Supplementary Appendix

Transcription

1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Curtin JA, Fridlyand J, Kageshita T, et al. Distinct sets of genetic alterations in melanoma. N Engl J Med 2005;353:

2 Supplementary Information Primary data processing: We acquired 16 bit 1024x1024 pixel DAPI, Cy3 and Cy5 images using a custom built CCD camera system and carried out image and data analysis using UCSF SPOT. 39 The arrays contain 2462 clones printed as triplicate spots. Ratios for each spot were calculated as the total background-corrected fluorescence intensity ratios for the test and reference channels. No other computational adjustments were applied to the data. We used the SPROC software to automatically filter the data based on quality criteria, average the log2 ratios of the replicate spots, and assign genome position. Only clones for which two or more spots passed quality criteria and for which the standard deviation of the replicates was less than 0.2 were included in the subsequent analysis. Data were normalized to set the median log2 ratio to zero. Prior to the statistical analysis, clones were removed if data was missing in more than 25% of the samples or in 50% of any individual melanoma sub-group. We used only clones that were mapped on the genome sequence and which did not detect copy number polymorphisms in normal samples. For classification and genome-wide testing, missing values were imputed using the lowess approach, 28 which predicts missing values on a given profile using values observed on the neighboring clones via piecewise linear regression. The procedure is implemented in the acgh package. 27 Definition of aberrations: A clone was declared aberrant if its absolute value exceeded the tumor specific threshold computed as 2.5 times the estimate of the standard deviation of the experimental noise for a given profile. 26 Amplifications were detected using the default parameters of the acgh package 27 which implements the algorithm described in Fridlyand et al (2004). 26 A clone was considered amplified if it belonged to a narrow

3 region with sufficiently higher log2 ratio compared to its neighbors. Specifically, the algorithm identifies regions, which are smaller than 10 Mbases and whose absolute log2 ratio exceeds 0.9 and also exceed the log2 ratio of its immediately flanking regions by at least 0.5. The algorithm also identifies regions narrower than 10Mbases whose log2 ratios exceed immediately flanking segments by 0.9. The latter rule permits the detection of amplifications originating out of regions present at decreased copy number. Clones with log2 ratios less than -0.9 were considered homozygously deleted. These levels were chosen empirically due to the expected presence of normal cells in the specimens and tumor heterogeneity and are concordant with the thresholds used in the previously 40, 41 published array CGH articles. Classification: For all classification procedures, the DLDA prediction rule 30 (Linear Discriminant Analysis with covariance matrix identical for all groups and with zero-offdiagonal elements) was used and the grid of the number of variables (10 through 1000) was set. Each sample was repeatedly left out the entire classification procedure (including variable selection using an F-statistic and mean and covariance matrix estimation) and for a given number of variables the predictor was built using the remaining samples. The left-out sample was predicted using the resulting classifier. The procedure was repeated for all samples and all variables in a grid. The leave-one-out error rate was stable across a wide range of variables (at least 200 variables for 2-class problems and at least 400 variables for 4-class problem)

4 References 26. Fridlyand J, Snijders AM, Pinkel D, Albertson DG, Jain AN. Hidden Markov models approach to the analysis of array CGH data. Journal of Multivariate Analysis 2004; 90: Fridlyand J, Dimitrov P. acgh Package Cleveland WS. Lowess - a Program for Smoothing Scatterplots by Robust Locally Weighted Regression. American Statistician 1981; 35: Dudoit S, Fridlyand J, Speed TP. Comparison of discrimination methods for the classification of tumors using gene expression data. Journal of the American Statistical Association 2002; 97: Jain AN, Tokuyasu TA, Snijders AM, Segraves R, Albertson DG, Pinkel D. Fully automatic quantification of microarray image data. Genome Research 2002; 12: Mehta KR, Nakao K, Zuraek MB, et al. Fractional genomic alteration detected by array-based comparative genomic hybridization independently predicts survival after hepatic resection for metastatic colorectal cancer. Clinical Cancer Research 2005; 11: Nakao K, Mehta KR, Moore DH, et al. High-resolution analysis of DNA copy number alterations in colorectal cancer by array-based comparative genomic hybridization. Carcinogenesis 2004; 25:

