Patent Application: US-201213979309-A

Abstract:
the invention relates to a method for determining prognosis , predicting or monitoring response to therapy of patients affected by melanoma through the evaluation of the methylation of different genomic dna sequences , including the long interspersed nucleotide element - 1 repetitive dna sequences and / or the concomitant methylation profile of specific groups of genes defined as methylation signature .

Description:
methylation levels of the “ long interspersed nucleotide element - 1 ” repetitive sequences predict survival of melanoma patients the prognosis of cutaneous melanoma ( cm ) differs for patients with identical clinico - pathological stage , and no molecular markers discriminating the prognosis of stage iii individuals have been established . genome - wide alterations in dna methylation are a common event in cancer . this example defines the prognostic value of genomic dna methylation levels in stage iii cm patients . overall level of genomic dna methylation was measured using bisulfite pyrosequencing at three cpg sites ( cpg1 , cpg2 , cpg3 ) of the long interspersed nucleotide element - 1 ( line - 1 ) sequences in short - term cm cultures from 42 stage iiic patients . the impact of line - 1 methylation on overall survival ( os ) was assessed using cox regression and kaplan - meier analysis . here the authors provide the first evidence that the methylation level of line - 1 repetitive elements has a significant prognostic value in stage iiic cm patients . specifically , patients whose tumors exhibit more hypomethylated line - 1 sequences had a significantly longer overall survival . the reported data have significant prospective practical clinical implications . indeed , it can be expected that the evaluation of line - 1 methylation status will : i ) provide cm patients with improved clinico - pathological sub - staging information ; ii ) allow physicians to personalize follow - up procedures for cm patients ; iii ) guide the future selection and / or stratification of patients for trials of adjuvant treatment ; iv ) select patients who are most likely to benefit from therapy , including dna hypomethylating drugs . overall , the evaluation of line - 1 methylation in the routine clinico - pathological ascertainment of cm patients is anticipated to greatly help in personalizing their comprehensive clinical management . short - term cell cultures were established from metastatic lesions removed surgically from consecutive cm patients referred to the national cancer institute of aviano ( italy ) for stage iii surgery from 1991 to 2007 , as previously described ( altomonte et al ., 1993 ). autologous tumor cell cultures were successfully established from 30 % of patients . the micrometastatic nature of lymph - node tumor tissues from ajcc stage iiia patients precluded their use for cell culture generation , while short - term cm cultures were available only from 12 stage tim patients , and were excluded from the statistical analyses . thus , the planned studies were conducted on a total of 42 available short - term cultures , identified as having been generated from cm patients classified as ajcc stage iiic . short - term cm cell cultures were grown in rpmi 1640 medium ( biochrome kg , berlin , germany ) supplemented with 20 % heat - inactivated fetal calf serum ( biochrome kg ) and 2 mm l - glutamine ( biochrome kg ). four independent cultures of nrem were purchased from invitrogen ( milan , italy ), gentaur ( brussels , belgium ), provitro ( berlin , germany ), and sciencell ( carlsbad , calif ., usa ), and were maintained in m254 medium supplemented with human melanocyte growth supplement ( invitrogen ). to minimize alterations potentially arising with extended in vitro culturing , all cell cultures were utilized for molecular assays at the 6 th ex vivo passage . genomic dna was extracted from short - term cultures of cm cells by proteinase k treatment followed by standard phenol / chloroform extraction and ethanol precipitation ( ausubel et al ., 1998 ). bisulfite conversion was carried out on 500 ng genomic dna using ez dna methylation - gold ™ kit ( zymo research , orange , calif ., usa ), according to the manufacturer &# 39 ; s protocol . methylation analysis of the line - 1 elements was performed as previously described ( yang et al ., 2004 ), with minor modifications . line - 1 elements were amplified using 50 pmol each of forward primer 5 ′- ttttttgagttaggtgtggg - 3 ′ ( seq id no . 221 ) and reverse biotinylated primer 5 ′- tctcactaaaaaataccaaacaa - 3 ′ ( seq id no . 222 ) in a 50 μl reaction volume containing 2 . 5 ng of bisulfite - treated dna , 1 × pcr buffer , 1 . 5 mm mgcl 2 and 1 . 25 u of platinum taq dna polymerase ( invitrogen ). pcr thermal amplification profile consisted of an initial denaturation step of 5 min at 95 ° c ., followed by 50 cycles of 30 s at 95 ° c ., 30 s at 58 ° c ., and 1 min at 72 ° c . the pcr product was purified using streptavidin sepharose high performance beads ( amersham biosciences , uppsala , sweden ) and denatured using 0 . 2 mol / l of naoh solution . next , 0 . 3 μmol / l of the sequencing primer ( 5 ′- gggtgggagtgat - 3 ′) ( seq id no . 223 ) was annealed to the purified single - stranded pcr product and the pyrosequencing reaction was performed using the psq hs 96 pyrosequencing system ( pyrosequencing , inc ., westborough , mass .). the level of methylation for each of the 3 analyzed cpg sites ( cpg1 , cpg2 , cpg3 ) was expressed as the percentage of methylated cytosines over the sum of methylated and unmethylated cytosines ( fig1 ). the primary objective was to determine differences in survival among various line - 1 dna methylation level groups . line - 1 dna methylation levels were dichotomized using median value into the following categories : cpg1 (& lt ; 25 . 68 , ≧ 25 . 68 ), cpg2 (& lt ; 27 . 26 , ≧ 27 . 26 ), and cpg3 (& lt ; 40 . 46 , ≧ 40 . 46 ). the characteristics including age , gender , primary tumor localization , breslow thickness , clark level , and ulceration of the primary tumor , number of lymph nodes involved , and pre - operative serum ldh values were examined . survival time was calculated in months from the date of stage iiic diagnosis until the date of death . according with the specific goals of the analysis , the authors did not classify the deaths considering their cause . patients were censored at the last follow - up date or the last date the patient was last known to be alive . median survival duration was determined by the kaplan - meier method ( kaplan and meier , 1958 ). cumulative survival by dna methylation level was evaluated using the log - rank test . p values were two sided and values & lt ; 0 . 