Biomedical subjects

N C Cross

Publications and source records attributed to N C Cross.

At least 55 records · Page 3Linked to original sources

PubMed1998

Consistent fusion of ZNF198 to the fibroblast growth factor receptor-1 in the t(8;13)(p11;q12) myeloproliferative syndrome.

The 8p11 myeloproliferative syndrome is a rare, aggressive condition associated with reciprocal translocations of chromosome band 8p11, most commonly the t(8;13)(p11;q12). To identify the genes involved in this translocation, we used fluorescence in situ hybridization (FISH) analysis to show that the chromosome 8 breakpoints fell within YAC 899e2 and that the chromosome 13 breakpoints are clustered in a region flanked by YACs 929f11 and 911h8. FISH using chromosome 13 PAC clones indicated that the t(8;13) is not simply a reciprocal translocation but also involves an inversion of 13q11-12. Exon trapping of a PAC that spanned the chromosome 13 translocation breakpoints led to the identification of a gene, ZNF198, that detected rearranged bands when used as a probe against Southern blots of patient DNA. Conceptual translation of the full-length ZNF198 cDNA sequence predicts a protein of 1377 amino acids that shows significant homology to the DXS6673E/KIAA0385 and KIAA0425 proteins. Alignment of these three proteins revealed a novel, conserved Zn-finger-related motif (MYM domain) of the general form CX2C19-22CX3CX13-19CX2CX19-25FCX3CX3F/Y that is repeated five times in each protein. To identify the translocation partner gene on chromosome 8, 5' and 3' RACE polymerase chain reactions (PCRs) were performed on patient RNA with several combinations of ZNF198 primers. Clones were identified in which the ZNF198 was fused to exon 9 of the fibroblast growth factor receptor-1 (FGFR1), a gene known to map to 8p11. An identical ZNF198-FGFR1 fusion was detected in the three patients with a t(8;13) for whom RNA was available; reciprocal FGFR1-ZNF198 transcripts were not detected. Rearrangements of both ZNF198 and FGFR1 were found in two further patients by Southern blotting. ZNF198-FGFR1 includes the five MYM domains of ZNF198 and the intracellular tyrosine kinase domain of FGFR1. We hypothesize that this fusion leads to constitutive activation of the FGFR1 tyrosine kinase in a manner analogous to the activation of ABL by BCR in chronic myeloid leukemia.

Amino Acid Sequence↗

PubMed1998

A novel fusion between MOZ and the nuclear receptor coactivator TIF2 in acute myeloid leukemia.

Chromosomal abnormalities of band 8p11 are associated with a distinct subtype of acute myeloid leukemia with French-American-British M4/5 morphology and prominent erythrophagocytosis by the blast cells. This subtype is usually associated with the t(8;16)(p11;p13), a translocation that has recently been shown to result in a fusion between the MOZ and CBP genes. We have cloned the inv(8)(p11q13), an abnormality associated with the same leukemia phenotype, and found a novel fusion between MOZ and the nuclear receptor transcriptional coactivator TIF2/GRIP-1/NCoA-2. This gene has not previously been implicated in the pathogenesis of leukemia or other malignancies. MOZ-TIF2 retains the histone acetyltransferase homology domains of both proteins and also the CBP binding domain of TIF2. We speculate that the apparently identical leukemia cell phenotype observed in cases with the t(8;16) and the inv(8) arises by recruitment of CBP by MOZ-TIF2, resulting in modulation of the transcriptional activity of target genes by a mechanism involving abnormal histone acetylation.

Acetyltransferases↗

PubMed1998

Aberrant transcripts of the FHIT gene are expressed in normal and leukaemic haemopoietic cells.

