Follow-up Schedule for Patients With Sentinel Node-negative Cutaneous Melanoma (The MELFO Study): An International Phase III Randomized Clinical Trial.

OBJECTIVES AND DESIGN: The MELFO (MELanoma FOllow-up) study is an international phase III randomized controlled trial comparing an experimental low-intensity schedule against current national guidelines. BACKGROUND: Evidence-based guidelines for the follow-up of sentinel node-negative melanoma patients are lacking. METHODS: Overall, 388 adult patients diagnosed with sentinel node-negative primary melanoma patients were randomized in cancer centers in the Netherlands and United Kingdom between 2006 and 2016. The conventional schedule group (control: n=196) was reviewed as per current national guidelines. The experimental schedule group (n=192) was reviewed in a reduced-frequency schedule. Quality of life was the primary outcome measurement. Detection rates and survival outcomes were recorded. Patient satisfaction rates and compliance with allocated schedules were compared. RESULTS: At 5 years, both arms expressed high satisfaction with their regimens (>97%). This study found no significant group effect on any patient-reported outcome measure scores between the follow-up protocols. In total, 75/388 (19.4%) patients recurred, with no difference in incidence found between the 2 arms (hazard ratio=0.87, 95% confidence interval: 0.54-1.39, P =0.57). Self-examination was the method of detection for 25 experimental patients and 32 control patients (75.8% vs. 76.2%; P =0.41). This study found no difference in any survival outcomes between the 2 study arms (disease-free survival: hazard ratio=1.00, 95% confidence interval: 0.49-2.07, P =0.99). CONCLUSIONS: A reduced-intensity, American Joint Committee on Cancer (AJCC) stage-adjusted follow-up schedule for sentinel node-negative melanoma patients is a safe strategy, and patient self-examination is effective for recurrence detection with no evidence of diagnostic delay. Patients' acceptance is very high.

The MelFo Study UK: Effects of a Reduced-Frequency, Stage-Adjusted Follow-Up Schedule for Cutaneous Melanoma 1B to 2C Patients After 3-Years.

BACKGROUND: Evidence-based guidelines for follow-up treatment of American Joint Committee on Cancer (AJCC) stages 1B to 2C melanoma patients are lacking. The MELanoma FOllow-up study is an international phase 3 randomized trial, and the 3-year interim data were recently reported from the Netherlands. The study was undertaken concurrently with a British cohort for comparison and validation of the Dutch study. METHODS: The study enrolled and stratified 207 patients by AJCC stage. The conventional schedule group (CSG; n = 103) cohort was reviewed as per UK guidelines. The experimental schedule group (ESG; n = 104) cohort was reviewed in a reduced-frequency nurse-led, consultant-supervised clinic. Quality of life (QoL) was measured at baseline (T1), a 1 year (T2), and at 3 years (T3) using the State-Trait Anxiety Inventory, the Cancer Worry Scale, the Impact-of-Event Scale, and the Mental and Physical Component scales (PCS/MCS) of the RAND-36. RESULTS: Of the 207 QoL questionnaires, 170 (82.1%) were completed at T3. Both cohorts expressed high satisfaction (> 93%) with their regimens. At T3, no significant group effect was found on any patient-reported outcome measures scores, indicating no QoL difference between the follow-up protocols. Recurrence had developed in 33 patients Conventional follow-up (CFU), 16 [15.5%]; Experimental follow-up (EFU), 17 [16.3%]. Self-examination was the method of detection for 12 ESG patients (70.6%) and 11 CSG patients (68.8%). The melanoma-specific survival was identical. CONCLUSION: The UK 3-year data were consistent with the previous Dutch report. The reduced follow-up strategy was shown to be safe, with significant resource usage benefits for national cancer services. Patient anxiety levels were not increased by a less-intensive follow-up regimen, and acceptance was high. The study data indicate that patient self-examination is very effective for recurrence detection.

Tissue-specific PhBPBT expression is differentially regulated in response to endogenous ethylene.

Ethylene is a gaseous plant hormone involved in many physiological processes including senescence, fruit ripening, and defence. Here the effects of pollination and wound-induced ethylene signals on transcript accumulation of benzoyl CoA:benzyl alcohol/phenylethanol benzoyltransferase (PhBPBT) are shown in Petuniaxhybrida cv. Mitchell 'Diploid' (MD). In petunia, PhBPBT is responsible for the biosynthesis of both benzyl benzoate and phenylethyl benzoate from benzyl alcohol and phenylethanol, respectively. RNAi-silenced lines, with reduced PhBPBT transcript, displayed reduced benzyl benzoate emission, and increased benzyl alcohol levels. Detailed expression analysis showed that PhBPBT is regulated by both light and an endogenous circadian rhythm, while it is also differentially regulated in response to ethylene in a tissue-specific manner. Twenty-four hours following pollination of MD flowers, expression of PhBPBT decreases in the corolla, while it increases in the ovary after 48 h. This is caused by ethylene that is emitted from the flower coinciding with fertilization as this is not observed in transgenic ethylene-insensitive plants (CaMV35S::etr1-1; 44568). Ethylene is also emitted from vegetative tissue of petunia following mechanical wounding, resulting in an increase in PhBPBT expression in the leaves where expression is normally below detection levels. Indicative of this pattern of expression, we hypothesize that PhBPBT and subsequent benzyl benzoate production is involved in defence-related processes in the corolla prior to pollination, in the ovary immediately following fertilization, and in vegetative tissue in response to wounding.

Small cysteine-rich peptides resembling antimicrobial peptides have been under-predicted in plants.

