Genomic aberrations of myeloproliferative and myelodysplastic/myeloproliferative neoplasms in chronic phase and during disease progression.

INTRODUCTION: Myeloproliferative neoplasms (MPN) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) may transform into secondary myelofibrosis (MF) or evolve into acute myeloid leukemia (AML). The genetic mechanisms underlying disease progression in MPN and MDS/MPN patients remain unclear. The purpose of this study was to investigate sequential genomic aberrations identified by single nucleotide polymorphism array (SNP-A)-based karyotyping that can detect cryptic aberrations or copy neutral loss of heterozygosity (CN-LOH) in the chronic phase and during disease progression of MPN and MDS/MPN patients. METHODS: The study group included 13 MPN and four MDS/MPN patients (seven polycythemia vera (PV); four essential thrombocythemia (ET); two MPN-unclassifiable (MPN-U); one chronic myelomonocytic leukemia (CMML); one atypical chronic myeloid leukemia, BCR-ABL1 negative (aCML); and two MDS/MPN-unclassifiable (MDS/MPN-U)). Among them, five patients (two PV, two MPN-U, and one MDS/MPN-U) progressed to MF and three patients (one CMML, one aCML, and one MDS/MPN-U) transformed to AML. The median follow-up period was 70 months (range, 7-152). Whole-genome SNP-A (SNP 6.0; Affymetrix, Santa Clara, CA, USA)-based karyotyping and JAK2 mutation analysis were performed according to the manufacturer's instructions. RESULTS: SNP-A showed 19 kinds of genomic aberrations, including seven gains, eight deletions, and four CN-LOH. CN-LOH of 9p involving JAK2 was the most common aberration, followed by 5q deletion and 9p gain. The incidence of genomic changes identified by SNP was not different in patients with disease progression (75%), compared with those without disease progression (56%) (P = 0.4). However, when excluding 9p CN-LOH, the incidence of genomic changes was significantly higher in patients with disease progression than in patients without disease progression (63% and 0%, respectively, P = 0.01). Among eight patients with disease progression, two patients (two MPN-U) showed abnormal SNP-A results, whereas metaphase cytogenetics (MC) analysis showed normal results at diagnosis and during follow-up. In nine patients without disease progression, SNP-A did not show any genomic aberrations except for 9p CN-LOH. In three patients (one PV, one aCML, and one MDS/MPN-U), clonal evolutions were identified by both MC and SNP-A according to disease progression. One PV patient who progressed to MF at 45 months after diagnosis showed sequential genomic changes from 9p CN-LOH to 9p gain by SNP-A. Results of JAK2 mutation analysis were variable depending on the patient. Most of the patients with 9p CN-LOH or 9p gain showed more than 50% of the JAK2 mutant alleles. In one patient (MDS/MPN-U) evolving to AML, the number of JAK2 mutant alleles decreased according to disease progression. CONCLUSION: This study suggests sequential genomic changes identified by SNP-A may be associated with disease progression.

Various patterns of IgH deletion identified by FISH using combined IgH and IgH/CCND1 probes in multiple myeloma and chronic lymphocytic leukemia.

INTRODUCTION: Interphase fluorescence in situ hybridization (FISH) can identify submicroscopic deletions adjacent to the breakpoints of rearrangements undetected by conventional cytogenetics. In this study, the characteristics and frequency of the IgH deletion identified by interphase FISH were investigated in patients with multiple myeloma (MM) and chronic lymphocytic leukemia (CLL). METHODS: The study group included 29 patients with MM and eight patients with CLL. Interphase FISH was performed with the IgH dual color, break-apart rearrangement probe and the IgH/CCND1 dual color, dual fusion translocation probe. RESULTS: The IgH deletion was found in 14% (4/29) of patients with MM and 13% (1/8) of the patients with CLL. Four patients had deletions of the whole or variable region of IgH on the native chromosome 14, whereas one patient had a deletion of the IgH variable region on a der(11)t(11;14). In two patients, the IgH break-apart FISH showed both patterns with and without IgH deletions. In cases showing the same pattern by IgH break-apart FISH, the IgH/CCND1 FISH showed different patterns, and vice versa. CONCLUSION: A variety of patterns of the IgH deletion were identified by interphase FISH using IgH break-apart and IgH/CCND1 probes in patients with MM and CLL. The results of this study suggest that the integrated information obtained with IgH break-apart and IgH/CCND1 FISH was needed to interpret FISH results unambiguously.

