Identification of Genetic and Epigenetic Variations in a Rat Model for Neurodevelopmental Disorders A combination of genetic variations , epimutations and environmental factors may be involved in the etiology of complex neurodevelopmental disorders like schizophrenia . To study such disorders , we use apomorphine-unsusceptible -LRB- APO-UNSUS -RRB- Wistar rats and their phenotypic counterpart apomorphine-susceptible -LRB- APO-SUS -RRB- rats that display a complex phenotype remarkably similar to that of schizophrenic patients . As the molecular basis of the APO-SUS/UNSUS rat model , we recently identified a genomic rearrangement of the Aph-1b gene . Here , we discovered between the two rat lines differences other than the Aph-1b gene defect , including a remarkable cluster of genetic variations , two variants corresponding to topoisomerase II-based recombination hot spots and an epigenetic -LRB- DNA methylation -RRB- difference in cerebellum and -LRB- hypo -RRB- thalamic but not hippocampal genomic DNA . Furthermore , genetic variations were found to correlate with the degree of apomorphine susceptibility in unselected Wistar rats . Together , the results show that a number of genetic and epigenetic differences exist between the APO-SUS and - UNSUS rat genomes , raising the possibility that in addition to the Aph-1b gene defect the newly identified variations may also contribute to the complex APO-SUS phenotype . Introduction Schizophrenia is a neurodevelopmental disorder affecting nearly 1 % of the world 's population -LRB- Jablensky et al. 1987 -RRB- , and is characterized by positive and negative symptoms -LRB- Kay and Opler 1987 -RRB- . The aetiology of schizophrenia and other related disorders , such as schizoaffective and bipolar disorder , is still unclear . Twin , family and adoption studies have suggested that complex interactions at the genetic and environmental level underlie the aetiology of schizophrenia -LRB- Gottesman 1991 -RRB- . It is thought that gene variations by themselves do not result in schizophrenia , but they can establish a predisposition status that , when combined with environmental stressors , may lead to schizophrenia pathogenesis . Numerous environmental factors , such as viral infections -LRB- Mednick et al. 1988 -RRB- , insufficient folate and methionine levels -LRB- Regland 2005 -RRB- , or repeated psychological stress -LRB- Goldstein 1987 -RRB- , can influence brain development of prenatal or early postnatal individuals with a genetic predisposition for neuropsychiatric disorders . Due to the heterogeneity in genetic and environmental interactions , most of the genes and pathways for schizophrenia and for other complex disorders are still unknown . To get insight into the gene -LRB- or genes -RRB- that may be involved in schizophrenia pathogenesis , a rat model was developed with schizophrenia-like features . This model was based on the behavioural response of Wistar rats to the dopamine agonist apomorphine -LRB- Cools et al. 1990 -RRB- . The apomorphine-susceptible -LRB- APO-SUS -RRB- rat line displayed many features of psychopathology , with similar disturbances at the behavioural , physiological , endocrinological and pharmacological level as seen in schizophrenics -LRB- Ellenbroek and Cools 2002 -RRB- . For example , APO-SUS rats have a reduced prepulse inhibition and latent inhibition -LRB- Ellenbroek et al. 1995 -RRB- , display a higher plasma release of adrenocorticotropin -LRB- ACTH -RRB- and corticosteroids in response to novelty -LRB- Rots et al. 1995 -RRB- , are more sensitive to dopamimetic drugs -LRB- Ellenbroek et al. 2000 -RRB- , and have a higher susceptibility to inflammatory and infectious diseases when compared to apomorphine-unsusceptible -LRB- APO-UNSUS -RRB- rats -LRB- Kavelaars et al. 1997 -RRB- . We therefore wondered about the molecular-genetic basis underlying the APO-SUS / - UNSUS rat model and recently identified a genetic difference between the two rat lines -LRB- Coolen et al. 2005 -RRB- . Whereas APO-UNSUS rats harbour three gene copies of the γ-secretase component Aph-1b , APO-SUS rats have only one or two copies . This gene-dosage imbalance was due to an unequal crossing over event -LRB- nonallelic homologous recombination -RRB- between two direct repeats -LRB- a segmental duplication -RRB- within the Aph-1b locus . In addition , we observed a direct link between the Aph-1b genotypes and a number of phenotypic APO-SUS and - UNSUS characteristics -LRB- Coolen et al. 2005 -RRB- . Approximately 10 years after developing the APO-SUS and - UNSUS lines a second , independent breeding procedure was started that resulted in rats with features similar to those displayed by the original APO-SUS and - UNSUS rat lines -LRB- Ellenbroek and Cools 2002 -RRB- . Interestingly , the replicated rat lines also resulted in APO-UNSUS rats with three Aph-1b gene copy numbers and APO-SUS rats with only one or two gene copies -LRB- Coolen et al. 2005 -RRB- . In the present study , we wondered whether genetic variations other than the Aph-1b gene-dosage imbalance may be present between the APO-SUS and - UNSUS rats , and whether epigenetic factors may be involved as well . Epigenetics has been defined as heritable changes in gene expression that do not occur by changes in the DNA sequence , but by modifications in DNA methylation and chromatin remodeling -LRB- Wolffe and Matzke 1999 -RRB- , or , in its widest sense , as any change in an organism that is not due to genetic factors -LRB- Van de Vijver et al. 2002 -RRB- . Increasing evidence suggests that epigenetic modifications play a role in disease susceptibility -LRB- reviewed by Jirtle and Skinner 2007 -RRB- . We used the arbitrarily primed-polymerase chain reaction -LRB- AP-PCR -RRB- fingerprinting technique -LRB- Welsh and McClelland 1990 -RRB- to analyse the genomes and epigenomes -LRB- DNA methylation -RRB- of the APO-SUS and - UNSUS rats . Comparison of the AP-PCR fingerprints generated from the genomic DNAs of the two rat lines revealed genetic as well as epigenetic alterations and we conclude that , besides in the Aph-1b locus , a number of other variations are present in the APO-SUS and - UNSUS genomes and epigenomes . Materials and methods Experimental animals The generation of the APO-SUS and - UNSUS rat lines with a high or low susceptibility for apomorphine , respectively , has been described previously -LRB- Cools et al. 1990 -RRB- . The present experiments were performed with male APO-SUS and - UNSUS rats belonging to the 32nd -LRB- original lines -RRB- and 18th -LRB- replicate lines -RRB- generation . At post-natal day 60 -LRB- PND60 -RRB- , APO-SUS and - UNSUS rats were sacrificed and the hippocampus , cerebellum and the combined thalamus/hypothalamus -LRB- further denoted as -LRB- hypo -RRB- thalamus -RRB- were isolated . To establish their apomorphine susceptibility , unselected male Wistar rats of the Nijmegen outbred population -LRB- PND60 -RRB- were injected with apomorphine -LRB- 1.5 mg/kg s.c. -RRB- and gnawing scores were measured in a gnawing box for 45 min , as described previously -LRB- Cools et al. 1990 -RRB- . Immediately following the measurements , the rats were sacrificed and the same tissues -LRB- hippocampus , cerebellum and -LRB- hypo -RRB- thalamus -RRB- were removed . All rats were bred and reared in the Central Animal Facility of the Radboud University Nijmegen under approved animal protocols and in accordance with institutional guidelines . Arbitrarily primed-PCR Genomic DNAs were isolated from hippocampus , cerebellum and -LRB- hypo -RRB- thalamus using standard procedures involving the use of proteinase K and phenol extraction . Two micrograms of genomic DNA were digested with 20 units of RsaI , 20 units RsaI in combination with the methylation-sensitive enzyme HpaII , or 20 units RsaI and MspI -LRB- MBI Fermentas -RRB- in a total volume of 40 μl at 37 °C for 16 h. HpaII does not cut DNA if the internal cytosine -LRB- CCGG -RRB- is methylated , whereas MspI is insensitive to DNA methylation . Using such combinations of methylation-sensitive and - insensitive enzymes allows genome-wide screening for differences at the genetic level -LRB- single-nucleotide polymorphisms -- -- SNPs , duplications , insertions , deletions and recombinations -RRB- as well as the epigenetic -LRB- DNA methylation -RRB- level . Restriction enzymes were heat inactivated by incubating the reactions at 65 °C for 20 min . Digested DNA -LRB- 100 ng -RRB- was amplified using AP-PCR -LRB- Welsh and McClelland 1990 -RRB- with a single primer . PCRs were performed in a total volume of 25 μl containing 10 mM Tris-HCl -LRB- pH 8.3 -RRB- , 2.5 mM MgCl2 , 50 mM KCl , 0.001 % gelatin , 0.25 % Nonidet P-40 , 0.25 % Tween-20 , 200 μM each