05 were considered to be statistically significant . cox proportional hazard method ( cox , 1972 ) was used to examine the effect of dna methylation level on survival and results were presented as hazard ratios ( hr ) with corresponding 95 % confidence intervals ( ci ). line - 1 methylation was also entered in the model as a continuous variable with the unit set at 10 % of methylation . patient characteristics and clinical and pathologic features were examined by univariate cox model . the statistical analyses were carried out using the sas software version 9 . 13 ( sas institute inc ., cary , n . c ., usa ). the study was conducted on cm patients who underwent radical lymph node dissection for stage iii disease at the centro di riferimento oncologico national cancer institute between 1991 and 2007 . patients diagnosed with a stage iiic disease , and for whom a short - term cell culture had been successfully generated from the surgically removed autologous neoplastic tissue , were included in the study . table 5 summarizes the 42 patients under study and their clinico - pathologic characteristics at presentation . † low ldh is established as ldh values ≦ 0 . 8 times the upper limit of normal , high ldh is established as ldh values & gt ; 0 . 8 times the upper limit of normal to define if cm undergoes changes in the overall content of 5 - methylcytosine , bisulfite pyrosequencing analyses were utilized to compare the extent of methylation of line - 1 repetitive elements in the 42 short - term cm cell cultures under study to that of primary cultures of nrem . line - 1 sequences were heavily methylated at all investigated cpg sites in nrem ( cpg1 : median 62 . 82 %, range 60 . 43 %- 67 . 53 %; cpg2 : median 52 . 57 %, range 51 . 37 %- 52 . 87 %; cpg3 : median 65 . 77 %, range 62 . 40 %- 67 . 33 %; fig2 ). on the other hand , much lower and largely heterogeneous levels of methylation of the line - 1 elements were observed in cm cells from stage iiic patients ( cpg1 : median 25 . 68 %, range 12 . 45 %- 54 . 05 %; cpg2 : median 27 . 26 %, range 16 . 50 %- 49 . 43 %; cpg3 : median 40 . 46 %, range 28 . 10 %- 64 . 15 %; fig2 ), demonstrating that highly variable alterations in the overall level of genomic dna occur in cm . the highly heterogeneous levels of line - 1 methylation observed in cm cells from stage iiic patients led us to investigate whether they correlated with a different clinical outcome of patients under study . kaplan - meier analysis indicated that median os for stage iiic cm patients under analysis was 15 . 3 months ( 95 % ci , 11 . 0 - 31 . 5 ; fig3 a ). to evaluate the association between line - 1 methylation status and os , patients were divided according to the median value of methylation of each analyzed cpg site ( cpg1 = 25 . 68 %; cpg2 = 27 . 26 %; cpg3 = 40 . 46 %). patients were defined as having hypomethylated or hypermethylated line - 1 sequences , depending on the methylation level being below or above the median value for each group , respectively . kaplan - meier analysis showed a trend towards an increased os rate for patients with hypomethylated cpg1 , however , the difference did not reach statistical significance ( p = 0 . 22 , log - rank = 1 . 51 ; fig3 b ). on the other hand , a significant survival advantage was observed in patients with cpg2 & lt ; 27 . 26 % as compared to patients with cpg2 & gt ; 27 . 26 % ( p = 0 . 04 , log - rank = 4 . 14 ) ( fig3 c ). similarly , the survival rate of patients with cpg3 & lt ; 40 . 46 % was significantly higher than that of patients with cpg3 & gt ; 40 . 46 % ( p = 0 . 01 , log - rank = 6 . 39 ) ( fig3 d ). in line with these data , median os of patients with hypomethylated cpg1 , cpg2 and cpg3 sites was 24 . 3 , 31 . 5 , and 31 . 9 months , respectively , as compared to 15 . 3 , 11 . 5 , and 11 . 5 months of patients with hypermethylated line - 1 cpgs ( fig3 , table 6 ). cox univariate analysis was carried out to identify patient characteristics and clinico - pathologic factors that predicted survival . among all factors examined , including age , gender , localization of primary tumor , breslow thickness , clark level and ulceration of primary tumor , number of lymph nodes involved , and level of pre - operative ldh , only cpg2 methylation ( hr = 2 . 12 for cpg2 ≧ 27 . 26 % vs . cpg2 & lt ; 27 . 26 ; 95 % ci : 1 . 01 - 4 . 44 ; p = 0 . 04 ) and cpg3 methylation ( hr = 2 . 63 for cpg2 & gt ; 40 . 46 % vs . cpg2 & lt ; 40 . 46 ; 95 % ci : 1 . 21 - 5 . 69 ; p = 0 . 01 ) were associated with statistically significant differences in os ( table 7 ). when line - 1 methylation was analyzed as a continuous variable , a statistically significant inverse association emerged between os and an increase of 10 % of methylation of cpg1 ( hr = 1 . 51 ; 95 % ci : 1 . 06 - 2 . 16 ; p = 0 . 02 ), cpg2 ( hr = 1 . 60 ; 95 % ci : 1 . 02 - 2 . 52 ; p = 0 . 04 ) and cpg3 ( hr = 1 . 49 ; 95 % ci : 1 . 06 - 2 . 09 ; p = 0 . 02 ) ( table 7 ). † cox proportional hazard method was used to examine the effect of line - 1 methylation on os . results were presented as hazard ratios ( hr ) with corresponding 95 % confidence intervals ( ci ); ‡ line - 1 methylation was also evaluated as continuous variable . the hr value is that of the line - 1 methylation relative to an increase of 10 %; in the present invention , the authors demonstrate that the global level of line - 1 methylation of short - term tumor cell cultures grown from patients with nodal disease is a significant predictor of os in stage iiic cm patients . this finding is of remarkable clinical relevance , since , to the best of our knowledge , it provides the first evidence of a molecular marker capable of differentiating the prognosis of cm patients in this high - risk substage . these results are of particular emphasis given the conduct of this study in subjects within a single clinically well - defined clinico - pathological staging sub - group , which has become the focus of several ongoing clinical trials in the us and europe ( i . e ., ecog intergroup trial e4697 , eortc trial 18071 , gsk trial 111482 “ derma ”). genomic dna hypomethylation has been proposed to have an important impact on tumor biology through the generation of chromosomal instability , reactivation of transposable elements , and loss of imprinting ( esteller , 2008 ). thus , a negative correlation between genomic hypomethylation and survival of cm patients could have been expected . instead , the authors found that hypomethylation of line - 1 elements at cpg2 or cpg3 sites was associated with a significantly better os , as demonstrated by kaplan - meier analysis and log - rank test . the positive prognostic value of line - 1 hypomethylation the authors have identified in cm is in sharp contrast with data most recently obtained in colon and ovarian cancer patients , in which line - 1 hypomethylation in neoplastic tissues was associated with a poorer prognosis ( ogino et al ., 2008 ; pattamadilok et al ., 2008 ). since cm behaves differently from other solid tumors , the underlying biological effect ( s ) of line - 1 hypomethylation on patients &# 39 ; outcome could depend on the tumor histotype . nevertheless , it should be emphasized that our findings are generated from patients in the same clinico - pathological stage of disease , while the studies on ovarian and colon cancer were conducted on the heterogeneous patients population as a whole , and did not investigate the prognostic potential of line - 1 methylation in specific clinically defined stages of disease . the mechanism ( s ) through which line - 1 hypomethylation affects survival of cm patients remains to be fully explored ; however , some speculation can be made , based on recent data in the literature . tellez et al ( tellez et al ., 2009 ) have demonstrated that higher levels of line - 1 methylation correlate with an increased number of aberrantly hypermethylated tumor suppressor genes ( tsg ) in cultured melanoma cell lines . thus , epigenetic inactivation of tsg might account for more aggressive disease the authors have observed in patients with elevated line - 1 methylation in their neoplastic cells . this notion is in accordance with initial studies reporting a negative association between survival and the presence of hypermethylated er - α , rassf1a , rar - β2 , or mint31 dna in neoplastic tissues or sera of stage iii / iv cm patients ( mori et al ., 2006 ; mori et al ., 2005 ; tanemura et al ., 2009 ). on the other hand , hypomethylation , and consequent transcriptional activation , of line - 1 elements might per se reduce the tumorigenic potential of neoplastic cells by triggering apoptosis and a senescence - like state through the activity of the second open reading frame of line -/( wallace et al ., 2008 ). furthermore , genomic dna hypomethylation has been associated with the de novo expression of tumor associated antigens belonging to the cancer testis antigen ( cta ) class by neoplastic cells of different histotype ( de smet et al ., 1996 ; sigalotti et al ., 2002 ; woloszynska - read et al ., 2008 ), and the authors have recently identified a significant correlation between a hypomethylated status of line - 1 elements and increased presence , levels and total number of cta concomitantly expressed in short - term cultures of cm cells ( see example 2 ). thus , it is intriguing to speculate that a better immune recognition of line - 1 hypomethylated cm cells might contribute to the improved survival of these patients . irrespective of the underlying biological mechanism ( s ) triggered by line - 1 hypomethylation , the prognostic value of line - 1 methylation here identified for stage iiic cm patients bears several important practical clinical implications . among these , the goal to provide cm patients with improved clinico - pathological sub - stage and / or follow - up - procedures would be enhanced using line - 1 methylation status , and these findings might be used to select and / or stratify patients for adjuvant treatment based on the methylation level of line - 1 in their tumors . in addition , the significant positive prognosis of line - 1 hypomethylated patients should prompt the incorporation of this in new studies aimed at understanding whether pharmacologic dna hypomethylation ( sigalotti et al ., 2007 ) could be regarded as a feasible chemoprevention approach in the initial phases of disease and / or in patients at high - risk of disease recurrence . the authors &# 39 ; present findings will be further investigated in prospective multicenter studies in which the prognostic significance and the predictive value for different treatments of cm will be validated . providing further support to our data will undoubtedly add the evaluation of line - 1 methylation into the routine clinico - pathological ascertainment of cm patients , helping to personalize their comprehensive clinical management hypomethylation ( i . e ., methylation below median ) at cpg2 and cpg3 sites significantly associated with improved prognosis of cm , cpg3 showing the strongest association . patients with hypomethylated cpg3 had increased os ( p = 0 . 01 , log - rank = 6 . 39 ) by kaplan - meier analysis . median os of patients with hypomethylated or hypermethylated cpg3 were 31 . 9 and 11 . 5 months , respectively . the 5 year os for patients with hypomethylated cpg3 was 48 % compared to 7 % for patients with hypermethylated sequences . among the variables examined by cox regression analysis , line - 1 methylation at cpg2 and cpg3 was the only predictor of os ( hazard ratio = 2 . 63 , for hypermethylated cpg3 ; 95 % confidence interval : 1 . 21 - 5 . 69 ; p = 0 . 01 ). line - 1 methylation is identified as a molecular marker of prognosis for cm patients in stage iiic . evaluation of line - 1 promises to represent a key tool for driving the most appropriate clinical management of stage iii cm patients . methylation levels of the “ long interspersed nucleotide element - 1 ” repetitive sequences correlate with presence and levels of expression of cancer testis antigens in cutaneous melanoma cancer testis antigens ( cta ) represent an important class of immunogenic tumor antigens that are currently utilized as targets in several cancer immunotherapy approaches . the expression of cta has been shown to depend on the methylation status of their promoters . in view of the therapeutic relevance of cta expression and of its regulation by promoter methylation , the authors asked the question whether line - 1 methylation might correlate with cta expression in cm cells , thus representing a surrogate marker for their presence and levels of expression . to this end , line - 1 methylation was measured in cm cells by pyrosequencing assays and cta expression was evaluated by qualitative or quantitative rt - pcr analyses . the correlation between line - 1 methylation and cta expression was evaluated through spearman &# 39 ; s rank correlation test . the extent of methylation of line - 1 elements was measured in primary cultures of cm cells at the 6 th in vitro passage by bisulfite pyrosequencing as reported in example 1 . total rna was extracted from cm cells at the 6 th in vitro passage using the trizol reagent ( invitrogen , milan , italy ) following manufacturer &# 39 ; s recommendations . rt - pcr reactions were performed as previously described ( coral et al ., 1999 ). the oligonucleotide primer sequences and gene - specific pcr amplification programs utilized have been defined for mage - a1 , - a2 , - a3 , - a4 , - a10 ( de plaen et al ., 1994 ; rimoldi et al ., 1999 ), ny - eso - 1 ( jager et al ., 1998 ), ssx - 2 ( gure et al ., 1997 ). the integrity of each rna and oligo - dt - synthesized cdna sample was confirmed by the amplification of the ( 3 - actin housekeeping gene ( coral et al ., 1999 ). ten μl of each rt - pcr sample were run on a 2 % agarose gel and visualized by ethidium bromide staining . real - time quantitative rt - pcr analyses were performed as previously described ( calabro et al ., 2005 ). briefly , total rna was digested with rnase free dnase ( roche diagnostics , milan , italy ) to remove contaminating genomic dna . synthesis of cdna was performed on 1 μg total rna using mmlv reverse transcriptase ( invitrogen ) and random hexamer primers ( promega , milan , italy ), following manufacturers &# 39 ; instructions . taqman quantitative rt - pcr reactions were performed on 10 ng of retrotranscribed total rna in a final volume of 25 μl taqman universal master mix ( applyed biosystems , milan , italy ) at95 ° c . for 10 min , followed by 45 cycles at 95 ° c . for 15 sec and at 60 ° c . for 1 min . taqman primers / probe sets were as follows : β - actin forward cgagcgcggctacagctt ( seq id no . 224 ), β - actin reverse ccttaatgtcacgcacgatt ( seq id no . 225 ), β - actin probe fam - accaccacggccgagcgg - bhq1 ( seq id no . 226 ); mage - a3 forward tgtcgtcggaaattggcagtat ( seq id no . 227 ), mage - a3 reverse caaagaccagctgcaaggaact ( seq id no . 228 ), mage - a3 probe fam - tctttcctgtgatcttc - mgb ( seq id no . 229 ). measurement of gene expression was performed utilizing the abi prism 7000 sequence detection system ( applyed biosystems ) and the copy number of mage - a3 and of the reference gene β - actin was established in each sample by extrapolation of the standard curve . the number of mage - a3 cdna molecules in each sample was then normalized to the number of cdna molecules of β - actin . the correlation between line - 1 methylation at each cpg site and the number of expressed cta or the quantitative levels of expression of mage - a3 was evaluated through spearman &# 39 ; s rank correlation test . p values & lt ; 0 . 05 were considered to be statistically significant . results demonstrate a significant inverse association of line - 1 methylation with both number of cta concomitantly expressed ( fig4 ) and level of expression of the cta mage - a3 ( fig5 ). whole - genome methylation profiles , and derived methylation signatures , predict survival of melanoma patients cutaneous melanoma ( cm ) is a very aggressive neoplasm of growing incidence and mortality rates in industrialized countries , and the leading cause of skin cancer - related deaths worldwide ( mackie et al ., 2009 ). surgery , in early phases of disease has curative potential for patients ; for advanced cm conventional therapies have failed to prolong survival ( bhatia et al ., 2009 ). at present , the best predictor of 5 - year survival is the clinico - pathological stage of disease , which defines overall survival ( os ) rates ranging from 95 % to 7 % for stage i to iv patients , respectively ( balch et al ., 2001 ; balch et al ., 2009 ). however , within the same clinico - pathological stage category , patients often behave radically differently , and the current lack of prognostic molecular markers impairs our ability to identify cm patients with highly aggressive as opposed to more indolentcourses of disease ( jennings and murphy , 2009 ). methylation of genomic dna in mammals occurs at sc - position of the cytosine in the context of cpg dinucleotides , and results in gene silencing through different mechanisms ( sigalotti et al ., 2010 ). recent studies have demonstrated that alterations in genomic dna methylation patterns represent a hallmark of cancer cells , being proposed to actively contribute to cancer development and progression mainly through inactivation of tumor suppressor genes by aberrant promoter hypermethylation ( esteller , 2008 ). in particular , epigenetic alterations are emerging as important players in cm tumorigenic process , as demonstrated by the continuously growing list of genes that are transcriptionally inactivated by aberrant dna hypermethylation in cm , and which potentially affect key cellular pathways such as cellular proliferation / differentiation , apoptosis , dna repair , extracellular matrix interaction , signaling , metastatization and immune recognition ( sigalotti et al ., 2010 ). the increasing role of aberrant methylation in cm biology strongly suggests for the opportunity to test methylation markers as potential indicators of disease prognosis . along this line , preliminary investigations are suggesting a possible prognostic role of the methylation status of selected genes in cm patients . indeed , lahtz et al have most recently evaluated a total of 230 cm patients at stage 0 to iv of disease , demonstrating pten methylation to be an independent negative prognostic factor , though it did not outperform the strongest traditional markers of tumor thickness and ulceration ( lahtz et al ., 2010 ). similarly , methylation of the tumor suppressor gene tslc1 was found to be significantly increased in advancing tumor stage and to associate with a reduced disease - related survival of cm patients ( you et al ., 2010 ), and the methylation of the methylated in tumors ( mint ) locus mint31 has been recently shown to be significantly associated with advancing clinical stage in 107 stage i to iv cm patients , and to predict improved disease - free and overall survival in the 25 analyzed stage iii patients ( tanemura et al ., 2009 ). in line with the above reported information , initial studies have also investigated the methylation status of selected tumor suppressor genes in sera of cm patients . among the genes more frequently methylated in cm , a negative