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The recently revised World Health Organization (WHO) classification of myeloid neoplasms recognizes prefibrotic myelofibrosis (prePMF) as a distinct entity, characterized by well-defined histopathologic features together with minor clinical criteria (leukocytes, anemia, increased LDH, splenomegaly). The aim of the study was to examine the clinical relevance of distinguishing prePMF from essential thrombocythemia (ET). We identified in our database all patients affected with ET, prePMF and primary myelofibrosis (PMF) diagnosed according to 2008 WHO criteria with a bone marrow fibrosis grade 0-1 at diagnosis and one DNA sample to define the mutational status. The bone marrow morphology of all 404 identified patients was reviewed by an expert pathologist and patients were reclassified according to the 2016 WHO criteria. After reclassification, our cohort included 269 ET, 109 prePMF, and 26 myeloproliferative neoplasm unclassificable. In comparison with ET, patients with prePMF had higher leukocyte count, lower hemoglobin level, higher platelet count, higher LDH values, and higher number of circulating CD34-positive cells; they showed more frequently splenomegaly (all P values < ·001). CALR mutations were more frequent in prePMF than in ET (35·8% vs 17·8%, P < ·001). PrePMF patients had shorter overall survival (P < ·001) and a trend to a higher incidence of leukemic evolution (P ·067) compared to ET patients, while they did not differ in terms of thrombotic and bleeding complications. In conclusion, ET and prePMF diagnosed according to 2016 WHO criteria are two entities with a different clinical phenotype at diagnosis and a different clinical outcome.

// Chiara Cavalloni 1, * , Elisa Rumi 1, 2, * , Virginia V. Ferretti 2 , Daniela Pietra 2 , Elisa Roncoroni 2 , Marta Bellini 1 , Michele Ciboddo 1 , Ilaria C. Casetti 1 , Benedetta Landini 2 , Elena Fugazza 2 , Daniela Troletti 2 , Cesare Astori 2 , Mario Cazzola 1, 2 1 Department of Molecular Medicine, University of Pavia, Pavia, Italy 2 Department of Hematology Oncology, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico San Matteo, Pavia, Italy * These authors contributed equally to this work Correspondence to: Elisa Rumi, email: elisarumi@hotmail.com , elisa.rumi@unipv.it Keywords: myeloproliferative, burden, CALR, JAK2, sequential Received: January 21, 2017 Accepted: March 24, 2017 Published: April 03, 2017 ABSTRACT We investigated the variation of CALR -mutant burden during follow-up in 105 CALR -mutant MPN and compared it to the variation of JAK2 -mutant burden in 226 JAK2 -mutant MPN. The median allele burden at last evaluation was significantly higher than at first evaluation in essential thrombocythemia (ET) (49.5% vs 45%, P < .001) but not in primary myelofibrosis (PMF) (52% vs 51%, P 0.398). Median values of slope were positive both in ET (0.071) and in PMF (0.032). In CALR -mutant ET there was a difference between natural and therapy-related slope ( P 0.006). In the JAK2 -mutated cohort, the median allele burden at last evaluation was not different respect to that at first evaluation, neither in ET (22.9% vs 23.2%, P = 0.216) nor in PMF (50.5% vs 45.0%, P = 0.809), despite a positive slope. Multivariate analysis to evaluate the effect of mutation ( CALR vs JAK2 ) on the slope of mutant burden in not treated pts with a positive slope adjusting for diagnosis (ET vs PMF) showed a trend toward a higher increase of mutant burden in CALR vs JAK2 (β = 0.19, P = 0.061) with no difference between diagnosis ( P = 0.419). The findings of this study suggest that clonal expansion in CALR-mutant MPN is faster than that observed in JAK2 -mutant MPN.

The 2016 WHO classification comprises two stages of primary myelofibrosis (PMF): early/prefibrotic primary myelofibrosis (pre-PMF) and overt fibrotic PMF (overt PMF). Diagnostic criteria rely on bone marrow morphology, fibrosis grade (0-1 in pre-PMF, 2-3 in overt PMF), and clinical features (leukoerythroblastosis, anemia, leucocytosis, increased lactate dehydrogenase, and palpable splenomegaly). An accurate differentiation from essential thrombocythemia (ET) is pivotal because the two entities show different clinical presentation and outcome, in terms of survival, leukemic evolution, and rates of progression to overt myelofibrosis. Areas covered: The current review provides an overview on how to diagnose and stratify patients with pre-PMF, taking into account their definite and peculiar risk of vascular event, which is often neglected, and their milder disease course, compared with overt PMF, with the aim of improving and individualizing their counseling and management. Expert commentary: Pre-PMF is a new entity characterized by a unique combination of both a thrombo-hemorrhagic risk (that brings it closer to PV and ET) and a definite risk of disease evolution (that places pre-PMF somewhat closer to the overt PMF variant).

A quarter of patients with essential thrombocythemia or primary myelofibrosis carry a driver mutation of CALR, the calreticulin gene. A 52-bp deletion (type 1) and a 5-bp insertion (type 2 mutation) are the most frequent variants. These indels might differentially impair the calcium binding activity of mutant calreticulin. We studied the relationship between mutation subtype and biological/clinical features of the disease. Thirty-two different types of CALR variants were identified in 311 patients. Based on their predicted effect on calreticulin C-terminal, mutations were classified as: (i) type 1-like (65%); (ii) type 2-like (32%); and (iii) other types (3%). Corresponding CALR mutants had significantly different estimated isoelectric points. Patients with type 1 mutation, but not those with type 2, showed abnormal cytosolic calcium signals in cultured megakaryocytes. Type 1-like mutations were mainly associated with a myelofibrosis phenotype and a significantly higher risk of myelofibrotic transformation in essential thrombocythemia. Type 2-like CALR mutations were preferentially associated with an essential thrombocythemia phenotype, low risk of thrombosis despite very-high platelet counts and indolent clinical course. Thus, mutation subtype contributes to determining clinical phenotype and outcomes in CALR-mutant myeloproliferative neoplasms. CALR variants that markedly impair the calcium binding activity of mutant calreticulin are mainly associated with a myelofibrosis phenotype.

