Construction and validation of a scoring system to predict resistance to chemotherapeutic drugs using gene expression profiles in canine lymphoma.

The present study aimed to verify the changes in the expression levels of 13 candidate genes associated with chemotherapy resistance and to construct a scoring system to predict resistance to these drugs. The expression levels of the 13 candidate genes were compared between 20 dogs with lymphoma that were sensitive to drugs used in CHOP-based protocol and 16 dogs with lymphoma that were resistant to these drugs. The expression levels of six genes; ASNS, CCR3, CALCA, FCER1A, LOC448801, and EDNRB were significantly different between the two groups. A scoring system to predict resistance to cyclophosphamide, doxorubicin and vincristine, which are used in CHOP-based protocol, was constructed based on expression levels of the six genes in these 36 dogs using logistic regression models. After internal validation, sensitivity and specificity of the scoring system were 0.759 and 0.853, respectively. External validation was conducted in another cohort of 33 dogs with lymphoma, and sensitivity and specificity of the scoring system were 0.800 and 0.696, respectively. In conclusion, this study identified six genes associated with resistance to drugs used in CHOP-based protocol in canine lymphoma and proposed a novel scoring system to predict resistance to these drugs. This system might be beneficial in selecting the most appropriate chemotherapy protocol for individual dogs with lymphoma.

Clinical relevance of EMT and stem-like gene expression in circulating tumor cells of metastatic colorectal cancer patients.

Using approved methods, circulating tumor cells (CTCs) are only isolated from blood in 30%-50% of metastatic colorectal cancer (mCRC) patients. We previously validated a technique to isolate circulating tumor cells (CTCs) in a cohort of mCRC patients by combining immunomagnetic enrichment of EpCAM+/CD45- cells with qRT-PCR amplification of CK20 and survivin expression. Here, we examined the prognostic utility of CTC epithelial-mesenchymal transition (EMT) and stem cell gene expression. An 8 ml blood sample was collected from 78 consecutive mCRC patients before treatment with investigational and standard chemotherapeutics. The mRNA expression of EMT (PI3Kα, Akt-2, Twist1) and stem cell (ALDH1) markers was measured. Associations between CTC gene expression and progression-free survival (PFS) and overall survival (OS) were determined using Cox regression models. Among patients without CK20 or survivin-expressing CTCs (n=17), 55% had expression of ALDH1, PI3Kα and/or Akt-2. Patients with positive CTC Akt-2 expression had a significantly shorter median PFS (3.0 versus 4.0 months) compared with those without CTC Akt-2 expression in univariable (hazard ratio (HR)=1.61; log-rank P=0.034) and multivariable analyses (HR=1.70; adjusted P=0.041). In univariable analysis, CTC ALDH1 expression was associated with shorter OS (10.0 versus 38.6 months; HR=2.04, P=0.021). Patients with CTCs expressing ALDH1, PI3Kα and/or Akt-2 had a significantly inferior PFS (3.0 versus 7.7 months; HR=1.88, P=0.015) and OS (10.0 versus 26.8+ months; HR=2.25, P=0.050) in univariable, but not multivariable, analysis. CONCLUSIONS: CTC Akt-2 expression may serve as a clinically useful prognostic marker in mCRC patients and warrants further evaluation in prospective trials.

Impact of genetic variations in the MAPK signaling pathway on outcome in metastatic colorectal cancer patients treated with first-line FOLFIRI and bevacizumab: data from FIRE-3 and TRIBE trials.

