A B S T R A C T PurposeDiffuse large B-cell lymphoma (DLBCL) is curable in 60% of patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). MYC translocations, with or without BCL2 translocations, have been associated with inferior survival in DLBCL. We investigated whether expression of MYC protein, with or without BCL2 protein expression, could risk-stratify patients at diagnosis. Patients and MethodsWe determined the correlation between presence of MYC and BCL2 proteins by immunohistochemistry (IHC) with survival in two independent cohorts of patients with DLBCL treated with R-CHOP. We further determined if MYC protein expression correlated with high MYC mRNA and/or presence of MYC translocation. ResultsIn the training cohort (n ϭ 167), MYC and BCL2 proteins were detected in 29% and 44% of patients, respectively. Concurrent expression (MYC positive/BCL2 positive) was present in 21% of patients. MYC protein correlated with presence of high MYC mRNA and MYC translocation (both P Ͻ .001), but the latter was less frequent (both 11%). MYC protein expression was only associated with inferior overall and progression-free survival when BCL2 protein was coexpressed (P Ͻ .001). Importantly, the poor prognostic effect of MYC positive/BCL2 positive was validated in an independent cohort of 140 patients with DLBCL and remained significant (P Ͻ .05) after adjusting for presence of high-risk features in a multivariable model that included elevated international prognostic index score, activated B-cell molecular subtype, and presence of concurrent MYC and BCL2 translocations. ConclusionAssessment of MYC and BCL2 expression by IHC represents a robust, rapid, and inexpensive approach to risk-stratify patients with DLBCL at diagnosis.

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.

A 2-year-old girl was seen initially at the emergency department of another institution and presented with symptoms of intermittent disequilibrium and worsening gait that had been present for the previous 2-3 weeks. Her medical history disclosed no other relevant findings, and she had no known risk factors. The patient had no history of recent travel or exposure to illness, and before the onset of symptoms, she had been healthy. She was transferred to our facility for further evaluation because of abnormal findings at computed tomography (CT) of the brain. Imaging FindingsCT examination of the brain without the use of contrast material demonstrated multiple hypoattenuating areas within the cerebral hemispheres bilaterally; these areas had the typical appearance of vasogenic edema (Fig 1a). The imaging findings were suggestive of a nonspecific infectious or inflammatory process, such as cerebritis. In the right thalamus, a focal hypoattenuating round lesion was depicted that extended toward the right basal ganglia and had an appearance suggestive of an abscess or tumor (Fig 1b).

2005 Background: Concurrent translocations in MYC and BCL2 determined by fluorescence in-situ hybridization (FISH), have been associated with a poor outcome in diffuse large B cell lymphoma (DLBCL) patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). However, unlike immunohistochemistry (IHC), FISH is expensive and is not routinely available in all clinical laboratories. The aim of this study was to determine if MYC protein expression or expression of the proliferation marker Ki-67 by IHC could be used to identify samples that harbour MYC translocations. Methods: DLBCL samples, diagnosed by an expert panel of hematopathologists according to the WHO criteria of 2008 and derived from 167 patients treated with R-CHOP, have been subjected to gene expression profiling (GEP) and FISH using commercial break-apart probes for BCL2 and MYC. MYC mRNA expression was determined using log2 normalized expression values of probe set 202431_s_at. The presence of MYC, BCL2 and Ki-67 protein expression was determined by IHC using commercially available antibodies (clone Y69 (Epitomics), clone 124 (Dako) and clone Ki-67 (Dako) respectively). MYC protein is not normally expressed in germinal centers (tonsils, negative control), thus the % of tumour cells staining for MYC was noted in each case. Thresholds for BCL2 protein (50%) and MYC mRNA (>9.4) were determined using the statistical software X-tile which determines the optimal threshold based on its association with clinical outcome. For MYC protein expression, no threshold was significant by X-tile thus a 40% threshold was used based on the bimodal distribution of the data, with a through occurring at 40%. Protein expression for MYC and Ki-67 was correlated to the presence of MYC translocations, MYC mRNA expression and outcome including overall survival (OS) and progression free survival (PFS). Results: Over-expression of MYC was present in 56/167 (34%) of DLBCL samples (18/167 translocations, 19/167 high mRNA and 47/167 high protein expression) and was not specific to the germinal center B cell (GCB) or activated B cell (ABC) molecular subtype. MYC protein expression (in ≥40% of cells) captured 13/18 MYC translocations and in 4/5 remaining cases, MYC staining was present in 20–39% of cells. MYC protein expression, alone, was not associated with OS but the presence of a MYC translocation was associated with an inferior OS in the ABC subtype only. BCL2 protein expression was also associated with an inferior OS in this cohort (p=0.002). Concurrent expression of MYC and BCL2 protein by IHC was associated with a markedly inferior OS compared to MYC protein- or MYC protein+/BCL2 protein- (median OS of 2 years, 7 years and > 7 years respectively, p<0.001). In a Cox-multivariate model, the co-expression of MYC and BCL2 proteins by IHC maintained prognostic significance independent of the IPI (OS p=0.007, PFS p=0.008). In contrast, Ki-67 staining (>90%) identified only 5/18 cases with MYC translocations and was not associated with outcome alone or in combination with BCL2. Conclusions: MYC deregulation in DLBCL is more common than previously reported (34%) and can occur in the absence of a MYC translocation. MYC and BCL2 protein expression could be easily determined by routine IHC in most clinical laboratories and should be prospectively tested as potential predictive biomarkers in DLBCL patients treated with R-CHOP. Disclosures: Grogan: Ventana Roche: Employment, Equity Ownership.