Expression of B7-1 by highly metastatic mouse T lymphomas induces optimal natural killer cell-mediated cytotoxicity.

The interaction between B7-1 and CD28 provides costimulatory signals not only for T cells but also for natural killer (NK) cells. Highly metastatic mouse T lymphoma cells (BW-Li) can escape from NK cell-mediated killing by expressing H-2Dk molecules that negatively regulate NK lytic activity. We have analyzed whether B7-1:CD28 overrules the MHC class I-mediated inactivation of NK cells by transfecting BW-Li with the gene coding for B7-1. Expression of B7-1 rendered BW-Li cells sensitive toward NK cells. The experimental metastatic capacity of the B7-1 transfectants was drastically reduced in both syngeneic AKR and SCID mice but could be restored in SCID-bg mice. These results provide direct evidence that B7-1 expression leads to NK-mediated elimination of metastasizing, NK-resistant tumor cells.

In vivo induction of insulin-like growth factor-I receptor and CD44v6 confers homing and adhesion to murine multiple myeloma cells.

One of the main characteristics of multiple myeloma (MM) cells is their specific homing and growth in the bone marrow (BM). Differences between stroma-dependent and -independent MM cell lines may reveal key molecules that play important roles in their homing to the BM. We addressed this topic with a murine MM model, including the in vivo 5T33MM (5T33MMvv) stroma-dependent cell line and its in vitro stroma-independent variant (5T33MMvt). Fluorescence-activated cell-sorting analysis showed expression of insulin-like growth factor (IGF)-I receptor and CD44v6 on all 5T33MMvv cells but not on 5T33MMvt cells. Checkerboard analysis and adhesion assays revealed IGF-I-dependent chemotaxis toward BM-conditioned medium and involvement of CD44v6 in the adhesion to BM stroma of only 5T33MMvv cells. However, when 5T33MMvt cells were injected in vivo (5T33MMvt-vv), after 18 h the MM cells harvested from BM were IGF-I receptor and CD44v6 positive. This up-regulation was confirmed in 5T33MMvt-vv cells isolated from terminally diseased animals. These ST33MMvt-vv cells exhibited IGF-I-dependent chemotaxis and CD44v6-dependent adhesion to BM stroma. In vitro culture of the 5T33MMvt-vv cells could completely down-regulate IGF-I receptor and CD44v6. In fact, we could show that direct contact of 5T33MMvt cells with BM endothelial cells is a prerequisite for IGF-I receptor and CD44v6 up-regulation. These data indicate that the BM microenvironment is capable of up-regulating molecules such as IGF-I receptor and CD44v6, which facilitate homing of MM cells to the BM and support their adhesion to BM stroma.

A novel approach for BCR-ABL1 standardization to improve International Scale estimation.

INTRODUCTION: Standardization of BCR-ABL1 messenger RNA quantification by real-time PCR on the International Scale (IS) is critical for monitoring therapy response in chronic myelogenous leukaemia. Since 2006, BCR-ABL1 IS standardization is propagated along reference laboratories by calculating a laboratory-specific conversion factor (CF), co-ordinated in Europe through the European Treatment and Outcome Study project. Although this process has proven successful to some extent, it has not been achievable for all laboratories due to the complexity of the process and the stringent requirements in terms of numbers of samples to be exchanged. In addition, several BCR-ABL1 IS quantification methods and secondary reference materials became commercially available. However, it was observed that different IS methods generate consistently different results. METHODS: To overcome these difficulties, we have developed an alternative and simple approach of CF calculation, based on the retrospective analysis of existing external quality assessment (EQA) data. Our approach does not depend on the exchange of samples and is solely based on the mathematical CF calculation using EQA results. RESULTS AND CONCLUSION: We have demonstrated by thorough statistical validation that this approach performs well in converting BCR-ABL1 measurements to improve IS estimation. In expectation of a true golden standard method for BCR-ABL1 IS quantification, the proposed method is a valuable alternative.

