Tests for Hazard Transformation

The semiparametric Cox proportional hazards model is routinely adopted to model time-to-event data. Proportionality is a strong assumption, especially when follow-up time, or study duration, is long. Zeng and Lin (J. R. Stat. Soc., Ser. B, 69:1–30, 2007) proposed a useful generalisation through a family of transformation models which allow hazard ratios to vary over time. In this paper we explore a variety of tests for the need for transformation, arguing that the Cox model is so ubiquitous that it should be considered as the default model, to be discarded only if there is good evidence against the model assumptions. Since fitting an alternative transformation model is more complicated than fitting the Cox model, especially as procedures are not yet incorporated in standard software, we focus mainly on tests which require a Cox fit only. A score test is derived, and we also consider performance of omnibus goodness-of-fit tests based on Schoenfeld residuals. These tests can be extended to compare different transformation models. In addition we explore the consequences of fitting a misspecified Cox model to data generated under a true transformation model. Data on survival of 1043 leukaemia patients are used for illustration.     

Effect of tryptophan and of glucose on exercise capacity of horses

We hypothesized that central fatigue may have a role in limitingthe endurance capacity of horses. Therefore, we tested the effect ofinfusing tryptophan and/or glucose on endurance time and plasmaconcentrations of free tryptophan and other substrates thought toaffect tryptophan uptake into the brain of seven mares (3-4 yr ofage, 353-435 kg) that ran on a treadmill at 50% of maximalO₂ consumption to fatigue. Withuse of a counterbalanced crossover design, the horses were infused withtryptophan (100 mg/kg in saline solution) or a similar volume of salinesolution (placebo) before exercise. During exercise, horses receivedinfusions of glucose (2 g/min, 50% wt/vol) or a similar volume ofsaline. Thus the treatments were 1)tryptophan and glucose (T & G), 2) tryptophan and placebo (T & P), 3)placebo and glucose (P & G), and 4)placebo and placebo (P & P). Mean heart rate, hematocrit, andconcentration of plasma total solids before and during exercise weresimilar for all trials. Mean time to exhaustion was reduced (P < 0.05) for T & P and T & Gcompared with P & P [86.1 ± 6.9 and 87.1 ± 6.8 vs. 102.3 ± 10.3 (SE) min], whereas endurance for P & G(122.4 ± 11.9 min) was greater than for all other trials (P < 0.05). Compared withnontryptophan trials, during the tryptophan trials plasma prolactinincreased (P < 0.05) nearlythreefold before exercise and almost twofold early in exercise. Muscleglycogen concentrations were reduced(P < 0.05) below preexercise values in the P & G and P & P trials only. However, glucose infusions (P & G)did not affect (P > 0.05)concentrations of plasma free fatty acids or ratios of branched-chainamino acids to free tryptophan. In conclusion, tryptophan infusionreduced endurance time, which was consistent with the central fatiguehypothesis. The failure of glucose infusion to alleviate the effects oftryptophan and the absence of significant muscle glycogen reduction inthe tryptophan trials suggest that the early onset of fatigue in thetryptophan trials is not due to a lack of readily available substrate.

     

Cell Cycle Duration in the Root Meristem of Sonoran Desert Cactaceae as Estimated by Cell-flow and Rate-of-cell-production Methods

Slow rates of cactus growth in the Sonoran Desert and high productivityof some Cactaceae under cultivation suggest that relativelylow growth rates are not the consequence of a long cell divisioncycle but of short optimal periods for growth and adverse environmentalfactors. To verify this hypothesis, the duration of the celldivision cycle (T)in the root apical meristem of seedlings ofthree sympatric species from the Sonoran Desert [Ferocactuspeninsulae(F. A. C. Weber) Britton & Rose ‘Townsendianus’(Britton & Rose) N. P. Taylor, stat. nov.,Stenocereus gummosus(Engelm.)Gibson & Horak andPachycereus pringlei(S. Watson) Britton& Rose] was estimated with the rate-of-cell-production (RCP)and the cell-flow (colchicine) methods. Both methods were appliedduring the steady-state growth phase, which was relatively shortin the first two species because of the determinate patternof root growth. The RCP method permitted estimation ofTin eachroot individually. Durations of the cell division cycle wereinversely proportional to the rate of root growth (r²rangedfrom 0.42 to 0.88,P<0.05).T,determined by the cell-flow method,ranged from 14.4 to 19.3 h in these species and was within thesame range asTdetermined by the RCP method. The averageTdeterminedby the RCP method was 67 to 75% of that determined by the cell-flowmethod. Results obtained with both methods are compared andanalysed. The proposed hypothesis appears to be correct, indicatingthat these species can be more productive under cultivationthan in the wild due to the relatively short duration of thecell division cycle. Adaptive features of these findings arealso considered.Copyright 1998 Annals of Botany Company Cactaceae, cell division cycle, root growth, root meristem, Sonoran Desert     

