Patrizia Scaravaglio

Significant Correlation Between the Degree of WT1 Expression and the International Prognostic Scoring System Score in Patients With Myelodysplastic Syndromes

PURPOSE To determine whether pattern of WT1 gene expression is a useful marker for establishing prognosis and tracking disease progression in patients with myelodysplastic syndromes (MDS). PATIENTS AND METHODS We performed a quantitative assessment of the WT1 transcript amount by real-time quantitative polymerase chain reaction (RQ-PCR) in 173 samples (131 bone marrow samples and 42 peripheral-blood samples) from 131 patients with MDS (79 patients with refractory anemia [RA], 31 with RA with excess blasts [RAEB], 18 with secondary acute myeloid leukemia [s-AML] evolved from MDS, and three with deletion of 5q as the sole cytogenetic abnormality). Values obtained were correlated with the blast percentage and International Prognostic Scoring System (IPSS) score. RESULTS Sixty-five percent of BM and 78% of PB samples for RA and 100% of BM and PB samples of RAEB and s-AML expressed WT1 transcript amounts greater than the level observed in healthy volunteers. The degree of WT1 expression was highly correlated with the type of MDS, was much higher in RAEB and s-AML compared with RA, and increased during disease progression. Moreover, a significant correlation was found between WT1 expression levels, blast cell percentage, and the presence of cytogenetic abnormalities. Therefore, we found a significant correlation between the amount of WT1 transcripts and the IPSS score, which currently represents the most reliable risk index of disease progression available for MDS patients. CONCLUSION WT1 is a useful molecular marker for risk assessment in MDS patients.

A 76-kb duplicon maps close to the BCR gene on chromosome 22 and the ABL gene on chromosome 9: Possible involvement in the genesis of the Philadelphia chromosome translocation

A patient with a typical form of chronic myeloid leukemia was found to carry a large deletion on the derivative chromosome 9q+ and an unusual BCR-ABL transcript characterized by the insertion, between BCR exon 14 and ABL exon 2, of 126 bp derived from a region located on chromosome 9, 1.4 Mb 5′ to ABL. This sequence was contained in the bacterial artificial chromosome RP11-65J3, which in fluorescence in situ hybridization experiments on normal metaphases was found to detect, in addition to the predicted clear signal at 9q34, a faint but distinct signal at 22q11.2, where the BCR gene is located, suggesting the presence of a large region of homology between the two chromosomal regions. Indeed, blast analysis of the RP11-65J3 sequence against the entire human genome revealed the presence of a stretch of homology, about 76 kb long, located approximately 150 kb 3′ to the BCR gene, and containing the 126-bp insertion sequence. Evolutionary studies using fluorescence in situ hybridization identified the region as a duplicon, which transposed from the region orthologous to human 9q34 to chromosome 22 after the divergence of orangutan from the human-chimpanzee-gorilla common ancestor about 14 million years ago. Recent sequence analyses have disclosed an unpredicted extensive segmental duplication of our genome, and the impact of duplicons in triggering genomic disorders is becoming more and more apparent. The discovery of a large duplicon relatively close to the ABL and BCR genes and the finding that the 126-bp insertion is very close to the duplicon at 9q34 open the question of the possible involvement of the duplicon in the formation of the Philadelphia chromosome translocation.

Karyotypic analysis of gastric carcinoma cell lines carrying an amplified c-met oncogene

MKN 45 is a poorly differentiated gastric carcinoma cell line from which the subclone GTL 16 was obtained. Both lines carry an amplification unit derived from chromosome 7 sequences and containing an activated c-met oncogene. Karyotypic analysis showed that GTL 16 derived from a subclone of MKN 45 after endoreduplication. Several clonal abnormalities are evident in both lines; some are frequently observed in gastrointestinal tumors (loss of 17p and monosomy 18). Other consistent anomalies include 6q-, t(8;10) and t(5;8), and inv(16). A marker chromosome (M1), which was previously shown to contain the c-met amplification unit, is constantly duplicated in all GTL 16 metaphases; in contrast, most unidentified markers are retained in only a single copy in GTL 16 cells. These data are in agreement with the hypothesis that the c-met oncogene activation in these gastric cancer cell lines might be related to a gene dosage effect.

Myelodysplastic disorders carrying both isolated del(5q) and JAK2(V617F) mutation: concise review, with focus on lenalidomide therapy.

The concomitant presence of del(5q) and JAK2V617F mutation is an infrequent event which occurs in rare patients with peculiar cytogenetic, molecular, morphological and clinical features, resembling those of both myelodysplastic syndromes and myeloproliferative neoplasms. Lenalidomide may induce rapid, profound, and long-lasting responses in a subset of these patients. However, the mechanism(s) by which the drug acts in these conditions remain not completely elucidated. A new case report and a review of all cases published so far in this setting are provided. Furthermore, the possibility of categorizing – from a clinical, pathological, and biological point of view – for at least some of these patients as a potential distinct entity is discussed.