BACKGROUND: The MAPK-interacting kinase 1 (MKNK1) is localized downstream of the RAS/RAF/ERK and the MAP3K1/MKK/p38 signaling pathway. Through phosphorylation MKNK1 regulates the function of eukaryotic translation initiation factor 4E, a key player in translational control, whose expression is often upregulated in metastatic colorectal cancer patients (mCRC). Preclinical data suggest that MKNK1 increases angiogenesis by upregulating angiogenic factors. We therefore hypothesize that variations in the MKNK1 gene predict outcome in mCRC patients treated with first-line FOLFIRI and bevacizumab (bev). PATIENTS AND METHODS: A total of 567 patients with KRAS wild-type mCRC in the randomized phase III FIRE-3 and TRIBE trials treated with first-line FOLFIRI/bev (discovery and validation cohorts) or FOLFIRI and cetuximab (cet) (control cohort) were included in this study. Five single-nucleotide polymorphisms in the MAPK signaling pathway were analyzed. RESULTS: AA genotype carriers of the MKNK1 rs8602 single-nucleotide polymorphism treated with FOLFIRI/bev in the discovery cohort (FIRE-3) had a shorter progression-free survival (PFS) than those harboring any C (7.9 versus 10.3 months, Hazard ratio (HR) 1.73, P = 0.038). This association could be confirmed in the validation cohort (TRIBE) in multivariable analysis (PFS 9.0 versus 11.0 months, HR 3.04, P = 0.029). Furthermore, AA carriers in the validation cohort had a decreased overall response rate (25% versus 66%, P = 0.049). Conversely, AA genotype carriers in the control group receiving FOLFIRI/cet did not show a shorter PFS. By combining both FOLFIRI/bev cohorts the worse outcome among AA carriers became more significant (PFS 9.0 versus 10.5 months) in univariable (HR 1.74, P = 0.015) and multivariable analysis (HR 1.76, P = 0.022). Accordingly, AA carriers did also exhibit an inferior overall response rate compared with those harboring any C (36% versus 65%, P = 0.005). CONCLUSION: MKNK1 polymorphism rs8602 might serve as a predictive marker in KRAS wild-type mCRC patients treated with FOLFIRI/bev in the first-line setting. Additionally, MKNK1 might be a promising target for drug development.

Genetic variants of DNA repair-related genes predict efficacy of TAS-102 in patients with refractory metastatic colorectal cancer.

Background: Tri-phosphorylated trifluridine (FTD) incorporation into DNA is TAS-102's main anti-tumor action. We tested whether genetic polymorphisms in homologous recombination (HR) and cell cycle checkpoint pathway for DNA repair is associated with outcomes in refractory metastatic colorectal cancer (mCRC) patients treated with TAS-102. Patients and methods: We analyzed genomic DNA extracted from 233 samples of three cohorts: an evaluation cohort of 52 patients receiving TAS-102, a validation cohort of 129 patients receiving TAS-102 and a control cohort of 52 patients receiving regorafenib. Single nucleotide polymorphisms of genes involved in HR (ATM, BRCA1, BRCA2, XRCC3, FANCD2, H2AX, RAD51) and cell cycle checkpoint (ATR, CHEK1, CHEK2, CDKN1A, TP53, CHE1, PIN1, PCNA) were analyzed by PCR-based direct sequencing. Results: In univariate analysis for the evaluation cohort, patients with any G allele in ATM rs609429 had longer overall survival (OS) than those with the C/C variant (8.7 vs. 4.4 months, HR 0.37, 95% CI: 0.14-0.99, P = 0.022). Patients carrying any A allele in XRCC3 rs861539 had significantly longer progression-free survival (PFS) (3.8 vs. 2.3 months, HR 0.44, 95% CI: 0.21-0.92, P = 0.024) and OS (15.6 vs. 6.3 months, HR 0.25, 95% CI: 0.08-0.79, P = 0.012) than those with the G/G variant. In multivariable analysis, ATM rs609429 remained significant for OS (P = 0.020). In the validation cohort, patients having ATM rs609429 with any G allele showed longer OS and PFS; the G/A variant in XRCC3 rs861539 showed longer OS, though without statistical significance. Conclusion: Genetic variants in the HR pathway may predict clinical outcome in mCRC patients receiving TAS-102.

Evaluation of tuberculosis contact investigations in Japan.

SETTING: Although monitoring and evaluation using standardised indicators is an important aspect of tuberculosis (TB) contact investigation, no attempts have been made to systematically evaluate contact investigations in Japan. OBJECTIVE: To evaluate TB contact investigations conducted in public health centres (PHCs) by estimating the scores of selected indicators. DESIGN: A cross-sectional study was conducted in 2012 to estimate six selected indicator scores for contact tracing, contact evaluation and contact treatment at 11 PHCs using the TB registry and relevant contact investigation records of all forms of newly notified active TB cases. Indicator scores were compared across PHCs using median and interquartile range (IQR). RESULTS: A total of 2527 contacts of 313 index TB cases were identified; of these, 1874 were evaluated using the tuberculin skin test (TST) or interferon-gamma release assays (IGRAs). Of 187 contacts who were TST/IGRA-positive, 15 were diagnosed with active TB. Consistently higher medians and lower IQRs were obtained for indicators of contact tracing and contact evaluation than those of contact treatment. CONCLUSION: Our study is the first to evaluate the performance of TB contact investigations in Japan using standardised indicators; the study indicated performance gaps, especially in the treatment for latent tuberculous infection among contacts.