correlation between presence of circulating methylated dna for rassf1a and er - a and survival of cm patients receiving biochemotherapy was found , suggesting for the potential usefulness of soluble methylation markers as prognostic factors in this malignancy ( mori et al ., 2006 ; mori et al ., 2005 ). despite these promising initial results , the studies so far available have several limitations , including : i ) investigation of only single / few genes ; ii ) reduced number of patients evaluated ; iii ) rarely investigated the prognostic significance of methylation markers in patients at the same stage of disease . furthermore , to the best of our knowledge , no studies have investigated the influence of the genome - wide gene methylation profiles on cm prognosis . accordingly , the authors investigated whether whole - genome methylation profiles may account for the differing survival patterns of cm patients of identical clinico - pathological stage of disease . the study was conducted on a series of 59 consecutive stage iii cm patients for whom the autologous short - term cell cultures were available . the latter were analyzed early during in vitro passage , and utilized instead of tumor tissues to overcome possible alterations in the evaluation of genomic methylation profiles due to the unavoidable presence of contaminating normal cells . results demonstrated that whole - genome methylation profiling is a powerful tool to identify cm patients with a significantly different prognosis , and that methylation signatures of 17 to 98 genes can be utilized to assign cm patients to distinct prognostic groups . these findings demonstrate that evaluation of whole - genome - defined methylation signatures may greatly help in guiding the daily clinical management of cm patients , and provide a strong rationale for the development of a large prospective validation study . short - term cell cultures were established from metastatic lesions removed surgically from consecutive cm patients referred to the national cancer institute of aviano ( italy ) for stage iii surgery from 1991 to 2007 , as previously described ( altomonte et al ., 1993 ). autologous tumor cell cultures were successfully established from 30 % of patients . the micrometastatic nature of lymph - node tumor tissues from ajcc stage iiia patients precluded their use for cell culture generation . thus , the planned studies were conducted on a total of 59 available short - term cultures , identified as having been generated from cm patients classified as ajcc stage iiib or iiic . short - term cm cell cultures were grown in rpmi 1640 medium ( biochrome kg , berlin , germany ) supplemented with 20 % heat - inactivated fetal calf serum ( biochrome kg ) and 2 mm l - glutamine ( biochrome kg ). to minimize alterations potentially arising with extended in vitro culturing , all cell cultures were utilized for molecular assays at the 6 th ex vivo passage . genomic dna was extracted from short - term cultures of cm cells by proteinase k treatment followed by standard phenol / chloroform extraction and ethanol precipitation ( fratta et al ., 2010 ). the 59 samples under analysis were evaluated for genome - wide promoter methylation using the illumina infinium humanmethylation27 bead array ( illumina inc , san diego , calif . ), which allows the interrogation of 27 , 578 cpg dinucleotides , covering 14 , 495 genes . the assay is based on the conversion of unmethylated c - nucleotides into u ( t ) nucleotides by the bisulfite treatment , leaving unaltered methylated ones . accordingly , the ez dna methylation kit ( zymo research , orange , calif ., usa ) was used for bisulfite conversion of 500 ng of genomic dna , and the remaining assay steps were performed as per illumina protocol . briefly , the bisulfite - modified genomic dna was whole - genome amplified , enzymatically fragmented , precipitated , resuspended , and hybridized overnight to locus - specific oligonucleotide primers on the beadarray . each interrogated locus is represented by specific oligomers linked to two bead types : one representing the sequence for the methylated dna ( m ) and the other for unmethylated dna ( u ). after hybridization , the c or t nucleotides were detected on m - or u - beads , respectively , by single - base primer extension . the arrays were then imaged using a beadarray ™ reader . image processing and intensity data extraction were performed according to illumina &# 39 ; s instructions . the methylation status of a specifc cpg site is calculated from the intensity of the m - and u - beads , as the ratio of fluorescent signals : β = max ( m , 0 )/[ max ( m , 0 )+ max ( u , 0 )]. dna methylation β values are continuous variables between 0 ( no methylation ) and 1 ( completely methylated ) representing the ratio of combined locus intensity . total cellular rna was purified from short - term cultures of cm cells by trizol reagent ( invitrogen , milan , italy ) following manufacturer &# 39 ; s recommendations . gene expression profiling was conducted on illumina human wg - 6 version 3 arrays using standard illumina protocols . all statistical analyses were performed using the r , version 2 . 11 . 1 , statistical environment ( http :// www . r - project . org ) and bioconductor2 . 6 packages ( http :// www . bioconductor . org ). the authors used the methylumi package for importing and pre - processing the methylation data , the package lumi ( du et al ., 2008 ) for importing and pre - processing the gene expression data , the survival package for kaplan - meier estimates and the pamr package for the shrunken centroid supervised analysis . in brief , the methylation data exported from the illumina beadstudio software were imported in r , checked for quality and normalized using the methylumi bioconductor package . k - means partitioning clustering was used to divide the transformed data into respectively 2 and 3 groups . the survival times of each of the ⅔ groups were evaluated using a kaplan - meier analysis . the characteristics including age , gender , primary tumor localization , breslow thickness , clark level , and ulceration of the primary tumor , number of lymph nodes involved , and pre - operative serum ldh values were also examined . survival time was calculated in months from the date of stage iii or iiic diagnosis until the date of death . according with the specific goals of the analysis , the authors did not classify the deaths considering their cause . patients were censored at the last follow - up date or the last date the patient was last known to be alive . median survival duration was determined by the kaplan - meier method . cumulative survival was evaluated using the log - rank test . p values were two sided and values & lt ; 0 . 