Scoring of MYC Protein Expression in Diffuse Large B-cell Lymphomas

Concordance Rate Among Hematopathologists

Amer Z Mahmoud; Tracy I George; David R Czuchlewski; Qian-Yun Zhang; Carla S Wilson; Cordelia E Sever; Alexei G Bakhirev; Dahua Zhang; Nichole L Steidler; Kaaren K Reichard; Huining Kang; Kathryn Foucar; Mohammad A Vasef


Mod Pathol. 2015;28(4):545-551. 

In This Article

Abstract and Introduction


Recent studies have shown that immunohistochemical evaluation of MYC protein expression in diffuse large B-cell lymphoma is a useful prognostic tool with high concordance rate among pathologists. Concordance in these studies was assessed among few pathologists from one institution by scoring tissue microarrays. In daily practice, MYC evaluation is performed on entire tumor sections by a diverse group of pathologists. In our study, nine hematopathologists from two institutions scored whole-tissue sections of two sets of cases. The training set included 13 cases of diffuse large B-cell lymphoma and 4 cases of Burkitt lymphoma. The validation set included 18 cases of diffuse large B-cell lymphoma and 1 case of Burkitt lymphoma. MYC positivity was defined as ≥40% of tumor cells demonstrating nuclear staining similar to prior studies. The mean score for each case was used to determine MYC status with discrepant cases defined as having any score causing a different MYC status designation. Discrepant cases from the training set were characterized by staining heterogeneity, extensive necrosis or crush artifact and had mean scores within 15 percentage points of 40%. Cases from the validation set that demonstrated any of these features were scored twice on two different days. Overall concordance was moderate (Kappa score: 0.68, P-value <0.001) with no significant change between the two sets (Kappa scores: 0.69 vs 0.67). Thirty-nine percent of cases were discrepant. The findings indicate that a significant number of diffuse large B-cell lymphomas are inherently difficult to score due to staining heterogeneity. The effect of heterogeneity can be under-represented when concordance is measured among few pathologists scoring tissue microarrays. Careful scoring strategy in our study failed to improve concordance. In the absence of specific instructions on how to deal with heterogeneity, caution is advised when evaluating MYC expression in diffuse large B-cell lymphoma.


The MYC oncogene is involved in many types of human cancer. The discovery of a consistent balanced chromosomal translocation involving the MYC gene in Burkitt lymphoma was the first evidence to characterize MYC as a human oncogene.[1] Subsequently, MYC gene alterations have been discovered in B-cell neoplasms other than Burkitt lymphoma.[2] Among those neoplasms, diffuse large B-cell lymphoma is the most widely studied. The presence of MYC rearrangements in patients with diffuse large B-cell lymphoma treated with Rituximab, Cyclophosphamide, Doxorubicin, Vincristine and Prednisone (R-CHOP) has been shown to be associated with poor prognosis.[3,4] In particular, the so-called 'double-hit' lymphomas that are characterized by MYC rearrangement and a concurrent rearrangement of other B-cell lymphoma-associated genes such as BCL2 or BCL6 are associated with poor response to therapy, aggressive clinical course and dismal prognosis.[5–7] These lymphomas are classified as 'B-cell lymphoma, unclassifiable with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma' in the current 2008 WHO classification of hematopoietic neoplasms.[8]

Evaluation for MYC alterations in diffuse large B-cell lymphoma is typically performed by fluorescence in situ hybridization (FISH). FISH is capable of detecting MYC gene alterations that result from translocation or amplification of the gene. These genetic alterations result in MYC protein overexpression, which is ultimately responsible for the oncogenic effect.[1,2] MYC protein overexpression has also been found to occur as a consequence of other genomic events not detected by FISH.[9] Thus FISH might miss a subset of diffuse large B-cell lymphoma cases that demonstrate MYC protein overexpression.

MYC protein expression has been evaluated in formalin-fixed paraffin-embedded tissue by immunohistochemistry in multiple studies.[9–13] These studies have shown that cases of diffuse large B-cell lymphoma with concurrent overexpression of MYC and BCL2 proteins have a dismal prognosis similar to those with double-hit lymphomas. The rate of so-called double-hit lymphoma-like cases as determined by immunohistochemistry is larger than the one detected by FISH.[9,10] Evaluation of MYC expression in these studies is performed by estimating the percentage of MYC protein expressing tumor cells. In the majority of studies, a cutoff of ≥40% is used to define MYC protein overexpression.

Although high rates of inter-observer concordance have been reported in prior studies, scoring is performed on scant material in tissue microarrays.[9–13] Tissue microarrays sample only a small portion of the tumor and therefore may under-represent the heterogeneity of staining among tumor cells encountered in daily practice. The heterogeneity of MYC protein staining is not adequately addressed in these studies. Additionally, the concordance rate is assessed among only two[10,11,13] or three[9] pathologists that might underestimate inter-observer variability among practicing pathologists. We hypothesized that these concordance rates might not be reproducible in daily practice. In this study, we examined the concordance rate in MYC scoring among nine hematopathologists from two institutions when evaluating entire tumor sections and investigated whether scoring a tissue microarray-sized field instead of entire section will improve the concordance. We also identified some features that characterize discrepant cases and evaluated whether careful scoring of these cases can improve concordance. The impact of using an image analysis program was also assessed.