ALBERT

Description: Background Most patients with essential thromocythemia (ET) have somatic mutations in janus kinase 2 (JAK2), calreticulin (CALR) and thrombopoietin receptor (MPL) genes. The clinical correlates at the presentation of patients with different mutations has not been established as yet. Materials and methods We evaluated the clinical, laboratory, and molecular features of 150 consecutive patients (93 females/57 males; average age 59,0+/- 14,2 years) with ET who were diagnosed at the University Clinic for Hematology in Skopje based on the revised WHO criteria. DNA from all patients was isolated from peripheral blood obtained at diagnosis using Qiagene DNA exctraction kit. The presence of JAK2V617F mutation was evaluated with fluorescent allele specific PCR/capillary electrophoresis. The CALR exon 9 insertions or deletions were initially detected with a fluorescent PCR/capillary electrophoresis and characterized with Sanger sequencing. The presence of mutations in exon 10 of the MPL gene was analyzed with direct sequencing of PCR amplified fragments. Informant consent was obtained from all participants. Statistical analysis was performed by Wilcoxon rank- sum test. Results The basic hematological parameters at diagnosis for all patients were as follows; Plt = 962.1x109 ±351,2 x109/L; Hb = 126,8 ±39,2 g/L; Le = 10,2x103 ±4,2 x103/µL. No correlation was found between any of these parameters and age at diagnosis or sex of the patients. JAK2V617F, CALR and MPL mutations were found in 89 (59.3%), 42 (28%) and 2 (1.3%) patients, respectively, while 17 (11.4%) patients did not have a mutation in the 3 evaluated genes (triple negative). Twelve different mutations were found in CALR gene, the most common being 52-bp deletion (c.1092_1143del) and 5-bp insertions (c.1154_1155insTTGTC) which were present in 11 (26,2%) and 20 (47,6%) patients, respectively. Ten different mutations, including 6 insertions (c.1088insTTGTC; c.1088delinsTTTGTC; c.1113_1123delinsTTGT; c.1154delinsTATGTC; c.1126_1131delinsTGCGT; c.1154_1155insGTGTC) and 4 deletions (c.1092_1137del; c.1096_1129del; c.1089_1141del; c.1100_1145del), were detected in the other 11 patients with CALR mutation. The JAK2V617F mutation and triple negative status were more prevalent in female patients (67% and 68% respectively), while no sex preference was detected in patients with the CALR mutation. The average age of patients with JAK2V617F, CALR insertions, CALR deletions and triple negative status were 60,7±13,9, 54,5±12,9, 59,0±15,1 and 58,8+/-14,8 years, respectively. Patients with CALR mutation had a higher platelet count at the time of diagnosis (1134x109 ±372,9 x 109 /L) compared to both patients with mutated JAK2 (952 x109 ±335,6 x109/L) (P=0.047) and to triple negative patients (mean 886 x109/L±303,2 x109/L) (P=0.024). Patients who carried CALR insertions had higher platelet counts (1222,9 ±396,8 x109/L) than patients with CALR deletions (mean 956,8 ±287,8 x109/L ) (P=0.0405). White blood cells counts were the highest in patients with JAK2V617F mutation (mean 11.1±4,5x103/µL) compared to both CALR positive (mean 8,8±2,7x103/µL) and triple negative patients (mean 7,9±2,2x103/µL) (P=0.0049). No difference in Hb levels were present between patients with various mutations. Conclussion ET patients with CALR insertions present with higher platelet counts and at an earlier age than patients with JAK2V617F, CALR deletions or triple negative patients, which might translate to differences in prognosis in these groups of patients. Higher incidence of JAK2V617F mutation and triple negative status in female patients point to a common mechanism for predisposition to these conditions. Disclosures No relevant conflicts of interest to declare.

Description: Somatic activating mutations in the JAK2 gene are one of the most important characteristics of the myeloproliferative neoplasams (MPNs). Moreover, JAK2 germline mutations occur in familiar MPN. However, the same JAK2 mutations are associated with different phenotypic MPNs and their exact role in the pathogenesis of the MPNs is not clear and well established. Here, we report a family with a germline JAK2 G571S mutation associated with only one sporadic case of essential thrombocythaemia (ET). Our patient, male, born 1958, presented at the age of 54 with a marked and sustained thrombocythosis (platelets count higher than 1500x109per liter over a period of 3 months). The bone marrow biopsy sample revealed normocellular bone marrow with an increased number of megakaryocytes organized in clusters; the reticulin staining was negative. The molecular diagnostic work up towards MPNs included the analysis of BCR/ABL fusion transcript, JAK2, CALR and MPL mutations. These analyses showed that the patient had 2 variants which might account for the ET phenotype; a somatic CARL mutation (insertion p.K385fs*47) and a germline C-T substitution at codon 571 in exon 13 of the JAK2 gene. Extended family analysis showed that the G571 variant was also present in the patient’s father, brother and one daughter. All of them had normal hematological parameters and didn’t exhibit any abnormality in the serum ferritin, prolactin, lipids, fasting glucose levels, blood pressure, body mass index, skin and skeletal changes. The JAK2 G571S mutation was already described in MPNs with a frequency

Description: PTPN22 is a protein tyrosine phosphatase that modulates antigen-receptor signaling in T and B cells. A PTPN22 R620W polymorphic allele (C1858T, rs2476601) has been associated with increased risk for the development of multiple autoimmune diseases in patients originating from Northwestern Europe, whereas this association is less clear in patients originating from Southern Europe where the 620W (1858T) allele is less frequent. A higher frequency of the 620W risk allele has recently been reported in Chronic lymphocytic leukemia (CLL) patients originating from Northwest Europe. These data adds to the recent findings that PTPN22 is markedly overexpressed in the majority of CLL patients and possibly contributes to the pathogenesis of the disease by affecting apoptosis/survival of antigen stimulated CLL B-lymphocytes. In order to extend further those observations we conducted a retrospective study on the frequency of the PTPN22 R620W variant in 87 CLL patients and 108 age/sex matched normal controls without a history of malignant disease originating from the Republic of Macedonia (Southeastern Europe) and compared the association of the variant with various clinical/molecular correlates. Similar allele frequency of the minor T allele (0.06 and 0.063) and genotype distribution (0.88, 0.115 and 0.005 of CC, CT and TT genotypes) was found in CLL patients and normal controls, respectively. Both study groups were in Hardy-Weinberg equilibrium. Also, the PTPN22 R620W variant was not associated with any of the studied clinical and biological parameters, such as blood counts, CD38 expression, IGHV mutation status, incidence of autoimmune hemolytic anemia and autoimmune thrombocytopenia, immunoglobulin levels, time-to-treatment, progression free survival and overall survival. We conclude that the PTPN22 R620W variant is not a risk factor for the development or progression of CLL in patients originating from the Southeastern European region. The differences of our data from the reported findings for CLL patients originating from Northwest Europe mirrors the differences observed in patients with various autoimmune diseases, suggesting a possible gene-environmental factor that affects the influence of the R620W variant on lymphocyte homeostasis. Disclosures: No relevant conflicts of interest to declare.

