EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations.

PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.

Standardized MRD quantification in European ALL trials: proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008.

Assessment of minimal residual disease (MRD) has acquired a prominent position in European treatment protocols for patients with acute lymphoblastic leukemia (ALL), on the basis of its high prognostic value for predicting outcome and the possibilities for implementation of MRD diagnostics in treatment stratification. Therefore, there is an increasing need for standardization of methodologies and harmonization of terminology. For this purpose, a panel of representatives of all major European study groups on childhood and adult ALL and of international experts on PCR- and flow cytometry-based MRD assessment was built in the context of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008. The panel summarized the current state of MRD diagnostics in ALL and developed recommendations on the minimal technical requirements that should be fulfilled before implementation of MRD diagnostics into clinical trials. Finally, a common terminology for a standard description of MRD response and monitoring was established defining the terms 'complete MRD response', 'MRD persistence' and 'MRD reappearance'. The proposed MRD terminology may allow a refined and standardized assessment of response to treatment in adult and childhood ALL, and provides a sound basis for the comparison of MRD results between different treatment protocols.

PAX5 mutations occur frequently in adult B-cell progenitor acute lymphoblastic leukemia and PAX5 haploinsufficiency is associated with BCR-ABL1 and TCF3-PBX1 fusion genes: a GRAALL study.

Adult and child B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) differ in terms of incidence and prognosis. These disparities are mainly due to the molecular abnormalities associated with these two clinical entities. A genome-wide analysis using oligo SNP arrays recently demonstrated that PAX5 (paired-box domain 5) is the main target of somatic mutations in childhood BCP-ALL being altered in 38.9% of the cases. We report here the most extensive analysis of alterations of PAX5 coding sequence in 117 adult BCP-ALL patients in the unique clinical protocol GRAALL-2003/GRAAPH-2003. Our study demonstrates that PAX5 is mutated in 34% of adult BCP-ALL, mutations being partial or complete deletion, partial or complete amplification, point mutation or fusion gene. PAX5 alterations are heterogeneous consisting in complete loss in 17%, focal deletions in 10%, point mutations in 7% and translocations in 1% of the cases. PAX5 complete loss and PAX5 point mutations differ. PAX5 complete loss seems to be a secondary event and is significantly associated with BCR-ABL1 or TCF3-PBX1 fusion genes and a lower white blood cell count.

Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

The diagnosis of malignant lymphoma is a recognized difficult area in histopathology. Therefore, detection of clonality in a suspected lymphoproliferation is a valuable diagnostic criterion. We have developed primer sets for the detection of rearrangements in the B- and T-cell receptor genes as reliable tools for clonality assessment in lymphoproliferations suspected for lymphoma. In this issue of Leukemia, the participants of the BIOMED-2 Concerted Action CT98-3936 report on the validation of the newly developed clonality assays in various disease entities. Clonality was detected in 99% of all B-cell malignancies and in 94% of all T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The combined BIOMED-2 results are summarized in a guideline, which can now be implemented in routine lymphoma diagnostics. The use of this standardized approach in patients with a suspect lymphoproliferation will result in improved diagnosis of malignant lymphoma.

CALM-AF10+ T-ALL expression profiles are characterized by overexpression of HOXA and BMI1 oncogenes.

