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. 2013 Feb;139(2):242-54.
doi: 10.1309/AJCP2Z0TAGMUYJEB.

Colorimetric in situ hybridization identifies MYC gene signal clusters correlating with increased copy number, mRNA, and protein in diffuse large B-cell lymphoma

Affiliations

Colorimetric in situ hybridization identifies MYC gene signal clusters correlating with increased copy number, mRNA, and protein in diffuse large B-cell lymphoma

Carlo Valentino et al. Am J Clin Pathol. 2013 Feb.

Abstract

Abnormalities of the MYC oncogene on chromosome 8 are characteristic of Burkitt lymphoma and other aggressive B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL). We recently described a colorimetric in situ hybridization (CISH) method for detecting extra copies of the MYC gene in DLBCL and the frequent occurrence of excess copies of discrete MYC signals in the context of diploidy or polyploidy of chromosome 8, which correlated with increased mRNA signals. We further observed enlarged MYC signals, which were counted as a single gene copy but, by their dimension and unusual shape, likely consisted of "clusters" of MYC genes. In this study, we sought to further characterize these clusters of MYC signals by determining whether the presence of these correlated with other genetic features, mRNA levels, protein, and overall survival. We found that MYC clusters correlated with an abnormal MYC locus and with increased mRNA. MYC mRNA correlated with protein levels, and both increased mRNA and protein correlated with poorer overall survival. MYC clusters were seen in both the germinal center and activated B-cell subtypes of DLBCL. Clusters of MYC signals may be an underappreciated, but clinically important, feature of aggressive B-cell lymphomas with potential prognostic and therapeutic relevance.