of the four deoxynucleotide triphosphates , ∼ 1 μCi of -LSB- α-32P -RSB- dCTP -LRB- 3000 Ci/mmol , Amersham Corp. -RRB- , 25 pmol of primer -LRB- AP-1 : 5 ′ - AACCCTCACCCTAACCCCGG-3 ′ , AP-7 : 5 ′ - AACCCTCACCCTAAGGCGCG-3 ′ , AP-777 : 5 ′ - CACTCCTCTACAAGGTGCCG-3 ′ or Topo : 5 ′ - GCCTCCTTGCAGGTCTTT-3 ′ -RRB- , and 0.8 units of Taq polymerase -LRB- MBI Fermentas -RRB- . Reactions were carried out in a thermal cycler -LRB- Perkin-Elmer -RRB- with five cycles of low stringency -LRB- 94 °C for 30 s , 40 °C for 60 s , 72 °C for 1.5 min -RRB- , followed by 30 cycles of high stringency -LRB- 94 °C for 15 s , 55 °C for 15 s , 72 °C for 1 min -RRB- . Two microliters of the PCR products were analysed on high-resolution 5 % polyacrylamide gels under denaturing conditions -LRB- 7 M urea -RRB- for 4 -- 4.5 h at 70 W. Gels were dried and radiolabelled DNA was visualized by autoradiography at − 70 °C -LRB- CEA AB , Sweden -RRB- . Cloning and sequencing of AP-PCR fragments AP-PCR fragments generated from APO-SUS and - UNSUS rat genomic DNAs were excised from the dried gels and incubated in 50 μl MilliQ at 80 °C for 10 min . The eluted DNA -LRB- two microliters -RRB- was reamplified with the same primer as used for the AP-PCR to generate sufficient amounts of template for subsequent cloning . The reactions were carried out for 40 cycles of 94 °C for 1 min , 55 °C for 30 s , 72 °C for 1 min , under the same conditions as described in the AP-PCR protocol -LRB- except that -LSB- α-32P -RSB- dCTP was not included -RRB- . The PCR products were purified , cloned into the pGEM-T easy vector -LRB- Promega -RRB- and sequenced with a T7 or Sp6 primer according to the manufacturer 's instructions using the ABI310 machine -LRB- Applied Biosystems -RRB- . Sequencing and genotyping of chromosomal region 9q22 A 1948-bp fragment that harbours the nucleotide sequence corresponding to product 3 was obtained by PCR on genomic DNA derived from -LRB- hypo -RRB- thalamic tissue of an APO-UNSUS rat using forward primer 5 ′ - GGGAAGCAACGCATCCTG-3 ′ and reverse primer 5 ′ - CATATCAAAGCACCAAGTCCACAG-3 ′ . The DNA was subsequently purified and directly sequenced using the ABI310 machine -LRB- Applied Biosystems -RRB- . Genotyping of chromosomal region 9q22 was performed with PCR using primers specific for either the APO-SUS or APO-UNSUS genomic sequence . Briefly , PCRs were performed in a total volume of 20 μl containing 50 ng genomic DNA , 10 mM Tris-HCl -LRB- pH 8.3 -RRB- , 2.5 mM MgCl2 , 50 mM KCl , 0.001 % gelatin , 0.25 % Nonidet P-40 , 0.25 % Tween-20 , 200 μM each of the four deoxynucleotide triphosphates , 0.6 μM of each primer -LRB- FW : 5 ′ - AACACTTGGACTCATTCTCACTGG - -LSB- G -LRB- SUS -RRB- or T -LRB- UNSUS -RRB- -RSB- -3 ′ and RV : 5 ′ - CCTGGATGGAATGTTGACAC - -LSB- C -LRB- SUS -RRB- or T -LRB- UNSUS -RRB- -RSB- -3 ′ -RRB- , and 0.8 units of Taq polymerase -LRB- MBI Fermentas -RRB- . Reactions were carried out at 94 °C for 60 s , 58 °C for 60 s and 72 °C for 60 s for 35 cycles . Products were analysed on a 1 % agarose gel . Quantification and statistics Quantification of AP-PCR products was performed using the Labworks 4.0 program -LRB- UVP BioImaging Systems , Cambridge , UK -RRB- and statistical evaluation was performed by means of an unpaired Student 's t-test . Results AP-PCR DNA fingerprint patterns of the APO-SUS and APO-UNSUS rat genomes and epigenomes In order to identify differences between the genomes and epigenomes of APO-SUS and - UNSUS rats , we performed a comparative analysis of fingerprints of AP-PCR products generated from genomic DNAs of the two rat lines . Initially , genomic DNAs isolated from APO-SUS and - UNSUS -LRB- hypo -RRB- thalamic tissue and digested with RsaI in combination with the methylation-sensitive restriction enzyme HpaII -LRB- CCGG -RRB- was analysed using arbitrary primers AP-1 , AP-7 or AP-777 . These primers were selected from a total set of ten primers because they gave fingerprints with reproducible and discrete products -LRB- data not shown -RRB- . Typical AP-PCR fingerprints obtained with the three selected arbitrary primers are shown in Fig. 1 . With each arbitrary primer ∼ 30 chromosomal fragments were reproducibly amplified . DNAs digested with RsaI and the methylation-insensitive enzyme MspI served as controls to determine whether the observed differences were due to a differential methylation of the CCGG sequence or a genetic polymorphism in this sequence . AP-PCR analysis with primer AP-1 revealed 16 products corresponding to fragments without an HpaII site -LRB- `` genetic fragments '' -RRB- and 23 products corresponding to fragments containing an HpaII site -LRB- `` epigenetic fragments '' -RRB- . Analysis with AP-PCR primer AP-7 showed 17 genetic and 11 epigenetic fragments , and with AP-777 primer 22 genetic and 6 epigenetic fragments . Fig. 1AP-PCR analysis of genomic DNAs from APO-UNSUS -LRB- U -RRB- and APO-SUS -LRB- S -RRB- -LRB- hypo -RRB- thalamus . AP-PCR was performed with primers AP-1 , AP-7 and AP-777 using genomic DNAs digested with RsaI and HpaII -LRB- H -RRB- or RsaI and MspI -LRB- M -RRB- as templates . Epigenetic products -LRB- methylation-sensitive and thus absent in the MspI lanes -RRB- are indicated by closed arrows and genetic products -LRB- methylation-insensitive and thus present in the MspI lanes -RRB- by open arrows Genetic variations between the APO-SUS and APO-UNSUS rat genomes Comparison of the genetic fingerprints generated with primers AP-1 , -7 and -777 revealed three reproducible variations between the genomic DNAs from the original -LRB- F32 -RRB- APO-SUS and - UNSUS rats , designated products 1 , 2 and 3 -LRB- Fig. 2a -RRB- . Product 1 was less prominent in the APO-SUS than in the APO-UNSUS rats , product 2 was found only in APO-SUS , while the level of product 3 was higher in the APO-SUS than - UNSUS rats . Interestingly , the three products were also present in the replicate -LRB- F18 -RRB- lines and at the same levels , indicating that the replication of the APO-SUS and - UNSUS lines had resulted in a similar genotypic distribution . Next , digestions using RsaI in combination with MspI were used to examine whether the observed differences were due to a genetic or an epigenetic alteration . Following digestion with RsaI and MspI , products 1 , 2 and 3 were still found , indicating that the presence of the three AP-PCR products was due to genetic differences -LRB- Fig. 2b -RRB- . Fig. 2Genetic variations in genomic DNAs from APO-SUS -LRB- S -RRB- and APO-UNSUS -LRB- U -RRB- -LRB- hypo -RRB- thalamus . Rats were from the original -LRB- F32 -RRB- or the replicate -LRB- F18 -RRB- rat lines . -LRB- A -RRB- Products generated by AP-PCR using primers AP-1 -LRB- product 1 -RRB- , AP-7 -LRB- product 2 -RRB- or AP-777 -LRB- product 3 -RRB- on genomic DNAs digested with the methylation-insensitive enzyme RsaI and the methylation-sensitive enzyme HpaII . ND = not determined . -LRB- B -RRB- Products 1 , 2 and 3 generated by AP-PCR on genomic DNAs digested with RsaI and HpaII -LRB- H -RRB- or with the two methylation-insensitive enzymes RsaI and MspI -LRB- M -RRB- . MspI products served as controls to determine whether products 1 , 2 and 3 were due to differential methylation or to a genetic polymorphism . The fact that products 1 , 2 and 3 were still present following MspI digestion indicates that they represent products without an HpaII site -LRB- `` genetic fragments '' -RRB- . -LRB- C -RRB- Products generated by AP-PCR using a topoisomerase II binding site consensus sequence -LRB- 5 ′ - GCCTCCTTGCAGGTCTTT-3 ′ -RRB- on genomic DNAs digested with the methylation-insensitive enzymes EcoRI -LRB- product 4 -RRB- or MboI -LRB- product 5 -RRB- . Arrows indicate increased amounts of the AP-PCR products We previously discovered that the Aph-1b gene-dosage imbalance between the APO-SUS and - UNSUS rats is the result of a DNA recombination event between the two Aph-1b genes . Furthermore , we identified the region in which the recombination occurred , namely in a region of 1106 nucleotides that is identical between the two genes and encompasses exon 5 -LRB- Coolen et al. 2005 -RRB- . In the present study , we decided to examine in detail the site of recombination and found a topoisomerase II binding site -LRB- 5 ′ - ACCCACCTGCTGGTGTCC-3 ′ -RRB- in the DNA region harbouring the recombination site . Topoisomerase II binding sites -LRB- with the vertebrate consensus sequence 5 ′ - RNYNNCNNGYNGKTNYNY-3 ′ -RRB- -LRB- Spitzner and Muller 1988 -RRB- are known to be hotspots where DNA recombination events occur easily -LRB- Craig and Nash 1983 -RRB- . We therefore wondered whether other topoisomerase II binding sites could have led to additional differences between the APO-SUS and - UNSUS rat genomes . Interestingly , using a primer based on the topoisomerase II binding site consensus for PCR analysis of genomic DNAs digested with EcoRI or MboI revealed two differences between the APO-SUS -LRB- n = 3 -RRB- and - UNSUS -LRB- n = 2 -RRB- rat genomes of the original -LRB- F32 -RRB- lines , designated products 4 and 5 -LRB- Fig. 2c -RRB- . Product 4 was present in APO-SUS but not in APO-UNSUS rat genomic DNA . In the replicate APO-SUS and - UNSUS lines -LRB- F18 -RRB- , the genomes of two APO-UNSUS rats did also not contain product 4 , whereas it was present in two of the four APO-SUS rats tested . Product 5 was present in three of the four APO-UNSUS rats examined -LRB- in both the original and the replicate lines -RRB- , whereas it was not observed in the seven APO-SUS rats tested -LRB- Fig. 2c -RRB- . Epigenetic variations between the APO-SUS and APO-UNSUS rat genomes We then wondered whether , besides the five genetic variations , also epigenetic variations would be present between the APO-SUS and - UNSUS rat lines . AP-PCR analysis using primer AP-1 on RsaI - and HpaII-digested genomic DNAs from APO-SUS and - UNSUS -LRB- hypo -RRB- thalamus revealed one epigenetic variation , designated the E1-product -LRB- Fig. 3a -RRB- . The difference was observed in both the original APO-SUS and - UNSUS rats as well as the replicated lines . An ∼ 1.4-fold reduced amount of the E1-product was observed in APO-SUS when compared with APO-UNSUS genomic DNAs -LRB- n = 12 , P < 0.05 -RRB- . Since DNA methylation may be tissue specific , we decided to examine the E1-product in two other brain tissues . The level of the E1-product was ∼ 2-fold reduced in genomic DNAs from the cerebellum of APO-SUS compared to APO-UNSUS rats -LRB- n = 4 , P < 0.01 -RRB- , while no difference in the E1-levels was found in the hippocampus of the APO-SUS and - UNSUS rats -LRB- n = 4 -RRB- . Fig. 3Epigenetic variation in genomic DNAs from APO-SUS -LRB- S -RRB- and APO-UNSUS -LRB- U -RRB- -LRB- hypo -RRB- thalamus . Rats were from the original -LRB- F32 -RRB- or the replicate -LRB- F18 -RRB- rat lines . -LRB- A -RRB- Product E1 generated by AP-PCR using primer AP-1 on genomic DNAs digested with RsaI and HpaII . Arrow indicates a representative example of an AP-PCR product that did not show variable amounts using primer AP-1 -LRB- randomly chosen out of 38 products -RRB- and was used for normalization . -LRB- B -RRB- The AP-PCR E1-product generated from genomic DNAs from cerebellum , hippocampus and -LRB- hypo -RRB- thalamus . All rats were from different nests . The products indicated by the arrow were used for normalisation . Amounts for the epigenetic E1-product were significantly different between the APO-SUS and APO-UNSUS rats in the cerebellum -LRB- ** P < 0.01 ; n = 4 , plus s.e.m. -RRB- and -LRB- hypo -RRB- thalamus -LRB- * P < 0.05 ; n = 12 , plus s.e.m. -RRB- , but not in the hippocampus -LRB- n = 4 , plus s.e.m. -RRB- Chromosomal localizations of the genetic and epigenetic variations between the APO-SUS and - UNSUS rat genomes To identify the locations of the genetic and epigenetic variations within the APO-SUS and - UNSUS rat genomes , AP-PCR fragments 1 , 2 , 3 and E1 were excised from the gel and each fragment was reamplified with the primer used for the original AP-PCR reaction . DNA sequence analysis of the amplified PCR fragments and database searches with the obtained nucleotide sequences revealed the chromosomal localisations of the four fragments ; product 1 was located on chromosome 19q11 , ∼ 3.3 kb upstream of the first exon of the GAIP-interacting protein , C terminus -LRB- GIPC -RRB- gene ; product 2 was part of a repeat sequence located on chromosome 2q34 ; product 3 was located in the first intron of the myosin 1b gene on chromosome 9q22 ; the epigenetic variation E1 was located on chromosome 6q31 , downstream of the Jun dimerization protein 2 -LRB- NP_446346.1 -RRB- and upstream of the ATF-like basic leucine zipper transcriptional factor B-ATF -LRB- SF-HT-activated gene 2 ; XP_216745.2 -RRB- . Since product 3 was localized within a gene , we decided to analyse this chromosomal region in more detail . Sequence analysis of the DNA region corresponding to AP-PCR fragment 3 and its surrounding region revealed a remarkably high number of genetic variations : 10 of the 1948 base pairs analysed were different between the APO-SUS and - UNSUS genomes -LRB- Fig. 4 -RRB- . Comparison of the nucleotide sequences of the APO-SUS and - UNSUS DNA regions with the corresponding database sequence -LRB- geneID : 117057 -RRB- revealed a 100 % identity between the database and the APO-UNSUS sequences , indicating that the APO-SUS genome has diverged from the database sequence . Next , more APO-SUS and - UNSUS rats were genotyped for this region . All APO-UNSUS rats tested -LRB- n = 5 -RRB- indeed showed the database sequence , whereas the APO-SUS rats tested -LRB- n = 5 -RRB- all contained the relatively high number of variations in this chromosomal region . Fig. 4Nucleotide sequence of chromosomal region 9q22 , corresponding to product 3 , in APO-SUS and APO-UNSUS rats . APO-SUS / - UNSUS genomic variations are indicated between brackets ; the first nucleotide represents the APO-UNSUS sequence , the second nucleotide the APO-SUS sequence . The nucleotide sequences corresponding to the annealing sites of arbitrary primer AP-777 are underlined The newly identified genetic and epigenetic variations , and apomorphine susceptibility in Wistar rats We wondered whether the molecular-genetic difference between the APO-SUS and - UNSUS rats -LRB- the Aph-1b gene-dosage imbalance -RRB- would also be present in the Nijmegen outbred population of Wistar rats , or if the imbalance was generated during the breeding of the rat lines . PCR analysis of the Aph-1b locus in the 50 Wistar rats examined revealed in all cases the presence of three copies of the Aph-1b gene , suggesting that the reduction of Aph-1b copies in the APO-SUS rats had been induced during the breeding of the rats . We next wondered whether the newly identified genetic and epigenetic variations in the APO-SUS and - UNSUS genomes were also induced during the breeding of the two rat lines , or if these variants are already present in Wistar rats . For this purpose , we performed AP-PCR analysis of -LRB- hypo -RRB- thalamic genomic DNAs from Wistar rats using primers AP-1 , -7 and -777 . The fingerprints revealed the presence of the APO-SUS as well as the - UNSUS variants of the genetic products 1 , 2 and 3 -LRB- n = 8 -RRB- and the epigenetic E1-product -LRB- n = 4 -RRB- in Wistar rats -LRB- Fig. 5 -RRB- , indicating that the newly identified variations were not induced during breeding of the APO-SUS and - UNSUS lines , but were already present in the original Wistar population . Fig. 5AP-PCR analysis -LRB- products 1 , 2 and E1 -RRB- and specific PCR -LRB- product 3 -RRB- analysis of genomic DNAs from Wistar rats with low or high apomorphine susceptibility . -LRB- A -RRB- The presence or absence of products 1 , 2 and 3 -LRB- genetic differences -RRB- was analysed in genomic DNAs from the -LRB- hypo -RRB- thalamus of Wistar rats with low -LRB- < 10 gnaws in 45 min -RRB- or high -LRB- > 500 gnaws in 45 min -RRB- apomorphine susceptibility . U : APO-UNSUS , S : APO-SUS . Lower panel : the amounts of the three products present in the Wistar rats were compared with the amounts found in the APO-SUS and - UNSUS rats . -LRB- ■ -RRB- the genotype of the genetic products 1 , 2 or 3 in the Wistar rats with low apomorphine susceptibility is similar to the products 1 , 2 and 3 genotype of the APO-UNSUS rats ; -LRB- □ -RRB- the genotype in the Wistar rats with low apomorphine susceptibility deviates from the APO-UNSUS genotype ; -LRB- -RRB- the genotype in the Wistar rats with high apomorphine susceptibility is comparable with the genotype in the APO-SUS rat ; -LRB- -RRB- the genotype in the Wistar rats with high apomorphine susceptibility deviates from the genotype in the APO-SUS rats . -LRB- B -RRB- AP-PCR analysis of the epigenetic E1-product -LRB- E1 -RRB- on genomic DNAs from cerebellum and hippocampus of four Wistar rats . The product indicated by the arrow is an example of a product that did not show variable amounts and was used for normalisation of the E1-product . The amounts of the E1-products generated from the cerebellum or hippocampus were not different between the two Wistar rats with low gnawing scores -LRB- < 10 gnaws in 45 min -RRB- and the two Wistar rats with high gnawing scores -LRB- > 500 gnaws in 45 min -RRB- To study whether the presence or absence of the genetic AP-PCR products observed in the APO-SUS and - UNSUS rats -LRB- products 1 , 2 and 3 -RRB- was linked to the apomorphine susceptibility of Wistar