Carla Consoli

Clonal selection of 11q CN-LOH and CBL gene mutation in a serially studied patient during MDS progression to AML

By conventional metaphase and SNP array cytogenetics we serially studied a patient affected by high-risk myelodysplastic syndrome (MDS), documenting the conversion from partial trisomy 8q to trisomy 8 and partial tetrasomy 8q during progression to acute myeloid leukemia (AML). Moreover, the serial application of high resolution genomic array analysis at different disease stages allowed the description of cryptic abnormalities and the demonstration of their enrichment in the AML phase. In particular the detection and quantification of a copy-neutral loss of heterozygosity region located in chromosome 11q guided the search for point mutations in the CBL gene, thus allowing the escription of the novel missense mutation K382E and the demonstration of its selection during progression to secondary AML.

Influence of complex variant chromosomal translocations in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors

Abstract Cytogenetic variants of the Philadelphia (Ph) chromosome can be observed in 5–8% of patients diagnosed with Chronic Myelogenous Leukemia (CML), and usually involve at least one chromosome other than 9 and 22. Despite the genetically heterogeneous nature of these alterations, available data indicate that CML patients displaying complex variant translocations (CVTs) do not exhibit a less favorable outcome as compared to individuals presenting conventional Ph-positive CML. Patients and methods. We report our experience with 10 CML patients carrying CVTs among 153 newly diagnosed cases followed at our Institution. Results and discussion. Unlike previously published reports, in our series only two CML patients exhibiting CVTs achieved an optimal response to tyrosine kinase inhibitors (TKI) treatment. The remaining eight patients obtained either a suboptimal response or failed drug therapy. Our data suggest that the presence of CVTs at diagnosis might confer an unfavorable clinical outcome, as these genetic alterations might be markers of genomic instability and indicate a higher likelihood of disease progression.

Broad copy neutral‐loss of heterozygosity regions and rare recurring copy number abnormalities in normal karyotype‐acute myeloid leukemia genomes

We analyzed, by the latest high‐resolution SNP arrays, 19 Normal Karyotype (NK)‐AML patients at diagnosis (Dx) and remission (R) phases, to determine the number of tumor‐associated copy number abnormalities (CNAs) and copy neutral‐loss of heterozygosity (CN‐LOH) regions per patient and to identify possible recurring genomic abnormalities. The number of tumor‐associated CNAs was determined after comparison of matched Dx/R samples using stringent conditions able to reduce the number of false positive CNAs. With the exception of a single outlier case, a low number of CNAs per patient was detected (median value of 1 somatic loss or gain per patient). However, a high prevalence of CNAs (60–70% of the patients showed at least one tumor‐associated gain or loss) and few recurring CNAs were observed, thus providing new hints towards identification of cooperating mutations. An extensive search of all tumor‐associated CN‐LOH regions >1 Mb revealed only three broad regions (terminal 12Mb of 22q, terminal 27Mb of 1p and the whole chromosome 21) in three patients out of 19 (16%). CN‐LOH of the whole chromosome 21 was responsible for homozygosity of a missense mutation (R80C) of RUNX1/AML1. Our study suggests that a relative submicroscopic copy number stability NK‐AML genomes is associated with low recurrence of specific CNAs and CN‐LOH in NK‐AML patient population. Sequencing of candidate genes in the identified CNAs and CN‐LOH regions should be considered a priority in the search of novel driver mutations of AML.

SPARC expression in CML is associated to imatinib treatment and to inhibition of leukemia cell proliferation

BackgroundSPARC is a matricellular glycoprotein with growth-inhibitory and antiangiogenic activity in some cell types. The study of this protein in hematopoietic malignancies led to conflicting reports about its role as a tumor suppressor or promoter, depending on its different functions in the tumor microenvironment. In this study we investigated the variations in SPARC production by peripheral blood cells from chronic myeloid leukemia (CML) patients at diagnosis and after treatment and we identified the subpopulation of cells that are the prevalent source of SPARC.MethodsWe evaluated SPARC expression using real-time PCR and western blotting. SPARC serum levels were detected by ELISA assay. Finally we analyzed the interaction between exogenous SPARC and imatinib (IM), in vitro, using ATP-lite and cell cycle analysis.ResultsOur study shows that the CML cells of patients at diagnosis have a low mRNA and protein expression of SPARC. Low serum levels of this protein are also recorded in CML patients at diagnosis. However, after IM treatment we observed an increase of SPARC mRNA, protein, and serum level in the peripheral blood of these patients that had already started at 3 months and was maintained for at least the 18 months of observation. This SPARC increase was predominantly due to monocyte production. In addition, exogenous SPARC protein reduced the growth of K562 cell line and synergized in vitro with IM by inhibiting cell cycle progression from G1 to S phase.ConclusionOur results suggest that low endogenous SPARC expression is a constant feature of BCR/ABL positive cells and that IM treatment induces SPARC overproduction by normal cells. This exogenous SPARC may inhibit CML cell proliferation and may synergize with IM activity against CML.

Prognostic value of CD34+ peak in peripheral blood during mobilization in intermediate-risk AML patients treated in first CR by autologous or allogeneic transplantation.

