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The anthocyanin synthetic pathway is regulated centrally by an MYB-bHLH-WD40 (MBW) complex. Anthocyanin pigmentation is an important fruit quality trait in red-fleshed kiwifruit; however, the underlying regulatory mechanisms involving the MBW complex are not well understood. In this study, one R2R3MYB (AcMYBF110 expressed in fruit characteristically), one bHLH (AcbHLH1), two upstream regulators of AcbHLH1 (AcbHLH4 and AcbHLH5), and one WDR (AcWDR1) are characterized as being involved in the regulation of anthocyanin synthesis in kiwifruit. AcMYBF110 plays an important role in the regulation of anthocyanin accumulation by specifically activating the promoters of several anthocyanin pathway genes including AcCHS, AcF3'H, AcANS, AcUFGT3a, AcUFGT6b, and AcGST1. Coexpression of AcbHLH1, AcbHLH4, or AcbHLH5 together with AcMYBF110 induces much greater anthocyanin accumulation in both tobacco leaves and in Actinidia arguta fruit compared with AcMYBF110 alone. Moreover, this activation is further enhanced by adding AcWDR1. We found that both AcMYBF110 and AcWDR1 interact with all three AcbHLH factors, while AcMYBF110 also interacts with AcWDR1 to form three different MBW complexes that have different regulatory roles in anthocyanin accumulation of kiwifruit. The AcMYBF110-AcbHLH1-AcWDR1 complex directly targets the promoters of anthocyanin synthetic genes. Other features of the regulatory pathways identified include promotion of AcMYBF110, AcbHLH1,and AcWDR1 activities by this MBW complex, providing for both reinforcement and feedback regulation, whereas the AcMYBF110-AcbHLH4/5-AcWDR1 complex is indirectly involved in the regulation of anthocyanin synthesis by activating the promoters of AcbHLH1 and AcWDR1 to amplify the regulation signals of the first MBW complex.

Purpose Combining small-molecule inhibitors of different targets was shown to be synergistic in preclinical studies. Testing this concept in clinical trials is, however, daunting due to challenges in toxicity management and efficacy assessment. This study attempted to evaluate the safety and efficacy of vatalanib plus everolimus in patients with advanced solid tumors and explore the utility of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) studies as a predictive biomarker. Patients and Methods This single-center, phase I trial containing 70 evaluable patients consisted of a dose escalation proportion based on the traditional "3 + 3" design (cohort IA and IB) and a dose expansion proportion (cohort IIA and IIB). Toxicity was evaluated using the Common Terminology Criteria of Adverse Events. Antitumor activity was assessed using the Modified Response Evaluation Criteria in Solid Tumors. Results The maximum tolerated doses were determined to be vatalanib 1250 mg once daily or 750 mg twice daily in combination with everolimus 10 mg once daily. No treatment-related death occurred. The most common toxicities were hypertriglyceridemia, hypercholesterolemia, fatigue, vomiting, nausea and diarrhea. There was no complete response. Nine patients (12.9%) had partial response (PR) and 41 (58.6%) had stable disease (SD). Significant antitumor activity was observed in neuroendocrine tumors with a disease-control rate (PR + SD) of 66.7% and other tumor types including renal cancer, melanoma, and non-small-cell lung cancer. Conclusions The combination of vatalanib and everolimus demonstrated reasonable toxicity and clinical activity. Future studies combining targeted therapies and incorporating biomarker analysis are warranted based on this phase I trial.

Background/Objectives Interplay between the Hedgehog (HH) and epidermal growth factor receptor (EGFR) pathways modulating the outcome of their signaling activity have been reported in various cancers including pancreatic ductal adenocarcinoma (PDAC). Therefore, simultaneous targeting of these pathways may be clinically beneficial. This Phase I study combined HH and EGFR inhibition in metastatic PDAC patients. Methods Combined effects of HH and EGFR inhibition using Vismodegib and Erlotinib with or without gemcitabine in metastatic solid tumors were assessed by CT. Another cohort of patients with metastatic PDAC was evaluated by FDG-PET and tumor biopsies-derived biomarkers. Results Treatment was well tolerated with the maximum tolerated dose cohort experiencing no grade 4 toxicities though 25% experienced grade 3 adverse effects. Recommended phase II dose of Vismodegib and Erlotinib were each 150 mg daily. No tumor responses were observed although 16 patients achieved stable disease for 2–7 cycles. Paired biopsy analysis before and after first cycle of therapy in PDAC patients showed reduced GLI1 mRNA, phospho-GLI1 and associated HH target genes in all cases. However, only half of the cases showed reduced levels of desmoplasia or changes in fibroblast markers. Most patients had decreased phospho-EGFR levels. Conclusions Vismodegib and Erlotinib combination was well-tolerated although overall outcome in patients with metastatic PDAC was not significantly impacted by combination treatment. Biomarker analysis suggests direct targets inhibition without significantly affecting the stromal compartment. These findings conflict with pre-clinical mouse models, and thus warrant further investigation into how upstream inhibition of these pathways is circumvented in PDAC.

