Modeling Genetic and Environmental Factors to Increase Heritability and Ease the Identification of Candidate Genes for Birth Weight: A Twin Study

Heritability estimates of birth weight have been inconsistent. Possible explanations are heritability changes during gestational age or the influence of covariates (e.g. chorionicity). The aim of this study was to model birth weights of twins across gestational age and to quantify the genetic and environmental components. We intended to reduce the common environmental variance to increase heritability and thereby the chance of identifying candidate genes influencing the genetic variance of birth weight. Perinatal data were obtained from 4232 live-born twin pairs from the East Flanders Prospective Twin Survey, Belgium. Heritability of birth weights across gestational ages was estimated using a non-linear multivariate Gaussian regression with covariates in the means model and in covariance structure. Maternal, twin-specific, and placental factors were considered as covariates. Heritability of birth weight decreased during gestation from 25 to 42 weeks. However, adjusting for covariates increased the heritability over this time period, with the highest heritability for first-born twins of multipara with separate placentas, who were staying alive (from 52% at 25 weeks to 30% at 42 weeks). Twin-specific factors revealed latent genetic components, whereas placental factors explained common and unique environmental factors. The number of placentas and site of the insertion of the umbilical cord masked the effect of chorionicity. Modeling genetic and environmental factors leads to a better estimate of their role in growth during gestation. For birth weight, mainly environmental factors were explained, resulting in an increase of the heritability and thereby the chance of finding genes influencing birth weight in linkage and association studies.
Introduction
Birth weight is a complex trait in which both environmental and genetic factors are involved. Low birth weight is not only associated with higher neonatal morbidity and mortality (Kramer et al. 2005), but also with post-natal psychopathology (Wichers et al. 2002). It is even stated that birth weight is a causal risk factor for child problem behavior and that the effects may well extend into adulthood (van Os et al. 2001).
Heritability estimates for birth weight using twin pairs, based on structural equation modeling or study of the offspring of twins, have given inconsistent results ranging from 10 to 40% (Baird et al. 2001; Clausson et al. 2000; Hur et al. 2005; Luciano et al. 2004; Magnus et al. 2001; van Baal and Boomsma 1998; van Dommelen et al. 2004; Vlietinck et al. 1989; Whitfield et al. 2001). The relatively low heritability estimates may be explained by unequal sharing of nutrients or unequal placental blood supply to the twin pair (Machin 1996). Vlietinck et.al. (Vlietinck et al. 1989) revealed that chorionicity explained 12% of the variance of birth weight with DC twins having a significantly higher similarity than MC twins. Random environment explained 23%, additive genetic factors 22.5%, and maternal age 0.7% of the total variance. But, the largest source of variation on birth weight in this study was gestational age (42%). Until now however, no study has focused on the change of variance of birth weight during gestation. We have shown that this variance increases during gestation (Gielen et al. 2007). As a consequent, heritability of birth weight may change over gestational age or different covariates may explain different parts of the variance-covariance structure. For example, it is more likely that at 37 weeks of gestation, nutrient factors (environmental factor) will explain a larger part of the covariance matrix as compared to 13 weeks of gestation.
The advantage of twin studies, is that the total variance can be split up into genetic (A), shared or common environmental (C) and unique environmental (residual, E) components, enabling an accurate estimation of heritability (= genetic variance/total variance) (Boomsma et al. 2002). The degree of heritability determines the power to localize and identify the genes involved (Snieder et al. 2003). For linkage and association studies a high degree of heritability is preferable. Since heritability is the proportion of phenotypic variation of a trait that can be attributed to genetic variation (Boomsma et al. 2002), reducing environmental and residual components will give an increase of the heritability. The latter can be achieved by adjusting for covariates that explain part of the environmental factors in the analysis. Additionally, a covariate may also explain part of the genetic variance and as a result this may provide an indication for finding genes for this covariate, but of course the drawback is that with this covariate in the model the success of finding the latent gene (s) will be reduced. Consequently, it is mandatory to determine how each covariate influences the genetic, common environmental and residual factors of the variance. Covariates that influence birth weight of twins are gestational age, maternal factors (parity and maternal age), twin specific factors (zygosity, sex, birth order), and placental factors (chorionicity, fusion of placentas and insertion of the umbilical cord) (Gielen et al. 2007).
The aim of this twin study was to model birth weight across gestational ages and to quantify the genetic and environmental components in order to explain the effects of covariates on the variance of birth weight. We hypothesize that heritability changes during gestation and that covariates mainly explain the common environmental and residual factors of the variance. By entering these covariates in the means model and in the covariance matrix, heritability will increase and, as a consequence, the power to find genes by linkage and association studies will increase, although the effects of each covariate on the genetic and environmental component may be different.
Materials and methods
Subjects
The study sample consisted of live born twin pairs selected from the East Flanders Prospective Twin Survey (EFPTS), Belgium (Loos et al. 1998). Between July 1964 and the end of December 2002, the EFPTS registered 6315 twin pairs who met the World Health Organization criteria for live born infants (birth weight ≥ 500 grams or gestational age ≥ 22 weeks, if birth weight unknown). The vast majority consisted of twins of Caucasian origin. Methods of data collection have previously been described in detail (Gielen et al. 2006; Gielen et al. 2007; Loos et al. 2005). Briefly, a defined set of obstetric and perinatal data were recorded (including gestational age, mode of conception, maternal age, birth order, parity, sex and neonatal survival) and placentas were examined within 24 hours after delivery according to a standardized protocol. Based on the site of the cord insertion two groups were distinguished: (1) central insertion (central and eccentric insertions), and (2) peripheral insertion. Gestational age was reported by the obstetrician, based on the last menstruation or a first trimester ultrasound investigation, and was calculated as the number of completed weeks of pregnancy. Zygosity was determined through sequential analysis based on sex, fetal membranes, umbilical cord blood groups (ABO, Rh, CcDEe, MNSs, Duffy, Kell), placental alkaline phosphatase (Decorte et al. 1990; Vlietinck 1986), and, since 1982, DNA fingerprints. Zygosity and chorionicity were determined with an accuracy of 99%.
Twin pairs of whom one or both children were stillborn (205 pairs) or suffered from major congenital malformation (120 pairs) were excluded (Loos et al. 2001). Two MC pairs were excluded because they were recorded as being MC but at birth had two separate placentas. Twin pairs with missing values in one or more covariates for one or both twins (birth weight, gestational age, parity, chorionicity, mode of conception, unknown whether staying alive postnatally, maternal age, placental type and weight, site of insertion of the umbilical cord) were excluded (1756 pairs) (Gielen et al. 2007). Finally, 8464 twins (4232 pairs) with complete datasets were analyzed.
Statistical analysis
The birth weights of pregnancies of different gestational ages were analyzed using a non-linear multivariate Gaussian regression, MVN ((μ, Σ2) where μ is the means model and Σ2 is the covariance matrix.
Means model
Growth was best modeled by a logistic growth curve (non-linear multivariate Gaussian regression) in which the time of conception (gestational age minus two weeks) was chosen as time point zero ( where β1 t/m β3 are the coefficients of the logistic growth curve and Time is time since conception; unadjusted model) (Neale and McArdle 2000). Furthermore, the two first weeks of pregnancy (the time between the last menstrual period and conception) were set to be zero. The logistic growth curve was modified to allow a change of growth rate ( where β4 is the change in growth rate after the time point described by coefficient β5). The following covariates were considered: sex of the individual and of the co-twin, chorionicity, number of placentas, site of umbilical cord insertion, birth order, maternal age, mode of conception, parity, and neonatal death (Gielen et al. 2007). Because in 1964 all gestational ages were based on the last menstruation, while in 2002 almost all gestational ages were based on first trimester ultrasound and this might influence gestational age, we controlled for birth year. The covariates were consecutively added in the logistic growth curve,
where X represents possible covariates and their interactions included in the model and β represents the corresponding coefficients. Finally, members of different groups were allowed to have different growth rates after a certain point in time as shown in the following equation
where Z is a binary covariate (indicator for belonging to group Z) and β6 is the additional change in growth rate after the time point described by coefficient β7 for members of group Z. (adjusted model). Examples of how to use the estimates can be found elsewhere (Gielen et al. 2007).
Covariance matrix
The twin model assumes that the total phenotypic variance can be decomposed into additive genetic effects (A), dominance genetic effects (D), effects of common environment (C) and effects of unique environment (E) (Neale and Maes 2002). The common environmental variance is assumed to be the same for both the MZ and DZ twin pairs and will account for the intra pair correlation between twin pair weights. MZ twins are assumed to share the same genetic variance (A and D), whereas DZ twins share half of the additive genetic variance and a quarter of the dominance genetic variance. In the case of a covariance matrix containing an additive genetic effect, common environment, and unique environment (ACE), the covariance matrix is respectively  and  for the MZ and DZ pairs. Because former analyses showed that a model in which the variance allowed to change linearly during gestational age fitted best (Gielen et al. 2007), we allowed the variance and covariance to change over time (for example, A = eα1 + eα2Time, C = eδ1 + eδ2Time, and ln(E) = σ1 + σ2Time, where Time is gestational age, not time since conception).
First, models containing the ACE and ADE components were fitted separately. Both models were fitted with only gestational age in the mean regression (unadjusted model) and with covariates in the mean regression (adjusted model). The best fitting unadjusted and adjusted models were tested with the (co-)variance being allowed to change during gestation. Next, the covariates of the mean regression of the adjusted model were entered as moderators in the covariance matrix the same way the mean regression of the adjusted model was built. This model is called the full model (for example, ln(E) = σ1 + σ2 Time + σ3X where σ3 is the coefficient of covariate X), which corresponds to the method described by Shaun Purcell (Purcell 2002). In this way, the covariance of the first-born twin was allowed to differ from the second born twin. The full model will show how heritability, A/(A + C + E), depends on the representative state of the covariates in the model.
For example, let us assume a model in which genetic, common and unique environmental components are defined as
A = e7.403 × Time, where the intercept α1 is set to zero
C = e6.688 × Time, where the intercept δ1 is set to zero
In(E) = 5.4054 + 0.1435 × Time + 0.4324 × SB + 0.724 × ND − 0.163 × MP + 0.2441 × OPM, where SB is the second born, ND is neonatal death, MP is multiparity, and OPM is one placental mass. The heritability for a first-born twin with a separate placenta, staying alive after one week of birth, of a multiparous mother, in case of two separate placentas would be at 25 weeks
and at 40 weeks of gestation
Model selection
Models were compared with the Akaike information criterion (AIC) (Akaike 1973; Lindsey and Jones 1998). Because the modeling process is exploratory, the inference criterion used for comparing the models under consideration is their ability to fit the observed data; i.e. models are compared directly through their minimized minus log likelihood. When the numbers of parameters in models differ, they are penalized by adding the number of estimated parameters. Smaller values indicate more preferable models. This criterion allows direct comparison among models, which are not required to be nested.
