The molecular abnormalities present in immature T-cell neoplasms and pediatric NHL are heterogeneous, involving different components of cellular regulation. The aim of this study was to investigate the molecular pathogenesis of T-LBL, with particular emphasis on whether detection of protein overexpression by IHC correlates with underlying molecular abnormalities by FISH. In T-ALL, conventional cytogenetic techniques have shown that translocations commonly involve regulatory elements of the TCR α/δ (14q11.2) or TCR β (7q34) loci and various cellular oncogenes such as TAL1, c-Myc, LMO2, NOTCH1, and unrecognized partner genes that may or may not be normally expressed during T-cell development [6
]. Multiple secondary abnormalities usually occur, with early abnormalities providing a proliferation advantage and/or differentiation block that combines with later events to result in transformation [9
]. Activation of cellular oncogenes, production of abnormal fusion genes, and deletions of tumor suppressor genes may cause malignancy [6
]. Identification of abnormalities is often done by conventional cytogenetic analysis, but genetic abnormalities may be cryptic [6
]. Use of FISH analysis or IHC staining to detect overexpression or aberrant expression of proteins have been used as a surrogate for cytogenetic analysis. Using this strategy, we evaluated expression of a broad panel of proteins associated with NHL, including c-Myc, p53, bcl-2, and bcl-6 by IHC and FISH in cases of T-LBL to determine whether possible molecular abnormalities can be reliably predicted by IHC.
Analysis of the data indicated that correlation between IHC and FISH results was weak, suggesting that IHC staining alone is not sufficient to identify specific molecular abnormalities, but may better reflect aberrant protein production that arises from a variety of mechanisms. For example, strong expression of c-Myc protein by IHC was present in the majority of our cases (77%) but often did not correlate with an identifiable c-Myc abnormality by FISH (72% normal by FISH); perhaps suggesting that upstream or downstream genetic abnormalities, such as NOTCH-1 mediated signaling, are contributing to dysregulation of c-Myc, as c-myc is an important downstream target of NOTCH-1 [6
Similarly, the tumor suppressor p53 was positive in most cases but showed variable staining, with most cases having staining in <50% of tumor cells. However, only 16% of cases were abnormal by FISH (gains), supporting the concept that protein overexpression is not directly or exclusively due to FISH-detectable molecular abnormalities [17
]. Reported expression of bcl-2 in T-LBL has been variable [18
], and we observed variable bcl-2 expression in 73% of cases with strong staining in only 23%. However, 82% of FISH results were normal, and of the three abnormal cases by FISH, only 2 cases had any detectable IHC staining for bcl-2.
Other important regulators of lymphomas, such as bcl-6, are often overexpressed predominantly due to rearrangements placing the bcl-6 gene adjacent to an exogenous promoter. Interestingly, although it is most often associated with B-cell neoplasms, bcl-6 was recently shown to be overexpressed in a subset (8 of 16) of T-LBL cases, although, subsequent molecular analysis showed germline bcl-6 configuration [20
]. Our bcl-6 results confirmed bcl-6 overexpression in T-LBL (14% of cases) and predominantly normal FISH (75% of cases).
The results of this study confirm that pediatric T-LBL, in general, has a high proliferative rate and increased c-Myc expression. Low expression of other important lymphoma –associated proteins such as p53, bcl-2, and skp2, and almost no expression of bcl-6 and TCL-1 were observed by immunohistochemistry. Furthermore, this initial study has demonstrated that immunohistochemical staining is an unreliable method for identifying protein overexpression arising secondary to underlying FISH molecular abnormalities. Although limited by a small number of cases, no correlation was observed between protein overexpression by IHC and FISH detection of molecular abnormalities, using appropriate controls and cutoffs. Although FISH studies may be less sensitive in paraffin-embedded tissue, our results were normal in the majority of cases and suggest chromosomal copy number changes in the abnormal cases, rather than translocations involving the target genes. Future studies could be designed to specifically relate T-ALL and T-LBL using FISH probes more specific to these tumors, and additional studies to further elucidate the molecular abnormalities in T-LBL, with particular focus on utilizing oligonucleotide microarray and/or array comparative genomic hybridizations, may correlate with aberrant protein expression and help better elucidate the molecular pathogenesis of childhood T-LBL.