Skin greasiness is a common postharvest disorder of apple (Malus × domestica). However, the molecular mechanism of skin greasiness is unclear. In this study, fruits of 'Golden Delicious' (GD), 'Granny Smith', and 'Fuji' with distinct characteristics of greasiness were used for greasiness scoring, wax morphology, wax metabolite, and RNA-seq analyses. Additionally, GD fruit were treated with 1-methylcyclopropene (1-MCP), which repressed greasiness. A partial least squares discriminant analysis (PLS-DA) revealed that wax esters were the critical wax fraction for skin greasiness. Among these wax esters, liquid linoleate esters of short-chain alcohols (C4-C6) and farnesol showed increased contents with increasing greasiness. Their concentrations were significantly correlated with greasiness score. To identify the genes encoding key enzymes for the synthesis of liquid linoleate esters, a weighted gene co-expression network analysis was conducted. MdDCR1, encoding an acyltransferase (defective in cuticular ridges, DCR), was selected as a candidate gene. MdDCR1 was significantly upregulated in greasy skin, and significantly suppressed by 1-MCP treatment. MdDCR1 silencing suppressed the accumulation of liquid linoleate esters of short-chain alcohols, including butyl linoleate, pentyl linoleate, and hexyl linoleate, in GD skin. These results provide insights into the molecular mechanisms of cuticular wax metabolism related to skin greasiness in apple. Our results show that transcriptional regulation of MdDCR1, encoding an acyltransferase that catalyzes the biosynthesis of liquid linoleate esters of short-chain alcohols (C4-C6) via an independent side branch of the C18:2 CoA pathway, regulates the formation of greasiness.

Fruit acidity determines the organoleptic quality and nutritive value of most fruits. In litchi, although the organic acid composition of pulps is known, the molecular mechanisms and genes underlying variation in fruit acidity remain elusive. Herein, developing pulps of two contrasting litchi varieties, Huaizhi (HZ, low-acidity) and Boye_No.8 (B8, high-acidity), were subjected to metabolomics and transcriptomics, and the dynamic metabolome and transcriptional changes were determined. Measurements revealed that the dominant acidity-related organic acid in litchi pulps is malate, followed in low levels by citrate and tartrate. Variation in litchi pulps’ acidity is mainly associated with significant differences in malate and citrate metabolisms during fruit development. Malic acid content decreased by 91.43% and 72.28% during fruit ripening in HZ and B8, respectively. The content of citric acid increased significantly in B8, while in HZ it was reduced considerably. Differentially accumulated metabolites and differentially expressed genes analyses unveiled fumarate, succinate, 2-oxoglutarate, GABA (γ-aminobutyric acid), phosphoenolpyruvate, and citrate metabolisms as the key driving pathways of litchi fruits’ acidity variation. The drastic malate and citrate degradation in HZ was linked to higher induction of fumarate and GABA biosynthesis, respectively. Thirty candidate genes, including three key genes (LITCHI026501.m2, fumarase; LITCHI020148.m5, glutamate decarboxylase; and LITCHI003343.m3, glutamate dehydrogenase), were identified for functional studies toward genetic modulation of litchi fruit acidity. Our findings provide insights into the molecular basis of acidity variation in litchi and provide valuable resources for fruit quality improvement.

The postharvest development of greasiness in apple skin has negative effects on fruit appearance and market quality and causes significant economic losses. The fluid wax constituents responsible for skin greasiness comprise mainly oleate and linoleate esters of (E,E)-farnesol and short-chain alcohols (C3-C5). Butyl esters are also key components. It is believed that accumulations of volatile alcohols affect the development of skin greasiness by offering substrates for the biosynthesis of the greasy esters. To demonstrate the putative role of volatile alcohols on apple skin greasiness, ‘Cripps Pink’ apples were treated with either 10 or 25 μL L−1 n-butanol (NBA) and then stored at 20 °C. The composition of each cuticular wax was analyzed, and the expression levels of the related genes were recorded. There was no significant difference in the time course of fruit ripening or senescence among the two NBA treatments and the control. As expected, accumulations of the fluid wax constituents were indeed promoted in the NBA-treated fruit. On day 42, compared with the control, the butyl ester contents of the skins of the 10 and 25 μL L−1 NBA-treated fruits were 1.7 and 2.9 times higher, respectively. Moreover, the 25 μL L−1 NBA-treated fruit showed higher greasiness levels and looked shinier than the fruit exposed to the lower NBA treatment or the control. Several genes were more highly expressed in the NBA-treated fruit than in the control. These included MdKASIII, MdSAD6, MdWSD1 (related to wax biosynthesis) and MdLTPG1 (related to wax export). We conclude that NBA had no significant effects on fruit ripening or senescence but promoted the development of skin greasiness.

Anthocyanins are responsible for the red color in red-fleshed kiwifruits. In most of these cyanidin 3-O-xyl-galactoside is the predominant anthocyanin. This requires a second glycosylation modification which attaches the UDP-xylose to the cyanidin 3-O-galactoside. We report here the functional characterization of two second glycosyltransferases from ‘Hongyang’ kiwifruit, AcUFGT6b and AcUFGT7c. These were found to cluster with other plant GGTs. In vitro, recombinant AcUFGT6b recognized 3-O-glycosylated anthocyanins and UDP-xylose, while AcUFGT7c showed no or negligible activity. In tobacco leaves, co-expression of AcMYBF110, AcUFGT3a and AcUFGT6b resulted in the accumulation of three new products: cyanidin 3-O-xyl-galactoside, cyanidin 3-O-xyl-glucoside and cyanidin 3-O-xyl-rutoside. RNAi assays showed that a lack of AcUFGT6b and AcUFGT7c in immature fruits of Actinidia arguta prevents the formation of cyanidin 3-O-xyl-galactoside. Also, the content of cyanidin 3-O-xyl-galactoside was obviously increased when AcUFGT6b was injected together with AcMYBF110 and AcUFGT6b7c-RNAi. In contrast, for AcUFGT7c, as well as in vitro, we show no activity in either tobacco leaves or kiwifruit. Both AcUFGT6b and AcUFGT7c are located in the ER and their promoters can be positively activated by AcMYBF110, indicating the functional differences of them are due neither to their cell localization nor to the upstream regulation of AcMYBF110. Interestingly, the deletion in the C-terminal of AcUFGT7c is likely to change its 3D protein structure compared with AcUFGT6b protein, resulting in it having no capacity to attract the UDP sugar moiety. These results suggest that AcUFGT6b, but not AcUFGT7c, is responsible for the end-product of the anthocyanins biosynthesis pathway in red-fleshed kiwifruit.

