Michele Mario Greco

High rate of remissions in chronic myelomonocytic leukemia treated with 5-azacytidine: results of an Italian retrospective study

1 Istituto di Ematologia, Universit à Cattolica del Sacro Cuore, Roma, Italy, 2 Dipartimento di Biotecnologie Cellulari ed Ematologia, Universit à La Sapienza, Roma, Italy, 3 Istituto di Ematologia, Fondazione Policlinico Tor Vergata, Roma, Italy, 4 Ematologia, A.O. SS Antonio e Biagio e Cesare Arrigo, Alessandria, Italy, 5 Dipartimento di Onco-Hematology, IRCCS, Centro di Riferimento Oncologico della Basilicata, Rionero in Vulture, Italy, 6 Divisione di Ematologia, Dipartimento di Medicina Traslazionale, Universit à del Piemonte Orientale Amedeo Avogadro, Novara, Italy, 7 S.Orsola-Malpighi University Hospital, Department of Hematology and Oncological Sciences “Ser à gnoli”, Bologna, Italy, 8 Dipartimento di Ematologia, Ospedale Sant ’ Andrea, Universit à La Sapienza, Roma, Italy and 9 Ematologia, AOU Careggi, Universit à di Firenze, Firenze, Italy

Treatment of “Poor Risk” Acute Myeloid Leukemia with Fludarabine, Cytarabine and G-CSF (Flag Regimen): A Single Center Study

We describe a single center experience of 41 consecutive patients with poor prognosis acute myeloid leukemia (AML) who received a single course of FLAG regimen consisting of Fludarabine 30 mg/m2/day plus Cytarabine 2 gr/ m2/day (days 1–5) and G-CSF 5 mg/Kg/day (from day 0 to polymorphonuclear recovery) as salvage therapy. Eleven patients were primarily refractory to previous chemotherapy, 10 patients were in first relapse, 2 patients in second relapse and 7 patients in relapse after transplants. Eleven cases were defined as secondary AML (diagnosis of AML made after a preexisting diagnosis of myelodysplastic syndrome). The median age was 52.6 years (range 16–72); 29 patients were males and 12 females. Overall, 23 (56%) patients reached complete remission (CR), 3 patients died of infection (2) or hemorrhage (1) during induction, and 15 (36%) patients had resistant disease. The highest CR rates (80%) were obtained in relapsed cases; de novo and secondary AML registered 60% and 45% of CR rates, respectively. Patients achieving CR received a second FLAG course as consolidation and were submitted to an individualized program post-remission therapy, depending on the age and performance status. Hematological and non hematological toxicities were acceptable. In conclusion, our data confirm that FLAG is a an high effective treatment for poor prognosis AML and in young patients allows intensive post remissional therapy including allogeneic BMT.

Minimally differentiated acute myeloid leukemia (AML M0): clinico-biological findings of 29 cases.

Twenty-nine cases of minimally differentiated acute myeloid leukemia or AML MO identified among 441 AML diagnosed in the last 12 years are reported. In all cases, flow cytometric analysis using a large panel of monoclonal antibodies and cytogenetic and molecular studies (IgH, TcRβ, BCR/ABL, AML1/ETO and CBFB-MYH11 rearrangements) were performed. Of the 29 patients, 27 were treated with intensive chemotherapy based on GIMEMA protocols. We noted a greater incidence of older (over 60 years) and male patients (52% and 65%, respectively). CD33, CD13, CD7 and TdT were expressed in 79.3%, 82.7%, 58.6% and 42.8% of cases, respectively. Antigenic MPO was present in 17 of 22 cases (77.3%). Most cases expressed CD34 (93.1%), HLA-DR (93.1%), CD117 (80%) and CD45RA (87%). CD45RO and CD90 were consistently negative. In all cases, we observed an up-expression of bcl-2 and a down-expression of CD95 with an inverse trend between the two markers (r -5253; p 0.03). Karyotypic abnormalities were demonstrated in 53.6% of cases. Of these, 6 involved chromosomes 5, 7 and 8, t(9;22), confirmed by the BCR/ABL transcript, was detected in one case. Rearrangements of the TcRβ and IgH chains were observed in 3 and 2 cases, respectively. No AML1/ETO and CBFB-MYH 11 transcripts were found. Twelve out of 27 patients (44%) achieved a complete remission (CR) (in 2 cases after rescue therapy). Seven early (range 1–9 months) and one late (32 months) relapses were observed. Five patients are alive, but only the 4 who underwent bone marrow transplantation are in persistent first CR. In conclusion, AML MO is a subtype of AML antigenically well detectable, endowed with many adverse parameters (older age, TdT and CD34 expression, resistance to apoptosis, unfavorable cytogenetic abnormalities) and poor prognosis. A very aggressive consolidation treatment can be useful to improve the outcome.