In vitro and in vivo wound healing-promoting activities of beta-lapachone

Impaired wound healing is a serious problem for diabetic patients. Wound healing is a complex process that requires the cooperation of many cell types, including keratinocytes, fibroblasts, endothelial cells, and macrophages. β-Lapachone, a natural compound extracted from the bark of the lapacho tree (Tabebuia avellanedae), is well known for its antitumor, antiinflammatory, and antineoplastic effects at different concentrations and conditions, but its effects on wound healing have not been studied. The purpose of the present study was to investigate the effects of β-lapachone on wound healing and its underlying mechanism. In the present study, we demonstrated that a low dose of β-lapachone enhanced the proliferation in several cells, facilitated the migration of mouse 3T3 fibroblasts and human endothelial EAhy926 cells through different MAPK signaling pathways, and accelerated scrape-wound healing in vitro. Application of ointment with or without β-lapachone to a punched wound in normal and diabetic (db/db) mice showed that the healing process was faster in β-lapachone-treated animals than in those treated with vehicle only. In addition, β-lapachone induced macrophages to release VEGF and EGF, which are beneficial for growth of many cells. Our results showed that β-lapachone can increase cell proliferation, including keratinocytes, fibroblasts, and endothelial cells, and migration of fibroblasts and endothelial cells and thus accelerate wound healing. Therefore, we suggest that β-lapachone may have potential for therapeutic use for wound healing. cell proliferation; mitogen-activated protein kinase signaling pathways     

A Flexible Alternative to the Cox Proportional Hazards Model for Assessing the Prognostic Accuracy of Hospice Patient Survival

Prognostic models are often used to estimate the length of patient survival. The Cox proportional hazards model has traditionally been applied to assess the accuracy of prognostic models. However, it may be suboptimal due to the inflexibility to model the baseline survival function and when the proportional hazards assumption is violated. The aim of this study was to use internal validation to compare the predictive power of a flexible Royston-Parmar family of survival functions with the Cox proportional hazards model. We applied the Palliative Performance Scale on a dataset of 590 hospice patients at the time of hospice admission. The retrospective data were obtained from the Lifepath Hospice and Palliative Care center in Hillsborough County, Florida, USA. The criteria used to evaluate and compare the models'' predictive performance were the explained variation statistic R², scaled Brier score, and the discrimination slope. The explained variation statistic demonstrated that overall the Royston-Parmar family of survival functions provided a better fit (R² = 0.298; 95% CI: 0.236–0.358) than the Cox model (R² = 0.156; 95% CI: 0.111–0.203). The scaled Brier scores and discrimination slopes were consistently higher under the Royston-Parmar model. Researchers involved in prognosticating patient survival are encouraged to consider the Royston-Parmar model as an alternative to Cox.     

Anthropological consideration on prevalence and fitness of β C and β S genotypes in Burkina Faso (a survey in the public schools)