Effects of 8-residue ß sheet breaker peptides on aged Aß40-induced memory impairment and Aß40 expression in rat brain and serum following intraamygdaloid injection.

Amyloidß-protein (Aß) assembly into toxic fibrillar structures is seminal in development of senile plaques, the pathological hallmark of Alzheimer's disease. Blocking this process could have a therapeutic value. ß-sheet breaker peptides (ßSBP) decrease Aß fibrillogenesis and neurotoxicity by preventing or dissolving misfolded Aß aggregates. The present study investigated the effects of ßSBPs on Aß40-related neuropathology, memory impairment in 8-armed radial maze and expression of Aß40 in brain and serum. Aß40 was injected into amygdaloid nucleus followed 8 days later by octapeptideßSBPs 15-22, 16-23 and 17-24. Aß40 was detected not only in amygdala, but also in serum. Aß40 induced cellular changes in amygdala and additionally in hippocampus. Aß40 decreased correct choices, whereas increased errors (both number of arms revisited and total number of revisits) and latency of completing the maze test. The ßSBPs decreased Aß40-induced pathological changes, memory impairment and Aß40 expression in serum. The ßSBP15-22 distinctively decreased the total errors on day 14. The present results show that octapeptide ßSBPs corrected Aß40-induced memory impairment, and support investigation of ßSBPs as a promising treatment of diseases characterized by neurodegeneration and memory impairment such as Alzheimer's disease.

Upper-extremity deep vein thrombosis related to central venous port systems implanted in cancer patients.

The purpose of the current study was to evaluate the incidence and course of upper-extremity deep vein thrombosis (UEDVT) related to an implanted central venous port (CV-port) system in cancer patients. From July 2007 to July 2008, 92 consecutive patients who underwent implantation of a CV-port for chemotherapy for colorectal cancer were prospectively enrolled in the study. All patients were examined at prescribed intervals by ultrasonography (US) to estimate the incidence of catheter-related venous thrombosis. We categorised ultrasound diagnosis into three types: Type 0, no thrombus; Type I, thrombi around catheter without obstruction of venous flow; Type II: thrombi with obstruction of venous flow. Upon initial ultrasound examination, 25 cases (27%) were categorised as Type 0, 64 (70%) as Type I and III (3%) as Type II. Of the 64 Type-I cases, 4 cases worsened to Type II within a month, and 3 others (including 1 patient who developed pulmonary embolism) became Type II after 1 month. Of the other Type-I cases, 12 cases improved to Type 0 and 45 cases remained Type I. All 10 patients categorised as Type II underwent anticoagulant therapy and resumed their chemotherapy without exacerbations of thrombosis. In cancer patients undergoing long-term chemotherapy, there is an unexpectedly high prevalence of catheter-related UEDVT, which can be detected by ultrasound at an early stage after implantation of a CV-port. Given that cancer patients with UEDVT may have worse outcomes than those without, clinicians should consider careful monitoring for UEDVT and introducing anticoagulant therapy.

Cognitive impairment in spinocerebellar degeneration.

It has been reported that patients with spinocerebellar degenerations (SCDs) have cognitive dysfunction as well as limb and truncal ataxia, dysarthria and dysphagia. We review cognitive dysfunction in common types of SCD, including spinocerebellar ataxia types 1, 2, 3, 6, and 17, dentatorubral-pallidoluysian atrophy, Friedreich's ataxia, and multiple system atrophy. There are few studies that address cognitive function in SCD. Although there are few comparison studies among the various SCDs, cognitive dysfunction may be more common and severe in spinocerebellar ataxia type 17 and dentatorubral-pallidoluysian atrophy. While cognitive dysfunction in SCD appears to represent frontal dysfunction, the mechanisms of cognitive dysfunction have not been directly clarified. Nevertheless, various lesions, including those in the cerebrocerebellar circuitry, cortico-striatal-thalamocortical circuitry, and the frontal lobe, may influence cognitive function to various degrees for each disease.

