Correction: Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

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Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10-3 and 36/67 (53%) and 53/67 (79%) at 10-4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.

PML-RARA-associated cooperating mutations belong to a transcriptional network that is deregulated in myeloid leukemias.

It has been shown that individual acute myeloid leukemia (AML) patients are characterized by one of few initiating DNA mutations and 5-10 cooperating mutations not yet defined among hundreds identified by massive sequencing of AML genomes. We report an in vivo insertional-mutagenesis screen for genes cooperating with one AML initiating mutations (PML-RARA, oncogene of acute promyelocytic leukemia, APL), which allowed identification of hundreds of genetic cooperators. The cooperators are mutated at low frequency in APL or AML patients but are always abnormally expressed in a cohort of 182 APLs and AMLs analyzed. These deregulations appear non-randomly distributed and present in all samples, regardless of their associated genomic mutations. Reverse-engineering approaches showed that these cooperators belong to a single transcriptional gene network, enriched in genes mutated in AMLs, where perturbation of single genes modifies expression of others. Their gene-ontology analysis showed enrichment of genes directly involved in cell proliferation control. Therefore, the pool of PML-RARA cooperating mutations appears large and heterogeneous, but functionally equivalent and deregulated in the majority of APLs and AMLs. Our data suggest that the high heterogeneity of DNA mutations in APLs and AMLs can be reduced to patterns of gene expression deregulation of a single 'mutated' gene network.

Chimeric antigen receptors against CD33/CD123 antigens efficiently target primary acute myeloid leukemia cells in vivo.

As significant numbers of acute myeloid leukemia (AML) patients are still refractory to conventional therapies or experience relapse, immunotherapy using T cells expressing chimeric antigen receptors (CARs) might represent a valid treatment option. AML cells frequently overexpress the myeloid antigens CD33 and CD123, for which specific CARs can be generated. However, CD33 is also expressed on normal hematopoietic stem/progenitor cells (HSPCs), and its targeting could potentially impair normal hematopoiesis. In contrast, CD123 is widely expressed by AML, while low expression is detected on HSPCs, making it a much more attractive target. In this study we describe the in vivo efficacy and safety of using cytokine-induced killer (CIK) cells genetically modified to express anti-CD33 or anti-CD123 CAR to target AML. We show that both these modified T cells are very efficient in reducing leukemia burden in vivo, but only the anti-CD123 CAR has limited killing on normal HSPCs, thus making it a very attractive immunotherapeutic tool for AML treatment.

Modern therapy of young and adult Ph-ALL.

Acute lymphoblastic leukemia (ALL) in adults is currently associated with an overall survival rate of around 40% at 5 years. This is an unsatisfactory result that makes it imperative to dissect further the biology of the disease in order to identify highly specific therapeutic targets to implement selectively the cure rate. The recognition of discrete ALL subsets followed by the application of risk-oriented therapies has been a major achievement over the past 30 years.

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.

PubMed reloaded: new interface, enhanced discovery.

The aim of this contribution was to present the redesigned version of the worldwide known PubMed interface. The changes relate to a simplified visualization, in order to require less efforts to find resources; they do not concern the functionalities or the search processing. This brief article will focus on the main differences between the old and the new version.

The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2(V617F).

We investigated the activity of ITF2357, a novel histone deacetylase inhibitor (HDACi) with antitumor activity, on cells carrying the JAK2(V617F) mutation obtained from polycythemia vera (PV) and essential thrombocythemia (ET) patients as well as the HEL cell line. The clonogenic activity of JAK2(V617F) mutated cells was inhibited by low concentrations of ITF2357 (IC(50) 0.001-0.01 microM), 100- to 250-fold lower than required to inhibit growth of normal or tumor cells lacking this mutation. Under these conditions, ITF2357 allowed a seven fold increase in the outgrowth of unmutated over mutated colonies. By western blotting we showed that in HEL cells, ITF2357 led to the disappearance of total and phosphorylated JAK2(V617F) as well as pSTAT5 and pSTAT3, but it did not affect the wild-type JAK2 or STAT proteins in the control K562 cell line. By real-time PCR, we showed that, upon exposure to ITF2357, JAK2(V617F) mRNA was not modified in granulocytes from PV patients while the expression of the PRV-1 gene, a known target of JAK2, was rapidly downmodulated. Altogether, the data presented suggest that ITF2357 inhibits proliferation of cells bearing the JAK2(V617F) mutation through a specific downmodulation of the JAK2(V617F) protein and inhibition of its downstream signaling.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays: towards new standards for gene expression measurements.

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

Glucocorticoid-induced apoptosis in lymphoid organs is associated with a delayed increase in circulating deoxyribonucleic acid.

Pathological processes like cancer, chronic inflammation and autoimmune phenomena, all of which involve massive cell death, are associated with significant increases in circulating DNA. In order to clarify whether massive apoptosis occurring under physiological circumstances also causes DNA release into the circulation, we correlated the time-course of dexamethasone-induced intra thymic cell apoptosis with plasma DNA dynamics in rats. Animals were given 10 mg/l dexamethasone in their drinking water for up to 7 days. Sequential plasma samples were obtained during the treatment and DNA was quantitated by a micro fluorometric assay. Thymus and spleen weight as well as apoptotic cell levels were assessed at different times. Seven days of glucocorticoid treatment reduced thymic and spleen mass by 82 and 31%, respectively. Intra thymic apoptosis was maximal 24 h after the beginning of glucocorticoid treatment, declining markedly by 48 h. Very little apoptosis was observed in the spleen. Plasma DNA increased steadily during the first 4 days of glucocorticoid treatment (11.8 +/- 1.2 microg/ml on day 0; 24.2 +/- 1.6 microg/ml on day 4) beginning to decline afterward. Thymectomy but not splenectomy, drastically reduced the glucocorticoid-induced increase in plasma DNA. It is concluded that hormone-induced massive intra thymic cell death is followed by a delayed release of nucleosomal DNA into the circulation.

Can breast cancer Hsp 27 (Heat Shock Protein 27000) expression influence axillary lymph node status?

The expression of heat shock protein 27 (Hsp 27) in breast cancers correlates with stage of disease, the lower the stage the higher the expression, and with the presence or absence of lymph node metastases; lymph node negative patients being more likely to express Hsp 27 (P<0.04).

Need for an accurate molecular diagnosis to assess the donor origin of leukemia relapse after allogeneic stem cell transplantation.

BACKGROUND AND OBJECTIVES: Leukemia relapse occurring in donor cells after allogeneic hematopoietic stem cell transplantation has been reported in rare cases. Cytogenetic analysis and molecular probing of variable number of tandem repeats (VNTRs) have been used to confirm this unusual event in the few cases so far reported in the literature. The aim of this study was to demonstrate that extensive molecular characterization of leukemic cells at diagnosis and relapse may be necessary to avoid many technical pitfalls possibly leading to an erroneous diagnosis of leukemia relapse in donor cells after allogeneic transplantation. DESIGN AND METHODS: We report the case of a 49- year old man who received an allogeneic transplantation from his HLA-identical sister because of BCR-ABL+ acute lymphoblastic leukemia (ALL). After having achieved complete hematologic and molecular remission, two years later an overt leukemia relapse occurred with cytogenetic findings suggesting a leukemia relapse in donor cells. The donor or patient origin of leukemic cells at relapse was further investigated by fluorescence in situ hybridization (FISH) karyotyping, reverse transcription (RT) polymerase chain reaction (PCR) analysis of BCR-ABL chimeric transcripts, PCR amplification of several VNTRs and the Y chromosome-specific DYS14 sequence and finally by amplification, cloning and sequencing of the CDRIII region of the immunoglobulin heavy chain (IgH) gene. RESULTS: At the time of relapse, conventional and FISH karyotyping revealed the presence of a Phl+ chromosome and a female karyotype in all the 25 metaphases analyzed and PCR amplification of the Y chromosome-specific DYS14 sequence was negative. Moreover, the molecular evaluation of hematopoietic chimerism performed by the NZ-22 VNTR allowed us to demonstrate that at the time of relapse, a consistent proportion of hematopoietic cells was of donor origin. However, the molecular cloning and sequencing of the CDRIII region of the immunoglobuin heavy chain (IgH) gene rearrangement in leukemic blasts at diagnosis and relapse demonstrated their identity thus formally proving the patient origin of both leukemic clones. INTERPRETATION AND CONCLUSIONS: While the simplest interpretation of the apparent female karyotype at relapse is the consequence of a loss of the Y chromosome which in leukemic blasts took place along with duplication of an X-chromosome, this case strongly emphasizes the need for accurate and extensive molecular characterization to prove the donor origin of a leukemia relapse after allogeneic transplantation.