Effect of each separate covariate on the genetic and environmental component
To explain the effect of each covariate on the (co-)variance of birth weight at different gestational ages, we removed a significant covariate completely from the full model (out of the mean regression and out of the covariance matrix) and compared the (co-)variances, so that the influence of that specific covariate became apparent.
All analyses were conducted with R (Ihaka and Gentleman 1996) version 2.0.1 using the twin library (Lindsey 2001).
Results
Heritability decreases during gestation
For both the unadjusted model with only gestational age in the mean regression, and the adjusted model with covariates in the mean regression, the ACE model with additive genetic (A), common environmental (C), and unique environmental (E) variances fitted best. Allowing the covariance to change over time improved the fit (Table 1).Table 1Model fit statistics of ACE and ADE models for the unadjusted model with only gestational age in the mean regression, the adjusted model with covariates in the mean regression, and the full model with covariates in the mean regression and in the covariance matrix. The best fitting model (lowest AIC) is presented in boldUnadjusted model Adjusted model−Log likelihooddfAIC−Log likelihoodDfAICACE61696.84845761703.8461350.49844161373.49ADE61727.45845761734.4561380.6844161403.6CE61711.88845861717.8861364.28844261386.28AE61727.45845861733.4561380.58844261402.58E62189.72845962194.7261826.93844361847.93ACE; A and C allowed to change over time61688.44845761695.4461343.61844161366.61Full modelACE; A and C allowed to change over time and covariates in covariance matrix61297.26843761324.26A: Additive genetic effects, D: Dominance genetic effects, C: Effects of common environment, E: Effects of unique environment
For both the unadjusted and adjusted ACE model, the common environmental variance was higher than the genetic variance. Although the variances of the adjusted model were lower, the proportions of the standardized variances of both models remained the same, and therefore heritability (standardized A-variance). Heritability decreased from 38% at 25 weeks to 15% at 42 weeks (Fig. 1). Allowing the covariance to change during gestation increased the genetic and common environmental variances from 25 to 42 weeks. The common environmental variance was only a little bit higher than the genetic variance, thereby increasing heritability at the end of gestation (from 36% at 25 weeks to 18% at 42 weeks). The model without an intercept for the genetic (α1) and common environmental variance (δ1) had a better fit, because it imposes these variance components to be zero at conception (Table 2).Fig. 1Genetic (A), common environmental (C) and unique environmental (E) variance and standardized variance of birth weight of the unadjusted (unadj) model and the adjusted model (adj). E-variance was allowed to change over time, but the A- and C-variance notTable 2Regression coefficients of the covariance matrix of the unadjusted model, the adjusted model, and the full modelACLog(E)Log(estimate)SELog(estimate)SEestimateSEUnadjustedbIntercept10.3700 0.173510.5800 0.11546.00550.3462Gestational age––0.14050.0094UnadjustedaIntercept0.0000–0.0000–6.30610.3468Gestational age6.89300.16046.9420.12500.13170.0094AdjustedbIntercept10.28000.185710.48000.12226.14360.3495Gestational age––0.13470.0094AdjustedaIntercept0.0000–0.0000–6.45520.3489Gestational age6.78300.17376.86000.12970.12560.0094Full modelIntercept0.0000–0.0000–5.40540.3963Gestational age7.04300.14216.68800.15710.14350.0103Second born0.43240.0642Neonatal death0.72400.1981Multiparity−0.16300.0491One placental mass0.24410.0636Unadjusted:model with only gestational age in the mean regressionAdjusted:model with covariates in the mean regressionFull model:model with covariates in the mean regression and in the covariance matrixThe unadjusted and adjusted models are presented witha and withoutb A and C allowed changing during gestation  and  where Time is gestational age and X represent the covariates with the corresponding estimates β.
Effect of covariates in the covariance matrix on heritability
The full model with also covariates in the covariance matrix was the best fitting model (Table 1; for estimates of the means model see Table 3). The genetic variance became larger than the common environmental variance (Fig. 3; Table 2). Not all covariates of the mean regression explained part of the covariance matrix (Table 2 and Table 3). The genetic and environmental variance were only influenced by gestation, the unique environmental variance by parity, birth order, neonatal survival and number of placentas (estimates in Table 2, path diagram in Fig. 2). Although these dichotomous covariates were only present in the unique environmental variance, heritability will nevertheless change, because heritability is the proportion of the total variance that can be explained by genetic variation.Table 3Regression coefficients of the full model with covariates in the mean regression and in the covariance matrixEstimateSEMeans modelCoefficients of the logistic growth curveb1 = saturation level at which growth stops3688.098084.9100b2 = growth rate5.80610.1762b3 = location parameter−0.18420.0068b4 = decrease in weight after inflection point0.95630.3017b5 = inflection pointa37.59340.3216b61 = decrease in weight for second bornb0.10540.0021b71 = inflection point for second borna30.72210.8399b62 = decrease in weight for neonatal deathb0.57330.1846b72 = inflection point for neonatal deatha29.00001.098b63= decrease in weight for MZ MCb1.14380.5059b73 = inflection point for MZ MCa37.78690.4227CovariatesMultiparity0.11650.0148Male0.14050.0118Birth year (form 0 [1964] to 38 [2002])0.00180.0007One placental mass−0.05250.0127Peripheral umbilical cord insertion−0.05930.0227Maternal age0.00460.0014Interaction termsBirth yeara Peripheral umbilical cord insertion−0.00270.0009Multiparitya Second born0.03170.0131aWeeks since conceptionbAfter inflection pointMZ MC = monozygotic monochorionic. Inflection point = weeks of gestational age since conception (Time). Equation: b1 t/m b7 coefficients, Z = second born, Neonatal death or MZ MC, X = covariates, and β = coefficient of covariatesFig. 2Partial path diagram of the full model shown for one twin only. For the estimates and interactions, see table 1 the adjusted model. BWT1 = Birth weight twin 1, Circle = latent variables, square = measured variables, triangle = definition variables in the means model, MZ MC = monozygotic monochorionic, A = additive genetic effects, C = effects of common environment, E = effects of unique environment
First-born twins of multipara with two separate placentas, who stay alive neonatally, have the lowest unique environmental variance (Table 2, Fig. 3: Lowest) and therefore the highest heritability (from 52% at 25 weeks to 30% at 42 weeks) (Fig. 4: Lowest). Allowing a dichotomous variable to be fixed to another representative state showed that the unique environmental variance remained lower than in the adjusted model, except for twins who died neonatally (Fig. 3). However, heritability was always higher in the unadjusted model than in the adjusted model (Fig. 4). When the covariates were chosen to represent second born twins of primipara with one placental mass and who died neonatally, the unique environmental variance became extremely high (Fig. 3: Highest) and heritability became lower than that of the adjusted model (Fig. 4: Highest).
Fig. 3Genetic (A), environmental (C) and residual (E) variance of birth weight of the adjusted (Adj) model and the full model (Full). Legend: Lowest: E-variance for a first-born twin of a multipara with two separate placentas who is staying alive. Primipara: E-variance for a first-born twin of a primipara with two separate placentas who is staying alive. One placenta: E-variance for a first-born twin of a multipara with one placental mass who is staying alive. Second born: E-variance for a second born twin of a multipara with two separate placentas who is staying alive. Neonatal death (ND): E-variance a first-born twin of a multipara with two separate placentas who died neonatally. Highest: E-variance for a second born twin of a primipara with one placental mass who died neonatallyFig. 4Heritability (standardized A-variance) of birth weight of the adjusted (Adj) and full model (Full) of Fig. 3. Legend: Lowest: Standardized E-variance for a first-born twin of a multipara with two separate placentas who is staying alive. Primipara: Standardized E-variance for a first-born twin of a primipara with two separate placentas who is staying alive. One placenta: Standardized E-variance for a first-born twin of a multipara with one placental mass who is staying alive. Second born: Standardized E-variance for a second born twin of a multipara with two separate placentas who is staying alive. Neonatal death (ND): Standardized E-variance a first-born twin of a multipara with two separate placentas who died neonatally. Highest: Standardized E-variance for a second born twin of a primipara with one placental mass who died neonatally
Effect of each separate covariate on the genetic and environmental component
Comparing the variances of the full model with and without a specific covariate showed that maternal factors (age and parity) had minor influence on the genetic variance, but no influence on heritability. Removing sex, birth order and neonatal survival gave an increase in genetic variance. However, only for sex, heritability increased if removed from the model. Removing the placental factors from the full means model resulted in a decrease of the genetic variance and a decrease of heritability. Insertion of the umbilical cord (unique for each twin) explained mainly the unique environmental variance, whereas number of placentas also explained common environment.
Chorionicity or other placental factors
Since chorionicity, number of placentas and insertion of the umbilical cord are related (MC twins always have one placenta and have more often a peripheral cord insertion), as an additional step we built a model in which we replaced cord insertion and number of placentas with chorionicity (chorionicity model). Although the AIC of the chorionicity model was higher, it roughly explained as much of the genetic and unique environmental variances but not of the common environmental variance (Fig. 5). Removing of either the two placental factors or chorionicity resulted in a decrease of genetic variance and decrease of heritability. The variances of the DC twins were comparable to the variances of two separate placentas, whereas variances of the MC twins were comparable to the variances of one placental mass (Fig. 5).
Fig. 5Effect of either placental factors (site of insertion of umbilical cord and number of placentas), or chorionicity on the variance and heritability of birth weight The curves represent the differences between the full model and the model without the specific covariate. The presented curves are the curves for the first born twin of a primipara, who is staying alive
Discussion
The aim of this study was to model birth weight at different gestational ages and quantify the genetic and environmental components. To our knowledge we are the first to show that heritability of birth weight seems to decrease during gestation, in our case from 38% at 25 weeks to 15% at 42 weeks. Entering the covariates of the mean regression as moderators in the covariance matrix, explained more of the common environmental variance than of the genetic variance. However, the unique environmental variance depended upon the representative state of the covariates and was lowest for first-born twins of multipara with two separate placentas, who stayed alive neonatally. Therefore these twins have the highest heritability: from 52% at 25 weeks to 30% at 42 weeks.
Part of the explanation for a decrease in heritability during gestation could be the increasing demand for nutrients and oxygen in twin gestation, especially in the third trimester (Loos 2001). After about 29 weeks of gestation, intrauterine growth of twins deviates from growth of singletons, (Loos et al. 2005; Luke et al. 1993; Soucie et al. 2006), resulting in a lower birth weight and placental weight (per twin) (Gielen et al. 2006; Loos et al. 2005; Pinar et al. 1996). In addition, placental factors can be involved. In twin pregnancies, a lower placental weight, monochorionicity, one placental mass (one placenta or two fused), and a peripheral cord insertion result in a lower birth weight (this study) (Gielen et al. 2007; Loos et al. 2001). Also, vascular anastomoses in MC twins result in higher discordancy rates (Machin et al. 1996). These placental factors can partly determine the (unequal) blood supply to the fetus and therefore the amount of nutrients and oxygen for each fetus, resulting in a higher degree of discordancy (Machin 1996) and low heritability estimates in twins in late gestation. In this study, first-born twins of multipara with two separate placentas, who stay alive, were genetically the most homogeneous group. This group also had the highest birth weights and placental weights (Gielen et al. 2006; Gielen et al. 2007), suggesting that they may have minimal environmental restriction and therefore the highest heritability.
