Efficacy of DSP30-IL2/TPA for detection of cytogenetic abnormalities in chronic lymphocytic leukaemia/small lymphocytic lymphoma.

INTRODUCTION: Chronic lymphocytic leukaemia (CLL) is the most prevalent leukaemia in the Western Hemisphere. Cytogenetic abnormalities in CLL are used for diagnosis, prognosis and treatment. However, detecting these is difficult because mature B cells do not readily divide in culture. Here, we present data on two mitogen cocktails: CpG-oligonucleotide DSP30/Interleukin-2 (IL-2) and DSP30/IL-2 in combination with 12-O-tetradecanoylphorbol-13-acetate (TPA). METHODS: We analysed 165 cases of CLL with FISH and cytogenetics from January 2011 to June 2013. In 2011, three cultures were set-up: unstimulated, DSP30/IL-2-stimulated and TPA-stimulated. In 2012-2013, two cultures were set-up: unstimulated and stimulated with TPA/DSP30/IL-2. RESULTS: In 2011, FISH had a detection rate of 91% and cytogenetics using DSP30/IL2 had a detection rate of 91% (n = 22). In 2012-2013, FISH had a detection rate of 79% and cytogenetics using TPA/DSP30/IL-2 had a detection rate of 98% (n = 40). The percentage of cases with normal FISH but abnormal cytogenetics increased from 9% in 2011 to 21% in 2012-2013. The TPA/DSP30/IL-2 cultures in 2012-2013 detected more novel abnormalities (n = 5) as compared to DSP30/IL-2 alone (n = 3). CONCLUSIONS: TPA/DSP30/IL2 was as good as or better than DSP30/IL2 alone. TPA/DSP30/IL-2 offers a high detection rate for CLL abnormalities with a single stimulated culture and may increase detection of clinically significant abnormalities.

A microplate-based screening assay for neuraminidase inhibitors.

Neuraminidase (NA) represents a highly promising new target for drug development in influenza virus genes. Rapid screening of enzyme inhibitors is a key method for the identification of leading compounds. In order to speed up the screening for enzyme inhibitors of natural and synthetic origin, effective and fast assays are needed. 2'-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MUNANA) was selected as substrate for development of a microplate-based assay. The enzymatic reaction conditions were optimized as follows: in a 100 µL reaction mixture, the final concentrations were 32.5 mM sodium acetate (pH 3.5), 20 µM 4-MUNANA, 0.005% (w/v) bovine serum albumin, and 0.42 µg/mL NA. In the study, the doseresponse relationship of oseltamivir carboxylate to NA activity was observed. In addition, an overall Z' value of 0.8 proved the systems robustness and potential for screening. The assay system developed will be a valuable tool to discover new structures for the therapeutic inhibition of NA used to treat Influenza.

The main functions and structural modifications of tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) as a chemotactic factor.

Gram negative bacteria-derived and synthetic N-formyl peptides play a key role in host defense as chemotactic factors for phagocytic leukocytes. The first compound to be identified was N-formylmethionyl-leucyl-phenylalanine (fMLP) which contains highly potent leukocyte chemoattractant. Natural fMLP was subsequently purified and identified in supernatants of gram negative bacteria. Recently, much more attention has been focused on the human formyl peptide receptor (FPR) and its variant formyl peptide receptor-like 1 (FPRL1) and formyl peptide receptor-like 2 (FPRL2). Chemotactic factors such as fMLP interact with their specific cell surface receptors, which results in multiple biological responses through a G protein-coupled signal pathway. In this review, the functions and structural modifications of fMLP are discussed in view of future drug development.

Molecular and cytogenetic characterization of a novel rearrangement involving chromosomes 9, 12, and 17 resulting in ETV6 (TEL) and ABL fusion.

We performed chromosome analysis on the bone marrow of a patient with BCR/ABL negative chronic myelogenous leukemia (CML). By interphase fluorescence in situ hybridization (FISH), an extra ABL signal was present in interphase nuclei and appeared to be located at 17p in the metaphase cells. Chromosome analysis showed a subtle abnormality at 17p13 and 12p13 but no visible rearrangement at 9q34 (ABL). Additional FISH experiments disclosed a rearrangement between the short arms of chromosomes 12 and 17 at approximately bands 12p13 and 17p13, respectively. In addition, subtelomeric FISH analysis confirmed the presence of terminal 12p at 17p13 and showed terminal 9q34 to be intact on each chromosome 9. Taken together, these results indicated a rearrangement involving chromosomes 9, 12, and 17 that suggested the possibility of juxtaposition of part of the ETV6 (also known as TEL) locus (12p13) with a portion of ABL (9q34) together at 17p13. The ETV6/ABL fusion was confirmed by RT-PCR, which showed that the first 5 exons of ETV6 were fused in frame with ABL at exon 2. Wild-type ETV6 and ABL were also expressed, in accordance with the FISH results that showed no loss of the second ETV6 or ABL allele.