Patients with essential thrombocythemia may carry JAK2 (V617F), an MPL substitution, or a calreticulin gene (CALR) mutation. We studied biologic and clinical features of essential thrombocythemia according to JAK2 or CALR mutation status and in relation to those of polycythemia vera. The mutant allele burden was lower in JAK2-mutated than in CALR-mutated essential thrombocythemia. Patients with JAK2 (V617F) were older, had a higher hemoglobin level and white blood cell count, and lower platelet count and serum erythropoietin than those with CALR mutation. Hematologic parameters of patients with JAK2-mutated essential thrombocythemia or polycythemia vera were related to the mutant allele burden. While no polycythemic transformation was observed in CALR-mutated patients, the cumulative risk was 29% at 15 years in those with JAK2-mutated essential thrombocythemia. There was no significant difference in myelofibrotic transformation between the 2 subtypes of essential thrombocythemia. Patients with JAK2-mutated essential thrombocythemia and those with polycythemia vera had a similar risk of thrombosis, which was twice that of patients with the CALR mutation. These observations are consistent with the notion that JAK2-mutated essential thrombocythemia and polycythemia vera represent different phenotypes of a single myeloproliferative neoplasm, whereas CALR-mutated essential thrombocythemia is a distinct disease entity.

To the editor: Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis, have in most instances a sporadic occurrence, but familial clustering of MPNs has been reported and familial cases are about 7% to 8% of all MPN patients

Summary Splenic marginal zone B-cell lymphoma is characterized by high genetic heterogeneity, and hepatitis C virus infection seems to be involved in a subset of patients. The aims of the analysis were to identify potential genetic alterations related to hepatitis C virus status, IgV H gene mutational status, and prognostic categories identified in a multicenter study ( Blood 2006;107:4643). Genome-wide array comparative genomic hybridization at a 100-kilobase (kb) resolution was performed in 34 patients with splenic marginal zone B-cell lymphoma, 12 of whom were hepatitis C virus positive. Array-comparative genomic hybridization experiments revealed no copy number alterations in 10 patients (4 were hepatitis C virus positive). A median of 5.6 and 3.8 copy number alterations were detected in hepatitis C virus–positive and in hepatitis C virus–negative patients, respectively. The most frequent copy number alterations involved chromosomes 7 and 17 (21% and 24%, respectively). Except for Xp gain ( P = .01), no differences in common alterations were found between hepatitis C virus–positive and hepatitis C virus–negative cases. Unmutated status of the IgV H gene was related to del(7q) ( P = .04) and dup(12q) ( P = .03). The high-risk group identified according to the new splenic marginal zone B-cell lymphoma prognostic score was associated with del(7q) ( P = .01) and del(17p) ( P = .02). Hepatitis C virus–positive splenic marginal zone B-cell lymphoma patients have no specific chromosome alterations. Patients with poor prognosis are characterized by distinctive imbalances.

Immunoglobulin gene usage and somatic mutation patterns were studied in 59 patients with splenic marginal zone lymphoma and were correlated with clinical characteristics. Fifty-nine IGHV rearrangements were amplified. IGHV1, IGHV3, and IGHV4 subgroups accounted for 30%, 56%, and 14% of sequences, respectively. IGHV genes most frequently used were IGHV1-2 (n=12), IGHV3-23 (n=15), IGHV3-30 (n=7) and IGHV4-34 (n=5). IGHV was unmutated in 25%. Villous lymphocytes >10% were detected in 50% of patients belonging to the IGHV1-2 group, in 21% of the IGHV3-23 group, and in no patient of the IGHV3-30 group (p=0.05). Liver involvement was present in 50% of the IGHV3-30 group, in 9% of the IGHV3-23 group, and in no patient of the IGHV1-2 group (p=0.04). HCV-serology was positive in 50% of the IGHV3-30 group, in 7% of the IGHV3-23 group, and in 17% of the IGHV1-2 group (p=0.04). The proportion of intermediate and high risk patients according to the SMZL score was higher in the unmutated respect to the mutated group (69% vs 32%, p=0.05). In conclusion, IGHV rearrangement analysis in splenic marginal zone B-cell lymphoma reveals a non-random preference for use of IGHV1-2, IGHV3-23 and IGHV3-30 genes, whose presence differs according to clinical features and prognostic category.

We report a case of a patient with myelofibrosis with myeloid metaplasia (MMM) who presented with progressive dyspnea of unexplained origin. Splenomegaly, blood smear, and bone marrow findings allowed diagnosis of MMM. High-resolution CT chest scan revealed diffuse septal thickening, while echocardiography and electrocardiogram showed no indirect evidence of pulmonary hypertension. Finally, lung biopsy revealed irregularly distributed interstitial fibrosis with islands of erythroblasts, immature granulocytic elements, and dysplastic megakaryocytes, allowing diagnosis of pulmonary extramedullary hematopoiesis (EMH). The patient received hydroxyurea as cytoreductive agent, obtaining a good hematologic response and an improvement of dyspnea. Note that, in this patient, dyspnea was the first clinical symptom of MMM; the dyspnea was not associated with pulmonary hypertension and improved following cytoreductive treatment. This case points to the importance of suspecting pulmonary EMH when unexplained progressive dyspnea occurs in a patient with MMM. Early recognition of pulmonary EMH may prevent PH and favor a better response to therapy.

One-third of patients with monoclonal gammopathy of undetermined significance (MGUS) may progress to multiple myeloma (MM) and may develop a long arm deletion of chromosome 13 (13q-). As the incidence of 13q-, time of development and prognostic impact in MGUS patients is still under debate, we decided to perform serial sequential conventional cytogenetics (CC) and metaphase/interphase fluorescence in situ hybridization (FISH) analyses on bone marrow mononuclear cells obtained from 18 asymptomatic, untreated MGUS patients. Median follow up was 30 months (range 6-72). Interphase FISH identified a 13q14 deletion in five out of 18 patients (on clinical diagnosis in one patient and during the follow up in the remaining four patients). Subsequently, metaphase FISH and CC also identified the deletion in four out of five patients. All five of the patients progressed to MM 6-12 months after 13q- identification, without developing any FISH determined JH rearrangements. MM progression also occurred in two other karyotypically normal patients. We conclude that: (i) the extent of the 13q deletion does not vary during the clinical outcome; (ii)13q- plays a crucial role in MGUS/MM pathogenesis and confers a proliferative advantage to clonal plasma cells being initially demonstrated by interphase FISH and only afterwards by metaphase FISH and CC; and (iii) association of 13q- with t(4;14)(p16.3;q32) remains to be demonstrated. However, a transition from MGUS to MM may also occur in patients with normal karyotypes or other abnormalities, suggesting the possibility of distinct pathogenetic pathways.

