Endothelial precursors and mature endothelial cells are increased in the peripheral blood of myelodysplastic syndromes.

Increased angiogenesis has been demonstrated to be a significant prognostic factor in many solid tumors. In the oncohematological setting, it has been associated with myelodysplastic syndromes (MDS), chronic myeloid leukemia, acute lymphoid, and myeloid leukemias. Recently, increased circulating endothelial cells (CECs) have been associated with breast cancer and non-Hodgkin lymphoma (NHL). Based on these premises we analysed total and activated CECs, and endothelial precursors (CEPs) in 50 MDS patients and 20 healthy donors. CECs and CEPs were quantified by flow cytometry. CEC levels were compared with bone marrow (BM) microvessel density (MVD). In addition, some angiogenic factors, namely vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and soluble VEGF-Receptor2 (VEGFR2), were tested in the sera from 25 MDS patients. Total, activated CECs and CEPs were significantly increased in MDS when compared to control group (p<0.0001); whereas in the MDS cases no association was found with French--American--British (FAB), International Prognostic Scoring System (IPSS) subtypes or survival. Patients with higher CECs also showed higher MVD. Among the cytokines analysed, sVEGFR2 was significantly higher in the lower IPSS risk classes, while the levels of bFGF directly correlated with total and activated CECs. Taken together these data strengthen the hypothesis of a possible role of angiogenesis in MDS pathogenesis.

Central venous catheter-related complications in patients with hematological malignancies: a retrospective analysis of risk factors and prophylactic measures.

UNLABELLED: We retrospectively analyzed the incidence of thrombotic and infectious complications in relation with the use of central venous catheters (CVCs), in a series of patients with hematological malignancies and low platelet and leucocyte counts. PATIENTS AND METHODS: 126 patients with hematological malignancies were analyzed. A total of 207 CVCs were implanted: 137 centrally (CICCs) and 70 peripherally (PICCs). The median duration of the CVCs was 19 days for a total of 4051 catheter-days. Antithrombotic prophylaxis was unfractionated heparin (UFH), 2,500 IU daily by 24 h continuous infusion in 169 CVCs, low molecular weight heparin (LMWH), 3,800 IU daily by single bolus intravenous injection (i.v.) in 21 and warfarin in one. No prophylaxis was given in 16 CVCs. Thrombotic complications developed in 15.5% of the CVCs (7.9 events/1000 catheter days), and the frequency of infectious complications was 10.6% (5.2 events/1000 catheter days). On multivariate analysis thromboses were more frequent and earlier with PICCs than CICCs (p = 0.0001), and in patients on UFH (16.6%) than in LMWH prophylaxis (4.7%), but the last difference was not statistically significant. In conclusions the incidence of thrombotic complications in our series was comparable to that observed in non-thrombocytopenic patients and was significantly higher in those carrying PICC than CICC (p = 0.0001). There were fewer thrombotic events in the patients receiving i.v. LMWH prophylaxis than in those receiving i.v. UFH. The use of anticoagulants was safe and not associated with hemorrhages.

FISH characterization of t(8;12)(q12;p13) observed as the sole karyotypic anomaly in a myelodysplastic syndrome patient.

We report a t(8;12)(q12; p13) as the sole cytogenetic anomaly in a patient with a myelodysplastic syndrome (MDS). By means of FISH, we mapped the genomic region involved in the breakpoint (bkp) on both chromosomes. The 12p13 bkp mapped between markers WI-664 and WI-9218, immediately distal to the breakpoint cluster region frequently involved in hematological neoplasms targeted by y964C10. The 8q12 bkp (not yet investigated by FISH) was characterized and found to occur between markers WI-3263 and D8S524 within the region recognized by y874E10.

Hyperhomocysteinemia in myelodysplastic syndromes: specific association with autoimmunity and cardiovascular disease.