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Figures

Figure 1
Figure 1
MYC clustering in diffuse large B-cell lymphoma (DLBCL) cases with MYC gene amplification. The DLBCL tumor sections were divided into 3 groups according to MYC and centromere 8 (CEN8) copy number. Group 1 consisted of cases with MYC and CEN8 copy number equal or less than 44 (normal MYC), group 2 with MYC copy number greater than 44 and CEN8 less than 44 (MYC amplification), and group 3 with MYC copy number and CEN8 greater than 44 (chromosome 8 polysomy). Lines represent the mean. The number of cells that contained clustering of MYC colorimetric in situ hybridization signal was counted for a total of 20 cells per section. A 1-way analysis of variance was used to determine significance (P < .0001).
Figure 2
Figure 2
Total MYC copy number and messenger RNA (mRNA) levels in cases with normal or abnormal MYC and in the presence or absence of MYC clustering. The total MYC copy number (A, B) and log2-transformed MYC mRNA levels (C, D) were compared between diffuse large B-cell lymphoma patient cases with normal (not increased) and abnormal (increased MYC and/or CEN8 copy number or MYC translocation positive) MYC (B, D) and in the absence or presence of MYC colorimetric in situ hybridization signal clustering (A, C). MYC mRNA levels were obtained from the Affymetrix U133 Plus 2.0 Chip (Affymetrix, Santa Clara, CA). Lines represent the mean. The total MYC copy number was counted from 20 cells per case. The Mann-Whitney 2-tailed t test was used to determine significance when the 2 groups differed in variances; otherwise, an unpaired t test was used.
Figure 3
Figure 3
MYC protein expression in cases that differ in MYC and chromosome 8 copy number and correlated to MYC messenger RNA (mRNA) and patient overall survival. The percent MYC protein positive cells as determined by immunohistochemistry was compared among the 3 different MYC and chromosome 8 copy number groups using a 1-way analysis of variance (A). MYC protein positive and negative cases were evaluated for level of MYC mRNA (log2 transformed) (B). Lines represent the mean. A case was defined as positive for MYC protein if at least 40% of cells were stained within a 300-cell count. The Mann-Whitney 2-tailed t test was used to determine significance.
Figure 4
Figure 4
MYC clustering, copy number, and messenger RNA (mRNA) levels in germinal center B-cell (GCB) and activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL) subtypes. The DLBCL cases were previously stratified into GCB-like or ABC-like subtypes based on gene expression profiling and analyzed for MYC clustering (A), total MYC copy number (B), and MYC mRNA levels (C). Lines represent the mean. The Mann-Whitney 2-tailed t test was used to determine significance when the 2 groups differed in variances; otherwise, an unpaired t test was used.
Figure 5
Figure 5
Diffuse large B-cell lymphoma (DLBCL) patient survival following R-CHOP (rituximab-cyclophosphamide, doxorubicin, vincristine, and prednisone). Kaplan-Meier overall survival curves of DLBCL patients according to MYC messenger RNA levels as detected by the Affymetrix U133 Plus 2.0 DNA microarray (Affymetrix, Santa Clara, CA) for the entire cohort (A), MYC colorimetric in situ hybridization signal clustering for group 2 cases (B), and MYC protein of patients dichotomized based on the 80th percentile (C). A, P = .0095; hazard ratio (HR), 0.5042; 95% CI, 0.2226-0.8117. B, P = .1701; HR, 0.5467; 95% CI, 0.2312-1.295. C, P = .0029; HR, 0.3646; 95% CI, 0.09051-0.6100.
Image 1
Image 1
MYC immunohistochemistry (IHC), colorimetric in situ hybridization (CISH), and CISH clusters. A-C, MYC CISH: MYC = blue, CEN8 = red A. Diffuse large B-cell lymphoma (DLBCL) MYC CISH ×1,000. This case is a representative example of group 1 (MYC <44 copies/20 cells; CEN8 <44 copies/20 cells). B, DLBCL MYC CISH ×1,000. An example of group 2 (MYC ≥44 copies/20 cells; CEN8 ≤44 copies/20 cells). The large cell (arrow) was interpreted as 6 blue signals and not as a clustered signal. C, DLBCL MYC CISH × 1,000. An example of group 3 (MYC ≥44 copies/20 cells; CEN8 >44 copies/20 cells). D-G, MYC CISH signal clusters: DLBCL MYC CISH × 1,000. The images show various patterns of clustering found in multiple cases examined. Frequently, the signal patterns wrapped around the CEN8 (red) signal, as exemplified in Image 1F (arrow). H-J, MYC IHC. H, Tonsil control, ×400. The image shows expected staining of the proliferative layer of epithelium but not the more superficial layers of epithelium (lower left). The lymphatic endothelial cells are negative, while scattered positivity is seen in the underlying lymphocytes (upper right). I, Tonsil control, ×400. Close-up view of the germinal center showing scattered positive centroblasts, negative follicular dendritic cells (arrow), and negative histiocytes. J, DLBCL, ×400. Strong positive staining in nearly all nuclei in a t(8;14)-positive case.
Image 1
Image 1
MYC immunohistochemistry (IHC), colorimetric in situ hybridization (CISH), and CISH clusters. A-C, MYC CISH: MYC = blue, CEN8 = red A. Diffuse large B-cell lymphoma (DLBCL) MYC CISH ×1,000. This case is a representative example of group 1 (MYC <44 copies/20 cells; CEN8 <44 copies/20 cells). B, DLBCL MYC CISH ×1,000. An example of group 2 (MYC ≥44 copies/20 cells; CEN8 ≤44 copies/20 cells). The large cell (arrow) was interpreted as 6 blue signals and not as a clustered signal. C, DLBCL MYC CISH × 1,000. An example of group 3 (MYC ≥44 copies/20 cells; CEN8 >44 copies/20 cells). D-G, MYC CISH signal clusters: DLBCL MYC CISH × 1,000. The images show various patterns of clustering found in multiple cases examined. Frequently, the signal patterns wrapped around the CEN8 (red) signal, as exemplified in Image 1F (arrow). H-J, MYC IHC. H, Tonsil control, ×400. The image shows expected staining of the proliferative layer of epithelium but not the more superficial layers of epithelium (lower left). The lymphatic endothelial cells are negative, while scattered positivity is seen in the underlying lymphocytes (upper right). I, Tonsil control, ×400. Close-up view of the germinal center showing scattered positive centroblasts, negative follicular dendritic cells (arrow), and negative histiocytes. J, DLBCL, ×400. Strong positive staining in nearly all nuclei in a t(8;14)-positive case.

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