Studies of the lungs in diabetes mellitus. II. Phospholipid analyses on the surfactant from broncho-alveolar lavage fluid of alloxan-induced diabetic rats

Structurally diverse anions (folate, 5-formyltetrahydrofolate, AMP, ADP, thiamine pyrophosphate, phosphate, sulfate, and chloride) that are competitive inhibitors of methotrexate influx in L1210 cells also enhance the efflux of methotrexate from these cells. The increase in efflux reaches a maximum of 2- to 4-fold depending upon the anion employed, and the anion concentrations required for half-maximal stimulation of efflux are similar to their K_i values for inhibition of methotrexate influx. A competitive inhibitor of methotrexate uptake (fluorescein-diaminopentane-methotrexate) that is not transported by this system, does not increase methotrexate efflux. These results suggest that the efflux of intracellular methotrexate is coupled to the concomitant uptake of an extracellular anion.     

Isolation of Stem-Like Cancer Cells in Primary Endometrial Cancer Using Cell Surface Markers CD133 and CXCR4

Endometrial cancer (EC) is the most common familiar gynecologic malignant tumor identified in the female reproductive system and has been increasing yearly. In this study, we will identify the surface markers and stem cell markers related with cancer stem cells (CSCs) of EC. Tissue samples were obtained from endometrial cancer patients during surgical procedures. Single cells were isolated from the tissues for culturing, transfection into nude mice, and histopathology analysis. RT-PCR demonstrated that the cultured cells strongly expressed stemness-related genes, such as c-Myc, Sox-2, Nanog, Oct 4A, ABCG2, BMI-1, CK-18, Nestin and β-actin. The expression of surface markers CD24, CD133, CD47, CD29, CD44, CXCR4, SSEA3 and SSEA4, CD24, and CD133 and chemokine markers such as CXCR4 were measured by flow cytometry. Then the double percentage of CD133+CXCR4+ cells constituted 7.2% and 9.3% in EC cells originated from two different patients, respectively. The CD133+CXCR4+ primary endometrial cancer cells grew faster, exhibited high expression of mRNA of stemness-related genes, produced more spheres, and had higher clonogenic ability than other subpopulations. They are also more resistant to anti-cancer drugs than other subpopulations. These findings indicate that CD133+CXCR4+ cells may possess some characteristics of CSCs in primary endometrial cancer. These cell surface markers may be useful for the development of drugs against CSC molecular targets or as a predictive marker for poor prognosis in primary endometrial cancer.     

Detection of KRAS Exon 2 Mutations in Circulating Tumor Cells Isolated by the ISET System from Patients with RAS Wild Type Metastatic Colorectal Cancer

INTRODUCTION: The presence of KRAS mutations in patients with metastatic colorectal cancer (mCRC) predicts poor response to agents targeting the EGFR. Even in patients with RAS wild type (WT) tumors, resistance eventually develops due to multiple mechanisms, including the expansion of previously undetected KRAS mutated clones. In this feasibility study, we aimed to detect KRAS exon 2 mutations in serial samples of circulating tumor cells (CTCs) of RAS WT patients with mCRC captured by the Isolation by Size of Epithelial Tumor cells (ISET) system. METHODS: CTC isolation using the ISET system was performed from prospectively collected blood samples obtained from patients with RAS and BRAF WT mCRC prior to first-line therapy initiation, at first imaging assessment and on disease progression. CTCs were enumerated using hematoxylin & eosin and CD45 double stain on a single membrane spot. DNA was extracted from 5 spots and KRAS exon 2 mutations were detected using a custom quantitative Polymerase Chain Reaction (qPCR) assay. RESULTS: Fifteen patients were enrolled and 28 blood samples were analyzed. In 9 (60%) patients, at least one sample was positive for the presence of a KRAS exon 2 mutation. In 11 out of 28 samples (39.2%) with detectable CTCs a KRAS mutation was detected; the corresponding percentages for baseline and on progression samples were 27% and 37.5%, respectively. The most commonly detected mutations were G13D and G12C (n = 3). The presence of KRAS mutated CTCs at baseline was not prognostic for either PFS (P = .950) or OS (P = .383). CTC kinetics did not follow tumor response patterns. CONCLUSION: The results demonstrate that using a qPCR-based assay, KRAS exon 2 mutations could be detected in CTCs captured by the ISET system from patients with RAS WT primary tumors. However, the clinical relevance of these CTCs remains to be determined in future studies.     