Description: Background Rituximab maintenance has been shown to be effective in patients with follicular lymphoma. The optimal duration of maintenance treatment remains unknown. Methods 270 patients (median age 57 years: range 25-82) with either untreated, relapsed, stable or chemotherapy resistant follicular lymphoma of all grades were treated with 4 weekly doses of rituximab monotherapy (375 mg/m²). Patients responding to the induction treatment (partial or complete response) received rituximab (375 mg/ m²) maintenance and were randomized either to a short maintenance consisting of four administrations every two months (arm A), or a prolonged maintenance for a maximum of five years or until disease progression or unacceptable toxicity (arm B). The primary endpoint was event-free survival (EFS) from randomization. Sample size calculation allowed detecting a median EFS increase with prolonged maintenance from 2.5 to 4.5 years with 80% power. Secondary endpoints included progression-free survival (PFS), overall survival (OS) and objective response. Comparisons between the two treatment arms were performed using the log-rank test for survival endpoints. Results From October 2004 to November 2007 165 patients were randomized, 82 in arm A and 83 in arm B. 124 patients were chemotherapy-naïve. The median EFS is 3.4 years (95% CI 2.1-5.3) in arm A and 5.3 years (95% CI 3.5-NA) in arm B. Using the prespecified log-rank test this difference is statistically not significant (p=0.14). We observed an unexplained difference in disease progression and relapse during the first 8 months after randomization (3 in arm A vs. 10 in arm B) when treatment in both arms was the same which led to an early crossing of the EFS curves at 18 months. Considering only patients at risk after 8 months from randomization EFS in arm B is significantly prolonged compared to arm A (median EFS 7.1 (95% CI 4.4-NA) vs. 2.9 years (95% CI 1.8-4.8); log-rank test p=0.004). Median PFS is significantly longer in arm B (7.4 (95% CI 5.1-NA) vs. 3.5 years (95% CI 2.1-5.9); log-rank test p=0.04). There is no significant difference in OS and in the observed best response. Maintenance treatment was stopped due to unacceptable toxicity in no patient in arm A vs. 3 in arm B. Six subsequent cancers developed in arm A and 8 in arm B. One infection grade ≥3 was reported in arm A whereas seven infections grade ≥3 occurred in 5 patients in arm B. Conclusions EFS, the primary endpoint was not met which is mainly due to the early separation of the survival curves favouring arm A, at a time when the treatment in both arms was the same. However, a retrospectively defined analysis considering only EFS events from the time when treatment was different in the two arms, shows a statistically significant increase in EFS with long-term maintenance compared to the 8 months maintenance regimen. Long-term rituximab maintenance also doubles the median PFS without leading to increased undue toxicity. Disclosures: Off Label Use: Rituximab for 5 years. Ghielmini:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Description: Abstract 1802 Background: Rituximab maintenance has been shown to be effective in patients with follicular lymphoma. The optimal duration of maintenance remains unknown. Methods: We prospectively registered 270 patients with untreated, chemotherapy resistant or relapsed follicular lymphoma. All patients received rituximab induction consisting of 4 weekly doses (375 mg/m2). Responding patients (PR and CR) were randomized to a short maintenance consisting of four doses of rituximab (375 mg/m2) every two months (arm A) or prolonged maintenance consisting of rituximab every two months for a maximum of five years or until disease progression or unacceptable toxicity (arm B). Primary endpoint is event-free survival. Here we present the safety analysis results after a median long-term maintenance period of 3.3 years. Results: From October 2004 to November 2007 165 patients were randomized, 82 in arm A and 83 in arm B. The median follow-up time is 3.2 years for arms A and B combined. While receiving maintenance therapy a total of 899 hematological and non-hematological adverse events were observed, 28 of grade 3 and 6 of grade 4. After randomization five patients experienced subsequent cancers. Seven grade 3 and 4 infections were reported. Two grade 3 infections occurred after 2 years of maintenance. Grade 3 and 4 neutropenia occurred in 6 (3.6 %) patients, decreased levels of IgG were observed in 24 (14.6 %) patients. In arm B, maintenance was stopped due to unacceptable toxicity in 2 patients after 16 and 42 months respectively and due to subsequent breast cancer in 1 patient after 20 months. One patient died 4 months after randomization because of ileus and consecutive peritonitis, considered to be unrelated to therapy. Sixty-three patients are on maintenance for two or more years of which 48 patients are on for three or more years. Two patients have completed the 5 years of maintenance. Conclusions: Rituximab maintenance beyond two years is feasible without evidence for increased toxicity. However, close follow up of patients under prolonged rituximab maintenance is still necessary. The trial has been closed for accrual but there are still patients on treatment. Disclosures: Taverna: Roche: Membership on an entity's Board of Directors or advisory committees. Ghielmini:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Description: The discovery of the activating V617F mutation in the JAK2 tyrosine kinase gene in patients with myloproliferative disorders (MPD) provides a major breakthrough in the understanding of the pathogenesis, proving clonality and securing diagnosis of these diseases. The JAK2 V617F allele is an acquired somatic disease allele that arises in hematopoietic progenitors and confers a selective growth advantage. The mutation has been traced to a primitive stem cell that is capable of erythroid and myeloid differentiation. The data for the potential involvement of the B and T lymphocytes with the JAK2V617F mutation are still inconsistent. We present the results from the study designed the evaluate the prevalence of the JAK2V617F mutation in MPDs patients in our population and to investigate whether MPD patients that carry the JAK2V617F mutation differ in clinical course and outcome from JAK2V617F negative MPD patients. The study group consisted of 64 living MPD patients diagnosed according to standard WHO criteria for diagnosis of MPD (26 patients were diagnosed as polycythemia vera (PV), 34 as essential thrombocythemia (ET), 6 as idiopatic myelofibrosis (MF) and 8 were classified as atypical MPD) with the median follow-up of 7,4 years. DNA samples were obtained from unfractionated blood samples and the frequency of V617F JAK2 mutation was analyzed by allele-specific PCR assay. The mutant allele burden in mutation positive samples was analyzed by DNA sequencing. Our results showed that the JAK2 V617F mutation was present in 79% of patients with PV (36% were homozygous for the mutation), 58% with ET (11% homozygous), 69% with MF (28% homozygous) and in 33% of patients with atypical MPD. The mutant JAK2V617F allele burden was greater than 95% in two PV patients, which in the presence of 27% lymphocytes in the peripheral blood of the patients indicate lymphocytes involvement with the mutation. The high frequency of observed homozygosity for JAK2V617F in our study group was probably due to the long disease duration (median follow-up 11,4 years) and favors the theory of a time dependent increase in clonal dominance. Correlations of clinical and laboratory features at diagnosis and subsequent follow-up, including incidence of thrombo-hemorrhagic events, disease transformation and survival of JAK2V617F-positive and JAK2V617F-negative patients did not reveal significant differences except for the incidence of thrombotic complications. The JAK2V617F positive group had a higher incidence of thrombotic complications (30%) compared with the JAK2-V617F-negative group (14%, P〈 0.05). Although we observed a disparity in the incidence of the JAK2V617F mutation in different MPD entities in our population with respect to the expected frequency of JAK2V617F from the literature, our results confirm the diagnostic significance of the JAK2V617F mutation in MPDs and support the notion that patients with this mutation should be classified in a new entity of MPDs. Identification of homozygous JAK2V617F mutation in unfractionated blood samples in a substantial proportion of long term follow-up MPD patients, together with the identification of the mutant JAK2V617F allele burden greater than 95% in two PV patients, suggests that acquisition of the JAK2V617F mutation arises in early multilineage hematopoietic progenitors and warrants further investigation.