CD34, RAB20, PU.1 and GFI1 mRNA expression in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) with hypocellular bone marrow (BM) is often difficult to distinguish from aplastic anemia (AA). Furthermore, the diagnosis of MDS with low blast counts and normal karyotype may be problematic. These issues highlight the need for a reliable marker for the diagnosis of MDS. This study was conducted to determine if changes of mRNA expression in any of the four selected genes would be useful markers for differentiation of hypoplastic MDS from AA, and MDS from benign disease, as well as to investigate whether mRNA expressions differ between MDS risk subgroups. Thirty-five patients diagnosed with MDS, 27 patients with AA and 17 patients with benign diseases were included. The CD34, RAB20, PU.1 and GFI1 mRNA levels were measured by real-time RT-PCR. The CD34 mRNA expressions in hypoplastic MDS were higher than those found in AA. PU.1 and GFI1 mRNA expressions were significantly lower in MDS with low blast counts and normal karyotype than those of benign disease. High-risk MDS showed higher CD34 expressions than those of low-risk MDS. This study suggests that measurement of CD34 and GFI1 mRNA expressions could be useful as a diagnostic and prognostic marker for MDS.

A new HLA-A*33 variant, HLA-A*3314, identified in a Korean individual.

The sequence of human leukocyte antigen (HLA)-A*3314 is identical to that of HLA-A*330301 except for a single-nucleotide substitution at codon 49 (GCG-->GGG) resulting in an amino acid change from Ala to Gly.

A prospective randomized study on the mobilization of CD34+ cells comparing continuous intravenous vs subcutaneous administration of rhG-CSF in normal donors.

The efficacy of mobilizing peripheral blood progenitor cells (PBPC) with continuous intravenous (c.i.v.) administration of rhG-CSF was randomly compared to subcutaneous (s.c.) administration, in 15 normal donors in each arm of the study for 6 days. The percentage and absolute numbers of CD34+ cells in the c.i.v. and s.c. groups increased maximally at day 3 and 5, respectively, when compared with the steady-state (day 0) level. Peak CD34+ cell levels were achieved on day 3 in the c.i.v. group, with more rapid results than in the s.c. group (49.3/microl vs 35.9/microl, P=0.043). Plasma rhG-CSF levels declined progressively during mobilization in each group as the WBC increased. The serum level of rhG-CSF did not correlate with CD34+ cell counts in the peripheral blood. Toxicity profiles in the c.i.v. and s.c. groups were similar. Each regimen was effective in successfully mobilizing the target CD34 cell number.

Inhibition of breast cancer growth in vivo by antiangiogenesis gene therapy with adenovirus-mediated antisense-VEGF.

Increased expression of VEGF in several types of tumours has been shown to correlate with poor prognosis. We used a replication-deficient adenoviral vector containing antisense VEGF cDNA (Ad5CMV-alphaVEGF) to down-regulate VEGF expression and increase the efficiency of delivery of the antisense sequence in the human breast cancer cell line MDA231-MB. Transfection of these cells with Ad5CMV-alphaVEGF in vitro reduced secreted levels of VEGF protein without affecting cell growth. Moreover, injection of the Ad5CMV-alphaVEGF vector into intramammary xenografts of these cells established in nude mice inhibited tumour growth and reduced the amount of VEGF protein and the density of microvessels in those tumours relative to tumours treated with the control vector Ad5(dl312). Our results showed that antisense VEGF(165)cDNA was efficiently delivered in vivo via an adenoviral vector and that this treatment significantly inhibited the growth of established experimental breast tumours. The Ad5CMV-alphaVEGF vector may be useful in targeting the tumour vasculature in the treatment of breast cancer.

