Nadia Cecconi

Unexpected cardiotoxicity in haematological bortezomib treated patients

Bortezomib is an antitumor compound that inhibits proteasome activity. It is able to impair the activation of nuclear factor (NF)-jB, blocking the degradation of inhibitory jB (IjB), which is required for NF-jB translocation into the nucleus and activation of target genes. NF-jB is important for cell survival, regulating the expression of genes involved in apoptosis, such as BCL2 and BCL2L1, cell cycle progression, inflammation and angiogenesis (Richardson et al, 2003). In 2006, we treated 69 patients with bortezomib, either alone or as combination therapy. Bortezomib therapy is known to be associated with neurological side effects and thrombocytopenia (Richardson et al, 2003); however, we noticed an unexpected increase of cardiac complications, ranging from heart failure to onset of arrhythmias. Only one single case of heart failure (Voortman & Giaccone, 2006) and one asymptomatic arrhythmia (Berenson et al, 2007) following bortezomib therapy have been reported to date. All except one of our bortezomib-treated patients were receiving at least a second line therapy, and some of them had previously received anthracycline-containing regimens. Eight (11Æ6%) of the 69 patients developed serious cardiac side effects requiring medication, hospitalisation or the implant of a pacemaker (see Table I). All patients underwent cardiovascular screening before receiving bortezomib and no clinically significant abnormalities were present. The only conditions that were identified as common to all the patients were bortezomib administration and age >60 years. All eight patients who experienced cardiotoxicity underwent at least four cycles of bortezomib, strengthening the hypothesis of a direct connection with the drug. Each cycle included bortezomib 1Æ3 mg/m on days 1, 4, 8 and 11 and the recycling period was 3 weeks. The onset of cardiac symptoms was never recorded before a cumulative dose of 20Æ8 mg/m of bortezomib. The ubiquitin-proteasome system is responsible for nonlysosomal degradation of intracellular proteins, including key regulators of cell cycle, angiogenesis and apoptosis, and transcription factors, which regulate crucial mechanisms of plaque formation and rupture. Additionally, the presence of a reduced proteasome activity is associated with an increased rate of apoptosis in smooth muscle cells, determining atherosclerotic plaque instability by weakening of the fibrous cap and enlargement of the necrotic core (Versari et al, 2006). Furthermore, NF-jB activation plays an essential role in the intracellular signal transduction of the second window of protection of delayed ischaemic preconditioning in the myocardium, leading to myocardial cytoprotection (Jancso et al, 2005). Therefore, bortezomib may simultaneously cause atherosclerotic plaque progression and tendency to rupture, and facilitate ischaemic heart complications by reducing/abrogating myocardial preconditioning. Additionally, although bortezomib was found to reduce the onset of delayed arrhythmias following coronary ligation in the canine model by upregulating

Rituximab as treatment for minimal residual disease in hairy cell leukaemia

Abstract:  Purine analogues have dramatically improved the outcome of patients affected by hairy cell leukemia (HCL), although complete eradication of disease was achieved in few cases. The purpose of this study was to evaluate the role of Rituximab in eradicating minimal residual disease (MRD) in HCL patients after a pre‐treatment with 2‐chloro‐deoxy‐adenosine (2‐CdA). Ten patients received four cycles of Rituximab after administration of Cladribrine. Before starting anti‐CD20 antibody, two patients were in complete remission, six in partial remission and two showed no significant response to Cladribrine. All cases resulted IgH‐positive. Median time from the last 2‐CdA infusion was 5.7 months. Eight of 10 patients [four in partial remission (PR), two in complete remission (CR) and two unresponsive after 2‐CdA] were evaluable for response. Two months after the end of anti‐CD20 therapy, all evaluated patients presented a complete haematological remission. Moreover, Rituximab increased percentage of molecular remission up to 100% 1 yr after the end of treatment. Interestingly, in all cases but one, including those persistently polymerase chain reaction (PCR)‐positive, semi‐quantitative molecular analyses showed MRD levels lower than those found before Rituximab administration. Toxicity was very mild. The present results not only confirm the therapeutic effect of Rituximab, but also show its relevance in eradicating MRD in HCL.

