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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.

In a case of immunoblastic lymphoma we observed the presence of either a deletion of the long arm of chromosome 6 or of an isochromosome, i(6p), which occurred alternatively in metaphase cells. This suggest a selective pressure for loss of heterozygosity of genes located on 6q and is in accordance with the hypothesis that one or more tumor suppressor genes might be located on the long arm chromosome 6.

The corner stone of the WHO classification and prognostic scores of myelodysplastic syndromes (MDS) is the blast count in bone marrow. The standard cytology evaluation of at least 500 bone marrow cells is easy to perform, but some concerns arise about reproducibility of this method. Nowadays bone marrow trephine biopsy and flow cytometry are frequently considered for the diagnosis of MDS. However there is so far paucity of data comparing cytology, histology and flow cytometry in quantifying bone marrow blasts in order to differentiate non RAEB from BAEB-I and RAEB-II cases.The Aim of the work was to analyse the differences and the prognostic impact of cytology, histology and flow cytometry in differentiating non RAEB from BAEB-I and RAEB-II.Since 1999, clinical and laboratory data from 1256 new cases of MDS were prospectively recorded into the Piemonte MDS Registry. Blast count could be performed with the three different methods: BMC (bone marrow cytology) has been performed in 844 cases, BMH (bone marrow histology) in 874 cases, and BMF (bone marrow flow cytometry) in 636. In order to quantify blasts, immune-histochemistry evaluation of CD34+ cells was used in BMH, while both CD34+ and CD117+ cells were considered in BMF. Out of the total of the 636 patients analysed by BMF only 420 had an accurate and complete registration of CD34 and CD117 positivity and were considered for the present analysis. In two hundred and thirty six cases all three evaluations were contemporary available. The concordance of each diagnostic method with the others and their prognostic value were evaluated in both univariate and multivariate analyses. A comparison between BMC and BMH was available in 571 cases, between BMC and BMF in 228 cases, and between BMH and BMF in 279 cases.The disagreement in classifying patients as non-RAEB or RAEB-I or RAEB-II between BMC and BMH was 156/571 (27%), with BMH over-evaluating blasts in 114/571 cases (20%) and under-evaluating blasts in 42/571 cases (7%). The disagreement between BMC and BMF was 80/228 (35%), with BMF over-evaluating and under-evaluating blast percentage in comparison to BMC in 53/228 (23%) and in 27/228 (12%) cases respectively. The disagreement between BMH and BMF was present in 113/279 (41%), with BMF over-evaluating and under-evaluating blast percentage in comparison to BMH in 44/279 (16%) and in 69/279 (25%) cases respectively. In univariate analysis all three methods of quantifing blasts and differentiating non-RAEB from RAEB-I and RAEB-II retained an important prognostic value for both leukemic evolution and survival. However when the three models were tested in multivariate analysis in order to define the best predictor of leukemic evolution, BMC retained the best predictive value.When BMH or BMF are used instead of BMC in order differentiate non-RAEB from RAEB-I and RAEB-II, the shift to a different WHO category is evident in at least 30% of patients and BMH and BMF do not play the same role as BMC. BMC still remain the standard method to quantify blasts for classification and prognostic evaluation of MDS.Off Label Use: Lenalidomide in Mantle Cell Lymphoma. Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Saglio:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract 4028