05 were considered to be statistically significant . methylation patterns ( signatures ) related to the discovered groups were determined using the nearest shrunken centroid classification algorithm ( pam ). as the algorithm requires a threshold to balance between the number of sample correctly classified and the subset of features representing the methylation patterns , the authors determined this threshold by cross - validation as recommended by the inventors of the algorithm ( tibshirani et al ., 2002 ). the study was conducted on cm patients who underwent radical lymph node dissection for stage iii disease at the centro di riferimento oncologico national cancer institute between 1991 and 2007 . patients diagnosed with a stage iii disease , and for whom a short - term cell culture had been successfully generated from the surgically removed autologous neoplastic tissue , were included in the study . table 8 summarizes the 59 patients under study and their clinico - pathologic characteristics at presentation . in light of the likely influence of altered genome - wide methylation profiles on cm biology , the authors sought to investigate whether distinct methylation patterns might associate with a different clinical outcome of cm patients . genome - wide gene methylation profiles were evaluated in the 59 short - term cm cell cultures under study using the illumina humanmethylation27 bead - chip whole - genome assay , which interrogates the methylation status of 27 , 578 cpg sites , corresponding to 14 , 495 genes . patients were then grouped according to their whole genome methylation profile by applying a k - means clustering , which is a non hierarchical unsupervised method that allows partitioning of data into a predetermined number ( k ) of groups ( fig6 ). k - means clustering algorithm was applied by requiring a separation of all stage iii cm patients under analysis into 2 or 3 subgroups ( fig6 ). accordingly , the classification split the population in either 2 subgroups ( group k2 - 1 and k2 - 2 ), consisting of 35 and 24 patients , respectively , or in 3 subgroups ( group k3 - 1 , k3 - 2 , k3 - 3 ), counting 27 , 24 and 8 patients , respectively ( fig6 ). the prognostic relevance of the genome - wide methylation profile - based classification was assessed by evaluating the obtained subgroups of patients for overall survival ( os ) through kaplan - meier analysis . results demonstrated a significant survival advantage for patients classified as k2 - 1 as compared to those belonging to group k2 - 2 ( p = 0 . 022 , log - rank = 5 . 2 ) ( fig6 a ). the statistically significant difference in os among groups was maintained also when splitting the population in 3 classes ( p = 0 . 047 , log - rank = 6 . 1 ) ( fig6 b ). in line with these data , an increased median os was observed for patients classified as k2 - 1 ( 31 . 5 months ) as compared to patients in the k2 - 2 group ( 14 . 8 months ) ( table 9 ). similarly , patients in the k3 - 1 class had a superior median os ( 31 . 9 months ) as compared to k3 - 3 ( 18 . 8 months ) and k3 - 2 ( 14 . 8 months ) ( table 9 ). accordingly , the 5 year os of patients classified as k2 - 1 and k2 - 2 was 41 . 8 % and 5 . 6 %, respectively , while that of patients classified as k3 - 1 , k3 - 3 and k3 - 2 was 43 . 7 %, 37 . 5 %, and 5 . 6 %, respectively ( table 9 ). in light of the above reported prognostic significance of genome - wide methylation profiles in stage iii cm patients , the authors asked the question whether methylation profiling could allow to identify different survival classes also in sub - stage iiic patients , which are characterized by highly homogeneous clinico - pathological characteristics . thus , the same analytical procedures employed for the whole population of stage iii patients were applied to the subgroup of 45 stage iiic patients . k - means classification split the population in either 2 subgroups ( group 3c - k2 - 1 and 3c - k2 - 2 ), counting of 33 and 12 patients , respectively , or in 3 subgroups ( group 3c - k3 - 1 , 3c - k3 - 2 , 3c - k3 - 3 ), including 11 , 27 and 7 patients , respectively ( fig6 ). kaplan - meier analysis demonstrated a significant survival advantage for patients classified as 3c - k2 - 1 as compared to group 3c - k2 - 2 ( p = 0 . 001 , log - rank = 10 . 2 ) ( fig6 c ). similarly , a significant difference in os was also observed among groups 3c - k3 - 3 , 3c - k3 - 2 , and 3c - k3 - 1 ( p = 0 . 021 , log - rank = 7 . 8 ) ( fig6 d ). an increased median os was observed for patients classified as 3c - k2 - 1 ( 31 . 5 months ) as compared to patients in the 3c - k2 - 2 group ( 10 . 4 months ) ( table 9 ). similarly , patients in the 3c - k3 - 2 class had a superior median os ( 31 . 5 months ) as compared to 3c - k3 - 3 ( 24 . 5 months ) and 3c - k3 - 1 ( 11 months ) ( table 9 ). accordingly , the 5 year os of patients classified as 3c - k2 - 1 and 3c - k2 - 2 was 41 . 2 % and 0 %, respectively , while that of patients classified as 3c - k3 - 3 , 3c - k3 - 2 and 3c - k3 - 1 was 42 . 9 %, 39 . 9 %, and 0 %, respectively ( table 9 ). laying on the above reported information , cox univariate analysis was carried out to identify patient characteristics and clinico - pathologic factors that predicted survival . among all examined factors , including age , gender , localization of primary tumor , breslow thickness , clark level and ulceration of primary tumor , number of lymph nodes involved , and level of pre - operative ldh , only the classes defined by k - means algorithm on the genome - wide methylation profiles were associated with statistically significant differences in os , both when evaluating the entire population of stage iii cm patients ( hr = 1 . 99 for group k2 - 2 vs . k2 - 1 ; 95 % ci : 1 . 09 - 3 . 64 ; p = 0 . 025 ), and the sole substage iiic patients ( hr = 3 . 2 for group 3c - k2 - 2 vs . 3c - k2 - 1 ; 95 % ci : 1 . 51 - 6 . 79 ; 0 . 0023 ) ( table 10 ). to define the methylation signature , that is the minimal number of methylation markers succinctly characterizing each of the cm patient prognostic group defined by the unsupervised clustering analysis , the authors have applied the “ nearest shrunken centroid ” algorithm ( tibshirani et al ., 2002 ) ( fig7 , 8 ). using this approach the authors have identified different methylation signatures able to correctly classify patients into the previously defined prognostic classes , with acceptable error rates ( fig7 , 8 ). indeed , methylation signature a , consisting of 24 genes , had an overall error rate of 0 . 