A case of infantile acute lymphoblastic leukemia presenting with rearrangement of MLL at 11q23 and apparent insertion or translocation at 10p12.

We report the case of an 11-month-old patient with a clinical diagnosis of infantile acute lymphoblastic leukemia and an MLL (11q23) rearrangement in 69% of nuclei, revealed with interphase fluorescence in situ hybridization (FISH). Routine chromosome analysis of the bone marrow showed a very subtle rearrangement involving the short arm of chromosome 10 and the long arm of chromosome 11 in the abnormal cells. To clarify the nature of this rearrangement, we hybridized the MLL break-apart probe to previously G-banded slides. The rearrangement was interpreted as a small inversion within the band 11q23, separating the 5' MLL from the 3' MLL region. This segment on the long arm of chromosome 11 containing the rearranged MLL locus was either inserted in or translocated to the short arm of chromosome 10 at approximately band 10p12. The inversion affecting MLL may have followed insertion or preceded it. Molecular characterization of this rearrangement was not possible, due to limited sample material. There have been previous reports of rearrangements of MLL with the MLLT10 (alias AF10) gene locus at 10p12, including an interstitial inverted insertion of 11q13q23 in one case and insertion of 11q14q23 at 10p12 in another. These both resulted in a large derivative chromosome 10 and transcription of an MLL/MLLT10 fusion product. To our knowledge, the novel and cryptic rearrangement detected in our patient has not been described previously. A follow-up study of the patient's bone marrow at the end of induction therapy showed no morphologic evidence of residual leukemia and both FISH and chromosome analyses were normal.

Value of CD23 determination by flow cytometry in differentiating mantle cell lymphoma from chronic lymphocytic leukemia/small lymphocytic lymphoma.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) share many morphologic and immunophenotypic features. In addition to histomorphologic examination, it is customary to use the absence of CD23 to differentiate MCL from CLL/SLL, based primarily on reported comparisons of immunohistochemical staining of tissue sections. These findings are widely extrapolated to flow cytometric analysis, although available data are contradictory and not sufficiently detailed. We compared expression of CD23 by flow cytometry in 22 cases of MCL and 25 cases of CLL/SLL. Lymphoma cells in 12 of 22 MCLs were negative for CD23, and 10 showed dim expression. In contrast, none of 25 CLL/SLLs were negative for CD23, 4 were dimly positive, and 21 were moderately or brightly positive. Thus, a significant proportion of MCL exhibited overlap of CD23 expression in the low-intensity range with CLL/SLL. Clinically, there was no correlation between the intensity of CD23 expression and clinical stage at diagnosis or survival. These findings emphasize that by flow cytometry, MCL can be differentiated reliably from CLL/SLL using CD23 if negative expression is observed. However, with dimly positive expression, interpretation should be cautious.

Castleman's disease confined to the leptomeninges.

We report a rare case of the plasma cell variant of Castleman's disease confined to the leptomeninges in a 42-year-old female. Flow cytometry demonstrated a minor monoclonal kappa light chain population, and conventional Southern blotting confirmed clonal rearrangement of the J(H) immunoglobulin heavy-chain gene. Polymerase chain reaction for Epstein-Barr virus and Kaposi's sarcoma-associated herpes virus was negative. The patient is disease-free five years after surgical resection. To our knowledge, clonal gene rearrangement has not been previously reported in the plasma cell variant of localized intracranial Castleman's disease.

Pleomorphic large cell sarcoma of the spleen with rhabdomyosarcomatous differentiation.

An unusual case is reported of pleomorphic large cell sarcoma of the spleen with rhabdomyosarcomatous differentiation in a 34-year old male. According to our knowledge, such a neoplasm has never been reported in the literature.

Detection of immunoglobulin kappa light chain rearrangements by polymerase chain reaction. An improved method for detecting clonal B-cell lymphoproliferative disorders.

The clonal determination of B-cell lymphoproliferative disorders by immunoglobulin heavy chain (IgH) rearrangement by polymerase chain reaction (PCR) is widely used. However, few attempts have been made to detect immunoglobulin kappa light chain (Igkappa) gene rearrangement using PCR. We studied 145 cases of B-cell neoplasms, along with 58 atypical and 18 reactive lymphoproliferative disorders, using newly designed degenerate oligoprimers recognizing the framework 3 (FR3kappa) and the joint (Jkappa) regions of the Igkappa gene. PCR products were analyzed on nondenaturing polyacrylamide gel (ndPAGE). Clonal B-cell determination was further investigated using IgH rearrangement and t(11:14) or t(14:18). By combining these methods, we detected either clonality or translocation in 117 of 137 cases (85%) in mature B-cell neoplasms. The additional analysis of Igkappa rearrangement improved sensitivity from 66% to 85%. To investigate whether the Ig gene configuration could be characterized using Igkappa PCR in B-cell neoplasms showing severe breakdown of genomic DNA, 18 selected cases were analyzed. Successful amplification was detected in 72% of the cases using either FR3/2-JH and/or FR3Jkappa oligoprimers. Finally, clonality was detected in 21 of 58 atypical B-cell proliferations, and among them, the atypical marginal cell (54%) and atypical large cell (50%) proliferations showed the highest frequency of clonal immunoglobulin gene products. We concluded that PCR/ndPAGE analysis of Igkappa is a sensitive, rapid, and efficient method for assessing clonality in conjunction with IgH and specific translocation analysis. This approach is particularly useful in the characterization of B-cell lymphoproliferative disorders in archival material with poor preservation of the genomic DNA.

Deletion of cyclin-dependent kinase 4 inhibitor genes P15 and P16 in non-Hodgkin's lymphoma.

B-cell non-Hodgkin's lymphoma (NHL) is a heterogeneous lymphoid malignancy consisting of several histologic types. Alterations in proto-oncogenes caused by reciprocal chromosome translocations have been implicated in the etiology of specific histologic groups. In this study, we examined the contribution of the cell cycle inhibitor genes P15, P16, and P18 to pathogenesis in a large panel of 209 cytogenetically characterized B-cell NHL tumors representing varied histologic groups. We identified the homozygous deletion of P15 and P16 genes in 13 tumors from 12 patients, all belonging to diffuse large-cell histology; 10 had this diagnosis made on presentation, 1 had transformed from small lymphocytic lymphoma, and 1 had transformed from Hodgkin's disease. Tumor-specific point mutations were not identified in the coding regions of these genes. Cytogenetically, chromosome 9p was normal in all but one tumor. On the other hand, eight tumors hemizygous for 9p by cytogenetic analysis showed wild-type configuration of these genes. Our study, therefore, indicates that deletion of P15 and P16 occurs in about 15% of diffuse large-cell NHL and is not usually detected by cytogenetic analysis. P18 was wild-type in all tumors including the 13 tumors hemizygous for 1p.

Absence of somatic changes in p21 gene in non-Hodgkin's lymphoma and chronic myelogenous leukemia.

p21 is induced by and mediates the effects of p53 in response to DNA damage arresting the cell in G1 or G2, by inhibiting multiple cyclin-cyclin-dependent kinases (CDK) or binding to proliferating-cell nuclear antigen (PCNA), respectively. To determine whether p21 mutants occur in tumors we examined DNA from 188 primary non-Hodgkin's B-cell lymphoma (NHL) tumors and 84 chronic myelogenous leukemia samples for mutational changes in the coding region of p21 by single-strand conformation polymorphism (SSCP) analysis and direct sequencing of polymerase chain reaction (PCR)-amplified DNA. We did not find mutations in the coding region in these two tumor types. We identified a polymorphic nucleotide change in codon 31 in which a transversion from C to A substituted amino acid arginine for serine. Three of 188 NHL tumors were homozygous for this change, but they were not identified in 84 CMLs or in 97 normal controls. On the other hand, in one CML case a transition from G to A in codon 64 substituted amino acid threonine for alanine. These data do not indicate that derangements in the coding region of p21 contribute to the initiation and/or progression of these tumors.

Analysis of alterations of oncogenes and tumor suppressor genes in chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (B-CLL) represents the most frequent adult leukemia in the Western world. The molecular pathogenesis of B-CLL is largely unknown. Although initial reports on small panels of cases had suggested a role for Bcl-1 and Bcl-2 oncogene activation in B-CLL, later investigations failed to confirm these data. Among tumor suppressor genes, p53 mutations have been reported in a fraction of cases. In this study, we have attempted a conclusive definition of the involvement of dominantly acting oncogenes (Bcl-1 and Bcl-2) and tumor suppressor loci (p53, 6q-) in 100 cases of B-CLL selected for their CD5 positivity and Rai's stage (0 to IV). Rearrangements of Bcl-1 and Bcl-2 and deletions of 6q and 17p were analyzed by Southern blot using multiple probes. Mutational analysis (single strand conformation polymorphism and polymerase chain reaction direct sequencing) was used to assay p53 inactivation. No alterations of Bcl-1 or Bcl-2 were detected in the 100 cases tested. Mutations of p53 were found in 10/100 cases without any significant association with clinical stage. Deletions of 6q were present in 4/100 cases. Overall, our data indicate that: 1) contrary to previous reports, Bcl-1 and Bcl-2 rearrangements are not involved in CD5+ B-CLL pathogenesis and 2) p53 mutations are present in 10% of cases at all stages of the disease.

Multiple genetic lesions in acquired immunodeficiency syndrome-related non-Hodgkin's lymphoma.

Non-Hodgkin's lymphoma (NHL) develops in about 5% to 10% of acquired immunodeficiency syndrome (AIDS) patients. The vast majority of AIDS-NHL are clinically aggressive B-cell NHL that are histologically classified as small noncleaved cell lymphoma (SNCCL), large cell immunoblastic plasmacytoid lymphoma (LC-IBPL), and large noncleaved cell lymphoma (LNCCL). In an attempt to understand the molecular pathogenesis of these tumors, we have investigated the involvement of dominantly acting oncogenes (c-myc, N-, K-, H-Ras), tumor suppressor genes (p53, RB1), and Epstein-Barr virus (EBV) infection in 27 AIDS-NHL samples (16 SNCCL, 5 LC-IBP, and 6 LNCCL). The following lesions were detected in AIDS-NHL: EBV infection (10/24; 41.6%), c-myc rearrangement (19/24; 79.1%), Ras mutation (4/27; 14.8%), and p53 loss/mutation (10/27; 37.0%). These lesions are not uniformly distributed, but, rather, cluster with specific types of AIDS-NHL: EBV infection is preferentially associated with LC-IBPL (4/4; 100%), while it is present in only a fraction of SNCCL (5/16; 31.2%) and LNCCL (1/4; 25%); c-myc oncogene activation clusters with SNCCL (16/16; 100%), whereas it is less frequent in LC-IBPL (1/4; 25%) and LNCCL (2/4; 50%); p53 inactivation is restricted to SNCCL (10/16; 62.5%) and consistently associated with c-myc activation. These data show that AIDS-NHL are associated with multiple genetic lesions that involve both proto-oncogenes and tumor suppressor genes and may accumulate in the relatively short period of time (4 to 6 years) between human immunodeficiency virus infection and AIDS-NHL development. These genetic lesions differ in the various AIDS-NHL subtypes, suggesting the involvement of distinct molecular pathway.

p53 mutations in human lymphoid malignancies: association with Burkitt lymphoma and chronic lymphocytic leukemia.

We have investigated the frequency of p53 mutations in B- and T-cell human lymphoid malignancies, including acute lymphoblastic leukemia, the major subtypes of non-Hodgkin lymphoma, and chronic lymphocytic leukemia. p53 exons 5-9 were studied by using genomic DNA from 197 primary tumors and 27 cell lines by single-strand conformation polymorphism analysis and by direct sequencing of PCR-amplified fragments. Mutations were found associated with (i) Burkitt lymphoma (9/27 biopsies; 17/27 cell lines) and its leukemic counterpart L3-type B-cell acute lymphoblastic leukemia (5/9), both of which also carry activated c-myc oncogenes, and (ii) B-cell chronic lymphocytic leukemia (6/40) and, in particular, its stage of progression known as Richter's transformation (3/7). Mutations were not found at any significant frequency in other types of non-Hodgkin lymphoma or acute lymphoblastic leukemia. In many cases, only the mutated allele was detectable, implying loss of the normal allele. These results suggest that (i) significant differences in the frequency of p53 mutations are present among subtypes of neoplasms derived from the same tissue; (ii) p53 may play a role in tumor progression in B-cell chronic lymphocytic leukemia; (iii) the presence of both p53 loss/inactivation and c-myc oncogene activation may be important in the pathogenesis of Burkitt lymphoma and its leukemic form L3-type B-cell acute lymphoblastic leukemia.