BACKGROUND
genetic and epigenetic changes are among the hallmarks of cancer, with the illegitimate amplification of oncogenes and anti-apoptotic genes, and inactivation or deletion of tumour suppressors and pro-apoptotic genes as the main features.
positional cloning of such genes is dependent on high-resolution methods that can detect losses and gains of chromosome segments.
several techniques have been developed that can detect dna copy number changes.
fluorescence in situ hybridization  was introduced in the early 1980s  <cit> .
this is based on the use of chromosome region specific fluorescent-labelled dna probes.
it can dissect complex chromosomal aberrations, including very small sub-microscopic deletions and permits studies on interphase cells.
it can reveal heterogeneity while also giving a general view of the whole karyotype.
large-scale analysis is time consuming, however, since it requires separate hybridizations and individual microscope analysis.
comparative genomic hybridization  has facilitated the detection of dna copy number changes.
it was first restricted to metaphase chromosomes   <cit> .
however, recently high-resolution techniques have been developed using dna fragments instead of metaphase chromosomes   <cit> .
the resolution of metaphase cgh is about  <dig> mb, whereas array-cgh can resolve hundreds base pairs  or hundreds kilo bases  and permits the detection of cryptic copy number changes.
thus, the whole genome can be screened in a single experiment  <cit> .
array-cgh cannot detect balanced chromosomal translocations, insertions and inversions, however, and makes the analysis of heterogeneous and normal cell contaminated tumour samples difficult.
another drawback is that cgh only detects relative copy number changes and cannot be used to determine the real segmental copy number and overall ploidy  <cit> .
in order to interpret  complex cgh profiles and to describe the underlying chromosome rearrangements, additional information is required.
the array-cgh results may be validated by a reciprocal approach, fish, that analyzes tumour cells with locus specific dna.
in the present study, nine tumour cell lines were analyzed in order to establish rules that would facilitate the interpretation of array-cgh data.
we focused our analysis on chr <dig> since it is frequently affected in solid tumours  <cit> .
a  <dig> bac/pac based chr <dig> array-cgh was constructed, and the results of chr <dig> array analysis were compared with multipoint fish , using the same  <dig> bac/pac clones as probes for interphase and metaphase chromosome analysis.
on the basis of this analysis we propose a relatively simple approach to calculate locus specific copy number and ploidy.
we show that the method can detect single copy number changes in up to near-pentaploid cells.
RESULTS
development of mpfish and array-cgh for detailed chr <dig> analysis
we have analyzed ten carcinoma cell lines with cytogenetic methods.
the chromosome number was counted for a minimum  <dig> metaphase spreads in each cell line .
subsequently, all lines were examined by chr <dig> painting.
several chr <dig> fragments were identified on differently rearranged marker chromosomes .
all of them had a characteristic morphology that allowed their identification on metaphase plates .
to identify the composition of each chr <dig> fragment on the marker chromosomes we developed a modified fish method, called mpfish.
mpfish can be introduced as a macro-array fish, which is the reciprocal of micro-array cgh.
while in array-cgh the bac/pac dna is spotted in micro-dots on the slide and the tumour dna is hybridized to it, in the mpfish, the tumour nuclei and chromosomes are fixed and the different bac/pac dnas are hybridized to multiple areas on the slide , in pairs of two  colour labelled probes .
by this improved fish application, we reduced the amount of the probe to 100– <dig> ng of labelled probe dna for the analysis of 10– <dig> samples.
we could also analyze 20– <dig> probes in double-colour hybridisation on each slide in a single experiment.
this has led to a considerable reduction in cost and time.
a set of  <dig> commercially available, fish mapped, chr <dig> specific bac/pac probes was used to analyze the ten cancer cell lines using interphase and metaphase mpfish .
one cell line, as expected  <cit> , had normal chrs <dig> and it was excluded from further analysis.
in total  <dig> regions, which represent a row of neighbouring clones with the same copy number, were identified and characterized in the remaining nine cell lines .
the majority of the chr <dig> rearrangements were generated by unbalanced translocations, but we could also identify interstitial deletions, duplications and even complex patterns of amplifications .
the copy number of each region was calculated based on result of metaphase and interphase fish .
majority of the tumour cell lines, analyzed here, had almost homogeneous cell populations.
in the cases of heterogeneous tumour cell lines, like tk <dig>  the predominant population  copy number was used.
