Genetic alteration associated with chronic lymphocytic leukemia.

The genetics of B-cell chronic lymphocytic leukemia (B-CLL) differ considerably from most other forms of hematologic malignancy which are usually characterized by chromosome translocations. B-CLL typically contains chromosomal deletions and chromosomes 13q14 and 11q22-->q23 are the most common. These two regions appear to share a common ancestral origin (Auer et al., 2007b). Overall, chromosomal abnormalities can be found in the majority of patients with B-CLL when using sensitive techniques (Dohneret al., 2000) and possibly reflects an underlying predisposition, with a small but significant number of familial cases. Although single and consistent abnormalities are most common, multiple rearrangements can occur, often with disease progression (Feganetal., 1995; Dohner et al., 2000). Regions of recurrent deletion suggest the presence of tumor suppressor genes if following Knudson's theoretical 2-hit model. However, despite extensive sequencing analysis over the last decade and lack of pathogenic mutations identified, there has been a move away from this suggested hypothesis and alternative mechanisms of gene inactivation involving epigenetic silencing or haploinsufficiency may be considered as more likely in this disease. This review focuses on the common genetic abnormalities in B-CLL and relates them to some of the more recent hypotheses on inactivation of genes within these regions of deletion.

Isolation of chromosome-specific DNA sequences from an Alu polymerase chain reaction library to define the breakpoint in a patient with a constitutional translocation t(1;13) (q22;q12) and ganglioneuroblastoma.

We describe the cytogenetic and molecular characterization of a t(1;13)(q22;q12) constitutional rearrangement occurring in a patient with a relatively benign form of neuroblastoma, called ganglioneuroblastoma. Somatic cell hybrids were generated between mouse 3T3 cells and a lymphoblastoid cell line from this patient, D.G. One isolated subclone, DGF27C11, contained the derivative chromosome, 1pter-q22::13q12-qter, but no other material from either chromosome 1 or 13. Using available DNA probes the 13 breakpoint was assigned proximal to all reported markers. In order to generate flanking markers to define this translocation further, an Alu polymerase chain reaction library was constructed from a somatic cell hybrid containing only the proximal, 13pter-13q14, region of chromosome 13. Seven unique sequences have been isolated from the library, three of which lie below and four of which lie above the 13q12 breakpoint. More precise mapping of the distal markers was achieved using a panel of somatic cell hybrids with overlapping deletions of chromosome 13. The paucity of probes in the 1q22 region has made a precise assignment of this breakpoint difficult, however it has been shown to lie distal to c-SKI and proximal to APOA2. This refined characterization of the breakpoint is a prerequisite for its cloning, which may yield genes important in the pathogenesis of ganglioneuroblastoma.

Antisense oligonucleotides suppress B-cell lymphoma growth in a SCID-hu mouse model.

The t(14;18) translocation is found in the majority follicular lymphomas and some high grade B-cell lymphomas. This is results in deregulation of the BCL-2 gene and appears to play a role in oncogenesis. Various numbers of cells from a cell line derived spontaneously from a patient with B-cell lymphoma bearing the t(14;18) translocation and negative for the Epstein-Barr virus (EBV) were injected by IP, IV, and SC routes into SCID mice. The mice developed lymphoma bearing the t(14;18) translocation with as few as 5 x 10(6) cells within 28 days. This was determined by histological examination. The higher the cell inoculation the more rapidly the lymphoma developed. Engraftment of the tumour cells was determined by PCR for the t(14;18) breakpoint region on peripheral blood samples and could be detected prior to development of overt lymphoma. Having established a lymphoma model the cells were treated with antisense oligonucleotides to the first open reading frame of the BCL-2 gene prior to inoculation of the SCID mice. Control treatments with sense and nonsense oligonucleotides was also performed. At 28 days the sense, nonsense and untreated cell SCID mice had developed lymphoma, however, the antisense treated group failed to develop lymphoma. The findings demonstrate the modelling of B-cell lymphoma bearing the t(14;18) translocation and the ability to modify the lymphoma process with the use of antisense oligonucleotides to the BCL-2 gene. Reduction of the BCL2 protein suppresses the oncogenic potential of these lymphoma cells confirming that it plays an essential role in the development of malignancy.

Molecular cloning of a novel 11q23 breakpoint associated with non-Hodgkin's lymphoma.

Chromosomal analysis of a non-Hodgkin's lymphoma revealed a t(11;14)(q23;q32) translocation amongst other abnormalities. To investigate the molecular basis of this translocation, a cosmid library was constructed from the tumour DNA and the rearranged IGH locus was isolated in a single cosmid. Fluorescence in situ hybridization confirmed that the cloned region contained sequences from chromosome 11q23 fused to chromosome 14q32. Sequence analysis identified the breakpoint as a fusion between a region from the switch segment of the C gamma 4 gene of the IGH locus and an unknown sequence on chromosome 11. The chromosome 11 sequence maps proximal to the CD3 gene cluster and is therefore distinct from both the HTRX1 gene (rearranged in acute leukaemias) and the RCK gene (rearranged in a cell line derived from a histiocytic B-cell lymphoma). This newly identified region contains a cluster of rare cutting restriction enzyme sites located within 200 bases of the breakpoint, suggestive of a CpG island. Although this t(11;14)(q23;q32) translocation and that in the RC-K8 cell line affect different regions on chromosome 11, the breakpoints on chromosome 14 were found to have occurred at equivalent positions of S gamma 2 and S gamma 4 segments.

The significance of circulating cells carrying t(14;18) in long remission from follicular lymphoma.

Peripheral blood mononuclear cell fractions from 15 patients in continuous clinical remission from follicular lymphoma for longer than 10 years were examined for cells carrying the t(14;18) translocation using the polymerase chain reaction (PCR). The assay used was able to detect one positive cell in approximately 5 x 10(5) cells (a single 14q+ molecule in 2.5 micrograms DNA). Cells positive for t(14;18) were found in six of eight patients initially presenting with stage III or IV disease, compared with zero of seven of those with stage I or II disease (P less than .05). In two cases 14q+ junction regions were also successfully amplified from formalin-fixed biopsy material obtained at presentation 12 and 17 years previously. In both, sequence analysis demonstrated that the cells circulating in remission belonged to the original clone. These results indicate that cells bearing t(14;18) frequently persist in the peripheral blood in long remission of advanced follicular lymphoma and question the value of their presence as a predictor of relapse.

Detection of cells bearing the t(14;18) translocation following myeloablative treatment and autologous bone marrow transplantation for follicular lymphoma.

PURPOSE: To use the polymerase chain reaction (PCR) technique for molecular assessment of the results of myeloablative treatment of follicular lymphoma with autologous bone marrow transplantation. PATIENTS AND METHODS: Seventy-six patients with follicular or transformed follicular lymphoma were treated with cyclophosphamide 60 mg/kg x 2 and total-body irradiation 12 Gy, supported by autologous bone marrow transplantation. The bone marrow mononuclear cell fraction was treated in vitro with CD20 monoclonal antibody and baby rabbit complement. The PCR technique was used to identify 50 patients with amplifiable t(14; 18) translocations in biopsy material from lymph nodes or bone marrow infiltrated by lymphoma. RESULTS: Following treatment of the harvested bone marrow in vitro, 29 samples were tested by PCR to assess the efficacy of purging. In 25 cases, the same t(14; 18) sequences were amplified as from the patients' original biopsies, while in four cases, the marrow became PCR-negative. Three of the four patients treated with PCR-negative marrow subsequently developed recurrent lymphoma, compared with 11 of 25 in the PCR-positive group. Bone marrow and peripheral-blood mononuclear cell samples from 27 patients were studied during the follow-up period. All but one had the presence of the lymphoma-related t(14; 18) clone detectable by PCR and confirmed by direct sequencing from at least one sample between 3 months and 7 years after reinfusion of the bone marrow. With a median follow-up duration of 3 years, 13 patients developed recurrent disease, 13 remained in remission with the t(14; 18) still detectable, and one died of acute myeloid leukemia. CONCLUSION: This form of therapy does not eliminate the lymphoma-related t(14; 18)-bearing clone of cells, although the significance of its continued presence is uncertain. Improved methods for both treatment of the bone marrow in vitro and treatment of the lymphoma in vivo are required.

Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin's lymphoma.

PURPOSE: To evaluate the pharmacokinetics and toxicity of an antisense oligonucleotide targeting bcl-2 in patients with non-Hodgkin's lymphoma (NHL) and to determine efficacy using clinical and biologic end points. PATIENTS AND METHODS: Twenty-one patients with Bcl-2-positive relapsed NHL received a 14-day subcutaneous infusion of G3139, an 18-mer phosphorothioate oligonucleotide complementary to the first six codons of the bcl-2 open reading frame. Plasma pharmacokinetics were measured by anion exchange high-performance liquid chromatography. Response was assessed by computed tomography. Changes in Bcl-2 expression were measured by fluorescence-activated cell sorting of patients' tumor samples. RESULTS: Eight cohorts of patients received doses between 4. 6 and 195.8 mg/m(2)/d. No significant systemic toxicity was seen at doses up to 110.4 mg/m(2)/d. All patients displayed skin inflammation at the subcutaneous infusion site. Dose-limiting toxicities were thrombocytopenia, hypotension, fever, and asthenia. The maximum-tolerated dose was 147.2 mg/m(2)/d. Plasma levels of G3139 equivalent to the efficacious plasma concentration in in vivo models were produced with doses above 36.8 mg/m(2)/d. Plasma levels associated with dose-limiting toxicity were greater than 4 microg/mL. By standard criteria, there was one complete response, 2 minor responses, nine cases of stable disease, and nine cases of progressive disease. Bcl-2 protein was reduced in seven of 16 assessable patients. This reduction occurred in tumor cells derived from lymph nodes in two patients and from peripheral blood or bone marrow mononuclear cell populations in the remaining five patients. CONCLUSION: Bcl-2 antisense therapy is feasible and shows potential for antitumor activity in NHL. Downregulation of Bcl-2 protein suggests a specific antisense mechanism.

Monosomy 7 and 7q--associated with myeloid malignancy.

An association between the complete or partial loss of chromosome 7 and preleukaemic myelodysplasia or acute myeloid leukaemia has been recognized from the early days of tumour cytogenetic analysis. Detection of such abnormalities usually heralds a poor prognosis. The loss of DNA on chromosome 7 has led to speculation that tumour-suppressor genes may play a significant role in this form of leukaemogenesis, although it may be part of a multistep process. A further association with leukaemia secondary to carcinogen exposure including previous chemotherapy or a number of congenital anaemias has increased the interest in discovering the gene or genes on chromosome 7. Banded chromosome analysis has suggested that there are two broad critical regions on the long arm of chromosome 7 at bands 7q22 and 7q34-q36 that may contain the relevant genes. Initial molecular analysis has confirmed these two regions to be of significance. The advent of fluorescence in-situ hybridization techniques has facilitated some definition of the 7q22 region, with identification of candidate genes for further functional analysis. It is becoming clear that there will be more than one gene on chromosome 7 involved in the leukaemic process and with the definition of these genes it may be possible to look for associations with different phenotypes and prognosis. As for the reason for chromosome 7 showing a particular predisposition to total or partial loss we may speculate that the DNA sequence and structure may confer a 'fragility' on the chromosome. A greater understanding of the DNA structure of the long arm may provide real insight into the mechanisms of leukaemia. We would like to speculate in the long term that this could lead to the ability to screen for leukaemia susceptibility and avoidance of 'inducers' in those at risk.

Antisense therapy of hematologic malignancies.

Many tumor cells are inherently resistant to curative treatment due to an altered pattern of gene expression. It is an attractive and logical proposition to use this difference within the lymphoma cell to eradicate the malignant process. One such new therapeutic approach based on the "silencing" of genes involved in the prevention of apoptosis is Bcl-2 antisense oligonucleotide (AO) therapy. In the field of lymphoma, obvious targets included follicular lymphoma with the t(15;18) translocation, which results in deregulated expression of the Bcl-2 gene, chemoresistance, and subsequent protection against lymphoma cell death. Targeting the initiating codon of the Bcl-2 gene decreases both cell viability and Bcl-2 protein expression in lymphoma and leukemia cell lines that overexpress Bcl-2. Preclinical toxicity studies using a Bcl-2 AO G3139 (Genta, San Diego, CA) show good tolerance at a dose of 10 mg/kg, which is considerably higher than the dose required for good antilymphoma efficacy. In a phase I clinical study, G3139 was well tolerated with minimal toxicity in a dose escalation up to 147.2 mg/m2/d. Evidence of efficacy includes a responder with stage IVB follicular lymphoma who achieved complete clinical and radiologic response that has lasted more than 2 years. The main dose-limiting toxicity has been reversible thrombocytopenia related to the thioate backbone. Other antisense reagents are also in development to combat non-Hodgkin's lymphoma (NHL). These include oligonucleotides that target the messages of the Bcl-X(L) and protein kinase-Calpha (PKCalpha) genes. AOs may also have an application in tumors expressing mutant p53. AOs against MDM2 genes have shown the ability to restore wild-type p53 expression, suggesting that as oncogenic pathways are unraveled, normal cell growth and death patterns may be restored by molecular manipulation. Downregulation of antiapoptosis by AOs in the human setting has low toxicity and antilymphoma activity in cases in which conventional chemotherapy has failed. In the future, antisense therapy followed by chemotherapy may overcome chemoresistance to provide effective therapy for a range of malignancies.

Early postischemic dantrolene-induced amelioration of poly(ADP-ribose) polymerase-related bioenergetic failure in neonatal rat brain slices.

In the infant brain, ischemia-induced ionic and enzyme mechanisms may independently lead to cell death by energy depletion: resequestration of calcium mobilized from intracellular stores consumes ATP, and activated poly(ADP-ribose) polymerase (PARP) uses oxidized nicotinamide adenine dinucleotide to form polyADP-ribosyl nuclear proteins associated with DNA damage. Using 31P nuclear magnetic resonance spectroscopy, we have monitored intracellular pH and cellular energy metabolites in ex vivo neonatal rat cerebral cortex before, during, and after substrate and oxygen deprivation. In an insult that exhibited secondary energy failure and apoptosis we identified a relative 25% augmentation of high-energy phosphates at the end of recovery when the ryanodine-receptor antagonist, dantrolene, was introduced in the early (0- to 40-minute) but not late (40- to 120-minute) stage of recovery (P < 0.05). In contrast to the absence of a late dantrolene-sensitive effect, inhibition of PARP with 3-methoxybenzamide was as effective (P < 0.05) as early dantrolene, even when introduced after a 40-minute delay. The dantrolene and 3-methoxybenzamide effects on high-energy phosphates were not additive, rather the early dantrolene-sensitive effect nullified the potential 3-methoxybenzamide effect. Therefore, in this vascular-independent neonatal preparation, postischemic mobilization of calcium from intracellular stores is associated with PARP-related energy depletion. Inhibition of either of these processes confers improved postischemic bioenergetic recovery in the developing brain.

Extraction of DNA from small sections of frozen tissue with simultaneous histological examination.

Though analysis of small sections of biopsy material by molecular techniques permits increased sensitivity, it also requires accurate histological examination of the tissue in order to reduce sampling error. A technique for the extraction of DNA from small sections of frozen biopsy material with simultaneous histological examination from adjacent sections is described that may enhance the accuracy of characterisation of the tissue, particularly where there is focal variation. The quality of the DNA obtained enables a full range of molecular studies to be carried out.

Molecular genetics of extranodal marginal zone (MALT-type) B-cell lymphoma.

Mucosa-associated lymphoid tissue lymphoma is now classified as extranodal marginal zone B-cell lymphoma. We reviewed the current literature on the biological and genetic mechanisms that lead to the development and progression of this unusual lymphoma. Particular attention was given to the clinical and biological significance of the immunoglobulin genes rearrangement, that has been proposed and widely used both diagnostically and as a tool to monitor the response to antibiotic treatment.

Extranodal marginal zone B-cell lymphoma genotyping by Alu-polymerase chain reaction.

