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To better define the incidence and significance of cryptic chromosome lesions in acute myeloid leukemia (AML), fluorescence in situ hybridization (FISH) studies were performed in interphase cells and, when appropriate, in metaphase cells and in morphologically intact BM smears. Fifty-five adult de novo AML (group A) and 27 elderly AML or AML after myelodysplastic syndrome (AML-MDS) (group B) were tested using probes detecting the following anomalies: -5, -7, +8, deletions of 5q31, 7q31, 12p13/ETV6, 17p13/p53, 20ql1. All the patients had a normal karyotype in more than 20 cells and tested negative for the common AML-associated fusion genes. No patient in group A was found to carry occult chromosome anomalies, whereas 8/27 patients in group B (P < 0.0001) showed 5q31 or 7q31 deletion (three cases each), a 17p13/p53 deletion or trisomy 8 (one case each) in 33-60% interphase cells. Metaphase cells showed only one hybridization signal at 5q31 (three cases) and 7q31 (one case), whereas two normal signals at 7q31 and chromosome 8 centromeres were seen in two patients with 7q deletion and trisomy 8 in interphase cells. The majority of blast cells (76-94%) carried the chromosome anomaly in all cases; erythroid involvement in a minority of cells was seen in three patients. In group B, the presence of occult chromosome anomalies was associated with exposure to myelotoxic agents in the workplace (5/8 cases vs 3/19, P = 0.026) and with a lower complete remission rate (0/6 patients vs 7/12, P = 0.024). We arrived at the following conclusions: (1) cryptic chromosome deletions in the order of a few hundred kb magnitude may be found in a fraction of elderly AML or MDS-related AML and not in de novo adult AML with normal karyotype; (2) these chromosome lesions are usually represented by submicroscopic rearrangements; (3) they display a specific pattern of cell-lineage involvement arguing in favor of their role in the outgrowth of the leukemic blast cells; (4) they are associated... [ABSTRACT FROM AUTHOR]

We have studied the effects of naloxone benzoylhydrazone (NalBzoH) at recombinant human OP[sub4] receptors expressed in Chinese hamster ovary (CHO) cells (CHO[subhOP4] ) and native OP[sub4] sites in isolated tissues from various species. In CHO[subhOP4] membranes, nociceptin (NC) and NalBzoH displaced [[sup125]I]Tyr[sup14] -NC with pK[subi] values of 10.1 and 7.3. In the presence of 100 μM GDP, NC stimulated GTPγ[sup35] S binding (pEC[sub50] = 8.5). NalBzoH was ineffective but antagonized the effects of NC (pA[sub2] = 6.9). At 5 μM GDP, there was an increase in potency (pEC[sub50] = 9.3) and efficacy (4.3-fold) of NC. NalBzOH was a partial agonist (pEC[sub50] = 7.0, E[submax] = 13% relative to NC). In CHO[subhOP4] cells, NC and NalBzoH inhibited cAMP formation with pEC[sub50] and E[submax] values of 9.8 and 100% and 6.0 and 44%, respectively. In the rat vas deferens, NalBzoH (10 μM) did not modify electrically induced twitches but competitively antagonized the inhibitory action of NC (pA[sub2] = 6.2). In the mouse vas deferens (mVD) and guinea pig ileum (gpI), NalBzoH inhibited twitches with pEC[sub50] and E[submax] values of 7.6 and 78% and 8.5 and 77%, respectively. The effect of 3 μM NalBzoH was fully inhibited by 3 μM naloxone in mVD and 30 μM in gpI. Under these conditions, NalBzoH antagonized the actions of NC in both preparations with pA[sub2] values of 6.3 and 6.8, respectively. Collectively, these data demonstrate that NalBzoH is a nonselective OP[sub4] ligand with system-dependent behaviour. [ABSTRACT FROM AUTHOR]