rats , we examined a group of five rats with low susceptibility to apomorphine -LRB- < 10 gnaws per 45 min -RRB- and a highly susceptible group of three rats -LRB- > 500 gnaws per 45 min -RRB- . Product 1 , which was less abundant in APO-SUS than - UNSUS rats , was not present in the three Wistar rats with high gnawing scores , but was also not found in two of the five Wistar rats with low gnawing scores . Of the eight rats examined , the APO-SUS-specific product 2 was found in two Wistar rats with a high and only one rat with a low degree of apomorphine susceptibility . The DNA region corresponding to product 3 was found twice as the APO-UNSUS sequence in the group consisting of the low-apomorphine-susceptible Wistar rats and once in the group of the high-apomorphine-susceptible rats -LRB- Fig. 5a -RRB- . These results indicate that none of the three products was directly linked to the apomorphine susceptibility of the Wistar rats . However , apomorphine susceptibility may not be the result of only a single genetic variation , but rather of multiple genetic and epigenetic alterations . Interestingly , 67 % of the three genetic products present in the Wistar rats with low apomorphine susceptibility were also found in the APO-UNSUS genome , and 78 % of the three products in Wistar rats with high apomorphine susceptibility were APO-SUS-specific variants -LRB- Fig. 5a -RRB- , indicating that a combination of the three genetic products may well be linked to apomorphine susceptibility . To study the link between the epigenetic E1-product and apomorphine susceptibility , two Wistar rats with low gnawing scores and two with high gnawing scores were examined for the amount of the E1-product in the cerebellum . Similar amounts of the E1-product as detected in APO-UNSUS rats were found in one Wistar rat with a low and in one Wistar rat with a high gnawing score . The amount of the E1-product detected in APO-SUS rats was found in one Wistar rat with low and in one Wistar rat with high apomorphine susceptibility , indicating that no direct link exists between the E1-product and apomorphine susceptibility . As observed in the APO-SUS and - UNSUS rats , no variation was detected in the level of the E1-product in hippocampal genomic DNAs from the four Wistar rats tested -LRB- Fig. 5b -RRB- . Discussion In this study , we investigated the genetic and epigenetic background of the phenotypically well-characterized APO-SUS and - UNSUS rats . Unravelling the molecular basis of this rat model may help in our understanding of complex human neurodevelopmental disorders , since many of the characteristics of the APO-SUS rat line are also observed in schizophrenic patients -LRB- Ellenbroek and Cools 2002 -RRB- . We recently identified a gene-dosage imbalance in the Aph-1b locus of the APO-SUS and - UNSUS rats , leading to a reduced expression of the Aph-1b gene in APO-SUS rats and a segregation with a number of behavioural parameters -LRB- Coolen et al. 2005 -RRB- . Here , we used AP-PCR analysis as an approach to explore the presence of any other alterations in the two genomes and epigenomes . We indeed identified additional genetic and epigenetic variations , indicating that the Aph-1b locus might not be solely responsible for the observed phenotypes of the two rat lines and suggesting a multi-genetic and - epigenetic origin of the differences observed between the APO-SUS and - UNSUS rats . Importantly , we found similar AP-PCR patterns in both the original and replicate APO-SUS and - UNSUS lines . It is therefore highly unlikely that the observed variations between the APO-SUS and - UNSUS rat genomes were simply due to coincidence . Detailed analysis of one of the newly identified genetic alterations -LRB- in the DNA region corresponding to product 3 -RRB- revealed in APO-SUS rats a cluster of variations in the myosin 1b gene . Hence , this cluster appears to be a hotspot for genetic instability . Product 1 was located upstream of the GIPC gene encoding a protein interacting with membrane-associated and transmembrane proteins , including the dopamine receptors D2 and D3 -LRB- Jeanneteau et al. 2004 -RRB- . The genetic variation in this locus may therefore contribute to the differences in apomorphine susceptibility between the APO-SUS and - UNSUS rat lines . We also identified genetic alterations using a primer corresponding to a topoisomerase II binding site consensus . The rationale for this study was based on our present finding that a topoisomerase II binding site was located at the recombination site in the Aph-1b locus . The two newly identified , topoisomerase II-based genetic variations might point to a more general role for topoisomerase II binding sites in psychopathological mechanisms . We hypothesize that at these sites environmental factors , such as stress during early development , may cause an increase in the incidence of recombination and other mutagenic events , leading to brain dysfunction and affected behavior . Besides the genetic differences , one tissue-specific epigenetic variation was found between the APO-SUS and - UNSUS epigenomes . At present it is not clear what , if any , functional consequence should be attributed to the decrease in the methylation status of this CpG in the -LRB- hypo -RRB- thalamus and cerebellum , but not in the hippocampus , of APO-SUS relative to APO-UNSUS rats . Earlier microarray analysis of mRNA expression in the hippocampus of the APO-SUS and - UNSUS rats -LRB- ∼ 7000 full-length sequences and ∼ 1000 EST clusters -RRB- revealed that only Aph-1b was differentially expressed -LRB- Coolen et al. 2005 -RRB- . The newly identified variations open the possibility that for an explanation of the background of the rat model one has to consider more genes that operate together in a multi-genetic and - epigenetic setting with several susceptibility loci . Thus , based on our present findings , more than one locus may be responsible for the complex phenotype of APO-SUS rats . Recently , it has been established that the contribution of genetic modifiers is also of importance for the outcome of a phenotype , since they can modulate the severity of the affected phenotype and the phenotypic characteristics without having a clear effect on the normal situation -LRB- Nadeau 2001 ; Nadeau and Topol 2006 -RRB- . Hence , as part of the genetic and epigenetic background of the APO-SUS and - UNSUS rat lines , our newly identified variants can be genetic and epigenetic modifiers influencing the phenotypic expression of the model . Insight into the genetic and epigenetic background may provide diagnostic tools , and clues for mechanisms and pathways to explain complex disorders . Besides the multiple variations in the APO-SUS and - UNSUS rat genomes and epigenomes , Aph-1b will presumably be a major player in the development of the complex APO-SUS phenotype , since it has a broad cellular effect via tissue-specific cleavage of many different substrates -LRB- Coolen et al. 2006b -RRB- and is functional already during early development -LRB- Coolen et al. 2006a -RRB- . Remarkably , however , the number of Aph-1b gene copies was not linked to apomorphine susceptibility in Wistar rats . Whereas their apomorphine susceptibility varied , all unselected Wistar rats tested harboured three copies of the Aph-1b gene . Therefore , genetic and epigenetic factors other than Aph-1b will likely contribute to the susceptibility for apomorphine . We now indeed found a correlation between the newly identified genetic variations and the apomorphine susceptibility in the Wistar population , confirming our hypothesis that apomorphine susceptibility is caused by a number of genetic and epigenetic factors . A combination of the newly identified variations may have thus initially contributed to the degree of apomorphine susceptibility in the original Wistar rat population . Subsequent apomorphine injections to determine the susceptibility for the drug during the breeding of the APO-SUS and - UNSUS lines may have acted as an environmental stressor , triggering the Aph-1b recombination event at the topoisomerase II binding site only in rats with a high susceptibility for apomorphine . The induced gene-dosage imbalance of the Aph-1b gene , probably in combination with other genetic or epigenetic factors , could then have led to the complex phenotype observed in the APO-SUS rats . In conclusion , the present findings suggest that psychopathological disturbances may be the result of multiple genetic as well as epigenetic factors . We infer that our newly identified variations are susceptibility loci for schizophrenia-like features in the rat and may give new insights into the genetic and epigenetic background of complex neurodevelopmental disorders .