Detection of oncogene expression by fluorescent in situ hybridization in combination with immunofluorescent staining of cell surface markers.

To study oncogene expression in heterogeneous cell populations we developed and optimized a non-radioactive in situ hybridization technique using biotinylated single-stranded RNA probes and combined this technique with immunofluorescent staining of cell surface markers. As a model for our studies we used HL60 cells. In these cells we detected c-myc mRNA molecules by in situ hybridization following staining of the pan myeloid cell surface marker CD33, by a monoclonal antibody. Hybrids were detected by streptavidin-FITC and CD33 by a TRITC-conjugated antibody. Controls involved pretreatment with RNAase, hybridization with sense RNA probes and blocking with an excess of unlabeled antisense probes. The integrity of the RNA in the cell was shown by hybridization with the GAPDH antisense probe. Essential for successful double-labeling was the choice of a fixation procedure that was suitable for the in situ hybridization and mild enough not to destroy the cell surface marker staining. This fluorescent in situ hybridization in combination with cell surface marker staining will be useful for studying gene expression in phenotypically well-defined cell populations.

Amplification of the c-myc and the pvt-like region in human multiple myeloma.

Genetic alterations that lead to the clonal expansion of differentiated cells in multiple myeloma have still to be elucidated. Many chromosomal aberrations have been found, but until now, none of them is typically associated with multiple myeloma. In search for genetic defects in multiple myeloma we studied the structure and expression of the c-myc oncogene and the pvt-like region because of their frequent association with other B-cell malignancies. Here we report co-amplification of the c-myc oncogene and the 5' part of the pvt-like region in two out of 26 cases of multiple myeloma. In both cases only kappa-light chains were produced. The amplification also manifested itself at the RNA level. Total RNA was analysed in one of these two cases showing abundant c-myc mRNA. In the same RNA sample we also detected a strong hybridizing band of about 7 kb, when the pSS.4 probe, representing the 5' part of the pvt-like region, was used. This band was not present in total RNA from normal bone marrow cells or bone marrow from multiple myeloma patients without the amplification of c-myc and the pvt-like region. Until now, transcripts of the pvt-like region were only found in a few human cell lines ranging in size from 1 to 11 kb. This is the first case in which a high expression of a +/- 7 kb transcript of the pvt-like region has been found in freshly obtained tumor material probably due to a pvt-amplification. The occurrence of abnormalities in the c-myc and the pvt-like region in multiple myeloma is a rare event and may be associated with the progression of this type of tumor.

Differential gene expression of the neural cell adhesion molecule (N-CAM) in a panel of multiple myeloma cell lines.

A striking feature of myeloma plasma cells concerns their expression of the neural cell adhesion molecule (N-CAM). The regulation of this particular expression is, however, not known. In this study, the N-CAM (CD56) gene regulation was examined in a panel of multiple myeloma (MM) cell lines. In this panel, both N-CAM-positive and -negative cells were analysed, reflecting the in vivo situation where a minority of MM patients have CD56-negative plasma cells at diagnosis or where in cases of extramedullary involvement CD56 expression decreases. At least two N-CAM mRNAs were found in the cell lines expressing the 140 kDa isoform. With one exception, no N-CAM transcripts could be detected in the N-CAM-negative cell lines. No structural differences could be found in the genomic organization of the N-CAM gene, or in the regulatory promoter region when CD56-positive and -negative cell lines were compared. In transfection studies, however, transcriptional activity of the N-CAM promoter was observed in N-CAM-negative cells, leading us to conclude that the up-regulation of N-CAM in MM cannot be explained by a simple transcriptional gene activation.

Establishment and characterization of a human stroma-dependent myeloma cell line (MM5.1) and its stroma-independent variant (MM5.2).