Significant efficacy of 2-CdA with or without rituximab in the treatment of splenic marginal zone lymphoma (SMZL)

BACKGROUND Splenic marginal zone lymphoma (SMZL) with or without villous lymphocytes is an indolent lymphoma that typically affects elderly patients. Treatment is required in symptomatic cases. Splenectomy remains one of the first-line options in patients fit for surgery. The best therapeutic strategy has not yet been identified. Among different possible chemotherapeutic approaches, purine analogues, alone or in association with rituximab, seem to be a valid therapeutic choice. PATIENTS AND METHODS Fifty SMZL patients were treated with cladribine with or without anti-CD20 mAb. RESULTS Forty-six of 50 patients were assessable for response. Overall response rate was 87%: 24 of 46 patients (52%) achieved a complete hematological response (CR), 16 of 46 (35%) a partial response and 6 (13%) were unresponsive. Interestingly, 15 of 24 cases (62%) in CR also achieved a molecular remission. CONCLUSIONS The present results indicate that this schedule is a valid therapeutic approach in SMZL. Addition of rituximab significantly improved quality of response and consequently the outcome of the disease.

Rituximab as treatment for minimal residual disease in hairy cell leukaemia: extended follow-up

Hairy cell leukaemia (HCL) is a rare chronic lymphoproliferative disorder accounting for about 2% of adult leukaemias. Malignant cells present features of mature activated B-lymphocytes with bright expression of light-chain-restricted surface immunoglobulin (sIg), expression of numerous clonally related heavy-chain isotypes, IGHV mutation and co-expression of mature B cell CD markers. Treatment is indicated in the presence of significant cytopenia, symptomatic splenomegaly, recurrent infection or systemic symptoms. In recent years, therapeutic advances have brought about drastic changes in HCL prognosis. Purine analogues, especially 2-chlorodeoxyadenosine (Cladribine), drastically improved the survival rate, although complete eradication of disease can be rarely achieved (Piro et al, 1990; Tallman et al, 1996). Minimal residual disease (MRD) can be detected either by polymerase chain reaction (PCR) or immunohistochemical techniques on bone marrow samples of patients in complete haematological remission after purine analogue therapy. The persistence of MRD could thus explain the high relapse rate observed in HCL patients after long-term follow-up from the end of induction therapy (Wheaton et al, 1996). Rituximab could represent one attractive option in relapsed cases, because hairy cells typically express high levels of CD20 (Juliusson et al, 1994; Hoffman & Auerbach, 2000; Hagberg & Lundholm, 2001). The optimal schedule has not yet been defined, but some data suggest that extended dosing may improve patient outcome (Thomas. et al, 2003). Moreover, Rituximab could play an interesting role even in eradicating MRD (Cervetti et al, 2004; Ravandi et al, 2006). In our Institution, between May 2002 and November 2006, 27 HCL patients (25 male and 2 female; median age 60 years, range 39–72 years) were treated with anti-CD20 after a pretreatment with Cladribrine. Patients were considered eligible for the study if in partial remission or with persistence of MRD after induction. Other inclusion criteria were: age >18 years; life expectancy >6 months; absence of renal, hepatic and respiratory failure; written informed consent. Baseline clinical characteristics of patients are summarized in Table I. Cladribrine was started if patients presented at least one of the following indications: neutropenia (absolute neutrophil count, <1Æ5 · 10/l), anaemia (haemoglobin level, <100 g/l), thrombocytopenia (platelet count, <100 · 10/l), symptomatic splenomegaly, constitutional symptoms or documented repeated infections. In 17 patients older than 60 years, Cladibrine was administered IV at a dose of 5 mg/m/weekly for a total of six cycles, whereas in 10 younger patients, Cladribine was infused at the dose of 5 mg/m/d for five consecutive days. All patients received acyclovir, fluconazole and cotrimoxazole as antiinfective prophylaxis until 8 weeks after completion of Cladribine administration. Two months after the end of treatment, patients were evaluated for early response. Response criteria were those defined by the National Cancer Institute Working Group. At the time of enrolment in the study, overall response after Cladribrine was 89% [complete remission (CR) 26%, partial remission (PR) 63%; three cases were unresponsive]. The median time between the last Cladribine infusion and the beginning of this protocol was 4Æ3 months (range 1Æ5– 113 months). Minimal residual disease was evaluated by repeating PCR assays. PCR analyses were performed on DNA extracted from mononuclear cells separated by Ficoll/Hypaque gradient, as previously reported (Cervetti et al, 2004). By this method, all cases were proven to be still PCR-positive after induction. After demonstration of persistent MRD or detectable clinical disease, patients were treated by Rituximab (375 mg/m once a week for four doses). Statistical calculations were performed using the Statistical Package for the Social Sciences (spss) for Windows, release 15

Significant efficacy of 2-chlorodeoxyadenosine± rituximab in the treatment of splenic marginal zone lymphoma (SMZL): extended follow-up