Ninety-six AML patients in 1st CR were evaluated for peak CD34+ cell levels in peripheral blood (PB) during PBSC mobilization and harvest. Distribution of CD34+ cell peaks was determined and cases were grouped on the basis of 50th and 75th percentile: group A, those having a CD34+ cell peak ⩽70 × 109/L (n=48); group B, those having a CD34+ cell peak between 70 and 183 × 109/L (n=24); group C, those having a CD34+ cell peak >183 × 109/L (n=24). Irrespective of post-remission treatment received, group A had a disease free survival (DFS) of 73%, group B a DFS of 51% and group C of 30% (P=0.0003). In intermediate cytogenetic risk patients, those treated by autologous transplantation had a DFS of 68, 33 and 14% in the groups A, B and C, respectively, (P=0.01) whereas after allogeneic transplantation DFS was 87% in group A+B vs 50% in group C (P=0.009). The peak of CD34+ cells in PB, was an independent predictor for DFS in multivariate analysis.

Successful treatment of advanced idiopathic myelofibrosis with imatinib mesylate

To the Editor: Idiopathic myelofibrosis (IM) is a clonal myeloproliferative disorder characterized by bone marrow fibrosis, extramedullary hemopoiesis, splenomegaly and leuko-erythroblastic blood picture (1). Till date, conventional treatments for IM have been directed toward the alleviation of symptoms and have shown limited efficacy without improving overall survival (2). As such, investigations on new therapeutic strategies are warranted. Here we describe two cases of IM in the advanced stage of the disease who were successfully managed with imatinib mesylate (Glivec, Novartis, Basel, Switzerland). The patients were two males, aged 63 and 59 years, respectively, whose diagnosis was confirmed by standard criteria (2) and scored as high risk according to Lille scoring system (3). One of the patients (case 2) presented HBV-related hepatic cirrhosis also. Cytogenetic analysis of bone marrow samples revealed in both cases a normal karyotype and molecular analysis showed no finding of Bcr-Abl fusion gene. Both patients underwent conventional treatment with hydroxyurea; however, after a median time of 21.5 months, they become drugrefractory. Peripheral blood analysis showed uncontrollable hyperleukocytosis in the two patients (median WBC count: 59.8 · 10/L) with morphologic features of accelerated phase; case 1 showed a decrease in Hb concentration (Hb: 8.9 g/dL), while case 2 needed weekly red blood and platelet cell transfusions because of anemia (Hb: 7.2 g/dL) and severe platelet reduction (PLT: 9.0 · 10/L). All of them exhibited elevated lactate dehydrogenase (LDH) levels (median 900.5 U/L). The pictures of bone marrow specimens confirmed diagnosis of accelerated phase of the disease. Cytogenetic re-evaluation showed no clonal abnormalities in case 1, while case 2 presented monosomy 7 in 10% of the metaphases. Again, there was no finding of BcrAbl fusion gene. Therapy with imatinib mesylate, at a daily dosage ranging from 200 to 400 mg (according to toxicity) was then started. No other cytotoxic or supportive agents were combined to imatinib mesylate. Patients exhibited a prompt hematologic response within 2 wk of therapy. In particular, case 2 did not need any further cell transfusions. Table 1 shows clinical data at 3 months of therapy. Imatinib mesylate normalized or reduced the WBC count (case 1, WBC: 10.8 · 10/L; case 2, WBC: 16.2 · 10/L), leaded to an increase in both Hb concentration and platelet count, reduced LDH levels and the degree of hepato-splenomegaly. Bone marrow examination revealed a reduction in the count of myeloblasts and promyelocytes with pictures of hypercellular marrow. No other cytogenetic abnormalities were detected. Imatinib mesylate administration is still going on with minimal side effects (fluid retention and musculoskeletal pain). The therapeutic role of imatinib mesylate therapy in chronic myeloproliferative disorders other than CML remains to be explored. Few trials have been reported to date and, although clinical results were not pronounced, partial responses (spleen size reduction, improvement in Hb concentration and platelet count) were documented (4–6). Even if two

Inadvertent transplantation of haematopoietic stem cells carrying constitutional Robertsonian translocation from an apparently normal donor to an AML patient: a case report.

Inadvertent transplantation of haematopoietic stem cells carrying constitutional Robertsonian translocation from an apparently normal donor to an AML patient: a case report

Autologous Transplantation in 1st Cr Aml Patients with Purged or Unpurged Bone Marrow: Good Clinical Results in Favorable CytogeneticGroup and in those Infused with a Low Number of Marrow Cells

In a single institution, 31 patients affected with Acute Myeloid Leukaemia (AML) in 1st Complete Remission (CR) received autologous bone marrow transplantation (ABMT). Mafosfamide was employed in a non-randomized fashion to purge marrows, in 15 cases bone marrow cells were purged, while in 16 they were left unpurged. Dose of infused Total Nucleated Cells (TNC) was an important factor for myeloid engraftment (P=0.02). LFS was 58% in purged and 40% in unpurged groups (P=0.26). Patients having a good prognosis cariotype had a LFS of 100% while the group of all other patients had a LFS of 37.5%. Patients receiving a dose of TNC below to median had a LFS of 65% and those receiving a dose of TNC>median had a LFS of 28% (P=0.017). Purging significantly improved LFS in patients “not harbouring good cytogenetic abnormalities” (53% LFS in purged group versus 18% in unpurged group, P=0.05). In conclusion, ABMT is associated with excellent results in “good prognosis cytogenetic”. Purging may improve results in patients belonging to “intermediate cytogenetic group”. A high number of infused TNC produces a fast myeloid engraftment but a poor LFS.