At diagnosis, approximately half of myelodysplastic (MDS) patients presents a normal karyotype by conventional cytogenetic analysis (CCA). Fluorescent in situ hybridization (FISH) is more sensitive than CCA allowing for the detection of minor clones and of submicroscopic lesions. We have analyzed by FISH 101 MDS patients with normal karyotype for the occurrence of the abnormalities which are most frequently observed in MDS (ie -5/5q-, -7/7q-, +8, 17p-). In 18 patients, 15 to 32% of interphase cells were found to carry one FISH abnormality. Six patients presented trisomy 8, five had del(5)(q31), five del(7)(q31), one monosomy 7 and one del(17)(p13). FISH abnormalities were more frequently observed among patients with an increased percentage of bone marrow blasts (P = 0.001). FISH abnormalities were also associated with a higher rate of progression into AML (13/18 vs 12/83, P < 0.001) and were predictive for a worse prognosis (P < 0.001). Multivariate analysis indicated that FISH positivity and IPSS risk group were independent predictors for a poor survival (P = 0.0057 and 0.0123, respectively) and for leukemic transformation (P = 0.0006 and 0.035, respectively). Leukemic transformation in FISH-positive patients was associated in all cases with an expansion of the abnormal clone. Our data demonstrated that a significant proportion of MDS patients with normal karyotype presented, if analyzed by FISH, clones of cytogenetically abnormal cells which played a determinant role in the progression of the disease. The presence of FISH abnormalities identified a group of MDS patients with normal karyotype characterized by an inferior prognosis. [ABSTRACT FROM AUTHOR]

A diagnosis of pro-B acute lymphoblastic leukemia (ALL) with CD15+ was made in a 42-year-old woman, 12 months after the treatment of uterine adenocarcinoma by carboplatinum, anthracyclines, etoposide and radiotherapy. Molecular cytogenetic studies revealed a karyotype with multiple chromosome changes, including the t(4;11)(q21;q23) and a 17p-chromosome, with MLL disruption and 17p13/p53 gene deletion in 86% of the cells. A p53 exon 6 mutation was documented, resulting in p53 protein stabilization, with 20% of the cells reacting with the 1801 anti-p53 monoclonal antibody. Dual-color FISH using MLL and p53 probes was performed on peripheral blood smears, providing direct evidence of the involvement of the blast cells and of the granulocytic lineage. Only a partial, shortlasting response was obtained by induction treatment, confirming that a poor prognosis is associated with therapy-related ALL with the 4;11 translocation. [ABSTRACT FROM AUTHOR]

To define better the chromosomal profile of atypical chronic lymphocytic leukemia (aCLL), cytogenetic and interphase cytogenetic studies were performed in 43 cases, using mitogen-stimulated cultures and DNA probes detecting the two most frequently occurring aberrations in CLL, ie +12 and 13q14 deletions. All cases showed monoclonal CD5/CD19-positive lymphocytosis, with more than 10% large lymphocytes and/or prolymphocytes in peripheral blood smears and reactivity with FMC7, or bright expression of surface immunoglobulins in a fraction of the cases. Karyotype aberrations were detected in 27 of 43 cases (62.8%). Recurrent chromosome changes were +12 (nine cases), 13q14 aberrations (five cases), 11q anomalies (three cases), 6q21-q23 abnormalities and 4q anomalies with different breakpoints (two cases each). Additional chromosome changes were seen in four cases with +12, in three cases with 13q14 anomalies, in two cases with 11q anomalies, in one case with 6q and 4q anomalies. Trisomy 12 was associated with 13q14 anomalies in three cases, one of which also had an 11q abnormality; other associations, found in one case each, were: 13q14 deletion with a 6q anomaly, 11q anomaly with 13q- and 7q-, a 6q anomaly with 7q- and +12. Interphase cytogenetics confirmed the results of chromosome banding analysis and showed that six patients with normal karyotype or no mitosis in fact had concomitant +12 and 13q14 deletion in four cases and isolated +12 or 13q14 deletion in one case each, with a resultant 76% overall incidence of cytogenetic abnormalities. The presence of +12, 13q14 deletions, 11q, and 6q21-q23 anomalies in 19 cases was associated with a 2-month median interval between diagnosis and start of treatment, as compared with a 24-month median interval in 14 cases with normal karyotype or non-recurrent chromosome changes (P = 0.003). We conclude that aCLL is characterized by a relatively high incidence of chromosome anomalies, with recurrent chromosome changes, involving chromosomes 12, 13q14, 6q21q23, 11q, and, possibly, 4q. The presence of complex karyotypes, with concomitant abnormalities of 13q, +12, 6q, 11q, suggests that the development of sequential chromosome changes, rather than any single specific anomaly, may underlie leukemogenesis in this cytologic subset of CLL, partially accounting for the relatively aggressive clinical course. [ABSTRACT FROM AUTHOR]