BACKGROUND Splenic marginal zone lymphoma with or without villous lymphocytes (SLVL/SMZL) is an indolent lymphoma that typically affects elderly patients and that has a median survival >10 years. It presents with marked splenomegaly. Treatment is required in symptomatic cases. Splenectomy remains one of the first-line options in patients fit for surgery. The best pharmacological strategy has not yet been identified for poor surgical risk cases. Among different possible chemotherapeutic approaches, purine analogs, alone or in association with Rituximab, seem to be a valid therapeutic choice. PATIENTS AND METHODS Fifty SMZL patients were treated with Cladribine ± anti-CD20 monoclonal antibody. RESULTS Forty-seven of 50 patients were evaluable for response. ORR was 87%: 24 of 47 patients (51%) achieved a complete hematological response (CR), 17 of 47 (36%) a partial response (PR) and 6 (13%) resulted unresponsive. Interestingly, 15 of 24 cases (62%) in CR achieved also a molecular remission. After a median follow-up of 48 months, 7 of 41 responsive cases relapsed and the 5-year PFS was 80%. CONCLUSIONS These data confirm the efficacy of this schedule emphasizing the impact of minimal residual disease even in the outcome of SMZL patients.

1,25‐DIHYDROXY‐VITAMIN D3 (1,25(OH)2D3) IN THE TREATMENT OF 1DIOPATHIC MYELOFIBROSIS

It has been recently reported that 1,25(OH)~Dj may play a therapeutic role in idiopathic myelofibrosis. McCarthy et aZ(1984) suggested that 1,25(OH)~D3 inhibits the proliferation of megakaryocytes which promote collagen synthesis and increase the number and activity of macrophages that mediate the degradation of fibrous tissue. Some attempts to treat marrow fibrosis by vitamin Dj have been reported: three patients affected by thrombocythaemia, sub-acute myelomonocytic leukaemia and acute myelofibrosis were succesfully treated (Arlet et al, 1984), and two patients who had myelofibrosis (which in one case followed PRV) became polycythaemic (Lane et aI, 1985). On the other hand in a recent publication in this Journal, a 6-month course of 1,2 5(OH)2D3 in four patients affected by idiopathic myelofibrosis was without effect (Richard et al, 1986). Even if, as the authors suggest, the response to 1,25(OH)& in myelofibrosis is not uniform, and other regimens (in which the dose is alternated) might have some practical effect on the reversibility of myelofibrosis, we fear that such reports may discourage more extensive studies in this area. For this reason we describe now the preliminary results of an open study on the 1,25(OH)2D3 administration in five patients affected by chronic idiopathic myelofibrosis and in one by hairy cell leukaemia. The diagnosis was made on the basis of clinical examination, laboratory tests including LDH, full blood counts, bone marrow aspiration (when this did not result in ‘punctio sicca’) and bone marrow biopsy. In six cases ferrokinetics was also evaluated: in accordance with the diagnosis the half-time and the plasma iron turnover were increased the iron utilization was reduced: the uptake of the iron in the spleen was increased whereas the uptake of the iron in the bone marrow was reduced. 1 pg of 1,2 5(0H)2D3 was administrated and the values of Hb, RBC, platelets and LDH were evaluated weekly. Kidney function as well as the level of calcium and phosphorus were monitored. No diet restriction was imposed. The results are described in Table I. The patients

Quantitative molecular evaluation of minimal residual disease in patients with chronic lymphocytic leukemia: efficacy of in vivo purging by alemtuzumab (Campath-1H).

Although novel therapies for chronic lymphocytic leukemia have resulted in higher hematologic response rates, the complete eradication of disease rarely occurs. Alemtuzumab (Campath-1H) seems to be extremely effective in this role in pretreated patients. The authors used a molecular semiquantitative polymerase chain reaction (PCR) method to assess the ability of alemtuzumab to induce PCR negativity in eight patients pretreated with fludarabine. IgH rearrangement was coamplified with a housekeeping gene and fluorescent PCR products were analyzed on a DNA automatic sequencer. Each patient was evaluated at diagnosis, after fludarabine, and after Campath-1H. The median interval between the last therapy course with fludarabine and the start of Campath-1H was 14 weeks. Patients received subcutaneous doses up to 10 mg, three times a week, for 12 weeks, with a median dose of 190 mg. After six cycles with fludarabine, only one patient (12.5%) achieved molecular remission, and in three other patients IgH levels decreased by 0.5 to 1 log. At the beginning of Campath-1H administration, all patients were PCR positive, including the one previously found to be negative. At the end of treatment, five patients achieved molecular remission (62.5%), four of them within 1 month after the end of therapy. Seventy-two percent of responses, with 43% of complete responses, were documented on bone marrow smears. A significant reduction of lymph node and spleen diameters was noted in 50% and 33% of patients, respectively. Four patients showed grade 2 skin reaction at the site of the subcutaneous injection and grade 1 or 2 fever. Two patients developed neutropenia (grade 2 and 3) and two hemolytic episodes. Three patients showed cytomegalovirus and one herpes zoster and Epstein-Barr virus reactivation. These results show that Campath-1H represents an efficacious in vivo purging tool with a safe profile.