This study investigated the key carotenoids and their biosynthetic regulation mechanisms in persimmon fruit skin. Carotenoid content and expression patterns of nine carotenogenic genes in skins of ‘Jinping’, ‘Huoguan’ and ‘Heishi’ cultivars were analyzed during fruit development. As skin color changed, lutein content decreased and β,β-carotenoid (β-carotene, β-cryptoxanthin, zeaxanthin), lycopene and α-carotene accumulated especially in late development stages. At harvest, the major carotenoids in ‘Jinping’ and ‘Heishi’ were β-carotene, while the red-colored lycopene was predominant in the skin of ‘Huoguan’. The minor carotenoids in all three cultivars were free β-cryptoxanthin, lutein, α-carotene and zeaxanthin. Consistently, the lowered expression of LCYe and increased expression of LCYb also indicated a shift in carotenoid biosynthesis from the β,e-branch to the β,β-branch accompanied fruit development. Simultaneously increased expression levels of DkGGPS, DkPSY, DkPDS, DkZDS, DkLCYb and DkBCH resulted in elevated accumulations of carotenoids. Correlation analysis showed increased DkPSY expression positively correlated with increased lutein, α-carotene, lycopene and β-cryptocanthin, and negatively with zeaxanthin. Both DkZDS and DkBCH expressions showed positive correlations with zeaxanthin, α-carotene, lycopene and β-cryptocanthin accumulation, but a negative correlation with lutein. The expression of DkLCYb was positively correlated with β-carotene levels only. These results indicate that DkPSY, DkZDS, DkLCYb and DkBCH may play significant roles by contributing coordinately to carotenoid biosynthesis during persimmon fruit development.

The anthocyanin synthetic pathway is regulated centrally by an MYB-bHLH-WD40 (MBW) complex. Anthocyanin pigmentation is an important fruit quality trait in red-fleshed kiwifruit; however, the underlying regulatory mechanisms involving the MBW complex are not well understood. In this study, one R2R3MYB (

Some apple (Malus × domestica) cultivars can become greasy during ripening due to changes in their surface wax composition. Epicuticular waxes are derived by the de novo synthesis of fatty acids, catalyzed by fatty acid synthase (FAS). Epigallocatechin-3-gallate (EGCG) has previously been shown to be an inhibitor of FAS in both animals and bacteria. Therefore, we hypothesize it may also affect the development of postharvest skin greasiness in apples. In this study, ‘Jonagold’ apples were treated with EGCG and then stored at 20 °C. The composition of the surface waxes was analyzed and the expressions of genes related to wax synthesis were recorded. The results show that at these postharvest storage temperatures, the level of skin greasiness rises rapidly. This is consistent with measured increases in the content of the main fluid waxes - linoleate and oleate esters. Compared with the controls, the accumulation of fluid waxes was suppressed by EGCG at most stages during ambient storage. A number of genes related to fluid wax biosynthesis, including MdKASIII, MdKAR, MdFAD2 and MdWSD11, showed significant increases in expression during the development of skin greasiness. Meanwhile, the expressions of MdKASII and MdENR, the transport gene MdLTPG1 and the regulation gene MdSHN3 declined during storage. The expressions of all these genes were down-regulated by EGCG, while the fruit respiration and firmness were similar between the EGCG-treated and control fruit throughout the storage period. We suppose that the inhibitory effects of EGCG are to down-regulate expression levels of genes involved in wax biosynthesis. This results in reduced accumulation of the fluid wax components and delays and lowers the development of skin greasiness. Our results also indicate that MdFAD2 and MdWSD11 may be critical for formation of greasy esters in apple skins. This work is the first evaluation of the effects of EGCG on apple skin greasiness. The results supply useful information elucidating the mechanisms through which apple fruit skins can become greasy during postharvest storage.

The aim of the current study was to characterize the functional role of DkLCYb in carotenoid biosynthesis in persimmon fruit. The accumulation of carotenoids and the expression of carotenogenic genes were investigated during persimmon fruit development and postharvest ripening. DkLCYb was cloned and its functional role in carotenoid biosynthesis was characterized. The results showed that carotenoids constantly accumulated during persimmon fruit development and postharvest ripening. More carotenoids accumulated during postharvest storage than during fruit development. The carotenoid composition of unsaponified extracts of the persimmon fruit flesh showed that the major carotenoids were lycopene and β-carotene in ‘Huoguan’ and ‘Heishi’, respectively. Consistent with this result, the expression of six carotenogenic genes increased to their highest levels during postharvest storage. The selected carotenoid biosynthesis genes showed an increased expression pattern during fruit development and in the early period of postharvest storage. A significant decrease in carotenogenic genes was found after stage 6 (DkPSY, DkLCYb, DkZDS and DkBCH) or stage 5 (DkLCYe,). Both DkLCYe and DkBCH showed a steady increase from stage 1 to stage 5; however, dramatically decreased expression of DkLCYe and sharply increased expression of DkBCH were found at stage 6. Correlation analysis showed that the expression of DkPSY, DkPDS, DkZDS, DkLCYb and DkBCH was significantly correlated with the accumulation of one or more carotenoid compounds analysed in this study. However, a negative correlation was found between the expression of DkLCYe and the accumulation of lycopene, β-cryptoxanthin and zeaxanthin. Specifically, expression of DkLCYb showed the highest correlation (p