Modeling common and unique environmental factors can increase heritability and therefore optimize finding genes responsible for the genetic variance in combined linkage and association studies. By partitioning the association effects into between twin pair and within twin pair components, true from false associations can be distinguished in combined linkage and association studies (Fulker et al. 1999; Posthuma et al. 2004). As a rule, the power in statistical analysis will increase as the sample size increases, if a quantitative trait locus (QTL) explains a larger part of the variance it is easier to find, and if multivariate models are used (Peeters 2005). For example, if a QTL effect explains 0.05 of the variance (α = 0.05 β = 0.80), with genetic factors explaining 20%, and common and unique environmental factors each explaining 40% of the variance, 6422 sib-pairs are needed. If genetic factors explain a larger part of the variance e.g. 40%, and common and unique environmental factors each explaining 30% of the variance, only 3751 sib-pairs are needed. If in the latter case the QTL effect is 0.10 even less sib-pairs (n = 1080) are needed. Lack of statistical power may also be a reason for the inconsistent results of linkage studies to identify quantitative trait loci (QTLs) for birth weight. Only three genome wide linkage studies in different populations have identified QTLs on chromosome 6 (Arya et al. 2006), 7 (Fradin et al. 2006), and 11 (Lindsay et al. 2002) (LOD > 3) with relative small sample sizes (less then 413 sibships (Fradin et al. 2006), (Lindsay et al. 2002) and 840 individuals from families (Arya et al. 2006)) and only two covariates taken into account (sex and gestational age).
By building a means model and then entering the significant covariates in the covariance matrix, we controlled for environmental variables correlated with the genetic effects on the trait (gene environment correlation = rGE) (Purcell 2002). All the covariates with a unique environmental component, besides gestational age, must either explain differences between twins or describe gene environment interactions (Purcell 2002; van der Sluis et al. 2006). Parity is common for both twins, but because of the interaction with birth order in the mean regression, parity explains part of the unique environmental variance.
Covariates also contain useful information in order to identify genes, which may be associated with the trait. In this study, only sex contained latent genetic information. For all other covariates used in this study, either heritability did not change, or environmental factors were explained with heritability depending on the representative state of the covariate. Since sex explained part of heritability, entering sex in the means model will mask genes that act differently according to sex and it is better to have these genes in the model instead of sex.
In the past, Vlietinck et al (1989) showed an effect of chorionicity, although fusion of the placentas and site of insertion of the umbilical cord was not taken into account. Our analyses showed that only by replacing the placental factors cord insertion and number of placentas with chorionicity, the effect of chorionicity became visible, although the model fitted worse. Chorionicity certainly plays a role in growth (in mean regression), but mainly after 40 weeks of gestation if fusion of the placentas and insertion of the umbilical cord are taken into account (Gielen et al. 2007). Consequently, in order to achieve a higher heritability by explaining environmental factors, number of placentas and site of the insertion of the umbilical cord are preferable above chorionicity.
The strength of our study lies in the large sample size, accurate zygosity and chorionicity determination. This results in a decrease of the unique environmental variance and therefore a better estimation of heritability of birth weight. The large sample size further offers the opportunity to determine heritability at different gestational ages, although twins might not be representative for singletons due to differences in growth restriction. On the other hand, in studies without MZ and DZ twins, no difference can be made between genetic and common environmental factors, resulting in an overestimation of the genetic factors with, in case of birth weight, heritability estimates up to 80% (Arya et al. 2006; Clausson et al. 2000; Magnus et al. 1984; Nance et al. 1983).
Information on parental ethnicity was not always available. However, it has been shown that the genetic variance of birth weight does not differ between populations in which the majority (>95%) is Caucasian and Asian (Hur et al. 2005), suggesting that the genetic variance might be equal for populations. Since a vast majority of the inhabitants of East Flanders are Caucasians, we assume that our results are not biased by ethnicity. Finally, parental weight and height were not available. We assume however no biased results, since both traits are highly heritable (Carmichael and McGue 1995; Preece 1996; Silventoinen et al. 2001). Because the aim of linkage and association studies is to identify loci or alleles responsible for birth weight, adjusting for genetic factors will reduce the chance of finding them as stated above. But also, because we studied birth weight of twin pairs, rather than offspring of twins, the genes implicated in our study relate to fetal rather than maternal effects (Luciano et al. 2004). Therefore, maternal genes will be seen as common environment (Luciano et al. 2004).
In conclusion, modeling genetic and environmental factors gives a better insight in factors influencing growth during gestation, and therefore birth weight. Heritability of birth weight changed during gestation with a decrease from 25 to 42 weeks with highest heritability for twins with the least environmental restriction (first-born twins of multipara with two separate placentas who stayed alive neonatally). For this complex trait, modeling the covariance matrix with covariates, mainly environmental factors were explained, resulting in an increase of the heritability and subsequently the chance of finding genes by linkage and association studies.

heritability
birth weight
twins
gestational age
variance.
growth curves


Origin of renal myofibroblasts in the model of unilateral ureter obstruction in the rat

Tubulo-interstitial fibrosis is a constant feature of chronic renal failure and it is suspected to contribute importantly to the deterioration of renal function. In the fibrotic kidney there exists, besides normal fibroblasts, a large population of myofibroblasts, which are supposedly responsible for the increased production of intercellular matrix. It has been proposed that myofibroblasts in chronic renal failure originate from the transformation of tubular cells via epithelial–mesenchymal transition (EMT) or from infiltration by bone marrow-derived precursors. Little attention has been paid to the possibility of a transformation of resident fibroblasts into myofibroblasts in renal fibrosis. Therefore we examined the fate of resident fibroblasts in the initial phase of renal fibrosis in the classical model of unilateral ureter obstruction (UUO) in the rat. Rats were perfusion-fixed on days 1, 2, 3 and 4 after ligature of the right ureter. Starting from 1 day of UUO an increasing expression of alpha-smooth muscle actin (αSMA) in resident fibroblasts was revealed by immunofluorescence and confirmed by the observation of bundles of microfilaments and webs of intermediate filaments in the electron microscope. Inversely, there was a decreased expression of 5′-nucleotidase (5′NT), a marker of renal cortical fibroblasts. The RER became more voluminous, suggesting an increased synthesis of matrix. Intercellular junctions, a characteristic feature of myofibroblasts, became more frequent. The mitotic activity in fibroblasts was strongly increased. Renal tubules underwent severe regressive changes but the cells retained their epithelial characteristics and there was no sign of EMT. In conclusion, after ureter ligature, resident peritubular fibroblasts proliferated and they showed progressive alterations, suggesting a transformation in myofibroblasts. Thus the resident fibroblasts likely play a central role in fibrosis in that model.
Introduction
Tubulo-interstitial fibrosis is a landmark of chronic renal failure, regardless of the underlying disease. It has been proposed that fibrosis is not only a response to tubular injury but that it may be the main driving force of nephron loss (Remuzzi and Bertani 1998; Zeisberg et al. 2001).
The basic aspects of fibrosis are similar in various organs. The increased accumulation of matrix is associated with a change of the phenotype of interstitial cells. Myofibroblasts are found in addition to or in the place of the quiescent fibroblasts of healthy tissues (Liu 2004; Desmouliere et al. 2005; Mucsi and Rosivall 2007). Compared to quiescent fibroblasts myofibroblasts show, besides their increase of the production of matrix and of the synthesis of matrix-degrading enzymes, structural characteristics, such as frequent intercellular contacts and a unique set of cytoskeletal proteins, the most distinctive being smooth muscle α-actin (αSMA) (Desmouliere et al. 2005;Hinz 2007; Hinz et al. 2007;Essawy et al. 1997).
As discussed in recent reviews myofibroblasts may have different origins, possibly depending on the organ and on the pathology (Desmouliere et al. 2005; Hinz et al. 2007). They may originate from the differentiation of resident fibroblasts, from bone-marrow-derived precursors which infiltrate the injured tissue or from transformation of epithelial cells, the so-called epithelial–mesenchymal transition (EMT).
The origin of myofibroblasts in the kidney has been the object of intensive research. There is evidence of a role of bone marrow-derived precursors in models of adriamycin nephropathy (Li et al. 2007) and of ischemia/reperfusion injury (Broekema et al. 2007). In contrast no significant contribution of such precursors to fibrosis was found in a murine model of unilateral ureter obstruction (UUO) (Roufosse et al. 2006). Numerous research reports and review papers discuss the role of EMT in renal fibrosis (Lan 2003; Iwano and Neilson 2004; Li et al. 2007; Mucsi and Rosivall 2007). In contrast, the possibility of a transformation of resident fibroblasts into myofibroblasts in renal fibrosis has hardly been considered. There is little information on the behavior and fate of resident fibroblasts in fibrosis in the kidney, in particular whether they proliferate and differentiate into myofibroblasts.
The aim of our present investigation was to study the morphology of cortical peritubular interstitium, with a focus on the resident fibroblasts, during the first 4 days following ureteral ligature in rats. UUO is a commonly used experimental model for inducing renal fibrosis and is furthermore of outmost clinical relevance. We choose to study the early events after ureteral ligature because in early stages the identification of cell types and their putative phenotypical changes might be better traceable than in advanced stages of fibrosis. We include in our study also the early effects of UUO on tubular epithelia.
We performed our investigation by light and electron microscopy, as well as immunofluorescence, and we included morphometric quantification of tissue components and analysis of changes in the proliferation rates of interstitial cells.
We demonstrate that resident fibroblasts progressively gain the phenotype of myofibroblasts and massively increase their rate of cell division during the first days after ureteral ligature. Our data suggest that resident peritubular fibroblasts transform to myofibroblsts and participate importantly in the development of renal fibrosis.
Methods
Experimental design
For induction of UUO male 20 Sprague–Dawley rats weighing 160 ± 3 g were anesthetized using isoflurane and the abdominal cavity was opened by a midline incision. The right ureter was tied with 4/0 silk 3–5 mm below the renal hilum. The rats of the control group (four rats) were also laparatomized, the ureter exposed, but no ligature was made. After closure of the abdomen the animals were returned to the cage with free access to standard lab chow and water ad libitum.
The experimental procedure conformed to the Institutional Guidelines of Experimental Animal Care and Use and was approved by the Veterinary office of the Kanton Zürich.
Tissue fixation
After 1, 2, 3 and 4 days of the operation, five ureter-ligated rats were fixed by vascular perfusion through the abdominal aorta as described previously (Dawson et al. 1989). The control group was perfused on day 4. The occluded renal pelvis was cut open just before perfusion to allow outflow of the fluid from the kidney. The fixative solution was composed of 3% paraformaldehyde, 0.01% glutardialdehyde and 0.05% picric acid in a 6:4 mixture of 0.1 M cacodylate buffer (pH 7.4, adjusted to 300 mOsm with sucrose) and 10% hydroxyethyl starch in saline (HAES steril TM; Fresenius AG, Germany). Five minutes after onset of the fixation both kidneys were removed.
