Maria Immacolata Ferreri

WT1 expression levels at diagnosis could predict long-term time-to-progression in adult patients affected by acute myeloid leukaemia and myelodysplastic syndromes.

Mutations of Wilms’ tumour gene (WT1) are reported in 10% of acute myeloid leukemias (AML) with normal karyotype, with reduction in both relapse-free-survival and overall survival (Virappane et al, 2008). WT1 is highly expressed in acute leukemias and the myelodysplastic syndromes (MDS) (Rosenfeld et al, 2003) where it is associated with poorer prognosis (Cilloni et al, 2008; Candoni et al, 2009). About 65% of low-risk and up to 100% of high-risk MDS cases express high WT1 transcripts, correlated with higher risk of progression (Tamaki et al, 1999; Cilloni et al, 2003). To date, no studies have evaluated whether diagnostic WT1 mRNA levels influence the long-term time-to-progression (TTP) in MDS and AML. Moreover, no significant data have been produced concerning WT1 and MDS cases that have been classified according to the newer World Health Organisation Prognostic Scoring System (WPSS) score (Malcovati et al, 2007). Thus, in the present study, we evaluated the possible impact on long-term TTP exerted by WT1 mRNA levels measured at diagnosis in a series of 54 cases (24 AML and 30 MDS). WT1 transcript was quantified with the ProfileQUANT TM kit (Ipsogen, Marseille, France) on total RNA isolated using RNeasy Mini kit (QIAGEN, Valencia, CA, USA). This method estimates the ‘normal’ WT1 copies/ABL1 · 10 copies ratio to be between 3 and 180. Clinical and demographic characteristics of the entire series are reported in Table I. Patients were stratified in two categories (WT1-low and -high) when the WT1 copies/ABL1 · 10 copies ratio was lower or higher than 180 respectively; the chi-square and logistic regression tests were used to assess eventual differences in clinical and demographic data. t-test was adopted for comparing mean values; Kaplan–Meier life tables were constructed for survival data and compared by means of the logrank test, with surviving patients being censored at 15 June 2009. All statistical analyses were performed with the Statistical Package for the Social Sciences (spss) software, version 17.0 (SPSS Italia, Bologna, Italy). P values <0Æ05 were considered significant. All low-risk MDS patients received epoietins and/or additional blood transfusion support; the high-risk MDS group included patients who received azacitidine 75 mg/m, 6 d a week for almost four cycles. For AML cases, induction therapy included idarubicin, less often doxorubicin with aracytin, according to the ‘3 + 7’ or ‘2 + 5’ scheme, on the basis of age (£ or >65 years). Fourteen transplanted patients were censored before stem cell infusion. At diagnosis, WT1 expression was high in 9 out of the 30 MDS cases (30%) (four in low-risk and five in high-risk group), and in 15 of the 24 patients (62Æ5%) affected by AML. Mean and standard deviation values were: 333Æ19 WT1 copies/10 ABL1 copies ± 97Æ89 for low-risk MDS; 551Æ31 copies/10 ABL1 copies ± 72Æ02 for high-risk MDS; 2390Æ89 copies/10 ABL1 copies 10 ± 39Æ92 for AML. WT1 mRNAs were significantly higher in AML when compared to both low-risk (P = 0Æ02) and high-risk MDS (P = 0Æ04). On the contrary, no significant difference in WT1 expression was found between the two risk score groups in MDS (P > 0Æ05). In our series of 30 MDS patients, the 36-monthTTP was 65% (median not reached at 5 years); it was not significantly affected by age, sex, performance status, white blood cell count (WBC), haemoglobin level (Hb), and platelet count (PLT) at diagnosis, blast percentage, cytogenetic features, or spleen dimension. Even the WPSS risk score in our series did not affect the TTP (36-month TTP 71% for low-risk versus 61% high-risk patients, P = 0Æ71). Similarly, the probability of progression was also not significantly affected by these analysed parameters. In contrast, the probability of progression was influenced by WT1 level: it was 14% for patients expressing low WT1 levels versus 56% for those with high WT1 mRNA (P = 0Æ03). Moreover, WT1 expression levels at diagnosis also significantly affected the 36-month TTP (Fig 1B): 73% of patients with normal WT1 expression were progression-free versus 19% of cases with elevated WT1 (P < 0Æ01). Noteworthy, this prognostic role of WT1 high expression was evident both in WPSS low-risk (Fig 1C) and high-risk categories (Fig 1d) (36-month TTP 78% vs. 5% in low-risk cases and 67% vs. 37% in the high-risk group; P < 0Æ01). In AML, the 36-month TTP was 46% (median = 23 months) and was not significantly conditioned by performance status, sex, WBC, Hb, PLT at diagnosis, blast per centage, French-American-British (FAB) subtype, cytogenetic features, presence/absence of FLT3 mutations, or spleen dimension. TTP was much lower for older patients (36-month TTP 32% vs. 60% for younger patients), but it was not statistically significantly different (P = 0Æ12). Even in AML, the probability of progression was not significantly affected by the analysed demographic/clinical correspondence