The t(10;11)(p13;q14-21) is found in T-ALL and acute myeloid leukemia and fuses CALM (Clathrin-Assembly protein-like Lymphoid-Myeloid leukaemia gene) to AF10. In order to gain insight into the transcriptional consequences of this fusion, microarray-based comparison of CALM-AF10+ vs CALM-AF10- T-ALL was performed. This analysis showed upregulation of HOXA5, HOXA9, HOXA10 and BMI1 in the CALM-AF10+ cases. Microarray results were validated by quantitative RT-PCR on an independent group of T-ALL and compared to mixed lineage leukemia-translocated acute leukemias (MLL-t AL). The overexpression of HOXA genes was associated with overexpression of its cofactor MEIS1 in CALM-AF10+ T-ALL, reaching levels of expression similar to those observed in MLL-t AL. Consequently, CALM-AF10+ T-ALL and MLL-t AL share a specific HOXA overexpression, indicating they activate common oncogenic pathways. In addition, BMI1, located close to AF10 breakpoint, was overexpressed only in CALM-AF10+ T-ALL and not in MLL-t AL. BMI1 controls cellular proliferation through suppression of the tumor suppressors encoded by the CDKN2A locus. This locus, often deleted in T-ALL, was conserved in CALM-AF10+ T-ALL. This suggests that decreased CDKN2A activity, as a result of BMI1 overexpression, contributes to leukemogenesis in CALM-AF10+ T-ALL. We propose to define a HOXA+ leukemia group composed of at least MLL-t, CALM-AF10 and HOXA-t AL, which may benefit from adapted management.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Host oyster tissue extracts modulate in vitro protease expression and cellular differentiation in the protozoan parasite, Perkinsus marinus.

Perkinsus marinus is responsible for a chronic disease (Dermo) of the Eastern oyster, Crassostrea virginica. In order to simulate the in vivo environment more closely, a chemically defined medium (JL-ODRP-3) was supplemented with tissue homogenate extracts or plasma from oysters possessing varying degrees of susceptibility to P. marinus infection. In media supplemented with extracts from highly susceptible oysters (C. virginica), P. marinus cells secreted elevated amounts of a set of low molecular weight serine proteases (LMP: 30-45 kDa) as assessed by enhanced digestion within gelatin-substrate SDS-PAGE gels. Oyster species of low susceptibility (C. gigas and C. ariakensis) did not exhibit this ability to upregulate P. marinus LMP expression. Oyster extract supplementation also led to pronounced changes in P. marinus cellular morphology, such that the cells were comparable to those observed within naturally infected oysters.

Rapid, multifluorescent TCRG Vgamma and Jgamma typing: application to T cell acute lymphoblastic leukemia and to the detection of minor clonal populations.

Detection of clonal T cell receptor gamma (TCRG) gene rearrangements by PCR is widely used in both the diagnostic assessment of lymphoproliferative disorders and the follow-up of acute lymphoblastic leukaemia (ALL), when residual positivity in excess of 10(-3) at morphological complete remission is increasingly recognised to be an independent marker of poor prognosis. This is largely based on specific detection of V-J rearrangements from childhood cases. We describe rapid, multifluorescent Vgamma and Jgamma PCR typing of multiplex amplified diagnostic samples, as applied to 46 T-ALL. These strategies allow selected analysis of appropriate cases, immediate identification of Vgamma and Jgamma segments in over 95% of alleles, improved resolution and precision sizing and a sensitivity of detection at the 10(-2)-10(-3) level. We demonstrate preferential V-J combinations but no difference in V-J usage between children and adults, nor between SIL-TAL1-negative and -positive cases. A combination of fluorescent multiplex and Vgamma-Jgamma-specific monoplex follow-up, as described here, will allow detection of both significant clonal evolution and of the diagnostic clone at a level of prognostic significance, by techniques which can readily be applied to large-scale prospective studies for which real-time analysis is required.

Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia.