Protein tyrosine kinases play a crucial role in normal and neoplastic cell development. Recently, a unique clonal mutation in the JAK2 gene that results in a valine to phenylalanine substitution at position 617 (V617F), leading to constitutive JAK2 activation and abnormal signaling, was found in the majority of BCR-ABL negative chronic myeloproliferative disorders (CMPD) patients. Although JAK2 activation by tyrosine phosphorilation is involved in the BCR-ABL transformation of CML cells, V617F mutation is not usually observed in Ph-positive leukemias.We describe two patients with BCR/ABL+ (p190) CML which followed a Ph- CMPD characterized by JAK2 mutation. Patient 1 was diagnosed as having a idiopathic myelofibrosis in Oct 1997. At that time a Ph-chromosome was not detected. The patient remained untreated until March 1999, when WBC started to increase up to 90.000/cmm with a parallel drop in platelet count and increase in spleen size. Hydroxiurea was started with partial hematological remission. In Jan 2000 the presence of a BCR/ABL transcript (p190 type) and a Ph-chromosome in 84% of the cells were observed. Imatinib treatment resulted in a initial improvement, soon followed by progressive increase in the WBC and platelet counts, in spite of a reduction of Ph+ cells to 4%. Hydroxyurea was added but hematological control remained unsatisfactory and imatinib treatment was stopped after 6 months. The patient continued on hydroxyurea for the following year. In 2002 he developed a myeloid blast crisis; karyotype was normal, 4.8% of the cells were Ph+ at FISH and the p190 transcript was still present at low levels. Patient 2 was diagnosed as essential throbocythemia in 1990; cytogenetic analysis showed a normal karyotype. A good control of the thrombocytosis was obtained with hydroxyurea for more than 10 years. In 2003 the hematological data showed leukocytosis (up to WBC 42.900/cmm), immature myeloid cells and 16% monocytes in PB. A masked Ph chromosome was observed by FISH in 47% of cells. RT-PCR was positive for a p190 transcript. The patient started therapy with imatinib which induced a rapid decrease in WBC count; however, the platelet count rised up to 977.000/cmm and hydroxiurea was added again. In subsequent bone marrow samples a progressive decrease of BCR/ABL positive cells to 2% was observed and a BCR-ABL/ABL ratio of 0.18 % was reached in January 2006. However, continuous hydroxyurea therapy was needed to partially control thrombocytosis. Both patients were tested for the presence of JAK2 mutation using the ABI Prims 3100 genetic analyzer and a JAK2-V617F mutation was present. Presence of two clones, possibly derived from a common abnormal stem cell, can be hypothesized in these cases. The possibility of a multistep patogenesis for CML has been widely discussed; in these patients, CML apparently developed as a late event, partially replacing a Ph-CMPD cell population characterized by the JAK2 mutation. Imatinib treatment allowed the reexpansion of the Ph- cells resulting in a overall poor hematological control. Both patients only displayed p190 BCR/ABL transcript, a quite unusual finding in chronic phase CML. It is not clear if any relationship may sussist between this molecular event and the unusual clinical history of these patients.

In vitro growth of acute myelogenous leukemia blasts as an established cell line is an uncommon event and molecular mechanisms allowing permanent in vitro proliferation have not been defined. In particular, few acute monoblastic leukemia (AMoL) cell lines have been described. Here we report a new AMoL cell line expressing a combination of growth factors and growth factor receptor that might be involved in an autocrine stimulation loop. AMoL (M5a according to F.A.B) was diagnosed in November 2004 in a 74 old woman. Caryotypic analysis revealed a combination of 46,XX, 47,XX,+8, 47,XX,+8,6q− caryotypes. A complete remission was achieved by an age- adapted FLAG-Ida regimen but caryotype analysis still revealed about 50% mitosis with 47,XX,+8 and in February 2005 the patient presented an isolated cutaneous relapse. In spite of treatment with low dose cytarabine, etoposide and retinoids, hematologic progression occurred in May 2005. Further treatments with vinblastine, hydroxyurea and gentuzumab-ozogamycine were only temporary effective and in June 2005 the patient died, with hyperleukocytosis, extensive cutaneous and possibly CNS involvement. A 10 ml peripheral blood sample was taken, after informed consent, at the time of the last progression and mononuclear cells were collected. Part the cells were cryopreserved and part cultured in RPMI medium + 15% fetal bovine serum (FBS). Cultured cells survived without significant expansion for 4 weeks, then progressive growth started. After a few passage the cell line (named PI-BR) has been established, with a doubling time of approximately 48 hours. During early passages cell growth was density dependent, impaired at concentrations lower than 3 ×10e4/ml. Morphology and cytochemical stains were in agreement to M5a F.A.B. subtype. Immunophenotypic analysis revealed an antigenic expression comparable to that of patient’s blasts: CD13++, CD33++, HLA-DR ++, CD64++, CD117 +, CD15+/−, CD34−, CD19−, CD9−. Caryotypic analysis was performed on patient’s cells cryopreserved when culture was initiated: only 4 mitosis were found: 2 with normal 46,XX, 2 with 47,XX,+8 caryotype. Caryotypic analysis, repeated on PI-BR cell line at passage 9, revealed a mixture of mitosis with 47,XX,+8, 47,XX,+ ring, 48,XX,+8,+ ring, normal 46,XX and hypodiploid (42–44 chromosome) caryotype. Therefore, although some chromosome abnormalities were acquired during in vitro culture, they do not seem responsible for the capability of continuous in vitro growth, since about 55% of cells at passage 9 maintained the same caryotypes of patient’s cells in vivo. Real time PCR revealed expression of genes codifying for some monocytic cytokines (IL6, IL8, IL10, IL18, TGF-beta1), Vascular Endotelium Growth Factor (VEGF)-A, VEGF-D and its receptor Flt-4. Immunoistochemistry confirmed the synthesis of Flt-4, VEGF-A and VEGF-D. VEGF-D was also detected by ELISA assay in cell line supernatant (14.7 pg/ml) at early passages. VEGF A and C and VEGF receptors have been reported to be expressed in some AML cell lines and in some fresh AML cell samples, displaying anti-apoptotic and proliferative activity. In the case of PI-BR cell line, synthesis of VEGF-D and of its receptor Flt-4 may have established an autocrine loop involved in continuous cell growth.

