R Anagnostopoulos

B C Neuhaus

H Gärtner

H P Pohl

S Müller

M Jonas

S Papp-Vary

Oertel

R Trappe
email: ralf.trappe@charite.de

H Riess

I Anagnostopoulos

R Neuhaus

B C Gärtner

H P Müller

S Jonas

M Papp-Vary

S Oertel

Efficiency of antiviral therapy plus IVIG in a case of primary EBV infection associated PTLD refractory to rituximab, chemotherapy, and antiviral therapy alone

Efficiency of antiviral therapy plus IVIG in a case of primary EBV infection associated PTLD refractory to rituximab, chemotherapy, and antiviral therapy alone

Dear Editor, Epstein-Barr virus (EBV) incompatibility in transplantation or EBV infection early after transplantation indicated by posttransplant seroconversion is considered a significant risk factor for the development of posttransplant lymphoproliferative disorder (PTLD). About 50% of patients with PTLD that do not respond to an initial reduction of immunosuppression and that are treated first-line with single-agent rituximab require additional treatment. CHOP salvage therapy after upfront treatment with rituximab achieves high remission rates. Antiviral therapy is a further treatment approach in the primary infection setting but little is known on the additional use of polyvalent intravenous immunoglobulins (IVIG).

We report on a young liver transplant recipient presenting with PTLD with high fever, anemia, thrombopenia, hypofibrinogenemia, high lactate dehydrogenase (LDH) levels, and gastrointestinal lymphoma manifestation resulting in massive hemorrhagic diarrhea. Rituximab, CHOPbased chemotherapy, and antiviral therapy alone were not successful to treat PTLD, but the additional therapeutic use of IVIG combined with antiviral therapy resulted in longlasting control of PTLD. The patient is a 28-year-old male who underwent orthotropic liver transplantation for primary sclerosing cholangitis in October 2004. The transplantation was preemptive and time to reperfusion was 10 h 28 min. For induction, the patient received 20 mg anti-CD25 antibody basiliximab at days 0 and +4, followed by a baseline standard immunosuppression of tacrolimus (FK506) 6 mg/day and prednisolone 25 mg/day. Cytomegalovirus (CMV) serology was negative for donor and recipient. The recipient was also negative for EBV, but as data on the EBV status of the donor were unavailable, prophylactic antiviral therapy (acyclovir) was administered for 6 months. There were no infections or rejection episodes.

After 16 months, the patient was admitted with a 4-week history of fever, night sweats, abdominal pain, weight loss (3 kg), and hemorrhagic diarrhea. At this time, immunosuppression comprised tacrolimus 3 mg/day and prednisolone 7.5 mg/day. LDH levels were elevated (339 U/l), but liver function was normal. Hemoglobin was slightly decreased (10.2 g/dl), but platelet and total white blood cell counts were normal, as were the CD4, CD8, and CD19 cell counts (359, 1,042, and 65 per microliter, respectively) and the serum immunoglobulin levels (IgG 1,041 mg/dl, IgA 73 mg/dl, IgM 55 mg/dl). Ultrasound showed thickening, hyperperfusion, and structural transformation of the colon and terminal ileum, corresponding with inflammatory changes in colonoscopy. EBV titers indicated a primary infection (positive EBV-IgM and EBV-IgG and negative EBNA1-IgG). Quantitative EBV polymerase chain reaction (PCR) in peripheral whole blood showed an extremely high viral load (900,000 copies EBV per milliliter), but CMV-PCR and CMV serology remained negative. A whole-body computed tomography (CT) scan showed some mesenterial lymphadenopathy at the right colon flexure without any other abnormalities. Bone marrow biopsy, cerebral CT scan, cerebrospinal fluid cytology, and EBV-PCR were unremarkable. Histopathological examination of colonic mucosa biopsies revealed an EBV-and CD20-positive polymorphic B-cell PTLD (Fig. 1). At diagnosis of PTLD, immunosuppression was reduced by reduction of tacrolimus to 1 mg/day; prednisone was continued with 7.5 mg/ day. Fever, diarrhea, and EBV viral load remained unchanged, so sequential immunochemotherapy (four doses of rituximab at days 1, 8, 15, 22 followed by four cycles of cytotoxic chemotherapy with CHOP-21) was initiated according to the PTLD-1 protocol 1 week later [START_REF] Trappe | Sequential treatment with rituximab and chop chemotherapy in b-cell ptld-a new standard in therapy[END_REF] (Fig. 2).