5 Supplementary Table 1. Case Group Sex Age Histogenetic Type Anatomic Site Thickness BRAF NRAS [years] [mm] AM32 acral NA NA ALM NA 1.9 NA NA AM41 acral M 62 NC palm 1 Mut Wt 105 acral M 69 ALM sole 2.8 NA NA 60 acral F 82 ALM sole 5.2 NA NA AM107 acral M 79 ALM sole 1.6 Wt Wt AM108 acral M 63 ALM sole 1.8 Wt Wt AM112 acral M 77 ALM sole 8 Wt Wt AM114 acral M 77 ALM sole 8 Mut Wt AM12 acral NA NA ALM sole 4.3 Wt Mut AM130 acral F 69 ALM sole 3 NA NA AM137 acral M 71 ALM sole 4.8 Wt Wt AM165 acral F 74 ALM sole 3.1 Wt Wt AM47 acral F 75 ALM sole 1.7 Wt Wt AM49 acral M 56 ALM sole 1.4 Wt Wt AM50 acral M NA ALM sole 2.4 NA NA AM9 acral NA NA ALM sole 3.4 Wt Wt D196 acral M NA ALM sole 4.2 NA NA 51 acral F 82 NC sole 3.6 Wt Wt AM17 acral F 77 NC sole 1.2 Wt Wt AM18 acral F 90 NC sole 2 Wt Mut AM19 acral M 85 NC sole 2.1 Mut Wt AM38 acral F 90 NC sole NC Wt Mut AM125 acral M 83 NM sole 3.6 Wt Wt AM133 acral F 84 SSM sole 1.8 Wt Wt AM169 acral M 66 SSM sole 1.9 Wt Wt AM22 acral F 68 SSM sole 2.2 Mut Wt AM40 acral F 34 SSM sole 2.1 Wt Wt AM74/1 acral M NA SSM sole 3.8 Wt Wt AM45 acral M 59 ALM sole 3.4 Wt Wt AM60 acral M 50 ALM subungual 9.6 Mut Wt AM23 acral M 42 ALM subungual 2 Mut Wt 20 acral M 73 ALM subungual 4.2 Wt Wt AM126 acral M 77 ALM subungual 4.6 Wt Wt

6 AM132 AM160 AM Mx15 Mx41 Mx58 Mx /1 136 Mx Mx Mx4 Mx7 AM175 AM170 AM171 acral M 57 ALM subungual NC Mut Wt acral F 66 ALM subungual 10 Wt Wt acral F 72 ALM subungual NC Wt Wt CSD M 72 LMM head 3 Wt Wt CSD M 82 LMM head 7.5 Wt Wt CSD F NA LMM head 1.05 Wt Mut CSD F 84 LMM head 5 Wt Wt CSD M 86 LMM head 5.3 Wt Wt CSD F 67 LMM head 5 Wt Wt CSD F 94 LMM head 1.2 Wt Wt CSD F 66 LMM head 1 Mut Wt CSD F 52 LMM head 1.5 Wt Mut CSD F 84 LMM head 6.8 Wt Wt CSD F 87 LMM head 1.1 Wt Wt CSD F 63 LMM head 2.9 NA NA CSD NA 76 LMM head 1.2 Wt Wt CSD NA 73 LMM head 1.8 Wt Wt CSD M 63 LMM head 5 Wt Wt CSD M 88 LMM head 1.5 Wt Mut CSD M 81 NC head 4 Wt Wt CSD F 69 NM head 3 Wt Mut CSD M 78 LMM head 0.96 Mut Wt CSD M NA LMM head 3.5 Wt Wt CSD M 73 NC head 4.5 Wt Wt CSD F 73 LMM NC 3.8 Wt Wt CSD M 82 LMM NC 3 Wt Wt CSD NA NA LMM NC 2 Mut Wt CSD M 76 LMM Trunk 1.65 Wt Wt CSD M 78 NC Trunk 6.2 NA NA CSD M 84 NM Trunk 1.4 NA NA CSD F 73 LMM Upper Extremity 3.8 Wt Wt CSD M 83 LMM Upper Extremity 6 Wt Wt CSD M 67 LMM Upper Extremity 3.3 Wt Wt mucosal F 76 NC genital 12 Wt Wt mucosal F 63 NC genital 10 Wt Wt mucosal F 83 NC genital 3.8 Wt Wt