in parallel with the mpfish analysis, a chr <dig> cgh array was set up covering both arms.
it contained the same set of  <dig> chr <dig> bac/pac clones as used for the mpfish experiments.
six chrx and  <dig> autosomal clones were used as controls.
dop-pcr amplified probes, generated by three different universal primers  <cit>  were followed by aminolinker pcr, pooled and spotted on code-link slides.
array-cgh was performed on the same nine cancer cell lines.
the ratios of the mean fluorescence intensity of test dna versus reference dna were calculated for each clone.
the average ratio of the autosomal controls was used for normalization.
each dot on the array-cgh charts  represents the normalised fluorescent ratio .
the anilfr  and standard deviation  values were calculated for each of the  <dig> mpfish defined rearranged chr <dig> regions  in order to assess the interlocus variation .
mpfish identifies mismapped clones
detailed analysis of array-cgh results is always required to exclude suspicious copy number changes.
we could see several clones with extreme values in the majority of array-cgh profiles, which needed more detailed investigation.
bac/pac clones were ordered on each array-cgh profile according to their mb position on chr <dig> in the ucsc database  <cit> .
by mpfish, we verified the localization of the clones on normal chr <dig>  and clones with wrong database localization could be readily identified.
for example, clone rp11-129g <dig> has two locations in the ucsc database  <cit> :  <dig>  –  <dig>  mb  and  <dig>  mb ; mpfish confirmed only the 3q <dig> localisation.
this was in agreement with our array-cgh data where the copy number of this clone indicated that it was localized on 3q.
mpfish validated also the localization of rp11-732m <dig> at 3p24- <dig>  while the ucsc database  <cit>  placed it at 3q <dig>  .
there was a second group of clones that according to database information had well defined localisations on chr <dig>  but they gave additional signals on other chromosomes upon mpfish analysis and had extreme hybridization ratios on the array-cgh.
these clones appeared to represent parts of segmental duplications detected by blastn .
these segmental duplications were not represented in the ucsc database  <cit> , because the similarity was just below the database threshold .
the third category of clones was eliminated because they were localized on chromosomes other than chr <dig>  altogether  <dig> clones  were eliminated from evaluation .
chr <dig> aberrations by array-cgh and mpfish
mpfish analysis of the nine cell lines identified  <dig> regions of different copy number on chr <dig>  based on array-cgh results, we assessed the anilfr and standard deviation for each region .
the anilfr values were different for regions with different copy numbers.
gains were found in  <dig> regions , losses in  <dig> .
losses were more frequent in 3p  while gains were more frequent on 3q .
we could visualize six interstitial losses .
only two were interstitial deletions ; the other four were generated by two translocations .
an example of interstitial loss is provided by the homozygous deletion , previously characterized in a diploid small cell lung cancer cell line, u <dig>  <cit> .
on the array-cgh profile, the homozygous deletion is identified by clones that show a value of anilfr ±  <dig> sd;  <dig>  ±  <dig>  .
mpfish using these probes could not detect any nuclear signal, due to a complete loss of this chr <dig> segment in u <dig> .
reconstitution of different chr <dig> fragments on the marker chromosomes confirmed that the same region was lost .
the analysis made also clear that the u <dig> deletion was generated by three different translocations.
only one of eight interstitial gains  detected on the array-cgh profiles was due to a simple duplication .
the others were due to translocations .
all three terminal losses  and six terminal gains  originated from unbalanced translocations.
an example of tg is shown on the array-cgh profile of uok <dig> where the copy number of the whole 3q increases by one added copy, due to a translocation with breakpoint at the centromere .
sequential, stepwise changes were most frequent .
they were generated by intermediate copy number changes leading to interstitial or terminal gains/losses.
they were initiated by translocations, followed by secondary rearrangements that generated further changes in copy number .
such stepwise changes have led to cumulative segmental amplifications in caki <dig> and hone <dig> .
the array-cgh profile of copy number gain at 3q <dig> in hone <dig> resulted from a complex rearrangement leading to the amplification of this segment .
increase in copy number can be measured by interphase mpfish and the rearrangements can be reconstituted by analyzing metaphase chromosomes in the same culture.
in addition to the rearrangements mentioned, we found three chr <dig> aberrations by mpfish that were not detected by array-cgh: two insertions and one balanced translocation.