Protein tyrosine kinases play a crucial role in normal and neoplastic cell development. Recently, a unique clonal mutation in the JAK2 gene that results in a valine to phenylalanine substitution at position 617 (V617F), leading to constitutive JAK2 activation and abnormal signaling, was found in the majority of BCR-ABL negative chronic myeloproliferative disorders (CMPD) patients. Although JAK2 activation by tyrosine phosphorilation is involved in the BCR-ABL transformation of CML cells, V617F mutation is not usually observed in Ph-positive leukemias.We describe two patients with BCR/ABL+ (p190) CML which followed a Ph- CMPD characterized by JAK2 mutation.

In vitro growth of acute myelogenous leukemia blasts as an established cell line is an uncommon event and molecular mechanisms allowing permanent in vitro proliferation have not been defined. In particular, few acute monoblastic leukemia (AMoL) cell lines have been described. Here we report a new AMoL cell line expressing a combination of growth factors and growth factor receptor that might be involved in an autocrine stimulation loop. AMoL (M5a according to F.A.B) was diagnosed in November 2004 in a 74 old woman. Caryotypic analysis revealed a combination of 46,XX, 47,XX,+8, 47,XX,+8,6q− caryotypes. A complete remission was achieved by an age- adapted FLAG-Ida regimen but caryotype analysis still revealed about 50% mitosis with 47,XX,+8 and in February 2005 the patient presented an isolated cutaneous relapse. In spite of treatment with low dose cytarabine, etoposide and retinoids, hematologic progression occurred in May 2005. Further treatments with vinblastine, hydroxyurea and gentuzumab-ozogamycine were only temporary effective and in June 2005 the patient died, with hyperleukocytosis, extensive cutaneous and possibly CNS involvement. A 10 ml peripheral blood sample was taken, after informed consent, at the time of the last progression and mononuclear cells were collected. Part the cells were cryopreserved and part cultured in RPMI medium + 15% fetal bovine serum (FBS). Cultured cells survived without significant expansion for 4 weeks, then progressive growth started. After a few passage the cell line (named PI-BR) has been established, with a doubling time of approximately 48 hours. During early passages cell growth was density dependent, impaired at concentrations lower than 3 ×10e4/ml. Morphology and cytochemical stains were in agreement to M5a F.A.B. subtype. Immunophenotypic analysis revealed an antigenic expression comparable to that of patient's blasts: CD13++, CD33++, HLA-DR ++, CD64++, CD117 +, CD15+/−, CD34−, CD19−, CD9−. Caryotypic analysis was performed on patient's cells cryopreserved when culture was initiated: only 4 mitosis were found: 2 with normal 46,XX, 2 with 47,XX,+8 caryotype. Caryotypic analysis, repeated on PI-BR cell line at passage 9, revealed a mixture of mitosis with 47,XX,+8, 47,XX,+ ring, 48,XX,+8,+ ring, normal 46,XX and hypodiploid (42–44 chromosome) caryotype. Therefore, although some chromosome abnormalities were acquired during in vitro culture, they do not seem responsible for the capability of continuous in vitro growth, since about 55% of cells at passage 9 maintained the same caryotypes of patient's cells in vivo. Real time PCR revealed expression of genes codifying for some monocytic cytokines (IL6, IL8, IL10, IL18, TGF-beta1), Vascular Endotelium Growth Factor (VEGF)-A, VEGF-D and its receptor Flt-4. Immunoistochemistry confirmed the synthesis of Flt-4, VEGF-A and VEGF-D. VEGF-D was also detected by ELISA assay in cell line supernatant (14.7 pg/ml) at early passages. VEGF A and C and VEGF receptors have been reported to be expressed in some AML cell lines and in some fresh AML cell samples, displaying anti-apoptotic and proliferative activity. In the case of PI-BR cell line, synthesis of VEGF-D and of its receptor Flt-4 may have established an autocrine loop involved in continuous cell growth.