O O B I I I O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O

genetic and epigenetic variations
neurodevelopmental disorders


Isolated

Limb

Perfusion

with

Tumor

Necrosis

Factor

Alpha

and

Melphalan

for

Locally

Advanced

Soft

Tissue

Sarcoma

Three

Time

Periods

at

Risk

for

Amputation

Background

The

aim

of

this

study

was

to

investigate

the

long-term

limb

salvage

rate

and

overall

survival

after

isolated

limb

perfusion

-LRB-

ILP

-RRB-

with

tumor

necrosis

factor

alpha

and

melphalan

for

locally

advanced

soft

tissue

sarcoma

-LRB-

STS

-RRB-

Limb

salvage

in

patients

with

locally

advanced

extremity

soft

tissue

sarcoma

-LRB-

STS

-RRB-

continues

to

be

a

challenge

Survival

in

these

patients

is

determined

by

the

development

of

distant

metastases

and

is

not

improved

with

the

amputation

of

the

affected

limb

Besides

amputation

an

extensive

surgical

procedure

followed

by

radiotherapy

is

a

treatment

option

Rosenberg

et

al.

showed

the

same

disease-free

and

overall

survival

as

amputation

in

the

early

with

this

treatment

regimen

Preoperative

therapies

to

improve

limb

salvage

rates

have

been

proposed

Suit

et

al.

reported

in

on

the

use

of

preoperative

radiotherapy

Eilber

et

al.

combined

preoperative

-LRB-

intra-arterial

or

systemic

-RRB-

chemotherapy

and

radiotherapy

to

improve

resectability

rates

In

a

randomized

trial

O'Sullivan

et

al.

reported

a

greater

risk

of

wound

complications

in

the

preoperative

radiotherapy

group

compared

with

the

postoperative

radiotherapy

group

The

use

of

brachytherapy

may

also

improve

local

control

and

avoid

amputation

The

current

treatment

strategy

of

high-grade

limb

sarcomas

is

wide

local

resection

with

the

goal

of

achieving

a

resection

with

a

margin

If

the

margin

is

cm

or

if

a

resection

-LRB-

microscopically

involved

margin

-RRB-

is

performed

adjuvant

radiotherapy

with

Gy

is

indicated

to

reduce

the

risk

of

local

failure

The

question

whether

radiotherapy

should

be

given

before

or

after

surgery

is

still

unanswered

Another

strategy

for

limb

salvage

in

locally

advanced

extremity

STS

is

to

perform

an

isolated

limb

perfusion

-LRB-

ILP

-RRB-

with

cytostatic

agents

This

procedure

which

was

originally

developed

for

the

treatment

of

melanoma

of

the

limb

in

was

also

applied

to

the

treatment

of

STS

of

the

limb

In

their

first

experience

Krementz

et

al.

showed

an

early

response

rate

of

with

melphalan

alone

however

complete

regression

of

the

tumor

was

rarely

seen

Other

perfusion

agents

in

the

treatment

of

limb

STS

were

therefore

investigated

Rossi

et

al.

claimed

that

doxorubicin

was

efficacious

another

showed

that

doxorubicin

alone

was

ineffective

and

that

combined

with

melphalan

it

was

too

toxic

Cisplatin

also

proved

to

be

less

effective

than

melphalan

in

the

limb

perfusion

setting

of

sarcomas

and

carboplatin

was

too

neurotoxic

With

the

addition

of

tumor

necrosis

factor

alpha

-LRB-

TNF-α

-RRB-

to

the

perfusion

circuit

Lienard

et

al.