Hyperhomocysteinemia (HH) has been associated with cardiovascular and autoimmune diseases and oxidative cell damage. Myelodysplastic syndromes (MDS) are associated with autoimmunity (AI) and increased oxidative stress. We tested the association of HH and oxidative stress in 33 MDS patients, by measuring plasma homocysteine and malondialdehyde (MDA). HH was found in 42% of cases, (4/5) cases with associated cardiovascular events (CVE)(80%), and 9/15 cases with associated AI (60%). Thus in MDS, HH was significantly associated with AI/CVE (chi(2) : p=0.0011), and this association seems to be specific, as demonstrated by the comparison of MDS presenting AI/CVE with the ischemic cardiopathy/rheumatoid arthritis control group (13/20, 65% vs 19/69, 27%; chi(2) : p=0.0021). The levels of MDA indicated increased oxidative stress. Our data may suggest that in a subset of MDS, HH may simultaneously contribute to bone marrow myelodysplasia, CVE and AI pathogenesis, possibly through oxidative cell damage.

Biological and molecular characterization of PNH-like lymphocytes emerging after Campath-1H therapy.

Campath-1H, an anti-CD52 monoclonal antibody, is therapeutically active in lymphoproliferative and autoimmune diseases. After Campath-1H therapy, lymphocytes with a paroxysmal nocturnal haemoglobinuria (PNH) phenotype have been reported to emerge. We characterized a PNH-like lymphocyte population emerging after Campath-1H therapy, in a patient with fludarabine refractory B-cell chronic lymphocytic leukaemia (B-CLL). We demonstrated a reduction in PIG-A mRNA levels compared with controls, and of all cytokines tested [interleukin (IL)-4, IL-13, IL-2, interferon(IFN)-gamma, IL-6, IL-10, and tumour necrosis factor (TNF)-alpha], except transforming growth factor (TGF)-beta. Given the inhibitory activity of TGF-beta, its elevated levels may contribute to the selective pressure of Campath-1H, leading to the emergence of PNH-like lymphocytes.

Immunohistochemical and molecular analysis of bax, bcl-2 and p53 genes in laryngeal squamous cell carcinomas.

BACKGROUND: We investigated the role of apoptosis and its potential alterations in laryngeal squamous cell carcinomas (LSCCs) by evaluating bax, bcl-2 and p53 protein expression in 50 cases and by characterising the molecular status of the bax and p53 genes. MATERIAL AND METHODS: p53 and bax gene mutations were investigated by means of PCR/SSCP and direct DNA sequencing, and bax, bcl-2 and p53 protein expression by means of immunohistochemistry. RESULTS: We identified p53 gene mutations in 17/50 cases (34%); p53 expression in 26 of the 50 cases (52%); bcl-2 expression in 5/50 cases (10%); bax expression in 32/47 cases (68%). 18/33 cases with a wild type p53 gene overexpressed p53 protein: 12 cases (approximately 66%) were bax+/bcl-2-. Of the remaining cases without p53 protein expression, seven cases (approximately 47%) were bax+/bcl-2-. CONCLUSIONS: Our observations suggest that the overexpression of p53 may contribute to the repression of bcl-2 and the induction of bax expression in LSCCs. However, the fact that a number of cases not expressing p53 did not present any clear up-regulation of bax or down-regulation of bcl-2 suggests that bcl-2 and bax may be regulated by various mechanisms other than p53.

Poor prognosis in non-villous splenic marginal zone cell lymphoma is associated with p53 mutations.

We have recently reported a series of 15 non-villous splenic marginal zone lymphoma patients, six of whom showed p53 mutations (40%). This molecular alteration did not correlate with any particular clinico-pathologic feature at diagnosis. After a median follow-up of 56 months, four cases evolved into aggressive fatal non-Hodgkin's lymphoma (NHL) and two had refractory progressive disease; interestingly, p53 mutations were demonstrated in five of these patients at diagnosis. As the patients with wild-type p53 presented responsive or indolent disease, this genetic alteration may be an early marker of aggressive transformation or refractoriness. p53 evaluation at diagnosis could be advisable in this particular subset of NHL.

Cloning and gene mapping of the chromosome 13q14 region deleted in chronic lymphocytic leukemia.