Although IL-6 has been identified as a major growth factor in multiple myeloma (MM), it is believed that maintenance of tumor growth in vivo depends on one or more additional stroma-derived factors. We describe a new human myeloma cell line (MM5.1) that can be maintained in the presence of bone marrow-derived stromal cell layers, and not only when cultured with exogeneous IL-6. This cell line expresses the same immunoglobulin kappa light chain RNA sequence as the patient's original tumor cells, has a plasma cell morphology and expresses plasma cell antigens (cytoplasmic kappa light chain, CD38, BB4). Without the presence of stromal factors, MM5.1 cells become apoptotic. A low proliferative effect was observed in the presence of oncostatin M (OSM) but other cytokines (IL-10, IL-11, stem cell factor (SCF) and leukemia inhibitory factor (LIF)) had no effect at all. We observed that MM5.1 cells also grow when physically separated from stromal cell layers by a 0.45 microm microporous membrane or when cultured in conditioned medium from stromal marrow cells. Unexpectedly, the growth in stromal supernatants was markedly inhibited by an anti-IL-6 antiserum and an anti-IL-6 receptor transducer chain (gp130) mAb in a dose-dependent manner. This implies that MM5.1 cells are IL-6 responsive only when exposed to one or more additional soluble factor(s) derived from bone marrow stroma. Coculturing MM5.1 cells with IL-6 and cytokines that were described to increase the IL-6 responsiveness of myeloma cells (G-CSF, GM-CSF and IL-3) had no effect on the growth or survival. A strong proliferative effect was observed when MM5.1 cells were cultured with IL-6 and soluble IL-6 receptor (sgp80). However no sgp80 could be detected in stromal supernatants using a sensitive immunoassay. This indicates that sustained proliferation of the MM5.1 cell line depends on a combination of IL6 and at least one, thus far unidentified, stroma-derived factor. After more than 1 year in continuous culture, we could obtain a variant of the line (MM5.2) that shows an improved growth rate and grows stroma independently. Molecular analysis revealed clonal identity with the early passage form and Epstein-Barr virus antigen expression was negative. The two variants of this cell line offer a useful model to identify molecular mechanisms involved in clonal evolution towards stroma-independent growth of myeloma cells.

ETV6 is the target of chromosome 12p deletions in t(12;21) childhood acute lymphocytic leukemia.

The presence of ETV6 deletions was investigated in 215 children with acute lymphoblastic leukemia (ALL) using the loss of heterozygosity (LOH) approach. We used four intragenic or juxtagenic microsatellite markers to detect allelic deletions. In this series of unselected patients, LOH of ETV6 markers was found in 23% of cases (6% of T-ALL and 26% of B lineage ALL) confirming that chromosome 12p12-13 deletions represent a major genetic alteration in childhood ALL, frequently missed by cytogenetic analysis. The presence of a t(12;21)(p13;q22) was studied by RT-PCR and/or FISH in a total of 134 patients (125 B lineage ALL, nine T-ALL) including 42 cases with LOH. Thirty-four out of 44 patients (77%) for whom a t(12;21) was observed displayed LOH of the ETV6 markers. When associated with a t(12;21), ETV6 is very likely to be the target of deletions as indicated by the detection of intragenic deletions in three patients. Although deletion of ETV6 and t(12;21) were associated in most patients, in eight cases (six B lineage and two T-ALL) LOH was detected at the ETV6 locus without ETV6-AML1 hybrid RNA. FISH studies conducted in five of these eight patients confirmed the absence of translocation involving ETV6. In such patients, the other allele of ETV6 could be disrupted by either a small deletion, a point mutation, or an epigenetic modification and it will be of interest to study the structure and expression of the remaining allele of ETV6 in these cases. Alternatively, a tumor suppressor gene located close to ETV6 and CDKN1B could be the target of deletions.

CD40 triggering of chronic lymphocytic leukemia B cells results in efficient alloantigen presentation and cytotoxic T lymphocyte induction by up-regulation of CD80 and CD86 costimulatory molecules.