Objective: To study the incidence of hemoglobinopathies (Hb C and Hb S) we have examined 15,367 students, aged 11.4+/-4.64 years (median 11; range 1–26), living in Burkina Faso (12,019 were students of 23 public schools of Ouagadougou and 3348 students of 7 public schools situated in six villages about 12–35 Km from Ouagadougou). Results: In all schools, we found a prevalence of females (54.2%) over males (45.8%) with a high incidence of Hb AC (19.16%), over Hb AS (8.35%). As expected, the gene frequency of β C in the schools of Ouagadougou was 0.112+/-0.015 and of β S 0.049+/-0.012, and this was similar in village schools (0.128+/-0.010 and 0.049+/-0.09 respectively), irrespective of town latitude. In all groups studied, β S and β C gene frequencies were age dependent since the advantage of HbS carriers in a malarial region is prevalently expressed in the first years of life. In fact, β C gene frequency increases, and β S decreases with age. The Mossi, living mainly in Ouagadougou, show a gene frequency which is similar to the Bissa ethnic groups, where the β C gene frequencies (0.116 and 0.118) are higher than the β S (0.049 and 0.044 respectively). On the contrary in the Peuhl ethnic group β C and β S gene frequencies (0.049 and 0.049) are the same, while in the Yorouba ethnic group immigrated from Nigeria β S gene frequency is higher (0.117) than the β C (0.068), showing that different gene frequencies are found in different ethnic groups. Conclusion: The difference in β S and β C genes frequencies (0.049 and 0.116) found in comparison with a previously performed study (Labie et al, 1984) in the same region (0.03 for the β S gene and 0.14 for the β C gene), may be due to migration flow of β S genes from the near Sahel region or to changes in life expectancy of children with β C and β S phenotypes. Moreover, the high percentage of SC (1,06%) and SS (0.188%) individuals detected with the present screening, compared with the lower percentages found by Labie et al in 1984, suggest that a modified environment can play an important role in reducing the morbility and lethality of β S genotype. However, even today the number of students affected by HbSC and Hb SS attending the schools remains low. Stricter controls of correlated diseases are necessary to guarantee healthier life conditions.     

Analysis of Time to Event Outcomes in Randomized Controlled Trials by Generalized Additive Models

Background

Randomized Controlled Trials almost invariably utilize the hazard ratio calculated with a Cox proportional hazard model as a treatment efficacy measure. Despite the widespread adoption of HRs, these provide a limited understanding of the treatment effect and may even provide a biased estimate when the assumption of proportional hazards in the Cox model is not verified by the trial data. Additional treatment effect measures on the survival probability or the time scale may be used to supplement HRs but a framework for the simultaneous generation of these measures is lacking.

Methods

By splitting follow-up time at the nodes of a Gauss Lobatto numerical quadrature rule, techniques for Poisson Generalized Additive Models (PGAM) can be adopted for flexible hazard modeling. Straightforward simulation post-estimation transforms PGAM estimates for the log hazard into estimates of the survival function. These in turn were used to calculate relative and absolute risks or even differences in restricted mean survival time between treatment arms. We illustrate our approach with extensive simulations and in two trials: IPASS (in which the proportionality of hazards was violated) and HEMO a long duration study conducted under evolving standards of care on a heterogeneous patient population.

Findings

PGAM can generate estimates of the survival function and the hazard ratio that are essentially identical to those obtained by Kaplan Meier curve analysis and the Cox model. PGAMs can simultaneously provide multiple measures of treatment efficacy after a single data pass. Furthermore, supported unadjusted (overall treatment effect) but also subgroup and adjusted analyses, while incorporating multiple time scales and accounting for non-proportional hazards in survival data.

Conclusions

By augmenting the HR conventionally reported, PGAMs have the potential to support the inferential goals of multiple stakeholders involved in the evaluation and appraisal of clinical trial results under proportional and non-proportional hazards.     

Effect of photoinhibition on algal photosynthesis: a dynamic model

Recent evidence from algal physiology and molecular biologyconfirms that photoinhibition is directly related to D1 proteindamage and recovery, and D1 protein damage leads to a decreasein electron transfer or an increase in turnover time of theelectron transfer chain. In this study, the turnover time ofthe electron transfer chain is defined as a function of therelative concentration of D1 protein in reaction centre II andthe photoinhibition processes due to D1 protein degradationare incorporated into a model of photosynthesis, initiated byDubinsky et al. (Plant Cell Physiol., 27, 1335–1349, 1986)and developed by Sakshaug et al. (Limnol. Oceanogr., 34, 198–205,1989). D1 protein damage is assumed to be both light and D1protein concentration dependent, and to be proportional to thecross-section of PSII (     

ON SOME SPECIES OF PALAEONEILO HALL & WHITFIELD, 1869, FROM THE JURASSIC ROCKS OF HABO HILL, DISTRICT KUTCH (GUJARAT)

Three species of Palaeoneilo Hall & Whitfield, namely P.belaeensis Cox, P. indicus Cox and P. rajnathi n. sp., are describedfrom the Jurassic rocks of Habo Hill in Kutch. The right, valveof belaeensis has been discovered. Its previous record fromthe Bathonian (Middle Jurassic) appears to be erroneous. Bothbelaeensis and indicus were unknown from the present area. (Received 11 November 1983;     

