to 10,000 Da, 14 peaks were significantly different in the NSCLC samples of the training group, as compared with the controls (P<0.000,001; AUC≥0.9); these included 2 higher peaks and 12 lower peaks. The classification model was established, and the test group was verified for only 3 peptide peaks (7,478.59, 2,271.44 and 4,468.38 Da), which were selected by the statistical software. Blind testing revealed that the proposed method had 100% sensitivity, 96.9% specificity and 98.5% accuracy. CONCLUSIONS: Our results showed that the serum peptide levels were significantly different between NSCLC patients and healthy individuals. A serum peptide-based classification of NSCLC patients was established using an automated MALDI-TOF-MS system. This method demonstrated high sensitivity and specificity in a small-scale test. Future studies should test the proposed model through mass validation. The model could be compared or combined with traditional diagnostic methods to establish novel techniques for the early diagnosis of patients with NSCLC.

[Clinic characteristics and prognosis in 102 non-small cell lung cancer patients less than 40 years old].

BACKGROUND AND OBJECTIVE: The incidence of young non-small cell lung cancer (NSCLC) annually increases. The aim of this study is to analyze the clinical pathological characteristics of young (less than 40 years old) NSCLC patients. METHODS: The data of 102 young NSCLC were retrospectively analyzed. RESULTS: Among the 102 patients, 43.1% were women and 29.4% were smokers. The male-to-female ratio was 1.32:1. The most frequent histologic type was adenocarcinoma (77.5%). Tumor differentiation was mostly poor (64.1%), and 87.8% had stages IIIb and IV diseases. The median recurrence time of 6 patients who had tumor resection was 13.5 months. The objective response rate (ORR) of 87 patients who received first-line chemotherapy was 46.0%, the disease control rate (DCR) was 79.3%, and the median time to progression (TTP) was 5.0 months. The ORR of 38 patients who received epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) therapy was 40.0%, with a DCR of 65.7% and a median TTP of 5.5 months. The DCR of 12 patients who received EGFR-TKI twice or more times was 66.7%, with a median TTP of 3.0 months. CONCLUSIONS: The time from the first presenting symptom until diagnosis was usually long. The female proportion presented an upward trend and the correlation became attenuated between young NSCLC patients and smoking. Most of the young NSCLC patients had adenocarcinoma and poor tumor differentiation. Multidisciplinary and systematic therapies were needed to improve the poor prognosis of the young NSCLC patients.

[Clinical observation of EGFR-TKI as a first-line therapy on advanced non-small cell lung cancer].

BACKGROUND AND OBJECTIVE: It has been proven that epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) significantly benefits advanced non-small cell lung cancer (NSCLC) patients harboring EGFR mutations in progression-free survival time with better tolerance. This study is undertaken to analyze efficacy and tolerance of advanced NSCLC patients harboring EGFR mutations taking EGFR-TKI as a first-line therapy. METHODS: Tumor samples from 54 patients with advanced NSCLC were examined for EGFR activating mutations (deletion mutation in exon 19 and the L858R point mutation in exon 21) by direct sequencing. The patients were first-line treated with oral administration of EGFR-TKI until disease progression. The efficacy and adverse events were observed, and survival was followed up. RESULTS: Among the patients, 61% (33 of 54) had EGFR exon 19 deletion, and 39% (21 of 54) had EGFR L858R point mutation. All patients received first-line TKI therapy. The total response rate was 96%, median progression free survival (PFS) was 8.3 months and median survival was 19.5 months. The patients with EGFR exon 19 deletion had significantly longer median PFS (9 versus 7 months, P=0.002) and longer median overall survival (OS)(25 versus 16 months, P=0.001) than patients with EGFR L858R point mutation. There is no significance in efficacy between gefitinib and erlotinib, and gefitinib is safer than erlotinib. The most common adverse events were rash and diarrhea. Two (4%) grade 4 skin toxity effects, two (4%) grade 3 aminotransferase level elevations, and one (1) grade 3 stomatitis were observed. CONCLUSION: The first-line EGFR-TKI treatment in advanced NSCLC patients harboring EGFR mutations is efficient and safe, which is more efficient in patients with EGFR exon 19 deletion than those with EGFR L858R mutation.

[Clinical investigation of efficacy of third-line and beyond pemetrexed treatment in advanced non-squamous non-small cell lung cancer].

BACKGROUND AND OBJECTIVE: Pemetrexed in combination with platinum or a single-agent has been approved for the first- and second-line treatment of non-small cell lung cancer (NSCLC). However, the role of pemetrexed therapy in the third-line and beyond treatment of NSCLC has yet to be generally accepted. The present retrospective study reports the efficacy and safety of pemetrexed in the third-line and beyond treatment of advanced non-squamous NSCLC. METHODS: A total of 46 patients with advanced non-squamous NSCLC received a combination of pemetrexed plus platinum or a single-agent after multi-line treatment failed to yield positive results. RESULTS: Of the 46 patients who participated in the study, 7 achieved partial responses, 20 reached a stage of stable disease, and 19 reached a stage of progressive disease. The over-all object response rate was 15.2% and the disease control rate (DCR) was 58.7%. The median progression-free survival time was 3.0 months. Pemetrexed in combination with platinum yielded a higher DCR than the pemetrexed plus single-agent treatment (P=0.043). Common adverse events included nausea, vomiting and myelosuppression. CONCLUSIONS: Administration of pemetrexed after failure of multi-line treatment is clinically beneficial to patients with advanced non-squamous NSCLC. The toxic effects of the treatment appear to be tolerable.