Description: Lymphocyte-predominant Hodgkin disease (LPHD) presents as early-stage disease with slow disease progression and excellent initial response to conventional chemotherapy. Although 96% of LPHD patients experience a complete remission (CR) upon first line treatment, many relapses occur. Residual tumor cells in Hodgkin’s patients can survive standard therapy and cause relapse. Thus, the elimination of minimal residual disease after first line treatment might improve the outcome in Hodgkin disease. CD20+ is strongly expressed by malignant cells in LPHD, but so far there is limited information regarding the efficacy of Rituximab in this patient population. A 66 year-old woman presented with bilateral inguinal lymphadenopathy in 2004 and complained on malaise and weight loss. Lymph node biopsy confirmed a diagnosis of LPHD and immunohistochemistry confirmed that CD20 antigen was expressed on more than 30% of malignant cells. Staging revealed II B disease. There were lymph node enlargements in the pelvis on CT. Her blood count, erythrocyte sedimentation rate, and albumin were normal with elevation of and lactate dehydrogenase and alkaline phoshatase. She was treated with ABVD regiment 8 cycles and CR was achieved. After standard chemotherapy patient with CD20+ LPHD received four weekly doses of Rituximab at 375 mg/m2 and maintenance therapy with additional four doses of Rituximab every three months for the next year. Treatment was well tolerated without any complications. Evaluation of disease assessment included physical examination and computed tomography, bone marrow biopsy and routine analyses, after the end of treatment and every 3 months after for the first two years, and on six months for the following year. There were no abnormalities on CT after the end of the treatment. Patient was in complete remission. CT after 36 months showed no detectible tumor mass. Patient remains still in complete remission three years after chemotherapy without detectible tumor mass. Our case report suggests that Rituximab is both safe and effective in patients with CD20+ LPHD. The optimal treatment for patients with LPHD remains uncertain. Current research aims to identify alternative treatment approaches that possess significant activity but less toxicity. Radiotherapy for early-stage patients and chemotherapy or combined modality treatment for advanced-stage patients remains to be standard of care. Initial observed benefit of Rituximab treatment in this group of patients suggests that there is a role for monoclonal antibodies in optimal treatment approach, especially in improving the outcome. Further studies with Rituximab are warranted in this patient population.

Description: Neuroblastoma is an embryonic neoplasm of the sympathetic nervous system derived from the primitive neural crest cells. It is the second most common extra cranial malignant tumor of childhood and the most common solid tumor of infancy, comprising 8 to 10 percents of pediatric cancers. Forty percent of patients who present with clinical symptoms at diagnosis are under 1 year of age, and less than 5% with clinical symptoms are over the age of 10 years. The tumor very rarely occurs in adult patient (defined as 20 years of age and older) and usually has an indolent course with poor survival. Up until June 2008, no more than 65 neuroblastomas in adults had been reported in the literature. Majority of the reports describe an indolent and longer course of the disease among adults with different biological characteristics when compared to children, but ultimately poor outcome regardless of the initial stage of the disease. Here, we report a case of aggressive adult neuroblastoma mimicking acute leukemia with fulminant course and fatal outcome. The patient, a previously healthy 38 years old Caucasian male, presented at our department with a 4 months history of hip pain, headaches, malaise, weakness, persistent diarrhea, poor appetite and weight loss. On admission he was febrile, prostrated, pale, fixed in bed. His abdomen was distended with present bowel movements. The liver and spleen were 3 cm and 5 cm below the costal margins respectively. No abdominal mass was palpable. Neurological examination showed right Horner’s syndrome, reduction of the superficial sensitivity on the Th1 and Th2 level and loss of the tendon-muscle reflexes of the lower limbs. He had paraplegia, indicating possible spinal cord compression. The complete blood counts revealed pancytopenia and presence of 11 % peculiar circulating blastoid cells. The LDH was 3552U/l. Under suspicion of acute leukemia bone marrow aspirate and biopsy was performed. Immunophenotyping of the bone marrow mononuclear cells showed presence of around 50% CD45negative, CD56 and CD9 positive cells, indicating a possible involvement of the bone marrow with neuroblastoma. A routine second opinion from a reference pathologist was also interpreted as neuroblastoma and confirmed with positive immunohistochemical staining for NSE, chromogranin, synaptophysin and negative for LCA. A work-up for neuroblastoma was done and investigations confirmed disseminated stage 4 neuroblastoma. In spite of the appropriate treatment with intensive chemotherapy regimen, the patient failed to improve and died of progressive disease. Our case report illustrate the usefulness of including CD56 marker in flow-cytometry acute leukemia assays, especially in cases when CD45 expression is absent, as initial screening tool for recognizing cases of neroblastoma which simulate acute leukemia.

