Molecular Classification of MYC-Driven B-Cell Lymphomas by Targeted Gene Expression Profiling of Fixed Biopsy Specimens  Christopher D. Carey, Daniel Gusenleitner, Bjoern Chapuy, Alexandra E. Kovach, Michael J. Kluk, Heather H. Sun, Rachel E. Crossland, Chris M. Bacon, Vikki Rand, Paola Dal Cin, Long P. Le, Donna Neuberg, Aliyah R. Sohani, Margaret A. Shipp, Stefano Monti, Scott J. Rodig  The Journal of Molecular Diagnostics  Volume 17, Issue 1, Pages 19-30 (January 2015) DOI: 10.1016/j.jmoldx.2014.08.006 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Target gene selection and the generation of molecular classifiers. A: Schematic showing the distribution of gene transcripts that were assayed in the initial and final profiling panels. B: Schematic outlining the protocols for the molecular classification of all aggressive B-cell lymphomas and cases with the pathological diagnosis of diffuse large B-cell lymphoma (DLBCL) and B-cell lymphoma unclassifiable (BCL-U). Twenty-one genes were used for the Diagnostic classifier, and 61 genes were used for the MYC activity classifier. In addition, eight genes were common to both classifiers, and there were six housekeeping genes (HK). The Journal of Molecular Diagnostics 2015 17, 19-30DOI: (10.1016/j.jmoldx.2014.08.006) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Unsupervised clustering of the normalized transcript values from 42 tumors comprising the training cohort and including all target probes in the initial profiling panel (one case later failed quality control during classification). The original pathological diagnosis and relative gene expression for the 185 genes comprising the initial profiling panel (heatmap) are shown (15 housekeeping genes were excluded). Max, maximum; min, minimum. The Journal of Molecular Diagnostics 2015 17, 19-30DOI: (10.1016/j.jmoldx.2014.08.006) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 A: Leave-one-out cross-validation of the final profiling panel and diagnostic classifier for the training cohort: Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). B: Results of the diagnostic classifier for the test cohort: BL, B-cell lymphoma unclassifiable (BCL-U), and DLBCL cases categorized according to the original pathological diagnosis, the assigned molecular diagnosis (diagnostic scores of 0.25 to 0.75 categorized as molecularly intermediate), diagnostic score (line graph, intermediate values shaded), the relative expression of the indicated transcripts (heat map), including the relative contribution of each to the classifier (horizontal shaded bar graphs), and MYC-rearrangement status. The cases of genetic double hit lymphomas are numbered, and additional gene rearrangements are indicated by arrowheads (BCL2-) or a circle (BCL6-). The single-hit DLBCL, with MYC-rearrangement only, is indicated by an asterisk. Max, maximum; min, minimum. The Journal of Molecular Diagnostics 2015 17, 19-30DOI: (10.1016/j.jmoldx.2014.08.006) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 A: Scatterplot shows the mean TCF3 signature (seven genes, x axis) and mean MYC signature (10 genes, y axis) for each tumor from the test cohort. The mean values for each signature are derived from transcript counts from these genes, as originally used in the diagnostic classifier. Colors indicate the pathological/genetic diagnoses [black for Burkitt lymphoma (BL), gray for diffuse large B-cell lymphoma (DLBCL), and yellow for genetic double hit lymphomas (DHLs)]. Shapes indicate the molecular classification assigned by the diagnostic classifier [triangle for molecular (m)BL, circle for molecular DLBCL (mDLBCL), and square for molecularly intermediate]. B: Histomorphological features of lymphomas with a MYC rearrangement and either a BCL2- or BCL6- rearrangement (genetic DHL). H&E-stained sections of DHL classified as mBL and mDLBCL. Unique identifiers and details of relevant translocations are shown. The tumors classified as mDLBCL have insets highlighting nuclear morphological characteristics. Original magnification, ×1000 (main images and insets). The Journal of Molecular Diagnostics 2015 17, 19-30DOI: (10.1016/j.jmoldx.2014.08.006) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 A: Leave-one-out cross-validation of the final profiling panel and MYC activity classifier for the training cohort. Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are segregated by pathological diagnosis, MYC activity score (line graph), the relative expression of the indicated transcripts (heat map), including the relative contribution of each to the classifier (horizontal, shaded bar graphs), MYC IHC class (MYC IHC-Low ≤50%, IHC-High >50), and MYC rearrangement status. Inset: The correlation between MYC IHC and MYC activity score for DLBCL only (Spearman r = 0.80; 95% CI, 0.6–0.9). B: Results of the final profiling panel and MYC activity classifier for the test cohort, BL, DLBCL, and B-cell lymphoma unclassifiable (BCL-U), are segregated by pathological diagnosis, MYC activity score (line graph), the relative expression of the indicated transcripts (heat map), including the relative contribution of each to the classifier (horizontal, shaded bar graphs), MYC IHC class (MYC IHC-Low ≤50%, IHC-High >50%), and MYC rearrangement status. Genetic double hit lymphomas are numbered and additional gene rearrangements are indicated by arrowheads (BCL2-) or a circle (BCL6-). The single-hit DLBCL, with MYC-rearrangement only, is indicated by an asterisk. Inset: The correlation between MYC IHC and MYC activity score for non-BL only (Spearman r = 0.66; 95% CI, 0.44–0.8). Max, maximum; min, minimum. The Journal of Molecular Diagnostics 2015 17, 19-30DOI: (10.1016/j.jmoldx.2014.08.006) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Results of the MYC classifier and overall survival among patients with primary diffuse large B-cell lymphoma treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone)–based chemotherapy. A: The correlation between MYC score and MYC IHC for the outcome series (Spearman r = 0.64; 95% CI, 0.4–0.8). B: Kaplan-Meier curve shows OS for the outcome series with a MYC score >0.5 (red line) and a MYC score <0.5 (black line). The Journal of Molecular Diagnostics 2015 17, 19-30DOI: (10.1016/j.jmoldx.2014.08.006) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions