Search

Background: Fluorescence in situ hybridisation (FISH) is useful for detecting specific chromosomal abnormalities in various tumours. In lymphomas, diagnosis is frequently made using paraffin wax embedded tissue. However, FISH performed under these conditions presents potential technical problems and difficulties in interpretation. Aims: To show that FISH using tissue imprints and cytopreps or alternatively, bone marrow (BM) smears, constitutes an easy and rapid strategy to overcome these constraints. Methods: The study comprised 46 patients with lymphoma. Sixty nine tissue imprints, cytopreps, or BM smears were analysed by FISH. Dual colour, dual fusion FISH probes were used to detect the t(8;1 4), t(11 ;1 4), and t(14;18) translocations, whereas a dual colour breakapart FISH probe was used to detect chromosomal translocations involving the BCL6 gene. Results: Tissue imprints and cytopreps were successfully hybridised in all 52 cases, whereas hybridisation was successful in 16 of 17 archival BM smears. All patients could be analysed to identify either the presence or absence of chromosomal translocations. Conclusions: The use of tissue imprints, cytopreps, or BM smears to identify chromosomal abnormalities by FISH is a rapid and useful ancillary approach for diagnostic purposes. Therefore, it could be used on a routine basis whenever fresh samples are available. [ABSTRACT FROM AUTHOR]

In situ digestion with the restriction endonuclease (RE) Sau3A (Sau3A REISD) uncovers a polymorphism for the pericentromeric heterochromatin of human chromosome 3, which can be positively stained (3[sup +] ) or not (3[sup –] ), and has proven useful to differentiate donor and recipient cells after sex-matched bone marrow transplantation and to analyze the so-called hemopoietic chimerism. The aim of the present investigation was to obtain insight into the molecular basis of such polymorphism to optimize its use for chimerism quantification using methodological approaches other than REISD. To this end, fluorescence in situ hybridization (FISH) assays using probes for the satellite DNA sequences that mainly constitute chromosome 3 pericentromeric heterochromatin (α-satellite and satellite 1 DNA) were performed on control and Sau3A-digested chromosomes. The results obtained suggest that chromosome 3 α-satellite DNA is digested in all individuals studied, irrespective of the karyotype obtained by Sau3A REISD (3[sup ++] , 3[sup +–] , 3[sup --] ), and thus it does not seem to be involved in the polymorphism uncovered by Sau3A on this chromosome. Satellite 1 DNA is not digested in any case, and shows a polymorphism for its domain size, which correlates with the polymorphism uncovered by Sau3A in such a way that 3[sup +] chromosomes show a large domain (3[sup L] ) and 3[sup –] chromosomes show a small domain (3[sup S] ). It seems, therefore, that the cause of the polymorphism uncovered by Sau3A on the pericentromeric region of chromosome 3 is a difference in the size of the satellite 1 DNA domain. Small satellite 1 DNA domains fall under the resolution level of REISD technique and are identified as 3[sup –] .Key words: heterochromatin, α-satellite DNA, classical satellite DNA, satellite 1 DNA, restriction endonucleases, FISH.La digestion in situ à l'aide de l'enzyme de restriction (RE) Sau3A (Sau3A REISD) révèle un polymorphisme au niveau de l'hétérochromatine péricentromérique du chromosome 3 chez l'humain. Cette hétérochromatine laquelle peut être colorée de façon positive (3[sup +] ) ou négative (3[sup –] ). Cela s'est avéré utile pour distinguer les cellules donatrices et réceptrices après une transplantation de la moelle osseuse et pour analyser le mosaïcisme hématopoiétique. Le but de la présente étude était d'investiguer les bases moléculaires de ce polymorphisme afin d'optimiser son utilisation lors de la quantification du mosaïcisme avec des méthodes autres que le REISD. À cette fin, des analyses FISH (hybridation in situ en fluorescence) employant comme sondes des séquences d'ADN satellite qui constituent une bonne part de l'hétérochromatine péricentromérique du chromosome 3 (les ADN satellite α et 1) ont été réalisées sur des chromosomes digérés ou non (contrôle) avec Sau3A. Les résultats obtenus suggèrent que l'ADN satellite du chromosome 3 est digéré chez tous les individus sans égard au caryotype obtenu par la technique Sau3A REISD (3[sup ++] , 3[sup +–] , 3[sup --] ). Ainsi, ce satellite ne semble pas impliqué dans le polymorphisme révélé par Sau3A chez ce chromosome. L'ADN satellite 1 n'est digéré dans aucun cas et montre du polymorphisme quant à la taille du domaine contenant ce satellite. Cette variation est corrélée avec le polymorphisme révélé par Sau3A puisque les chromosomes 3[sup +] montrent un domaine de grande taille (3[sup L] ) tandis que les chromosomes 3[sup –] possèdent un petit domaine (3[sup S] ). Il semblerait donc que le fondement moléculaire du polymorphisme révélé par Sau3A dans la région péricentromérique du chromosome 3 soit une différence quant à la taille du domaine contenant l'ADN satellite 1. Des domaines d'ADN satellite 1 de petite taille peuvent être résolus par la technique de digestion enzymatique in situ et sont identifiés 3[sup –] .Mots clés : hétérochromatine, ADN satellite α, ADN satellite classique, ADN satellite 1, endonucléase de restriction, FISH.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article about the loss of the Y chromosome in leukocyte lineages of donor origin after stem cell transplantation is presented.

EZH2 is mutated in nearly 25% of follicular lymphoma (FL) cases. Little is known about how EZH2 affects patients' response to therapy. In this context, the aim of this study was to retrospectively analyze the frequency of mutations in EZH2 at diagnosis in tissue and ctDNA in patients with FL and to assess the patients' outcomes after receiving immunochemotherapy, depending on the EZH2 mutation status. Among the 154 patients included in the study, 27% had mutated EZH2 (46% with high-grade and 26% with low-grade FL). Of the mutated tissue samples, the mutation in ctDNA was identified in 44% of cases. EZH2 mutation in ctDNA was not identified in any patient unmutated in the tissue.Unmutated patients who received R-CHOP had significantly more relapses than patients who received R-Bendamustine (16/49 vs. 2/23, p = 0.040). Furthermore, our results show that patients with mutated EZH2 treated with R-CHOP vs. those treated with R-Bendamustine present a lower incidence of relapse (10% vs. 42% p = 0.09 at 4 years), a higher PFS (92% vs. 40% p = 0.039 at 4 years), and higher OS (100% vs. 78% p = 0.039 at 4 years). Based on these data, RCHOP could be a more suitable regimen for mutated patients, and R-bendamustine for unmutated patients. These findings could mean the first-time identification of a useful biomarker to guide upfront therapy in FL. [ABSTRACT FROM AUTHOR]

Mantle-cell lymphoma (MCL) has a poorer prognosis than other small B-cell lymphomas, thus a definitive diagnosis is essential. The t(11;14)(q13;q32) associated with MCL juxtaposes portions of CCND1 (11q13) and IGH (14q32), resulting in over-expression of cyclin D1. In this study, a highly sensitive two-colour fluorescence in situ hybridization (FISH) method was developed to detect t(11;14)(q13;q32) in nuclei isolated from paraffin-embedded tissue. Twenty-three MCLs, 13 normal controls and nine small B-cell lymphomas other than MCL were studied by FISH. Each MCL had been previously investigated to detect genomic IGH–CCND1 fusion by polymerase chain reaction (PCR) using DNA extracted from frozen tissue. The IGH–CCND1 fusion detection rate in the MCLs was 96% by FISH compared with 35% by PCR. By FISH, one MCL and three small B-cell lymphomas other than MCL harboured abnormalities involving only IGH. Less than 1% of cells showed false-positive IGH–CCND1 fusion in normal specimens by FISH. Thus, this highly sensitive FISH assay is very useful in confirming the diagnosis of MCL, has wide applicability as it may be performed on both paraffin-embedded and fresh tissue, and may also facilitate detection of translocations involving these loci in tumours other than MCL. [ABSTRACT FROM AUTHOR]

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a very rare disease that currently lacks genomic and genetic biomarkers to assist in its clinical management. We performed whole-exome sequencing (WES) of three BPDCN cases. Based on these data, we designed a resequencing approach to identify mutations in 38 selected genes in 25 BPDCN samples. WES revealed 37-99 deleterious gene mutations per exome with no common affected genes between patients, but with clear overlap in terms of molecular and disease pathways (hematological and dermatological disease). We identified for the first time deleterious mutations in IKZF3, HOXB9, UBE2G2 and ZEB2 in human leukemia. Target sequencing identified 29 recurring genes, ranging in prevalence from 36% for previously known genes, such as TET2, to 12-16% for newly identified genes, such as IKZF3 or ZEB2. Half of the tumors had mutations affecting either the DNA methylation or chromatin remodeling pathways. The clinical analysis revealed that patients with mutations in DNA methylation pathway had a significantly reduced overall survival (P=0.047). We provide the first mutational profiling of BPDCN. The data support the current WHO classification of the disease as a myeloid disorder and provide a biological rationale for the incorporation of epigenetic therapies for its treatment. [ABSTRACT FROM AUTHOR]

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a very rare disease that currently lacks genomic and genetic biomarkers to assist in its clinical management. We performed whole-exome sequencing (WES) of three BPDCN cases. Based on these data, we designed a resequencing approach to identify mutations in 38 selected genes in 25 BPDCN samples. WES revealed 37-99 deleterious gene mutations per exome with no common affected genes between patients, but with clear overlap in terms of molecular and disease pathways (hematological and dermatological disease). We identified for the first time deleterious mutations in IKZF3, HOXB9, UBE2G2 and ZEB2 in human leukemia. Target sequencing identified 29 recurring genes, ranging in prevalence from 36% for previously known genes, such as TET2, to 12-16% for newly identified genes, such as IKZF3 or ZEB2. Half of the tumors had mutations affecting either the DNA methylation or chromatin remodeling pathways. The clinical analysis revealed that patients with mutations in DNA methylation pathway had a significantly reduced overall survival (P=0.047). We provide the first mutational profiling of BPDCN. The data support the current WHO classification of the disease as a myeloid disorder and provide a biological rationale for the incorporation of epigenetic therapies for its treatment. [ABSTRACT FROM AUTHOR]

Single-unit umbilical cord blood (CB) SCT is limited by low total nucleated cell (TNC) dose. Co-infusion of CD34+ cells from a third party HLA-mismatched donor, known as dual or haplo-cord transplant, reduces the period of post-transplant neutropenia and related complications. The aim of this study was to analyze the value of early post-transplant peripheral blood (PB) and T cell chimerism after 28 dual transplants regarding CB engraftment. Cumulative incidence of myeloid engraftment at 30 days was 93% with a median time to engraftment of 14 days (10-29). Patients who developed CB graft failure (n=5) showed very low percentages of CB cells on days +14, +21 and +28 with decreasing dynamics. On the other hand, percentages of CB cells in patients who achieved CB engraftment increased over time. Interestingly, such patients showed two distinct chimerism dynamics in PB, but all of them showed a predominance of CB T cells early after SCT with increasing dynamics over time. Early post-transplant chimerism dynamics in PB and T cells predicts CB graft failure enabling rapid therapeutic measures to be applied. On the other hand, early increasing percentages of CB T cells correlates with ultimate CB engraftment. [ABSTRACT FROM AUTHOR]

Allo-SCT has a strong curative potential for AML patients mainly due to a GVL effect. Unfortunately, GvL and GVHD are intimately linked. IFN regulatory factor-3 (IRF3), by modulating innate immune reactions, could impact on the incidence and intensity of GVL and GVHD. We analyzed two gene variants in IRF3 (rs7251 and rs2304205) on the clinical outcome of 249 AML patients submitted to HLA-identical sibling allo-SCT. Patients with a donor carrying the dominant GG gene variant in rs7251 had, as compared with GC and CC variants, a lower acute GVHD (aGVHD) III-IV incidence (4% vs 11% vs 27%; P=0.0078), a higher relapse incidence (49% vs 35% vs 26%; P=0.018), and lower TRM (7% vs 24% vs 18%; P=0.0065). In functional studies, the GG variant was associated with lower production of IFN-γ, decreased lymphocyte proliferation after antigen presentation by DCs, and lower cytotoxic response of mature natural killer cells. Patients carrying the AA dominant variant in rs2304205 had higher relapse incidence (50% vs 39% vs 18%, P=0.0068). The presence of both variants (GG in rs7251 and AA in rs2304205) in donors and patients resulted in a stronger clinical impact. [ABSTRACT FROM AUTHOR]

Increasing mixed chimerism (MC) after allogeneic stem cell transplantation (SCT) has been associated with a high risk of relapse in acute leukemia. We evaluated a new method for chimerism detection, based on the quantitative real-time PCR (qrt-PCR) amplification of null alleles or insertion/deletion polymorphisms (indels). All qrt-PCR assays with null alleles and indels attained a sensitivity of at least 10-4, as well as good intra- and interassay concordance, and a high accuracy in experiments with cell mixtures. Informativeness was found in 80.3%of the 61 donor/recipient pairs tested. Nonrelapsed patients showed a progressive decrease in peripheral blood chimerism to values below 0.01%(complete chimerism (CC)). Bone marrow chimerism failed to reach CC more than 4 years after SCT. Increasing MC was observed prior to relapse in 88.2%of patients. Compared with conventional PCR amplification of variable number of tandem repeats, qrt-PCR predicted a significantly higher number of relapses (88.2 vs 44.4%) with a median anticipation period of 58 days. In conclusion, chimerism determination by qrt-PCR amplification of null alleles and indels constitutes a useful tool for the follow-up of patients with acute leukemia after SCT, showing better results than those obtained with conventional PCR.Leukemia (2005) 19, 336-343. doi:10.1038/sj.leu.2403622 Published online 20 January 2005 [ABSTRACT FROM AUTHOR]

Graft rejection is a major cause of treatment failure after T-cell-depleted stem cell transplantation (TCD-SCT) and remains a therapeutic challenge. Donor leukocyte infusions (DLI5) have an efficient graft versus host effect, which has been successfully used to treat recipient relapses. We hypothesized that this effect could be exploited to counteract the host versus graft reactions responsible for graft rejection. We report two adult patients with haematological malignancies who underwent sex-mismatched TCD-SCT from HLA-identical sibling donors. Peripheral blood (PB) counts and bone marrow (BM) cellularity were studied on a serial basis. Sequential chimaerism and minimal residual disease analysis were performed by FISH on PB and BM samples as well as on leukocyte lineages (T and B lymphocytes and myeloid cells) purified from PB using immunomagnetic technology. Both patients were diagnosed with incipient graft rejection 2-3 months after engraftment, based on persistently decreasing PB counts and BM cellularity together with the observation of decreasing mixed chimaerism (increasing percentage of recipient cells), mostly in whole PB and T lymphocytes. Both patients were successfully treated with a single DLI (1 × 107 CD3 + cells/kg), thereafter achieving normal PB counts and BM cellularity as well as complete chimaerism. Interestingly, the only side effect observed was mild graft versus host disease that did not require treatment. In conclusion, provided that an early diagnosis is made, the graft versus host lymphohaemopoietic effect harboured by immunocompetent donor cells can be successfully used for the treatment of incipient graft rejection. [ABSTRACT FROM AUTHOR]

The article discusses research which retrospectively examines patient-donor pairs to determine the impact of mismatching for UTA2-1 immunogenic peptide on clinical outcome after allo hematopoietic stem cell transplantation (allo-HSCT) from an HLA-identical sibling donor. It reveals the aspect of UTA2-1 mismatch as an infrequent condition in allo-HSCT. Further, it indicates that the presence of UTA2-1 mismatch does not seem to be relevant to the clinical outcome after allo-HSCT.

A letter to the editor is presented in response to the article about mesemeric inflammatory veno-occlusive disease (MIVOD) after allogeneic peripheral blood stem cell transplantation (PBSCT) published in the previous issue.

The article features a letter to the editor on cases of Listeria monocytogenes meningitis in two allogeneic hematopoietic stem cell transplant recipients.

Presents a correction to the article 'Quantification of Donor and Recipient Hemopoietic Cells by Real-Time PCR of Single-Nucleotide Polymorphisms,' by F. Maas, N. Schaap, S. Kolen, Z. Zoetbrood, I. Buno, H. Dolstra, T. de White, A. Schattenberg and E. van de Wiel-van Kemenade.