Frequent deletions and loss of heterozygosity in a segment of chromosome 13 (13q14) in cases of B-cell chronic lymphocytic leukemia (CLL) have suggested that this malignancy is caused by inactivation of an unknown tumor suppressor gene located in this region. Toward the identification of the putative CLL tumor suppressor, we have constructed a high-resolution physical map of YAC, PAC, and cosmid contigs covering 600 kb of the 13q14 genomic region. In addition to densely positioned genetic markers and STSs, this map was further annotated by localization of 32 transcribed sequences (ESTs) using a combination of exon trapping, direct cDNA selection, sample sequencing of cosmids and PACs, and homology searches. On the basis of these mapping data, allelic loss analyses at 13q14 using CLL tumor samples allowed narrowing of the genomic segment encompassing the putative CLL gene to <300 kb. Twenty-three ESTs located within this minimally deleted region are candidate exons for the CLL-associated tumor suppressor gene.

Molecular and immunohistochemical analysis of the bcl-1/cyclin D1 gene in laryngeal squamous cell carcinomas: correlation of protein expression with lymph node metastases and advanced clinical stage.

BACKGROUND: The molecular pathogenesis of laryngeal squamous cell carcinomas (LSCCs) is still only partially understood, although genetic alterations affecting various protooncogenes or tumor suppressor genes have often been detected. METHODS: To improve their understanding of the role of cyclin D1 in the pathogenesis of LSCCs, the authors investigated the expression of cyclin D1 protein and the amplification status of the bcl-1/cyclin D1 locus in a panel of 58 pathologic samples. RESULTS: Expression of cyclin D1 protein was detected in 23 of the 58 patients (approximately 39%), 14 of whom had lymph node metastases (approximately 61%); of the remaining 35 patients without any detectable cyclin D1 expression, 7 had lymph node metastases (20%). Expression of cyclin D1 was detectable in 5% of the specimens of normal mucosa, 13% of those with mild-to-moderate dysplasia, and 25% of those with severe dysplasia. Amplification of the bcl-1/cyclin D1 locus was detected in 12 of the 49 LSCCs investigated (approximately 24%), 7 of which had lymph node metastases (approximately 58%); of the remaining 37 LSCCs with an apparently normal copy number of the cyclin D1 locus, 12 had lymph node metastases (approximately 32%). The authors found almost complete concordance between locus amplification and protein expression. Statistical analysis showed a correlation between cyclin D1 expression and both the presence of lymph node metastases (P < 0.01) and advanced clinical stage (P < 0.02). CONCLUSIONS: The authors' observations suggest that the deregulation of cyclin D1 expression may be involved in the pathogenesis of more aggressive LSCCs.

Identification of a tumor-associated mutant form of the NF-kappaB RelA gene with reduced DNA-binding and transactivating activities.

Alterations of NF-kappaB family members have been found to be associated with various forms of lymphoid malignancies. In order to determine whether alterations of the RelA gene are involved in lymphomagenesis, we analysed a large and representative panel (200 cases) of such tumors. Southern blot analysis did not reveal any rearrangements or locus amplification, suggesting that structural alterations of the RelA gene may represent rare events in lymphoid neoplasia. By means of PCR-SSCP analysis, we were able to identify a single point mutation leading to amino acid substitution (codon 494, Glu-Asp) in the transactivating (TA) domain in one case of multiple myeloma. The mutated allele was expressed in the pathological bone marrow sample but not in the peripheral blood cells of the patient. We demonstrate that the RelA protein carrying this specific mutation (called RelA494D) has less transactivating ability than the normal RelA protein. Interestingly, the mutated protein has a lower affinity for kappaB binding sites both as a homodimer or in association with the NFKB1/p50 subunit. Transfection experiments using a Gal4-RelA494D fusion protein indicated that the mutation does not alter the intrinsic transactivating ability of the TA domain of RelA. Furthermore, in vitro translated RelA494D is able to dimerize efficiently with other NF-kappaB members, such as p50, cREL and Ikappa Balpha. Our data therefore suggest that this mutation may alter the specific structural conformation needed for the DNA interaction of RelA, and provide insights into the amino acid sequences involved in mediating the biological activities of RelA.