Freshly collected chronic lymphocytic leukemia B cells (B-CLL cells) are known to be inefficient at stimulating allogeneic T cells, and to lack significant expression of B7 (CD80 and CD86) costimulatory molecules. We investigated the potential of CD40 triggering to up-regulate the expression of adhesion and costimulatory molecules on B-CLL cells, and to enhance their immunogenicity towards allogeneic T cells. B-CLL cells cocultured with human CD40 ligand-expressing mouse fibroblasts rapidly up-regulated CD54 and CD58 adhesion molecules and B7-1 (CD80) and B7-2 (CD86) costimulatory molecules, and acquired a strong stimulatory capacity towards CD4+ as well as isolated CD8+ allogeneic T cells. Costimulation by both CD80 and CD86 proved critical for allogeneic T cell proliferation and CD25 and HLA-DR expression, since these were strongly inhibited by anti-CD80 or anti-CD86 monoclonal antibodies, and completely abrogated by CTLA4-Ig fusion protein, which blocks both CD80 and CD86. B7 costimulation also proved critical for restimulation of primed B-CLL-reactive T cells. Most importantly, priming of purified CD8+ T cells with CD40-triggered allogeneic B-CLL cells resulted in cytotoxic activity against the unstimulated B-CLL cells. These findings raise the possibility that CD40 triggering of B-CLL cells might be exploited in immunotherapeutic protocols.

Myeloma isotype-switch variants in the murine 5T myeloma model: evidence that myeloma IgM and IgA expressing subclones can originate from the IgG expressing tumour.

Isotype-switch variants can easily be detected in a significant proportion of multiple myeloma (MM) patients. The biological significance of these isotype-switch variants remains obscure. Therefore, we studied the appearance of these isotype-switch variants in two murine MM models, 5T2MM and 5T33MM, both of IgG isotype. With a MM-specific PCR assay we could detect isotype-switch variants in the bone marrow of both the 5T2MM and the 5T33MM bearing mice, reflecting again the close resemblance of this mouse model to the human MM. These isotype-switch variants were not found in an in vitro stroma-independent variant of the 5T33MM line. However, when this 5T33MMvitro line was injected into young syngeneic mice, isotype-switch variants appeared thereafter in the isolated tumour cells. These isotype-switch variants could only originate from the MM-IgG expressing cell since IgG subclones from the 5T33MMvitro line again gave rise to isotype-switch variants. The appearance of IgA cells can be explained by down-stream switching of IgG to IgA, while the emergence of IgM cells have to occur via trans-switching to the sister chromatid as the Cmu region is deleted from the CIS-chromosome. This study demonstrates that isotype-switch variants originate from the major tumour clone suggesting no role for the MM-IgM expressing cell as a pre-switch precursor MM cell. The appearance of isotype-switch variants should be considered as a rare but normal event now becoming visible due to the high number of clonal cells present in MM.

Immunoglobulins D and M multiple myeloma variants are heavily mutated.

Multiple myeloma (MM) is a B-cell malignancy characterized by the expansion of malignant plasma cells within the bone marrow. Previous studies that have examined the Ig VH genes of IgG and IgA MMs have shown the presence of somatic mutations, suggesting that in these cases, the myeloma precursor cell passed through the phase of antigenic selection within the germinal center but is no longer exposed to the somatic mutation process. However, no information about this matter is available in the rare IgD and IgM MM variants. Therefore, we have analyzed the Ig VH genes of three IgD, one IgM, and one biclonal (IgG and IgM) MM for the presence of somatic mutations. Our study demonstrates that all of these myeloma clones have accumulated a high number of somatic mutations within their Ig VH genes but show no intraclonal variation. Moreover, proof that the clone sustained a strong antigenic selection pressure could be provided in three cases (one IgD and two IgMs). Therefore, this study strongly implies that IgD and IgM MMs emerge from a postgerminal center preswitched B cell that is no longer exposed to the somatic mutation process or able to undergo further isotype switching in vivo.

IKZF1 deletion is an independent prognostic marker in childhood B-cell precursor acute lymphoblastic leukemia, and distinguishes patients benefiting from pulses during maintenance therapy: results of the EORTC Children's Leukemia Group study 58951.

