Bio-inspired supramolecular materials by orthogonal self-assembly of hydrogelators and phospholipids.

The orthogonal self-assembly of multiple components is a powerful strategy towards the formation of complex biomimetic architectures, but so far the rules for designing such systems are unclear. Here we show how to identify orthogonal self-assembly at the supramolecular level and describe guidelines to achieve self-sorting in self-assembled mixed systems. By investigating multicomponent self-assembled systems consisting of low molecular weight gelators and phospholipids, both at a molecular and a supramolecular level, we found that orthogonal self-assembly can only take place if the entities assemble via a strong and distinct set of interactions. The resulting supramolecular architectures consist of fibrillar networks that coexist with liposomes and thereby provide additional levels of compartmentalization and enhanced stability as compared to self-assembled systems of gelators or phospholipids alone.

Detection of monosomy 7 and trisomies 8 and 11 in myelodysplastic disorders by interphase fluorescent in situ hybridization. Comparison with acute non-lymphocytic leukemias.

Cells from 50 patients with myelodysplastic syndrome (MDS) and 20 patients with acute non-lymphoblastic leukemia (ANLL) were studied by fluorescent in situ hybridization (FISH) using alphoid biotinylated probes to detect numerical chromosome 7, 8 and 11 aberrations in interphase nuclei. FISH data were compared with cytogenetic results. Both methods were in agreement in 25/50 MDS and 20/20 ANLL cases. Trisomy 11 was found neither by cytogenetic study nor by FISH. In 11 MDS patients the percentage of abnormal cells was higher by FISH than by classical cytogenetic analysis. FISH revealed monosomy 7 which was undetectable by karyotypic study in 5-22% cells from 15 MDS patients. It also allowed the identification of two small markers and a ring chromosome in two MDS cases. FISH hence appears to be useful for the detection of minor abnormal clones and is a convenient complement to conventional cytogenetic analysis in the study of MDS.

Identification of several genes differentially expressed during progression of chronic myelogenous leukemia.

The Bcr-Abl fusion protein plays a crucial role in the initiation and maintenance of chronic myelogenous leukemia (CML). However, additional events are necessary for the transition from the chronic phase to the terminal phase of the disease. To identify genes involved in the disease progression, we constructed a subtractive library from enriched K562 cell mRNA. We obtained 1084 cDNA clones. After a specific hybridization of these clones with a cDNA probe from either chronic phase or K562 cells, 43 clones which present a differential hybridization level have been selected. Among them, several clones corresponded to ribosomal protein genes showing an increased transcription level during the blast crisis. We observed variations in the expression of a cellular adhesion molecule, a laminin-binding protein. An increased transcription level of the MAZ gene has been shown in the terminal phase of the disease. This gene encodes a protein that regulates the transcription of myc.

Abnormalities of the short arm of chromosome 12 in T cell prolymphocytic leukemia.

Abnormalities of the short arm of chromosome 12 are nonrandom events in T cell prolymphocytic leukemia (T-PLL). Fluorescence in situ hybridization (FISH) studies were performed in three patients with T-PLL and one patient with T cell peripheral lymphoma and rearrangement of 12p. Whereas the rearrangements of 12p were different in the four patients, a breakpoint centromeric to the ETV6 gene was present in the three T-PLL patients. In addition, loss of heterozygosity for a chromosomal segment telomeric to ETV6 with loss of the RAD52 locus was also shown by FISH studies. In contrast, the breakpoint was telomeric to ETV6 in the patient with peripheral lymphoma.

Glutathione S transferase theta 1 gene defects in myelodysplastic syndromes and their correlation with karyotype and exposure to potential carcinogens.

Glutathione S transferase theta 1 (GSTT1) is implicated in the detoxification of different substances, including carcinogens. Recently, an increased incidence of GSTT1 null genotype was found in myelodysplastic syndromes (MDS) by comparison with a control population. We analyzed GSTT1 gene by PCR in 174 MDS cases and 100 controls. The incidence of GSTT1 null genotype was 22% in MDS in 19% in controls (P = 0.53). The incidence of GSTT1 null genotype in MDS did not differ according to gender, FAB classification, karyotype and whether MDS were therapy related or 'de novo'. In 86 of the de novo cases, data on previous occupational and environmental exposure to a list of 170 substances were available. In those MDS patients, a significantly lower frequency of GSTT1 null genotype was seen in cases with previous jobs exposed to chemicals, and with previous exposure to mineral dusts and exhaust gases. A lower frequency (but with only borderline significance) was seen in MDS patients who had been coal miners and those who had been exposed to any of the 70 substances analyzed. Overall, GSTT1 null genotype occurred at a similar incidence (19%) in controls and in MDS cases previously exposed to any substance, but tended to be higher in unexposed MDS patients (40%, P = 0.07). Our results do not confirm the higher incidence of GSTT1 null genotype observed in MDS. The lower incidence of GSTT1 null genotype in MDS cases exposed to some compounds previously found associated with MDS is apparently unexpected. However, it could be explained by the fact that GSTT1 enzyme, which has a detoxification role for some compounds, could also have an activating role for other substances, including solvents.