Prefrontal hypoperfusion and cognitive dysfunction correlates in spinocerebellar ataxia type 6.

OBJECTIVE: The aim of this study is to evaluate the correlation between brain perfusion and cognitive dysfunction in spinocerebellar ataxia type 6 (SCA6) patients. METHODS: Thirteen genetically confirmed SCA6 patients and 21 age- and education-matched control subjects were subjected to single photon emission computed tomography (SPECT) and neuropsychological tests. Brain perfusion was examined with SPECT analysis, while general cognition, verbal and visual memory, attention, visuospatial ability, language, executive function, depression, and anxiety were examined with the neuropsychological tests. RESULTS: SCA6 patients showed prefrontal hypoperfusion, and impairments of visual memory, verbal fluency, and executive function compared to control subjects. These neuropsychological impairments in SCA6 patients were significantly correlated with a decrease in prefrontal perfusion. This relation was not correlated to other factors, such as age, education and severity of cerebellar ataxia, which are possible relevant factors associated with cognitive performance. CONCLUSIONS: SCA6 patients have mild cognitive impairment, and correlating prefrontal hypoperfusion. These results indicate cognitive impairment in SCA6 patients resulting from prefrontal hypoperfusion.

Cognitive impairments in multiple system atrophy: MSA-C vs MSA-P.

OBJECTIVE: We evaluated comprehensive neuropsychological tests and regional brain blood flow to compare cognitive dysfunction between two types of multiple system atrophy: predominant cerebellar ataxia (MSA-C) and predominant parkinsonism (MSA-P). METHODS: Twenty-one patients with MSA-C, 14 patients with MSA-P, and 21 age- and education-matched control subjects were subjected to neuropsychological tests and SPECT. The neuropsychological tests examined general cognition, verbal and visual memory, working memory, visuospatial and constructional ability, language, executive function, depression, and anxiety, while SPECT analysis examined brain perfusion. RESULTS: Patients with MSA-P showed severe involvement of visuospatial and constructional function, verbal fluency, and executive function compared with control subjects. Patients with MSA-C showed involvement only in visuospatial and constructional function compared with control subjects and a milder degree of involvement compared with patients with MSA-P. Patients with MSA-P tended toward a wide and severe impairment in cognitive function compared with patients with MSA-C. In addition, neuropsychological impairment in patients with MSA-P was significantly correlated with a decrease in prefrontal perfusion. This significant relation was not correlated to other factors such as age, education, and severity of cerebellar ataxia and parkinsonism, which are relevant factors associated with cognitive performance. CONCLUSIONS: Patients with multiple system atrophy-parkinsonism show more severe and more widespread cognitive dysfunctions than patients with multiple system atrophy-cerebellar ataxia. Our results also indicate that cognitive dysfunction in patients with multiple system atrophy-parkinsonism may be associated with prefrontal involvement.

Cognitive impairment in spinocerebellar ataxia type 6.

OBJECTIVE: The aim of this study was to evaluate cognitive impairment in patients with spinocerebellar ataxia type 6 (SCA6) and to verify the role of cerebellar involvement in intellectual abilities. METHODS: Cognitive function was examined in 18 patients with genetically confirmed SCA6 and in 21 age and education matched controls using a test battery for attention, verbal and visuospatial memory, as well as executive function. RESULTS: Verbal fluency and immediate visual memory task were markedly impaired in SCA6 compared with the control group (p = 0.007, 0.004 and 0.014, respectively). The results of the Rule Shift Cards Test was reduced in patients with SCA6, but the reduction was not significant. These cognitive dysfunctions did not correlated with CAG repeat length, age at onset, ataxic motor dysfunctional scale or depression. CONCLUSIONS: Our results demonstrate that specific cognitive deficits occur in patients with SCA6, independent of ataxic motor dysfunction. These deficits may reflect disruption of cortico-cerebellar circuits.

Characteristics and distribution of endogenous RFamide-related peptide-1.