Summary. Essential thrombocythaemia (ET) is a disease associated with an elevated risk of thrombosis. This study evaluated the efficacy and safety of pipobroman (PB) in the long-term control of ET patients who had, at diagnosis, one or more of the following currently known risk factors for thrombosis or haemorrhage (high-risk patients): age > 60 years, history of thrombosis or haemorrhage, platelets > 1000 × 109/l. From 1978 to 2000, with a median follow-up of 10 years, 118 previously untreated high-risk ET patients (median age 62 years, range 25–82), were treated with PB at the starting dose of 0·8–1 mg/kg/d. All patients reached a platelet count

We describe a 73-year-old man diagnosed with acute myelomonocytic leukemia (AML-M4) following myelodysplasia with trisomy 11 and with a t(11;11;22). This is the first case with both abnormalities present in the same cells and with the t(11;11;22) involving a chromosome 11 already duplicated at 11q23. This band contains the MLL gene that undergoes partial tandem duplication in patients with +11, which is "promiscuous," being translocated with a large number of genetic partners. Our patient had a complex karyotype that was completely defined by in situ hybridization. This technique demonstrated that the t(11;11;22) derivative with a duplication of band 11q23 carried from three to four copies of MLL. Two copies of the gene were close to each other and centromeric to the break-point region. Therefore, a partial tandem duplication of the MLL gene might have happened before the occurrence of t(11;11;22). Considering the associated chromosome defects, the monosomy for the long arm of chromosome 7, due to an unbalanced translocation t(7;17), further underlines the possibility that a partial tandem duplication of the MLL gene might have taken place.

Fludarabine is effective in chronic lymphocytic leukaemia (CLL) and low-grade non-Hodgkin's lymphoma (NHL). A major side-effect of this purine analogue is immunosuppression which may favour opportunistic infections. Additionally, impairment of immunosurveillance might promote Epstein-Barr virus (EBV) reactivation and possibly favour transformation to high-grade malignancy. The aim of this study was to evaluate the immunosuppression-related effects of the fludarabine-based combination Flucyd in advanced low-grade NHL or CLL by serially monitoring T-lymphocyte subsets, opportunistic infections, EBV-reactivation, and histologic transformation. 24 patients with advanced NHL (n = 21) or CLL (n = 3) received fludarabine 25 mg/m2/d + cyclophosphamide 350 mg/m2/d + dexamethasone 20 mg/d in 3 d courses for a maximum of six courses. The overall response rate was 79% (eight CR, 11 PR, five failures); 11 patients relapsed or progressed between 3 and 19 months from response, and eight are in CR or PR at 3-27 months. The CD4+ lymphocyte counts decreased significantly during therapy from a median of 484/microliter pre-treatment (range 142-1865) to a median of 198/microliter (71-367). In 19 responders monitored off therapy every 3 months until relapse/progression, CD4+ counts were persistently low with minimal recovery over time. During treatment, 16 infections occurred in 11/24 patients. No delayed opportunistic infections occurred in responders while off therapy. The circulating EBV DNA load serially measured in 19 patients by a quantitative PCR assay showed an increase in four patients during treatment. A lymph node biopsy performed in two of these was PCR positive for EBV DNA, whereas LMP1 and EBERs were negative. Six NHL patients evolved into high-grade B-cell NHL. In conclusion, fludarabine combined with cyclophosphamide and dexamethasone is an effective therapy for recurrent indolent lymphoma. This combination produces prolonged T-lymphocytopenia and has the potential to reactivate a latent EBV infection. T-cell dysfunction, however, is not associated with higher incidence of clinical opportunistic infections and does not adversely influence clinical outcome.

Complete or partial monosomy for the long arms of chromosomes 5 or 7 or both is frequently observed in therapy-related myelodysplastic syndromes and acute nonlymphocytic leukemia. Sporadic cases have been reported in which partial monosomy is due to unbalanced translocations. The patient described herein carries one such rearrangement. 46,XY,t(1;2) (q32;p23),del(5)(q13),der(7)(5qter-->5q22::7p15-->7 q21:),del(12)(p12), resulting in partial monosomy for the long arms of chromosomes 5 and 7 and in partial monosomy for the short arm of chromosome 7.

Somatic mutations in the calreticulin (CALR) gene were recently discovered in patients with sporadic essential thrombocythemia (ET) and primary myelofibrosis (PMF) lacking JAK2 and MPL mutations. We studied CALR mutation status in familial cases of myeloproliferative neoplasm. In a cohort of 127 patients, CALR indels were identified in 6 of 55 (11%) subjects with ET and in 6 of 20 (30%) with PMF, whereas 52 cases of polycythemia vera had nonmutated CALR. All CALR mutations were somatic, found in granulocytes but not in T lymphocytes. Patients with CALR-mutated ET showed a higher platelet count (P = .017) and a lower cumulative incidence of thrombosis (P = .036) and of disease progression (P = .047) compared with those with JAK2 (V617F). In conclusion, a significant proportion of familial ET and PMF nonmutated for JAK2 carry a somatic mutation of CALR.

We evaluated the presenting clinical characteristics and outcome of 68 patients with nodular lymphocyte predominance Hodgkin's disease (NLP-HD), in order to delineate the pattern of evolution of the disease. The male to female ratio was 46/22 and median age 35 yrs (range, 14-86). Eight patients had a history of benign hyperplasia on lymph node biopsies performed 6 to 36 months before the diagnosis of NLP-HD. Early stage disease accounted for 75% of cases. One patient had a coexistent non-Hodgkin's lymphoma (NHL). Treatment was as follows: radiotherapy in 26, chemotherapy in 23, combined modality in 19. CR rate was 93% (63/68). 18 patients relapsed as HD and 5 developed NHL. The cumulative risk of NHL was 9% at 10 yrs. During remission, 4 patients had 5 episodes of follicular hyperplasia histologically documented. Overall survival rate was 71% at 10 yrs and and 63% at 15 yrs. Freedom from progression (FFP) declined from 67% at 5 yrs to 45% at 10 yrs, because of late relapses. Localized disease predicted for a better FFP (p = 0.01), but was not associated with a reduced risk of recurrence over time. NLP-HD is characterized by an indolent course with a constant pattern of relapse over time, also in patients with early stage disease at diagnosis. In addition to relapse as NLP-HD, patients may evolve into a NHL or develop benign lymph nodal hyperplasia. Careful long-term follow up is needed for these patients.

We studied mutations of MPL exon 10 in patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF), first investigating a cohort of 892 consecutive patients. MPL mutation scanning was performed on granulocyte genomic DNA by using a high-resolution melt assay, and the mutant allele burden was evaluated by using deep sequencing. Somatic mutations of MPL, all but one involving codon W515, were detected in 26/661 (4%) patients with ET, 10/187 (5%) with PMF, and 7/44 (16%) patients with post-ET myelofibrosis. Comparison of JAK2 (V617F)-mutated and MPL-mutated patients showed only minor phenotypic differences. In an extended group of 62 MPL-mutated patients, the granulocyte mutant allele burden ranged from 1% to 95% and was significantly higher in patients with PMF or post-ET myelofibrosis compared with those with ET. Patients with higher mutation burdens had evidence of acquired copy-neutral loss of heterozygosity (CN-LOH) of chromosome 1p in granulocytes, consistent with a transition from heterozygosity to homozygosity for the MPL mutation in clonal cells. A significant association was found between MPL-mutant allele burden greater than 50% and marrow fibrosis. These observations suggest that acquired CN-LOH of chromosome 1p involving the MPL location may represent a molecular mechanism of fibrotic transformation in MPL-mutated myeloproliferative neoplasms.