Bcl-2 protein expression and p53 gene mutation in chronic lymphocytic leukemia: correlation with in vitro sensitivity to chlorambucil and purine analogs.

BACKGROUND AND OBJECTIVE: Bcl-2 oncogenic protein expression plays a major role in blocking the apoptotic mechanism. p53 gene mutations have also been suggested to account for the chlorambucil resistance in CLL. Thus we studied the relationship between bcl-2 protein expression, p53 gene mutations and in vitro drug sensitivity in CLL. METHODS: Fifty-three samples from untreated CLL patients in early disease stages were tested in vitro for chemosensitivity to chlorambucil (CLB), fludarabine (FAMP) and 2-chlorodeoxyadenosine (2-CDA) using the MTT assay. Intracellular bcl-2 protein expression was evaluated by flow cytometry analysis. p53 gene mutations were detected by using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis. RESULTS: The median LD50 values were 1.55 microM, 4.41 microM and 58.2 microM for 2-CDA, FAMP and CLB, respectively. About 23%, 41% and 11% of samples were defined as being sensitive to FAMP, 2-CDA and CLB, respectively, when samples were clustered for LD50 threshold values corresponding to the plasmatic levels of the drug. No statistically significant difference in bcl-2 protein expression was noted between sensitive and resistant samples for each drug. A p53 gene mutation was detected in 4 of the 30 cases studies and all of them were among samples resistant to CLB. INTERPRETATION AND CONCLUSIONS: Bcl-2 expression is not an indicator of in vitro response to drugs in CLL; similarly, although the four cases showing a p53 gene mutation were associated with CLB resistance, drug resistant samples were also observed in the group of patients showing wild type p53, suggesting multiple mechanisms of drug resistance in CLL.

Identification of a novel gene, PSD, adjacent to NFKB2/lyt-10, which contains Sec7 and pleckstrin-homology domains.

We have identified a novel human gene on chromosome 10q24 located contiguously to the 3' end of the NFKB2/lyt-10 gene in a tail to tail arrangement. We describe here a cDNA of 4307 bp, isolated from an adult human brain cDNA library, which contains an open reading frame encoding a putative protein of 645 amino acids with a predicted molecular weight of 71 kDa. Database homology searches indicate that this is a novel gene coding for a putative protein containing two discrete domains with significant homology to the Sec7 and pleckstrin-homology (PH) domains, respectively. We named this gene PSD (plekstrin-Sec7 domains gene). Northern blot analysis of a panel of RNAs from normal human tissues using the PSD cDNA as probe revealed the presence of three different tissue-specific transcripts of approximately 4.3, 2.3, and 1.8 kb, the longest of which is expressed only in brain. Our data suggest that the PSD gene may code for a protein related to a recently identified protein family containing both the Sec7 and the PH domains thought to be involved in signaling transduction processes.

The involvement of the candidate proto-oncogene NFKB2/lyt-10 in lymphoid malignancies.

NF-kappa B transcription factors regulate the expression of a variety of genes involved in immune responses and cell growth. In higher vertebrates, the NF-kappa B family encompasses five distinct members. Three NF-kappa B proteins, p65/RelA, RelB, and c-rel/Rel, have high transactivating potential in addition to their DNA binding activity. Two subunits, NF-kappa B1p50 and NF-kappa B2p52, coded respectively by the NFKB1 and NFKB2 genes, may only have DNA binding activity. Moreover, p50 and p52 subunits are translated as precursors, respectively p105 and p100, which can be processed into the mature active forms by the removal of their carboxy-terminal ankyrin domain. The five proteins share a homologous amino-terminal domain (rel domain) involved in DNA binding, dimerization, nuclear transport, and binding of regulatory subunits. All members form homo- and heterodimeric complexes with different DNA binding specificity and transactivating potential. Structural alterations of some members of the NF-kappa B gene family have been observed in lymphoid malignancies. In particular, the NFKB2 gene, localized on chromosome 10q24, represents a candidate proto-oncogene, since it has been found rearranged in certain types of lymphoma and more commonly in cutaneous lymphoma. Molecular analysis indicated that these rearrangements may occur as a consequence of chromosomal translocations or small internal chromosomal deletions. Rearrangements cluster within the carboxy-terminal ankyrin domain of the NFKB2 gene leading to the production of carboxy-terminally truncated proteins which, in some cases, are fused to heterologous protein domains. Experimental data showed that these abnormal proteins are constitutively localized in the nucleus, have lost the transcriptional repressor functions typical of normal NF-kappa B2p52 and may be capable of transactivation activity. These findings suggest that abnormal NFKB2 proteins may contribute to lymphomagenesis by altering the NF-kappa B system, both quantitatively and qualitatively, and leading to the activation of specific subsets of kappa B-controlled genes.