DNA amplification by polymerase chain reaction (PCR) with primers designed on the widely distributed Alu sequences allows the production of specific inter-Alu DNA-fingerprints. Amplification of tumour and matched normal DNA can show differences due to genetic alterations within the tumour genome. We applied this approach to study low-grade extranodal marginal zone B-cell lymphoma (of MALT type). After digestion with restriction enzymes, DNA samples were separately amplified by PCR with three different Alu-primers. A comparison between the fingerprint pattern from lymphoma and normal samples was made. Inter-Alu bands differing between the two samples were excised from the gel, cloned and sequenced. Nine cases of low-grade MALT-lymphomas have been analysed, giving seventeen different bands between tumour and normal. DNA sequence analysis showed identities for three of them with sequences available at the GenBank. The methodology of Alu-PCR to detect DNA-based abnormalities, in addition or combination with RNA-based methods, is a powerful tool to identify candidate regions frequently altered in tumours. With the increased available genomic sequences through the Human Genome Project, there will be an increasing probability of picking up perfect homologies with these sequences using cloned differential Alu-PCR bands in BLAST searches through genome databases.

RT-PCR Analysis of Breakpoints Involving the MLL Gene Located at 11q23 in Acute Leukemia.

Chromosome rearrangements of chromosome 11 at band 1 1q23 are detected in a high proportion of infant leukemias (

Stochastic modelling of apoptosis kinetics.

Robust quantitative estimation of average whole cell mitochondrial dysfunction is a useful tool for assessing sensitivity to apoptotic stimuli induced either by novel agents, or following manipulation of apoptotic threshold by pharmacological or functional genomics approaches. We have mathematically modelled the kinetics of whole cell mitochondrial membrane potential depolarisation within a population of cells as a Bernouli transition. An exponential distribution enables the median latency preceding mitochondrial membrane potential dissipation to be derived. The kinetic model can be fitted to in vitro single cell resolution data derived from kinetic flow cytometric studies by non-linear regression. We propose that kinetic determination of cumulative frequency distributions provides a useful approach for estimating apoptosis sensitivity across cell populations over short time-frames.

Molecular pathology of lymphomas.

Chromosomal translocations appear to be the initiating step in the development of malignancy in both B cell and T cell lymphomas. These results in the juxtapositioning of proto-oncogenes and transcriptional factors to the IG or TCR genes and lead to the deregulation of their expression. A survival advantage is provided to the lymphoid cells and contributes to neoplasm by permitting a clone to persist until other oncogenes such as c-myc are activated. This suggests a multistep progression to tumorigenesis. The occurrence of these translocations is assisted by obligatory DNA breaks and rearrangements of IG and TCR that have to occur for B and T cells to mature and function. The molecular alterations specific to lymphomas provide a potential specific target for therapy, including molecular manipulations. Animal modelling of lymphoma will provide the greatest possibilities for investigating the pathogenesis of lymphoma and future treatment modalities.

Antisense Oligonucleotides: Considerations for Lymphoma Therapy.

The majority of lymphomas are of B-cell lineage in origin. Translocations involving the q32 region of chromosome 14, the site of the immunoglobulin heavy chain gene (IGH), are most often observed. Rearrangement of one of the two alleles of this IGH gene is essential for development of the pre-B-cell into a functional B-cell and takes place normally under the influence of a DNA recombinase enzyme system. B-cell lymphomas predominantly involve the deregulation of proto-oncogenes following their juxtaposition to immunoglobulin genes. Their occurrence in part must be due to their obligate DNA breaks and rearrangement within the IG loci and probably involves a mistake mediated by the recombinase enzyme system responsible for normal IG rearrangement.(1,2) The overall result of these changes is a failure of the malignant cell to die in a programmed manner (apoptosis). Conventional treatments are not targeted to these molecular changes and often fail to effect a cure due to an inability to induce apoptosis. Antisense oligonucleotides (ASO) consisting of short sequences of DNA complementary to aberrantly expressed genes in tumours could potentially 'switch off' the inappropriate gene with a consequent antitumour effect by the induction of apoptosis. This represents the underlying basis of antisense therapy in malignant lymphoma. The binding of a sequence-specific oligonucleotide to a targeted length of mRNA occurs with a high level of specificity. Formation of an mRNA-DNA duplex should in theory suppress the translation of the targeted message into protein. If the production of that protein is essential for the survival, or malignant potential, of the cell, then blocking its production will negate the oncogenicity of the cell. Antisense oligonucleotides have been reported to inhibit gene expression as far back as 1978 with the inhibition of the Rous sarcoma virus in transfected chick embryo fibroblasts by a 13-mer oligonucleotide.(3).