Clinical Features and Prognosis of Lung Cancer with Brain Metastasis.

PURPOSE: Brain metastasis is estimated to occur in 20~40% of solid tumor patients and the most common primary tumor is lung cancer. Even though the prognosis of brain metastasis is grave and the 1-year survival rate is only 15%, symptom palliations are made with whole brain radiation therapy. We retrospectively evaluated the clinical features and prognostic factors of lung cancer with brain metastasis. MATERIALS AND METHODS: From January 1987 to October 1999, 50 lung cancer patients with brain metastasis underwent whole brain radiation therapy. We reviewed the improvement in neurologic symptoms and survival according to the following parameters; performance status, histological type, presence of brain metastasis at the initial diagnosis of lung cancer, presence of extracranial metastasis, multiplicity of brain lesion, presence of primary lung symptom and treatment modalities. RESULTS: The most frequent symptom with brain metastasis was a headache (50%). Palliation of the headache and other symptoms was achieved in 81% of the patients. Median overall survival after brain metastasis was 21 weeks and the 1 year survival rate was 15%. Patients without extracranial metastasis had a longer median survival than those with, 38 weeks versus 15 weeks, respectively (p=0.01). CONCLUSION: In lung cancer with brain metastasis, neurologic symptoms can be palliated with whole brain radiation therapy, and in this study among such patients, absence of extracranial metastasis can be a good prognostic factor.

Mobilization kinetics of CD34(+) cells in association with modulation of CD44 and CD31 expression during continuous intravenous administration of G-CSF in normal donors.

The aim of the present study is to evaluate the kinetics of CD34(+) cells and investigate the potential modulation of CD44 and CD31 expression on CD34(+) cells during continuous i.v. administration of G-CSF, thus to elucidate the possible mechanism of peripheral blood progenitor cell (PBPC) mobilization. Fifteen healthy donors were enrolled in this study. G-CSF (10 microg/kg/day) was administered for four consecutive days through continuous 24-h i.v. infusion. For measurement of complete blood counts, CD34(+) cell levels and their expression of CD44 and CD31, PB sampling was performed immediately before the administration of G-CSF (steady-state) and after 4, 8, 24, 48, 72, 96, and 120 h of G-CSF administration. The percentage and absolute number of CD34(+) cells significantly increased at day 3 (0. 55 +/- 0.09%, 51.12 +/- 24.83 x 10(3)/ml) and day 4 (0.47 +/- 0.09%, 46.66 +/- 24.93 x 10(3)/ml), compared to the steady-state level (0. 06 +/- 0.09%, 2.03 +/- 5.69 x 10(3)/ml). At day 3 to day 5 following the onset of G-CSF administration, a strong decrease of CD44 and CD31 expression was observed on mobilized CD34(+) cells compared to controls: the relative fluorescence intensity of CD44 and CD31 was, respectively, 50%-70% and 40%-90% lower than that of controls. We conclude that continuous i.v. administration of G-CSF apparently results in more rapid mobilization of CD34(+) cells, and downregulation of CD44 and CD31 on CD34(+) cells is likely to be involved in the mobilization of PBPC after treatment with G-CSF.

Early detection of relapse by hypermetaphase fluorescence in situ hybridization after allogeneic bone marrow transplantation for chronic myeloid leukemia.

PURPOSE: Standard G-band cytogenetic analysis (CG) provides information on approximately 25 metaphases for monitoring the presence of Philadelphia chromosome positive (Ph+) cells in chronic myelogenous leukemia (CML) patients, making the detection of a low frequency of Ph+ cells problematic. The purpose of this study was to improve the detection of a low frequency of Ph+ cells. PATIENTS AND METHODS: We combined fluorescence in situ hybridization (FISH) with long-term colcemid exposure, capturing several hundred metaphases in bone marrow cultures (hypermetaphase FISH [HMF]). Using probes that identify Ph+ cells, HMF was compared with CG analysis in the follow-up evaluations of 51 patients with CML at various time points after allogeneic bone marrow transplant (BMT). RESULTS: Thirty-five patients never showed the presence of Ph+ cells by either method. In four patients, high frequencies of Ph+ cells were detected by both methods. In the remaining 12 patients, Ph+ cells were detected by HMF at time points after BMT when they were not detected by CG. In seven of the 12 patients, low but statistically significant frequencies of Ph+ cells (0.37% to 5.20%) were detected 3 months or later after BMT, and when no intervention was initiated, all seven patients later relapsed. Based on those data, an eighth patient with mixed chimerism and a similar HMF-detected Ph+ frequency (1.8% at 27 months after BMT) was reinfused with donor lymphocytes and achieved remission with 0% Ph+ cells studied by HMF (up to 50 months after BMT). Ph+ cells detected by HMF but not by CG less than 3 months after BMT disappeared on later examination in two of four patients. After detection of Ph+ cells by HMF only, the median time to cytogenetic progression (detection of Ph+ cells by CG) was 101 days. CONCLUSION: The results demonstrate the ability of HMF to detect low but clinically relevant levels of leukemic cells not detected by CG in transplant patients. The data indicate that HMF can detect low levels of Ph+ cells before standard cytogenetics at a time that may be useful in monitoring disease status and planning clinical interventions.

Putative chromosomal deletions on 9P, 9Q and 22Q occur preferentially in malignant gastrointestinal stromal tumors.

To characterize the type of genetic alterations in gastrointestinal stromal tumors (GISTs), we performed a comprehensive allelotype study of 14 GISTs (2 benign, 7 borderline and 5 malignant) by polymerase-chain-reaction and loss-of-heterozygosity (PCR-LOH) analysis using 102 microsatellite markers, and compared the results with comparative-genomic-hybridization (CGH) analysis. Among the 38 evaluated chromosomal arms, 16 (42.1%) showed LOH in at least one patient. Most frequent LOH was observed at chromosome 14p and 14q (9/14, 64%) and this was demonstrated in all types of GISTs (50% in benign, 71% in borderline and 80% in malignant). Additional chromosomal deletions were found in several chromosomal arms. Among them, deletions on chromosomal arms of 22q (3/14, 21.4%), 9p (2/14, 14.3%) and 9q (2/14, 14.3%) were the most frequent, and were detected only in malignant GISTs both by PCR-LOH and by CGH analysis. Additionally, 2 malignant GISTs with LOH on 9p showed homozygous deletions in the restricted area of 9p by multiplex PCR-LOH analysis. Thus, several putative chromosomal changes were preferentially present in malignant GISTs but rare in benign and borderline GISTs. These findings suggest that accumulated chromosomal changes may contribute to the progression and/or malignant transformation of GISTs.

Myeloid differentiation of human cord blood CD34+ cells during ex vivo expansion using thrombopoietin, flt3-ligand and/or granulocyte-colony stimulating factor.

We investigated the phenotypic changes of human umbilical cord blood (CB) CD34+ cells during ex vivo expansion using thrombopoietin (TPO), flt3-ligand (FL), and/or granulocyte-colony stimulating factor (G-CSF). During ex vivo expansion of CD34+ cells isolated from human CB for up to 5 weeks, surface expression of molecules on the cultured cells including CD64 (Fc gammaRI), CD32 (Fc gammaRII), CD16 (Fc gammaRII), CD11b (MAC-1) and CD18 (beta2-integrin) was analysed by flow cytometry along with simultaneous measurement of apoptosis by 7-aminoactinomycin D staining method. CD64, CD32 and/or CD18 expressing cells appeared in the cultures both with and without the addition of G-CSF until the tenth day. However, without G-CSF, CD16+ fractions did not appear and CD11b+ fractions were not maintained. With G-CSF, the CD16+ or CD11b+ fractions appeared only from the second week. These results suggest that G-CSF is necessary for the late stage of myeloid maturation during which CD16 and CD11b are expressed.

Distinct patterns of apoptosis in association with modulation of CD44 induced by thrombopoietin and granulocyte-colony stimulating factor during ex vivo expansion of human cord blood CD34+ cells.

The insufficient number of haemopoietic stem cells (HSCs) in cord blood (CB) is the major potential limitation to widespread use of CB for marrow replacement. Cytokine-mediated ex vivo expansion has been proposed as a means of increasing the number of CB HSCs for transplantation. However, the biology of CB HSCs during cytokine-mediated ex vivo expansion, such as apoptosis or expression of adhesion molecules, has not yet been elucidated. We have investigated the patterns of apoptosis and CD44 expression on human CB CD34+ cells during ex vivo expansion. CD34+ cells isolated from human CB were cultured in a stroma-free liquid culture system with thrombopoietin (TPO), flt3-ligand (FL), stem cell factor (SCF), and/or granulocyte-colony stimulating factor (G-CSF). During the culture, for up to 5 weeks, apoptosis was measured by staining with 7-amino-actinomycin D (7-AAD) along with concurrent immunophenotyping of CD34 and CD44 with three-colour flow cytometry. In the cultures with TPO, an apoptotic fraction with down-regulated CD44 appeared from the fourth day up to the second week. G-CSF also induced apoptosis but in a different manner; the apoptotic fraction without down-regulation of CD44 appeared unremittingly for up to 5 weeks. FL did not induce apoptosis or down-regulation of CD44. These findings show that apoptosis is indeed involved in the regulation of CB CD34+ cells in ex vivo expansion and the patterns of apoptosis are dependent on the type of cytokines used. The distinct patterns of apoptosis suggest different mechanisms of TPO and G-CSF in inducing apoptosis, which still remains to be elucidated.

Bioisosterism: interchange of 4-OH to 4-NH2 in vanillin or homovanillin ring of capsaicinoids.

A series of 4-amino Capsaicin analogs 15, 17 and 19 were prepared to investigate the bioisosteric effect of 4-amino group, and all these compounds exhibited moderate or weak potency from their analgesic test. From our previous results and others, 4-hydroxyl group as well as 3-methoxy substituent could be crucial for high analgesic activity. This biological result also shows that the activity is sensitive to alkyl chain length in hydrophobic region and the phenylacetic amides 19 are more active than the corresponding urea derivatives 17.

4-Substituted-kynurenic acid derivatives: A novel class of NMDA receptor glycine site antagonists.

A series of 4-substituted-kynurenic acid derivatives possessing several different substituents at C4-position which are consisted of both a flexible propyloxy chain and an adjunct several type of carbonyl groups has been synthesized and evaluated for theirin vitro antagonist activity at the glycine site on the NMDA receptor. Of them, N-benzoylthiourea15c and N-phenylthiourea15a were found to have the bestin vitro binding affinity with IC(50) of 3.95 and 6.04 muM, respectively. On the other hand, in compounds12a approximately c and13 the displacement of a thiourea group to an amide or a carbamate caused a significant decrease of thein vitro binding affinity. In the SAR study of the 4-substituted kynurenic acid derivatives, it was realized that the terminal substitution pattern on a flexible C4-propyloxy chain of kynurenic acid nucleus significantly influences on the binding affinity for glycine site; the binding affinity to the NMDA receptor might be increased by the introduction of a suitable electron rich substituent at C4 of kynurenic acid nucleus.

Design and structural requirements of potent peptidomimetic inhibitors of p21ras farnesyltransferase.