From Genes to Therapy: The Case of Philadelphia Chromosome-Positive Leukemias

Abstract: The Philadelphia chromosome (Ph‐chromosome) has long represented the only cytogenetic abnormality known to be associated with a specific malignant disease in humans, being present in more than 95% of patients with chronic myelogenous leukemia. This abnormality is the result of a reciprocal translocation between the long arms of chromosome 9 and 22, t(9;22)(q34;q11), and its presence is not restricted to chronic myelogenous leukemia, but can also be found in 30% of cases of acute lymphoblastic leukemia in adults. In the 1980s, the molecular counterpart of the chromosomal rearrangement was identified to consist of the juxtaposition of parts of the BCR and ABL genes to form a BCR‐ABL hybrid gene. The resulting chimeric proteins (P210 and P190), which retain constitutively activated tyrosine kinase activity, have demonstrated a causative role in the genesis of the leukemic process. Although many aspects of the BCR‐ABL driven transformation remain unsolved, great advances in understanding the molecular pathology of Ph‐positive leukemias resulted in meaningful improvement in the clinical setting. Molecular tools to diagnose disease (PCR, FISH, and southern blot) and to monitor minimal residual disease after potential curative treatment are now in current practice, and new powerful therapeutic tools have emerged that target the molecular oncogenic pathways activated in Ph‐positive cells. Among them, specific ABL tyrosine kinase inhibitors recently obtained extraordinary results in many clinical protocols. This review summarizes the most recent advances in this field with special focus on the putative mechanisms of the transformation and progression of chronic myelogenous leukemia and on the major impact that understanding the molecular biology of these diseases is having in clinical practice.

Detection of minimal residual disease in peripheral blood stem cells from two acute myeloid leukemia patients with trisomy 8 predicts early relapse after autologous bone marrow transplantation

We report two cases of acute myeloid leukemia (AML) French-American-British M4 classification with trisomy 8 at diagnosis as the sole chromosome abnormality. Both patients were treated with the GIMEMA AML-10 protocol and underwent autologous bone marrow transplantation (ABMT) in hematologic remission. Peripheral blood stem cells (PBSC), and bone marrow in one patient, were collected after consolidation therapy and tested by fluorescence in situ hybridization (FISH) analysis with an alpha-satellite probe for chromosome 8. It revealed that all samples were positive for minimal residual disease (MRD) as the value of trisomic cells exceeded the mean +3 standard deviations of the controls. ABMT was done following a myeloablative regimen (busulphan/cyclophosphamide) and PBSC were reinfused. Both patients relapsed, 4 and 2 months, respectively, after autotransplant. Although more data are needed, these results suggest that the persistence of MRD, as detected by FISH, in stem cell collections, is associated with a poor outcome in AML patients with trisomy 8 undergoing ABMT.

Trisomy 8 and an unbalanced t(5;17)(q11;p11) characterize two karyotypically independent clones in a case of idiopathic myelofibrosis evolving to acute nonlymphoid leukemia

In a patient with idiopathic myelofibrosis (MFI) that had progressed to acute nonlymphoid leukemia (ANLL) after a long-lasting cytotoxic treatment, we observed two karyotypically independent cell populations, one showing trisomy of chromosome 8 as the only anomaly and one with an unbalanced translocation t(5;17)(q11) resulting in partial monosomy of 5q and 17p. The overall karyotypic configuration suggested that chromosome changes occurred as secondary events during the multistep process of leukemogenesis. The probable sequence of cytogenetic events in this patient and a review of the literature indicated that the t(5;17) may represent a therapy-induced abnormality nonrandomly related to the terminal phase of myeloid disorders.

Translocation t(11;21)(q24;q11.2) is a new nonrandomly occurring chromosome change in myelodysplastic syndromes

An identical translocation, t(11;21)(q24;q11.2), has been observed in three patients with a myelodysplastic syndrome. In all cases, duplication of the 11q+ marker and loss of the normal chromosome 11 were observed either at diagnosis or during the evolution of the disease. This apparently characteristic chromosome abnormality has not been previously described.

Effect of recombinant human IL-3 on the mitotic index and karyotype of hemopoietic cells

The proliferative induction by hemopoietic growth factors may provide a useful tool to improve the mitotic yield of hemopoietic cells, allowing a more accurate cytogenetic analysis in hematologic malignancies. For such a purpose, we studied the effects of the recombinant human IL-3 (rhIL-3) on the mitotic index and the karyotype of bone marrow cells from 14 patients with myelodysplastic (MDS) and myeloproliferative syndromes (MPS). The mitotic response to IL-3 of normal bone marrow samples was also evaluated. Total bone marrow cells were cultured for 24 to 72 hours either in presence or absence of rhIL-3. In most cases, IL-3--stimulated samples showed a considerably higher (4-70 times) mitotic index than unstimulated controls. Although a great patient-to-patient variability was observed, a common pattern of mitogenic response to IL-3 emerged among MPS, MDS, and normal cases. At 48 hours of incubation, the mean mitotic index from MPS and MDS cases stimulated with IL-3 was significantly higher (p less than 0.01) than unstimulated controls, whereas the mean mitotic increase from normal samples did not reach statistical significance (p greater than 0.1). Even though not statistically evaluable, a similar trend of response was observed at 24 and 72 hours of culture. Chromosome studies of MPS and MDS cases showed the same karyotype either in stimulated and unstimulated samples.

Chromosome-abnormalities Involving Heterochromatic Regions in Monocytic Leukemia

We report two cases of monocytic leukemia associated with cytogenetic changes involving the juxtacentromeric heterochromatin of different chromosomes. In a patient with chronic myelomonocytic leukemia (CMMoL) we describe a translocation t(1;9)(q12;q13) in which the duplicated derivative chromosome 9q + showed a huge centromeric C-band, derived by fusion of the heterochromatic regions of chromosomes 1 and 9. The constitutional karyotype showed two heterochromatin polymorphisms, 1qh + and inv(9qh). In the second case, an acute monoblastic leukemia was associated with an abnormally elongated juxtacentromeric heterochromatic region of chromosome 4 that was not constitutionally present.