Description: Background: Follicular lymphoma is usually a disase with a prolonged course and a chemotherapy-free regimen might be a favourable treatment strategy. SAKK 35/03 investigated two different durations of rituximab maintenance (5 years vs. 6 months) in patients with follicular lymphoma after induction with 4 weekly doses of rituximab monotherapy. With a median follow-up of 6.4 years we were not able to show a benefit with long-term rituximab maintenance up to five years in event-free survival (EFS) or overall survival (OS ). Here we report the final results with a median follow-up of 10 years. Methods: 270 patients (median age 57 years: range 25-82) with untreated, relapsed, stable or chemotherapy resistant follicular lymphoma were treated with 4 doses of rituximab monotherapy in weekly intervals (375 mg/m²). Patients achieving at least a partial response were randomly assigned to receive maintenance therapy with one infusion of rituximab every 2 months, either on a short-term schedule (four administrations) or a long-term schedule (maximum of five years or until disease progression or unacceptable toxicity). The primary endpoint was EFS . Progression-free survival (PFS), OS, and toxicity were secondary endpoints. Comparisons between the two treatment arms were performed using the log-rank test for survival endpoints. Results: 165 patients were randomly assigned to short-term (n=82) or long-term (n=83) maintenance. At a median follow-up period of 10 years, the median EFS is 3.4 years (95% CI 2.1-5.5) in the short-term arm and 5.3 years (95% CI 3.5-7.5) in the long-term arm. Using the pre-specified log-rank test this difference is statistically not significant (p=0.39 ). There is no significant difference in PFS and OS. Median OS in the short-term arm is 11.0 years (95% CI 11.0, NA ) and not reached in the long-term arm (p=0.80). The incidence of subsequent cancers increased in both arms over time, nine patients developed a subsequent cancer in the short-term maintenance arm and 10 in the long-term maintenance arm. There was no major additional toxicity with longer follow-up. Conclusions : Even with a long follow-up of 10 years we were not able to show a significant benefit of long-term compared to short-term rituximab maintenance in EFS, PFS and OS. Treatment strategies and study designs need to take these results into consideration in order to guarantee optimal medical and scientific results. Disclosures Zucca: Celltrion: Consultancy; AstraZeneca: Consultancy. Ghielmini:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Description: Purpose: Clinical and pharmacokinetic data suggest that high serum concentrations of rituximab and prolonged exposure to rituximab are associated with higher response rates and improved quality of response. We used rituximab-maintenance therapy to target minimal residual disease, with the aim of increasing the duration of remissions achieved following initial treatment. Methods: During 2001–2005, 14 patients with CD20+ B-cell non-Hodgkin’s lymphoma (seven with diffuse large B cell lymphoma [DLBCL] and seven with follicular lymphoma [FL]) were enrolled in the study. Patients with DLBCL and FL were required to have a CR or PR, respectively, following initial treatment. The most common initial treatments were R-CHOP, R-FC or CHOP. Rituximab-maintenance therapy was administered as 375mg/m2 doses every 3 months for 2 years. Three patients with aggressive DLBCL were treated with high-dose chemotherapy/autologous stem-cell transplantation (HDT/ASCT) and received rituximab-maintenance therapy every 2 months for four doses in total. Results: Most patients had disease of Ann Arbor stage ≥ 3 (two FL patients stage I). In patients with DLBCL, median event-free and overall survivals were 22.1 months and 22.3 months, respectively, with the corresponding values being 20.6 months and 26.1 months in patients with FL. To date, 13 patients are in continuous clinical remission and only one patient has relapsed. Conclusions: Rituximab-maintenance therapy is effective and well tolerated in this setting. Recruitment for this study is ongoing and evaluation of a larger patient population, together with a longer follow-up, will determine whether this treatment approach has curative potential.