Deletions and apparent transcriptional abnormalities of the FHIT gene at 3p14.2 have recently been reported in a wide variety of solid tumours. To determine whether lesions of this gene also occur in leukaemia, we have analysed a total of 97 patients (chronic myeloid leukaemia, CML, in chronic phase or blast crisis, n = 71; de novo acute leukaemia, n = 26) and 16 normal individuals. Intact FHIT transcripts from all cases were amplified using RT-PCR. In addition, smaller size bands that were less intense than the full-length products were amplified from several samples from patients with leukaemia and also from normal leucocytes. Sequencing of the small products revealed that they were derived from FHIT transcripts lacking whole exons. Using single-strand conformation polymorphism analysis, no mutations in the coding sequence were detected in any patient. Furthermore, loss of heterozygosity was not seen in any of 36 informative patients at D3S1300 or D3S1481, markers located within the FHIT locus. We conclude that the FHIT gene and other uncharacterized tumour-suppressor genes at 3p14.2 are unlikely to be involved in the pathogenesis of acute leukaemia or progression of CML from chronic phase to blast crisis. Moreover, low-abundance FHIT transcripts that lack whole exons are not specific to malignant cells and should not be taken as evidence of an abnormality in the absence of demonstrable genomic DNA lesions.

Acid Anhydride Hydrolases↗

PubMed1998

Adoptive immunotherapy for relapse of chronic myeloid leukemia after allogeneic bone marrow transplant: equal efficacy of lymphocytes from sibling and matched unrelated donors.

Lymphocyte transfusion from the marrow donor (DLT) is well established as an effective therapy for relapse of CML post allogeneic BMT. Reports thus far have been mostly limited to patients who received DLT from a matched sibling donor. We compared the efficacy and toxicity of DLT in 30 patients who were treated with cells from their HLA-identical sibling (n = 18) or from their phenotypically HLA-matched unrelated marrow donor (n = 12). The overall probability of obtaining a cytogenetic remission was 69% (95%CI: 51-83%) and was not significantly different between the two groups. The disease stage at the time of DLT was the only factor associated with cytogenetic remission by multivariate analysis; patients treated in cytogenetic or molecular relapse (n = 11) were seven times more likely (RR = 7.4, 95%CI: 2.4-22.4, P = 0.0005) to respond compared to patients treated for hematologic relapse (n = 19). There was a trend towards more acute GVHD II-IV in the unrelated donor group (58 vs 39%, P = 0.09), but the probability of developing extensive chronic GVHD was not significantly different (56 vs 39%, P = 0.4). We conclude that transfusion of donor cells from HLA-matched volunteer donors does not appreciably increase the risk of GVHD compared with transfusion of cells from HLA-identical siblings in patients with CML who relapse following allogeneic BMT. Conversely, there is no evidence for an increased graft-versus-leukemia effect after DLT from volunteer donors.

Adult↗

PubMed1998

BCR-ABL-positive progenitors in chronic myeloid leukaemia patients in complete cytogenetic remission after treatment with interferon-alpha.

To determine the source of residual disease detected in patients with chronic myeloid leukaemia (CML) in complete cytogenetic remission (n=8) after treatment with interferon-alpha (IFN-alpha), we have tested CFU-GM colonies grown from bone marrow mononuclear cells or from plastic-adherent (Pdelta) cells for BCR-ABL mRNA using a nested multiplex RT-PCR. We compared our results with those obtained by analysis of colonies from newly diagnosed patients (n=4) and patients achieving no cytogenetic response (n=1) or incomplete cytogenetic response to treatment with IFN-alpha (n=5). A total of 1239 informative colonies were analysed. A small proportion of BCR-ABL-positive colonies was detected in all eight patients in complete cytogenetic remission, suggesting the persistence of leukaemia that could potentially lead to relapse. The overall proportion of BCR-ABL-positive colonies in patients achieving a cytogenetic response to IFN-alpha correlated with the levels of BCR-ABL transcripts detected in the peripheral blood by competitive RT-PCR (P=0.004). We conclude that residual disease detected in the peripheral blood of complete cytogenetic responders to IFN-alpha is at least partly derived from clonogenic myeloid cells. It is probable that the leukaemia clone in CML is only very rarely or never entirely eradicated by treatment with IFN-alpha.

Adult↗

PubMed1998

A novel BCR-ABL fusion gene (e2/1a) in a patient with Philadelphia-positive chronic myelogenous leukaemia and an aggressive clinical course.

A novel variant BCR-ABL mRNA transcript was detected by reverse transcription polymerase chain reaction (RT-PCR) in a patient with Philadelphia positive (Ph+ve) chronic myelogenous leukaemia (CML) who had an aggressive clinical course. Sequence analysis of the amplified cDNA fragment revealed an in-frame fusion with part of BCR exon e2 joined to part of ABL exon 1a. PCR of genomic DNA from this patient demonstrated that this unusual chimaeric mRNA resulted from breakpoints that fell within each of these exons. This is the first report of breakpoints occurring within both ABL and BCR exons and the first fusion to involve ABL exon 1a.

Base Sequence↗

PubMed1998

Minimal residual disease in chronic myeloid leukaemia.

The principle aim of residual disease analysis in patients with chronic myeloid leukaemia (CML) is to gauge patient response to treatment and, in patients after allogeneic BMT, to enable early diagnosis of relapse. RT-PCR is by far the most sensitive assay to detect residual disease in CML and can enable a single leukaemia cell to be detected in a background of 10(5)-10(6) normal cells. This is approximately 1000 x greater than the routine detection limit of the other methods. After allogeneic BMT, many CML patients are BCR-ABL positive for prolonged periods of time without subsequently relapsing. Thus the simple presence or absence of residual BCR-ABL transcripts in patients' leukocytes is of little value in the management of individual cases. Quantitative PCR techniques can distinguish between those PCR positive patients who have low or falling BCR-ABL levels on sequential analysis from those who have levels that are increasing. Provided assays are performed frequently enough, rising or persistently high numbers of BCR-ABL transcripts can be detected prior to frank relapse and this information may be used for early therapeutic intervention. Most patients who respond to treatment for relapse by donor lymphocyte infusion (DLI) achieve durable molecular remission. Quantitative PCR is also useful to gauge the response of CML patients to IFN-alpha. We have found that the great majority of patients in complete cytogenetic remission after treatment with IFN-alpha remain PCR positive and harbour a minority population of BCR-ABL positive myeloid precursor cells. It is unlikely therefore this treatment modality completely eliminates the disease in any patient.

Bone Marrow Transplantation↗

PubMed1997

Abnormalities of chromosome band 8p11 in leukemia: two clinical syndromes can be distinguished on the basis of MOZ involvement.

Two distinct leukemia syndromes are associated with abnormalities of chromosome band 8p11. First, a myeloproliferative disorder with features characteristic of both chronic myeloid leukemia and non-Hodgkin's lymphoma and second, an acute myeloid leukemia (AML) with French-American-British (FAB) M4/5 morphology and prominent erythrophagocytosis. The two syndromes are exemplified by a t(8;13)(p11;q12) and a t(8;16)(p11;p13), respectively, but cytogenetic variants of both have been described. Recently, the t(8;16) has been cloned and shown to fuse the MOZ gene at 8p11 to the CBP gene at 16p13. We have used fluorescence in situ hybridization (FISH), Southern blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) to refine the 8p11 breakpoint in three cases with t(8;13)(p11;q12) and in a single case of AML-M5 with a clinical picture apparently identical to that found in patients with a t(8;16), but characterized by an inv(8)(p11q13). FISH analysis was performed with several 8p11 CEPH yeast artificial chromosome (YAC) clones. YAC 782H11 was centromeric to the one case with t(8;13) tested, but was telomeric to the inv(8). YAC 847B12 was telomeric to both the t(8;13) and the inv(8), whereas YAC 829D12 was centromeric to the t(8;13), but split by the inv(8). Southern blotting and PCR of YAC 829D12 showed that it contained the MOZ gene. A 900-bp MOZ fragment encompassing the published t(8;16) breakpoint was amplified by PCR from normal peripheral blood leukocyte cDNA and used to probe Southern blots of patient DNA. A rearrangement was detected in the case with inv(8), but not in any of the three cases with t(8;13). Southern blotting with a CBP probe and RT-PCR with MOZ and CBP primers suggested that the inv(8) does not result in a cryptic MOZ-CBP fusion. It is likely, therefore, that MOZ is fused to a novel gene at 8q13 in this case. We conclude that the t(8;13) breakpoint is flanked by YACs 782H11 and 847B12 and is at least 1 Mb telomeric to MOZ. MOZ is involved, however, in a new variant of the t(8;16).

Acetyltransferases↗

PubMed1997

Assessing residual leukaemia.

For the great majority of patients with chronic myeloid leukaemia (CML), the Philadelphia (Ph) chromosome is a specific marker of the malignant clone. The standard method to assess the quality of remission in these patients is cytogenetic analysis of bone marrow derived metaphases. However, the molecular definition of the t(9;22) and its consequences has enabled other tests to be developed that can specifically detect CML cells. Fluorescence in situ hybridization (FISH) analyses chromosomes to detect either the juxtaposition of BCR and ABL sequences or the disruption of these genes; Southern blotting analyses genomic DNA to determine whether the BCR gene is rearranged; reverse-transcriptase polymerase chain reaction (RT-PCR) analyses RNA to determine the presence or absence of BCR-ABL transcripts; Western blotting analyses cell lysates to determine the presence or absence of BCR-ABL protein. Each of these techniques has particular advantages and pitfalls but in general they may be used to replace or at least to reduce the frequency of conventional cytogenetic analysis. Partly because of economic factors and the lack of standardization or effective quality control, these assays are still largely restricted to research laboratories. The sensitivity with which residual leukaemia can be detected suggests that FISH, Southern blotting and Western blotting are likely to be most useful in assessing patient response to interferon-alpha or other forms of treatment that typically induce partial remission. RT-PCR is by far the most sensitive assay and is probably most appropriate for monitoring patients who are in complete remission.

Biomarkers, Tumor↗

PubMed1997

The commonly deleted region at 9p21-22 in lymphoblastic leukemias spans at least 400 kb and includes p16 but not p15 or the IFN gene cluster.

Chromosome band 9p21-22 is one of the most common targets for deletions in cancer. The p16 tumor suppressor gene maps to this region and is inactivated in a wide variety of tumor cell lines and primary tumors. However, in some of the neoplasms with frequent 9p21 loss of heterozygosity (LOH), a structurally and functionally normal p16 is found suggesting that this gene might not be the primary or only target for inactivation. To define the smallest region of overlap of deletions at chromosome band 9p21-22 and to clarify the involvement of p16, p15 and the IFN cluster gene in lymphoblastic leukemias, we used a multiplex polymerase chain reaction to construct a detailed map of deletions at 9p21-22. We studied DNA from 30 lymphoblastic leukemia patients and nine cell lines using 10 genes/markers that map to this region, including four STS markers located between p16 and D9S171 (STS1, STS2) or between p16 and IFNalpha (STS3, STS4). We found that the size of the deletions in this region is variable and that the commonly deleted region spans at least 400 kb; it includes the p16 gene but excludes the IFN gene cluster and the p15 gene. The identification of this commonly deleted region enables a more focused search for other putative tumor suppressor gene at 9p21 which could be relevant to leukemogenesis.

Acute Disease↗

PubMed1997

An extremely delayed cytogenetic response to interferon-alpha in a patient with chronic myeloid leukaemia.

In chronic myeloid leukaemia (CML), treatment with interferon alpha IFN-alpha results in loss of the Ph' chromosome in a significant proportion of patients. Most cytogenetic responses occur early at a median of 9 months after initiation of treatment and failure to detect a cytogenetic response within a predetermined period may be a reason for IFN-alpha withdrawal. We report a patient in whom IFN-alpha dosage was initially severely limited by bone marrow suppression but in whom continuing treatment led to a first cytogenetic response only after 53 months. Increasing Ph' negativity over a further 2 years was associated with improving haematological tolerance which permitted IFN-alpha dose escalation and complete cytogenetic remission was achieved at 7 years after diagnosis. This remission has been sustained and has thus followed the most delayed cytogenetic response to IFN-alpha so far reported.

Antineoplastic Agents↗

PubMed1997

Rarity of dominant-negative mutations of the G-CSF receptor in patients with blast crisis of chronic myeloid leukemia or de novo acute leukemia.

It is likely that leukemia results, at least in part, from mutations that lead to a block in the normal process of differentiation. A defined region of the cytoplasmic domain of the granulocyte colony-stimulating factor receptor (G-CSF-R) transmits signals for maturation or differentiation of myeloid progenitor cells. Mutations in this region have been found in some patients with severe congenital neutropenia (SCN) who subsequently evolved to acute myeloid leukemia (AML). To determine if mutations of the G-CSF-R are more widespread in hematological malignancies, we have investigated a total of 47 patients, including 29 patients with blast crisis of chronic myeloid leukemia (CML-BC) and 18 patients with de novo acute leukemia as well as 19 normal controls, by RT-PCR and SSCP analysis. Two point mutations were found in a single individual with secondary AML (FAB type M1). The first was heterozygous and is predicted to replace the normal glutamine at position 718 with a stop codon, leading to a truncated protein. An identical mutation has been described previously and shown to act in a dominant negative manner. The second mutation was homozygous and would substitute a lysine for the normal glutamic acid at position 785. No mutations were found in any other patient or control samples. We conclude that mutations in the cytoplasmic domain of the G-CSF-R are infrequent in CML-BC or acute leukemia but may contribute to malignant transformation in some cases.

Blast Crisis↗

PubMed1997

Molecular response of CML patients treated with interferon-alpha monitored by quantitative Southern blot analysis. German chronic myeloid leukaemia (CML) Study Group.

We analysed 459 samples from 206 chronic myeloid leukaemia (CML) patients at diagnosis and during or after treatment with interferon-alpha (IFN-alpha) by quantitative Southern blot analysis for BCR rearrangement. In a minority (2%) of Ph-positive patients, no BCR rearrangement was detectable due to breakpoints outside the major breakpoint cluster region (M-bcr) or possibly due to M-bcr deletions. Results from 235 samples were compared with the proportion of Ph-positive metaphases found in contemporaneous bone marrow specimens analysed by conventional cytogenetics. The rank correlation between both methods was 0.82 (P < 0.001). The proportion of CML cells in samples determined by Southern blot analysis (BCR ratio) was significantly different between cytogenetically-defined minor, partial, and complete response groups (P < 0.001). Empirically-derived cut-off points in the BCR ratio were introduced in order to define molecular response groups for comparison to standard cytogenetic response groups: a BCR ratio of 0% was defined as complete molecular response and ratios of 1-24%, 25-50%, and > 50% were defined as partial, minor, and no molecular response, respectively. Using these cut-off points the concordance between both methods was 67% (P < 0.0001), a major cytogenetic response could be predicted or excluded in more than 90% of cases (P < 0.0001). Our findings demonstrated that quantitative Southern blot was as sensitive as cytogenetics and as peripheral blood samples are suitable for this technique it should be considered as the method of choice for routine monitoring IFN-alpha therapy in CML patients.

Adolescent↗

PubMed1997

The rate and kinetics of molecular response to donor leucocyte transfusions in chronic myeloid leukaemia patients treated for relapse after allogeneic bone marrow transplantation.

We have assessed the molecular response of 30 consecutive patients with chronic myeloid leukaemia (CML) treated for relapse after allogeneic bone marrow transplantation (BMT) by donor leucocyte transfusions (DLT). Response was evaluated by qualitative nested and quantitative competitive RT-PCR for BCR-ABL mRNA at various time intervals before and after DLT. The probability of attaining molecular remission at 2 years was 61% (95% CI 42-78%). Disease state at the time of DLT was significantly associated with response: molecular remission was achieved for 9/10 (90%) patients treated early (cytogenetic or molecular relapse) compared to only 8/20 (40%) patients treated late (haematological relapse; P = 0.009). The Kaplan-Meier estimates of molecular remission at 2 years post DLT for patients treated in early or late relapse were 86.6% and 47.3% respectively (P = 0.004). The median time interval from DLT to molecular remission was 11.0 months (range 2.5-32). Molecular remissions were durable for most (15/17) patients (median follow-up 21.2 months; range 0-55). Two patterns of molecular response were found: a very rapid decline after an initial lag phase or a more gradual decline over a period of several months. We conclude that molecular monitoring is a sensitive indicator of response to DLT; different kinetics of molecular response may reflect disease heterogeneity or differences in the mode of action of DLT.

Bone Marrow Transplantation↗

PubMed1997

Dominant-negative mutations of the Wilms' tumour predisposing gene (WT1) are infrequent in CML blast crisis and de novo acute leukaemia.

