Pathological Features of MYC IHC-High DLBCL.

<p>Pathological Features of MYC IHC-High DLBCL.</p

Comparison of MYC IHC score and Ki67 proliferation index for all cases analyzed.

<p>Cases with a <i>MYC</i>-translocation are colored in red and cases without a <i>MYC</i>-translocation are colored in black. The two biomarkers demonstrate a weak but positive correlation (Spearman <i>r</i> = 0.33, p = 0.003).</p

GSEA of MYC target genes in tumors characterized for MYC protein expression;

<p>(<b>A</b>). Gene Set Enrichment Analysis (GSEA) of all primary DLBCL cases reveals coordinate upregulation of MYC and MYC target genes (black vertical lines) in tumors with >50% MYC positive tumor nuclei (including <i>MYC</i> translocation-positive and –negative cases) (See Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033813#pone.0033813.s006" target="_blank">Methods S1</a> for additional details). <b>Gene Set Enrichment Analysis (GSEA) of MYC-target genes in </b><b><i>MYC</i></b><b> translocation–negative Primary DLBCL tumors;</b> (<b>B</b>) <i>MYC</i> translocation-negative primary DLBCL cases with >50% MYC positive tumor nuclei have coordinate upregulation of MYC and MYC target genes (black vertical lines).</p

Immunohistochemical detection of MYC in representative Primary DLBCLs.

<p>Photomicrographs of select tumors and reactive tissue stained for MYC (positive staining = brown nuclei). Positive control (Burkitt lymphoma with a confirmed <i>MYC</i> translocation) revealed uniform, intense staining in >90% of tumor cells (Burkitt). In contrast, reactive lymphoid tissue revealed variable staining in only 10% of normal lymphocyte nuclei (Tonsil). Representative images from DLBCL cases and associated percent MYC+ tumor nuclei: Case 1, 90% MYC+; Case 7, 70% MYC+; and Cases 35 and 38, 30% MYC+. MYC staining was exclusively nuclear in all cases under the described staining conditions.</p

Comparison of MYC protein expression and <i>MYC</i> translocation status in cases of Primary <i>(de novo)</i> DLBCL;

<p>(<b>A</b>). Comparison of percent MYC positive tumor nuclei for Burkitt lymphoma, reactive tonsil and 56 primary DLBCLs with their corresponding <i>MYC</i> translocation status (bar graph; pink = <i>MYC</i> translocation, green = no <i>MYC</i> translocation, gray = not determined). Tumors with an IHC score of >50% are indicated in red at left. Tumors are arranged by IHC-determined percentage of MYC-positive nuclei. <b>Comparison of MYC protein expression and </b><b><i>MYC</i></b><b> translocation in cases of Secondary DLBCL;</b> (<b>B</b>). Comparison of percent MYC positive tumor nuclei for Burkitt lymphoma, reactive tonsil and 21 cases of recurrent or transformed DLBCLs with their corresponding <i>MYC</i> translocation status (bar graph; pink = <i>MYC</i> translocation, green = no <i>MYC</i> translocation). Tumors with an IHC score of >50% are indicated in red at left. Tumors are arranged by IHC-determined percentage of MYC-positive nuclei.</p

Morphological features of select MYC IHC-High DLBCLs.

<p>Hematoxylin and Eosin stained sections (all 1000× original magnification) of (A) Relapse case #2, DLBCL NOS; (B). Relapse case #4, DLBCL NOS; (C) Primary case #1, DLBCL, immunoblastic variant; (D) Primary case #3, DLBCL, NOS, with some morphological features that might suggest B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (see text for details).</p

Characteristics of R-CHOP treated Patient Cohort used for Survival Analysis.

1)<p>MYC High cases show positive staining for MYC in >50% of tumor nuclei; MYC Low cases show positive staining for MYC in ≤50% tumor nuclei.</p>2)<p>Cases classified as MYC IHC-High are associated with a higher IPI than cases classified as MYC IHC-Low (p = 0.06, Kruskal-Wallis test).</p

Outcome analysis according to MYC protein expression in Primary (<i>de novo</i>) DLBCL.

<p>Kaplan-Meier curve depicting the statistically significant (p = 0.01, log rank test) difference in the overall survival for the 38 primary DLBCL patients treated with R-CHOP. Tumors were grouped by MYC IHC score >50% (n = 6) or ≤50% (n = 32).</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