metaphase mpfish on figure 4a shows that the marker chromosome m <dig> in uok <dig> had a chromosome fragment inserted in the pericentromeric region.
this breakpoint was not identified by array-cgh , because the copy number remained unchanged.
in the case of properly selected probes, interphase mpfish can detect insertion .
the change detected by array-cgh at this region was loss of the short arm of chr <dig> as a result of an unbalanced translocation in m <dig> .
it should be also mentioned that changes detected by array-cgh based on single clone data  should be always confirmed by additional methods, like fish .
correlation between anilfr and segment copy number
we compared anilfr identified by array-cgh for regions that were determined by > <dig> clones, with copy number assessed by mpfish .
figure  <dig> shows the relationship between these two values.
each region with a certain copy number was represented by a point on the graph where the x-coordinate corresponded to the region copy number obtained from mpfish analysis, while the y-coordinate corresponded to its anilfr.
the number of data points was  <dig> in uok <dig>  uok115;  <dig> in caki <dig>  a <dig> and tk164;  <dig> in u2020;  <dig> in caki2;  <dig> in uok <dig> and  <dig> in hone <dig> equivalent to the number of regions with different copy numbers of the respective cell line.
based on these data points, the trend lines for each cell line  were calculated in excel.
all nine trend lines followed the linear function of y = kx+b; where y is the anilfr determined by array-cgh; x is the copy number from mpfish.
the constant b is equal to y =  <dig> , corresponding to the crossing of all trend lines on y-axis.
this value was equivalent also to anilfr of the homozygous deletion in u <dig>  thus reflecting the background anilfr.
the other constant, k, reflects the slope of the trend line and is determined by the background anilfr  and ploidy level  of the cell line: k = /p .
the fact that normalization was made according to the autosomal controls means that the value of x corresponding to y =  <dig> represents the average autosomal segment copy number, which corresponds to ploidy of the cell line .
this formula shows that the copy number resolution of array-cgh  is inversely proportional to the ploidy level.
the expected copy number of a region  was calculated using the formula y = kx+b, taking into account the ploidy from chromosome counts  and the background anilfr from homozygous deletion in u <dig>  these expected values corresponded well to the copy numbers determined by mpfish  with few exceptions .
the exceptional regions r <dig> and r <dig> in tk <dig> showed high levels of heterogeneity by mpfish.
sixty percent of the cells had four r <dig> and five r <dig> copies.
this was considered as a major abnormality and included in table  <dig>  however 40% of cells contained five r <dig> and six r <dig> copies.
taking this in consideration, we corrected the data points for this cell line, using as x-values, the average copy numbers between subpopulations .
the exceptional amplified region r <dig> in caki <dig> showed also heterogeneity by fish.
the other exceptional regions were defined by too few  clones.
the expected ploidy was estimated  as x-coordinate of trend lines at y =  <dig> .
a good correlation was found between these expected values and real ploidy based on chromosome counts.
copy number resolution of array-cgh
for  <dig> out of  <dig> regions anilfr ±  <dig> sd value was different from adjacent ones, , showing that copy number change was detected by array-cgh in up to pentaploid cells.
in attempts to generalise the results, we calculated the expected anilfr difference for single copy number change using y = kx+b formula.
this difference in cell lines of ploidy  <dig>   <dig>   <dig> and  <dig> would be  <dig> ;  <dig> ;  <dig>  and  <dig>  respectively.
median sd for all regions was  <dig> .
the difference between the double sd  of two regions allowed a distinction between these regions.
more than  <dig>  differences characterised single copy number change in all cells irrespective of ploidy, making it possible to identify single copy number changes in array-cgh.
taking into account the fact that the vast majority  of the sd values lied in a range up to  <dig> , we concluded that single copy number change can be determined highly efficiently in di- and triploid cells and less efficiently in tetra and pentaploid cells.
the vast majority of anilfr values were fitting in the +/- 2sd interval .
therefore the precise location of the breakpoint can be made generally at the transition, where anilfr changes more than 4x median sd .
such difference is characteristic for single copy number changes in diploid cell lines.
for tri- tetra- and pentaploid cells, it would be difficult to identify breakpoints of single copy number transitions .
the difference of more than  <dig>  characterised double copy number changes, therefore breakpoints could be identified with high confidence for 2-copy transitions .