Light and electron microscopy
From the unligated left and from the ligated right kidney large tissue blocks extending from the renal capsule to at least the upper third of the inner zone were immersed for at least 24 h in the fixative solution described above to which 1% glutardialdehyde was added. This tissue was then embedded into epoxy resin and used for light and electron microscopy. Semithin (1 μm) sections and ultrathin (80 nm) sections were cut with an ultramicrotome. Semithin sections were stained with 1% methylene blue and 1% azure II and were studied with a Polyvar microscope (Reichert Jung, Vienna, Austria). Ultrathin sections were postfixed with osmium tetroxyde and contrasted with uranyl acetate and studied with a CM100 Philips electron microscope.
Immunofluorescence microscopy
Coronal slices across the right and left kidney, comprising the cortex and the outer medulla were frozen in liquid propane, cooled with liquid nitrogen. From these slices, 2–3 μm thick sections were cut in a cryostat (Microm, Walldorf, Germany). The sections were pre-treated with 5% normal goat serum diluted in PBS with 1%BSA, which was also the diluent for all other antibodies. They were incubated at 4°C overnight in a humidified chamber with primary antibodies described in Table 1. Binding sites of the primary antibodies from rabbit origin were detected using Alexa 555-conjugated goat anti-rabbit (Invitrogen, Carlsbad, CA, USA) and the primary antibody from mouse origin was detected using Alexa 488-conjugated goat anti-mouse (Invitrogen). For chromatin staining 4,6-diamidino-2-phenylindole (DAPI; Sigma, St Louis, MO, USA) was added to the secondary antibodies. The slides were mounted in DAKO-glycergel (Dakopatts, Glostrup, Denmark) containing 2.5% of 1,4-diazabicyclo (2.2.2)octane (DABCO; Sigma, St Louis, MO, USA) as fading retardant.Table 1List of primary antibodiesPrimary antibodiesHostSourceS100A4RabbitDako, Glostrup, DenmarkEcto-5′nucleotidase (5′NT)RabbitM. Le HirEcto-5′nucleotidase (5′NT)MouseBD Biosciences, Franklin Lakes, NJ, USAAlpha smooth muscle actin (αSMA)MouseDako, Glostrup, DenmarkAlpha smooth muscle actin (αSMA)RabbitAbcam, Cambridge, UKRat MHC class IIMouseClone OX 6Phospho-S6-kinaseRabbitCell Signaling, Danvers, MA, USACollagen Type IMouseMD Biosciences, Zurich, SwitzerlandVimentinMouseChemicon, Temecula, CA, USA
Sections were studied by epifluorescence with a Polyvar microscope (Reichert Jung, Vienna, Austria), and digital images were acquired with a CCD camera.
Quantitative evaluation
In order to quantify changes in the structural composition of the renal cortex and to assess the mitotic rates of interstitial cells within this area, a morphometrical analysis was undertaken on sections of kidneys from three rats in each of the five experimental groups.
In 1 μm thick epon sections with a surface of approximately 2 × 4 mm, a randomly selected cortical area of approximately 1 mm2 was photographed at 100× magnification using a Leica DMRBE microscope (Leica Microsystems, Heerbrugg, Switzerland) connected to a Olympus SIS colorview camera (Olympus Soft imaging systems, Münster, Germany). The individual micrographs were merged to one large image and the exact size of the total image area was subsequently determined by analySIS software (Olympus Soft imaging systems, Münster, Germany).
The images were overlaid with a 15 × 15 grid to assign the histological structures covered by each of the 225 intersections to one of five categories, namely tubules, capillaries, interstitium, renal corpuscles and large vessels. The fractional areas of these histological structures were defined as the ratio of the number of category-specific intersections to the total number of intersections (225).
Furthermore, the total numbers of peritubular interstitial cells in the image were counted and the numbers of mitotic cells in the cortical interstitium were recorded. This allowed calculating the fraction of mitotic cells in relation to all interstitial cells. The identity of each mitotic cell in the cortical interstitium was attributed to one of the two categories “fibroblasts” or “mononuclear cells” (dendritic cells, infiltrating leucocytes) independently by two investigators. Mitotic cells that could not be assigned to one of these two categories or that were classified differently by the two investigators recorded as ‘non-identified’.
Statistics
Numerical data are expressed as mean values (n = 3) ± standard deviation (SD).
Results
Macroscopy
The ligature of the right ureter resulted invariably in each rat in a large expansion of the renal pelvis within the renal sinus and of the ureter above the ligature. The enlargement appeared more prominent at the first and second day after the operation than after the third and fourth day. The fluid contained in the expanded renal pelvis was clear up to the second day and appeared trouble and viscous the third and fourth day. In sham operated rats no expansion of the renal pelvis was observed.
Microscopy
Tubular epithelia
In control and contra-lateral kidneys the tubules were patent with rather homogeneous luminal diameter (Figs. 1a, 2a).Fig. 1Rat renal cortex of contra lateral (a, e) and of ureter ligated (b–d, f–h) kidneys; 1 μm epon sections. Overviews (a–d) show the dilatation of distal segments after 1 day, the collapse and shrinkage of predominantly proximal tubules after 2–4 days and the progressive enlargement of the interstitium. Higher magnifications (d–h) reveal the increasing complexity of interstitial cells; asterisk fibroblasts; D distal segment, P proximal tubule (cut tangentially in H); arrow fibroblast in prophase of mitosis, arrowhead mononuclear cell in mitosis. Bara–d ~100 μm, e–h ~10 μmFig. 2Proximal tubules in the cortex of a sham operated kidney (a) and of a ureter-ligated kidney on day 3 (b, c). 80 nm epon sections. In b and c the outer diameters of the tubules as well as the width of their lumina are decreased and the fractional area of nuclei in the epithelium is abnormally high. d Higher magnification of the area framed in c, revealing junctional complexes (arrowheads) and microvilli. Bars in a, b, c (same magnification): 10 μm; in d 1 μm
Starting from day 1 the most striking feature in tubules of kidneys with occluded ureter was the heterogeneity of tubular diameter.
Proximal tubules
After 1 day (Fig. 1b) most proximal tubular profiles appeared similar as in controls, only a few proximal tubular profiles were markedly smaller in diameter than in controls. As soon as after 2 days (Fig. 1c) the majority of proximal tubules displayed narrowed lumina and decreased outer diameters. That tubular shrinkage progressed through the 4 days of experiment (Figs. 1d, 2). The density of nuclei was strikingly increased in shrunken tubules (Fig. 2), thus, the decreased epithelial volume reflected cellular atrophy rather than cell death. The brush border remained well visible even after nearly complete collapse of the lumen (Figs. 1g, 2c, d). Although proximal and distal tubules in the cortex revealed severe regressive changes no disintegration of the epithelial layer was detected either by light or by electron microscopy. In all cases the proximal tubule were lined by a continuous epithelium showing ultra-structural evidence of polarity like microvilli at the luminal aspect and morphologically intact junctional complexes. We found no instance of disruption of the tubular basement membrane. The polarized expression of 5′NT at the luminal pole of all proximal tubules further evidenced the preservation of epithelial architecture in proximal tubules. Tubular cell death was not conspicuous in the cortex. Mitotic cells were rather frequently observed in proximal tubules from day 2 on. At the same time epithelial cells, including mitotic cells, were occasionally seen in the lumen, indicating some degree of anoikosis. From day 3 on an increasing number of proximal tubular profiles bound anti collagen 1-antibodies. Even in those profiles the individual epithelial cells were still recognizable.
Distal segments and collecting ducts
After 1 day dilation of the luminal diameters of most distal segments was eye-catching (Fig. 1b). Occasionally their lumen was filled with hyaline material. From day 2 on cellular hypotrophy associated with a collapsed lumen, as described above for proximal tubules, became apparent also in distal segments. At day 4 many distal profiles were massively shrunken and atrophic (Figs. 1d, h, 3). However, some profiles remained histologically almost normal up to day 4. The diameters of collecting duct profiles were dilated from day 1 on and their largely open lumina were strikingly prominent among the shrunken tubular profiles in the cortex at day 3 and 4 (Fig. 1c, d).Fig. 3Thick ascending limb in the cortex of a sham operated kidney (a) and of a ureter ligated-kidney on day 2 (b); 80 nm epon sections. In b the outer diameters of the tubule as well as the width of the lumen is decreased. c Higher magnification of the area framed in b, revealing junctional complexes (arrowheads) and microvilli. Bars in a, b (same magnification): 5 μm, in d 1 μm
Quantitative estimation of changes of cortical tubular volume and peritubular spaces
The fractional area of tubules, capillaries and the interstitium was assessed in 1 μm epon sections of perfusion fixed kidneys (Fig. 4). The epithelial shrinkage of most proximal tubules and of some distal tubules was reflected in significantly decreased fractional volume of tubules from day 2 (Fig.4).Fig. 4Quantitative analysis of the structural changes in rat renal cortex in response to UUO. The fractional area of the tubular system decreased while that of the interstitium increased continuously during the first 3 days after UUO. Tubular and interstitial areas differ significantly from control values from day 2 on. The fractional area of capillaries did not change significantly during the 4 days of UUO. Values are means ± SD, n = 3
The peritubular interstitial space is defined as the space between the tubular, glomerular and capillary basement membranes. After ligature of the ureter its fractional volume increased significantly from about 11% in control kidneys (day 0) to more than twice this value on days 3 and 4 of UUO (Figs. 1d, 4). The fractional volume of capillaries did not change significantly (Fig. 4).
Cortical peritubular interstitium
The major cellular constituents of the peritubular interstitium are peritubular fibroblasts (Dawson et al. 1989; Gandhi et al. 1990; Kaissling and Le Hir 1994; Kaissling et al. 1996) and dendritic cells (Kaissling and Le Hir 1994; Dong et al. 2005; Soos et al. 2006). In addition a few lymphocytes and rare granulocytes may be encountered (Kaissling and Le Hir 1994). The precise delimitation of the individual interstitial cells within the narrow interstitial space is difficult in conventional light microscopic preparations. Fixation by vascular perfusion resulted in unfolded intercellular spaces and in thin sections (80 nm and 1 μm epon sections, 3 μm cryostat sections) delimitation of the cytoplasm of individual interstitial cells is possible.