Myelodysplastic syndromes: advantages of a combined cytogenetic and molecular diagnostic workup

In this study we present a new diagnostic workup for the myelodysplastic syndromes (MDS) including FISH, aCGH, and somatic mutation assays in addition to the conventional cytogenetics (CC). We analyzed 61 patients by CC, FISH for chromosome 5, 7, 8 and PDGFR rearrangements, aCGH, and PCR for ASXL1, EZH2, TP53, TET2, RUNX1, DNMT3A, SF3B1 somatic mutations. Moreover, we quantified WT1 and RPS14 gene expression levels, in order to find their possible adjunctive value and their possible clinical impact. CC analysis showed 32% of patients with at least one aberration. FISH analysis detected chromosomal aberrations in 24% of patients and recovered 5 cases (13.5%) at normal karyotype (two 5q- syndromes, one del(7) case, two cases with PDGFR rearrangement). The aGCH detected 10 “new” unbalanced cases in respect of the CC, including one with alteration of the ETV6 gene. After mutational analysis, 33 patients (54%) presented at least one mutation and represented the only marker of clonality in 36% of all patients. The statistical analysis confirmed the prognostic role of CC either on overall or on progression-free-survival. In addition, deletions detected by aCGH and WT1 over-expression negatively conditioned survival. In conclusion, our work showed that 1) the addition of FISH (at least for chr. 5 and 7) can improve the definition of the risk score; 2) mutational analysis, especially for the TP53 and SF3B1, could better define the type of MDS and represent a “clinical warning”; 3) the aCGH use could be probably applied to selected cases (with suboptimal response or failure).

Discordant lymphoma consisting of splenic mantle cell lymphoma and marginal zone lymphoma involving the bone marrow and peripheral blood: a case report

IntroductionDiscordant lymphomas are rare entities characterized by the simultaneous presence of two distinct types of lymphomas in different anatomic sites. We describe a very rare case of simultaneous occurrence of splenic mantle cell lymphoma and marginal zone lymphoma involving the bone marrow and peripheral blood.Case presentationWe report the case of a 60-year-old asymptomatic Caucasian woman in whom discordant lymphomas were discovered when a slight lymphocytosis and a conspicuous splenomegaly were observed. The different morphological, immunophenotypical and immunohistochemical features found in the different pathologic samples obtained from peripheral blood, bone marrow and spleen sections made it possible to differentiate two types of non-Hodgkin B-cell lymphomas: a mantle cell lymphoma infiltrating the spleen and a marginal zone lymphoma involving both the bone marrow and peripheral blood. Since a similar IgH gene rearrangement was found both in the bone marrow and in the spleen, the hypothesis of a common origin, followed by a different clonal selection of the neoplastic lymphocytes may be taken into consideration.ConclusionOur case emphasizes the usefulness of investigating simultaneous specimens from different anatomic sites from the same patient and the relevant diagnostic role of splenectomy.

Reduced circulating B-lymphocytes and altered B-cell compartments in patients suffering from chronic myeloid leukaemia undergoing therapy with Imatinib

*p versus normals and versus CML-1= 0.001. Data are expressed as means ± SD. To the Editor Imatinib mesylate, an inhibitor of several tyrosine kinases, such as Abelson gene (ABL), Breakpoint Cluster RegionAbelson gene (BCR-ABL1), c-KIT and platelet-derived growth factor receptors [1] is used to treat chronic myeloid leukaemia (CML) in chronic phase and is able to interfere with some immunologic pathways. Imatinib modifies some biological aspects of B-lymphocyte, being able to induce hypogammaglobulinemia and to alter the phenotype of bone marrow plasma cells [2,3]. A recent report by De Lavallade et al. [4] has shed new light on the mechanisms by which Imatinib and other tyrosine kinase inhibitors can affect B-lymphocyte immune response. We report our experience on 34 patients with CML undergoing Imatinib therapy. We evaluated the circulating B-lymphocyte compartments by means of multiparameter flow cytometry. Samples from 34 CML patients (23 men, 11 women, age 37–83 years), undergoing Imatinib treatment (Glivec, Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA), 400mg/daily) as the first line therapy, were studied at the time of periodical (every 3months) monitoring of response to therapy. At the time of the study, the patients were not undergoing therapy with other drugs. None of the patients had a clinical history of recurrent infections. Twenty-three normal subjects (age 19–65 years) were evaluated as control group. The following blood parameters were registered: white blood cells (WBC), total lymphocyte percentage and absolute number, γ-globulin levels. The disease was monitored by conventional chromosome analysis (quinacrine and Giemsa banding), FISH analysis (LSI BCR-ABLdual-colour dual-fusion translocation probe), polymerase chain reaction (PCR) for the BCR/ABL1 translocation using the standardized PCR protocol of the European BIOMED-1 project and the standard operating procedures of LabNet guidelines [5]. Responders were defined as patients who obtained at least a three-log reduction in the BCR-ABL1messenger RNA level (MMR> 3). Major response was defined as MMR> 4 and complete molecular response as MMR> 5. Immunophenotyping was carried out by a FacsCanto II cytometer equipped with two lasers (488 and 633 nm), using a five-colour method with the following monoclonal antibodies: CD20/PerCP-Cy5.5, CD19/PE-Cy.7, CD27/APC and CD45/APC-Cy7.7 (Becton Dickinson, Franklin Lakes, NJ, USA). The antibody panel was completed by a F(ab′)2