A Simple Method for Making a Vibrating Probe System

A simple and easy method for making the vibrating probe systemfirst developed by Jaffe and Nuccitelli [(1974) J. Cell Biol.63: 614] is described. The best performance of the probe insea water was 30.7 µA cm^-2 per one µV output, 0.4µA cm^-2 peak-to-peak output-noise and negligible output-driftat 30-µm vibrating amplitude and 3-s amplifier time constant.Sample applications were made for both freshwater and marinealgae. (Received March 22, 1984; Accepted May 28, 1984)     

Purification and Characterization of Rice Embryo BAPAase (Benzoyl-L-arginine p-nitroanilide Hydrolase)

Benzoyl-L-arginine p-nitroanilide hydrolase (BAPAase), whichhas both endopeptidase and carboxypeptidase activity towardthe Arg-X or Lys-X bond of small peptides [Shibata and Doi (1984)Plant & Cell Physiol. 25: 1421], was purified from riceembryos by ammonium sulfate and polymin fractionations and byion exchange, gel exclusion and hydrophobic chromatographies.The purified enzyme was homogeneous when analyzed by polyacrylamidegel electrophoresis. It was unstable in the absence of surface-activereagents such as Triton X-100. Maximum activity for benzoyl-Largininep-nitroanilide (L-BAPA) or carboxypeptidase activity towardbutoxycarbonyl-Gly-Lys-Leu was obtained at pH 9.0. L-BAPA athigh concentrations inhibited the enzyme's activity. Di-isopropylphosphofluoridate, N-tosyl-L-lysine chloromethyl ketone, leupeptinand antipain, which are specific inhibitors of trypsin, inhibitedBAPAase activity, but soybean and rice bran trypsin inhibitorhad no effect on it. Sulfhydryl reagents strongly inhibitedthe BAPAase activity. (Received May 26, 1984; Accepted August 29, 1984)     

A Mathematical Model to Utilize the Logistic Function in Germination and Seedling Growth

Several models have been proposed to describe germination rates,but most are limited in statistical analysis and biologicalmeaning of indices. Therefore, a mathematical model is proposedto utilize the logistic function. The function was defined asan overall response including time, temperature, and the interactionbetween time and temperature. Cumulative germination percentagesover time were used to develop the model. Germination tests were conducted on indiangrass (Sorghastrumnutans (L.) Nash) strain ‘IG-2C-F1’, at constanttemperatures of 9, 12, 15, 20, 25, and 30 °C. The functionfitted the observed data over six temperatures at r² = 0.99.Time to reach 10% of final germination (Gt₁₀) increased from2.5 d at 30 °C to 44.0 d at 9 °C, and Gt₅₀ (time toreach 50% of final germination) increased from 3.6 d at 30 °Cto 53.8 d at 9 °C. True germination rate (% d^–1) foreach temperature was maximum at Gt₅₀. A linear model of 1/Gt₅₀versus temperature was used to estimate the base temperatureof 8.3 °C for germination. An Arrhenius plot indicated achange occurred between 20 °C and 25 °C for temperatureresponse of germination. Published data on hypocotyl growthof Cucumis melo L. were recalculated using the model. Absolutegrowth rates showed a temperature response similar to the publishedweighted-mean elongation rates. Base temperature for hypocotylgrowth of C. melo was estimated as 8.8 °C. The proposedmodel proved to be useful in calculating and interpreting germinationand growth kinetics. Key words: Indiangrass, Sorghastrum nutans (L.) Nash, Germination rate, Threshold temperature, Arrhenius plot, Growth rate, Cucumis melo L     

Observation of multiple folding pathways of beta-hairpin trpzip2 from independent continuous folding trajectories

Motivation: After 10-year investigations, the folding mechanismsof β-hairpins are still under debate. Experiments stronglysupport zip-out pathway, while most simulations prefer the hydrophobiccollapse model (including middle-out and zip-in pathways). Inthis article, we show that all pathways can occur during thefolding of β-hairpins but with different probabilities.The zip-out pathway is the most probable one. This is in agreementwith the experimental results. We came to our conclusions by38 100-ns room-temperature all-atom molecular dynamics simulationsof the β-hairpin trpzip2. Our results may help to clarifythe inconsistencies in the current pictures of β-hairpinfolding mechanisms. Contact: yxiao{at}mail.hust.edu.cn Supplementary information: Supplementary data are availableat Bioinformatics online. Associate Editor: Anna Tramontano     