‘Royal Gala’ apple fruit (Malus domestica) is one of the popular apple fruit consumed world-wide. A great amount literature was accumulated for this classical apple variety about the molecular mechanisms of individual gene or gene family transcript changes during ripening, yet few of them on the large scale at transcriptomic levels. The present study was designed to use RNA-Seq to compare expression profiles between commercial ripening and physiological ripening stages of apple fruit in order to provide a deeper whole view for understanding the molecular control of ‘Royal Gala’ fruit ripening on transcript level. 13.78 G and 12.28 G clean reads were obtained for the commercial ripening and physiological ripening stages apple fruit, respectively acquired and were blasted against the apple genome. 78,553 genes were expressed in ripening apple fruit and among them 860 genes were differentially expressed between the two maturity stages, with 199 up-regulated and 661 down-regulated. The enrichment of differentially expressed genes (DEGs) in gene ontology (GO) pathways revealed that genes in 8 GO terms were more activated at physiological maturity stage. Meanwhile, KEGG enrichment showed that secondary metabolism contained more up-regulated DEGs at physiological maturity stage than at commercial maturity stage. Furthermore, genes working on ripening-related processes were examined. For transcription factors regulating ethylene biosynthesis such as ERFs and HBs, the expression levels were significantly different between the two samples. Regarding fruit firmness decline, expression variation happened to genes encoding enzymes involving in cell wall metabolism, such as polygalacturonase PG1, xyloglucan endotransglucosylase XTHs and EXP3, as well as cell wall structure protein gene FLA. In conclusion, the RNA-Seq data presents the global expression changes of ripening related transcripts in ‘Royal Gala’ apple and novel identified genes and/or transcription factors may provide new clues to illuminate this complicated event.

Bitter pit (BP) is a physiological disorder of calcium deficiency in apple fruit and has not been fully characterized. The Ca2+/H+ exchanger (CAX) is a kind of Ca2+ transport protein. In this study, we analysed mineral contents in different parts of fruit, the expression levels of genes related to Ca transport and the functional roles of MdCAXs. The results showed lower water-soluble Ca content and significantly higher ratios of water-soluble P/Ca and (K + Mg)/Ca in the calyx-end flesh of BP and normal fruit than the peduncle-end flesh of them. The expression level of MdCAX11 increased as the severity of BP increased, and the expression profile was negatively correlated with the water-soluble Ca content. Yeast growth assays showed that both MdCAX11 and MdCAX5 have the function of transporting Ca to vacuoles. Subcellular localization analysis showed that MdCAX11-GFP and MdCAX5-GFP were colocalized on the vacuole membrane with a tonoplast marker. Taken together, the results of this study indicate that high expression levels of MdCAX11 and MdCAX5 may cause an influx of Ca from the cytosol into vacuoles, which may be related to the occurrence of BP.

In this study, the melanin in persimmon and its formation were investigated. Melanin was found to be deposited on the cell walls of the upper epidermis and subepidermal cells in persimmon skin and the isolated pigment appears to have lamellar structures. Diagnostic analysis of the isolated pigment showed results that were similar to those of melanin from other sources. Ultraviolet-visible spectroscopy revealed that the extracted skin pigment displayed a broadband, structureless absorption profile that increased progressively towards shorter wavelengths. The Fourier transform infrared spectroscopy assay revealed that melanin in persimmon skin exhibits many characteristic absorption peaks. The phenolic profile analysis suggested that the precursors of this pigment may include gallic acid, procyanidin B1, procyanidin B2, ferulic acid and epigallocatechin gallate. The PPO activity and DkPPO expression significantly increased during melanin formation, and transient overexpression of DkPPO promoted melanin synthesis. These results indicate that the isolated pigment was a type of melanin and that PPO plays a critical role in its formation.

Red-fleshed kiwifruits are receiving increasing attention because of their high phenolic contents. However, detailed information on their phenolic compounds and antioxidant capacities remains scarce. Here, six red-fleshed and six green-fleshed kiwifruits were investigated to determine their contents of phenolic compounds and their antioxidant capacities. The results showed chlorogenic acid, p-coumaric acid and ferulic acid were the main phenolic compounds found in kiwifruit. Most of red-fleshed kiwifruits contain higher amounts of total phenolics and anthocyanins, as well as higher activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). Moreover, they exhibited stronger antioxidant capacities than green-fleshed kiwifruits in ABTS, DPPH and FRAP assays. Furthermore, the reactive oxygen species (ROS) inhibition assay showed the phenolics extracted from red-fleshed kiwifruit can better protect tobacco leaves against hydrogen peroxide (H2O2)-induced oxidative damage. This is because of their abundant anthocyanins which in vitro contribute more to H2O2 scavenging than the other phenolic compounds. Based on these findings, it is fair to conclude the red-fleshed kiwifruits are promising sources of antioxidants in human nutrition.