Phenotypical changes of peritubular fibroblasts
In control animals the morphology of fibroblasts (Fig. 1e) and dendritic cells was as described previously (Kaissling et al. 1996). In fibroblasts the nucleus was generally angular and surrounded by a sparse cytoplasmic rim, most organelles being located in the extended cytoplasmic processes (Figs. 1e; 5a). The latter showed connections with tubules and capillaries and to each other, as seen in electron microscopy (EM; Figs. 5a, 6b, c). Thereby fibroblasts establish a continuous framework within the peritubular interstitium (Kaissling and Le Hir 1994; Kaissling et al. 1996).Fig. 5Characteristic fibroblasts in a sham-operated kidney (a) and in a ureter-obstructed kidney on day 2 (b). In a the RER (arrow) is flat and concentrated in a cell process whereas the cytoplasmic rim around the angular nucleus is narrow and virtually organelle-free. The fibroblast displays the characteristic narrow association with a tubule and a blood capillary. Collagen fibrils are hardly detectable. The fibroblasts in b reveal inflated cisterns of RER (arrows) distributed all over the cytoplasm, including the direct surroundings of the oval nucleus. In B most collagen fibrils are cross-sectioned and appear as dots (arrowheads). They are abundant along the basement membranes of tubules (lower right and upper left corners). Bars in a and b (same magnification): 2 μmFig. 6Ultrastructural details of fibroblasts in ureter-ligated kidney (a–c: day 2). a A bundle of actin filaments displays dense bodies (arrows). b Adherent contact between two fibroblasts. c Intercellular contact between two fibroblasts that display different types of RER, inflated on the left, flat as in controls on the right. d Fibroblast in a ureter-ligated kidney (day 4). The profile of the cell reveals large irregularly formed cytoplasmic processes. Different types of RER are visible even within the same cell; in the pericaryon the cisterns are flat (arrowheads), in the large process they are inflated (arrows), masses of collagen fibrils are seen in the immediate vicinity of the process. Bars 0.5 μm and in d 5 μm
The most conspicuous organelles in the cytoplasm of fibroblasts were the large, mostly flattened, cisterns of rough endoplasmic reticulum (RER) (Fig. 4a). Actin filaments were found exclusively in a narrow rim of cytoplasm underlying the plasma membrane, most frequently at sites of contact with tubules and vessels (Kaissling et al. 1996).
Starting from day 1 (Fig. 1f–h) the appearance of fibroblasts became strikingly heterogeneous. Whereas a small fraction almost retained the morphology found in control kidneys, others displayed profound changes. In general the nucleus was rather rounded (Fig. 5b), often with indentations, and it was surrounded with a broad cytoplasmic rim. The cytoplasmic processes were often extensively branched (Fig. 1f–h). The RER was more abundant than in controls. In a large fraction of fibroblasts the RER cisterns were inflated and filled with flocculent material (Figs. 5b, 6c) consistent with enhanced synthesis of matrix. Indeed, massive accumulations of collagen fibrils were seen in the immediate vicinity of fibroblasts with such inflated cisterns of RER. In other cells the RER cisterns were flat (Fig. 6c) as in controls. The two forms could be found in neighboring fibroblasts (Fig. 6c) and even occasionally within a single cell (Fig. 6d). Intermediate forms of the RER were also found suggesting a progressive activation of the fibroblasts. Bundles of actin filaments were observed in the cytoplasmic processes. The presence of dense bodies (Fig. 6a) identified them as αSMA filaments. There was no obvious correlation between the abundance of bundles of actin filaments and the abundance or shape of the RER. In some fibroblasts of obstructed, but not of control kidneys intermediate filaments were seen (not shown). Adherent junctions between fibroblasts were sparse in controls but were common in obstructed kidneys (Fig. 6b, c).They sometimes connected a fibroblast with a flattened RER with one with an inflated RER but never a fibroblast with a mononuclear cell or two mononuclear cells with each other.
The study of the peritubular interstitium in the renal cortex by immunofluorescence takes advantage of the fact that fibroblasts in healthy kidneys of rats and mice strongly express 5′NT in their plasma membrane (Dawson et al. 1989; Kaissling and Le Hir 1994). Only a very small population of lymphocytes that are rarely encountered in the interstitium and easily distinguished from fibroblasts on account of their different shape may weakly express 5′NT (=CD 73) (Kaissling and Le Hir 1994).
Alpha SMA is the hallmark of myofibroblasts. In the kidney of adult healthy controls there is no trace of αSMA, as well as of vimentin (Essawy et al. 1997) in peritubular 5’NT-positive fibroblasts (Marxer-Meier et al. 1998). Alpha SMA was apparent in the peritubular interstitium as soon as after one day of ureteral obstruction. Double labeling for 5′NT and αSMA (Figs. 7, 8) revealed that αSMA was localized consistently within 5’NT-positive fibroblasts (Fig. 7d–f), in agreement with the observation of bundles of actin filaments and dense bodies in fibroblasts in the electron microscope. Alpha SMA was detectable in the majority of 5′NT-positive fibroblasts on day one, though in highly variable intensity, and in virtually all of them on day 4 of UUO (Fig. 8). The average intensity of immunolabeling for αSMA in individual fibroblasts progressively increased during the 4 days. The intensity of labeling of 5′NT and of αSMA seemed to be reciprocal. In fibroblasts strongly positive for αSMA the 5′NT labeling was weaker than in normal fibroblasts and the staining pattern for 5′NT changed from a sharp outline of the plasma membrane to a slightly diffuse, granular pattern over the plasma membrane and cytoplasm (Fig. 8, see also Fig.14). Large areas with strongly αSMA- and weakly 5′NT-positive fibroblasts were found around shrunken tubules, whereas prominent 5′NT and weakly αSMA-positive cells were found in areas where the tubules seemed to be rather intact (Fig. 8).Fig. 7Renal cortex in sham-operated (a) and in ureter-ligeted kidneys (b, c). 3 μm cryostat sections; immunofluorescence; ecto-5′nucleotidase (5′NT)—red channel, alpha smooth muscle actin (αSMA)—green channel); chromatin staining by DAPI—blue channel. In controls (a) 5′NT strongly labels cells in the interstitium and the brush border of proximal tubules, αSMA labels exclusively smooth muscle cells in arterial vessels (a). In the interstitium of ureter-ligated kidneys (b, c) 5′NT staining decreases, whereas that of αSMA appears and becomes increasingly prominent. d–f interstitial fibroblast in ureter-ligated kidney after 2 days. 5′NT is distributed over the plasma membrane and cytoplasm in a granular manner, αSMA is apparent along the plasma membrane and in the cellular processes. Bara–c ~100 μm, d–f ~10 μmFig. 8Renal cortex after 4 days of ureter ligation; 3 μm cryostat sections; immunofluorescence; ecto-5′ nucleotidase (5′NT)—red channel, alpha smooth muscle actin (αSMA)—green channel); chromatin staining by DAPI—blue channel. a Merge of channels, b 5′NT, c αSMA; the framed area 1 comprises interstitium with high 5′NT and faint αSMA staining, adjacent to rather intact tubules, the framed area 2 comprises interstitium with strong αSMA and faint 5′NT staining, adjacent to collapsed tubules. Bara–c ~100 μm; 1, 2: 10 μmFig. 9Renal cortex after ureter ligation; 3 μm cryostat section, immunofluorescence for αSMA—green channel and collagen type I (Col I)—red channel; chromatin staining by DAPI—blue channel; a arteriole. a–c Overview on the renal cortex after three days of ureter ligation; localization and intensity of αSMA and of Col I staining in the intertstitium coincide; heavy collagen deposits are seen around collapsed tubules (see also Fig. 13 where the ring of collagen is shown by TEM). d–f: a single fibroblast with distinct αSMA under the plasmalemm and immediately adjacent collagen deposits 2 days after ureter-ligature. Bar ~50 μmFig. 10MHC II-positive interstitial cells in the renal cortex. a–e 3 μm cryostat sections, immunofluorescence for MHC II, f 1 μm epon section. In controls faintly stained MHC II-positive cells are present throughout the interstitial spaces, a few are seen also in the glomerulus (G). The amount and size of MHC II-positive cells in the peritubular interstitium increases after 2 and even more after 4 days of ureter ligation, a few MHC II-positive cells are seen in the Glomerulus (G); d–f characteristic morphology of dendritic cells (asterisks) with a smooth, rounded or ovoid nucleus and lace veil-shaped large processes; in (f) two mononuclear cells in mitosis are seen in left half. Barsa–c ~100 μm; d–f ~10 μmFig. 11FSP1/S100A4-positive cells in the renal cortex. 3 μm cryostat sections, immonofluorescence; 5′NT—red channel; FSP 1/S100A4—green channel. In controls a very few FSP1/S100A4-positive cells are seen in the peritubular interstitium among the 5’NT-positive interstitial cells; the abundance of FSP1/S100A4 positive cells in the interstitial spaces increases at day 2 and 3 after ureteral obstruction; a few are found in glomeruli (G); arterioles (arrow) are weakly labeled by the antibody to FSP1/S100A4, tubular epithelia are negative for FSP1/S100A4. Bar ~100 μmFig. 12FSP1/S100A4 and αSMA in renal cortex after 4 days of ureteral obstruction. 3 μm cryostat sections, immonofluorescence; red channel—FSP1/S100A4, green channel—αSMA; FSP1/S100A4-positive cells in the interstitial space never stained for αSMA; tubular profiles never stained for FSP1/S100A4 or αSMA; (G)—glomerulus, insert: at higher magnification the shapes of FSP1/S100A4-positive cells suggest that they are lymphocytes and not fibroblasts. Bar ~100 μm; insert ~10 μmFig. 13Mitotic figures in ureter-ligated kidneys on day 2; 80 nm epon sections. The fibroblast in a (prophase) displays a rather normal morphology with its close association with a capillary (upper left corner); the RER appears abnormally abundant but the shape of the cisterns is not altered. The fibroblasts in b shows inflated cisterns of RER. Note the abundance of collagen fibrils along the basement membrane of the tubule (arrowheads, compare with Fig. 9). The mononuclear cell in c cannot be precisely identified. d Proximal tubular cell. Bars 4 μm. Bars ~100 μm and 10 μmFig. 14Mitotic cells in the peritubular interstitium in ureter-ligated kidneys. 3 μm cryostat sections, immonofluorescence; αSMA—green channel; 5′NT, or phopho-S6 kinase (p-S6 K)—red channel, chromatin staining by DAPI—blue channel. The mitotic cell in a heavily expresses 5′NT and faintly αSMA, the adjacent cell shows strong αSMA and faint 5′NT staining. The two mitotic cells in B display strong staining for αSMA along the plasma membrane and in the cell processes, 5′NT staining appears granular and is seen over the cytoplasm. The mitotic cell in C shows strong αSMA along the plasma membrane, in the cell processes and in a reticulated manner also in the cytoplasm; p-S6 K is up regulated in the cytoplasm of mitotic cells, whereas in quiescent cells the nucleus shows weak staining. Bar ~10 μm
Accumulation of collagen type I in the peritubular interstitium
Accumulation of collagen I (Col I) in the interstitial space is a hallmark of renal fibrosis. Collagen type I is produced by (myo)fibroblasts. By immunofluorescence Col I was virtually absent in the cortical peritubular interstitial space of controls. A modest immunofluorescence was apparent along the tubular profiles.