discussion
most solid tumours have complex karyotypes with several chromosomal rearrangements.
the combination of high-resolution methods can describe these changes efficiently.
in this study, we have chosen nine cell lines with complex karyotypes to evaluate the effectiveness of different methods.
array-cgh, interphase and metaphase mpfish gave corresponding results.
in the case of complex rearrangements not followed by copy number changes ; array-cgh and interphase mpfish could not detect the rearrangements in contrast with metaphase mpfish that did.
however one drawback is that metaphase mpfish requires high mitotic frequency in the analyzed cell population, which limits its application for tumour biopsy analysis.
thus combining metaphase, interphase mpfish and array-cgh permitted the detailed characterization of the reorganized chr <dig> segments.
the losses and especially gains seen on array-cgh profiles reflected complicated and clearly sequential chromosome rearrangements.
this is shown by the high incidence of cumulative changes in our analysis corresponding to sequential unbalanced translocations, leading to the accumulation of fragments carrying a gain-region, often in the terminal part of 3q.
even high copy number amplifications detected in hone <dig> and caki <dig> were the consequences of cumulative changes of different types .
each of these stepwise changes should provide the cell with selective growth advantage, supporting the importance of single copy number changes in cancer development.
array-cgh may have its limitations in determining ploidy and it is controversial whether it can determine the exact copy number of different chromosome regions  <cit> .
some publications claim a linear relation between the theoretical and measured copy number changes  <cit> .
in our present study we set on to analyse the relationship between copy number and anilfr for several regions in cell lines of different ploidy.
based on a comparison of seven cell lines having > <dig> data points, we found strong support for a linear relation .
we have also shown that the trend line functions can be described by an equation that permits the estimation of the copy number of a certain chromosome region, which corresponded to our experimental results.
the actual copy number is the function of parameters that include the variable anilfr, and two constants, the ploidy and the background.
background anilfr can vary depending on the array-cgh platform  <cit>  and experimental conditions in a particular lab.
it can be determined by using a cell line  containing a large homozygous deletion.
in order to estimate the real copy number for each anilfr, it would thus be important to know the cell ploidy.
if this is missing, at least one locus copy number has to be determined by fish.
it may be concluded from the proposed formula /p is ploidy), that the array-cgh results would be mainly influenced by cell ploidy under certain conditions.
we estimated the copy number resolution of our pac/bac based array-cgh and showed that single copy number changes can be detected even in pentaploid cells, although the most reliable results are obtained in di- and triploid samples.
however it may be more difficult to identify a breakpoint position since it can be mapped precisely only in diploid cells.
the heterogeneity and the normal cell contamination of tumours need to be considered as well.
heterogeneity and the presence of subpopulations occur frequently even in cell lines.
the level of heterogeneity can influence the interpretation of copy number changes in array-cgh as we have shown for the tk <dig> cell line.
in contrast, mpfish determines the copy number at single cell level and thus allows identification of subpopulations.
normal cell contamination needs to be considered as well.
it is useful to use log <dig> graphical representations of array-cgh that visualize small differences in fluorescence signal ratios more accurately.
mpfish counts on single cell level provide the most accurate analysis.
CONCLUSIONS
we have evaluated the effectiveness of array-cgh and mpfish in characterizing chr <dig> rearrangements in nine karyotypically complex cell lines.
we have found the following:
<dig>  the losses and gains seen on array-cgh profiles are projections of complex sequential chromosome rearrangements leading to the loss or accumulation of specific fragments.
<dig>  the analysis of the correlation between real copy number from mpfish and the anilfr value detected by array-cgh showed that the copy number is a linear function of parameters that include the variable, anilfr, and two constants, ploidy and background normalized fluorescence ratio.
<dig>  with our  <dig> mb pac/bac based chr <dig> array-cgh, a single copy number change can be detected even in pentaploid cells, but the most reliable results are obtained in di- and triploid samples.
it is more difficult to identify the breakpoint position which can only be mapped precisely in diploid cells.
<dig>  in heterogeneous or normal cell contaminated samples the most accurate analysis can be made by mpfish.