Fruits of kiwifruit cultivars (Actinidia chinensis and A. deliciosa) generally have green or yellow flesh when ripe. A small number of genotypes have red flesh but this coloration is usually restricted to the inner pericarp. Three kiwifruit cultivars having red (‘Hongyang’), or yellow (‘Jinnong-2’), or green (‘Hayward’) flesh were investigated for their color characteristics and pigment contents during development and ripening. The results show the yellow of the ‘Jinnong-2’ fruit is due to the combined effects of chlorophyll degradation and of beta-carotene accumulation. The red inner pericarps of ‘Hongyang’ fruit are due to anthocyanin accumulation. Expression differences of the pathway genes in the inner pericarps of the three different kiwifruits suggest that stay-green (SGR) controls the degradation of chlorophylls, while lycopene beta-cyclase (LCY-β) controls the biosynthesis of beta-carotene. The abundance of anthocyanin in the inner pericarps of the ‘Hongyang’ fruit is the results of high expressions of UDP flavonoid glycosyltransferases (UFGT). At the same time, expressions of anthocyanin transcription factors show that AcMYBF110 expression parallels changes in anthocyanin concentration, so seems to be a key R2R3 MYB, regulating anthocyanin biosynthesis. Further, transient color assays reveal that AcMYBF110 can autonomously induce anthocyanin accumulation in Nicotiana tabacum leaves by activating the transcription of dihydroflavonol 4-reductase (NtDFR), anthocyanidin synthase (NtANS) and NtUFGT. For basic helix-loop-helix proteins (bHLHs) and WD-repeat proteins (WD40s), expression differences show these may depend on AcMYBF110 forming a MYB-bHLH-WD40 complex to regulate anthocyanin biosynthesis, instead of it having a direct involvement.

Much of the diversity of anthocyanin pigmentation in plant tissues is due to the action of glycosyltransferases, which attach sugar moieties to the anthocyanin aglycone. This step can increase both their solubility and stability. We investigated the pigmentation of the outer and inner pericarps of developing fruits of the red-fleshed kiwifruit Actinidia chinensis cv. 'Hongyang'. The results show that the red color of the inner pericarp is due to anthocyanin. Based on expression analyses of structural genes, AcUFGT was shown to be the key gene involved in the anthocyanin biosynthetic pathway. Expression of AcUFGT in developing fruit paralleled changes in anthocyanin concentration. Thirteen putative UFGT genes, including different transcripts, were identified in the genome of 'Hongyang'. Among these, only the expression of AcUFGT3a was found to be highly consistent with anthocyanin accumulation. Fruit infiltrated with virus-induced gene silencing showed delayed red colorations, lower anthocyanin contents and lower expressions of AcUFGT3a. At the same time, transient overexpression of AcUFGT3a in both Actinidia arguta and green apple fruit resulted in higher anthocyanin contents and deeper red coloration. In vitro biochemical assays revealed that recombinant AcUFGT3a recognized only anthocyanidins as substrate but not flavonols. Also, UDP-galactose was used preferentially as the sugar donor. These results indicate AcUFGT3a is the key enzyme regulating anthocyanin accumulation in red-fleshed kiwifruit.

Background We hypothesized that the combination of bevacizumab, carboplatin, and pemetrexed will be an effective first-line regimen in fit, elderly patients with nonsquamous non–small-cell lung cancer. Methods Treatment-naive, stage IIIB/IV nonsquamous non–small-cell lung cancer patients more than 70 years old with good performance status (Eastern Cooperative Oncology Group performance status 0–1) and adequate organ function were eligible. Carboplatin area under the curve 6, pemetrexed 500 mg/m 2 , and bevacizumab 15 mg/kg were administered on day 1 of each 21-day cycle (up to six cycles) followed by maintenance pemetrexed and bevacizumab. The primary end point of 6-month progression-free survival rate of at least 70% was assessed using a one-stage binomial design. Quality of life (QOL) questionnaires were administered. Polymorphisms in genes encoding relevant proteins (drug targets, transport, and metabolism proteins) were correlated with treatment outcome. Results Fifty-seven eligible patients were enrolled. Median age was 74.5 years. Median treatment cycles received was 6. The most common grade 3 or higher non-hematologic adverse events were fatigue (26%) and hypertension (11%); 16% had grade 4 neutropenia and 6.5% had grade 4 thrombocytopenia. Three patients experienced grade 3/4 hemorrhagic events (one pulmonary, two gastrointestinal). Primary end point of PFS6 was 60% (95% confidence interval [CI]: 45.9–73%). Median PFS was 7.0 months (95% CI: 5.9–10.1), median overall survival was 13.7 months (95% CI: 9.4–16.8). Polymorphic KDR and VEGFA variants correlated with survival and toxicity, respectively. There was no significant change in overall QOL scores over time. Conclusion This regimen is feasible and did not decrease the QOL in this study population. However, it did not meet the primary efficacy end point.

BACKGROUND: In patients with advanced lung cancer, overall survival is largely influenced by progression status. Because progression-free survival (PFS)-based endpoints are controversial, the authors evaluated the impact of the progression date (PD) determination approach on PFS estimates. METHODS: Individual patient data from 21 trials (14 North Central Cancer Treatment Group trials and 7 Southwest Oncology Group trials) were used. The reported PD (RPD) was defined as either the radiographic scan date or the clinical deterioration date. PD was determined using Method 1 (M1), the RPD; M2, 1 day after the last progression-free scan; M3, midpoint between the last progression-free scan and the RPD; and M4, an interval-censoring approach. PFS was estimated using Kaplan-Meier (M1-M3), and maximum-likelihood (M4) methods. Simulation studies were performed to understand the impact of the length of time elapsed between the last progression-free scan and the PD on time-to-progression estimates. RESULTS: PFS estimates using the RPD were the highest, and M2 was the most conservative. M3 and M4 were similar because the majority of progressions occurred during treatment (ie, frequent disease assessments). M3 was influenced less by the length of the assessment schedules (percentage difference from the true time-to-progression