Analysis of Chemopredictive Assay for Targeting Cancer Stem Cells in Glioblastoma Patients

Introduction: The prognosis of glioblastoma (GBM) treated with standard-of-care maximal surgical resection and concurrent adjuvant temozolomide (TMZ)/radiotherapy remains very poor (less than 15 months). GBMs have been found to contain a small population of cancer stem cells (CSCs) that contribute to tumor propagation, maintenance, and treatment resistance. The highly invasive nature of high-grade gliomas and their inherent resistance to therapy lead to very high rates of recurrence. For these reasons, not all patients with similar diagnoses respond to the same chemotherapy, schedule, or dose. Administration of ineffective anticancer therapy is not only costly but more importantly burdens the patient with unnecessary toxicity and selects for the development of resistant cancer cell clones. We have developed a drug response assay (ChemoID) that identifies the most effective chemotherapy against CSCs and bulk of tumor cells from of a panel of potential treatments, offering great promise for individualized cancer management. Providing the treating physician with drug response information on a panel of approved drugs will aid in personalized therapy selections of the most effective chemotherapy for individual patients, thereby improving outcomes. A prospective study was conducted evaluating the use of the ChemoID drug response assay in GBM patients treated with standard of care. Methods: Forty-one GBM patients (mean age 54 years, 59% male), all eligible for a surgical biopsy, were enrolled in an Institutional Review Board–approved protocol, and fresh tissue samples were collected for drug sensitivity testing. Patients were all treated with standard-of-care TMZ plus radiation with or without maximal surgery, depending on the status of the disease. Patients were prospectively monitored for tumor response, time to recurrence, progression-free survival (PFS), and overall survival (OS). Odds ratio (OR) associations of 12-month recurrence, PFS, and OS outcomes were estimated for CSC, bulk tumor, and combined assay responses for the standard-of-care TMZ treatment; sensitivities/specificities, areas under the curve (AUCs), and risk reclassification components were examined. Results: Median follow-up was 8 months (range 3-49 months). For every 5% increase in in vitro CSC cell kill by TMZ, 12-month patient response (nonrecurrence of cancer) increased two-fold, OR = 2.2 (P = .016). Similar but somewhat less supported associations with the bulk tumor test were seen, OR = 2.75 (P = .07) for each 5% bulk tumor cell kill by TMZ. Combining CSC and bulk tumor assay results in a single model yielded a statistically supported CSC association, OR = 2.36 (P = .036), but a much attenuated remaining bulk tumor association, OR = 1.46 (P = .472). AUCs and [sensitivity/specificity] at optimal outpoints (>40% CSC cell kill and >55% bulk tumor cell kill) were AUC = 0.989 [sensitivity = 100/specificity = 97], 0.972 [100/89], and 0.989 [100/97] for the CSC only, bulk tumor only, and combined models, respectively. Risk categorization of patients was improved by 11% when using the CSC test in conjunction with the bulk test (risk reclassification nonevent net reclassification improvement [NRI] and overall NRI = 0.111, P = .030). Median recurrence time was 20 months for patients with a positive (>40% cell kill) CSC test versus only 3 months for those with a negative CSC test, whereas median recurrence time was 13 months versus 4 months for patients with a positive (>55% cell kill) bulk test versus negative. Similar favorable results for the CSC test were observed for PFS and OS outcomes. Panel results across 14 potential other treatments indicated that 34/41 (83%) potentially more optimal alternative therapies may have been chosen using CSC results, whereas 27/41 (66%) alternative therapies may have been chosen using bulk tumor results. Conclusions: The ChemoID CSC drug response assay has the potential to increase the accuracy of bulk tumor assays to help guide individualized chemotherapy choices. GBM cancer recurrence may occur quickly if the CSC test has a low in vitro cell kill rate even if the bulk tumor test cell kill rate is high.     