Role of Low-Dose 2-CdA in Refractory or Resistant Lymphoplasmocytic Lymphoma

Abstract Cladribrine (2-CdA), a purine analogue active on both dividing and resting lymphocytes, plays an important role in the treatment of indolent lymphoproliferative malignancies such as Hairy Cell Leukemia (HCL), Chronic Lymphocytic Leukemia (CLL), Lymphoplasmocytic Lymphoma (LPL), Waldenströms Macroglobulinemia (WM). With the aim of evaluating the efficacy and toxicity of low dose 2-CdA, 15 lymphoplasmocytic lymphoma patients, not eligible for more aggressive or standard therapies, because of age or poor performance status, were treated with the drug at a dose of 5 mg/m2, once a week for six total courses. All patients showed disease progression. Fourteen patients were valuable for response. In eleven out of these 14 (85.7%) disease progression stopped, with 21% having good hematological responses (one CR and two PR). The treatment was generally well tolerated, without serious infectious events. This schedule may be appropriate for the management of patients where the aim of the treatment is control of disease progression.

Efficacy and Toxicity of Liposomal Daunorubicin Included in PVABEC Regimen for Aggressive NHL of the Elderly

Liposomes used for delivering antineoplastic drugs to sites of disease are able to minimize side effects and enhance therapeutic efficacy. Liposomal Daunorubicin (Daunoxome ® ; DNX) has a selective and higher accumulation in neoplastic tissues and seems to be able to escape Multi-Drug Resistance (MDR). We treated 35 elderly patients with aggressive non-Hodgkins lymphoma (NHL) with PVBECDNX, a regimen analogue to P-VABEC in which doxorubicin is replaced with DNX at a dose of 50 mg (first 13 patients) and thereafter 50 mg/m 2. Twenty-six out of 35 patients were evaluable for response; 15 obtained a CR, 5 a PR (overall response rate of 77%). After a median follow-up of 13 months the 2-years actuarial overall survival was 75% and the failure-free survival was 71%. Two patients out of six no responders died because of progression of disease, and one died in CR because of pre-existing cardiovascular disorders. Eight patients did not tolerate DNX infusion (back pain). Non-haematological toxicity was mild. This study confirms that PVABEC-like regimens are able to induce a high overall response rate in a percentage of patients affected by aggressive lymphoma and shows that DNX is as effective as Daunorubicin in these disorders, but its acute toxicity is reduced.

The Droplet Digital PCR: A New Valid Molecular Approach for the Assessment of B-RAF V600E Mutation in Hairy Cell Leukemia

Hairy cell leukemia (HCL) is a chronic lymphoproliferative B-cell disorder where the B-RAF V600E mutation has been recently detected, as reported for solid neoplasias but not for other B-cell lymphomas. The digital droplet PCR (dd-PCR) is a molecular technique that, without standard references, is able to accurately quantitate DNA mutations. ddPCR could be an useful instrument for the detection of the B-RAF V600E mutation in HCL, where the minimal residual disease monitoring is fundamental for planning a patients-targeted treatment in the era of new anti-CD20 and anti-RAF compounds. This retrospective study enrolled 47 patients observed at the Hematology Unit of the University of Pisa, Italy, from January 2005 to January 2014: 27 patients were affected by “classic” HCL, two by the variant HCL (vHCL), and 18 by splenic marginal zone lymphoma (SMZL). The aim of the study was to compare dd-PCR to “classic” quantitative PCR (QT-PCR) in terms of sensitivity and specificity and to demonstrate its possible use in HCL. Results showed that: (1) the sensitivity of dd-PCR is about half a logarithm superior to QT-PCR (5 × 10-5 vs. 2.5 × 10-4), (2) the specificity of the dd-PCR is comparable to QT-PCR (no patient with marginal splenic lymphoma or HCL variant resulted mutated), (3) its high sensitivity would allow to use dd-PCR in the monitoring of MRD. At the end of treatment, among patients in complete remission, 33% were still MRD-positive by dd-PCR versus 28% by QT-PCR versus 11% by the evaluation of the B-cell clonality, after 12 months, dd-PCR was comparable to QT-PCR and both detected the B-RAF mutation in 15% of cases defined as MRD-negative by IgH rearrangement. Moreover, (4) the feasibility and the costs of dd-PCR are comparable to those of QT-PCR. In conclusion, our study supports the introduction of dd-PCR in the scenario of HCL, also during the follow-up.