Prospective studies on the detection of minimal residual disease (MRD) in acute leukemia patients have shown that large-scale MRD studies are feasible and that clinically relevant MRD-based risk group classification can be achieved and can now be used for designing new treatment protocols. However, multicenter international treatment protocols with MRD-based stratification of treatment need careful standardization and quality control of the MRD techniques. This was the aim of the European BIOMED-1 Concerted Action 'Investigation of minimal residual disease in acute leukemia: international standardization and clinical evaluation' with participants of 14 laboratories in eight European countries (ES, NL, PT, IT, DE, FR, SE and AT). Standardization and quality control was performed for the three main types of MRD techniques, ie flow cytometric immunophenotyping, PCR analysis of antigen receptor genes, and RT-PCR analysis of well-defined chromosomal aberrations. This study focussed on the latter MRD technique. A total of nine well-defined chromosome aberrations with fusion gene transcripts were selected: t(1;19) with E2A-PBX1, t(4;11) with MLL-AF4, t(8;21) with AML1-ETO, t(9;22) with BCR-ABL p190 and BCR-ABL p210, t(12;21) with TEL-AML1, t(15;17) with PML-RARA, inv (16) with CBFB-MYH11, and microdeletion 1p32 with SIL-TAL1. PCR primers were designed according to predefined criteria for single PCR (external primers A <--> B) and nested PCR (internal primers C <--> D) as well as for 'shifted' PCR with a primer upstream (E5' primer) or downstream (E3' primer) of the external A <--> B primers. The 'shifted' E primers were designed for performing an independent PCR together with one of the internal primers for confirmation (or exclusion) of positive results. Various local RT and PCR protocols were compared and subsequently a common protocol was designed, tested and adapted, resulting in a standardized RT-PCR protocol. After initial testing (with adaptations whenever necessary) and approval by two or three laboratories, the primers were tested by all participating laboratories, using 17 cell lines and patient samples as positive controls. This testing included comparison with local protocols and primers as well as sensitivity testing via dilution experiments. The collaborative efforts resulted in standardized primer sets with a minimal target sensitivity of 10-2 for virtually all single PCR analyses, whereas the nested PCR analyses generally reached the minimal target sensitivity of 10-4. The standardized RT-PCR protocol and primer sets can now be used for molecular classification of acute leukemia at diagnosis and for MRD detection during follow-up to evaluate treatment effectiveness.

NG2 expression in MLL rearranged acute myeloid leukaemia is restricted to monoblastic cases.

Expression of NG2 has been reported in the majority of paediatric acute leukaemia (AL) cases with MLL rearrangement. We demonstrated 7. 1 positivity in 2/3 paediatric and 4/11 adult MLL rearranged acute myeloid leukaemia (AML) but in 0/28 adult AML without MLL rearrangement, thus extending the 100% specificity to adult cases. Positivity correlated with stage of maturation arrest since it was found in 0/6 immature AML but in 6/8 monoblastic cases. These data demonstrate that, if NG2 expression in AL is the (in)direct result of MLL rearrangement, such activation is restricted to a monoblastic population in AML. They also have practical implications for NG2 diagnostic screening strategies.

Molecular approaches to the diagnosis and evaluation of lymphoid malignancies.

The processes of somatic immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement that occur in lymphoid precursors provide insights into the pathogenesis and molecular analysis of lymphoid malignancies, in addition to the more universal molecular oncogenic mechanisms. Detection of lymphoid clonality can help distinguish polyclonal reactive disorders from clonal, predominantly, but not exclusively, malignant proliferations. Ig/TCR V-(D)-J polymerase chain reaction (PCR) amplification has largely replaced Southern blotting, and the techniques of PCR product analysis are evolving rapidly. V-(D)-J errors are often involved in genetic abnormalities leading to lymphoid malignancies, with consequent deregulated expression of the associated proto-oncogenes. Genetic abnormalities producing fusion transcripts and chimeric proteins are also frequent, particularly in acute lymphoblastic leukemia (ALL). A variety of molecular techniques, including reverse-transcriptase (RT)-PCR, Southern blotting, and fluorescence in situ hybridization (FISH) are finding an increasingly established place in the diagnosis, prognostic evaluation, therapeutic stratification, and follow-up of lymphoblastic leukemias, and it is likely that the same methods will be applied to non-Hodgkin's lymphoma (NHL) and to chronic leukemias.

Homing receptor alpha4beta7 integrin expression predicts digestive tract involvement in mantle cell lymphoma.