Plasma Protein Profiling Reveal Osteoprotegerin as a Marker of Prognostic Impact for Colorectal Cancer

BACKGROUND: Due to difficulties in predicting recurrences in colorectal cancer stages II and III, reliable prognostic biomarkers could be a breakthrough for individualized treatment and follow-up. OBJECTIVE: To find potential prognostic protein biomarkers in colorectal cancer, using the proximity extension assays. METHODS: A panel of 92 oncology-related proteins was analyzed with proximity extension assays, in plasma from a cohort of 261 colorectal cancer patients with stage II-IV. The survival analyses were corrected for disease stage and age, and the recurrence analyses were corrected for disease stage. The significance threshold was adjusted for multiple comparisons. RESULTS: The plasma proteins expression levels had a greater prognostic relevance in disease stage III colorectal cancer than in disease stage II, and for overall survival than for time to recurrence. Osteoprotegerin was the only biomarker candidate in the protein panel that had a statistical significant association with overall survival (P = .00029). None of the proteins were statistically significantly associated with time to recurrence. CONCLUSIONS: Of the 92 analyzed plasma proteins, osteoprotegerin showed the strongest prognostic impact in patients with colorectal cancer, and therefore osteoprotegerin is a potential predictive marker, and it also could be a target for treatments.     

Genetic and Clinical Characteristics of Phyllodes Tumors of the Breast

PURPOSE: Phyllodes tumors (PTs) of the breast are rare, accounting for less than 1% of all breast tumors. Among PTs, malignant PTs (MPTs) have malignant characteristics and distant metastases occur in about 20% to 30% of MPTs. However, there is no effective treatment for MPTs with distant metastasis, resulting in an abject prognosis. We performed targeted deep sequencing on PTs to identify the associations between genetic alterations and clinical prognosis. METHODS: We performed targeted deep sequencing to evaluate the genetic characteristics of PTs and analyzed the relationships between clinical and genetic characteristics. RESULTS: A total of 17 PTs were collected between 2001 and 2012. Histologic review was performed by pathologists. The samples included three benign PTs, one borderline PT, and 13 MPTs. The most frequently detected genetic alteration occurred in the TERT promoter region (70.6%), followed by MED12 (64.7%). EGFR amplification and TP53 alteration were detected in four MPTs without genetic alterations in MED12 and TERT promoter regions. Genetic alterations of RARA and ZNF703 were repeatedly found in PTs with local recurrence, and genetic alterations of SETD2, BRCA2, and TSC1 were detected in PTs with distant metastasis. Especially, MPT harboring PTEN and RB1 copy number deletion showed rapid disease progression. CONCLUSIONS: In this study, we provide genetic characterization and potential therapeutic target for this rare, potentially lethal disease. Further large-scale comprehensive genetic study and functional validation are warranted.     

Prognostic Value of Molecular Breast Cancer Subtypes based on Her2, ESR1, PGR and Ki67 mRNA-Expression in Muscle Invasive Bladder Cancer

INTRODUCTION: Gene expression analyses have identified similarities between bladder and breast cancer, where clinical risk stratification is based on Her2, ESR1, PGR and Ki67 expression. The aim of the study was to assess the respective marker gene expression in patients treated with radical cystectomy for muscle-invasive bladder cancer (MIBC) and to evaluate the applicability of breast cancer subtypes for MIBC risk stratification. MATERIALS & METHODS: 102 patients treated with radical cystectomy for MIBC were assessed. Using routine FFPE tissue and an IVD validated kit, mRNA expression was measured by single step RT-qPCR. Partition test were employed to define cut-off values for high or low marker gene expression. Association of expression with outcome was assessed using Kaplan-Meier analysis and multivariate cox regression analysis. Finally, we performed validation of our results in the MD-Anderson cohort (n = 57). RESULTS: Cancer specific survival (CSS) was impaired in patients with high gene expression of Her2 (P = 0.0009) and ESR1 (P = 0.04). In the multivariate regression model Her2 expression remained significant for the prediction of CSS (HR = 2.11, CI 1.11-4.21, P = 0.024). Furthermore, molecular stratification by breast cancer subgroups was significant (P = 0.023) for CSS prediction. Especially the differentiation between Her2-positive and Luminal A (HR = 4.41, CI 1.53-18.71, P = 0.004) and Luminal B (HR = 1.96, CI 0.99-4.08, P = 0.053) respectively was an independent prognostic parameter for CSS. External validation resulted in comparable risk stratification with differences in fractional subgroups distribution. CONCLUSION: Gene expression of Her2, ESR1, PGR, Ki67 and corresponding breast cancer subtypes allow a risk-stratification in MIBC, whereby Her2 overexpressing tumors reveal a particularly poor prognosis.     