The added value of IKZF1 gene deletion (IKZF1(del)) as a stratifying criterion in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is still debated. We performed a comprehensive analysis of the impact of IKZF1(del) in a large cohort of children (n=1223) with BCR-ABL1-negative BCP-ALL treated in the EORTC-CLG trial 58951. Patients with IKZF1(del) had a lower 8-year event-free survival (EFS, 67.7% versus 86.5%; hazard ratio (HR)=2.41; 95% confidence interval (CI)=1.75-3.32; P<0.001). Importantly, despite association with high-risk features such as high minimal residual disease, IKZF1(del) remained significantly predictive in multivariate analyses. Analysis by genetic subtype showed that IKZF1(del) increased risk only in the high hyperdiploid ALLs (HR=2.57; 95% CI=1.19-5.55; P=0.013) and in 'B-other' ALLs, that is, lacking classifying genetic lesions (HR=2.22; 95% CI=1.45-3.39; P<0.001), the latter having then a dramatically low 8-year EFS (56.4; 95% CI=44.6-66.7). Among IKZF1(del)-positive patients randomized for vincristine-steroid pulses during maintenance, those receiving pulses had a significantly higher 8-year EFS (93.3; 95% CI=61.3-99.0 versus 42.1; 95% CI=20.4-62.5). Thus, IKZF1(del) retains independent prognostic significance in the context of current risk-adapted protocols, and is associated with a dismal outcome in 'B-other' ALL. Addition of vincristine-steroid pulses during maintenance may specifically benefit to IKZF1(del) patients in preventing relapses.

Interleukin-1 and interleukin-6 in psoriasis.

We report on the levels of expression of IL-1 and IL-6 in skin from psoriasis patients. Different approaches were pursued. Initially, the levels of IL-1 beta and IL-6 were measured in suction blister fluid from lesional and uninvolved skin from psoriasis patients, using a sensitive enzyme-linked immunoabsorbent assay (ELISA) and bio-assay. Skin sections were also examined for the presence of IL-1 and IL-6 using IL-1 beta- and IL-6-specific antibodies. Finally, the expression of IL-1 and IL-6 mRNA was determined in cultured keratinocytes (KC) and fibroblasts from psoriasis skin. Suction blister fluid from lesional and uninvolved psoriasis skin and from skin of healthy individuals did not contain detectable levels (greater than 100 pg/ml) of IL-1 beta. Blister fluid from psoriasis lesions contained low but significant levels of IL-6, whereas the serum levels of IL-6 in these patients was undetectable. Using cryostat skin sections and an IL-1 beta-specific monoclonal antibody (MoAb) in an indirect immunoperoxidase technique, a diffuse staining in the entire epidermis was observed in sections of uninvolved skin from psoriasis patients. In cryostat sections of psoriasis lesions, a faint diffuse staining of the epidermis and a pronounced "dot-like" intracellular staining pattern was observed. On the other hand, the same IL-1 beta-specific MoAb showed, in a indirect immunofluorescence technique using unfixed epidermal cells, bright membrane staining in epidermal cell suspensions from psoriasis lesions. Slightly elevated levels of IL-1 beta and IL-1 alpha mRNA were observed in cultured KC from psoriasis lesions as compared to those in normal KC and in the HEp-2 cell line. Very low levels of IL-6 mRNA were expressed in KC from psoriasis lesions and healthy individuals. Fibroblasts from psoriasis lesions expressed extremely low levels of IL-1 alpha and IL-1 beta, but high levels of IL-6 mRNA. The results point to a paradoxical situation in psoriatic skin: blister fluid from psoriasis lesions contains no IL-1 beta, whereas IL-1 beta is overexpressed on the plasma membrane and in the intracellular compartment of epidermal cells. This finding may help in explaining the observed absence of IL-1 in aqueous extracts of psoriatic scales. Because cultured KC from psoriasis lesions express minimal levels of IL-6 mRNA. dermal fibroblasts, probably together with the inflammatory infiltrate, may represent a major source of IL-6 in psoriasis lesions in vivo.