 genetic and epigenetic changes are among the hallmarks of cancer, with the illegitimate amplification of oncogenes and anti-apoptotic genes, and inactivation or deletion of tumour suppressors and pro-apoptotic genes as the main features. positional cloning of such genes is dependent on high-resolution methods that can detect losses and gains of chromosome segments. several techniques have been developed that can detect dna copy number changes. fluorescence in situ hybridization was introduced in the early 1980s. this is based on the use of chromosome region specific fluorescent-labelled dna probes. it can dissect complex chromosomal aberrations, including very small sub-microscopic deletions and permits studies on interphase cells. it can reveal heterogeneity while also giving a general view of the whole karyotype. large-scale analysis is time consuming, however, since it requires separate hybridizations and individual microscope analysis. comparative genomic hybridization has facilitated the detection of dna copy number changes. it was first restricted to metaphase chromosomes. however, recently high-resolution techniques have been developed using dna fragments instead of metaphase chromosomes. the resolution of metaphase cgh is about mb, whereas array-cgh can resolve hundreds base pairs or hundreds kilo bases and permits the detection of cryptic copy number changes. thus, the whole genome can be screened in a single experiment. array-cgh cannot detect balanced chromosomal translocations, insertions and inversions, however, and makes the analysis of heterogeneous and normal cell contaminated tumour samples difficult. another drawback is that cgh only detects relative copy number changes and cannot be used to determine the real segmental copy number and overall ploidy. in order to interpret complex cgh profiles and to describe the underlying chromosome rearrangements, additional information is required. the array-cgh results may be validated by a reciprocal approach, fish, that analyzes tumour cells with locus specific dna. in the present study, nine tumour cell lines were analyzed in order to establish rules that would facilitate the interpretation of array-cgh data. we focused our analysis on chr since it is frequently affected in solid tumours. a bac/pac based chr array-cgh was constructed, and the results of chr array analysis were compared with multipoint fish, using the same bac/pac clones as probes for interphase and metaphase chromosome analysis. on the basis of this analysis we propose a relatively simple approach to calculate locus specific copy number and ploidy. we show that the method can detect single copy number changes in up to near-pentaploid cells.  development of mpfish and array-cgh for detailed chr analysis we have analyzed ten carcinoma cell lines with cytogenetic methods. the chromosome number was counted for a minimum metaphase spreads in each cell line. subsequently, all lines were examined by chr painting. several chr fragments were identified on differently rearranged marker chromosomes. all of them had a characteristic morphology that allowed their identification on metaphase plates. to identify the composition of each chr fragment on the marker chromosomes we developed a modified fish method, called mpfish. mpfish can be introduced as a macro-array fish, which is the reciprocal of micro-array cgh. while in array-cgh the bac/pac dna is spotted in micro-dots on the slide and the tumour dna is hybridized to it, in the mpfish, the tumour nuclei and chromosomes are fixed and the different bac/pac dnas are hybridized to multiple areas on the slide, in pairs of two colour labelled probes. by this improved fish application, we reduced the amount of the probe to 100– ng of labelled probe dna for the analysis of 10– samples. we could also analyze 20– probes in double-colour hybridisation on each slide in a single experiment. this has led to a considerable reduction in cost and time. a set of commercially available, fish mapped, chr specific bac/pac probes was used to analyze the ten cancer cell lines using interphase and metaphase mpfish. one cell line, as expected, had normal chrs and it was excluded from further analysis. in total regions, which represent a row of neighbouring clones with the same copy number, were identified and characterized in the remaining nine cell lines. the majority of the chr rearrangements were generated by unbalanced translocations, but we could also identify interstitial deletions, duplications and even complex patterns of amplifications. the copy number of each region was calculated based on result of metaphase and interphase fish. majority of the tumour cell lines, analyzed here, had almost homogeneous cell populations. in the cases of heterogeneous tumour cell lines, like tk the predominant population copy number was used. in parallel with the mpfish analysis, a chr cgh array was set up covering both arms. it contained the same set of chr bac/pac clones as used for the mpfish experiments. six chrx and autosomal clones were used as controls. dop-pcr amplified probes, generated by three different universal primers were followed by aminolinker pcr, pooled and spotted on code-link slides. array-cgh was performed on the same nine cancer cell lines. the ratios of the mean fluorescence intensity of test dna versus reference dna were calculated for each clone. the average ratio of the autosomal controls was used for normalization. each dot on the array-cgh charts represents the normalised fluorescent ratio. the anilfr and standard deviation values were calculated