The clinical and hematological characteristics of 5 patients affected with both acute myeloid leukemia (AML) and diabetes insipidus (DI) are described. Banded chromosomal analysis demonstrated monosomy 7 in 2 patients and a complex cytogenetic rearrangement in another. No patient entered complete remission with standard induction chemotherapy. These data confirm that in patients with AML, the association of DI (with or without monosomy 7) is an unfavorable prognostic factor.

One hundred eighty-eight unselected consecutive patients with "de novo" myelodysplastic syndrome (MDS) were studied cytogenetically. They were subclassified as 4 refractory anemia with ringed sideroblasts (RARS), 67 refractory anemia (RA), 58 refractory anemia with excess of blasts (RAEB), 40 RAEB in transformation (RAEB-t), and 19 chronic myelomonocytic leukemia (CMML). The overall incidence of chromosome abnormalities was 69%. The RAEB and RAEB-t patients showed karyotypic changes, more often than RA and CMML (76% and 100% vs. 56% and 42%, respectively). The most frequent single anomaly was del(5)(q13-q22q33) (22 cases), followed by monosomy 7 or del 7q (11 cases), del(11) (q14q23) (8 cases), trisomy 8 (4 cases). Complex karyotypes (defined by the presence of three or more structural or numerical abnormalities) were detected in 33 patients. With regard to the FAB classification, del (5)(q13q33) was associated with RA, and complex rearrangements with RAEB and RAEB-t. Leukemic transformation occurred in 66 patients (46%), none with a normal karyotype or del(11)(q14q23) as single abnormality. In patients carrying 5q- alone, acute evolution correlated with proximal breakpoint localization, being found in no case with del(5)(q13q33) but in three out of four cases with del(5)(q22q33). Acute leukemia (AL) progression happened in all cases with complex rearrangements and monosomy 7 or del(7q). Two of the four trisomy eight patients evolved in AL. By using the Cox proportional hazard regression analysis it was demonstrated that the karyotype abnormality was a significant predictor of leukemic transformation (P < 0.001). Patients with abnormal karyotypes without complex abnormalities had a survival (median survival 12 months) shorter than that of cases with only normal metaphases (median 83 months) (P < 0.001); patients with a mixture of normal/abnormal metaphases had a median survival of 31 months. The median survival for complex karyotypes was 7 months. Among cases with single defects, del(5)(q13q33) showed the best survival (64 months), monosomy 7 and del(7q) the worst (7 months) (P < 0.001).

Summary Background A prospective study was conducted to assess the efficacy and toxicity of a salvage regimen consisting of CCNU, Melphalan, and VP-16 (CAV) given at 28-day intervals in patients with Hodgkin's disease (HD) relapsing after primary therapy or refractory to the alternating MOPP/ABVD regimen. Patients and methods This study included 58 patients (median age: 34 years), with resistant or relapsing HD. Primary therapy had consisted of alternating MOPP/ABVD (81%) or MOPP alone (19%); 38% of patients were relapsing from prior complete remission (CR) while 62% had resistant disease. Extranodal disease was present in 55% and B-symp-toms in 72% of patients; one-fifth had bulky disease and/or bone marrow involvement. The CAV was used as first salvage in half of the patients. Results Complete remission was obtained in 17 patients (29%); unfavorable factors for CR in univariate analysis were the presence of bulky disease and the failure to achieve CR with prior therapy. Nine patients (53% of remitters) have subsequently relapsed with a 10-month median duration of CR. The 3-year overall survival after CAV was 25% with an 18-month median survival; significant differences in survival were found according to the extent of disease, the presence of B-symptoms and the HD status (prior sensitive or resistant disease, first or subsequent relapse). Seven patients are long-term remitters (12%), and one of them has been given high-dose chemotherapy and autologous bone marrow transplantation at relapse after CAV. The CAV toxicity was mostly hematological; severe pancytopenia occurred in six cases with two cases of fatal infections and one of fatal hemorrhage. Conclusion CAV therapy was moderately effective as third-line salvage in patients with HD resistant to alternating MOPP/ABVD or previously given two different regimens for relapse; the toxicity was mostly hematological and supportive therapy was needed in one-third of the patients.

Summary Background A prospective study was conducted to assess (a) the long-term results and toxicity of the alternating MOPP/ABVD regimen in advanced Hodgkin's disease; (b) the prognostic value of pretreatment variables and of drug dose intensity. Patients and methods A total 138 consecutive patients with advanced Hodgkin's disease entered this study; patient selection included stages IIB (33% of total), IIIB (26%), IV (25%), and stages EA-IIIA (16%) with bulky disease and pulmonary hilum involvement. The MOPP/ABVD program was delivered in an 8-month program; adjuvant radiotherapy on sites of bulky disease was delivered in 24 patients. Results Complete remission was obtained in 106 (77%) patients; significant factors for CR in univariate analysis were stage, symptoms, histology, and bone marrow involvement. The five-year relapse-free survival (RFS) was 83%; in a multivariate analysis, histology only correlated with RFS (p = 0.04). The five-year freedom from tumor mortality and overall survival (OS) were 79% and 67%, respectively. An adverse prognostic significance for OS was observed for B symptoms and bone marrow involvement. The median percentage of relative dose intensity (RDI) was as follows: Adriamycin 86, mechlorethamine 85, vincristine 73, vinblas-tine 84, bleomycin 79, procarbazine 74, dacarbazine 81. No significant association was found between RDI and clinical outcome. No severe pancytopenia or life-threatening complications occurred during therapy. Conclusions Alternating MOPP and ABVD cured more than 65% of patients with advanced HD; acute and late tox-icity were acceptable. Prognostic analysis defined subgroups with a lower chance of cure which may deserve a more intensive initial therapy.

TET2 haplo-insufficiency occurs through different molecular mechanisms and is promptly revealed by array comparative genomic hybridization, single nucleotide polymorphism (SNP) array, and next-generation sequencing (NGS). Fluorescence in situ hybridization (FISH) can effectively demonstrate TET2 deletions and is often used to validate molecular results. In the present study 41 MDS patients with and without 4q abnormalities were analyzed with a series of bacterial artificial chromosome (BAC) probes spanning the 4q22.3-q25 region. On conventional cytogenetic (CC) studies, a structural defect of the long arm of chromosome 4 (4q) was observed in seven patients. In three, one each with a t(1;4)(p21;q24), an ins(5;4)(q23;q24qter), and a t(4;17)(q31;p13) as the sole chromosomal abnormality, FISH with the RP11-356L5 and RP11-16G16 probes, which cover the TET2 locus, produced one signal only. Unexpectedly, this same result was achieved in 3 of the remaining 34 patients. Thus, a TET2 deletion was observed in a total of six patients (14.6%). TET2 deletion was not correlated with any particular clinical findings or outcome. These findings demonstrate that 1) FISH is an effective and economical method to reveal cryptic abnormalities of band 4q22-q24 resulting in TET2 deletions; 2) in these patients, TET2 deletion is the unifying genetic event; and 3) the different breakpoints within the 4q22-q25 region suggest that deletions are not mediated by repetitive sequences.