05 and classified stage iii patients into classes k2 - 1 and k2 - 2 with a total of 3 errors , while methylation signature b , including 98 genes , had an overall error rate of 0 . 034 and classified stage iii patients into classes k3 - 1 , k3 - 2 , and k3 - 3 with a total of 2 errors ( table 3 , fig7 ). the same approach conducted on sub - stage iiic patients led to the identification of a methylation signature of 16 genes ( signature c ) able to sort stage 111c patients into groups 3c - k2 - 1 and 3c - k2 - 2 without any misinterpretation , and a methylation signature of 47 genes ( signature d ) able to sort stage iiic patients into groups 3c - k3 - 1 , 3c - k3 - 2 , and 3c - k3 - 3 with a total of 1 error ( overall error rate of 0 . 022 ) ( table 3 , fig8 ). the initial analysis was further refined in the attempt to identify methylation signatures that were likely to be directly associated to a biologic effect through the transcriptional regulation of the respective genes . to this end , a spearman &# 39 ; s correlation analysis was conducted comparing genome - wide methylation data and gene expression profiles obtained from neoplastic cells of the stage iii cm patients under study . this analysis yielded a set of 820 methylation probes which methylation status was significantly correlated ( p & lt ; 0 . 05 ) with the expression of the respective gene . by restricting the application of the “ nearest shrunken centroid ” algorithm to this sub - set of methylation signals , the authors were able to identify methylation signatures capable to correctly classify stage iiic patients into the previously defined prognostic classes ( table 3 , fig9 ). of these , a 51 gene methylation signature ( signature e ) was able to assign stage iiic patients to groups 3c - k2 - 1 or 3c - k2 - 2 , committing only one error ( overall error rate 0 . 022 ), while a 73 gene methylation signature ( signature f ) was able to sort stage iiic patients into groups 3c - k3 - 1 , 3c - k3 - 2 , and 3c - k3 - 3 without any misinterpretation ( table 3 , fig9 ). when , among the genes having a significant ( p & lt ; 0 . 05 ) association between expression and methylation status , only those with a coefficient of correlation rho & lt ;− 0 . 4 were taken into account , an additional signature of 35 genes ( signature g ) was defined , which was able to assign stage iiic patients to groups 3c - k2 - 1 or 3c - k2 - 2 , committing only one error ( overall error rate 0 . 022 ) ( table 3 , fig1 ). whole genome methylation profiles identified classes with significantly different os in stage iii cm patients . as an example , stage iiic patients with a “ favorable ” methylation profile had increased os ( p = 0 . 001 , log - rank = 10 . 2 ) by kaplan - meier analysis . median os of stage iiic patients with a “ favorable ” vs “ unfavorable ” methylation profile were 31 . 5 and 10 . 4 months , respectively . the 5 year os for stage iiic patients with a “ favorable ” methylation profile was 41 . 2 % as compared to 0 % for patients with an “ unfavorable ” methylation profile . among the variables examined by cox regression analysis , classification defined by methylation profile was the only predictor of os ( hazard ratio = 3 . 2 , for “ unfavorable ” methylation profile ; 95 % confidence interval : 1 . 51 - 6 . 79 ; p = 0 . 002 ). different methylation signatures , consisting of 17 to 98 genes , able to correctly assign ( overall error rate & lt ; 0 . 05 ) stage iii or iiic patients to distinct methylation - defined prognostic groups , were identified . discrete whole - genome methylation signatures have been identified as molecular markers of prognosis for cm patients in stage iii . their use in the daily practice will drive the most appropriate clinical management of stage iii cm patients . prognostic significance of the methylation status of single genes taken from whole - genome defined prognostic methylation signatures in light of the prognostic relevance of whole - genome methylation profiles that the authors have identified ( see example 3 above ), the authors have addressed the question whether specific genes taken from the prognostic methylation signatures may retain a prognostic information . to this end , the authors selected genes alox12b , ctag2 , fgf4 , igll1 , ociad2 , pages , s100a9 , slc6a11 , slc6a18 , and tub from signature e and g as playing a major contribution in the classification tasks . the 45 stage iiic patients under study were scored accordingly to the methylation of each gene in their tumor cells being & lt ; 33 %, between 33 % and 66 %, and & gt ; 66 %, or & lt ; and & gt ; 50 %. the impact on patients &# 39 ; survival of alox12b , ctag2 , fgf4 , igll1 , ociad2 , pages , s100a9 , slc6a11 , slc6a18 , and tub methylation levels was evaluated by kaplan - meier analysis and log - rank test , showing a significantly improved survival for patients with unmethylated vs methylated genes ( table 11 , fig1 ). similar results were found for other tested genes , including : batf , bgn , crhr1 , csag1 , ctsk , dennd2d , flj33860 , gibs , grm4 , ly96 , mageal magea10 , mgc35206 , mlstd1 , slc18a2 , sorbs2 , and trim40 ( data not shown ). * patients were divided according to the % of methylation of genes selected from prognostic methylation signatures in their tumor cells being & lt ; 33 %, between 33 % and 66 %, and & gt ; 66 %, or & lt ; and ≧ 50 %. † survival functions were calculated by the kaplan - meier method . data are reported as median os in months , together with the corresponding 95 % confidence intervals ( ci ). prognostic utility of methylation scores defined by whole - genome methylation profiles in melanoma in light of the prognostic relevance of whole - genome methylation profiles that the authors have identified ( see example 3 above ), the authors have addressed the question whether simple scores summarizing the whole genome methylation profiles could retain a prognostic information . to this end , the genome - wide methylation profile of tumor cells from each patient ( defined by illumina infinium humanmethylation27 bead array , see example 3 above ) was summarized with a “ methylation score ” as follows : methylation for each gene among the patients was standardized by the z