Quantitative and functional study of breast cancer axillary lymph nodes and those draining other human malignant tumors.

Lymphocyte functional activity from lymph nodes draining human malignancies reflects the host immune response against tumour. Breast cancer is the neoplasia with the greatest amount of identified antigens but a weak inducer of a host efficient immune response. In our study we compared the mitogen stimulated-proliferative response of cells isolated from metastases-free lymph nodes draining breast cancer (Group 1), other malignant tumours (Group 2), and those obtained from patients without malignancies (Control group). A significant decrease of the proliferative response in cells isolated from lymph nodes draining breast cancer was observed comparing it to the other groups. Quantitative analysis of B and T cells showed a higher number of B cells than T cells in Groups 1 and 2. Moreover, Group 1 presented a two fold increase of T cells compared with Group 2. Our results suggest that the immunosuppression observed in lymph nodes draining breast cancer is higher than the inmunosuppression presented in other malignant tumours and that impaired function is not correlated with the increased number of T cells.

Leucocyte alkaline phosphatase identifies terminally differentiated normal neutrophils and its lack in chronic myelogenous leukaemia is not dependent on p210 tyrosine kinase activity.

Leucocyte alkaline phosphatase (LAP) is a marker of post-mitotic granulocytes and its activity is reduced or absent in chronic myelogenous leukaemia (CML) granulocytes as a consequence of LAP messenger RNA (mRNA) deficiency. We provide evidence that along the granulocytic maturation in normal marrow, the acquisition of LAP surface expression, identified by the monoclonal antibody 1B12.1, was restricted to CD11bbright/CD16bright positive cells. Moreover, in normal granulocytes, exposure to granulocyte colony-stimulating factor (G-CSF) in vitro and in vivo increased the cell surface expression of LAP. Although G-CSF was able to induce the LAP surface expression in CML granulocytes, the inhibition of p210 tyrosine kinase activity by genistein or CGP75148B failed to restore LAP mRNA expression and LAP protein synthesis. In conclusion, the acquisition of LAP protein on the cell surface of granulocytes follows CD16 antigen expression and can be considered as the last marker of terminally differentiated neutrophils. G-CSF is a potent regulator of the LAP mRNA expression and protein synthesis in normal and CML-derived neutrophils. The lack of direct activity of p210 tyrosine kinase on LAP mRNA expression in CML neutrophils supports the notion that the LAP defect in this disease could be related to a precocious and uncontrolled release of white blood cells from the bone marrow into the blood stream.

[Biology of heat shock proteins]. / Biologia de las proteinas del shock térmico.

Hsp (Heat shock proteins) are a family of constitutive proteins of all pro and eukariotic cells that play different physiological roles: they promote the folding (acquisition of tertiary structure) assembly, translocation and secretion of newly synthesized polypeptides and participate in the removal or repairing of denatured proteins acting as molecular chaperons. This family of proteins is composed by numerous members grouped according to their molecular weight. When cells are subjected to different stresses such as hyperthermic shock, radiation, toxins, viral infections, etc., Hsp are overexpressed. In this way, they exert a cytoprotective effect, making the cells resistant to apoptosis. In humans, Hsp are overexpressed in cancer cells from ovary, endometrium, breast, prostate, digestive tract, etc. In some cases, overexpression is correlated with an unfavorable outcome because these proteins could favour metastatic disease. Some authors associate them not only with proliferation but also with differentiation of the neoplastic tissue. Recent studies show their influence in resistance to chemotherapeutic drugs. In autoimmune diseases like rheumatoid arthritis, Hsp can suppress the inflammatory response. Nevertheless, their role in the immune system has not been well established.

[Dendritic cells and their role in pathology]. / Células dendríticas y su papel en patologia.

The aim of this review is to update the knowledge on dendritic cells (CD), as potent antigen-presenting cells (APC) expressing class II major histocompatibility (MHC) antigen. The different types of DC are derived from a common bone marrow precursor. They differentiate and migrate to lymphoid and non-lymphoid tissues under the influence of diverse stimuli. After binding antigen in their periphery they move to the lymph node activating T cells. Depending on the microenvironment, DC express several surface markers and secrete cytokines such as IL-12, Il-1 and TNF alpha. DC play a role in the pathogenesis of autoimmune and viral diseases being relevant in AIDS. These cells also infiltrate human tumors where they could be involved in the induction of anti-tumor immune response. The immunostimulatory properties of DC are currently applied in DC-based therapies of melanoma and lymphoma patients.