for each of the mpfish defined rearranged chr regions in order to assess the interlocus variation. mpfish identifies mismapped clones detailed analysis of array-cgh results is always required to exclude suspicious copy number changes. we could see several clones with extreme values in the majority of array-cgh profiles, which needed more detailed investigation. bac/pac clones were ordered on each array-cgh profile according to their mb position on chr in the ucsc database. by mpfish, we verified the localization of the clones on normal chr and clones with wrong database localization could be readily identified. for example, clone rp11-129g has two locations in the ucsc database: – mb and mb; mpfish confirmed only the 3q localisation. this was in agreement with our array-cgh data where the copy number of this clone indicated that it was localized on 3q. mpfish validated also the localization of rp11-732m at 3p24- while the ucsc database placed it at 3q. there was a second group of clones that according to database information had well defined localisations on chr but they gave additional signals on other chromosomes upon mpfish analysis and had extreme hybridization ratios on the array-cgh. these clones appeared to represent parts of segmental duplications detected by blastn. these segmental duplications were not represented in the ucsc database, because the similarity was just below the database threshold. the third category of clones was eliminated because they were localized on chromosomes other than chr altogether clones were eliminated from evaluation. chr aberrations by array-cgh and mpfish mpfish analysis of the nine cell lines identified regions of different copy number on chr based on array-cgh results, we assessed the anilfr and standard deviation for each region. the anilfr values were different for regions with different copy numbers. gains were found in regions, losses in. losses were more frequent in 3p while gains were more frequent on 3q. we could visualize six interstitial losses. only two were interstitial deletions; the other four were generated by two translocations. an example of interstitial loss is provided by the homozygous deletion, previously characterized in a diploid small cell lung cancer cell line, u. on the array-cgh profile, the homozygous deletion is identified by clones that show a value of anilfr ± sd; ±. mpfish using these probes could not detect any nuclear signal, due to a complete loss of this chr segment in u. reconstitution of different chr fragments on the marker chromosomes confirmed that the same region was lost. the analysis made also clear that the u deletion was generated by three different translocations. only one of eight interstitial gains detected on the array-cgh profiles was due to a simple duplication. the others were due to translocations. all three terminal losses and six terminal gains originated from unbalanced translocations. an example of tg is shown on the array-cgh profile of uok where the copy number of the whole 3q increases by one added copy, due to a translocation with breakpoint at the centromere. sequential, stepwise changes were most frequent. they were generated by intermediate copy number changes leading to interstitial or terminal gains/losses. they were initiated by translocations, followed by secondary rearrangements that generated further changes in copy number. such stepwise changes have led to cumulative segmental amplifications in caki and hone. the array-cgh profile of copy number gain at 3q in hone resulted from a complex rearrangement leading to the amplification of this segment. increase in copy number can be measured by interphase mpfish and the rearrangements can be reconstituted by analyzing metaphase chromosomes in the same culture. in addition to the rearrangements mentioned, we found three chr aberrations by mpfish that were not detected by array-cgh: two insertions and one balanced translocation. metaphase mpfish on figure 4a shows that the marker chromosome m in uok had a chromosome fragment inserted in the pericentromeric region. this breakpoint was not identified by array-cgh, because the copy number remained unchanged. in the case of properly selected probes, interphase mpfish can detect insertion. the change detected by array-cgh at this region was loss of the short arm of chr as a result of an unbalanced translocation in m. it should be also mentioned that changes detected by array-cgh based on single clone data should be always confirmed by additional methods, like fish. correlation between anilfr and segment copy number we compared anilfr identified by array-cgh for regions that were determined by > clones, with copy number assessed by mpfish. figure shows the relationship between these two values. each region with a certain copy number was represented by a point on the graph where the x-coordinate corresponded to the region copy number obtained from mpfish analysis, while the y-coordinate corresponded to its anilfr. the number of data points was in uok uok115; in caki a and tk164; in u2020; in caki2; in uok and in hone equivalent to the number of regions with different copy numbers of the respective cell line. based on these data points, the trend lines for each cell line were calculated in excel. all nine trend lines followed the linear function of y = kx+b; where y is the anilfr determined by array-cgh; x is the copy number from mpfish. the constant b is equal to y =, corresponding to the crossing of all trend lines on y-axis. this value was equivalent also to anilfr of the homozygous deletion in u thus