p53 protein expression in laryngeal squamous cell carcinomas bearing wild-type and mutated p53 gene.

We performed an immunohistochemical analysis to investigate the expression of p53 protein in a panel of 18 laryngeal squamous cell carcinomas, 15 primary tumours and three in relapse, previously analysed by us for the presence of p53 gene mutations. Dysplastic and/or normal surrounding mucosa was evaluated in 15 different tumours. The results of our study are the following: (1) expression of p53 protein was observed in one out of five tumours positive for p53 gene mutations (20%) and in 10 out of 13 (80%) negative cases; (2), p53 protein over-expression was frequently observed in normal and/or dysplastic mucosa surrounding either wild-type (7/11) or mutated p53 tumours (2/4); (3), p53 immunoreactive cells showed a pattern of distribution in normal and mildly/ moderately dysplastic mucosa (basal layers), different from that in severely dysplastic mucosa (whole thickness). These data further support the hypothesis that p53 protein over-expression may be a marker of the earliest phases of multistep tumorigenesis in laryngeal squamous cell carcinoma.

p53 tumour suppressor gene and RAS oncogenes: molecular analysis in the chronic and leukaemic phases of essential thrombocythaemia.

A panel of 51 cases of essential thrombocythaemia (ET), in chronic or leukaemic phase, was investigated for p53 gene and RAS oncogenes mutations by PCR-SSCP-direct sequencing. No RAS oncogenes mutations were detected, but p53 mutations were identified in three cases: 1/27 cases (approximately 4%) in chronic phase not undergoing chemotherapy, 1/19 cases (approximately 5%) in chronic phase undergoing chemotherapy, and 1/5 cases (20%) which had progressed to leukaemia. Our results suggest that: (1) p53 gene mutations occur sporadically in the chronic phase of ET, independent of chemotherapy, and may contribute to the progression to the leukaemic phase in a limited number of ET patients; (2) the RAS genes family does not seem to be involved in the pathogenesis of ET, unlike other bcr/abl negative chronic myeloproliferative diseases (CMPDs).

Molecular analysis of cutaneous B- and T-cell lymphomas.

Among extranodal non-Hodgkin's lymphomas, primary cutaneous lymphomas (CLs) represent a consistent group of B- and T-cell malignancies. We investigated the arrangement of Ig and T-cell receptor (TCR) genes, together with the involvement of several oncogenes and the tumor-suppressor gene p53, in a panel of primary cutaneous B- and T-cell lymphomas (CBCLs and CTCLs). Southern blot analysis was performed to detect rearrangements of the Ig, c-myc, bcl-1, bcl-2, bcl-3, bcl-6, and the NFKB2/lyt-10 genes in 52 cases of CBCLs and of the TCR, bcl-3, and NFKB2/lyt-10 genes in 38 cases of CTCLs. tal-1 gene deletions were analyzed in CTCLs by means of polymerase chain reaction (PCR). p53 gene mutations were assayed using PCR, single-strand conformation polymorphism analysis, and direct DNA sequencing in CBCL and CTCL cases. Clonal rearrangements of Ig genes or oncogenes were found in 25 of the 52 CBCLs. In particular, we detected rearrangements of the bcl-1 locus (2 cases), the bcl-2 gene (2 cases), the NFKB2/lyt-10 gene (2 cases), and the bcl-6 gene (1 case); interestingly, 4 of these cases showed a germline arrangement of the Ig genes. Clonal rearrangements of TCR genes were detected in 37 of the 38 CTCLs. Rearrangements of the NFKB2/lyt-10 gene were present in 2 cases and tal-1 gene deletions in 3 CTCL cases; p53 gene mutations were detected in 1 CTCL case. Overall, our data indicate that (1) clonal rearrangement of Ig genes is frequently undetectable by means of Southern blot in CBCLs (60%); (2) genetic lesions are involved in a limited but significant fraction of primary CLs showing a molecular marker of clonality (13/62; 20%); and (3) rearrangements of the bcl-1, bcl-2, or bcl-6 loci, associated with specific subsets of nodal lymphoid neoplasias, are rarely observed in CBCLs. Moreover, our results suggest that tal-1 gene deletions may play a pathogenetic role in non-acute T-cell malignancies and that, in the context of lymphoid malignancies, CLs may represent a favorable target for the possible oncogenic potential of the NFKB2/lyt-10 gene.