made

a

step

forward

in

the

treatment

of

locally

advanced

extremity

STS

A

large

European

multicenter

study

proved

the

ILP

concept

in

the

limb

salvage

procedures

for

locally

advanced

STS

with

TNF-α

and

melphalan

The

objective

response

rate

was

and

a

limb

salvage

rate

of

was

achieved

with

minimal

treatment-related

morbidity

Since

patients

with

locally

advanced

STS

of

the

limbs

have

been

treated

at

the

University

Medical

Center

Groningen

by

ILP

with

TNF-α

and

melphalan

with

or

without

interferon

gamma

as

perfusion

agents

followed

by

delayed

surgical

excision

and

postoperative

radiotherapy

if

a

marginal

resection

or

nonradical

resection

was

performed

Recently

we

encountered

long-term

local

morbidity

and

therefore

the

aim

of

the

present

study

was

to

analyze

the

limb

salvage

rate

and

survival

in

patients

with

locally

advanced

STS

of

the

extremities

that

were

treated

in

our

center

and

to

report

the

late

effects

of

this

treatment

modality

PATIENTS

AND

METHODS

Patient

Characteristics

During

a

total

of

patients

with

STS

of

the

extremity

underwent

perfusions

with

a

combination

of

TNF-α

and

melphalan

with

-LRB-

n

-RRB-

or

without

-LRB-

n

-RRB-

interferon

gamma

Thirty-six

men

and

women

with

a

median

age

of

-LRB-

range

-RRB-

years

were

treated

Tumors

were

considered

unresectable

because

of

size

their

multicentricity

in

the

limb

or

fixation

to

the

neurovascular

bundle

and/or

bone

and

therefore

amputation

was

the

only

treatment

option

Perfusion

was

performed

at

the

iliac

level

in

cases

-LRB-

-RRB-

at

the

popliteal

level

in

cases

-LRB-

-RRB-

and

at

the

femoral

and

axillary

level

in

cases

each

-LRB-

-RRB-

There

were

primary

-LRB-

-RRB-

and

recurrent

-LRB-

-RRB-

sarcomas

Sixty-two

sarcomas

were

located

in

the

leg

-LRB-

-RRB-

and

were

located

in

the

arm

-LRB-

-RRB-

All

patients

were

treated

after

informed

consent

was

obtained

according

to

institutional

guidelines

Nineteen

different

histological

types

of

STS

were

distinguished

The

pathological

grade

of

the

tumor

was

scored

following

the

criteria

of

Coindre

et

al.

and

the

stage

of

the

tumor

was

scored

according

to

the

American

Joint

Committee

on

Cancer

-LRB-

AJCC

-RRB-

-LRB-

Table

-RRB-

Median

tumor

size

was

-LRB-

range

-RRB-

cm

In

case

of

multifocal

disease

the

largest

diameter

was

used

TABLE

Histological

grade

and

stage

of

tumorsaCharacteristicn

Grade

Grade

according

to

Coindre

et

al.

and

stage

according

to

American

Joint

Committee

on

Cancer

Perfusion

Technique

The

perfusion

technique

used

at

the

University

Medical

Center

Groningen

is

based

on

the

technique

developed

by

Creech

et

al.

and

described

elsewhere

The

major

modifications

during

the

last

years

were

the

use

of

modern

thermal

blankets

improvement

in

leakage

monitoring

and

the

introduction

of

a

membrane

oxygenator

and

heat

exchanger

to

ensure

optimal

perfusion

at

°C

Because

extensive

washing

with

L

of

saline

is

used

systemic

inflammatory

response

syndrome

-LRB-

SIRS

-RRB-

is

rarely

seen

After

surgery

patients

can

be

monitored

on

the

recovery

ward

instead

of

the

intensive

care

unit

Assessment

of

Tumor

Response

Tumor

Remnant

and

Follow-up

Responses

were

assessed

by

standardized

World

Health

Organization

criteria

and

on

the

basis

of

physical

examination

and/or

imaging

investigations

-LRB-

magnetic

resonance

imaging

and

computed

tomographic

scans

-RRB-

Complete

response

was

defined

as

the

disappearance

of

all

measurable

disease

in

the

limb

for

weeks

partial

response

as

regression

of

the

tumor

size

by

of

the

largest

diameter

for

weeks

and

no

change

as

regression

of

of

the

tumor

in

the

limb

or

progression

of

for

longer

than

weeks

Resection

of

the

tumor

remnants

was

performed

weeks

-LRB-

median

weeks

-RRB-

after

perfusion

After

resection

response

was

also

assessed

by

pathological

examination

The

tumor

remnants

were

measured

in

three

dimensions

and

the

percentage

of

necrosis

estimated

in

relation

to

the

complete

tumor

volume

Representative

tumor

sections

were

taken

encompassing

macroscopically

different

tumor

areas

including

necrosis

As

a

general

rule

one

section

per

centimeter

largest

diameter

with

a

minimum

of

three

was

taken

On

the

basis

of

an

integration

of

gross

and

microscopic

findings

a

final

estimate

of

the

percentages

of

viable

and

necrotic

or

regressive

tumor

was

made

Excision

margins

were

also

evaluated

on

pathological

examination

and

classified

as

radical

when

the

resection

margins

were

free

of

tumor

cells

-LRB-

complete

resection

-RRB-

as

when

resection

margins

were

microscopically

involved

or

as

when

resection

margins

were

macroscopically

positive

involved

Postoperative

radiotherapy

-LRB-

Gy

-RRB-

was

considered

indicated

in

case

of

necrosis

on

pathological

examination

of

the

tumor

or

with

marginal

or

microscopically

positive

resection

margins

All

patients

were

followed

after

perfusion

treatment

in

a

standardized

protocol

Median

follow-up

was

-LRB-

range

-RRB-

months

Statistical

Analysis

Survival

and

limb

salvage

curves

were

calculated

according

to

the

Kaplan-Meier

method

and

log

rank

test

Values

of

P

were

considered

to

be

statistically

significant

GraphPad

Prism

version

for

Windows

statistical

software

was

used

RESULTS

Tumor

Response

A

clinical

complete

response

was

observed

after

ILPs

-LRB-

-RRB-

a

partial

response

after

ILPs

-LRB-

-RRB-

and

no

change

after

ILPs

-LRB-

-RRB-

local

progression

was

never

observed

Resection

of

the

remnant

tumor

was

performed

in

patients

-LRB-

-RRB-

The

pathological

response

is

illustrated

in

Fig.

After

ILPs

-LRB-

-RRB-

no

viable

tumor

cells

were

found

on

pathological

examination

In

ILPs

-LRB-

-RRB-

necrosis

was

found

on

pathological

examination

When

both

groups

were

added

together

a

good

response

to

ILP

was

found

in

of

the

patients

In

ILPs

-LRB-

-RRB-

an

intermediate

response

was

found

on

pathological

examination

-LRB-

necrosis

-RRB-

After

eight

ILPs

-LRB-

-RRB-

of

necrosis

or

no

necrosis

was

found

on

pathological

examination

In

five

patients

-LRB-

-RRB-

tumor

response

was

not

assessed

because

of

progression

of

distant

metastases

in

four

patients

and

a

local

recurrence

in

one

patient

that

necessitated

a

second

perfusion

resulting

in

necrosis

of

the

tumor

No

correlation

could

be

demonstrated

between

grade

and

percentage

of

tumor

necrosis

after

perfusion

-LRB-

Pearson

's

correlation

-RRB-

Postoperative

radiotherapy

-LRB-

total

dose

Gy

Gy

daily

and

Gy

boost

-RRB-

was

given

in

patients

with

microscopically

involved

or

marginally

free

resection

margins

Radiotherapy

was

initiated

within

weeks

after

tumor

resection

Radiation

treatment

was

delivered

through

a

multiple-field

technique

with

computed

tomographic

treatment

planning

on

a

linear

accelerator

MV

FIG.