Within a cohort of 1,915 consecutive patients with myeloproliferative neoplasm followed for a median time of 5.2 years (range 0-33.3), we investigated the occurrence of lymphoid neoplasm with the aim of defining this risk and to investigate the role of genetic predisposing factors. We identified 22 patients with myeloproliferative neoplasm who developed lymphoid neoplasm over their lifetime. We found that the risk of developing lymphoid neoplasm was 2.79-fold higher (95% CI, 1.80-4.33; P

PURPOSE To evaluate the clinical features of presentation, the morphologic and immunohistochemical pattern, the modality of spread, and the response to current treatments of patients with primary mediastinal B-cell lymphoma, a recently documented subtype of non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS Thirty consecutive patients (14 males, 16 females; median age, 26 years) with primary mediastinal B-cell lymphoma with sclerosis were studied. RESULTS The clinical aspects were largely homogeneous: 93% presented with chest symptoms of a rapidly enlarging mass of the anterior mediastinum; the tumor was bulky in 73%, and superior vena cava syndrome (SVCS) was present in 57%. Also, patients without SVCS symptoms showed subclinical venacaval compression at computed tomographic (CT) scan, for a total incidence of caval obstruction of 80%. Intrathoracic extension to adjacent organs was seen in 47% of patients. Despite its invasive behavior, only four patients showed extrathoracic spread at diagnosis. In 23 cases, the tumor presented with morphologic features that resembled follicular center-cell lymphomas. In seven, the neoplastic population was composed mainly of centrocyte-like cells with abundant clear cytoplasm not referable to any known B-cell lymphoma subtype. All cases showed huge sclerosis. Of 29 patients assessable for response, 16 (55%) achieved a complete response (CR): five of 14 (36%) treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), and 11 of 15 (73%) treated with methotrexate plus leucovarin, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (MACOP-B) or etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (VACOP-B) (P = .047). We could identify no clinical, biologic, or histopathologic features significantly correlated with response. After chemotherapy, 14 of 16 remitters received consolidation radiotherapy to the mediastinum. At 3 years, the actuarial survival rate is 38% for all cases and 72% for remitters. None of the 13 patients who did not achieve CR responded to salvage treatments. CONCLUSION This study shows that primary mediastinal B-cell lymphoma with sclerosis is a distinctive subtype of NHL with unique clinicopathologic aspects and aggressive behavior. Prompt recognition and aggressive treatment may provide long survival in a good proportion of cases. However, a subset of patients is extremely refractory to first- and second-line treatment. Conventional prognostic factors seem inadequate to identify these very-poor-risk cases.

We studied the relationship between JAK2 (V617F) mutant allele burden and clinical phenotype, disease progression and survival in patients with polycythemia vera (PV). The percentage of granulocyte mutant alleles was evaluated using a quantitative real-time polymerase chain reaction-based allelic discrimination assay. Of the 338 patients enrolled in this prospective study, 320 (94.7%) carried the JAK2 (V617F) mutation. Direct relationships were found between mutant allele burden and hemoglobin concentration (P=0.001), white blood cell count (P=0.001), spleen size (P=0.001) and age-adjusted bone marrow cellularity (P=0.002), while an inverse relationship was found with platelet count (P0.001). During the study period, eight patients progressed to post-PV myelofibrosis (MF) (all carrying50% mutant alleles), while 10 patients developed acute myeloid leukemia (AML). The mutant allele burden was significantly related to the risk of developing myelofibrosis (P=0.029) and retained its significant effect also in multivariable analysis (P=0.03). By contrast, the risk of developing AML as well as that of thrombosis was not significantly related to mutant allele burden. Leukocytosis did not affect thrombosis, MF, leukemia or survival. In conclusion, a JAK2 (V617F) allele burden50% represents a risk factor for progression to MF in PV.

A novel t(Y;11) translocation with MLL gene rearrangement in a case of acute myelomonocytic leukemia (AML-M4)

Background Pegylated filgrastim (PEG-f), a long-lasting granulocyte-colony-stimulating factor, has been used in different hematologic conditions to shorten chemotherapy-induced neutropenia and to mobilize peripheral blood stem cells. Data on mobilization efficacy in patients with multiple myeloma are, however, still limited. Study design and methods The feasibility and mobilizing capacity of DCEP chemotherapy followed by a single subcutaneous dose of 6 mg of PEG-f in 23 myeloma patients (11 females and 12 males) whose median age was 55 years (range, 31-67 years) were investigated. Results The median number of CD34+ cells collected was 5.72 x 10(6) per kg body weight with a range between 0 x 10(6) and 29.4 x 10(6) per kg body weight. Twenty patients (87%) yielded more than 2 x 10(6) per kg body weight CD34+ cells. Among the 22 patients who mobilized some CD34+ cells, 27 leukapheresis procedures were carried out (a single leukapheresis procedure in 17 patients and 2 leukapheresis procedures in 5). The median interval between the start of chemotherapy and the first leukapheresis procedure was 12 days (range, 11-16 days). With regard to tolerability, 7 patients complained of mild to moderate back pain, controlled with oral analgesics. No patient was hospitalized, and no fever or infections occurred. Conclusion These results, compared with those previously reported for the DCEP-filgrastim combination, suggest that DCEP chemotherapy followed by PEG-f is a promising combination to mobilize peripheral blood stem cells in myeloma patients.

From 2000 to 2004, 152 patients with multiple myeloma agedor=65 years, enrolled in high-dose programs, were treated with two schedules of DCEP (dexamethasone, cyclophosphamide, etoposide, and cisplatin): 106 patients (group I) were mobilized with the infusional version of DCEP (infusional-DCEP), and 46 patients (group II) with a shorter version (DCEP-short). The median number of CD34(+) cells collected was similar in the two groups as was the percentage of patients yieldingor=4 x 10(6) cells/kg. The proportion of patients in whom mobilization failed was similar in the two groups. The incidence of WHO grade III neutropenia was higher in group II, although the difference was not statistically significant; the percentage of patients requiring hospitalization for severe infections was similar in the two groups. The incidence of WHO grade IV thrombocytopenia did not differ between the two groups. The response rate was 72% in group I and 80% in group II with similar percentages of patients achieving good responses. DCEP-short is a good mobilizing regimen, sharing the same characteristics as infusional-DCEP: high mobilizing efficacy, low toxicity and good antitumor activity. This new schedule of DCEP does, however, allow complete outpatient management and so could be advantageously included in any high-dose therapy program.