Interleukin-4 -590T/C polymorphism influences the susceptibility to nonsmall cell lung cancer.

Lung cancer is the leading cause of cancer mortality worldwide. Nonsmall cell lung cancer (NSCLC) accounts for most of these cases. Interleukin 4 (IL-4) is a typical pleiotropic T helper 2 cytokine and plays crucial roles in tumor immunology. IL-4 gene -590T/C polymorphism has been shown to be associated with different autoimmune diseases and cancers. The present study evaluated the correlation between this polymorphism and the susceptibility to NSCLC in the Chinese population. The IL-4 variant -590T/C was detected by polymerase chain reaction-restriction fragment length polymorphism in 1072 NSCLC cases and 1126 age-matched healthy controls. Data were analyzed using the Chi-square test. Results showed that frequencies of IL-4 -590 TC, CC genotype, and -590 C allele were significantly lower in patients with NSCLC than in healthy controls (odds ratio [OR]=0.77, 95% confidence interval [CI] 0.64-0.93, p=0.006; OR=0.54, 95% CI 0.38-0.76, p=0.0004; and OR=0.72, 95% CI 0.62-0.83, p=1.1 × 10(-5), respectively). Our data suggest that the -590T/C polymorphism of the IL-4 gene is associated with a decreased susceptibility to NSCLC.

A comparison of ARMS and direct sequencing for EGFR mutation analysis and tyrosine kinase inhibitors treatment prediction in body fluid samples of non-small-cell lung cancer patients.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutation is strongly associated with the therapeutic effect of tyrosine kinase inhibitors (TKIs) in patients with non-small-cell lung cancer (NSCLC). Nevertheless, tumor tissue that needed for mutation analysis is frequently unavailable. Body fluid was considered to be a feasible substitute for the analysis, but arising problems in clinical practice such as relatively lower mutation rate and poor clinical correlation are not yet fully resolved. METHOD: In this study, 50 patients (32 pleural fluids and 18 plasmas) with TKIs therapy experience and with direct sequencing results were selected from 220 patients for further analysis. The EGFR mutation status was re-evaluated by Amplification Refractory Mutation System (ARMS), and the clinical outcomes of TKIs were analyzed retrospectively. RESULTS: As compared with direct sequencing, 16 positive and 23 negative patients were confirmed by ARMS, and the other 11 former negative patients (6 pleural fluids and 5 plasmas) were redefined as positive, with a fairly well clinical outcome (7 PR, 3 SD, and 1 PD). The objective response rate (ORR) of positive patients was significant, 81.3% (direct sequencing) and 72.7% (ARMS) for pleural fluids, and 80% (ARMS) for plasma. Notably, even reclassified by ARMS, the ORR for negative patients was still relatively high, 60% for pleural fluids and 46.2% for plasma. CONCLUSIONS: When using body fluids for EGFR mutation analysis, positive result is consistently a good indicator for TKIs therapy, and the predictive effect was no less than that of tumor tissue, no matter what method was employed. However, even reclassified by ARMS, the correlation between negative results and clinical outcome of TKIs was still unsatisfied. The results indicated that false negative mutation still existed, which may be settled by using method with sensitivity to single DNA molecule or by optimizing the extraction procedure with RNA or CTC to ensure adequate amount of tumor-derived nucleic acid for the test.

[Expression of carbonic anhydrase IX in NSCLC and its relationship with VEGF and Ki67 expression].

BACKGROUND AND OBJECTIVE: It has been shown that carbonic anhydrase IX (CAIX) was closely related to tumor hypoxia. Proliferating cell nuclear antigen (Ki67) is considered to be an objective indicator which could reliable and comprehensive response to cell population proliferation; vascular endothelial growth factor (VEGF) was positively correlated with tumor angiogenesis. The aim of this study is to investigate the significance of the expression of CAIX in non-small cell lung cancer (NSCLC) tissues by analyzing the correlation between CAIX and clinical characteristics of patients with lung cancer and VEGF, Ki67 expressions. METHODS: CAIX, VEGF and Ki67 protein were detected in 76 cases of NSCLC and 10 cases of lung inflammatory pseudotumor by immunohistochemistry. RESULTS: CAIX was expressed in 45 of 64 cases of NSCLC and not expressed in benign lesion pulmonary tissues. VEGF was expressed in 55 of 76 cases and Ki67 was expressed in 30 of 76 cases. The expression of CAIX in squamous cell carcinoma (SC) was higher than that in adenocarcinoma (AC) (69.7% vs 27.9%, P=0.001). In 34 cases who received radiotherapy, the objective response rates of CAIX positive and negative groups were 27.8% and 62.5% respectively; the expression of CAIX was positively correlated with VEGF (r=0.231, P=0.043) and was not correlated with Ki67 (r=0.064, P=0.583). CONCLUSIONS: Compared with benign lesion pulmonary tissue, the expression of CAIX in NSCLC tissues is significantly increased and is positively correlated with VEGF. The expression of CAIX was also correlated with the objective response rate of radiotherapy, which provided new evidence about that hypoxia can increase NSCLC radiotherapy resistance.