Purpose Activation of EGFR can stimulate proliferative and survival signaling through mTOR. Preclinical data demonstrates synergistic activity of combined EGFR and mTOR inhibition. We undertook a phase I trial of temsirolimus (T, an mTOR inhibitor) and EKB-569 (E, an EGFR inhibitor) to determine the safety and tolerability. Methods The primary aim was to determine the maximally tolerated dose (MTD) of this combination in adults with solid tumors. Following the dose-escalation phase, (Cohort A), two subsequent cohorts were used to assess any pharmacokinetic (PK) interaction between the agents. Results Forty eight patients were enrolled. The MTD of this combination was E, 35 mg daily and T, 30 mg on days 1-3 and 15-17 using a 28-day cycle. The most common toxicities were nausea, diarrhea, fatigue, anorexia, stomatitis, rash, anemia, neutropenia, thrombocytopenia, and hypertriglyceridemia. Sixteen patients (36%) had at least one grade 3 toxicity. The most frequent grade 3/4 toxicities were diarrhea, dehydration, and nausea and vomiting (19% each). No grade 5 events were seen. Four patients had a partial response and 15 had stable disease. Clinical benefit was seen across a range of tumor types and in all cohorts. PK analysis revealed no significant interaction between E and T. Conclusions This combination of agents is associated with tolerable toxicities at doses that induced responses. PK studies revealed no interaction between the drugs. Further investigations of this targeting strategy may be attractive in renal cell carcinoma, non-small cell lung cancer, alveolar sarcoma, and carcinoid tumor.

Purpose We investigated the putative surrogate endpoints (PSEs) of best response (BR), complete response (CR), confirmed response (CoR), and progression-free survival (PFS) for associations with Overall Survival (OS), and as possible surrogate endpoints for OS.

HypothesisWe conducted this pooled analysis to assess the prognostic value of pretreatment Quality of Life (QOL) assessments on overall survival (OS) in advanced non-small cell lung cancer (NSCLC).MethodsFour hundred twenty patients with advanced NSCLC (stages IIIB with pleural effusion and IV) from six North Central Cancer Treatment Group trials were included in this study. QOL assessments included the single-item Uniscale (355 patients), Lung Cancer Symptom Scale (217 patients), and Functional Assessment of Cancer Therapy-Lung (197 patients). QOL scores were transformed to a 0 to 100 scale with higher scores representing better status and categorized using the sample median or clinically deficient score (CDS, ≤50 versus >50). Cox proportional hazards models stratified by study were used to evaluate the prognostic importance of QOL on OS alone and in the presence of other prognostic factors such as performance status, age, gender, body mass index, and laboratory parameters.ResultsPretreatment QOL accessed by Uniscale was significantly associated with OS univariately (p < 0.0001). Uniscale (p < 0.0001; hazard ratio = 1.6 for the sample median and 2.0 for the CDS categorization) and body mass index were the only significant predictors of OS multivariately. The median survival of patients who had a Uniscale score less than or equal to the CDS (≤50) was 5.7 versus 11.1 months for the >50 group; and 7.8 versus 13 months for the less than or equal to sample median (≤83) group and >83 group, respectively. The Lung Cancer Symptom Scale and the Functional Assessment of Cancer Therapy-Lung total scores were not significant predictors of OS.ConclusionsPretreatment QOL measured by Uniscale is a significant and an independent prognostic factor for OS, and QOL should be routinely integrated as a stratification factor in advanced NSCLC trials.

Background A Phase I trial of the 2-drug regimen of everolimus plus gemcitabine (Cohort I) and the 3-drug regimen of everolimus plus gemcitabine and cisplatin (Cohort II) was performed to determine the maximally tolerated dose (MTD) of both combinations. An expansion cohort (Cohort III) of patients with cholangiocarcinoma or gallbladder carcinoma was treated at the MTD. Methods A standard 3 + 3 design dose escalation was used. Everolimus was given on Monday/Wednesday/Friday or daily depending upon the dose level. Gemcitabine and cisplatin were administered on days 1 and 8 of each 21 day cycle. Results Twelve patients were entered in Cohort I and 15 in Cohort II. The MTD for Cohort I was everolimus 5 mg on Monday/Wednesday/Friday and gemcitabine 800 mg/m2. For Cohort II, it was everolimus 5 mg on Monday/Wednesday/Friday, gemcitabine 600 mg/m2, and cisplatin 12.5 mg/m2. All DLTs in this study were hematologic. Complete responses (CR) were seen in a patient with primary peritoneal carcinoma and another with recurrent pancreatic cancer. Partial responses (PR) were seen in 3 patients: breast, ampullary carcinoma and pheochromocytoma. Of 10 patients enrolled in Cohort III, six patients had stable disease and 4 had progressive disease. Conclusions This Phase I clinical trial has demonstrated that these 2-drug and 3-drug combinations are generally well tolerated and safely administered. The main DLTs in both regimens were hematologic, specifically thrombocytopenia. The 3-drug combination can be considered as a platform for future studies in specific tumor types.