One-third of patients with monoclonal gammopathy of undetermined significance (MGUS) may progress to multiple myeloma (MM) and may develop a long arm deletion of chromosome 13 (13q-). As the incidence of 13q-, time of development and prognostic impact in MGUS patients is still under debate, we decided to perform serial sequential conventional cytogenetics (CC) and metaphase/interphase fluorescence in situ hybridization (FISH) analyses on bone marrow mononuclear cells obtained from 18 asymptomatic, untreated MGUS patients. Median follow up was 30 months (range 6-72). Interphase FISH identified a 13q14 deletion in five out of 18 patients (on clinical diagnosis in one patient and during the follow up in the remaining four patients). Subsequently, metaphase FISH and CC also identified the deletion in four out of five patients. All five of the patients progressed to MM 6-12 months after 13q- identification, without developing any FISH determined JH rearrangements. MM progression also occurred in two other karyotypically normal patients. We conclude that: (i) the extent of the 13q deletion does not vary during the clinical outcome; (ii)13q- plays a crucial role in MGUS/MM pathogenesis and confers a proliferative advantage to clonal plasma cells being initially demonstrated by interphase FISH and only afterwards by metaphase FISH and CC; and (iii) association of 13q- with t(4;14)(p16.3;q32) remains to be demonstrated. However, a transition from MGUS to MM may also occur in patients with normal karyotypes or other abnormalities, suggesting the possibility of distinct pathogenetic pathways.

A group of 11 heavily pretreated patients receiving low-dose thalidomide was compared with a similar group of 10 patients with refractory myeloma treated with a convention-al oral chemotherapy. This study shows that thalidomide is not only effective in controlling the neoplastic clone but more-over, thanks to its low toxicity, allows out-patient management of these subjects.

Summary. Options for relapsed/refractory indolent lymphoma include chemotherapy, immunotherapy and high-dose therapy with autologous support. The best combination of these approaches, however, is not defined. We treated 10 patients with relapsed/refractory follicular (n = 7) or mantle cell lymphoma (n = 3) using chemotherapy, immunotherapy, high-dose therapy and autotransplant in a sequence of four phases, each designed to play a specific role in tumour eradication. After the debulking with VACOP-B (doxorubicin, cyclophosphamide, etoposide, vincristine, prednisone, bleomycin) (phase 1), 9/10 patients responded but none achieved a molecular response. After the immuno-chemotherapy phase, which combined Rituximab with vincristine and cyclophosphamide, seven patients were in complete response (CR) and three in good partial response (PR), and all those with a molecular marker of disease showed a disappearance of the signal from marrow and blood. Phase 3, which coupled high-dose cytarabine with Rituximab, was effective in mobilizing an adequate number of progenitor cells that were polymerase chain reaction negative in all informative cases. Phase 4 consisted of high-dose therapy with autologous support followed by two doses of Rituximab. Autograft was performed in nine patients. The haematopoietic recovery was as expected. This sequence of chemotherapy, immuno-chemotherapy, stem cell mobilization with in vivo purging and autotransplant, organized in four blocks of treatment, was simple to administer and devoid of toxic effects. It permits rapid attainment of clinical and molecular response and enables the harvest of lymphoma-free peripheral blood progenitor cells even in heavily pretreated patients with relapsed or refractory disease.

Rituximab is a chimeric monoclonal antibody (MAb) directed against the B-cell CD20 antigen that has been approved for therapy of relapsed and resistant follicular non-Hodgkin's lymphoma (NHL). This study describes the development and validation of a highly sensitive, rapid, accurate, precise enzyme-linked immunosorbent assay (ELISA) to measure Rituximab serum concentrations. This study also describes the application of the ELISA method to a pharmacokinetic study in a homogeneous group of patients with follicular lymphoma who received 4 weekly doses of MAb at the standard dose of 375 mg/m2 as consolidation of chemotherapy. In the patients in this study, the median Rituximab serum concentrations increased during therapy, and showed a slow decline during the posttreatment period. The Rituximab elimination half-life of approximately 20 days accounts for the demonstrated accumulation of MAb in serum samples. Because previous pharmacokinetic studies showed a correlation between Rituximab serum levels and tumor response, the ELISA method used in this study, which allows a precise control of serum concentrations, could be useful for predicting the final response to the MAb and for selecting patients able to benefit from higher dosage or repeated drug administration.

PURPOSE Fludarabine phosphate is effective as a single agent in low-grade non-Hodgkin's lymphoma (NHL). Combined with other antineoplastic agents it enhances the antitumor effect. Our aim was to define the therapeutic efficacy and toxicity of a combination of fludarabine, cyclophosphamide and dexamethasone (FluCyD) in patients with advanced low-grade lymphoma. PATIENTS AND METHODS Twenty-five adults with pretreated advanced-stage low-grade NHL were treated with three-day courses of fludarabine 25 mg/m2/day, cyclophosphamide 350 mg/m2/day, and dexamethasone 20 mg/day, every four weeks for a maximum of six courses. RESULTS Of the 25 patients, 18 (72%) responded, 8 (32%) achieving CR and 10 (40%) PR. Seven were failures. The median follow-up was 21 months (5-26). Eight CR patients remain in CR after 5-21 months. Of 10 PR patients, 3 are in continuous PR without further treatment after 12, 17 and 18 months. Myelosuppression was the most prevalent toxic effect. Although severe granulocytopenia (granulocyte count nadir < 500/microliter) and thrombocytopenia (platelet count nadir < 50,000/microliter) occurred in only 10% and 16% of courses, respectively, slow granulocyte or platelet count recovery caused delay of 40% of the courses. Nine patients (36%) required discontinuation of therapy because of persistent granulocytopenia and/or thrombocytopenia: three after one course, three after 2-4 courses, and three after five courses. Thirteen infectious episodes in 11 patients complicated 11% of courses. Two of 10 patients monitored for the circulating EBV load showed increased viral load. One of these developed aggressive lymphoma. CD4+ lymphocytes declined from a pre-therapy median value of 425/microliter to 141/microliter post-treatment (P = 0.001). Non-hematologic toxicities were rare and mild. CONCLUSIONS The combination of fludarabine with cyclophosphamide and dexamethasone is effective in pretreated advanced-stage low-grade NHL. It may broaden the range of therapeutic options in the salvage treatment of these patients. The main toxicity of this combination is prolonged myelosuppression that may cause treatment delay or withdrawal. The benefit of adding granulocyte colony-stimulating factor, particularly in patients with poor marrow reserve, needs to be investigated.