score method , calculated by ( x − μ )/ σ where x stands for methylation data of each gene in each sample , μstands for mean of methylation of each gene among all samples , and σstands for the standard deviation . each patient was then assigned a “ methylation score ” consisting of the average of z scores for all genes . the 45 stage iiic patients under study were divided accordingly to the “ methylation score ” of their tumor cells being below or above the median value of the patients &# 39 ; population . the impact on patients &# 39 ; survival of the whole genome methylation profiling - derived “ methylation score ” was evaluated by kaplan - meier analysis , log - rank test and cox regression analysis , showing a significantly improved survival for patients with low vs high “ methylation score ” ( table 12 , fig1 ). ‡ cox proportional hazard method was used to examine the effect of line - 1 methylation on os . results were presented as hazard ratios ( hr ) with corresponding 95 % confidence intervals ( ci ); prognostic significance of combined evaluation of line - 1 methylation levels and multigene methylation signatures in stage iii cutaneous melanoma patients in light of the prognostic relevance of line - 1 methylation status and of whole - genome methylation profiles that the authors have identified ( see examples 1 , 3 above ), the authors have addressed the question whether combining these two markers would result in an additional prognostic information . to this end , 59 stage iii melanoma patients were scored according to their whole genome methylation profile as k2 - 1 or k2 - 2 ( stage iii patients ) or s3 - k2 - 1 or s3 - k2 - 2 ( substage iiic patients ), and as having hypomethylated or hypermethylated line - 1 sequences , as detailed in examples 1 and 3 . the impact on patients &# 39 ; survival of the combined evaluation of the 2 markers was evaluated by kaplan - meier analysis and log - rank test ( fig1 , table 13 ). results demonstrated that the combined use of the 2 markers allowed to further discriminate survival groups within the population under study ( p & lt ; 0 . 05 ). * patients were scored by k - means clustering algorithm as k2 - 1 or k2 - 2 ( stage iii patients ) or as s3 - k2 - 1 or s3 - k2 - 2 ( sub - stage iiic patients ), according to their genome - wide methylation profile , and as having hypomethylated (& lt ; median value ) or hypermethylated (≧ median value ) line - 1 sequences . combined evaluation of this 2 markers resulted in the definition of 4 classes . † survival functions were calculated by the kaplan - meier method . data are reported as median os in months , together with the corresponding 95 % confidence intervals ( ci ). dna was extracted from neoplastic cells and evaluated for line - 1 methylation at 3 cpg sites ( cpg1 , 2 , 3 ) by pyrosequencing or for mean line - 1 methylation by quantitative methylation - specific pcr ( qmsp ). neoplastic cells were purified using anti - hmw - maa antibody - coated magnetic beads from cryopreserved cell suspensions obtained by mechanical and enzymatic dissection of neoplastic lesions surgically removed from cm patients . surgically - removed neoplastic tissues from cm patients were flesh - frozen in liquid nitrogen and maintained at − 80 ° c . until use . surgically - removed neoplastic tissues from cm patients were formalin - fixed and paraffin - embedded . ten micrometer sections ( total 2 to 6 ) were cut from paraffin embedded lesions and used for evaluation of line - 1 methylation . neoplastic tissue of different extensions ( area 9 - 100 mm2 ) was scraped from 15 micrometer sections of formalin - fixed paraffin - embedded cm tissues , and used for evaluation of line - 1 methylation . in a further technical validation , neoplastic cells were purified from hematoxylin and eosin stained formalin - fixed paraffin - embedded cm tissues by laser capture microdissection ( lcm ) using an arcturus system ( applied biosystems ). genomic dna was extracted from lcm tumor cells using the picopure dna kit ( applied biosystems ) and subjected to bisulfite modification by the ez dna methylation - gold kit ( zymo research ). the extent of methylation of line - 1 elements was measured by a qmsp assay using primers : l1m fwd cgcgagtcgaagtagggc ( seq id no . 230 ) and l1m rev acccgattttccaaatacgaccg ( seq id no . 231 ), specific for the methylated sequences ; and l1u fwd tgtgtgtgagttgaagtagggt ( seq id no . 232 ) and l1u rev acccaattttccaaatacaaccatca ( seq id no . 233 ), specific for the umethylated sequences . sybr green qmsp reactions were performed with methylated - or unmethylated - specific primer pairs on 2 μl of bisulfite - modified genomic dna in a final volume of 25 μl 1x power sybr green mastermix ( applyed biosystems ) at 95 ° c . for 10 min , followed by 45 cycles of 15 sec at 95 ° c . and 1 min at 60 ° c . real - time measurement of fluorescence intensity was performed utilizing the abi prism 7000 sequence detection system ( applyed biosystems ), and the copy number of methylated or unmethylated sequences for each target gene was established in each sample by extrapolation from the standard curves generated with plasmids carrying fully methylated or unmethylated line - 1 sequences . the % of methylation was defined as the ratio between methylated molecules and the sum of methylated and unmethylated molecules . † neoplastic cells were purified using anti - hmw - maa antibody - coated magnetic beads from cryopreserved cell suspensions obtained by mechanical and enzymatic dissection of neoplastic lesions surgically removed from cm patients ; ‡ surgically - removed neoplastic tissues from cm patients were flesh - frozen in liquid nitrogen and maintained at − 80 ° c . until use ; § surgically - removed neoplastic tissues from cm patients were formalin - fixed and paraffin - embedded . ten micrometer sections ( total 2 to 6 ) were cut from paraffin embedded lesions and used for evaluation of line - 1 methylation ; †† neoplastic tissue of different extensions ( area 9 - 100 mm2 ) was scraped from 15 micrometer sections of formalin - fixed paraffin - embedded cm tissues , and used for evaluation of line - 1 methylation . § correlation between % line - 1 values obtained from 6 th passage neoplastic cells and autologous lcm neoplastic cells was evaluated by the pearson &# 39 ; s correlation test . coefficient of correlation is reported with the corresponding 95 % confidence interval ( ci ). reported p value is 2 - 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