reflecting the background anilfr. the other constant, k, reflects the slope of the trend line and is determined by the background anilfr and ploidy level of the cell line: k = /p. the fact that normalization was made according to the autosomal controls means that the value of x corresponding to y = represents the average autosomal segment copy number, which corresponds to ploidy of the cell line. this formula shows that the copy number resolution of array-cgh is inversely proportional to the ploidy level. the expected copy number of a region was calculated using the formula y = kx+b, taking into account the ploidy from chromosome counts and the background anilfr from homozygous deletion in u these expected values corresponded well to the copy numbers determined by mpfish with few exceptions. the exceptional regions r and r in tk showed high levels of heterogeneity by mpfish. sixty percent of the cells had four r and five r copies. this was considered as a major abnormality and included in table however 40% of cells contained five r and six r copies. taking this in consideration, we corrected the data points for this cell line, using as x-values, the average copy numbers between subpopulations. the exceptional amplified region r in caki showed also heterogeneity by fish. the other exceptional regions were defined by too few clones. the expected ploidy was estimated as x-coordinate of trend lines at y =. a good correlation was found between these expected values and real ploidy based on chromosome counts. copy number resolution of array-cgh for out of regions anilfr ± sd value was different from adjacent ones,, showing that copy number change was detected by array-cgh in up to pentaploid cells. in attempts to generalise the results, we calculated the expected anilfr difference for single copy number change using y = kx+b formula. this difference in cell lines of ploidy and would be;; and respectively. median sd for all regions was. the difference between the double sd of two regions allowed a distinction between these regions. more than differences characterised single copy number change in all cells irrespective of ploidy, making it possible to identify single copy number changes in array-cgh. taking into account the fact that the vast majority of the sd values lied in a range up to, we concluded that single copy number change can be determined highly efficiently in di- and triploid cells and less efficiently in tetra and pentaploid cells. the vast majority of anilfr values were fitting in the +/- 2sd interval. therefore the precise location of the breakpoint can be made generally at the transition, where anilfr changes more than 4x median sd. such difference is characteristic for single copy number changes in diploid cell lines. for tri- tetra- and pentaploid cells, it would be difficult to identify breakpoints of single copy number transitions. the difference of more than characterised double copy number changes, therefore breakpoints could be identified with high confidence for 2-copy transitions. discussion most solid tumours have complex karyotypes with several chromosomal rearrangements. the combination of high-resolution methods can describe these changes efficiently. in this study, we have chosen nine cell lines with complex karyotypes to evaluate the effectiveness of different methods. array-cgh, interphase and metaphase mpfish gave corresponding results. in the case of complex rearrangements not followed by copy number changes; array-cgh and interphase mpfish could not detect the rearrangements in contrast with metaphase mpfish that did. however one drawback is that metaphase mpfish requires high mitotic frequency in the analyzed cell population, which limits its application for tumour biopsy analysis. thus combining metaphase, interphase mpfish and array-cgh permitted the detailed characterization of the reorganized chr segments. the losses and especially gains seen on array-cgh profiles reflected complicated and clearly sequential chromosome rearrangements. this is shown by the high incidence of cumulative changes in our analysis corresponding to sequential unbalanced translocations, leading to the accumulation of fragments carrying a gain-region, often in the terminal part of 3q. even high copy number amplifications detected in hone and caki were the consequences of cumulative changes of different types. each of these stepwise changes should provide the cell with selective growth advantage, supporting the importance of single copy number changes in cancer development. array-cgh may have its limitations in determining ploidy and it is controversial whether it can determine the exact copy number of different chromosome regions. some publications claim a linear relation between the theoretical and measured copy number changes. in our present study we set on to analyse the relationship between copy number and anilfr for several regions in cell lines of different ploidy. based on a comparison of seven cell lines having > data points, we found strong support for a linear relation. we have also shown that the trend line functions can be described by an equation that permits the estimation of the copy number of a certain chromosome region, which corresponded to our experimental results. the actual copy number is the function of parameters that include the variable anilfr, and two constants, the ploidy and the background. background anilfr can vary depending on the array-cgh platform and experimental conditions in a particular lab. it can be determined by using a cell line containing a large homozygous