Two additional cases of t dic(9:12) in acute lymphocytic leukemia (ALL): prognosis in ALL with dic(9:12).

We report two occurrences of dic(9;12) in acute lymphoblastic leukemia and review previous cases. Cases of dic(9;12) share common features with cases of 9p and 12p rearrangements, but prognosis seems particularly good in cases of dic(9;12). The persistence of a specific dicentric in stable clones is remarkable and points to unusual centromeric behavior and/or marked selective advantage of the anomaly.

Cytogenetic heterogeneity in t(11;19) acute leukemia: clinical, hematological and cytogenetic analyses of 48 patients--updated published cases and 16 new observations.

We report on 16 cases of t(11;19) acute leukemia and review data of published observations: altogether updated data of 48 patients are analyzed. Four hematological groups could be distinguished: (i) 13 cases of acute lymphoblastic leukemia (ALL) of B lineage, mostly CD19+; (ii) eight cases of biphenotypic leukemia: CD19+ (most often) ALL but with simultaneous or inducible expression of differentiation marker of monocytic lineage. The B lineage and biphenotypic leukemias were predominantly found in female infants; (iii) four cases of T-ALL in children; and (iv) 23 acute non-lymphocytic leukemia (ANLL) cases generally of M4 or M5 subtype, predominantly in males. Cytogenetically, at least two subtypes were observed with possibly an identical breakpoint on 11q23 but discrete breakpoints on 19p: lymphoid, biphenotypic, and most congenital myeloid cases showed a distal breakpoint on 19p13 producing 11q- and 19p+ derivatives, while most older myeloid cases showed 11q+ and 19p- derivatives as a result of a more proximal breakpoint on 19p12 or p13.1. The latter type was clearly detected using R bands but barely visible using Q or G bands while the other translocation was easy to detect with G bands but could be missed with R bands. The white blood cell count is usually high in these t(11;19) acute leukemias and prognosis is poor, except for T-ALL cases.

Does BCR-ABL genomic rearrangement persist in CML patients in complete remission after interferon alpha therapy?

Cytogenetic, interphase fluorescent in situ hybridization (FISH) and RT-PCR methods were used to study minimal residual disease in peripheral blood stem cells collected for autografting in three chronic myeloid leukemia (CML) patients in sustained complete cytogenetic remission after treatment with interferon alpha (IFNalpha). Karyotypic analysis failed to reveal Ph-positive metaphases. FISH detected 9-16% nuclei with a BCR-ABL fusion gene, contrasting with RT-PCR, performed in two cases, which was negative in one case and weakly positive in the other. RT-PCR was also subsequently weakly positive in the third patient. This discrepancy suggests that the BCR-ABL genomic rearrangement persists unexpressed in quiescent cells. These preliminary results, which need to be confirmed in larger series, suggest that monitoring residual disease in CML should be performed both at DNA and RNA levels. Moreover, autografting following IFNalpha therapy should be considered with caution because of the persistence of the BCR-ABL genomic rearrangement in a sizeable proportion of the cells.

Quantitative HOX expression in chromosomally defined subsets of acute myelogenous leukemia.

We used a degenerate RT-PCR screen and subsequent real-time quantitative RT-PCR assays to examine the expression of HOX and TALE-family genes in 34 cases of chromosomally defined AML for which outcome data were available. AMLs with favorable cytogenetic features were associated with low overall HOX gene expression whereas poor prognostic cases had high levels. Characteristically, multiple HOXA family members including HOXA3-HOXA10 were jointly overexpressed in conjunction with HOXB3, HOXB6, MEIS1 and PBX3. Higher levels of expression were also observed in the FAB subtype, AML-M1. Spearmann correlation coefficients indicated that the expression levels for many of these genes were highly inter-related. While we did not detect any significant correlations between HOX expression and complete response rates or age in this limited set of patients, there was a significant correlation between event-free survival and HOXA7 with a trend toward significance for HoxA9, HoxA4 and HoxA5. While patients with elevated HOX expression did worse, there were notable exceptions. Thus, although HOX overexpression and clinical resistance to chemotherapy often coincide, they are not inextricably linked. Our results indicate that quantitative HOX analysis has the potential to add new information to the management of patients with AML, especially where characteristic chromosomal alterations are lacking.