We have recently identified RFamide-related peptide (RFRP) gene that would encode three peptides (i.e., RFRP-1, -2, and -3) in human and bovine, and demonstrated that synthetic RFRP-1 and -3 act as specific agonists for a G protein-coupled receptor OT7T022. However, molecular characteristics and tissue distribution of endogenous RFRPs have not been determined yet. In this study, we prepared a monoclonal antibody for the C-terminal portion of rat RFRP-1. As this antibody could recognize a consensus sequence among the C-terminal portions of rat, human, and bovine RFRP-1, we purified endogenous RFRP-1 from bovine hypothalamus on the basis of immunoreactivity to the antibody. The purified bovine endogenous RFRP-1 was found to have 35-amino-acid length that corresponds to 37-amino-acid length in human and rat. We subsequently constructed a sandwich enzyme immunoassay using the monoclonal antibody and a polyclonal antibody for the N-terminal portion of rat RFRP-1, and analyzed the tissue distribution of endogenous RFRP-1 in rats. Significant levels of RFRP-1 were detected only in the central nervous system, and the highest concentration of RFRP-1 was detected in the hypothalamus. RFRP-1-positive nerve cells were detected in the rat hypothalamus by immunohistochemical analyses using the monoclonal antibody. In culture, RFRP-1 lowered cAMP production in Chinese hamster ovary cells expressing OT7T022 and it was abolished by pre-treatment with pertussis toxin, suggesting that OT7T022 couples G(i)/G(o) in the signal transduction pathway.

Metastin suppresses the motility and growth of CHO cells transfected with its receptor.

We recently reported having identified of the ligand for an orphan G-protein-coupled receptor, hOT7T175, as the gene product (68-121)-amide of the metastasis suppressor gene KiSS-1. We further showed that the ligand, which we named "metastin," inhibits chemotaxis and invasion of Chinese hamster ovary (CHO) cells transfected with hOT7T175 cDNA (CHO/h175) in vitro, and pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. In the present study, we investigated the activity of metastin in CHO/h175 cells in greater detail. Metastin significantly suppressed motility in a chemotaxis assay and wound healing assay at 10-100 nM order concentrations. Two N-terminally truncated peptides, metastin(40-54) and metastin(45-54) inhibited the migration of CHO/h175 cells as potently as metastin itself. Metastin also inhibited the spreading, monolayer growth and colony formation in agar (0.8%) of CHO/h175 cells at 10-100 nM concentrations. These results indicate that metastin is a potent inhibitor of cell motility, leading to suppression of cell growth and antimetastatic activity, and suggest that low molecular chemical compounds could replace its activity as a novel antimetastatic agent.

Novel betacellulin derivatives. Separation of the differentiation activity from the mitogenic activity.

Betacellulin (BTC) is a member of the epidermal growth factor family. It has two biological activities: mitogenic activity in fibroblasts and vascular smooth muscle cells, and differentiation activity for the differentiation of pancreatic acinar AR42J cells into insulin-secreting cells. The previous finding that recombinant BTC promotes the neogenesis of beta-cells in a mouse model supports the possibility that BTC is a therapeutic protein. However, the mitogenic activity of BTC may not be needed for differentiation into beta-cells and may cause a side effect in clinical use. We prepared several derivatives of BTC to segregate the two activities, to decrease the mitogenic activity, and to maintain the differentiation activity. We succeeded in obtaining BTC derivatives segregated by the two biological activities by preparing truncated-type derivatives. A derivative of BTC, BTC24-76, with a truncated N-terminal 23 amino acids and C-terminal 4 amino acids, was 2.5-fold more active in differentiation and had one-tenth of the mitogenic activity. The derivatives described in the present study should be helpful in future applications as therapeutic proteins and in basic research for discovery of a BTC-specific receptor.

Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor.

Metastasis is a major cause of death in cancer patients and involves a multistep process including detachment of cancer cells from a primary cancer, invasion of surrounding tissue, spread through circulation, re-invasion and proliferation in distant organs. KiSS-1 is a human metastasis suppressor gene, that suppresses metastases of human melanomas and breast carcinomas without affecting tumorigenicity. However, its gene product and functional mechanisms have not been elucidated. Here we show that KiSS-1 (refs 1, 4) encodes a carboxy-terminally amidated peptide with 54 amino-acid residues, which we have isolated from human placenta as the endogenous ligand of an orphan G-protein-coupled receptor (hOT7T175) and have named 'metastin'. Metastin inhibits chemotaxis and invasion of hOT7T175-transfected CHO cells in vitro and attenuates pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. The results suggest possible mechanisms of action for KiSS-1 and a potential new therapeutic approach.