Percentage

of

necrosis

estimated

at

pathological

examination

of

resected

tumor

remnant

in

relation

to

number

of

patients

Amputations

and

Limb

Salvage

A

total

of

amputations

-LRB-

-RRB-

were

performed

Table

presents

the

time

interval

between

ILP

and

amputation

and

the

rationale

for

amputation

Overall

and

years

limb

salvage

was

and

respectively

-LRB-

Fig.

-RRB-

When

we

analyzed

the

limb

salvage

curve

we

found

that

the

risk

for

amputation

could

be

divided

into

three

time

episodes

The

first

period

occurs

within

the

first

year

or

year

and

a

half

after

perfusion

-LRB-

n

patients

-RRB-

with

amputation

performed

to

treat

massive

perfusion-induced

necrosis

of

the

tumor

and

overlying

skin

which

resulted

in

a

soft

tissue

deficit

-LRB-

n

-RRB-

tumor

recurrence

after

perfusion

-LRB-

n

-RRB-

wound

complications

after

ILP

followed

by

radiotherapy

-LRB-

n

-RRB-

a

microscopically

involved

resection

margin

with

the

rejection

of

the

patient

for

adjuvant

radiotherapy

of

the

foot

-LRB-

n

-RRB-

insufficient

clinical

response

-LRB-

one

patient

-RRB-

and

arterial

thrombosis

with

no

vascular

reconstruction

possibilities

months

after

resection

of

a

local

recurrence

in

the

groin

-LRB-

n

-RRB-

TABLE

Amputations

performed

in

patients

according

to

interval

durationPatient

no.DiagnosisAge

-LRB-

y

-RRB-

Interval

-LRB-

mo

-RRB-

ResectionRTRationale

for

amputationCurrent

recurrenceDOD

necrosisNED

necrosisDOD

necrosisDOD

clinical

responseDOD

resection

RT

not

possibleAWD

recurrenceDOD

necrosisDOD

necrosisNED

recurrenceDOD

resection

RT

not

possibleNED

recurrenceaDOD

complications

after

ILP

with

radiotherapyaAWD

complications

after

ILP

with

radiotherapyaNED

occlusionAWD

recurrenceNED

recurrenceDOD

recurrenceDOD

leg

ischemiaNED

leg

ischemiaNED

monthsPUS

pleomorphic

undifferentiated

sarcoma

RT

radiotherapy

NED

no

evidence

of

disease

AWD

alive

with

disease

DOD

death

of

disease

PNET

malignant

peripheral

nerve

sheath

tumor

resection

microscopically

involved

resection

margin

ILP

isolated

limb

perfusion.a

Patients

treated

for

second

ILP

with

tumor

necrosis

factor

alpha

and

melphalan.FIG

Limb

salvage

curve

in

patients

with

locally

advanced

soft

tissue

sarcoma

treated

with

tumor

necrosis

factor

alpha

melphalan

and

isolated

limb

perfusion

The

second

time

period

was

within

years

after

ILP

with

two

amputations

performed

for

late

local

recurrent

disease

-LRB-

and

months

after

perfusion

-RRB-

The

third

episode

occurred

approximately

years

after

perfusion

Amputation

was

performed

for

critical

leg

ischemia

with

neuropathy

due

to

treatment-induced

atherosclerosis

of

the

remaining

tibial

artery

that

was

not

suitable

for

arterial

reconstruction

-LRB-

and

months

after

perfusion

-RRB-

An

example

of

the

clinical

appearance

of

patient

is

shown

in

Fig.

In

this

patient

a

popliteal

ILP

was

performed

at

the

age

of

years

for

a

chondrosarcoma

After

marginal

resection

this

patient

received

Gy

adjuvant

radiotherapy

Ten

years

after

ILP

an

amputation

was

performed

because

of

critical

leg

ischemia

No

recurrent

disease

was

found

on

pathological

examination

of

the

amputated

specimen

Another

two

patients

developed

a

pathological

fracture

of

the

femur

due

to

radiation-induced

osteonecrosis

-LRB-

and

months

after

perfusion

-RRB-

These

four

patients

with

late

post-ILP

complications

received

high-dose

postperfusion

radiotherapy

-LRB-

Gy

-RRB-

FIG.

Clinical

appearance

of

lower

leg

of

patient

-LRB-

Table

-RRB-

before

amputation

for

critical

leg

ischemia

Systemic

Metastases

and

Survival

Twelve

patients

presented

with

distant

metastases

at

time

of

ILP

-LRB-

stage

IV

AJCC

-RRB-

half

of

these

patients

had

lung

metastases

and

the

other

half

had

lymph

node

metastases

Eleven

of

these

patients

died

of

disease

after

a

median

period

of

-LRB-

range

-RRB-

months

one

patient

is

alive

with

no

evidence

of

disease

after

months

During

follow-up

patients

-LRB-

-RRB-

developed

distant

metastases

at

a

median

interval

of

-LRB-

range

-RRB-

months

A

statistically

significant

difference

-LRB-

P

-RRB-

was

observed

between

patients

with

no

distant

metastases

at

the

time

of

ILP

compared

with

patients

with

metastases

at

the

time

of

ILP

-LRB-

Fig.

-RRB-

Overall

and

years

survival

for

all

patients

was

and

respectively

-LRB-

Fig.

-RRB-

FIG.

Overall

survival

in

patients

with

locally

advanced

soft

tissue

sarcoma

treated

with

isolated

limb

perfusion

-LRB-

ILP

-RRB-

tumor

necrosis

factor

alpha

and

melphalan

Statistically

significant

difference

was

observed

between

patients

with

no

distant

metastases

at

time

of

ILP

-LRB-

mets

-RRB-

compared

with

patients

with

metastases

at

time

of

ILP

-LRB-

mets

-RRB-

DISCUSSION

The

results

of

a

European

multicenter

trial

performed

in

the

led

to

the

approval

of

the

use

of

TNF-α

for

ILP

in

patients

with

locally

advanced

extremity

sarcomas

by

the

European

Medicine

Evaluation

Agency

ILP

with

TNF-α

is

currently

available

in

more

than

centers

and

in

a

total

of

so-called

TNF-α

perfusions

were

performed

As

one

of

the

first

centers

that

participated

in

the

TNF-α

ILP

experience

and

with

more

than

a

decade

's

experience

we

recently

encountered

long-term

treatment-related

morbidity

necessitating

amputation

of

the

perfused

limb

years

after

treatment

For

this

reason

we

analyzed

our

results

of

ILP

with

TNF-α

and

melphalan

and

describe

our

results

in

the

present

study

We

observed

an

overall

response

rate

of

which

is

in

the

range

of

the

response

rates

reported

in

the

literature

Although

a

suggestion

has

been

made

for

a

relation

between

the

grade

of

sarcoma

and

the

response

to

TNF-α

ILP

we

could

not

demonstrate

a

correlation

between

grade

and

the

percentage

of

necrosis

after

ILP

with

TNF-α

This

is

in

concordance

with

the

results

of

the

Amsterdam

group

Various

reports

have

shown

that

a

limb

salvage

rate

of

can

be

achieved

in

patients

with

locally

advanced

limb

sarcoma

An

independent

review

committee

reconsidered

the

unresectability

criteria

of

all

patients

enrolled

onto

the

European

study

Eighty

percent

of

the

patients

in

this

study

met

the

criteria

for

unresectability

and

survival

curves

based

on

a

match-control

study

with

cases

of

the

Scandinavian

Soft

Tissue

Sarcoma

Databank

showed

that

TNF-α

ILP

had

no

negative

effect

on

survival

We

used

the

Kaplan-Meier

method

to

calculate

limb

salvage

because

this

method

adjusts

for

censored

observations

i.e.