Minimal residual disease (MRD) in remission t(8;21) AML and in vivo differentiation detected by FISH and CD34+ cell sorting.

Many patients with t(8;21) AML have residual positive cells during remission. We previously developed D-FISH probes that detect both derivative chromosomes and the normal alleles. In negative controls, only 2/44,000 (0.0045%) positive signals were observed. To investigate MRD, we examined specimens from 29 patients who had initially obtained CR. In remission patients, 61% had 1-4/2000 positive cells (0.05-0.19%). Higher frequencies were found in two patients in early relapse and in one patient in early remission. However, a negative test did not exclude relapse. Since false positives were negligible and because most t(8;21) AMLs express CD34, we asked whether cell sorting combined with FISH would increase the sensitivity. In one patient, we observed that 80% of CD34+ cells were t(8;21)+ at 2 months from initial clinical and cytogenetic remission. However, by 5 months the pre- and post-sorted populations contained 0.15% and 0.06% t(8;21) cells, respectively. Whereas essentially all t(8;21) cells in the initial specimen expressed CD34, only 0.6% were subsequently CD34+. These results are consistent with in vitro assays showing that residual t(8;21) cells undergo differentiation. Thus, FISH can identify MRD in a majority of t(8;21) patients and, combined with CD34+ selection, may provide an indirect assessment of the differentiation state of residual t(8;21) cells.

Role of p53 and RB on in vitro growth of normal umbilical cord blood cells.

Human umbilical cord blood (UCB) is rich in hematopoietic stem cells and progenitors and recently has been used in the clinic as an alternative source for graft and marrow repopulation. We tried to determine in vitro the roles of wild-type (wt) p53 and wt RB tumor/growth suppressor genes in the regulation of proliferation and maturation of hematopoietic UCB cells. CD34+ cells, isolated from mononuclear cells of UCB, were cultured in semisolid medium under conditions that favor growth of hematopoietic cells. We studied the level of expression of p53 and RB mRNAs and proteins during cell culture by Northern blot and cytofluorometry analysis, respectively. Sense (S), antisense (AS), or scrambled (missense [MS]) p53 and RB oligodeoxynucleotides (ODNs) were used to study the behavior of these cells in the absence of expression of p53 and/or RB. Adequate doses of p53 or RB ODNs inducing maximal inhibitory effect were used to study the behavior of these cells in the absence of expression of p53 and/or RB. Adequate doses of p53 or RB ODNs inducing maximal inhibitory effect with minimal cellular toxicity were determined. Exposure of CD34+ cells to p53 or AS, RB AS, or both p53 and RB AS but not other ODNs (sense or missense) resulted in a significantly increased number of colony-forming units-granulocyte/macrophage (CFU-GM) induced by interleukin-3 (IL-3) and/or granulocyte-macrophage colony-stimulating factor (GM-CSF). The number of erythroid colonies (CFU-E) and burst-forming units (BFU-E) derived from CD34+ cells in the presence of erythropoietin (Epo) was not significantly increased, whereas the number of such colonies was markedly increased in the presence of IL-3 + EPO upon p53 AS and/or RB AS treatment with hypothesis that wt p53 and RB are proliferation suppressor genes that interfere with normal maturation of hematopoietic cells.

Complete variable region deletion in a mu heavy chain disease protein (ROUL). Correlation with light chain secretion.

In a patient affected with chronic lymphocytic leukemia with lymphocyte surface mu and kappa determinants and vacuolated bone marrow plasma cells, the serum contained polymers of a truncated mu chain and normal-sized kappa chains. These light chains were present as monomers and covalent dimers in studies performed under dissociating conditions, but they were linked by non-covalent bridges to a portion of the serum short mu chains. The patient's urine contained a kappa type Bence-Jones protein. Study of a messenger RNA and complementary DNA from blood cells showed the abnormal mu chain to lack the entire variable region, likely due to a direct splicing of the leader peptide exon onto the CH1 exon. The production of light chains, a rare event in heavy chain diseases, appears to correlate with the occurrence of a heavy chain deletion restricted to the variable domain, likely because the non-covalently linked light chains allow these unusual heavy chains to be secreted.

The 8p11 anomaly in "monoblastic" leukaemia.

We report on three cases of monoblastic leukaemia with a chromosomal breakpoint at 8p11. One of our cases exhibited a translocation t(8;16) as has been described in 12 previous cases reported in 1987. The two other cases showed respectively t(6;8) and t(8;19) and they seem to be the first two reports of variant translocation in this disease. The available cases serve to define the main characteristics of this new subtype of non lymphocytic acute leukaemia: phagocytosis in most of the cases, the possible involvement of a granulomonocytic precursor, and a common breakpoint in 8p11.

