Amedea Donelli

Autologous hematopoietic stem cell transplantation in multiple sclerosis A phase II trial

Objective: To assess in multiple sclerosis (MS) the effect of intense immunosuppression followed by autologous hematopoietic stem cells transplantation (AHSCT) vs mitoxantrone (MTX) on disease activity measured by MRI. Methods: We conducted a multicenter, phase II, randomized trial including patients with secondary progressive or relapsing-remitting MS, with a documented increase in the last year on the Expanded Disability Status Scale, in spite of conventional therapy, and presence of one or more gadolinium-enhancing (Gd+) areas. Patients were randomized to receive intense immunosuppression (mobilization with cyclophosphamide and filgrastim, conditioning with carmustine, cytosine-arabinoside, etoposide, melphalan, and anti-thymocyte globulin) followed by AHSCT or MTX 20 mg every month for 6 months. The primary endpoint was the cumulative number of new T2 lesions in the 4 years following randomization. Secondary endpoints were the cumulative number of Gd+ lesions, relapse rate, and disability progression. Safety and tolerability were also assessed. Twenty-one patients were randomized and 17 had postbaseline evaluable MRI scans. Results: AHSCT reduced by 79% the number of new T2 lesions as compared to MTX (rate ratio 0.21, p = 0.00016). It also reduced Gd+ lesions as well as the annualized relapse rate. No difference was found in the progression of disability. Conclusion: Intense immunosuppression followed by AHSCT is significantly superior to MTX in reducing MRI activity in severe cases of MS. These results strongly support further phase III studies with primary clinical endpoints. The study was registered as EUDRACT No. 2007-000064-24.

Study of the levels of expression of two oncogenes, c-myc and c-myb, in acute and chronic leukemias of both lymphoid and myeloid lineage

Total cellular RNA from a series of leukemic cell populations, both myeloid and lymphoid, as well as from normal circulating lymphocytes was analysed for the expression of two cellular oncogenes, c-myc and c-myb, by Northern blot hybridization assay. Expression of c-myc but not of c-myb was observed in unstimulated normal lymphocytes. Stimulation by PHA was shown to activate the expression of both genes. Remarkably different levels of expression of c-myc were observed in ALL, whereas in CLL the expression of c-myc was uniformly low or absent. Differential expression of c-myc was detected in AML as well as in CML, c-myb was differentially expressed in AML and ALL, and absent in CLL and CML. Other single cases of hemopoietic disorders were studied, but the expression of the two oncogenes was low or absent. Neither evident genome amplification nor genome rearrangements were detected in the cell DNAs digested with restriction endonucleases.

Cellular levels of mRNA from c‐myc, c‐myb and c‐fes onc‐genes in normal myeloid anderythroid precursors of human bone marrow: an in situ hybridization study

Summary. The expression of three onc‐genes, c‐myc, c‐myb and c‐fes, has been evaluated at the cellular level in myeloid and erythroid precursors of normal human bone marrow, by ‘in situ’ hybridization with tritium‐labelled probes. A relatively large amount of m‐RNA from the three onc‐genes was detected in myeloblasts and promyelocytes, but whereas the expression of c‐myb and c‐myb decreased in more advanced stage of maturation of the myeloid lineage, c‐fes mRNA remained at a relatively high level until the granulocyte stage, c‐myc and c‐myb were expressed at a fairly high level in basophilic erythroblasts, which also showed low levels of c‐fes mRNA. No expression of these onc‐genes was detectable in more mature erythroblasts, Megakaryocytes showed high levels of m‐RNA from all three onc‐genes.

Expression of human c‐fes one‐gene occurs at detectable levels in myeloid but not in lymphoid cell populations

Summary. Total cellular RNA from a variety of myeloid and lymphoid cell populations, normal and leukaemic, was analysed for the expression of a human cellular one‐gene, c‐fes, by northern blot hybridization assays. The probe used was a molecularly cloned human DNA sequence homologous to the 5′ terminal sequence of v‐fes. All the myeloid cellular populations expressed the c‐fes gene. In some cell populations at an advanced stage of differentiation (circulating leucocytes from Chronic myeloid leukaemia (CML), HL60 cells induced to differentiate by retinoic acid) the level of expression was even higher than in early stages of the myeloid lineage (blast cells from AML, uninduced HL60 cells). No transcript of the c‐fes gene was detected in the different lymphoid populations studied. The occurrence of an RNA complementary to the c‐fes sequence appears sufficiently characteristic of a myeloid population to distinguish it from a lymphoid population, normal and leukaemic.