Short progression‐free survival in myeloma patients receiving rituximab as maintenance therapy after autologous transplantation

undergoing PBSCT (using the same protocol, but without anti-CD20 antibody) was 38 months (unpublished observations), we considered these results unacceptable and the trial was stopped. Salvage therapy with single or multiple additional PBSCT (using a melphalan dose of 100 mg/m 2 ) or with a combination of thalidomide, dexamethasone and zoledronic acid, enabled a second remission to be achieved in all cases. Although the number of patients is too low to draw definitive conclusions, in our experience the use of rituximab as maintenance therapy after PBSCT was associated with an unexpectedly high rate of early relapse in MM patients. Recently, a case of plasma cell leukaemia treated with autologous transplantation and early relapse after consolidation therapy with anti-CD20 antibody, has been reported (Gemmel et al, 2002). The reasons of these findings are not clear. We hypothesize a possible role for rituximab in provoking a further decrease in the residual, normal B-cell activity within the context of the complex immunological network characterizing anti-tumour immune response. The therapeutic role of rituximab in MM remains elusive and requires ad hoc studies.

Rituximab for allo-SCT-associated thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) may rarely complicate Allo-SCT.1, 2 First-line therapy of TTP is plasma exchange.3 However, other therapeutic approaches, such as steroids, i.v. high-dose Ig and vincristine, are still used. Despite anecdotally, recently, the anti-CD20 monoclonal antibody rituximab has been reported to successfully cure SCT-associated thrombotic microangiopathy.4, 5, 6, 7

Morphologically Typical and Atypical B-Cell Chronic Lymphocytic Leukemias Display a Different Pattern of Surface Antigenic Density

Recent evidences suggest that B-cell chronic lymphocytic leukemia (B-CLL) may have heterogeneous biological and clinical features. Immunological phenotype may be useful for distinguishing these different forms of disease. We used a quantitative flow cytometric approach to analyze the expression of several membrane molecules (CD19, CD20, CD22, CD23, CD11c, CD5, CD79b) commonly used to diagnose and characterize B-CLL in a choort of 84 consecutive B-CLL patients diagnosed according to morphological and immunological findings. We found that morphologically so-called “atypical” B-CLL displayed a significantly higher number of CD20 and CD22 molecules than typical forms. On the other hand, CD19 was found to be more expressed in typical B-CLL, although without reaching statistical significance. Finally, no difference was detected with respect to CD23, CD79b, CD11c and CD5 number of molecules/per cell between typical and atypical B-CLL. Other clinico-biological features, such as surface membrane immunoglobulin density, percentage of CD79b and FMC7 expression, peripheral blood lymphocytosis, trisomy 12 and advanced clinical stages were also found to be more frequent in atypical B-CLL. In conclusion, our data confirm the hypothesis that atypical B-CLL is a disease sustained by more mature B-cells, closely related but, at the same time, clearly distincted from neoplastic cells of typical B-CLL.