Aggressive Phenotype of Cells Disseminated via Hematogenous and Lymphatic Route in Breast Cancer Patients

Intratumoral heterogeneity of breast cancer remains a major challenge in successful treatment. Failure of cancer therapies can also be accredited to inability to systemically eradicate cancer stem cells (CSCs). Recent evidence points to the role of epithelial-mesenchymal transition (EMT) in expanding the pool of tumor cells with CSCs features. Thus, we assessed expression level as well as heterogeneity of CSCs markers in primary tumors (PT), lymph node metastasis (LNM), and circulating tumor cells (CTCs)–enriched blood fractions in order to correlate them with signs of EMT activation as well as clinicopathological data of breast cancer patients. Level of CSCs markers (ALDH1, CD44, CD133, OCT-4, NANOG) and EMT markers was quantified in PT (N=107), LNM (N=56), and CTCs-enriched blood fractions (N=85). Heterogeneity of CSCs markers expression within each PT and LNM was assessed by calculating Gini Index. Percentage of ALDH1-positive cells was elevated in PT in comparison to LNM (P = .005). However, heterogeneity of the four CSCs markers: ALDH1 (P = .019), CD133 (P = .009), OCT-4 (P = .027), and CD44 (P < .001) was decreased in LNM. Samples classified as mesenchymal (post-EMT) showed elevated expression of CSCs markers (OCT-4 and CD44 in PT; OCT-4 in LNM; ALDH1, OCT-4, NANOG, CD44 in CTCs). Patients with mesenchymal-like CTCs had worse prognosis than patients with epithelial-like or no CTCs (P = .0025). CSCs markers are enriched in PT, LNM, and CTCs with mesenchymal features, but their heterogeneity is decreased in metastatic lymph nodes. Mesenchymal CTCs phenotype correlates with poor prognosis of the patients.     

Silencing of Discoidin Domain Receptor-1 (DDR1) Concurrently Inhibits Multiple Steps of Metastasis Cascade in Gastric Cancer

Accumulating evidence suggests that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), plays a role in cancer progression by interacting with the surrounding collagen matrix. In this study, we investigated the expression and role of DDR1 in human gastric cancer metastasis. Proliferation, migration, invasion, and tube formation assays were conducted in DDR1-expressing MKN74 gastric cancer cells and corresponding DDR1-silenced cells. The effects of DDR1 on tumor growth and metastasis were examined in orthotopically implanted and experimental liver metastasis models in nude mice. The expression of DDR1 in surgical specimens was analyzed by immunohistochemistry. DDR1 was expressed in human gastric cancer cell lines, and its expression in human gastric tumors was associated with poor prognosis. Among seven gastric cancer cell lines, MKN74 expressed the highest levels of DDR1. DDR1-silenced MKN74 cells showed unaltered proliferation activity. In contrast, migration, invasion, and tube formation were significantly reduced. When examined in an orthotopic nude mouse model, DDR1-silenced implanted tumors significantly reduced angiogenesis and lymphangiogenesis, thereby leading to reductions in lymph node metastasis and liver metastasis. In a model of experimental liver metastasis, DDR1-silenced cells almost completely inhibited liver colonization and metastasis. DDR1 deficiency led to reduced expression of the genes encoding vascular endothelial growth factor (VEGF)-A, VEGF-C, and platelet-derived growth factor-B. These results suggest that DDR1 is involved in gastric cancer tumor progression and that silencing of DDR1 inhibits multiple steps of the gastric cancer metastasis process.     