High-dose therapy and autologous stem cell transplantation versus conventional therapy for patients with advanced Hodgkin’s lymphoma responding to front-line therapy: long-term results

The inclusion of high-dose therapy/autologous stem cell transplantation (HDT/ASCT) in the initial treatment plan for patients with unfavorable Hodgkin’s lymphoma (HL) has been a matter of debate for the last two decades. In 1991, Carella et al .[1][1] published a pilot study of HDT and ASCT in

T‐cell receptor genes expression in B‐cell leukaemias

Summary. Using Northern‐blot analysis we have studied the expression of T‐cell receptor (TCR) α, β and γ chain genes in primary cells obtained from 36 leukaemic patients. Fifteen patients had myeloid leukaemias, two had T‐cell leukaemias, and 19 leukaemias corresponding to various stages of B‐lymphocyte differentiation. We observed that truncated TCR β mRNAs were produced in B‐cells at relatively high levels even in the absence of detectable gene rearrangements. Ti α mRNAs of abnormal size were also frequently found. Such transcripts were not detectable in total RNA extracted from leukaemic myeloid cells. Factors allowing Ti α and β gene transcription must be active in leukaemic pre‐B and B cells but not in myeloid cells. Neither B‐lineage nor myeloid leukaemias expressed TCR γ gene at detectable levels.

Differential patterns of expression of cell cycle-related genes in blast cells of acute myeloid leukemia

The expression of two G1-specific clones, p2A9 and p4F1, of two cell cycle-related oncogenes, c-myc and c-myb and of one S phase-specific gene, the H3 histone gene, was explored in 11 cases of acute myeloid leukemia. Both Northern blot analysis and in-situ hybridization were employed. Differential patterns of gene expression were observed. In 6 out of 11 cases a considerable or high expression of the p2A9 and p4F1 clones and of c-myc and c-myb oncogenes was observed. In 2 cases a high expression of c-myc was matched by low or absent expression of the other genes examined. In 3 cases the expression of 2A9, 4F1, c-myc and c-myb was very low or undetectable. In two of these cases a considerable expression of the H3 histone gene was observed.

Occurence of a novel t(11;19)(q13;q13.3) in complete remission of acute promyelocytic leukemia

Abstract A woman with t(15;17) and PMLIRARa positive acute promyelocytic leukemia (APL-M3v) achieved a complete remission (CR) with cytogenetic and molecular conversion, after one-month ATRA plus idarubicin treatment. During CR, less than one-month after consolidation therapy with topoisomerase II inhibitors, a novel t(11;19) (q13;q13.3) was detected in peripheral blood stem cells and later in harvest bone marrow cells. Persisting CR and the negativity for BCL1 and PRAD1 genes rearrangement, the autotransplantation was performed, with good outcome. The patient is still in CR eighteen months post-transplant, in spite of the persistence of a small t(11;19) clone in BM cells. The emergence of a novel chromosomal change during CR of acute leukemia is a rare phenomenon. This is the first t(11;19)(q13;q13.3) described in APL. This finding raises the issue of whether the abnormal karyotypes at remission might represent a risk of tumor recurrence. The meaning of this genomic instability is yet unknown.

Overexpression of the MPO gene occurring in a case of APL without unusual genotypic characteristics

Northern blot analysis of four typical cases of acute promyelocytic leukemia showed that one of the cell population examined was characterized by a very high level of expression of the myeloperoxidase (MPO) gene. Western blot analysis confirms that the protein content of the cells corresponded to the levels of the MPO mRNA. Southern blot studies of the DNA of this cell population ruled out the presence of any genome amplification or rearrangement. Chromosome hybridization studies in situ confirmed that the MPO gene was translocated on the long arm of chromosome 15. The observation that a typical genomic pattern may or may not be associated with the MPO overexpression leads us to believe that so far it is impossible to reach any conclusion about the significance of the translocation in the genesis of MPO overexpression.

Kappa light chain gene rearrangement in a T-cell lymphoma

Forty-three patients were studied to determine whether light chain gene rearrangements may occur in hematopoietic cells not pertaining to the B-lineage. In only one patient, affected by T-cell lymphoblastic lymphoma, one kappa light chain allele was rearranged. Neither at the protein level nor at the RNA level the rearranged gene was expressed. These data confirm that, although rarely, kappa light chain gene rearrangements may occur in neoplastic T-cells. Furthermore, as in our patient Ig heavy chain genes retained a germline configuration, the present data demonstrate that kappa light chain gene rearrangements may occur regardless of Ig heavy chain gene arrangement.