Growth factors (GF) are reported to play an important role in the therapy of myelodisplastic syndromes (MDS). After in vitro administration a consistent group of MDS may respond to GF but the possibility of differentiation, regulation or expansion of myelodisplastic clones following GF therapy is still a question to be answered as their optimum dose and combinations. To validate if in vivo treatment with GF, may promote the regulation or the recovery of myelopoiesis and/or modify the clonality of the responses, we gave G-CSF after intensive chemotherapy in high risk MDS and acute leukemia evolving from MDS patients. According to our data the use of G-CSF after intensive chemotherapy may improve the CR rate without increase of leukemic transformation. However the answer were clonal and the remission duration remained very short so we suggest to utilize this time to perform other therapeutic strategies such as, when possible, the BMT.

BACKGROUND CA 125 is a glycoprotein produced by epithelial ovarian tumors and by mesothelial cells; its levels also have been shown to be elevated in patients with non-Hodgkin's lymphoma (NHL). METHODS The authors evaluated serum CA 125 levels in patients with NHL to elucidate the frequency of this finding, its relationship with other presenting features, and its potential role as tumor marker. One hundred and fifty-seven patients underwent the first CA 125 assessment at onset, 54 at disease recurrence or progression, and 62 during complete remission (CR). RESULTS Of the 157 patients evaluated at diagnosis (median CA 125: 26 U/mL; range, 2-1400 U/mL), 63 (40%) had increased CA 125 values. Higher CA 125 levels were associated with advanced disease, aggressive histology, mediastinal and/or abdominal involvement, bulky tumor, high tumor burden, effusions, contiguous extranodal extension, high serum lactate dehydrogenase (LDH) activity, and elevated β2-microglobulin (β2-M) levels. Parameters identified by multivariate analysis to be independently associated with high CA 125 were: aggressive histology, mediastinal and/or abdominal disease, bulky tumor, high serum LDH activity and β2-M serum levels, and the presence of effusion (P = 0.0000; explained variation = 0.64). Of the patients presenting with abnormal CA 125 levels, all those who achieved a CR (35) and 3 of the 6 who achieved a partial response had normalization of CA 125 values by the end of treatment. Conversely, CA 125 remained above normal values in 18 nonresponders. All 62 patients evaluated during CR showed normal CA 125 levels. Among patients first evaluated at disease recurrence or progression, 22 of 54 (41%) showed increased CA 125 levels, which were associated with the same parameters of disease found in patients examined at diagnosis. CONCLUSIONS High serum CA 125 levels were found to correlate with mediastinal and/or abdominal involvement, high tumor mass, and effusions, reflecting the reaction of mesothelial cells to the tumor. Serum CA 125 is a reliable biologic marker for the staging and restaging of patients with lymphoma. Serial measurements are useful, in conjunction with other markers, for monitoring response to treatment. Cancer 1998;82:576-82. © 1998 American Cancer Society.

We describe a 28-year-old man with paroxysmal nocturnal haemoglobinuria (PNH) and a high transfusion requirement. Prior to and during therapy with recombinant human erythropoietin (rHuEpo), we evaluated the levels of ‘decay-accelerating-factor’, CD55, and ‘membrane-inhibitor-of-reactive-lysis’, CD59, as markers of the disease, whilst CD58, a marker present on leucocytes, was utilized to monitor normal haemopoietic activity. The patient became transfusion independent 1 month after beginning rHuEpo and remains well. The analysis of CD55, CD59 and CD58 suggests that the efficacy of rHuEpo was due to a selective rHuEpo action on normal erythroid clones.

Background About 95% of patients with polycythemia vera (PV) and 60-70% of those with essential thrombocythemia (ET) carry the unique JAK2 (V617F) mutation. Previous observations suggest that JAK2 (V617F)-positive ET and PV form a biological continuum, in which the degree of erythrocytosis is determined by physiological and genetic factors. Aims In this work, we studied the natural history of JAK2 (V617F)-positive ET and PV with the aim of establishing whether the two disorders indeed represent different phenotypic expression of a genotypic/phenotypic continuum. Methods We identified 1269 patients diagnosed with ET or PV at our Division between 1980 and 2012, for whom at least one DNA sample was available. The JAK2 (V617F) mutation was assessed using allele-specific quantitative PCR. As patients carrying JAK2 exon 12 or MPL mutations were excluded, the final study population included 1214 patients, 719 of whom with ET (463 JAK2 mutated, 256 JAK2 wild-type) and 495 with PV. Results I: presenting features The clinical phenotype of ET patients at diagnosis differed according to JAK2 mutational status. JAK2 mutated ET presented with older age at diagnosis, higher hemoglobin (Hb) level and white blood cell (WBC) count, lower platelet (PLT) count and erythropoietin level compared to JAK2 wild-type ET (Wilcoxon rank-sum test: P The median V617F allele burden was significantly lower in JAK2 mutated ET than in PV (18.4% vs 43.4%, P Results II: PV evolution Evolution to PV was observed in 53 JAK2 mutated ET patients (incidence 95% CI: 1.4-2.4 per 100 p-years) vs none of the 256 JAK2 wild-type ET (incidence 95% CI: 0-0.2 per 100 p-years), resulting in a significantly different occurrence. The median time to PV evolution was 54 months (range 3.5-220). The cumulative incidence of PV evolution in JAK2 mutated ET patients at 15 years was 28.8% (95% CI: 20.7-37.3; Figure 1). PV evolution was significantly associated with higher JAK2 allele burden at diagnosis (Cox regression HR=1.04, P Based on the hypothesis that PV patients might have had a silent “pre-PV phase”, we did an ad hoc search for any complete blood count (CBC) collected before diagnosis. Among PV patients, 177 (36%) had a previous CBC, collected at a median time of 22 months (range 1-305) before PV diagnosis. A normal CBC was observed in 15% of patients; the remaining subjects showed thrombocytosis (≥450 x 109/L) and/or leukocytosis (≥10 x 109/L) and/or erythrocytosis. The median time to PV onset was significantly shorter in patients showing at least one CBC abnormality than in those with normal CBC (24 vs 48 months, P=.011). Results III: clinical course The median follow-up was 5.1 years (range, 0-32 years). JAK2-mutated ET and PV did not differ in terms of cumulative incidence of thrombosis (25.3% vs 33.7% at 15 years, P=.35; Figure 2A) and had similar overall survival (OS) (90.3% vs 82.6% at 15 years, P=.29; Figure 2B). Conversely, JAK2 wild-type ET showed a better OS in comparison with both JAK2 mutated ET (P=.028) and PV (P=.004) and a lower incidence of thrombosis (12.7% at 15 years) than JAK2 mutated ET (P=.002) and PV (P A similar cumulative incidence of disease progression (leukemia and myelofibrosis) was observed in JAK2 mutated (11.7% at 15 years) and JAK2 wild-type ET (12.1% at 15 years), whereas a higher cumulative incidence was observed in PV (26% at 15 years; P=.011 and P=.007 when compared with JAK2 mutated ET and JAK2 wild-type ET respectively). Conclusions This study supports the hypothesis that JAK2 mutated ET and PV are different expressions of a genotypic/phenotypic continuum, in which the mutant allele burden contributes to determine the clinical phenotype. The risk of progression from JAK2 mutated ET to PV is about 2% per year. This work was supported by grant #1005 from Associazione Italiana per la Ricerca sul Cancro (AIRC) “Special Program Molecular Clinical Oncology 5x1000” to AGIMM (AIRC-Gruppo Italiano Malattie Mieloproliferative - http:www-progettoagimm.it). Disclosures: No relevant conflicts of interest to declare.