deletion. in order to estimate the real copy number for each anilfr, it would thus be important to know the cell ploidy. if this is missing, at least one locus copy number has to be determined by fish. it may be concluded from the proposed formula /p is ploidy), that the array-cgh results would be mainly influenced by cell ploidy under certain conditions. we estimated the copy number resolution of our pac/bac based array-cgh and showed that single copy number changes can be detected even in pentaploid cells, although the most reliable results are obtained in di- and triploid samples. however it may be more difficult to identify a breakpoint position since it can be mapped precisely only in diploid cells. the heterogeneity and the normal cell contamination of tumours need to be considered as well. heterogeneity and the presence of subpopulations occur frequently even in cell lines. the level of heterogeneity can influence the interpretation of copy number changes in array-cgh as we have shown for the tk cell line. in contrast, mpfish determines the copy number at single cell level and thus allows identification of subpopulations. normal cell contamination needs to be considered as well. it is useful to use log graphical representations of array-cgh that visualize small differences in fluorescence signal ratios more accurately. mpfish counts on single cell level provide the most accurate analysis.  we have evaluated the effectiveness of array-cgh and mpfish in characterizing chr rearrangements in nine karyotypically complex cell lines. we have found the following: the losses and gains seen on array-cgh profiles are projections of complex sequential chromosome rearrangements leading to the loss or accumulation of specific fragments. the analysis of the correlation between real copy number from mpfish and the anilfr value detected by array-cgh showed that the copy number is a linear function of parameters that include the variable, anilfr, and two constants, ploidy and background normalized fluorescence ratio. with our mb pac/bac based chr array-cgh, a single copy number change can be detected even in pentaploid cells, but the most reliable results are obtained in di- and triploid samples. it is more difficult to identify the breakpoint position which can only be mapped precisely in diploid cells. in heterogeneous or normal cell contaminated samples the most accurate analysis can be made by mpfish. 
the analysis of the correlation between real copy number from mpfish and the average normalized inter-locus fluorescence ratio value detected by array-cgh demonstrated that copy number is a linear function of parameters that include the variable, anilfr, and two constants, ploidy and background normalized fluorescence ratio. totally chr fragments were identified having copy numbers from to mpfish results from the bac/pac clones and array-cgh gave mainly corresponding results. chr locus copy number was assessed by interphase and metaphase mpfish. each copy number change on the array profile could be related to a specific chromosome aberration detected by metaphase mpfish. in most cases, the changes in copy number seen on array-cgh profiles reflected cumulative chromosome rearrangements. although our chr bac/pac array could identify single copy number changes even in pentaploid cells, mpfish provided a more accurate analysis in the dissection of complex karyotypes at high ploidy levels. in this study we have compared array-cgh and multipoint fish for their ability to characterize complex rearrangements on human chromosome in tumour cell lines. the ploidy of the tumour cell lines ranged from near-diploid to near-pentaploid. we have used bac/pac clones covering chr with an approximately mb resolution to analyze nine carcinoma lines. recently, several high-resolution methods of chromosome analysis have been developed.
 recently, several high-resolution methods of chromosome analysis have been developed. it is important to compare these methods and to select reliable combinations of techniques to analyze complex chromosomal rearrangements in tumours. in this study we have compared array-cgh and multipoint fish for their ability to characterize complex rearrangements on human chromosome in tumour cell lines. we have used bac/pac clones covering chr with an approximately mb resolution to analyze nine carcinoma lines. chr was chosen for analysis, because of its frequent rearrangements in human solid tumours.  the ploidy of the tumour cell lines ranged from near-diploid to near-pentaploid. chr locus copy number was assessed by interphase and metaphase mpfish. totally chr fragments were identified having copy numbers from to mpfish results from the bac/pac clones and array-cgh gave mainly corresponding results. each copy number change on the array profile could be related to a specific chromosome aberration detected by metaphase mpfish. the analysis of the correlation between real copy number from mpfish and the average normalized inter-locus fluorescence ratio value detected by array-cgh demonstrated that copy number is a linear function of parameters that include the variable, anilfr, and two constants, ploidy and background normalized fluorescence ratio.  in most cases, the changes in copy number seen on array-cgh profiles reflected cumulative chromosome rearrangements. most of them stemmed from unbalanced translocations. although our chr bac/pac array could identify single copy number changes even in pentaploid cells, mpfish provided a more accurate analysis in the dissection of complex karyotypes at high ploidy levels. 