Cysteine farnesylation of the ras oncogene product, p21ras, on its carboxyl-terminal CA1A2X box (C = cysteine, A = aliphatic, and X = methionine or serine) is required for its transforming activity. p21ras farnesyltransferase (FTase), the enzyme responsible or this important posttranslational modification can be inhibited by simple CA1A2X peptides. We have synthesized a family of CA1A2X peptidomimetics where the central 2 aliphatic amino acids are replaced by a variety of spacer groups with different shapes and conformational characteristics to investigate the structural requirements of these inhibitors. The biological activities of CA1A2X peptidomimetics, where the dipeptide "A1A2" is replaced by 3- or 4-aminomethylbenzoic acid (AMBA) and 3- or 4-aminobenzoic acid (ABA), are evaluated in a p21ras FTase inhibitory assay. Peptidomimetics Cys-4-ABA-Met and Cys-3-AMBA-Met contain spacers that provide good distance correspondence of the carboxylate and ammonium separation with that of the parent KB p21ras tetrapeptide, Cys-Val-Ile-Met, and are as potent FTase inhibitors (IC50 values of 50 and 100 nM, respectively). In contrast, replacing the central dipeptide with 4-AMBA reduced FTase inhibitory activity by 17-fold whereas replacement by 3-ABA reduces inhibitory activity of the peptidomimetics by 43-fold. Cys-4-ABA-Met (IC50 = 50 nM) is 128 times more potent as a p21ras FTase inhibitor than Cys-3-ABA-Met (IC50 = 6400 nM), yet these two peptidomimetics differ only in the substitution pattern around the phenyl ring. These results coupled with computer modeling studies demonstrate that the interaction between FTase and the peptidomimetics requires precise structural and conformational characteristics; in particular, correct positioning of the Cys and Met must be respected. Furthermore, Cys-3-AMBA-Met and Cys-4-ABA-Met are true inhibitors of p21ras FTase since they are not farnesylated by this enzyme, in contrast to Cys-Val-Ile-Met, which inhibits the enzyme by acting as alternative substrate. Computer modeling studies of the potent FTase inhibitor Cys-4-ABA-Met show that a folded conformation, where the thiol and carboxylate groups are close, is not possible. Therefore a beta-turn conformation that would result in the simultaneous coordination of the Cys-thiol and Met-carboxylate to zinc ion is not important for inhibition of p21ras FTase, as previously suggested.

Potent inhibition of human tumor p21ras farnesyltransferase by A1A2-lacking p21ras CA1A2X peptidomimetics.

The ras oncogene product p21ras requires farnesylation and subsequent plasma membrane association for its transforming activity. This key post-translational modification is catalyzed by p21ras farnesyltransferase, which transfers farnesyl from farnesylpyrophosphate to the cysteine of the CA1A2X carboxyl-terminal tetrapeptide of p21ras. In the present report, we describe potent inhibition of p21ras farnesyltransferase by CA1A2X peptidomimetics containing no peptidic amide bonds. We synthesized a series of CA1A2X analogues where the 2 aliphatic amino acids A1 and A2 were replaced by a hydrophobic spacer, 3-aminomethylbenzoic acid (AMBA). The peptidomimetic Cys-AMBA-Met, inhibits p21ras farnesyltransferase from human colon carcinoma (COLO-205) and Burkitt's lymphoma (Daudi) with IC50 values of 60 and 120 nM, respectively. Cys-AMBA-Met is 3-, 8-, and 9-fold (COLO-205) and 2-, 5-, and 7-fold (Daudi) more potent than the corresponding tetrapeptides of p21KB-ras (CVIM), p21N-ras (CVVM), and p21KA-ras (CIIM), respectively. Replacing methionine at the X position with negatively charged glutamate reduces its ability to inhibit the enzyme, whereas positively charged lysine at this position abolishes the inhibitory character of the peptidomimetic. A hydrophobic moiety at the X position, as in Cys-AMBA-Phe, retains potent inhibitory activity. Leucine in the X position of CA1A2X is a post-translational signal for protein geranylgeranylation rather than farnesylation, and, as expected, Cys-AMBA-Leu does not inhibit the enzyme. Furthermore, CVIM, CVVM, and CIIM are farnesylated by human p21ras farnesyltransferases and inhibit these enzymes by serving as alternative substrates. In contrast, the peptidomimetics described here are true p21ras farnesyltransferase inhibitors since none is farnesylated by this enzyme.