PCR-based analysis of rearranged immunoglobulin or T-cell receptor genes by GeneScan analysis or heteroduplex analysis for clonality assessment in lymphoma diagnostics.

The assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (Ig) or T-cell receptor (TCR) genes is a valuable technique in the diagnosis of suspect lymphoproliferative disorders. Furthermore this technique is more and more used to evaluate dissemination of non-Hodgkin lymphoma and/or the presence of (minimal) residual disease. In this chapter we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TCRB), and TCR gamma (TCRG) gene rearrangements. The described PCR protocol is based on the standardized multiplex PCRs as developed by the European BIOMED-2 collaborative study (Concerted Action BMH4-CT98-3936). Furthermore it also includes the pre-analytical DNA isolation step from various tissues (formalin fixed paraffin-embedded tissue, fresh tissues, body fluids, peripheral blood and bone marrow), GeneScan analysis of labeled PCR products on a genetic analyzer, heteroduplex analysis of unlabeled PCR products, and post-analytical guidelines for the interpretation of the obtained "molecular morphology" patterns.

Integrated transcript and genome analyses reveal NKX2-1 and MEF2C as potential oncogenes in T cell acute lymphoblastic leukemia.

To identify oncogenic pathways in T cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular-cytogenetic analyses including the Chromosome Conformation Capture on Chip (4C) method. Two T-ALL subtypes were identified that lacked rearrangements of known oncogenes. One subtype associated with cortical arrest, expression of cell cycle genes, and ectopic NKX2-1 or NKX2-2 expression for which rearrangements were identified. The second subtype associated with immature T cell development and high expression of the MEF2C transcription factor as consequence of rearrangements of MEF2C, transcription factors that target MEF2C, or MEF2C-associated cofactors. We propose NKX2-1, NKX2-2, and MEF2C as T-ALL oncogenes that are activated by various rearrangements.

Different chromosomal breakpoints impact the level of LMO2 expression in T-ALL.

The t(11;14)(p13;q11) is presumed to arise from an erroneous T-cell receptor delta TCRD V(D)J recombination and to result in LMO2 activation. However, the mechanisms underlying this translocation and the resulting LMO2 activation are poorly defined. We performed combined in vivo, ex vivo, and in silico analyses on 9 new t(11;14)(p13;q11)-positive T-cell acute lymphoblastic leukemia (T-ALL) as well as normal thymocytes. Our data support the involvement of 2 distinct t(11;14)(p13;q11) V(D)J-related translocation mechanisms. We provide compelling evidence that removal of a negative regulatory element from the LMO2 locus, rather than juxtaposition to the TCRD enhancer, is the main determinant for LMO2 activation in the majority of t(11;14)(p13;q11) translocations. Furthermore, the position of the LMO2 breakpoints in T-ALL in the light of the occurrence of TCRD-LMO2 translocations in normal thymocytes points to a critical role for the exact breakpoint location in determining LMO2 activation levels and the consequent pressure for T-ALL development.

The predictive value of lipoprotein lipase for survival in chronic lymphocytic leukemia.

BACKGROUND AND OBJECTIVES: The mutational status of the immunoglobulin heavy chain variable region genes (IGVH) is a strong indicator of prognosis in B-cell chronic lymphocytic leukaemia (CLL). Since the determination of the IGVH mutation status is very labor-intensive, alternative prognostically relevant markers would facilitate CLL diagnostics. DESIGN AND METHODS: Ten genes were selected from previously published gene expression profiling studies based on their differential expression in IGVH mutated versus unmutated cases of CLL, and tested with real-time quantitative polymerase chain reaction (RQ-PCR) in unpurified samples from 130 CLL patients. To ascertain potential contaminating effects by normal hematopoietic cells, the expression levels of the selected genes were determined in normal monocytes, B cells, T cells, NK cells and granulocytes. RESULTS: The selected genes, i.e., ZAP70, LPL, SPG20, ADAM29, NRIP1, AKAP12, DMD, SEPT10, TPM2 and CLECSF2, showed prognostic significance. In multivariate logistic regression analysis expression levels of LPL, ZAP70, ADAM29 and SEPT10 were the most predictive for IGVH mutational status. In univariate analysis the expression of LPL was the best predictor. For survival, expression of LPL was the strongest prognostic factor. In combination with the three cytogenetic markers associated with a poor prognosis, i.e., deletions 17p13, 11q22 and trisomy 12, expression of LPL and IGVH mutational status performed equally well with regard to their predictive value for survival, both being more predictive than ZAP70. INTERPRETATION AND CONCLUSIONS: This study demonstrates that LPL expression is a predictor for survival in CLL, and for this purpose is as good as IGVH mutational status and more reliable than ZAP70 expression when tested in unpurified CLL samples.