In the obstructed kidneys increasing amounts of Col I was found beginning with day 1 after ligature. In the cortex of ureter-ligated kidneys immunolabeling revealed heterogeneity in the abundance of Col I and this correlated closely with the intensity of immunolabeling for αSMA (Fig. 9). Thus interstitial Col I staining was the most abundant at sites with strongly αSMA-positive fibroblasts. At sites with moderate αSMA expression the collagen deposits could be attributed to single interstitial αSMA-positive cells (Fig. 9d, e). Col I staining became particularly strong around the tubular basement membranes of shrunken tubules, in agreement with the accumulation of collagen fibrils along the tubular basement membranes, seen in EM (Figs. 5b, 13b). Col I was more abundant in regions where the tubules were atrophic than in regions with apparently intact tubules. At these sites the tubular epithelium revealed some binding to antibodies to Col I.
Increases in mononuclear cells in the cortical interstitium
Mononuclear cells can be distinguished from fibroblasts on account of morphological criteria (Kaissling and Le Hir 1994; Kaissling et al. 1996) and in immunofluorescence by their absence of 5′NT and presence of specific marker proteins. In the healthy kidney the majority of mononuclear cells in the interstitial space are dendritic cells. The dendritic cells in rats can be recognized by their high expression of MHC class II. Morphologically these cells are distinguished from fibroblasts by their usually ovoid nucleus and extensive formation of cell projections that appear lighter than those of fibroblasts and that reveal frequent holes like in a lace-veil (Fig. 10) (Kaissling and Le Hir 1994; Kaissling et al. 1996). In sharp contrast to (myo)fibroblasts dendritic cells lack actin filaments and junctions with other cells.
Massive invasion of mononuclear cells in the obstructed kidney was observed from day 2 after ureter ligature. In the light- and the electron microscope most infiltrating cells in the occluded kidneys displayed the morphology of dendritic cells, of lymphocytes, or of macrophages. Frequency and size of dendritic cells increased dramatically during the 4 days of ureteral obstruction (Fig. 10).
FSP1/S100A4-positive cells (FSP1/S100A4)
FSP1/S100A4 has been implicated in development of renal fibrosis. It had been suggested that proximal tubular cells up-regulate FSP1/S100A4 and transform to myofibroblasts (Iwano et al. 2002). Immunostaining for FSP1/S100A4 (Figs. 11, 12) revealed cells in the interstitial space that displayed the smooth outlines of lymphocytes, as described previously (Le Hir et al. 2005). The abundance of these cells was low in control kidneys and increased sharply on day 2 after ligature and thereafter remained approximately stable (Fig. 10b, c). Occasionally these cells were found also within the lumen of capillaries and venules, as well as within glomeruli. In no instance we observed FSP1/S100A4 positive tubular cells or cells, double positive either for FSP1/S100A4 and 5’NT or for FSP1/S100A4 and αSMA (Fig. 12).
Proliferation of interstitial cells
Mitoses in interstitial cells are rarely encountered in control kidneys. Thus, the occurrence of numerous mitoses within the interstitial space after ureteral ligature was particularly striking. All stages of mitoses were found in the light- (Fig. 1) and electron microscope (Fig. 13) and by immunofluorescence (Fig. 14) in all types of interstitial cells. Immunostaining for phospho-S-kinase, which is up regulated in the cytoplasm in all stages of mitosis (Fig. 14c), was helpful for easy spotting of mitoses at low magnification (Schmidt et al. 2007).
Quantitative analysis of mitotic interstitial cells in 1 μm epon sections showed a sharp increase of the mitotic index in the peritubular interstitium on day 2 in UUO (Fig. 15a). Thereafter the mitotic index decreased but it remained significantly higher in the occluded kidney at days 3 and 4 than in controls. Even though up to 20% of mitotic cells could not be identified it was evident that fibroblasts and mononuclear cells contributed similarly to the increase. This was confirmed by electron microscopy (Fig. 13), since of a total of 32 mitotic cells found on days 2, 3 and 4 in the interstitium, 17 were fibroblasts and 15 mononuclear cells.Fig. 15Quantitative analysis of mitotic cells in the interstitium in response to UUO. a Overall mitotic rates in interstitial cells. Values on day 1, 2, 3 and 4 differ significantly from values of controls. b Relative contribution of fibroblasts and mononuclear cells to mitotic activity in the interstitium. Means ± standard deviation; n = 3
In the electron microscope mitotic figures were seen in fibroblasts with normal RER as well as in fibroblasts with inflated RER (Fig. 13). In fluorescence microscopy mitotic figures in fibroblasts were independent of the levels of expression of 5’NT and αSMA (Fig. 14). Thus, cell proliferation affected the various stages of transformation of fibroblasts in myofibroblasts.
Mitotic figures in tubules and in capillaries were rare in control kidneys. They were easily found in occluded kidneys on day 2, and less so on days 1, 3 and 4. Quantification was not carried out.
Discussion
The main purpose of the present study was to investigate the early response to ureter obstruction of resident fibroblasts in the renal cortex. Two major alterations were observed, starting from the first day after ligature of the ureter in the rat. Firstly, ultrastructural features and the expression of specific proteins suggested the progressive acquisition by virtually all fibroblasts of characteristic features of myofibroblasts. Because the transformation did not affect all fibroblasts simultaneously a large heterogeneity in the phenotypes of fibroblasts was present. Secondly, the incidence of mitosis of fibroblasts increased dramatically, affecting the various phenotypes of fibroblasts.
Transformation of resident fibroblasts to the myofibroblast phenotype
Immunofluorescence data as well as ultrastructural data showed the acquisition by resident fibroblasts of features of myofibroblasts. In immunofluorescence cortical interstitial fibroblasts were identified in the present study by their expression of 5′NT (Kaissling and Le Hir 1994; Kaissling et al. 1996; Le Hir et al. 2005). Co expression of 5’NT with αSMA was never observed in interstitial cells of control or contralateral kidneys but appeared already on day 1 after UUO, and became progressively more frequent up to day 4, where nearly all 5’NT-positive fibroblasts expressed more or less strongly αSMA. Thus, it appears that during the first days of UUO virtually all resident fibroblasts acquired the most characteristic feature of myofibroblats.
In the electron microscope the increased size of cisterns of RER suggested an increased production of extracellular matrix proteins by fibroblasts of obstructed kidneys. This also is a feature of the transformation of fibroblasts into myofibroblasts (Desmouliere et al. 2005; Hinz 2007; Hinz et al. 2007). Accordingly, the incidence of collagen fibrils was markedly increased in the interstitium after day 3 and 4 days of UUO. The presence of intercellular junctions is considered characteristic for myofibroblasts (Powell et al. 1999). It must be stressed that previously junctions were found between interstitial fibroblasts of normal rat kidneys (Kaissling and Le Hir 1994; Kaissling et al. 1996). However, in the present study they were much more easily found in obstructed kidneys compared to control or contralateral kidneys. These collected data suggest that the different shapes and phenotypes of fibroblasts seen in obstructed kidneys are different functional stages of one cell type. The striking differences between neighboring fibroblasts pertaining to the general cell morphology, to the abundance and shape of cisterns of RER and to the expression of 5′NT, αSMA and vimentin in obstructed kidneys suggest that alterations of fibroblasts following UUO are not attributable only to diffusible factors, for example secreted by infiltrating mononuclear cells. Forces exerted on individual cells might play a role as well. Indeed, a study of uranyl acetate-induced acute renal failure in rats (Fujigaki et al. 2005) suggested that forces exerted on fibroblasts are responsible for expression of αSMA in fibroblasts around dilated tubules and that under this condition myofibroblasts are derived from the progressive transformation of resident fibroblasts. Transformation of 5′NT-positive fibroblasts to the myofibroblast phenotype with expression of αSMA and vimentin was observed also around dilated, damaged distal convoluted tubules after treatment with thiazides (Loffing et al. 1996; Le Hir et al. 2005). The role of the tension exerted on stress fibers in the differentiation of myofibroblasts has been discussed recently (Hinz 2007).
Controversial identity of FSP1/S100A4-expressing cells
As found before (Anders et al. 2002; Iwano et al. 2002), in the present study UUO resulted in an increased incidence of FSP1/S100A4-positive cells in the renal cortical interstitium. In previous studies those cells were considered as fibroblasts/myofibroblasts (Anders et al. 2002; Iwano et al. 2002). That interpretation is not supported by our observations since we did not find cells double positive for FSP1 and αSMA or 5′NT in UUO. Similarly, in polycystic kidneys in mice (Okada et al. 2000) and in TGF over-expression-driven fibrosis in mice (Chai et al. 2003) FSP1-positive cells were generally negative for αSMA. Furthermore in the present study the FSP1-positive cells were generally ovoid with one or two plump cell processes. Finally frequent FSP1-positive cells adhered at the luminal aspect of endothelia. Thus FSP1-positive cells are probably not myofibroblasts but rather infiltrating leukocytes as found in a previous study (Le Hir et al. 2005).
Proliferation of interstitial cells
The increased abundance of fibroblasts in the UUO kidneys may be explained by the proliferation of resident cells, with no need for the extra-renal source proposed by others (Broekema et al. 2007; Li et al. 2007). Indeed, a peak of mitotic activity was observed on day 2 in tubular, vascular and interstitial cells. This is in agreement with the time course described in the same model in a previous study (Pat et al. 2005). Duymelinck et al. (Duymelinck et al. 2000) observed a peak in Ki-67-positive cells in αSMA-positive myofibroblasts in rats at day 5 after UUO. Also the nuclear incorporation (Hughes et al. 1999) of bromo-2′-deoxyuridine in αSMA-positive interstitial cells in a mouse model of UUO strongly suggested proliferation of myofibroblasts (Hughes et al. 1999). This is clearly confirmed by the detection of mitotic figures in the present study. Mitoses were found in cells with various levels of αSMA and 5′NT. In the electron microscope some mitotic fibroblasts displayed a normal RER, others an inflated RER. Thus, proliferation was not restricted to a specific stage of differentiation of fibroblasts. The origin of mitotic factors might be infiltrating cells but also tubular cells. The latter produce M-CSF in the UUO model in rats (Isbel et al. 2001).
Changes of tubular morphology
A further observation was made, which is highly relevant to the discussion about the role of EMT in renal fibrosis: whereas distal and proximal tubules in the cortex displayed profound regressive and atrophic alterations after ureter ligature, they preserved their epithelial architecture. In other words there was no morphological evidence of EMT during the 4 days following UUO. The dilatation of distal tubules following UUO has been reported before (Kida and Sato 2007). We found that the major morphological alterations in the proximal tubule are cellular atrophy accompanied by decreased luminal and outer diameters, affecting a majority of, but not all nephrons. The role of atrophic tubules in development of interstitial fibrosis has been pointed out in a rat model for renal failure, induced by disturbance of microcirculation following microsphere injection (Suzuki et al. 2001) and in a model using administration of aristolochic acid (Pozdzik et al. 2008). The signals, implicated in activation, proliferation and transformation of resident fibroblasts to myofibroblasts are among others TGFβ and PDGF-D, produced and released by damaged tubules (Suzuki et al. 2001; Taneda et al. 2003; Forino et al. 2006; Pozdzik et al. 2008). Tubular shrinkage affected also the distal segments of some nephrons. Virtually identical tubular alterations occurred in nephrons in which urine flow was decreased as a consequence of obstruction of the urinary pole by crescents in models of glomerular diseases (Le Hir and Besse-Eschmann 2003; Kriz and LeHir 2005).