Platelet‐derived growth factor beta receptor (PDGFRB) gene is rearranged in a significant percentage of myelodysplastic syndromes with normal karyotype

dirocco, M., Bolli, N., Martelli, M.F. & Falini, B. (2008) In human genome, generation of a nuclear export signal through duplication appears unique to nucleophosmin (NPM1) mutations and is restricted to AML. Leukemia, 22, 1285–1289. Mrozek, K., Marcucci, G., Paschka, P., Whitman, S.P. & Bloomfield, C.D. (2007) Clinical relevance of mutations and gene-expression changes in adult acute myeloid leukemia with normal cytogenetics: are we ready for a prognostically prioritized molecular classification? Blood, 109, 431–448. Nishimura, Y., Ohkubo, T., Furuichi, Y. & Umekawa, H. (2002) Tryptophans 286 and 288 in the C-terminal Region of Protein B23.1 are Important for Its Nucleolar Localization. Bioscience, Biotechnology, and Biochemistry, 66, 2239–2242. Noguera, N.I., Breccia, M., Divona, M., Diverio, D., Costa, V., De Santis, S., Avvisati, G., Pinazzi, M.B., Petti, M.C., Mandelli, F. & Lo Coco, F. (2002) Alteration of the FLT3 gene in acute promyelocytic leukemia: association with diagnostic characteristic and analysis of clinical outcome in patients treated with the Italian AIDA protocol. Leukemia, 16, 2185–2189. Pitiot, A.S., Santamarı́a, I., Garcı́a-Suárez, O., Centeno, I., Astudillo, A., Rayón, C. & Balbı́n, M. (2007) A new type of NPM1 gene mutation in AML leading to a C-terminal truncated protein. Leukemia, 21, 1564–1566.

A rare case of de novo CD5+ diffuse large B-cell lymphoma in leukemic phase and positive for CD13

We report a case of de novo diffuse large B-cell lymphoma (DLBCL) in leukemic phase, positive for both CD5 and CD13. Morphologic evaluation, flow cytometric immunophenotyping, karyotyping and polymerase chain reaction studies were performed. Neoplastic lymphocytes appeared as blast-like cells, positive for CD19, CD20, CD5, CD13, CD79a, HLADR, and with restriction for surface immunoglobulin K light chains. Rearrangement of IgH gene, BCL2 / IgH translocation and complex karyotype were found. The patient was treated with RCOMP regimen and achieved complete remission. However, only one month after the first restaging of disease, the patient presented with symptoms attributable to central nervous system involvement and her clinical conditions worsened rapidly. While both CD5 expression and leukemic presentation are uncommon findings in DLBCL, positivity for CD13 is very rare. The outcome of our patient shows the poor prognosis of CD5+ DLBCL with leukemic presentation. The possible role of CD13 coexpression is discussed.

A 14.8 Mb 12p Deletion Disrupting ETV6 in a Patient with Myelodysplastic Syndrome

We present on a new case of myelodysplastic syndrome characterized by array Comparative Genomic Hybridization. This technique confirmed the monosomy 7, detected by conventional cytogenetics, and revealed also a deletion on the short arm of chromosome 12. This deletion extends for about 14.8 Mb and breaks ETV6 gene. 12p deletion extents in hematological malignancies may vary, but the minimally deleted region almost invariably contains ETV6, that is considered the main candidate tumor suppressor genes within the region for tumor progression. It has been shown that levels of ETV6 were significantly decreased in cases with 12p13 deletions, whereas expression of other genes in the deleted region, like BCL2L14, LRP6, DUSP16 and GPRC5D, did not show any variation, independently of their copy number status. This observation strengthens the fact that ETV6 may play a potential role in the tumorigenesis process. The role of ETV6 in our patient myelodysplastic syndrome is showed by his clinical history and his poor prognosis.