Association Between Background Parenchymal Enhancement and Pathologic Complete Remission Throughout the Neoadjuvant Chemotherapy in Breast Cancer Patients

PURPOSE: To retrospectively investigate the quantitative background parenchymal enhancement (BPE) of the contralateral normal breast in patients with unilateral invasive breast cancer throughout multiple monitoring points of neoadjuvant chemotherapy (NAC) and to further determine whether BPE is associated with tumor response, especially at the early stage of NAC. MATERIALS AND METHODS: A total of 90 patients with unilateral breast cancer who then received six or eight cycles of NAC before surgery were analyzed retrospectively. BPE was measured in dynamic contrast-enhanced MRI at baseline and after 2nd, 4th, and 6th NAC, respectively. Correlation between BPE and tumor size was analyzed, and the association between pathologic complete remission (pCR) and BPE was also analyzed. RESULTS: The BPE of contralateral normal breast showed a constant reduction throughout NAC therapy regardless of the menopausal status (P < .001 in all). Both the BPEs and the changes of BPE in each of the three monitoring points were significantly correlated with those in tumor size (P < .05 in all), and the reduction of BPE after 2nd NAC had the largest diagnostic value for pCR (AUC = 0.726, P < .001), particularly in hormonal receptor (HR)-negative patients (OR = 0.243, 95%CI = 0.083 to 0.706, P = .009). CONCLUSION: The BPE of contralateral normal breast had a constant decreased tendency similar to the change of tumor size in NAC. Reduction of BPE at the early stage of NAC was positively associated with pCR, especially in HR-negative status.     

eEF1A1 Overexpression Enhances Tumor Progression and Indicates Poor Prognosis in Hepatocellular Carcinoma

Liver is a major contributor of protein production physiologically. The aberrant state of protein synthesis leads to tumor progression. Eukaryotic elongation factor 1 alpha 1 (eEF1A1) is a major member of the eukaryotic elongation factor family that regulates protein synthesis. Although eEF1A1 plays an essential role in controlling the cell fate, its clinical significance in tumor development and progression has not been reported. Here, we aimed to uncover the expression and prognostic significance of eEF1A1 in hepatocellular carcinoma (HCC). Our data indicated that eEF1A1 expression was elevated in HCC cell lines and clinical samples at both the mRNA and protein levels. Immunohistochemistry revealed that eEF1A1 expression was upregulated in HCC samples compared with corresponding non-tumorous tissues. In 50 HCC cases with portal vein embolus, higher eEF1A1 immunoreactivity was detected in tumor metastases compared with the primary lesions. Kaplan–Meier analysis indicated that increased eEF1A1 expression was closely associated with unfavorable post-surgical overall and disease-free survival in 453 HCC patients. Moreover, multivariate analysis indicated eEF1A1 as an independent predictor for overall and disease-free survival. Collectively, our study suggests eEF1A1 as a novel prognostic biomarker and potential therapeutic target for HCC patients.     

Continuous Delivery of Neutralizing Antibodies Elevate CCL2 Levels in Mice Bearing MCF10CA1d Breast Tumor Xenografts

Chemokines are small soluble molecules that play critical roles in wound healing, infection, and cancer progression. In particular, overexpression of the C-C motif chemokine ligand 2 (CCL2) in multiple cancer types correlates with poor patient prognosis. Animal studies have shown that CCL2 signals to macrophages and breast cancer cells to promote tumor growth, invasion, and metastasis, indicating that CCL2 is a promising therapeutic target. However, the effectiveness of human-specific neutralizing antibodies has not been fully evaluated. Furthermore, controversies remain on the use of neutralizing antibodies to target CCL2 and could be due to mode of drug delivery. Here, we investigated the effects of continuous delivery of human CCL2-neutralizing antibodies on breast cancer progression. Nude mice bearing MCF10CA1d breast tumor xenografts were implanted with osmotic pumps containing control IgG or anti-CCL2 and analyzed for CCL2 levels and tumor progression over 4 weeks. Despite inhibiting CCL2-induced migration in vitro, CCL2-neutralizing antibodies did not significantly affect tumor growth, invasion, macrophage recruitment, or tumor angiogenesis. CCL2 antibodies did not affect murine CCL2 levels but significantly increased human CCL2 levels in circulating blood and tumor interstitial fluid. CCL2-neutralizing antibodies reduced CCL2 levels in cultured cells short term at high concentrations. Enzyme-linked immunosorbent assay analysis of CCL2 in cultured fibroblasts and breast cancer cells revealed that the neutralizing antibodies sequestered CCL2 in the media. CCL2 levels were restored once the antibodies were removed. These studies reveal limitations in CCL2-neutralizing antibodies as a therapeutic agent, with important implications for translating CCL2 targeting to the clinic.     