Tumorigenicity of mouse T lymphoma cells is controlled by the level of major histocompatibility complex class I H-2Kk antigens.

We have previously found that an increased tumorigenicity and spontaneous metastatic potential of BW5147-derived T lymphoma cells was associated with a decrease in major histocompatibility complex (MHC) class I H-2Kk antigen expression. This suggested that H-2Kk antigens may control the tumorigenic potential of BW T lymphoma cells. Our current experiments aimed to prove this association by specifically altering H-2Kk expression by gene transfection. Transfected cells expressing a high level of H-2Kk antigens were significantly less tumorigenic and metastatic after subcutaneous inoculation. However, there was selection in vivo for cells expressing a reduced level of H-2Kk antigens, which concomitantly led to an increased tumorigenicity. These data further confirmed the strong association between H-2Kk expression and tumorigenicity. We subsequently tested whether the immune system is implicated in this phenomenon by inoculating the H-2Kk transfectants into irradiated, immunocompromised recipients. Our results indicate that the reduced tumorigenicity of the BW H-2Kk transfectants is due to an immune rejection mechanism, mediated by CD8+ immune effector cells, as revealed by in vivo depletion experiments with anti-CD8 antibodies. Hence, we hereby demonstrated that H-2Kk antigens increased the immunogenicity of BW cells, via a CD8-dependent mechanism, which consequently reduced their tumorigenicity.

The clonogenic precursor cell in multiple myeloma.

Multiple myeloma is characterized by the monoclonal expansion of plasma cells in the bone marrow. Although the predominant cell type is the plasma cell, the initial oncogenic transformation is considered to take place in a more immature B cell. There is still much controversy about this precursor cell type. Phenotypic analysis of bone marrow and peripheral blood revealed that in multiple myeloma a great diversity exists in the phenotype of the cells considered to be involved. Because of the lack of a myeloma specific genetic lesion it is very difficult to trace back the cell in which the transforming event, leading to multiple myeloma, took place. The only real clonal marker is the idiotype of the immunoglobulin molecule expressed by the myeloma cells. With recombinant DNA technology it is now possible to produce clonal markers for each individual myeloma patient which recognize only the immunoglobulin genes expressed by the myeloma cell and its precursors. The sequences of these myeloma immunoglobulin genes do reveal a lot of information about the stage in the B-cell differentiation pathway in which the oncogenic event might have taken place. The presence of somatic mutations in a non-random fashion without intraclonal variation leads to the conclusion that the precursor myeloma cell could not possibly be a pre-B cell or stem cell but has to be a mature B cell that has been in contact with antigen and has past through the phase of somatic mutation, like a memory B cell or plasmablast.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of Interleukin-1ß and Interleukin-6 in Human Multiple Myeloma by Fluorescent in situ Hybridization.

Using fluorescent in situ hybridization together with cell surface marker staining, we studied the expression of mRNA of IL-6 and mRNA of IL-1ß in bone marrow samples from human multiple myeloma patients. It is known that IL-6 can stimulate B cell growth and differentiation and recently it has been suggested that IL-6 is responsible for autocrine growth stimulation of myeloma cells and that IL-1 may play a role in bone resorption. These interleukins have previously been detected in the supernatants of cultured myeloma cells. Here we report the expression of IL-1ß mRNA by plasma cells, T cells and macrophages according to morphology and immunologic marker analysis, suggesting that not only myeloma cells but other cell types can also contribute to the production of IL-1ß and thus to bone-resorption. IL-6 mRNA could not be detected in plasma cells from bone marrow aspirates but were present in monocytes and T cells, suggesting that in vivo IL-6 stimulates the growth of myeloma cells in a paracrine instead of an autocrine way.