Minimal residual disease after allogeneic stem cell transplant: a comparison among multiparametric flow cytometry, Wilms tumor 1 expression and chimerism status (Complete chimerism versus Low Level Mixed Chimerism) in acute leukemia

Abstract Relapse represents the main cause of treatment failure after allogeneic stem cell transplant (allo-SCT). The detection of minimal residual disease (MRD) by multiparametric flow cytometry (MFC), chimerism, cytogenetics and molecular analysis may be critical to prevent relapse. Therefore, we assessed the overall agreement among chimerism (low level mixed chimerism [LL-MC] vs. complete chimerism [CC]), MFC and Wilms tumor 1 (WT1) mRNA to detect MRD and investigated the impact of MRD obtained from the three methods on patient outcome. Sixty-seven fresh bone marrow (BM) samples from 24 patients (17 acute myeloid leukemia [AML], seven acute lymphoblastic leukemia [ALL]) in complete remission (CR) after allo-SCT were investigated at different time points. A moderate agreement was found among the three techniques investigated. A higher concordance between positive results from MFC (75.0% vs. 32.7%, p = 0.010) and WT1 (58.3% vs. 29.1%, p = 0.090) was detected among LL-MC rather than CC samples. Relapse-free survival (RFS) and overall survival (OS) were found to be higher in MRD negative patients than in MRD positive patients analyzed with MFC and WT1. Our results discourage the use of low autologous signals as the only marker of MRD, and suggest the usefulness of MFC and WT1 real-time quantitative polymerase chain reaction (RQ-PCR) in stratifying patients with respect to risk of relapse.

Optimal time-points for minimal residual disease monitoring change on the basis of the method used in patients with acute myeloid leukemia who underwent allogeneic stem cell transplantation: A comparison between multiparameter flow cytometry and Wilms’ tumor 1 expression

Minimal residual disease (MRD) of 30 adult AML patients was monitored by multiparameter flow cytometry (MFC) and WT1 expression before and after allogeneic stem cell transplantation (allo-SCT). Diagnostic performance of pre-transplant MRD measured by MFC was higher than that obtained by WT1 expression. Comparable results were displayed at day +30 post-transplant, while better values by WT1 compared to MFC were found at day +90. Positive MRD by MFC predicted a shorter disease free survival (DFS) before and 1 month after transplant (p=0.006 and p=0.005), while only high WT1 levels at 1 month from the transplant significantly impacted on DFS (p=0.010). Our results support the idea that MRD monitoring by MFC should be suggested before and 30 days after the transplant, while WT1 expression should be preferred after this procedure. The assessment of MRD at day +30 from allo-SCT is recommended as post transplant check-point for the predictive role displayed, independently of the method used.

Gemtuzumab ozogamicin as maintenance therapy after autologous stem cell transplantation in elderly patients with acute myeloid leukaemia.