Multicellular organisms produce small cysteine-rich antimicrobial peptides as an innate defense against pathogens. While defensins, a well-known class of such peptides, are common among eukaryotes, there are other classes restricted to the plant kingdom. These include thionins, lipid transfer proteins and snakins. In earlier work, we identified several divergent classes of small putatively secreted cysteine-rich peptides (CRPs) in legumes [Graham et al. (2004)Plant Physiol. 135, 1179-97]. Here, we built sequence motif models for each of these classes of peptides, and iteratively searched for related sequences within the comprehensive UniProt protein dataset, the Institute for Genomic Research's 33 plant gene indices, and the entire genomes of the model dicot, Arabidopsis thaliana, and the model monocot and crop species, Oryza sativa (rice). Using this search strategy, we identified approximately 13,000 plant genes encoding peptides with common features: (i) an N-terminal signal peptide, (ii) a small divergent charged or polar mature peptide with conserved cysteines, (iii) a similar intron/exon structure, (iv) spatial clustering in the genomes studied, and (v) overrepresentation in expressed sequences from reproductive structures of specific taxa. The identified genes include classes of defensins, thionins, lipid transfer proteins, and snakins, plus other protease inhibitors, pollen allergens, and uncharacterized gene families. We estimate that these classes of genes account for approximately 2-3% of the gene repertoire of each model species. Although 24% of the genes identified were not annotated in the latest Arabidopsis genome releases (TIGR5, TAIR6), we confirmed expression via RT-PCR for 59% of the sequences attempted. These findings highlight limitations in current annotation procedures for small divergent peptide classes.

Ethylene-regulated floral volatile synthesis in petunia corollas.

In many flowering plants, such as petunia (Petunia x hybrida), ethylene produced in floral organs after pollination elicits a series of physiological and biochemical events, ultimately leading to senescence of petals and successful fertilization. Here, we demonstrate, using transgenic ethylene insensitive (44568) and Mitchell Diploid petunias, that multiple components of emission of volatile organic compounds (VOCs) are regulated by ethylene. Expression of benzoic acid/salicylic acid carboxyl methyltransferase (PhBSMT1 and 2) mRNA is temporally and spatially down-regulated in floral organs in a manner consistent with current models for post-pollination ethylene synthesis in petunia corollas. Emission of methylbenzoate and other VOCs after pollination and exogenous ethylene treatment parallels a reduction in PhBSMT1 and 2 mRNA levels. Under cyclic light conditions (day/night), PhBSMT mRNA levels are rhythmic and precede emission of methylbenzoate by approximately 6 h. When shifted into constant dark or light conditions, PhBSMT mRNA levels and subsequent methylbenzoate emission correspondingly decrease or increase to minimum or maximum levels observed during normal conditions, thus suggesting that light may be a more critical influence on cyclic emission of methylbenzoate than a circadian clock. Transgenic PhBSMT RNAi flowers with reduced PhBSMT mRNA levels show a 75% to 99% decrease in methylbenzoate emission, with minimal changes in other petunia VOCs. These results implicate PhBSMT1 and 2 as genes responsible for synthesis of methylbenzoate in petunia.

The central role of PhEIN2 in ethylene responses throughout plant development in petunia.

The plant hormone ethylene regulates many aspects of growth and development. Loss-of-function mutations in ETHYLENE INSENSITIVE2 (EIN2) result in ethylene insensitivity in Arabidopsis, indicating an essential role of EIN2 in ethylene signaling. However, little is known about the role of EIN2 in species other than Arabidopsis. To gain a better understanding of EIN2, a petunia (Petunia x hybrida cv Mitchell Diploid [MD]) homolog of the Arabidopsis EIN2 gene (PhEIN2) was isolated, and the role of PhEIN2 was analyzed in a wide range of plant responses to ethylene, many that do not occur in Arabidopsis. PhEIN2 mRNA was present at varying levels in tissues examined, and the PhEIN2 expression decreased after ethylene treatment in petals. These results indicate that expression of PhEIN2 mRNA is spatially and temporally regulated in petunia during plant development. Transgenic petunia plants with reduced PhEIN2 expression were compared to wild-type MD and ethylene-insensitive petunia plants expressing the Arabidopsis etr1-1 gene for several physiological processes. Both PhEIN2 and etr1-1 transgenic plants exhibited significant delays in flower senescence and fruit ripening, inhibited adventitious root and seedling root hair formation, premature death, and increased hypocotyl length in seedling ethylene response assays compared to MD. Moderate or strong levels of reduction in ethylene sensitivity were achieved with expression of both etr1-1 and PhEIN2 transgenes, as measured by downstream expression of PhEIL1. These results demonstrate that PhEIN2 mediates ethylene signals in a wide range of physiological processes and also indicate the central role of EIN2 in ethylene signal transduction.

Regulation of methylbenzoate emission after pollination in snapdragon and petunia flowers.

The molecular mechanisms responsible for postpollination changes in floral scent emission were investigated in snapdragon cv Maryland True Pink and petunia cv Mitchell flowers using a volatile ester, methylbenzoate, one of the major scent compounds emitted by these flowers, as an example. In both species, a 70 to 75% pollination-induced decrease in methylbenzoate emission begins only after pollen tubes reach the ovary, a process that takes between 35 and 40 h in snapdragon and approximately 32 h in petunia. This postpollination decrease in emission is not triggered by pollen deposition on the stigma. Petunia and snapdragon both synthesize methylbenzoate from benzoic acid and S-adenosyl-l-methionine (SAM); however, they use different mechanisms to downregulate its production after pollination. In petunia, expression of the gene responsible for methylbenzoate synthesis is suppressed by ethylene. In snapdragon, the decrease in methylbenzoate emission is the result of a decrease in both S-adenosyl-l-methionine:benzoic acid carboxyl methyltransferase (BAMT) activity and the ratio of SAM to S-adenosyl-l-homocysteine ("methylation index") after pollination, although the BAMT gene also is sensitive to ethylene.