Double Hit Lymphomas: Role of Immunohistochemistry in the Era of Florescent in-Situ Hybridization

Background

Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease. A subset of DLBCL patients with translocations involving MYC and BCL2 genes, called "Double-hit" lymphoma (DHL), are being identified to have poor prognosis after standard chemotherapy. Fluorescent in situ hybridization (FISH) technique which can identify these gene mutations is the gold standard for diagnosis of DHL. Immunohistochemistry (IHC) is a rapid and inexpensive test that can identify abnormal protein expression of these mutated genes. Two studies thus far have shown that MYC IHC can be used as a screening test to identify DHL cases with sensitivities of 74% and 100%, both multi-center studies conducted at university hospitals. We conducted this study at a single large community hospital to determine if these results can be generalized to our institution.

Objective

Our primary objective was to determine the association between protein and genetic expression of MYC and BCL2 in cases of de novo DLBCL by comparing the results of IHC and FISH. Our secondary objectives were to determine if MYC IHC could be used as a screening test to determine DHL status, to determine if the double hit biology is associated with any clinic-pathological features which might allow for risk stratification of patients and to determine the prevalence of DHL in our cohort of DLBCL patients.

Methods

Twenty-two patients who were diagnosed with primary DLBCL from February 2015 to February 2016 at Abington Hospital (AH) Jefferson Health were identified after applying exclusion and inclusion criteria. Clinico-pathological data was obtained by review of electronic medical records and biopsy specimens for IHC and FISH were obtained from the Department of Pathology at AH. The FISH test was performed by Quest Diagnostics™ and IHC was performed by the Chair of the Department of Pathology at our institution. Statistical analysis was performed using IBM SPSS Statistics for Windows, version 20.0 (Armonk, NY: IBM Corp).

Results

Double hit gene rearrangements by FISH were detected in 3/22 (13.6%) patients. MYC IHC was positive in 8/22 (36.3%) cases, and 3 of these 8 cases (37.5%) were FISH positive. Sensitivity, specificity and concordance of MYC IHC as a screening tool were 100%,73.6% and 72.27%, respectively. BCL2 staining was positive in 19/22 (86.3%) cases, and only 3 of the 19 cases (15.7%) were FISH positive. 5/22 (22.7%) cases were positive for both MYC and BCL2 by IHC (double protein expresser status) and 3 of these 5 (60%) cases were positive for DHL status by FISH. DHL biology did not show an association with any clinico-pathological parameters including age, elevated serum Lactate dehydrogenase, R-IPI (Revised International Prognostic Index) score, ECOG performance status, extra-nodal disease and cell-of-origin sub-type.

Conclusion

The association between MYC IHC and FISH in our study was not statistically significant but showed a trend towards association. Given that sensitivity of MYC in our study was 100%, we propose that it can be used as an effective screening test if similar results can be validated in larger studies. Our study supports the practice of routinely testing for MYC and BCL2 status in all cases of DLBCL, irrespective of clinical features of the disease, to identify the high-risk DHL subset of patients, who can be candidates for more intense newer chemotherapy regimens and for prognostic purposes