Loss of appetite and weight predict poor outcomes in patients with advanced cancer. Effective and affordable palliative strategies are lacking; but because an emerging non-cancer literature suggests that alcohol can increase appetite and weight, this study explored associations between alcohol and clinical outcomes in lung cancer patients. Among 404 consecutive lung cancer patients enrolled in the Mayo Clinic Lung Cancer Cohort between 2004 and 2008, alcohol consumption (within 6 mo of diagnosis) was as follows: 199 (49%) used none, 158 (14%) were moderate users (7 drinks per wk or less), and 47 (12%) were heavier consumers (more than 7 drinks per wk). Only heavier consumers had a lower likelihood of anorexia (odds ratio: 0.49; 95% CI: 0.25, 0.94; P = 0.03) and weight loss (odds ratio: 0.43; 95% CI: 0.20, 0.91; P = 0.03) compared to those who consumed no alcohol. These conclusions were sustained in multivariate analyses. Neither moderate nor heavier consumption was associated with better or worse survival, although, in univariate analyses, a drop in alcohol consumption was associated with worse survival. This report suggests a need for further study of alcohol as a palliative agent for cancer-associated loss of appetite and weight.

Continued cigarette smoking after small cell lung cancer (SCLC) diagnosis has been shown to shorten patients' survival, but little is known about the impact of smoking and cessation on quality of life (QOL) profile (e.g., overall QOL, pain, fatigue, cough, dyspnea, appetite change, and performance status) in SCLC survivors (who survived at least 6 months post initial diagnosis). In this study, we sought to evaluate the relationship between cigarette smoking and QOL profiles in SCLC patients.A total of 223 survivors were classified into five groups: never smokers, former smokers (quit more than 1 year prior to diagnosis), recent quitters (quit within 1 year surrounding diagnosis), late quitters (quit after 1 year post diagnosis) and never quitters. One hundred and sixty-eight of these survivors were matched with 334 lung-cancer-free controls on age, gender, and smoking status for comparative analysis. QOL scales were scored from 0 (worse) to 100 (best). Conditional logistic regression, linear mixed-effect models, and Wilcoxon signed rank tests were used.SCLC survivors consistently showed a significant deficit in QOL profile; e.g., mean overall QOL in patients was 17.5 points worse than the controls (p0.0001). Among all smokers, former smokers reported the best QOL profile, while late or never quitters reported the worst. The recent quitters showed an improving trend in QOL profile and lower percent of reduced appetite (an average of 43%) compared to the late or never quitters (58%).Our study confirmed the negative impact of smoking on SCLC survivors' QOL and found that smoking cessation surrounding the time of diagnosis could improve overall QOL and symptoms. The findings of this study provide evidence for oncologists to recommend smoking cessation to their SCLC patients.

Introduction: Although the association between alpha 1-antitrypsin deficiency (α 1 ATD) carriers and lung cancer risk has been found, the effects of α 1 ATD carriers and serum alpha 1-antitrypsin (α 1 AT) concentration on non-small cell lung cancer (NSCLC) survival remained unclear. Methods: Patients were selected from the Epidemiology and Genetics of Lung Cancer Study at Mayo Clinic with the criteria of (1) primary NSCLC diagnosis and (2) available α 1 ATD carrier status tested by isoelectric focusing serum α 1 AT concentration by immunonephelometry. The effects of carrier status and serum α 1 AT concentration on survival were evaluated by Cox proportional hazards models with (1) a landmark approach, where overall survival was defined from the time of blood draw to death from any cause and (2) included only patients with blood draw time before initial treatment. Results: One thousand three hundred twenty-one patients were included in this study, with 179 α 1 ATD carriers and 1142 noncarriers. No differences in overall survival by α 1 ATD carrier status were found (adjusted hazard ratio [AHR]: 0.98; 95% confidence interval [CI]: 0.82–1.18). Nevertheless, serum α 1 AT concentration was significantly associated with survival among all patients in the landmark model (AHR per 50 mg/dl increments: 1.15; 95% CI: 1.10–1.20) and among patients whose blood was drawn for serum α 1 AT level assessment before any treatment (AHR per 50 mg/dl increments: 1.44; 95% CI: 1.21–1.71). Conclusions: Being an α 1 ATD carrier had no significant effect on NSCLC survival. The increased serum α 1 AT concentration was a poor prognosis marker for NSCLC, regardless of carrier status.

The cancer anorexia/weight loss syndrome commonly occurs in patients with non-small cell lung cancer (NSCLC) and is characterized by loss of weight and appetite as well as diminished survival. The current study explored whether any of 22 single nucleotide polymorphisms (SNPs) of certain previously implicated inflammatory cytokines (interleukin-1 beta, interleukin-1RN, interleukin-6, and tumor necrosis factor) are associated with this syndrome.All NSCLC patients who had been enrolled in the Mayo Clinic Lung Cancer Cohort, had completed a health-related questionnaire approximately 6 months after enrollment, and had blood drawn were included in this study, thus yielding a sample size of 471 patients.Sixty-six (14%) patients manifested weight loss shortly after diagnosis, and 152 (32%) reported appetite loss. Only tumor necrosis factor alpha rs800629 was associated with anorexia (odds ratio: 0.46; 95% confidence interval: 0.29, 0.72; p0.001); patients who were heterozygous and minor homozygous were less likely to suffer anorexia. Otherwise, there were no statistically significant associations between any of the other 21 SNPs and weight loss and/or anorexia. In univariate analyses, weight loss, anorexia, more advanced cancer stage, and interleukin-1 beta rs1143627 were associated with a worse survival, and interleukin-6 rs2069835 was associated with better survival. However, in multivariate analyses, cancer stage and patient age were the only statistically significant predictors of worse survival.No specific SNP was associated with all aspects of the cancer anorexia/weight loss syndrome, but rs800629 may merit further study in cancer-associated anorexia.