Appropriate staging and evaluation of residual disease is critical to improving the treatment of patients with lymphoma. The specific expression of homing receptors may determine the preferential dissemination pattern of tumoral cells. We investigated the expression of the mucosal homing receptor alpha4beta7 on tumoral cells from peripheral lymph node in patients with newly diagnosed mantle cell lymphoma (MCL) to check whether it is associated with gastrointestinal involvement. Expression of the alpha4beta1 integrin and the peripheral lymph node addressin CD62L were also examined. Thirteen MCL patients presenting with peripheral lymphadenopathy were studied. Expression of the mucosal homing receptor integrin alpha4beta7 by peripheral lymph node lymphoma cells was found to be frequent (5/13) and associated with gastrointestinal involvement (5/7). In contrast, lymphoma cells from patients without gastrointestinal involvement did not express alpha4beta7 (6/6) (P = 0.03). These data suggest that alpha4beta7 integrin is expressed by a subset of MCLs and that its expression may predict digestive tract involvement in MCL, furnishing a basis for recognizing two distinct clinical and phenotypic forms, ie, "digestive homing (or digestive primitive)" versus "peripheral" MCL. Further studies on more patients will be needed to understand the impact of biological differences on the prognosis of these two clinical forms.

FISH detection of chromosome 14q32/IgH translocations: evaluation in follicular lymphoma.

A FISH strategy capable of detecting chromosome 14q32 rearrangements involving the IgH locus, including in interphase nuclei, was developed using Ig variable and constant region cosmids from the extremities of the locus in a dual hybridization approach, using signal splitting as evidence of rearrangement. The large size of the locus (1.3 Mb) and the propensity for internal deletion due to physiological VDJ recombination and isotype switching complicate analysis of this locus. We used the Ig10 cosmid, which hybridizes to C epsilon and C alpha2 at the 3' end of the constant region, in order to minimize deletion and/or splitting of the constant region probe. Cos Ig10 and the IgV18 VH probes were compared with a specific IgH-BCL2 FISH dual hybridization approach in follicular lymphoma (FL). Both were capable of detecting the t(14;18) in interphase nuclei, including in cases with no apparent abnormality by classic karyotype analysis, although the sensitivity of the IgH approach was slightly lower. We have also successfully applied these probes to whole cell cytospin preparations, rendering analysis of cryopreserved material possible, although interpretation should be limited to frequent events, particularly following cell manipulation. Analysis of flow cytometric sorted bone marrow fractions from three FL patients by FISH and FICTION showed that the t(14;18) was present in a much lower proportion of CD34 positive than negative cells but that the higher level of background hybridization limits use of these techniques for the reliable quantification of rare events.

Helix-loop-helix (E2-5, HEB, TAL1 and Id1) protein interaction with the TCRalphadelta enhancers.

In order to dissect the correlation between aberrant TAL1 basic-helix-loop-helix (b-HLH) expression and the exclusive development of T cell acute lymphoblastic leukemias (T-ALL) of the TCRalphabeta lineage, we have assessed the ability of class A b-HLH proteins to regulate the TCRalpha and delta enhancers. We demonstrate that E47S binds to TCRalpha but not to TCRdelta E-boxes in vitro. Despite this, neither E2-5 nor HEB transactivate the TCRalpha enhancer in NIH 3T3, nor did Id1 modify endogenously driven TCRalpha [alphaE1-4] activity in a TCRalphabeta cell line. We also demonstrate that TAL1 inhibits both binding of E47S to aE3 and aE4 and endogenous transactivation of the TCRalpha enhancer. Comparison of the activity of the minimal [alphaE1-2] fragment, which contains no E-boxes, with the accessory [aE3-4] fragment, which contains two, suggested some contribution from the latter to TCRalpha enhancer activity in HPB-ALL. TCR [alphaE1-2] activity was partially (40%) inhibited by TAL1 but not at all by Id1. In contrast, [alphaE3-4] activity was almost completely inhibited by TAL1 (80%) and slightly reduced by Id1 (15%). These data demonstrate that class A b-HLH regulation of the TCRalpha enhancer E-boxes differs from their B lymphoid Igmicro counterparts and suggest a novel mechanism of transcriptional inhibition by TAL1, which may be, at least partly, independent of E-box-mediated activation, as we currently recognize it. They also clearly demonstrate that the restriction of TAL1 deregulation to T-ALL of the TCRalphabeta lineage is not due to induction of TCRalpha enhancer activity by the TAL1 protein.