Acute myeloid leukaemia (AML) has a dismal prognosis, particularly in older patients who make up a large proportion of the AML population. Induction therapy, typically consisting of cytarabine and anthracycline combination regimens, is the cornerstone of treatment and achieves a complete remission (CR) rate of 45–55% in older patients and a median survival of only 8–12 months (Roboz, 2007). Unfortunately, aggressive, postremission consolidation chemotherapy does not appear to improve survival in older patients who are at increased risk of chemotherapy-related mortality (Stone, 2001). Gemtuzumab ozogamicin (GO) is a recombinant humanized monoclonal antibody conjugated to the antitumour antibiotic calicheamicin. The antibody component targets the CD33 cell surface marker that is expressed on cancerous cells in the majority of AML patients. GO is currently indicated for the treatment of AML in patients aged ‡60 years who are in their first relapse and are not considered suitable candidates for other types of chemotherapy. Gemtuzumab ozogamicin has also been investigated in the first-line treatment of AML, with more promising results seen when this agent is added to combination chemotherapy induction regimens (Fenton & Perry, 2005; Pagano et al, 2007). Clinical trials currently underway are investigating the role of GO in consolidation therapy. Studies evaluating the use of GO in conditioning regimens prior to haematopoietic SCT have indicated an increased risk of hepatic venoocclusive disease, though this risk appears to be greatly reduced if more than 3Æ5 months elapses between GO exposure and SCT (Wadleigh et al, 2003). Little or nothing is known about the usefulness of GO as maintenance therapy after autologous SCT (Tsimberidous et al, 2005) and no experience in this setting has been published to date. Gemtuzumab ozogamicin monotherapy has typically been administered as a 2-h infusion at a dose of 9 mg/m on days 1 and 15 of treatment. Favourable tolerability results following the administration of fractionated doses have recently been reported (Taksin et al, 2007). We hypothesized that GO could have beneficial effects as maintenance therapy after haematopoietic SCT in high-risk patients. Here, we report data on five elderly AML patients in CR who received four fractionated doses of GO as maintenance therapy following ASCT. This study was carried out under ethical approval. Our approach differs from previous reports in the literature in that, instead of using GO to induce a first remission or re-induce a second remission after relapse, we administered GO to patients while they remained in remission, 2 months after undergoing ASCT. Patient 1 was a 64-year-old male with 88% CD33 expression at diagnosis. He was in second CR following an induction regimen of two cycles of fludarabine + cytarabine + idarubicin. This patient had previously undergone an ASCT, using a conditioning regimen of busulphan + cyclophosphamide. Patient 2 was a female, aged 67 years and had 90% CD33 expression at diagnosis. She was in first CR following an induction regimen of two cycles of etoposide + cytarabine + idarubicin. Patients 3 and 4 were a 69-year-old male and a 68-year-old female with 95% and 93% CD33 expression at diagnosis respectively. They were in first CR following an induction regimen of two cycles of fludarabine + cytarabine. Patient 4 was still receiving maintenance therapy and, at that time, received two doses of GO. Finally, patient 5 was a 60-year-old male with 90% CD33 expression at diagnosis. He had an AML refractory to a standard induction chemotherapy (3 + 7 regimen) and was rescued with one MEC (mitoxantrone, etoposide and cytosine arabinoside) regimen. Histocompatible donors were not available for any of the patients. Three months after attaining CR, all the five patients received a myeloablative conditioning regimen and underwent ASCT. Two months after complete engraftment, patients initiated treatment with GO (see Table I for details of preASCT conditioning therapy and GO regimens). Disease outcomes and details of haematological adverse events are summarized in Table I. All the five patients remained alive and in CR at +21, +17, +15, +7, +13 months, respectively, while the overall survival at the time of reporting was +39, +19, +17, +8, +20 months. There were no cases of grade 3 or 4 liver toxicity and bleeding was not observed in any of the patients. Thrombocytopenia (50–100 · 10 cells/l) occurred in all the five patients and neutropenia (0Æ5– 1Æ0 · 10 cells/l) in two patients. In conclusion, despite the limited numbers, GO demonstrated good efficacy when administered in fractionated doses as maintenance therapy after ASCT in our series of older patients with CD33 AML in first or second CR. To our knowledge, this is the first report on the use of GO as maintenance therapy after ASCT. GO showed an acceptable tolerability profile, with no severe hepatotoxicity and no bleeding. Thrombocytopenia occurred in all the five patients Correspondence

TREATMENT OF PLASMA CELL LEUKAEMIA AND RESISTANT/RELAPSED MULTIPLE MYELOMA WITH VINCRISTINE, MITOXANTRONE AND DEXAMETHASONE (VMD PROTOCOL)

Broun. E.R.. Heerema. N.A. & Tricot, G. (1990) Spontaneous remission in myelodysplastic syndrome. A case report. Cancer Genetics and Cytogenetics, 46, 125-128. Lambertenghi-Deliliers. G., Orazi, A.. Luksch. R. , Annaloro. C. & Soligo, D. (1991) Myelodysplastic syndrome with increased marrow fibrosis: a distinct clinico-pathological entity. British journal o/Haematology. 78, 161-166. Narayanan, M.N. & Lewis, M.J. (1991) Spontaneous remission in acute myeloid leukaemia. Clinical and Laboratory Haematology (in press). Pagliucia. A.. Layton, D.M.. Manoharon. A.. Gordon, S.. Green, P.J. & Mufti. G J . (1989) Myelofibrosis in primary myelodysplastic syndromes: a clinical morphological study of 1 0 cases. British journal o/Haernatology. 71, 499-504. Takahashi. M., Koike. T., Nagayama. R. . Fujiwara. M., Koyama. S.. Ohnishi. M.. Nakamori, Y., Soga, N.. Aoki, S.. Tatewaki. W.. Wada. K.. Moriyama. Y. & Shibata, A. (1991) Myelodysplastic syndrome with myelofibrosis: myelodysplastic syndrome as a major primary disorder for acute myelofibrosis. Clinical and hboratory Haemato-