Also previous other studies showed that fibrosis developed and myofibroblasts appeared without contribution of EMT in nephritic syndrome in humans (Kuusniemi et al. 2005) and in laboratory animals (Duymelinck et al. 2000; Fujigaki et al. 2005; Pozdzik et al. 2008). A similar conclusion was drawn from a rat model of angiotensin II-induced renal fibrosis (Faulkner et al. 2005) and a murine model (Chai et al. 2003).
In other studies FSP1 was detected by immunofluorescence in tubules of obstructed kidneys in animal models of UUO (Iwano et al. 2002; Kida et al. 2007; Sakairi et al. 2007). This finding was interpreted as evidence of EMT. We never detected FSP1 in tubular cells in the present study. It must be noted that in the previous studies tubular immunoreactivity for FSP1 was shown only on day 7 after ureter ligature or later, whereas our study ended on day 4.
In conclusion, during the first days following ureter ligature the resident peritubular fibroblasts differentiate into myofibroblasts and they proliferate strongly. These events are likely essential to fibrosis in UUO.

myofibroblast
fibroblast
fibrosis
kidney
morphology
s100a4
fsp1
epithelial mesenchymal transition (emt)


Non-Radical Diagnostic Biopsies Do Not Negatively Influence Melanoma Patient Survival

Background In fair-skinned Caucasian populations both the incidence and mortality rates of cutaneous melanoma have been increasing over the past decades. With adjuvant therapies still being under investigation, early detection is the only way to improve melanoma patient survival. The influence of incisional biopsies on melanoma patient survival has been discussed for many years. This study investigates both the influence of diagnostic biopsy type and the presence of residual tumor cells in the re-excision specimen on disease free and overall survival.
In fair-skinned Caucasian populations cutaneous melanoma is an important growing public health problem, causing a heavy burden on healthcare services. Both its incidence and mortality rates have been increasing in Europe over the past decades.1 The absolute total number of new cases of melanoma in the Netherlands is expected to be more than 4800 in 2015, compared with around 2400 in 2000.2 The Netherlands, as many other countries, has a two-tiered medical care system in which patients need to seek a medical opinion initially from a general practitioner (GP) before referral if necessary, to a specialist.
The mean number needed to treat (NNT), defined as the mean number of pigmented lesions needed to be excised to identify one melanoma, among 468 GPs in Perth, Australia, was 29 and ranged from 83 in the youngest patients (≤19 years) to 11 in the oldest patients (≥70 years).3 Assuming that for each new case of melanoma another 20–50 patients with pigmented skin lesions will visit the GP, the demand for detection will increase quite markedly. With adjuvant therapies still being under investigation, early detection is the only way to improve melanoma patient survival.4 Dutch guidelines recommend pigmented skin lesions suspect for melanoma to be removed through a diagnostic excision biopsy with a minimal lateral clearance of 2 mm.5 This recommendation is inline with the advice of the National Institutes of Health (NIH) to remove any suspicious lesion through excisional biopsy with a narrow margin of normal-appearing skin.6 The influence of an incisional biopsy on melanoma patient survival has been discussed for many years and different investigators have found contradicting results.7–15 Two recent publications concluding incisional biopsies not to interfere with melanoma patient survival were not able to end this discussion since patient groups were not fully comparable16 or follow up (FU) was short.17
This study investigated both the influence of diagnostic biopsy type and the presence of residual tumor cells in the re-excision specimen on melanoma patient disease free survival (DFS) and overall survival (OS), in 471 patients with a mean FU of more than 5 years. Survival analysis was done using Cox’s proportional hazard model adjusted for; gender, age, site of primary melanoma, Breslow thickness, type of melanoma, ulceration, lymphatic invasion and sentinel node (SN) status. Both the diagnostic biopsy type and the presence of tumor cells in the re-excision specimen were found not to influence melanoma patient survival.
METHODS
Patients
Between August 1993 and September 2004, 551 patients were diagnosed with clinical stage I/II cutaneous melanoma according to criteria of the American Joint Committee on Cancer (AJCC) and underwent re-excision of the primary melanoma site and a SN biopsy. If patients were referred to us from another institution the pathologic characteristics of the primary melanoma were reviewed in our hospital before the SN procedure. All patients were treated according to the same protocol; re-excision margins were 1 cm for melanomas with a Breslow thickness of ≤ 2 mm and 2 cm for melanomas with a Breslow thickness of >2 mm. To identify and retrieve the SN, the triple technique was used as described previously.18–20 In short, the day before surgery patients underwent a dynamic and static lymphoscintigraphy to determine the lymphatic drainage pattern. Just prior to surgery, Patent Blue V (Laboratoire Guerbet, Aulnay-sous-Bois, France) was injected intradermally next to the initial site of the melanoma. During surgery, guided by a hand held gamma probe and the blue staining of the draining tissues, the SN was removed.
To investigate the influence on survival, patients were divided both according to their diagnostic biopsy type; wide excisional biopsy (lateral clearance ≥ 2 mm), narrow excisional biopsy (lateral clearance < 2 mm), excisional biopsy with positive margins and incisional biopsy (includes punch) and the presence of residual tumor cells in their re-excision specimen.
Statistical Analysis
Data were processed with the Statistical Package for the Social Sciences software for Windows 2000 (SPSS 11.5, Chicago, IL). Cox’s proportional hazard model was used for survival analysis. P values <0.05 were considered significant.
RESULTS
Patient Population
Between August 1993 and September 2004, 551 patients were diagnosed with clinical stage I/II cutaneous melanoma, 257 male (46.6%) and 294 female (53.4%) with a mean age of 49.9 years (Table 1). Most primary melanomas were located on the trunk (43.7%) or on the lower extremities (36.7%). Breslow thickness was categorized into four groups ( ≤ 1.00 mm; 1.01–2.00 mm; 2.01–4.00 mm; >4.01 mm), but due to spontaneous regression of the primary lesion remained unknown in 38 patients. The majority of patients had a superficial spreading melanoma (65.0%) or a nodular melanoma (26.7%). In 46 patients the type of melanoma was different or remained unknown (8.3%). Ulceration, defined as the absence of intact epidermis overlying the major portion of primary melanoma, was diagnosed in 80 patients (14.5%), unknown in 1 patient (0.2%) and absent in 470 patients (85.3%). Lymphatic invasion was present in 25 patients (4.5%), absent in 521 patients (94.6%) and remained unknown in 5 patients (0.9%). The SN was negative in 446 patients (80.9%) and positive in 94 patients (17.1%). In 11 patients the SN was not removed and the SN status remained unknown (2.0%). In total, there were 101 missing variables in 80 patients; all were excluded from the study.
SN Identification
In 11 of the 551 patients the SN status remained unknown (2.0%), in 5 of these patients the SN was located in the deep lobe of the parotid gland and in one patient the SN was located high in the left axilla, in all cases the decision was made not to remove the SN to avoid potential morbidity associated with the intervention. The SN was not identified in 3 cases due to non-visualization by preoperative lymphoscintigraphy. In one patient the SN was located in the right axilla and could not be removed because the patient was suffering from frozen shoulder syndrome, the physical condition of another patient did not allow further treatment. Therefore, the success rate of SN identification was 98% (540 of 551 patients). Two of the patients with the SN located in the deep lobe of the parotid gland experienced metastasis of the parotid gland, one patient is still alive with disease and one patient is dead of disease. The patient whose physical condition did not allow further treatment, passed away soon after re-excision of the primary melanoma site, from massive hematogenic and lymphogenic metastasis. The 8 remaining patients have shown no evidence of disease.
Diagnostic Biopsy Type and Survival
The influence of diagnostic biopsy type on DFS and OS was tested in 471 patients with a mean FU of more than 5 years; 279 patients (59.3%) underwent a wide excision biopsy, 109 patients (23.1%) a narrow excision biopsy, 52 patients (11.0%) an excision biopsy with positive margins and 31 patients (6.6%) an incision biopsy (Table 2A).
TABLE 1.Patient characteristicsCharacteristicsPatients (n = 551)Follow up (years)  Mean (SD)5.1 (2.8)Gender  Male257 (46.6%)  Female294 (53.4%)Age (years)  Mean (SD)49.9 (15.3)Site of primary melanoma  Lower extremity202 (36.7%)  Upper extremity 63 (11.4%)  Head/Neck45 (8.2%)  Trunk241 (43.7%)Breslow thickness (mm)  0 < x ≤ 1153 (27.8%)  1 < x ≤ 2207 (37.6%)  2 < x ≤ 4114 (20.7%)  > 439 (7.1%)  Unknown (regression)38 (6.9%)Type of melanoma  Superficial spreading358 (65.0%)  Nodular147 (26.7%)  Other/Unknown46 (8.3%)Ulceration  No470 (85.3%)  Yes80 (14.5%)  Unknown1 (0.2%)Lymphatic invasion  No521 (94.6%)  Yes25 (4.5%)  Unknown5 (0.9%)Sentinel node status  Negative446 (80.9%)  Positive94 (17.1%)  Unknown11 (2.0%)TABLE 2.Patient distribution according to (A) diagnostic biopsy type and (B) the presence of residual tumor cells in the re-excision specimenAPatients (n = 471)Follow up (years; mean ± SD)Diagnostic biopsy type  Wide excision biopsy ( ≥ 2 mm)279 (59.3%)5.0 ± 3.0  Narrow excision biopsy (0<x<2 mm)109 (23.1%)6.0 ± 2.7  Excision biopsy with positive margins52 (11.0%)5.8 ± 2.9  Incision biopsy31 (6.6%)5.4 ± 2.4BPatients (n = 441)Follow up (years; mean ± SD)Re excision specimen  No residual tumor cells400 (90.7%)5.1 ± 2.9  Residual tumor cells41 (9.3%)5.8 ± 2.6
In 91/471 patients (19.3%) the SN was positive, 58/279 patients (20.8%) after a wide excision biopsy, 14/109 patients (12.8%) after a narrow excision biopsy, 15/52 patients (28.8%) after an excision biopsy with positive margins and 4/31 patients (12.9%) after an incisional biopsy.
In 79/471 patients (16.8%) a recurrence was found during FU, 45/279 patients (16.1%) after a wide excision biopsy (21 locoregional skin, 8 SN basin and 16 systemic), 17/109 patients (15.5%) after a narrow excision biopsy (7 locoregional skin, 2 SN basin and 8 systemic), 10/52 patients (19.2%) after an excision biopsy with positive margins (5 locoregional skin, 2 SN basin and 3 systemic) and 7/31 patients (22.6%) after an incision biopsy (5 locoregional skin, and 2 systemic). But confounding factors were not equally distributed between the diagnostic biopsy groups. Therefore, survival analysis was done using Cox’s proportional hazard model adjusted for; gender, age, site of primary melanoma, Breslow thickness, type of melanoma, ulceration, lymphatic invasion and sentinel node (SN) status.
