Common regions of LOH in high-risk neuroblastoma and corresponding copy number change (A), and areas of copy number gain (B) identified by 10K SNP array analysis

<p>Common regions of LOH in high-risk neuroblastoma and corresponding copy number change (A), and areas of copy number gain (B) identified by 10K SNP array analysis</p

Figure 3

<p>LOH and copy number changes on chromosomes 11, 3, 4, 1, 17, and 7. Global view of common areas of LOH (left) and copy number change (right) in 22 primary neuroblastomas. Each sample is depicted as a series of vertical bars in both the LOH and copy number panels. Blue areas represent regions of LOH, while yellow denotes retention of heterozygosity. Copy number is marked by shades of red, with ≤1 copy in light red and ≥3 copies in dark red (see scale at the bottom of the panel).</p

Figure 1

<p>Frequency of chromosomal aberrations. Fraction of samples with copy number >2.8 (red bars above baseline), copy number <1.2 (grey bars below baseline), and LOH (blue bars below baseline). SNPs are mapped according to their chromosomal position, from chromosome 1 on the left to chromosome X on the right. (Copy number alterations for chromosome X in samples derived from males were counted if copy number was >1.4 or <0.6.)</p

Figure 2

<p>LOH and copy number changes on chromosome 11. SNP array analysis of neuroblastoma tumor samples and matched constitutional DNA showing LOH on the left and copy number on the right of the chromosome 11 ideogram. For each sample, chromosome 11 is depicted as a vertical bar in both the LOH and copy number panels. Blue areas represent regions of LOH, while yellow denotes retention of heterozygosity. Copy number is marked by shades of red, with ≤1 copy in light red and ≥3 copies in dark red (see scale at the bottom of the panel). Both chromosome 11q and 11p LOH, as well as gain of 11p, are shown.</p

Figure 5

<p>Amplifications on chromosome 2p. (A) SNP copy number analysis of a portion of chromosome arm 2p demonstrating amplification (represented by the darker red SNPs) at the <i>MYCN</i> gene locus (2p24.3) in three tumor samples and at the <i>ALK</i> gene locus (2p23.2) in one sample. (B) Individual SNP copy number assessment of the SNPs surrounding the <i>ALK</i> locus. The blue curve in the graph on the right indicates the degree of amplification of each SNP from 0 on the left to 40 on the right. The red vertical line indicates a copy number of 2. (C) FISH of neuroblastoma with the <i>MYCN</i> probe showing multiple copies of <i>MYCN</i> (left panel), and FISH of the same tumor using the <i>ALK</i> break apart probe showing amplification of <i>ALK</i> (right panel).</p

Figure 4

<p>Copy number and LOH analysis of chromosome 17. SNP array analysis of neuroblastoma tumor samples and matched constitutional DNA showing LOH on the left and copy number on the right of the chromosome 17 ideogram. For each sample, chromosome 17 is depicted as a vertical bar in both the LOH and copy number panels. Blue areas represent regions of LOH, while yellow denotes retention of heterozygosity. Copy number is marked by shades of red, with ≤1 copy in light red and ≥3 copies in dark red (see scale at the bottom of the panel). This figure depicts copy number gain of chromosome 17q in 21 out of 22 samples.</p

Deep sequencing deconvolution of multiple cDNA inserts.

<p><b>A.</b> Screening of the SK-MEL-23 melanoma cell line results in multiple clones with different patterns of cDNA inserts (upper) that all harbor NRAS based on an NRAS specific PCR (lower). <b>B.</b> The mutant NRAS allele harbors an A434T (G60G) and C435A (Q61K) dinucleotide substitution (underlined by thick black bar) that is present as 100% of sequence within a BaF3 transformant and as 50% or less in the SK-MEL-23 cDNA, plasmid acceptor library and retroviral library. <b>C.</b> Deep sequencing identified 12 distinct fusion sequences between the attB (black triangle) and 5′ UTR of NRAS from the SK-MEL-23 library. The number of individual reads for each of the sequences is provided. <b>D.</b> A subset of approximately 90,000 reads across the NRAS dinucleotide substitution are shown. The sequence is on the genomic negative strand and thus reversed from <b>C.</b> The C435A (G→T shown on the positive strand) substitution that results in Q61K is between the hashed marks. Gray reads have the paired-end on the same exon while brown reads have the paired-end on the next exon.</p

cDNA library screening identified an ALK translocation partner and resistance allele.

<p><b>A.</b> Immunohistochemistry using an antibody against ALK and karyotype of the anaplastic large cell lymphoma specimen 421. <b>B.</b> Multiple IL3-independent clones were recovered from screening of the cDNA library generated from 421, all of which contained ALK cDNA using primers within the flanking attB sites or within ALK itself. <b>C.</b> Immunoblot using antibody against ALK demonstrates ATIC-ALK expression in multiple clones but not in wild-type Ba/F3 cells. <b>D.</b> Effect on proliferation of 96 individual clones recovered from the 421 library with TAE684 300 nM, quantified using the ATP chemiluminescent agent CellTiterGlo. <b>E.</b> PCR amplification using the att-f and att-r primers with DNA from clone D3 demonstrates a full-length STAT5B allele that harbored a G210T (K70N) mutation.</p

Recovery of a transforming ERRB2 splice variant.

<p><b>A.</b> cDNA library screening of the melanoma cell lines 70 W (a sub-line of the MeWo) and HCC1569 resulted in IL3 independent Ba/F3 clones that contained ERBB2Δ16. <b>B.</b> Relative proliferation quantified using the ATP chemiluminescent agent CellTiterGlo. <b>C.</b> cDNA transcripts recovered from screening of MeWo, HCC1569 and 70 W cell lines. All of the transcripts lacked amino acids 2137–2184 that comprise exon 6. <b>D.</b> Expression of ERBB2 cDNA containing exon 16 using specific primers. Error bars indicate standard deviation. <b>E.</b> Expression of ERBB2Δ16 cDNA using specific primers, including a 5′ primer that overlaps exons 15 and 17 so does not amplify wild-type ERBB2.</p

cDNA library screening identifies EML4-ALK.

<p><b>A.</b> Screening of a library generated from H2228 cells generated 16 clones with distinct patterns of integrated cDNA. All clones contained one of two EML4-ALK transcripts that differed based on splicing of exon 6 and the length of the 5′ untranslated region, as indicated by the most 5′ nucleotide fused to the attB sequence. λ indicates size ladder. <b>B.</b> Screening of a library generated from H3122 cells generated Ba/F3 clones isolated at the indicated time point after withdrawal of IL3. PCR is shown using primers that amplify integrated cDNA. <b>C.</b> PCR amplification from eight clones generated from the H3122 library with different patterns of integrated cDNA. Five distinct EML4-ALK transcripts were identified based on differences in the length of the 5′ untranslated region, as in <b>A.</b></p