Oncoselective transduction of CD80 and CD86 in tumor cell lines using an autonomous recombinant parvovirus.

BACKGROUND: The aim of this study was to enhance selectively the immunostimulatory properties of tumor cells. Based on their oncotropic properties, we used autonomous recombinant parvoviruses to transduce the genes coding for the constimulatory molecules CD80 (B7-1) or CD86 (B7-2) specifically into tumor cells without transducing normal cells. MATERIALS AND METHODS: After infection of tumor cells by these viruses, surface expression of CD80 and CD86 molecules was assessed by FACS and enhancement of immunostimulatory properties was assessed in alloreactions with G-10 purified T cells. RESULTS: Infection of normal and transformed cells with recombinant MVM- B7-1 or B7-2 viruses leads to expression of costimulatory molecules only by tumor cells and confers on them the capacity to sensitize naive T cells in vitro. CONCLUSION: This approach should ultimately lead to selective expression of costimulatory molecules in tumor tissues in vivo without affecting normal cells.

Detection of minimal residual disease in multiple myeloma and acute leukaemia.

The importance of minimal residual disease detection has increased due to the advanced therapeutic protocols available for multiple myeloma and acute leukaemia. High-dose chemotherapy, followed by stem cell transplantation is often used in patients with multiple myeloma. But despite a longer disease-free period and overall survival, all patients relapse. In the treatment of acute leukaemia, there are similar problems. The present strategy is to give continuous chemotherapy to eradicate minimal residual disease. In this review, we consider the methods used to detect and quantify minimal residual disease. At present, the most effective seem to be those based on the use of polymerase chain reactions to detect the malignant cells.

An intragenic ERG deletion is a marker of an oncogenic subtype of B-cell precursor acute lymphoblastic leukemia with a favorable outcome despite frequent IKZF1 deletions.

Oncogenic subtypes in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are used for risk stratification. However, a significant number of BCP-ALL patients are still genetically unassigned. Using array-comparative genomic hybridization in a selected BCP-ALL cohort, we characterized a recurrent V(D)J-mediated intragenic deletion of the ERG gene (ERG(del)). A breakpoint-specific PCR assay was designed and used to screen an independent non-selected cohort of 897 children aged 1-17 years treated for BCP-ALL in the EORTC-CLG 58951 trial. ERG(del) was found in 29/897 patients (3.2%) and was mutually exclusive of known classifying genetic lesions, suggesting that it characterized a distinct leukemia entity. ERG(del) was associated with higher age (median 7.0 vs. 4.0 years, P=0.004), aberrant CD2 expression (43.5% vs. 3.7%, P<0.001) and frequent IKZF1 Δ4-7 deletions (37.9% vs. 5.3%, P<0.001). However, ERG(del) patients had a very good outcome, with an 8-year event-free survival (8-y EFS) and an 8-year overall survival of 86.4% and 95.6%, respectively, suggesting that the IKZF1 deletion had no impact on prognosis in this genetic subtype. Accordingly, within patients with an IKZF1 Δ4-7 deletion, those with ERG(del) had a better outcome (8-y EFS: 85.7% vs. 51.3%; hazard ratio: 0.16; 95% confidence interval: 0.02-1.20; P=0.04). These findings have implications for further stratification including IKZF1 status.

Reflections and proposals to assure quality in molecular diagnostics.

Molecular diagnostic testing has become an important tool in clinical laboratories. Accreditation according to the international quality standard ISO15189:2007 for medical laboratories is required for reimbursement of several molecular diagnostic tests in Belgium. Since the ISO15189:2007 standard applies to medical laboratories in general, the particular requirements for quality and competence are mentioned in general terms, not taking into account the specificities of molecular biology testing. Therefore, the working group "MolecularDiagnostics.be" described a consensus interpretation of chapter 5, Technical requirements, of the ISO standard for application in molecular diagnostic laboratories. The manuscript can be used as an instrument to prepare internal and external audits that meet the 15015189:2007 (chapter 5) criteria.