Cytogenetic and fluorescence in situ hybridization studies were successfully performed in 217 chronic lymphocytic leukemia (CLL). In all, 13 patients with 6q21 deletion were identified and characterized in comparison with 92 patients with ‘favourable’ karyotype (normal or 13q-), 69 cases with ‘intermediate risk’ (1-2 anomalies) and 43 cases with ‘unfavourable’ karyotype (complex, 11q- or 17p-). Six out of 13 cases with 6q- showed an excess of atypical lymphocytes, a finding confirmed at the histologic level; >20% CD38+ cells were seen in 5/6 cases. IGVH mutational status revealed >98% homology to the germline sequence in 4/10 cases. When compared with the ‘favourable’ group, patients with 6q- showed a higher white blood cell (WBC) count, frequent splenomegaly, atypical morphology, CD38+ and short time from diagnosis to first treatment and short survival. A higher median WBC count was found in the 6q- group vs the intermediate-risk group; survival was shorter in the unfavourable group. To ascertain if the 6q- anomaly was an independent factor predicting for an inferior outcome among those patients with ‘favourable’ cytogenetics, we performed an analysis of prognostic factors in 105 patients (92 ‘favourable’ plus 13 with 6q-), showing that the 6q- chromosome maintained its prognostic significance at multivariate analysis (P=0.02) along with stage (P=0.01). We conclude that CLL with 6q- is characterized by a high incidence of atypical morphology, classical immunophenotype with CD38 positivity and intermediate incidence of IGVH somatic hypermutation. Clinicobiological features and outcome show that this cytogenetic subset of CLL should be allocated in an intermediate-risk category.Leukemia (2004) 18, 476-483. doi:10.1038/sj.leu.2403242 Published online 18 December 2003 [ABSTRACT FROM AUTHOR]

A series of analogs of the ORL1 receptor antagonist [Nphe¹]-NC(1-13)-NH&sub2; was prepared and tested for agonistic and antagonistic activities in the mouse vas deferens, a preparation that shows high sensitivity to nociceptin and related peptides. The purpose of this study was to determine the role of the aromatic residue at the N-terminal for antagonism and eventually identify compounds with improved potency. Results indicated that all 23 compounds are inactive as agonists, and the antagonistic potency of the initial template [Nphe¹]-NC(1-13)-NH&sub2; is high (pΚ&subB; 6.43) compared with those of all other compounds except [(S)(βMe)Nphe¹]NC(1-13)-NH&sub2; (pΚ&subB; 6.48). The other 22 compounds can be divided into two groups: 10 show antagonistic potencies (pΚ&subB; ) ranging from 5.30 to 5.86, whereas the other 12 compounds are inactive. This study clearly shows that the aromatic ring of Nphe is very critical for the interaction with the ORL1 receptor and can not be enlarged or sterically modified without significant loss of antagonistic potency. [ABSTRACT FROM AUTHOR]

A series of analogs of the ORL1 receptor antagonist [Nphe&sub1;]-NC(1-13)-NH&sub2; was prepared and tested for agonistic and antagonistic activities in the mouse vas deferens, a preparation that shows high sensitivity to nociceptin and related peptides. The purpose of this study was to determine the role of the aromatic residue at the N-terminal for antagonism and eventually identify compounds with improved potency. Results indicated that all 23 compounds are inactive as agonists, and the antagonistic potency of the initial template [Nphe¹]-NC(1-13)-NH&sub2; is high (pKB 6.43) compared with those of all other compounds except [(S)(bMe)Nphe 1 ]NC(1-13)-NH2 (pKB 6.48). The other 22 compounds can be divided into two groups: 10 show antagonistic potencies (pKB ) ranging from 5.30 to 5.86, whereas the other 12 compounds are inactive. This study clearly shows that the aromatic ring of Nphe is very critical for the interaction with the ORL1 receptor and can not be enlarged or sterically modifed without significant loss of antagonistic potency. [ABSTRACT FROM AUTHOR]

