Oncogene Amplification as an Incidental Finding in FISH Testing for Gene Rearrangements in Lymphoid Hematopoietic Neoplasms

Abstract 2505

Oncogene amplification resulting in overexpression, although common in solid tumors, is rare in hematopoietic neoplasms. This is particularly true in lymphoid neoplasms compared to AML where MYC, MLL or RUNX1 (AML1) amplification has been mostly seen, and to CML where BCR/ABL fusion gene amplification has been also reported. Typically, lymphoid neoplasms are tested at diagnosis by FISH for specific reciprocal chromosome translocations that lead to overexpression of deregulated oncogenes such as BCL1, BCL2, BCL6 and MYC in B-cell lymphoma and myeloma or BCR/ABL gene fusion in ALL. Nevertheless, we have unexpectedly seen oncogene amplification from time to time in our FISH lab. To study the incidence of gene amplification and its diagnostic and clinical implications, we retrospectively analyzed FISH results routinely performed on paraffin embedded lymphoma tissues, lymph nodes, bone marrow aspirates and peripheral bloods in the past three and half years using translocation probes for BCL2/IGH, BCL1/IGH, MYC/IGH and BCR/ABL and break apart probes for BCL6 and MYC. The highest amplification rate seen was in BCL2/IGH testing: 11 of the 1,710 cases were positive (0.643%). In 2 cases with follicular lymphoma (FL), BCL2 amplification presented as homogenously staining regions (hsr), one case with diffuse large B-cell lymphoma (DLBCL) showed double minutes (dmin), and two cases with FL had a pattern of combined hsr and dmin. Five cases with low grade FL intriguingly showed a similar pattern of BCL2/IGH translocation and concomitant amplification of the rearranged BCL2 as a small hsr. The remaining case diagnosed as Burkitt lymphoma (BL) was positive for MYC/IGH translocation and for BCL2 amplification present as large hsr. BCL6 amplification was observed in 4 of 1,537 cases tested (0.26%). In all these cases, amplification presented as hsr. One case diagnosed as EBV+ DLBLC was positive for MYC/IGH translocation and 3' BCL6 high level amplification. Another case with FL showed BCL6 rearrangement and 5' BCL6 amplification. BCL1 amplification with translocation was seen as hsr in 1 case with mantle cell lymphoma out of 2,898 tested. BCL1 amplification was observed in another case with plasma cell myeloma as hsr out of 3,413 tests performed. MYC gene amplification was positive in 3 of 2,186 (0.137%). One case with BL showed MYC rearrangement with a concomitant 3' deletion and 5' amplification. The second case diagnosed as B-cell lymphoma with features intermediate between DLBCL and BL showed highly amplified MYC gene as hsr. The third case was DLBCL with 15–50 copies of MYC gene per cell as dmin. Among 530 BCR/ABL tests ordered for acute leukemia or ALL, 2 (0.377%) cases with T cell lymphoblastic leukemia/lymphoma (T-ALL/LBL) showed ABL amplification as episomes (4–8 copies in one case; 6–30 in another). Both were abnormal by cytogenetics analysis, but negative for t(9;22)(q34;q11.2) and without dmin or hsr identified. Although detection rates of oncogene amplification seem to be very low with limited specific translocation probes applied to lymphoid neoplasms, they may well be higher when FISH signal patterns are analyzed more carefully. In our study, 5 of the 11 FL cases tested for IGH/BCL2 showed a peculiar pattern positive for the t(14;18)-IGH/BCL2 and a concomitant red signal amplification of BCL2, the size of which is usually small and may be overlooked under microscope. All these 5 cases had low grade FL, suggesting that oncogene amplification can be an early genomic event in lymphomagenesis. MYC gene amplification is generally considered a late stage genomic alteration. When MYC is rearranged, amplification of BCL2, BCL6 or BCL1 may need to be taken into consideration for disease stratification, or vice versa. For instance, so called “double-hit” or “triple-hit” lymphomas currently require two or three concurrent translocations for diagnosis. Oncogene amplification, equally important for deregulation/overexpression as a translocation, should be considered as a second or a third hit in diagnosis and differential diagnosis of B-cell lymphoma unclassifiable with features intermediate between DLBCL and BL. FISH is a very useful tool to identify episomal oncogene amplification. Episomes, unlike cytogenetically evident dmin and hrs, are invisible by chromosome analysis, but their detection is important as reported for integration of targeted tyrosine kinase inhibitors into chemotherapeutic regimens.