To determine if mutations of the Wilms' tumor predisposing gene (WT1) are associated with haematological malignancies, we have investigated 65 cases of acute leukaemia, including 39 patients in blast crisis of chronic myeloid leukaemia (CML), by amplification of WT1 exons 7, 8 and 9 followed by single-strand conformation polymorphism analysis. WT1 transcripts were detected by RT-PCR in all samples. An exon 7 silent polymorphism (A-->G; Arg 313) was identified in 17 individuals, 5 of whom were homozygous, but no other lesions were found. In 1 sample from a patient with acute lymphoblastic leukaemia a smaller size transcript missing exon 9 was detected; a similar abnormality has been described previously in a patient with Wilms' tumour and the resultant protein shown to act in a dominant-negative manner. No mutations of the exon 9 donor or acceptor splice sites were found in this patient and the basis of the abnormal transcript remains obscure. We conclude that dominant-negative mutations of the zinc finger region of the WT1 gene are uncommon in CML blast crisis. Abnormalities of this gene may, however, contribute to a small proportion of cases of de novo acute leukaemia.

Acute Disease↗

PubMed1997

Monitoring the efficiency of interferon-alpha therapy in chronic myelogenous leukemia (CML) patients by competitive polymerase chain reaction.

Interferon alpha (IFN-alpha) induces cytogenetic responses of variable degree in patients with CML. We sought to establish the relationship between BCR-ABL transcript numbers measured by competitive two-step reverse transcription polymerase chain reaction (RT-PCR) and cytogenetic status in CML patients treated with IFN-alpha. All 398 samples from 163 patients investigated by RT-PCR were positive for BCR-ABL transcripts. In order to standardize results for variability in RNA and cDNA quality, we quantified total ABL transcripts in each sample as internal control. The BCR-ABL/ABL ratios correlated with the cytogenetic results. Quantitative nested PCR allowed the detection of residual BCR-ABL transcripts in all complete cytogenetic responders on IFN-alpha. We conclude that competitive PCR with internal controls is a reliable method for monitoring patients on IFN-alpha and reduces the need for repeated marrow investigations.

Bone Marrow↗

PubMed1997

Characterization of a t(10;12)(q24;p13) in a case of CML in transformation.

We have used Southern blotting and fluorescence in situ hybridization (FISH) to define the breakpoints of a reciprocal translocation, t(10;12)(q24;p13), acquired as a secondary abnormality in a patient with Philadelphia chromosome positive chronic myeloid leukemia (CML) in transformation. A YAC clone that spanned the breakpoint at 12p13 was identified; this YAC included the CDKN1B gene but did not include ETV6. Neither ETV6 nor CDKN1B was rearranged, as determined by FISH and Southern blotting; however, a small deletion encompassing the translocated CDKN1B allele was detected. Analysis of two candidate genes at 10q24, HOX11 and NFKB2 suggested that they are not involved in this translocation. The preliminary mapping of breakpoints in this case demonstrated that they are different from an apparently identical translocation identified previously in a patient with myelodysplastic syndrome. The identification of the split YAC and small deletion should enable a more focused search for a gene or genes that may contribute to progression from chronic phase to blast crisis in CML.

Blotting, Southern↗

PubMed1996

Interferon augments PML and PML/RAR alpha expression in normal myeloid and acute promyelocytic cells and cooperates with all-trans retinoic acid to induce maturation of a retinoid-resistant promyelocytic cell line.

The PML gene is fused to the retinoic acid receptor alpha gene (RAR alpha) in the acute promyelocytic leukemia (APL) 15; 17 translocation. PML is expressed in diverse tissues and cell lines and localized in the nucleus with a typical speckled pattern. In the bone marrow, it is preferentially expressed in myeloid cells. PML appears to be transcriptionally regulated by class I and II interferons, which raises the possibility that interferons modulate the function and growth and differentiation potential of normal myeloid cells and precursors by activating PML-dependent pathways. Similarly, interferons could act on APL cells, alone or in combination with all-trans retinoic acid (RA), especially if the PML/RAR alpha fusion transcript that results from the t(15; 17) is induced by interferon. We report here that PML is expressed at low levels or not expressed in normal circulating human monocytes, lymphocytes, and polymorphonucleate cells, but is markedly induced by interferon; that PML and PML/RAR alpha expression is augmented by interferon in the NB4 APL cell line, which carries the t(15; 17), and in APL blasts from patients; that interferon inhibits growth and survival of NB4 APL cells in cooperation with RA; that interferons alone have minimal maturation effect on NB4 cells; and, finally, that interferon gamma, but not alpha or beta, induces maturation and growth suppression of NB4 cells with de novo retinoid resistance, and partially restores RA response.

Cell Differentiation↗