BACKGROUND Extramedullary blast crisis (BC) of Ph’+ CML is infrequent and commonly affects bone, lymphoid tissue, skin and soft tissues and central or peripheral nervous system. Most of nodal lymphoid tumors occuring in the setting of CML derive from T-cell precursors and represent the evolution of CML to a lymphoid nodal BC. CASE HISTORY In February 2004, a 52 year-old patient underwent diagnostic wide biopsy of a nasopharingeal mass that caused severe acute respiratory symptoms. At the histological examination, the nasopharyngeal mucosa exhibited a diffuse pattern of infiltration by neoplastic cells with a characteristic single-file arrangement. The cells showed typical convoluted nuclei with one or two nucleoli and abundant cytoplasm (L2, lymphoblasts). The malignant cell population expressed a preT-cell immunophenotype: cytoplasmic CD3(+), CD43 (+), TdT (+/−), CD34(+), CD4(−), CD8(−). Thus, the diagnosis of T-cell LL was formulated. Whole-body CT scan revealed nasopharingeal mass, retropharyngeal, laterocervical, axillary, inguinal enlarged nodes and splenomegaly. Laboratory tests indicated leukocytosis (58,000/μl) with a differential WBC count typical of CML in chronic phase (myeloblasts <1%). This diagnosis was confirmed by a low alkaline phosphatase value (score: 1), histological features of bone marrow (BM) biopsy, classical cytogenetics (presence of the Ph’ in 100% of 25 metaphases analysed and absence of other cytogenetic abnormalities) and FISH evaluation (D-FISH bcr-abl in interphase and metaphase, Oncor probe). The nested RT-PCR (JQ Guo et al., Leukemia;2002,15:2447) disclosed the presence of the hybrid protein p210 (b2,a2), but not that of p190. A laterocervical node was excised to perform cytogenetic and molecular analyses in order to determine whether the T-cell LL was an unrelated disease or the expression of an extramedullary BC. The histology confirmed the presence of a uniform population of T-lymphoblasts in which classical cytogenetic analysis disclosed the following kariotype: 49,XY,t(9;22)(q34;q11),+9,+19,+der(22)t(9;22)(q34;q11) and 50–52,XY t(9;22)(q34;q11),+6+9,+9q+,+19,+20,+22q−. The FISH analysis showed the presence of bcr-abl gene in all cells analyzed and the presence of multiple copies of this gene as well as of double Ph’ chromosome. Nested RT-PCR showed the presence of both p210 and p190 transcripts. These findings indicated that the T-cell LL was an extramedullary BC of a CML simultaneously diagnosed in chronic phase in BM and peripheral blood. The patient was initially treated with vincristine, daunomicine, asparaginase and prednisone combined with Gleevec (800mg/die). Consolidation therapy, consisting of high-dose Ara-C (4g/m2 for 4 days) and mithoxantrone (10mg/m2 for 2 days), followed by a mieloablative course with mithoxantrone (60 mg/m2) and melphalan (180 mg/m2) and autologous stem cell support (ASCT) was administered after an initial clinical response. Gleevec was given during the entire treatment period. A fugacious complete clinical-hematological and a partial cytogenetic (FISH: 12% of cells bcr-abl+) and molecular (number of bcr-abl/104 ABL copies= 184, real-time quantitative RT-PCR: J Gabert et al., Leukemia;2003:1) remission was documented after ASCT. The patient refused further consolidation treatment with Gleevec and died, due to progressive disease, in December 2004.

BACKGROUND