Quantal Response of Seed Germination in Seven Genera of Cruciferae to White Light of Varying Photon Flux Density and Photoperiod

The response of the germination of seeds of Barbarea vema (Mill.)Aschers, Brassica chinensis L., Brassica juncea (L.) Czern.& Coss., Brassica oleracea L. var. gongylodes L., Camelinasaliva (L.) Crantz, Eruca saliva Mill., Lepidium sativum L.,Nasturtium officinale R. Br., and Rorippa palustris (L.) Besserto white fluorescent light of different photon flux densitiesapplied for different daily durations in a diurnal alternatingtemperature regime of 20 °C/30 °C (16 h/8 h) was quantifiedby linear relations between probit percentage germination andthe logarithm of photon dose, the product of photon flux densityand duration. The low energy reaction, in which increasing dosepromotes germination, was detected in all the seed populationsbut in Barbarea vema and Brassica Juncea the lowest photon doseapplied (10^–5–2 and 10^{–5 7} mol m^–2 d^–1,respectively) was sufficient to saturate the response. Comparisons,where possible, between photoperiods demonstrated reciprocity,i.e. germination was proportional to photon dose irrespectiveof photoperiod, for the low energy reaction in Brassica oleracea(1 min d^–1 to 1 h d^–1), Camelina saliva (1 min d^–1to 8 h d^–1), Eruca saliva (1 min d^–1 to 24 h d^–1),Lepidium sativum (I min d^–1 to 8 h d^–1) and Rorippapalustris (1 min d^–1 to 8 h d^–1), but not in Brassicachinensis and Nasturtium officinale. The high irradiance reaction,in which increasing dose inhibits germination, was detectedin Barbarea vema, Brassica chinensis, Brassica juncea, Brassicaoleracea, and Camelina saliva. The minimum dose at which inhibitionwas detected was lO^–0–3 mol m^–2 d^–1.These results are discussed in the context of devising optimallight regimes for laboratory tests intended to maximize germination The response of germination to photon dose was also quantifiedwith 3 x 10^–4 M GA₂, co-applied (Brassica chinensis, Camelinasaliva, and Lepidium sativum) and with 2 x 10^–2 M potassiumnitrate co-applied (Brassica chinensis). In the latter casepotassium nitrate had no effect in the dark and inhibited germinationin the light, but GA₂, promoted germination substantially inall three species. Variation amongst seeds in the minimum photondose required to stimulate germination was not affected by co-applicationof GA₂, in Brassica chinensis and Camelina saliva, whereas seedsof Lepidium salivum showed a narrower distribution of sensitivitiesto the low energy reaction in the presence of GA₂ Barbarea vema (Mill.) Aschers, Brassica chinensis L., Brassica juncea (L.) Czern. & Coss., Brassica oleracea L. var. gongylodes L., Camelina saliva (L.) Crantz, Eruca saliva Mill., Lepidium satiaum L., Nasturtium officinale R. Br., Rorippa palustris (L.) Besser, Cruciferae, light, gibberellic acid, seed germination, seed dormancy     

Patch leaving decision rules and the Marginal Value Theorem: an experimental analysis and a simulation model

The patch exploitation strategy of females of the insect parasitoid Trichogramma brassicae was studied on patches containing different proportions of hosts that were previously attacked by conspecificfemales. On average, T. brassicae females spent more time onpatches of higher quality, and all patches were reduced tothe same level of profitability before being left. This appearedto be in accordance to the optimal predictions of the CharnovMarginal Value Theorem. The proximate leaving mechanisms involvedwere analyzed by means of a Cox proportional hazards model.Each oviposition in a healthy host appeared to have an incremental influence on the patch residence time, whereas each rejectionof a healthy host or of a host that was previously attackedby the same female (i.e., self-superparasitism) had a decrementaleffect. These patch leaving mechanisms did not change accordingto the quality of the patch the females were exploiting. AMonte Carlo simulation was developed around the results of the Cox regression model. The results suggest that this set of patchleaving rules seems to provide the females with a sufficientway to reach the predictions of the Charnov model. Among thedifferent mechanisms involved, the incremental effect associatedwith each oviposition in a healthy host appeared to play the most important role. The relationship between the proximatemechanistic rules adopted by the females and the ultimate predictionof the Charnov model is discussed.     