BIOMED-2 multiplex immunoglobulin/T-cell receptor polymerase chain reaction protocols can reliably replace Southern blot analysis in routine clonality diagnostics.

To establish the most sensitive and efficient strategy of clonality diagnostics via immunoglobulin and T-cell receptor gene rearrangement studies in suspected lymphoproliferative disorders, we evaluated 300 samples (from 218 patients) submitted consecutively for routine diagnostics. All samples were studied using the BIOMED-2 multiplex polymerase chain reaction (PCR) protocol. In 176 samples, Southern blot (SB) data were also available, and the two types of molecular results were compared. Results of PCR and SB analysis of both T-cell receptor and immunoglobulin loci were concordant in 85% of samples. For discordant results, PCR results were more consistent with the final diagnosis in 73% of samples. No false-negative results were obtained by PCR analysis. In contrast, SB analysis failed to detect clonality in a relatively high number of samples, mainly in cases of low tumor burden. We conclude that the novel BIOMED-2 multiplex PCR strategy is of great value in diagnosing patients with suspected B- and T-cell proliferations. Because of its higher speed, efficiency, and sensitivity, it can reliably replace SB analysis in clonality diagnostics in a routine laboratory setting. Just as with SB results, PCR results should always be interpreted in the context of clinical, immunophenotypical, and histopathological data.

Unraveling the consecutive recombination events in the human IGK locus.

In addition to the classical Vkappa-Jkappa, Vkappa-kappa deleting element (Kde), and intron-Kde gene rearrangements, atypical recombinations involving Jkappa recombination signal sequence (RSS) or intronRSS elements can occur in the Igkappa (IGK) locus, as observed in human B cell malignancies. In-depth analysis revealed that atypical JkappaRSS-intronRSS, Vkappa-intronRSS, and JkappaRSS-Kde recombinations not only occur in B cell malignancies, but rather reflect physiological gene rearrangements present in normal human B cells as well. Excision circle analysis and recombination substrate assays can discriminate between single-step vs multistep rearrangements. Using this combined approach, we unraveled that the atypical Vkappa-intronRSS and JkappaRSS-Kde pseudohybrid joints most probably result from ongoing recombination following an initial aberrant JkappaRSS-intronRSS signal joint formation. Based on our observations in normal and malignant human B cells, a model is presented to describe the sequential (classical and atypical) recombination events in the human IGK locus and their estimated relative frequencies (0.2-1.0 vs < 0.03). The initial JkappaRSS-intronRSS signal joint formation (except for Jkappa1RSS-intronRSS) might be a side event of an active V(D)J recombination mechanism, but the subsequent formation of Vkappa-intronRSS and JkappaRSS-Kde pseudohybrid joints can represent an alternative pathway for IGK allele inactivation and allelic exclusion, in addition to classical Ckappa deletions. Although usage of this alternative pathway is limited, it seems essential for inactivation of those IGK alleles that have undergone initial aberrant recombinations, which might otherwise hamper selection of functional Ig L chain proteins.

Identification and characterization of the interaction between tuberin and 14-3-3zeta.

Tuberous sclerosis is caused by mutations to either the TSC1 or TSC2 tumor suppressor gene. The disease is characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction, and dermatological abnormalities. TSC1 encodes a 130-kDa protein called hamartin, and TSC2 encodes a 200-kDa protein called tuberin. Although it has been shown that hamartin and tuberin form a complex and mediate phosphoinositide 3-kinase/Akt-dependent phosphorylation of the ribosomal protein S6, it is not yet clear how inactivation of either protein leads to tuberous sclerosis. Therefore, to obtain additional insight into tuberin and hamartin function, yeast two-hybrid screening experiments were performed to identify proteins that interact with tuberin. One of the proteins identified was 14-3-3zeta, a member of the 14-3-3 protein family. The interaction between tuberin and 14-3-3zeta was confirmed in vitro and by co-immunoprecipitation; multiple sites within tuberin for 14-3-3zeta binding were identified; and it was determined that 14-3-3zeta associated with the tuberin-hamartin complex. Finally, it was shown that the tuberin/14-3-3zeta interaction is regulated by Akt-mediated phosphorylation of tuberin, providing insight into how tuberin may regulate phosphorylation of S6.