OBJECTIVE To describe the natural history of pelvic organ prolapse and risk factors for changes in vaginal descent in older women. METHODS This 4-year prospective observational study included 259 postmenopausal women with a uterus enrolled at one Women's Health Initiative clinical site who completed at least two annual pelvic organ prolapse quantification (POP-Q) examinations. We calculated 1-year and 3-year incidence and resolution risks for prolapse (defined as maximal vaginal descent to or beyond the hymen) and estimated progression and regression rates (1 cm or greater and 2 cm or greater changes in maximal vaginal descent) and risk factors. RESULTS Mean age was 68.1+/-5.5 years, and median vaginal parity was 4. Seventy-three (28%) women had four exams, 128 (49%) had three exams, and 58 (22%) had two exams. Prolapse waxed and waned yearly in individual women. Overall 1-year and 3-year prolapse incidences were 26% (95% confidence interval [CI] 20-33%) and 40% (95% CI 26-56%); 1-year and 3-year prolapse resolution risks were 21% (95% CI 11-33%) and 19% (95% CI 7-39%). Rates of any change in maximal vaginal descent over time varied depending on baseline measurements. Over 3 years, the maximal vaginal descent increased by at least 2 cm in 11.0% (95% CI 4.9-20.5%) of the women and decreased by at least 2 cm in 2.7% (95% CI 0.3-9.5%). Increasing body mass index and grand multiparity increased the risk for vaginal descent progression. CONCLUSION Prolapse progresses and regresses in older women, although rates of vaginal descent progression are slightly greater than regression overall. Obesity is a risk factor for progression in vaginal descent. LEVEL OF EVIDENCE III.

To evaluate the efficiency of sliding-window technique in extracting and analyzing somatosensory evoked potentials (SEP) from multichannel electroencephalogram (EEG) data.A time window of certain window size was moved along the time dimension of data sets. Values within the window were averaged for each trial, and then compared with a preset control window. The probability of randomly appeared significance resulting from repeated statistical comparison was calculated utilizing simulated EEG data sets. Cluster size (number of successive significant data points with given individual significance threshold) was determined to keep the general alpha value under 0.05. To test this procedure, multichannel EEG signals were recorded and analyzed from fourteen healthy right-handed volunteers, with painful and non-painful electrical stimuli delivered to the right middle fingers.Cluster size increased in parallel with window size and individual statistical threshold. The major SEP components of real EEG data, as well as the difference between pain and non-pain SEPs, were demonstrated to be significant with the sliding-window method.Sliding-window method is an effective tool for the analysis of SEP data.

7605 Background: Overall survival (OS) can be partitioned into progression-free survival (PFS) and PPS. PPS helps to understand trial results, especially when PFS and OS data on trial treatment effects are discordant. At ASCO 2011, we reported that the magnitude of difference in the PFS estimates using different DP date methods was large enough to alter trial conclusions. Here, we investigate the impact of the DP date method on 1) PPS estimates, and 2) predictive utility of DP metrics on subsequent OS (SOS). Methods: Individual patient (pt) data from 14 trials were pooled. DP date was determined using: reported progression date (RPD) (method 1, M1), one day after last progression-free (PF) scan (M2), and midpoint between last PF scan and RPD (M3). PPS was estimated using the method of Kaplan-Meier for the 3 DP date methods. A flexible landmark analysis at 2, 4, and 6 months (mos) using Cox proportional hazards model was used to assess the impact of DP status (progression versus no-progression) on SOS (using M1, M2, or M3). Results: Among NSCLC (SCLC), 87% (91%) of pts reported DP. As expected, the PPS estimates were the lowest for RPD, highest for M2, and in-between for M3 (a direct consequence of the DP date method); with no difference by arm for the randomized trials. Regardless of the DP date method, patients who were progression-free had improved SOS (NSCLC: Hazard ratio, HR

e13052 Background: The combination of GEM and CDDP has shown activity in a variety of cancers, including biliary tract/gallbladder. Preclinical testing shows a potential benefit to the addition of EV. Methods: Using a standard 3+3 design, the MTDs of GEM + EV (Cohort I) and GEM + CDDP + EV (Cohort II) were determined as shown in the table. The MTD was defined as the dose level below the lowest dose that induces a DLT in at least one-third of pts. At the MTD of Cohort II, 10 patients were enrolled with biliary tract/gallbladder cancer (Cohort III). A weekly CBC was obtained. Assessments occurred every 3 weeks and imaging for response at every other cycle. Results: In Cohort I (N=12), no DLT occurred at dose level 0, and in dose level 1, grade 3 thrombocytopenia was found in 2 of 6 pts. The MTD for Cohort I was determined to be dose level 0. Responses were seen in 3 pts: 2 CRs (primary peritoneal, pancreatic) and 1 PR (breast). In Cohort II (N=15) DLTs at dose level 0 were neutropenia and thrombocytopenia in 2 of 3 pts and at dose level -1, thrombocytopenia in 2 of 6 pts. At dose level -2, 1 of 6 pts experienced a DLT (grade 3 thrombocytopenia), establishing this dose level as the MTD. Responses were seen in 2 pts, both PRs (ampullary, pheochromocytoma). All 10 pts have been enrolled in Cohort III with 2 DLTs (neutropenia and thrombocytopenia). Stable disease seen in 5 of 7 evaluable pts. Conclusions: Gem + EV was well tolerated at dose level 0, though dose escalation was limited by thrombocytopenia. GEM + CDDP + EV had DLTs of neutropenia and thrombocytopenia leading to the MTD of dose level of -2. Cohort III is fully accrued and the 2 DLTs are hematologic. [Table: see text]