Exposure to ionizing radiations (Rx) has been implicated as a causative factor of chronic myelogenous leukemia (CML). We performed a retrospective study of 443 consecutive CML patients, looking for a history of significant exposure to Rx, and evaluated the clinical and hematological characteristics in order to find any difference between radiation-related CML patients and those with de novo CML. We identified 406 patients without known exposure to mutagens (group I) and 37 patients with prior significant exposure to Rx (group II). In comparison to patients of group I, those of group II showed particular clinical and hematological features: significantly lower incidence of bulky splenomegaly (p0.05) and hyperleukocytosis (WBC100 x 10(9)/l; p0.05); significantly higher incidence of anemia (Hb10 g/dl; p0.01). Patients with radiation-related CML had a significantly better survival than those with de novo CML (median survival 61 months vs 42 months; p0.05). In conclusion, this study of a large cohort of CML patients indicates that the subgroup of patients with a history of significant exposure to ionizing radiation has particular clinical and hematological features at onset (lower tumor burden, higher frequency of anemia) and a better survival.

Summary Background Controversy still exists over the optimal management of early-stage Hodgkin's disease (HD); presentation features may have a different prognostic impact according to initial therapy, and long-term toxicity must be fully evaluated. Patients and methods This study included 164 patients with stage IA-IIA HD treated with radiotherapy (RT) alone or combined radio- and chemotherapy (CT) according to presenting features and their attendant prognostic significance. The RT group included 88 patients with favorable prognostic features; the combined modality group included 76 patients with one or more unfavorable features. In the RT group, 85% of patients received extended-mantle or STNI; in the combined modality group, RT consisted of mantle-(49%), extended mantle- (37%), and involved-field irradiation (14%); CT consisted of 6 cycles of MOPP before 1984; 3 cycles of ABVD were substituted for MOPP thereafter. Results Complete remission was obtained in 94% and 99% of patients of the RT and combined modality groups, respectively. The 10-year actuarial relapse-free survival (RFS) in the RT group was 62% and was influenced by stage (p = 0.04) and histology (p = 0.01); in the combined modality group, RFS was 88% and was influenced by the presence of bulky disease. Overall survival and tumor mortality between the therapy groups were comparable. RT-related tox-icity consisted of mediastinal fibrosis (8 cases), myelitis (3), hypothyroidism (2); other long-term events included 2 cases of acute leukemia in the combined MOPP and RT group. Altogether, 8 of 20 patients who died were in their first complete remission. Conclusions In stage IA-IIA HD, the combined modality therapy reduced the risk of relapse compared to radiation alone; long-term toxicity of RT was not negligible and relapses could occur late.

Abstract 3037 Fotemustine (Muphoran), a nitrosourea alkylating agent approved for use in the treatment of metastatic melanoma, has proven to be effective as single agent in relapsed/refractory multiple mieloma (MM). We report preliminary data of a phase II single centre study exploring the feasibility and the efficacy of the combination bortezomib (B) + fotemustine (Mu) + dexamethasone (D) (B-MuD) in relapsed/refractory MM patients. This study has been approved by local ethical committee; all patients (pts) signed written informed consent before the enrolment. MM pts relapsed or refractory after at least one therapy were eligible for the study. Pts who received prior bortezomib-containing regimen were included only if not considered bortezomib-refractory. Fotemustine at the escalating doses of 80 and 100 mg/m2 i.v. on day 1 was associated to Bortezomib 1,3 mg/m2 i.v. on days 1,4,8,11 + Dexamethasone 20 mg orally on days 1–2, 4–5, 8–9, 11–12 of 21-day cycle for a total of 6 cycles. Protocol was amended after the enrolment of the first five pts due to a considerable toxicity. We observed 3 grade 3–4 peripheral neuropathy, 1 grade 3 pneumonia, 4 grade 4 thrombocytopenia and two pts dropped-out (one for grade 3 pneumonia at 2° cycle, and one for grade 4 peripheral neuropathy at 3° cycles). Thus the schedule was modified as following: Fotemustine at escalating doses of 80 and 100 mg/m2 i.v. on day 1, Bortezomib 1,3 mg/m2 i.v. once weekly on days 1, 8, 15, 22, Dexamethasone 20 mg i.v. on days 1, 8, 15, 22 for six 35-day cycles. An interim analysis of feasibility and efficacy was planned after the inclusion of the first two cohort of 6 pts each, treated with escalating dose of Fotemustine according to the amended schedule. Up to now, 18 pts have been enrolled (5 pts before and 13 after the amendment): M/F 10/8, median age 69 years (44-82), median number of previous therapies 2 (1-5). Previous treatments included autologous transplant in 10 pts (59%), bortezomib in 8 pts (44%), oral melphalan in 7 pts (41%) and thalidomide in 12 (71%). After the inclusion of 12 pts the MTD for Fotemustine was established to be 100 mg/m2. No drop-outs were registered after the amendment. Preliminary data on response are available in 10 pts. Nine pts (90%) obtained at least a PR, 8 pts (80%) registered ≥VGPR (CR 10%). At time of this analysis 79 cycles were delivered: 14 before, 65 after the amendment. Eighty-nine AE of any grade were observed, 43 hematological and 46 non-hematological. Thrombocytopenia was the most common AE either before and after the amendment. Need for dose reduction was significantly lower after the amendment. In detail fotemustine was reduced in 14% of cycles before and never after the amendment (p=0.0001), bortezomib dose reduction were performed in 36% of cycles before and 15% after the amendment (p=0.08), dexamethasone dose reduction occurred in 64% of cycles before and 13% after the amendment (p=0.0001). In conclusion, this interim analysis shows that fotemustine in combination with bortezomib and dexamethasone is safe and gives encouraging results in relapsed/refractory myeloma patients with 80% of ≥VGPR. Updated results will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.