7 AM174 AM121 AM120 AM123 AM124 AM138 AM139 AM145 AM146 AM141 AM142 AM143 AM144 AM147 AM148 AM149 AM AM mucosal F 38 NC genital 6.7 Wt Wt mucosal F 72 NC Nasopharyengal NC Wt Wt mucosal F 67 NC Nasopharyengal 22 Wt Wt mucosal M 41 NC Nasopharyengal NC Mut NA mucosal F 81 NC Nasopharyengal 45 Wt Wt mucosal M 47 NC Nasopharyengal 4.5 Wt Wt mucosal F 75 NC Nasopharyengal 5 Mut Wt mucosal F 44 NC Nasopharyengal NC Wt Wt mucosal F 78 NC Nasopharyengal 5 Wt Mut mucosal M 65 NC Nasopharyengal 5 Wt Wt mucosal F 72 NC Nasopharyengal 5 Wt Wt mucosal M 68 NC Nasopharyengal 10 Wt Wt mucosal F 44 NC Nasopharyengal 41 Wt Wt mucosal M 71 NC Nasopharyengal 5 NA NA mucosal M 54 NC Nasopharyengal 5 Wt Wt mucosal F 68 NC Nasopharyengal 5 Wt Wt mucosal M 55 NC Nasopharyengal 5 Wt Wt non-csd F 38 SSM head 2.7 Mut Wt non-csd F 81 SSM Lower Extremity 3 Wt Wt non-csd M 66 SSM Lower Extremity 1.82 Wt Wt non-csd F 21 NC Lower Extremity 2.47 Mut Wt non-csd M 65 SSM Lower Extremity 1.2 Wt Mut non-csd NA NA SSM Lower Extremity 3.1 Wt Wt non-csd M 76 NC Lower Extremity 5 Wt Mut non-csd F 72 SSM Lower Extremity 4 NA NA non-csd F 83 SSM Lower Extremity 3 Mut Wt non-csd F 53 SSM Lower Extremity 2.14 Mut Wt non-csd F 69 SSM Lower Extremity Mut Wt non-csd M 59 SSM Lower Extremity 1.2 Wt Wt non-csd F 84 SSM Lower Extremity 3.7 Wt Mut non-csd F 75 SSM NC 3.7 Mut Wt non-csd M 64 NC Trunk 7.5 NA NA non-csd M 74 SSM Trunk 3.1 Mut Wt non-csd F 80 NC Trunk 7 Mut Wt non-csd F 60 NC Trunk 7.5 Mut Wt non-csd F 67 SSM Trunk 2.25 Wt Mut

8 4 D22/1 26 D21/1 D D80/ D non-csd F 28 SSM Trunk 3.6 Mut Wt non-csd F 38 SSM Trunk 1.9 Mut Wt non-csd M 65 SSM Trunk 5.5 Mut Wt non-csd F 52 SSM Trunk 5.5 Mut Wt non-csd M 65 NC Trunk NC Wt Mut non-csd M 47 SSM Trunk 2.3 Mut Wt non-csd M 65 SSM Trunk 1.55 NA NA non-csd M 71 SSM Trunk 2.4 Wt Wt non-csd F 46 SSM Trunk 4.9 Mut Wt non-csd M 67 SSM Trunk 5 Mut Wt non-csd F 65 SSM Trunk 3.6 Mut Wt non-csd M 82 SSM Trunk 5.5 Wt Mut non-csd M 28 SSM Trunk 1.2 Mut Wt non-csd M 42 SSM Trunk 1.2 Mut Wt non-csd M 51 SSM Trunk 4.5 Mut Wt non-csd M 53 SSM Upper Extremity 11.7 Wt Mut non-csd F 74 SSM Upper Extremity 2.25 Mut Wt non-csd M 65 SSM Upper Extremity 4.75 Wt Wt non-csd F 82 NM Upper Extremity 1.8 Wt Mut non-csd M 11 NM Upper Extremity 3.3 Wt Wt non-csd M 85 NM Upper Extremity 9.5 Mut Wt Abbreviations used: NC: not classified; NA: not available; SSM: superficial spreading melanoma; NM: nodular melanoma; LMM: lentigo maligna melanoma; ALM: acral lentiginous melanoma; CSD: chronic sun damage

9 Supplementary Table 2: Case p16 p-erk p-akt CCND ND ND 0 19 ND ND ND 2 51 ND ND ND ND ND ND ND ND 81 3 ND ND 2 Mx15 3 ND ND 2 Mx7 2 3 ND AM ND Mx ND ND 102 ND 1 3 ND ND ND AM ND Mx ND 2 Mx ND AM ND AM ND ND ND

10 ND 0 0 ND ND ND ND AM ND AM ND AM ND AM ND AM ND AM ND AM ND AM ND AM ND AM47 ND 0 1 ND AM ND AM50 ND 0 ND ND AM74/1 ND 0 1 ND AM9 ND 0 0 ND Mx4 0 0 ND 2 Mx ND 1 Mx ND 2 Supplementary Table 2 legend: Expression of p16 was determined using the p16ink4a Ab-4 monoclonal antibody Cat# MS-887-P1 (Lab Vision Corporation, Freemont, CA). The phosphorylation status of pakt was determined with the Phospho-Akt (Ser473) polyclonal antibody Cat# 9277S (Cell Signaling Technology, Inc., Beverly, MA). The phosphorylation status of ERK (p-erk) was determined using the Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) monoclonal antibody Cat# 9106S (Cell Signaling). Expression of cyclin D1 was determined using the monoclonal antibody ASM29 from Zymed (South San Francisco, CA). All immunohistochemistry was performed according to standard procedures using 3-amino-9-ethylcarbazole as a chromagen. Scores were recorded semiquantitatively with intensity values from 0-4 where 0 equals no expression and 4 equals most intense expression.