Up-Regulation of PI 3-Kinases and the Activation of PI3K-Akt Signaling Pathway in Cancer Stem-Like Cells Through DNA Hypomethylation Mediated by the Cancer Microenvironment

Previously, we have succeeded in converting induced pluripotent stem cells (iPSCs) into cancer stem cells (CSCs) by treating the iPSCs with conditioned medium of Lewis lung carcinoma (LLC) cells. The converted CSCs, named miPS-LLCcm cells, exhibited the self-renewal, differentiation potential, and potential to form malignant tumors with metastasis. In this study, we further characterized miPS-LLCcm cells both in vivo and in vitro. The tumors formed by subcutaneous injection showed the structures with pathophysiological features consisting of undifferentiated and malignant phenotypes generally found in adenocarcinoma. Metastasis in the lung was also observed as nodule structures. Excising from the tumors, primary cultured cells from the tumor and the nodule showed self-renewal, differentiation potential as well as tumor forming ability, which are the essential characters of CSCs. We then characterized the epigenetic regulation occurring in the CSCs. By comparing the DNA methylation level of CG rich regions, the differentially methylated regions (DMRs) were evaluated in all stages of CSCs when compared with the parental iPSCs. In DMRs, hypomethylation was found superior to hypermethylation in the miPS-LLCcm cells and its derivatives. The hypo- and hypermethylated genes were used to nominate KEGG pathways related with CSC. As a result, several categories were defined in the KEGG pathways from which most related with cancers, significant and high expression of components was PI3K-AKT signaling pathway. Simultaneously, the AKT activation was also confirmed in the CSCs. The PI3K-Akt signaling pathway should be an important pathway for the CSCs established by the treatment with conditioned medium of LLC cells.     

SIRT1 Regulates the Chemoresistance and Invasiveness of Ovarian Carcinoma Cells

BACKGROUND: SIRT1 is a longevity gene that forestalls aging and age-related diseases including cancer, and has recently attracted widespread attention due to its overexpression in some cancers. We previously identified the overexpression of SIRT1 in ovarian carcinoma (OvCa) as a poor prognostic factor. However, mechanistic insights into the function of SIRT1 in OvCa have yet to be elucidated. METHODS: Quantitative real-time reverse PCR (qRT-PCR) and Western blotting were employed to examine the expression of SIRT1 in a panel of human OvCa cell lines. si-RNA or sh-RNA and cDNA technologies were utilized to knockdown or overexpress SIRT1, respectively. The effects of SIRT1 on proliferation and chemoresistance were examined using a WST-1 assay, and the underlying mechanisms were confirmed using an apoptotic assay, and the quantification of glutathione (GSH), and reactive oxygen species (ROS). The aggressiveness of SIRT1 was analyzed using in vitro invasion and migration assays. RESULTS: SIRT1 was more strongly expressed in OvCa cell lines than in the immortalized ovarian epithelium at the gene and protein levels. Stress up-regulated the expression of SIRT1 in dose- and time-dependent manners. SIRT1 significantly enhanced the proliferation (P < .05), chemoresistance (P < .05), and aggressiveness of OvCa cells by up-regulating multiple antioxidant pathways to inhibit oxidative stress. Further study into the overexpression of SIRT1 demonstrated the up-regulation of several stemness-associated genes and enrichment of CD44v9 via an as-yet-unidentified pathway. CONCLUSIONS: Our results suggest that SIRT1 plays a role in the acquisition of aggressiveness and chemoresistance by OvCa, and has potential as a therapeutic target for OvCa.     