In univariate analysis; gender, age, Breslow thickness, type of melanoma, ulceration, lymphatic invasion and SN status were all significantly related to both DFS and OS (Table 3). Site of primary melanoma on the head/neck was in univariate analysis only significantly related to DFS (Table 3). Diagnostic biopsy type did not have a significant relation with either DFS or OS (Table 3).
TABLE 3.Univariate and multivariate Cox regression analysis of disease-free survival and overall survival according to diagnostic biopsy typeDisease Free SurvivalOverall SurvivalUnivariateMultivariateUnivariateMultivariateTested variablesPHR95% CIPPHR95% CIPDiagnostic biopsy type  Wide excision biopsy0.6911.00–0.2040.5001.00–0.765  Narrow excision biopsy0.3600.710.39–1.280.2550.3150.740.37–1.510.411  Excision biopsy with positive margins0.8700.590.29–1.180.1320.7480.750.34–1.650.476  Incision biopsy0.5810.470.19–1.160.1010.3870.740.29–1.890.530Gender0.0181.150.70–1.890.5730.0041.440.81–2.570.215Age (years)0.0041.021.00–1.040.0180.0021.031.01–1.050.007Site of primary melanoma  Lower extremity0.0911.00–0.0230.153  Upper extremity0.8250.910.41–2.010.8090.071  Head/Neck0.0143.401.45–7.970.0050.095  Trunk0.7310.930.54–1.610.7900.063Breslow thickness (mm)  0 < x ≤ 1<0.0011.00–<0.001<0.0011.00–0.001  1 < x ≤ 20.00313.191.77–98.410.0120.0168.151.07–62.190.043  2 < x ≤ 4<0.00134.114.50–258.600.001<0.00121.432.79–164.840.003  > 4<0.00115.731.86–132.810.011<0.00111.851.35–103.700.025  Type of melanoma<0.0011.580.95–2.620.078<0.0011.570.88–2.790.128  Ulceration<0.0011.831.09–3.070.023<0.0011.640.89–3.040.116  Lymphatic invasion<0.0013.982.05–7.72<0.001<0.0012.191.07–4.480.032  Sentinel node status<0.0013.872.23–6.70<0.001<0.0013.191.75–5.81<0.001
Even though, diagnostic biopsy type did not have a significant relation with either DFS or OS in univariate Cox regression analysis it was also tested in multivariate Cox regression analysis together with all significant variables from univariate analysis, to investigate its influence on DFS and OS after correction for the confounding factors. In multivariate analysis, the only significant and independent predictors of both DFS and OS were; age, Breslow thickness, lymphatic invasion and SN status (Table 3). Site of primary melanoma on the lower extremities or head/neck and ulceration were in multivariate analysis only significant and independent predictors of DFS (Table 3). Also in multivariate analysis, diagnostic biopsy type did not have a significant relation with either DFS or OS (Fig. 1A, B).
FIG. 1.A Disease free survival rates and B overall survival rates according to the initial biopsy type.
The same analysis was done after combining the groups; the wide excision biopsy group was joined with the narrow excision biopsy group and compared to the excision biopsy group with positive margins joined with the incision biopsy group. Still, diagnostic biopsy type did not have a significant influence on either DFS or OS (data not shown).
Residual Tumor Cells in the Re-Excision Specimen and Survival
441/471 Patients (93.6%) underwent re-excision of the primary melanoma site and in 30 patients (6.4%) the diagnostic biopsy was found to be sufficient. In 41/441 patients (9.3%) residual tumor cells were found in the re-excision specimen (Table 2B). All 41 patients with residual tumor cells in the re-excision specimen underwent either an excision biopsy with positive margins or an incision biopsy. In none of the patients with a wide or narrow excision biopsy residual tumor cells were found.
In univariate analysis; gender, age, Breslow thickness, type of melanoma, ulceration, lymphatic invasion and SN status were all significantly related to both DFS and OS (Table 4). Site of primary melanoma on the lower extremity or head/neck was in univariate analysis significantly related to DFS and site of primary melanoma on the head/neck or trunk was significantly related to OS (Table 4). Residual tumor cells in the re-excision specimen did not have a significant relation with either DFS or OS (Table 4).
TABLE 4.Univariate and multivariate Cox regression analysis of disease-free survival and overall survival according to the presence of residual tumor cells in the re-excision specimenDisease Free SurvivalOverall SurvivalUnivariateMultivariateUnivariateMultivariateTested variablesPHR95% CIPPHR95% CIPResidual tumor cells0.0940.790.38–1.640.5320.2300.830.36–1.920.668Gender0.0251.140.68–1.920.6110.0091.220.65–2.280.531Age (years)0.0071.021.00–1.040.0380.0071.021.00–1.040.040Site of primary melanoma  Lower extremity0.0321.00–0.0330.1051.00–0.123  Upper extremity0.8300.950.41–2.200.9100.0692.210.85–5.710.102  Head/Neck0.0043.221.35–7.660.0080.0403.221.03–10.090.045  Trunk0.6470.960.54–1.740.9030.0492.020.98–4.200.059Breslow thickness (mm)  0 < x ≤ 1<0.0011.00–<0.001<0.0011.00–0.004  1 < x ≤ 20.00512.821.71–96.070.0130.0207.991.04–61.490.046  2 < x ≤ 4<0.00130.163.97–229.220.001<0.00120.132.59–156.250.004  > 4<0.00115.911.86–136.250.012<0.00111.841.32–106.540.028  Type of melanoma<0.0011.610.95–2.730.076<0.0011.630.87–3.040.128  Ulceration<0.0011.550.90–2.670.119<0.0011.610.85–3.050.141  Lymphatic invasion<0.0014.162.13–8.14<0.001<0.0012.241.04–4.800.038  Sentinel node status<0.0013.301.87–5.83<0.001<0.0013.051.56–5.960.001
Even though, residual tumor cells in the re-excision specimen did not have a significant relation with either DFS or OS in univariate Cox regression analysis it was also tested in multivariate Cox regression analysis together with all significant variables from univariate analysis, to investigate its influence on DFS and OS after correction for the confounding factors. In multivariate analysis, the only significant and independent predictors of both DFS and OS were; age, site of primary melanoma on the head/neck, Breslow thickness, lymphatic invasion and SN status (Table 4). Also in multivariate analysis, residual tumor cells in the re-excision specimen did not have a significant relation with either DFS or OS (Fig. 2A, B).
FIG. 2.A Disease free survival rates and B overall survival rates according to the presence of residual tumor cells in the re-excision specimen.
Consistent Confounders of Melanoma Patient Survival
Age, Breslow thickness, lymphatic invasion and SN status were the most consistent, independent and significant confounders of melanoma patient DFS and OS (Table 3, 4). Site of the primary melanoma was not always an independent and significant confounder of melanoma patient survival, but location on the head/neck region consistently carried the highest hazard ratio (HR) (Table 3, 4). Surprisingly, ulceration was not an independent and significant confounder of OS (Table 3, 4).
DISCUSSION
Numerous investigators have studied the influence of incisional biopsy on melanoma patient survival and found contradicting results. Fitzpatrick et al. found a five-year OS rate of 30% in the incisional biopsy group as compared to 48% in the excisional biopsy group but the different biopsy groups were not matched for important prognostic factors.8 Epstein et al. found a more favorable ten-year OS in the biopsy group (65%) as compared to the primary wide excision group (56%) but biopsy was loosely defined as less than optimal or complete surgical excision.9 Ironside et al. found a five-year OS rate of 66% in the excision- and 43% in the incision biopsy group but failed to describe the distribution of prognostic factors between both groups.10 Rampen et al. also found a worse prognosis for patients after an incision biopsy (14 patients) but the study was small and retrospective.11 Griffiths et al. found no difference in survival but 7/19 incisional biopsy patients were excluded because of missing histopathological data.12 Survival rates were not significantly different between the incision- and excision biopsy groups of Lederman et al., but patient groups were matched for Breslow thickness only.13 Lees et al. also indicated no significant averse effect of incisional biopsy in 96 patients, but 40% of the histopathological data was not assessable.14 Austin et al did find a significantly reduced survival in the incisional biopsy group, but patients in the incisional biopsy group were also significantly older.15 The two most recent publications found no negative influence of incisional biopsies on melanoma patient survival but in Bong et al. patients groups were not fully matched and in Martin et al. median FU was only 28 months.16,17
This study not only investigated the influence of diagnostic biopsy type but also the presence of residual tumor cells in the re-excision specimen on melanoma patient survival in patient groups adjusted for 8 important confounders of survival with a mean FU > 5 years. Excision biopsies with positive margins and incisional biopsies were found not to influence melanoma patient survival. Interestingly, DFS and OS even seemed slightly better in the non-radical biopsy groups (Fig. 1A, B). In line with this, patients with residual tumor cells in their re-excision specimen also had a slightly better survival as compared to patients without residual tumor cells in their re-excision specimen (Fig. 2A, B). Melanoma is the most immunogenic tumor identified to date; melanoma specific T cells are detectable both in the blood and in tumor draining lymph nodes from melanoma patients and their frequency can be increased by specific vaccination.21–23 This intrinsic immunogenicity makes melanoma lesions particularly amenable to therapeutic approaches aimed at strengthening tumor immune surveillance. Did residual tumor cells combined with biopsy induced wound healing trigger the melanoma patient’s immunesystem? In non-melanoma skin cancer (NMSC) biopsy induced tumor regression has previously been described, Swetter et al reported that 24% of NMSCs transected on the initial biopsy showed no residual tumor in the excision specimens and suggested biopsy induced wound healing to play an important role.24
Even though non-radical diagnostic biopsies and residual tumor cells in the re-excision specimen do not have a negative influence on melanoma patient survival, the routine use of incision biopsies is not recommended. Incisional biopsies often consist only of a small percentage of the surface area of the pigmented skin lesion making it difficult to sample a representative area within the tumor. Somach et al found that 40% of the histopathological features were more pronounced in the re-excision specimen as compared to the incision biopsy and in 20% areas of invasive melanoma not detected in the incisional biopsy were observed in the re-excision specimen.25 Further more when melanoma is diagnosed, attempting to evaluate the depth of invasion in an incisional biopsy is treacherous and may lead to over- or underestimation of the invasion.26,27 Of course these problems are less prominent in excision biopsies with positive margins, here the majority of the lesion has been removed and only the outer borders are compromised making a sampling error highly unlikely.
In conclusion; age, Breslow thickness, lymphatic invasion and SN status were the most consistent, independent and significant confounders of melanoma patient DFS and OS. The site of primary melanoma and ulceration were also important confounders of survival. Both the diagnostic biopsy type and the presence of residual tumor cells in the re-excision specimen did not have a negative influence on melanoma patient DFS and OS. With melanoma incidence rates rising1 and early detection of melanoma still being the only way to improve melanoma patient survival,4 it is important for all physicians to feel confident about removing a pigmented skin lesion suspect for melanoma. Incisional biopsies are not recommended but there is no cause for concern when an excision biopsy turns out to have positive margins.

diagnostic
melanoma
survival
initial
re-excision and biopsy