Chromosome 6p rearrangements appear to be secondary changes in various haematological malignancies.

We report on six cases of 6p rearrangement in various haematological malignancies. On reviewing the literature, we assume 6p rearrangements to be secondary anomalies in both myeloid and lymphoid malignancies, and confirm it to be strongly associated with -5/del (5q) in myelodysplastic syndromes.

Four additional cases of trisomy 14 as the sole anomaly in various haematological malignancies.

We report on four cases of trisomy 14 as the sole anomaly. Three cases were myelodysplastic syndromes and one was a non-Hodgkin's lymphoma. This anomaly is mainly in myeloid disorders and still remains to be well documented. On the other hand, we show this anomaly to be also a non-random anomaly in lymphoproliferative disorders.

Recurrent molecular deletion of the 12p13 region, centromeric to ETV6/TEL, in T-cell prolymphocytic leukemia.

INTRODUCTION: T-cell prolymphocytic leukemia is a rare form of mature leukemia which occurs in adults and in younger patients suffering ataxia telangiectasia. Among others, complex chromosome aberrations of chromosome 12 have been described in this disease. We searched for deletions of the 12p13 region as the result of these chromosome rearrangements. MATERIAL AND METHODS: Paired leukemic and non-leukemic cells were obtained from a series of 21 patients suffering T-cell prolymphocytic leukemia. Loss of heterozygosity was searched for by microsatellite typing using a fluorescent automated laser DNA sequencer to analyze the amplification products. Proteins were analyzed by Western blot. Southern blot analysis of one patient was conducted. RESULTS AND CONCLUSION: Loss of heterozygosity of the 12p13 region, including the ETV6 and CDKN1B genes, was detected in nine of these 21 cases (43%). Western and Southern blot analyses of one case demonstrated a biallelic deletion which did not include ETV6. Taken together, our results defined a minimal region of deletion of less than one Mb flanked by the markers b312C2T7 and D12S320, excluding ETV6 as a candidate gene. Deletion of the 12p13 region is thus a highly recurrent genetic event in T-cell prolymphocytic leukemia.

Allogeneic bone marrow transplantation for hypereosinophilic syndrome with advanced myelofibrosis.

A 33-year-old man with an atypical course of hypereosinophilic syndrome including malignant hypercalcemia, osteolytic lesions and evolution into severe myelofibrosis was treated by allogeneic bone marrow transplantation after conditioning with cytoxan and total body irradiation. As the transplant was sex-mismatched, chimerism was studied by means of cytogenetic analysis and Y chromosomal DNA amplification by PCR assay. Long-term complete remission has been assessed by normalization of blood cell counts, magnetic resonance imaging and karyotypic analysis. A relapse was observed 40 months after transplantation. The patient remains alive 44 months post-BMT. This case report is compared with those reported in the literature.

Molecular remission after allogeneic bone marrow transplantation for chronic lymphocytic leukemia.

A 51-year-old man with previously treated CLL received an allogeneic sex mismatched BMT after total body irradiation and high dose chemotherapy. Residual disease was studied at phenotypic and molecular levels including Y chromosome DNA amplification by PCR assay. The patient was clinically disease-free 20 months after BMT with disappearance of the leukemic clone assessed by the most sensitive methods of detection. Long-term follow-up is necessary to ascertain the relevance of Y DNA amplification in predicting outcome in this patient.

Translocation (8;9)(q12;p21). A new non-random rearrangement in lymphoid malignancies.

We report two cases of t(8;9) with probable breakpoints in 8q12 and 9p21 in malignant lymphoma. In a review of the literature, we found two cases of acute lymphocytic leukemia and one case of malignant lymphoma which probably share the same breakpoints.

11q13 rearrangement in B cell chronic lymphocytic leukemia.

Translocation t(11;14)(q13;q32) and/or 11q13 rearrangements have been reported in various B cell immunoproliferative disorders. They appear to be frequent in mantle cell zone lymphoma (MZL) and rare in B-cell chronic lymphocytic leukemia (B-CLL). Discrimination between MZL and B-CLL is sometimes uncertain on the basis of morphology and immunophenotype. To evaluate the incidence of 11q13 rearrangements in B-CLL, purified B cells from 59 untreated patients were studied by cytogenetic methods after short term stimulated culture. Abnormalities at band 11q13 were found in 2 cases only. Fluorescent in situ hybridization (FISH) study confirmed del(11)(q13) in one case and showed translocation t(11;13) in another one. Thus this rearrangement appears to be very rare in B-CLL and its finding should lead to a careful search for the characteristic features of MZL, namely, morphology, the expression of CD5 without CD23, high density monotypic SIg, together with t(11;14) and/or bcl-1/IgH rearrangement.