A novel recurrent translocation t(11;14)(p11;q32) was found in three patients with splenic marginal zone B cell lymphoma (MZBCL). Fluorescence in situ hybridization (FISH) studies with IgH probes revealed in all cases involvement of the IgH locus, with breakpoint downstream of the IGVH sequences. Partner genes at 11p11 were not identified. The translocation defined the stem line in two patients, who carried additional cytogenetic aberrations, including a 17p deletion, present in both cases. In one patient a 7q- chromosome was the primary cytogenetic defect, the t(11;14) having been found in four out of 11 abnormal metaphase cells at the time of transformation into high-grade MZBCL. Hematological features in all cases included splenomegaly with peripheral blood (PB) involvement by a monoclonal B cell population consisting of lymphocytes with villous projections and several blast-like cells. The immunophenotype was CD19+; CD22bright+; CD23-, CD10-, CD5-, surface Igbright+. A bone biopsy in one patient revealed an interstitial infiltration with an intrasinusoidal pattern of growth. Histological studies on spleen specimens in two patients showed an expanded marginal zone, with small lymphocytes and several blast-like cells. One patient had a therapy-demanding disease, with partial, short-term responses to cytotoxic treatment; one patient transformed into a high-grade MZBCL involving the gut, the PB and the bone marrow 2 years after diagnosis; one patient was unresponsive to cytotoxic treatment and underwent splenectomy. The t(11;14)(p11;q32) may define a subset of splenic MZBCL with a high-grade component and a relatively aggressive clinical behavior. [ABSTRACT FROM AUTHOR]

In order to analyze the efficiency of interphase FISH for the detection and monitoring of Ph+ cells in chronic myelogenous leukemia (CML) under interferon (IFN) treatment, the following experiments were performed: (1) 98 specimens derived from 32 patients were analyzed in parallel by dual-color FISH and by conventional chromosome analysis (CCA). A 300/200 kb BCR/ABL probe was used in all tests and a smaller 35.5/39 kb probe was tested in parallel in 22 BM samples; (2) 30 BM samples were prepared by direct harvest and by 24-h culture and were analyzed in parallel; (3) PB and BM samples obtained simultaneously from 11 patients were analyzed. The cut-off point for the recognition of BCR/ABL fusion was set at 2.4%, calculated as the mean percent of false positivity in 11 controls plus 3 s.d. A very close correlation was observed (r = 0.994, r2 = 0.988, P < 0.0001) between the percentages of ph+ cells as assessed by CCA and by interphase FISH in 98 samples (26 at diagnosis). There was a moderate overestimation of the frequency of Ph+ cells by FISH with respect to CCA, that was more evident at low-to-medium values of Ph positivity. Seven specimens without Ph+ metaphases (17–50 cells analyzed) were shown to carry 2.5–8% interphase cells with BCR/ABL fusion. Similar percentages of BCR/ABL+ nuclei were recorded in 22 samples hybridized using the 300/200 kb and the 35.5/39 kb probe-sets (variation range: 0–5%, mean 2.3%). A very good correlation between the frequency of Ph+ interphase cells was observed when analyzing in parallel BM preparations after direct harvest and after 24-h culture. Underestimation of the percentage of BCR/ABL+ cells was noted to occur in 2/11 PB samples, compared to BM samples, the remaining nine cases showing superimposable results at either sites. We arrived at the following conclusions: (1) dual-color FISH enables an accurate detection and monitoring of the size of the Ph-positive clone in CML at diagnosis and after IFN-therapy; (2) FISH is more accurate than CCA, especially at low levels of Ph-positive cells; (3) testing of directly harvested BM samples is feasible and accurate, giving the opportunity to perform centralized FISH analysis in the context of multicentre trials; (4) the percentage of BCR/ABL+ PB cells usually, though not invariably, reflects the frequency of mutated cells in the BM. [ABSTRACT FROM AUTHOR]