Multiple genetic lesions in laryngeal squamous cell carcinomas.

BACKGROUND: To understand the molecular pathogenesis of laryngeal squamous cell carcinomas (LSCCs), this study investigated the involvement of various protooncogene loci (bcl-1, int-2, c-erbB-1, c-myc, ras) and the p53 tumor suppressor gene in 18 patients with LSCC (15 at clinical presentation, 3 in clinical relapse). METHODS: For all patients, the mutations affecting the p53 and the H-, K-, and N-ras genes were evaluated by polymerase chain reaction (PCR), single-strand conformation polymorphism, and the direct sequencing of PCR-amplified fragments. The bcl-1, int-2, c-erbB-1, and c-myc loci of 15 patients were investigated using Southern blot analysis. RESULTS: A mutation of the p53 gene was detected in 5/18 patients (approximately 28%), bcl-1 locus amplification in 4/15 (approximately 26%), c-erbB-1 locus amplification in 2/15 (approximately 13%), and c-myc locus amplification in 1/15 (approximately 6%). The simultaneous presence of more than one genetic lesion was observed in four patients; two showed int-2/bcl-1 coamplification, and two int-2/c-erbB-1 coamplification, one of whom also showed a p53 gene mutation. A novel p53 mutation involving the splice acceptor site of exon 6 was detected in one patient. Two of the five patients positive for p53 mutations had clinical relapses of primary tumors. bcl-1 locus amplification only was observed in patients with lymph node metastases (4/6). All but one of the patients with molecular genetic lesions showed a peculiar infiltrating pattern. CONCLUSIONS: Overall, these results show that alterations of known protooncogenes and the p53 tumor suppressor gene are involved in a large proportion of LSCCs (11/18; approximately 60%) and may suggest that distinct molecular pathways occur in the pathogenesis of these tumors.

bcl-1, bcl-2, p53, c-myc, and lyt-10 analysis in cutaneous lymphomas.

In the present study we investigated the pathogenetic role of c-myc, bcl-2, and lyt-10 oncogenes, bcl-1 locus, and p53 suppressor gene in a representative panel of cutaneous lymphomas, including 25 cases of cutaneous B cell lymphoma (CBCL) and 29 cases of cutaneous T cell lymphoma (CTCL). In our analysis four cases of CBCL were found rearranged for bcl-2 and two for the bcl-1 locus. Two cases of CTCL and one case of CBCL were found rearranged for lyt-10. No rearrangements of c-myc oncogene were found in CBCL. Analysis of p53 gene showed mutation only in one case of mycosis fungoides in tumoral stage, at codon 163 of p53 gene (TAC-->CAC; Tyr--> Asp). Our data suggest that in primary CBCL bcl-2 oncogenes and bcl-1 locus are rarely involved. Furthermore, in primary CTCL p53 gene is not affected at significant frequency. The occurrence of p53 mutation in a patient affected by mycosis fungoides in tumoral stage may represent an involvement of p53 gene in tumor progression of CTCL, a finding observed in several types of human cancer.