patients

who

were

alive

and

well

at

the

time

of

last

contact

or

patients

who

died

of

distant

metastases

but

who

retained

preserved

limb

function

By

use

of

this

method

we

calculated

a

limb

salvage

rate

of

Amputations

were

performed

mostly

because

of

postperfusion-related

complications

or

early

local

recurrence

in

the

first

year

after

ILP

A

second

curve

in

limb

salvage

was

observed

within

years

after

TNF-α

ILP

in

two

patients

with

late

local

recurrences

A

third

bend

in

the

limb

salvage

curve

was

observed

approximately

years

after

ILP

This

was

a

new

observation

in

two

patients

who

had

critical

leg

ischemia

with

ulceration

and

continuous

pain

Besides

ILP

with

TNF-α

and

melphalan

both

patients

received

adjuvant

radiotherapy

-LRB-

and

Gy

-RRB-

after

marginal

tumor

resection

What

seems

to

be

the

cause

of

this

late

morbidity

Analysis

of

the

functional

and

long-term

morbidity

in

patients

with

stage

I

melanoma

treated

with

ILP

with

melphalan

as

the

sole

perfusion

agent

in

our

center

showed

after

a

median

follow-up

of

-LRB-

range

-RRB-

months

no

patients

had

critical

leg

ischemia

The

Rotterdam

and

Amsterdam

perfusion

group

reported

long-term

morbidity

consisting

of

muscle

atrophy

or

fibrosis

in

of

the

patients

after

ILP

with

melphalan

however

cases

of

critical

leg

ischemia

are

not

described

The

fact

that

in

our

series

no

muscle

atrophy

or

fibrosis

was

found

might

be

explained

by

the

fact

that

we

always

perform

a

lateral

fasciotomy

after

ILP

to

prevent

a

compartment

syndrome

When

we

performed

a

literature

search

for

late

morbidity

after

ILP

with

TNF-α

and

melphalan

no

studies

could

be

retrieved

The

clinical

importance

of

late

morbidity

after

radiotherapy

has

evolved

since

Eifel

et

al.

retrospectively

reviewed

the

medical

records

of

FIGO

stage

IB

patients

receiving

primary

radiotherapy

at

the

M.

D.

Anderson

Cancer

Center

between

and

She

showed

that

after

years

there

was

a

small

but

continuous

risk

of

experiencing

major

complications

of

radiotherapy

-LRB-

i.e.

urinary

rectal

and

small

bowel

complications

-RRB-

at

up

to

years

of

follow-up

Johansson

et

al.

described

a

high

occurrence

of

severe

neuropathy

closely

linked

to

the

development

of

fibrosis

around

the

nerve

trunks

after

aggressive

postoperative

telecobalt

therapy

received

in

in

a

group

of

patients

with

breast

cancer

who

were

initially

treated

with

modified

radical

mastectomy

Radiotherapy

damage

to

the

vascular

system

was

demonstrated

by

in

an

experimental

setting

arteries

of

hamster

cheek

pouch

showed

localized

constrictions

after

irradiation

These

constrictions

were

caused

by

clones

of

endothelial

cells

and

may

be

the

predominant

factor

influencing

the

degeneration

of

the

capillary

bed

after

radiotherapy

Evidence

of

this

occlusive

effect

of

vessels

by

proliferating

endothelial

cells

after

radiation

have

also

been

reported

by

other

investigators

Another

argument

to

explain

the

observed

late

morbidity

at

least

in

part

to

radiotherapy

is

the

fact

that

another

two

patients

who

received

adjuvant

radiotherapy

after

ILP

with

TNF-α

and

melphalan

developed

a

pathological

fracture

of

the

femur

-LRB-

and

months

after

ILP

with

TNF-α

and

melphalan

-RRB-

Radiotherapy-induced

osteonecrosis

is

a

well-known

phenomenon

Lin

et

al.

described

fractures

of

the

femur

after

surgery

and

irradiation

for

STS

of

the

thigh

Treatment

of

these

fractures

was

difficult

and

demanding

with

only

four

bony

unions

after

a

mean

follow-up

of

months

When

we

add

up

the

evidence

of

developing

fibrosis

after

ILP

with

melphalan

and

the

development

of

fibrosis

after

radiotherapy

the

combination

of

the

two

regimens

could

explain

the

observed

late

morbidity

rate

in

the

present

series

Overall

survival

for

all

patients

showed

a

steadily

decline

with

a

percentage

of

Even

after

and

months

patients

die

of

distant

metastases

Sixteen

percent

of

the

patients

had

metastases

at

the

time

of

ILP

A

marked

difference

in

survival

was

observed

when

comparing

patients

with

pulmonary

or

lymph

node

metastases

with

patients

lacking

metastases

at

time

of

ILP

Five-year

overall

survival

of

in

this

series

is

higher

than

the

reported

survival

of

in

the

Amsterdam

and

the

reported

of

Lejeune

et

al.

This

is

an

unexpected

observation

because

selection

criteria

for

ILP

with

TNF-α

between

the

institutes

are

comparable

High-grade

tumors

and

tumors

cm

in

diameter

have

a

great

tendency

to

metastasize

Patients

with

such

tumors

could

theoretically

benefit

from

neoadjuvant

chemotherapy

A

quantitative

meta-analysis

of

data

from

trials

of

doxorubicin-based

adjuvant

chemotherapy

showed

a

benefit

from

systemic

adjuvant

chemotherapy

of

for

local

relapse-free

interval

but

there

was

no

overall

survival

benefit

at

years

Delaney

et

al.

developed

a

regimen

of

preoperative

chemotherapy

consisting

of

mesna

Adriamycin

ifosfamide

and

dacarbazine

-LRB-

MAID

-RRB-

interspersed

with

radiotherapy

followed

by

resection

and

postoperative

chemotherapy

with

or

without

radiotherapy

to

improve

outcome

in

patients

with

high-grade

extremity

STS

Compared

with

a

historical

group

of

control

patients

outcome

in

the

MAID

group

was

better

In

an

update

of

patients

required

amputation

because

of

disease

had

unresectable

disease

and

patient

refused

surgery

Estimated

survival

and

local-regional

control

were

and

respectively

These

results

are

comparable

with

the

results

of

the

present

study

However

systemic

therapy

is

associated

with

systemic

toxic

effects

in

contrast

with

the

mild

systemic

side

effects

observed

after

ILP

with

TNF-α

and

melphalan

Since

we

have

not

changed

the

indication

for

TNF-α

perfusion

Patients

who

were

candidates

for

amputation

of

the

involved

limb

as

assessed

by

preoperative

magnetic

resonance

imaging

were

offered

an

ILP

with

TNF-α

and

melphalan

with

the

goal

of

preserving

the

limb

with

a

locally

advanced

STS

After

ILP

patients

received

a

delayed

surgical

resection

adjuvant

radiotherapy

was

provided

to

patients

with

marginal

or

microscopically

positive

resection

margins

This

treatment

resulted

in

a

high

limb

salvage

rate

in

patients

with

locally

advanced

STS

although

late

morbidity

can

occur

especially

when

adjuvant

postoperative

radiotherapy

is

applied

Therefore

continuous

follow-up

of

these

patients

is

warranted

B I I O B I I I O B O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O B I I O O O O B I I I O B O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O B O O B I I I O B O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I I O O O O B I I I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O

isolated limb perfusion
sarcoma
tumor necrosis factor alpha
melphalan
radiotherapy
amputation
late morbidity