Cytoplasmic TrkA Expression as a Screen for Detecting NTRK1 Fusions in Colorectal Cancer

NTRK1 gene fusions, the targets of multikinase inhibitors, are promising therapeutic targets for colorectal cancer (CRC). However, screening methods for detecting NTRK1 gene fusions in CRC tissues have not been reported. In this study, we investigated the potential use of immunohistochemistry (IHC) for detecting NTRK1 gene fusions. We performed and compared IHC with fluorescence in situ hybridization (FISH) in 80 CRC patients. TrkA immunostaining was observed to be both membranous and cytoplasmic and was scored semiquantitatively using staining intensity and proportions. The tumors were observed to be NTRK1 gene fusion-positive when ≥20 out of 100 nuclei in FISH. A significant correlation between the IHC and FISH results for determination of the NTRK1 gene fusions was observed. We measured the cytoplasmic TrkA expression, which showed an area under the receiver operating characteristic (ROC) curve of 0.926 (range: 0.864-0.987, 95% CI, P = .001). By choosing 4.5 (sum of the intensity and proportion scores of cytoplasmic TrkA expression) as the cut-off value for the positive and negative NTRK1 gene fusion groups, the sensitivity and specificity for predicting lymph node metastasis were 100 and 83.8%, respectively (P = .001). Specifically, high cytoplasmic TrkA expression (sum of intensity and proportion scores >4) was associated with the presence of NTRK1 gene fusions (P < .0001, r = 0.528). Taken together, our data showed that IHC for TrkA can be used as an efficient screening method for detecting NTRK1 gene fusions in CRC.     

A rapid and simple method for the determination of base substitution and frameshift specificity of mutagens

A rapid method for the determination of mutagenic specificity has been developed which makes use of the ochre mutation (TAA) in the his-4 gene of Escherichiacoli. Reversion to His⁺ may occur by suppressor mutation (Type I) or by mutation within the his-4 gene (Type II). The Type I mutations may be further subdivided with respect to the type of suppressor mutation by their ability to suppress nonsense mutants of bacteriophage T4, thus allowing the identification of the responsible base substitution (Kato et al., 1980). The system has the ability to identify mutagens which produce A:T → G:C transitions since only Type II mutants can arise through this base substitution; and in fact, the system confirms the A:T → G:C specificity of the mutagen, N⁴-hydroxycytidine (Janion and Glickman, 1980) since only Type II mutants were induced by treatment with this base analogue.When this system was further tested with several additional mutagens, the results indicate that ethyl methanesulphonate, methyl nitrosourea and ethyl nitrosourea produce primarily Type I revertants which were primarily G:C → A:T transitions. UV-light, γ-rays, 4NQO and methyl methanesulphonate produced all types of base substitutions. The tester strain was further improved by introducing a series of sequenced trp^? frameshift mutations, thus allowing the simultaneous monitoring of frameshift and base-substitution mutations.     

The Correlation Between Serum Chemokines and Clinical Outcome in Patients with Advanced Biliary Tract Cancer

BACKGROUND: Biliary tract cancers (BTCs) are known to have a dismal prognosis. A number of chemokines play important roles in the progress of BTCs. However, the serum levels of chemokines in BTCs have not yet been explored. METHODS: The sera of healthy donors (n = 8) and patients with BTCs who were enrolled in second line sunitinib trials (n = 27) were collected. The concentrations of three kinds of chemokines (CXCL5, CXCL8 and CXCL12) were measured using ELISA assay. The median concentrations of chemokines were compared between healthy donors and BTC patients and the role of chemokines as a prognostic biomarker was examined. RESULTS: BTC patients generally had higher serum levels of CXCL5 and CXCL12 compared to healthy donors. Patients with cholangiocarcinoma showed significantly higher levels of serum CXCL12 than patients with gallbladder cancer. In survival analysis, only CXCL12 level showed a prognostic impact on overall survival (median OS: 6.9 vs. 0.9 months in low CXCL12 vs. high CXCL12, respectively; P = .008). High CXCL5 levels were also correlated with poor survival without statistical insignificance (median OS: 6.2 vs. 2.0 months in low CXCL5 vs. high CXCL5, respectively; P = .070). CONCLUSIONS: There was a significant difference in OS according to the level of CXCL12, suggesting that serum CXCL12 levels may be a useful surrogate marker for clinical outcome in advanced BTCs.