Phase I study of second-line chemotherapy with docetaxel and carboplatin in advanced non-small-cell lung cancer.

PURPOSE: Docetaxel and carboplatin have a broad spectrum of antitumor activity. We conducted a phase I study of docetaxel and carboplatin as second-line chemotherapy in previously treated non-small-cell lung cancer (NSCLC). This study aimed to determine the maximum tolerated dose (MTD) and the dose-limiting toxicities in this second-line combination chemotherapy. METHODS: Patients with advanced NSCLC were treated with escalating docetaxel doses in combination with a fixed-target area under the concentration-time curve (AUC) of 5 mg min/ml of carboplatin on day 1 of a 3-4-week cycle. The carboplatin dose was determined by multiplying the AUC by the clearance predicted using the Chatelut formula. The docetaxel dose was escalated from 40 mg/m2 to the MTD by 10 mg/m2 increments. RESULTS: A total of 16 patients previously treated with anticancer drugs were enrolled through three dose levels (40, 50 and 60 mg/m2 of docetaxel). All patients were assessable for toxicity and response. The MTD was docetaxel 60 mg/m2 with a carboplatin target AUC of 5 mg min/ml, and the dose-limiting toxicities in two of four patients were neutropenia and thrombocytopenia. Overall, neutropenia and thrombocytopenia of grade 3/4 occurred in eight patients (50%) and three patients (19%), respectively. Four patients (25%) and two patients (13%) experienced both grade 1 diarrhea and dermatitis, respectively. Allergic reactions, fluid retention, pneumonitis, neurotoxicity and mucositis were not observed. Of 16 patients, 5 showed an objective response (response rate 31%; 95% CI 14-56%). CONCLUSIONS: The combination of docetaxel and carboplatin is a feasible and well-tolerated second-line chemotherapy regimen in the treatment of NSCLC. Docetaxel 50 mg/m2 under the carboplatin target AUC of 5 mg x min/ml using the Chatelut formula was the recommended dose for phase II study.

Birth cohort effects on incidence of lung cancers: a population-based study in Nagasaki, Japan.

Smoking prevalence remains high (around 60%) among Japanese males, but smoking initiation among males born in the 1930s decreased by approximately 10% due to economic difficulties following World War II. The present study was designed to examine whether this temporary decline in smoking initiation influenced the subsequent incidence of lung cancers, especially adenocarcinoma. Trends of lung cancer incidence by histological type in both sexes were investigated using data from the population-based cancer registry in Nagasaki, Japan, from 1986 through 1995. During this period, 5668 males and 2309 females were diagnosed as having lung cancer, and the overall incidence of lung cancers among both sexes remained stable. However, males aged 55 - 59 years showed a decrease in the age-specific incidence of adenocarcinoma and squamous-cell carcinoma (P < 0.05 and P < 0.01, respectively). In birth cohort analyses, the incidence of adenocarcinoma and squamous-cell carcinoma was lower in the 1935 - 1939 birth male cohort than in the successive cohorts. The incidence of lung cancers among females with low smoking prevalence did not change with birth cohort. The low smoking initiation among the 1935 - 1939 birth male cohort appeared to have resulted in a decreased incidence of adenocarcinoma and squamous cell carcinoma among middle-aged Japanese males. The present study suggests that smoking prevention has an effect in reducing the incidence of lung adenocarcinoma, as well as squamous-cell carcinoma, among smokers.

Interactions of ofloxacin and erythromycin with the multidrug resistance protein (MRP) in MRP-overexpressing human leukemia cells.

To investigate interactions between the multidrug resistance protein (MRP) and antimicrobial agents, we examined the effects of 12 agents on vincristine sensitivity and efflux of the calcein acetoxy-methyl ester (calcein-AM) of a MRP substrate in MRP-overexpressing cells. Only ofloxacin and erythromycin enhanced sensitivity with increased intracellular vincristine accumulation and inhibited the calcein-AM efflux. Our findings suggest that the two agents are possible MRP substrates and may competitively inhibit MRP function as a drug efflux pump.