A novel phytoplankton chlorophyll technique: toward automated analysis

Interest in the <1 µm picoplankton fraction of naturalwaters, especially the oligotrophic oceans, has generated atrend toward the use of smaller and smaller porosity membranefilters for particulate analyses. The controversy concerningthe suitability of glass fiber versus membrane filters for retainingchlorophyll-containing parlicles is re-examined and the inadequaciesof filtration discussed. Previous comparisons in the literaturefail to resolve this controversy as tests were performed inwaters of >1.0 µm/l chlorophyll. A novel phytoplanktonchlorophyll technique for fresh and marine waters is describedwhich eliminates the need to concentrate plankton on filters.Two commercially available instruments have been modified topermit fluorescence measurements of whole water extracts. Thelower limit of detection is 0.1 µg/l in situ concentrationor 0.02 µg/l extract concentration. The technique lendsitself well to automation with particular applications to insitu fluorometry, flow cytometry and continuous chlorophylldeterminations. ^*This paper is the result of a study made at the Group for AquaticPrimary Productivity (GAP), Second International Workshop heldat the National Oceanographic Institute, Haifa, Israel in April–May1984.     

Cause‐Specific Cumulative Incidence Estimation and the Fine and Gray Model Under Both Left Truncation and Right Censoring

Summary The standard estimator for the cause‐specific cumulative incidence function in a competing risks setting with left truncated and/or right censored data can be written in two alternative forms. One is a weighted empirical cumulative distribution function and the other a product‐limit estimator. This equivalence suggests an alternative view of the analysis of time‐to‐event data with left truncation and right censoring: individuals who are still at risk or experienced an earlier competing event receive weights from the censoring and truncation mechanisms. As a consequence, inference on the cumulative scale can be performed using weighted versions of standard procedures. This holds for estimation of the cause‐specific cumulative incidence function as well as for estimation of the regression parameters in the Fine and Gray proportional subdistribution hazards model. We show that, with the appropriate filtration, a martingale property holds that allows deriving asymptotic results for the proportional subdistribution hazards model in the same way as for the standard Cox proportional hazards model. Estimation of the cause‐specific cumulative incidence function and regression on the subdistribution hazard can be performed using standard software for survival analysis if the software allows for inclusion of time‐dependent weights. We show the implementation in the R statistical package. The proportional subdistribution hazards model is used to investigate the effect of calendar period as a deterministic external time varying covariate, which can be seen as a special case of left truncation, on AIDS related and non‐AIDS related cumulative mortality.     

Adhesion Pattern and Prognosis Studies of T4N0M0 Colorectal Cancer Following En Bloc Multivisceral Resection: Evaluation of T4 Subclassification

In current TNM stage system, T4 lesions represent a complex group and should be considered to further optimize the classification. This study evaluates the significance of adhesion pattern in T4 subclassification based on prognostic analysis of T4N0M0 colorectal cancer following en bloc multivisceral resection (MVR). Prospectively collected data (1992–2004) were analyzed for 278 patients with stage T4N0M0 lesions following MVR for colorectal cancer. Patients were divided into inflammatory adhesion (IA) and malignant invasion (MI) groups based on adhesion to adjacent organs. Survival was evaluated by Kaplan–Meier and Cox proportional hazards regression analyses. MI was detected in 249 of 460 (54.1%) resected organs and in 159 of 287 (55.40%) patients undergoing MVR. Compared with IA group, patients in MI group showed no significant difference in clinicopathological data except tumor differentiation (P = 0.0376). Cox proportional hazards regression showed that MI was independently associated with overall survival among both colon (HR = 2.028; P = 0.0001) and rectal (HR = 0.451; P = 0.0002) cancer patients. Kaplan–Meier analysis showed that MI patients had a significantly higher MVR compared with IA patients (colon cancer: P = 0.0018; rectal cancer: P = 0.0116). In conclusion, MI was validated as an adverse prognostic factor for stage T4N0M0 colorectal cancer following MVR suggesting that it may be classified as a T4-subgroup in order to reinforce practice guidelines.