After rituximab was commenced, the patient's clinical status rapidly improved; diarrhea was less frequent, fever disappeared, and LDH elevation was reduced. However, at day 37 (2 weeks after the last or fourth dose of rituximab), the patient was readmitted with high fever, significant hemorrhagic diarrhea, and LDH elevation (401 U/l). Hemoglobin was significantly decreased (8.5 g/dl), as were platelet and white blood cells counts (91,000 and 1,010 per microliter, respectively). Total fibrinogen was in the lower normal range (166 mg/dl). Due to rapid clinical deterioration indicating progressive disease (PD), CHOP was started immediately with 50% dose reduction of cyclophosphamide and doxorubicin due to the patient's reduced peripheral blood count.

After initiation of chemotherapy at day 38, the clinical status improved rapidly again, with still frequent but nonhemorrhagic diarrhea, disappearance of fever (at day 40), and reduction of LDH (179 U/l at day 51). Platelet and white blood cell counts increased to 251,000 and 3,070 per microliter, respectively, at day 44, and total fibrinogen was 568 mg/dl at day 50. Prior to the planned second application of CHOP, the patient presented with neutropenic fever and rising LDH levels. LDH peaked at 707 U/l on day 56 when the second cycle of CHOP chemotherapy (25% dose-reduced) was initiated prematurely. Again, fever resolved quickly and LDH levels reduced to a nadir of 217 U/l but increased to 582 U/l at day 73 when the third cycle of CHOP was administered prematurely. Although there was again an initial response to chemotherapy, fever reached 40°C at day 84 and was accompanied by hemorrhagic diarrhea, increasing LDH levels, marked tachycardia, and tachypnea. Ultrasound and CT scans demonstrated progressive lymphadenopathy and colonic infiltration. EBV viral load in peripheral blood was 400,000 copies per milliliter (Fig. 2). On the basis of previous experience of antiviral therapy in ZEBRA-positive PTLD [START_REF] Oertel | Identification of early antigen bzlf1/zebra protein of Epstein-Barr virus can predict the effectiveness of antiviral treatment in patients with post-transplant lymphoproliferative disease[END_REF], antiviral therapy with cidofovir was commenced.

Two days after commencing cidofovir 5 mg/kg at day 86, the patient was febrile (41°C) and had elevated LDH (2,003 U/L) and critical gastrointestinal bleeding due to hyperfibrinolysis (fibrinogen <40 m/dl, anti-thrombin-III activity 82%), requiring multiple daily erythrocyte transfusions. Secondary consumption of platelets and coagulation factors made intensive care management necessary. Antibiotics had no evident effect. Because colonoscopy and ultrasound examinations performed on days 91 and 92 confirmed a dramatic improvement of PTLD-associated lesions, cidofovir was continued at day 94. The patient recovered quickly and hyperfibrinolysis resolved, with fibrinogen levels reaching 153 and 397 mg/dl at days 95 and 105, respectively. Platelet counts stabilized (206,000 and 270,000 per microliter at days 96 and 103, respectively) and LDH decreased (328 U/l at day 105). From day 98 onwards, the patient was afebrile and diarrhea was less frequent. Monitoring of EBV DNA in peripheral blood showed an initial increase from 403,000 copies per milliliter prior to cidofovir to 956,000 and 1,400,000 copies per milliliter at days 92 and 96, respectively, with a final drop to 3,475 copies per milliliter at day 104 when the third course of cidofovir was administered (Fig. 2). However, from a nadir of 328 U/L at 105 day, LDH levels increased to 1,664 U per 763 l within 15 days. With increasing LDH levels, the patient again experienced clinical deterioration with high fever, tachycardia, tachypnea, and a second hyperfibrinolytic syndrome with severe gastrointestinal bleeding. Fibrinogen levels decreased from 475 mg/dl on day 110 to 84 mg/dl on day 124 and abdominal ultrasound on day 124 did not indicate PTLD within the colon, but peritoneal lymphomatosis (peritoneal thickening and presence of ascites) indicative of PD was suspected. EBV DNA in peripheral blood was 400,000 copies per milliliter on day 122.

As a last resort, we initiated IVIG 0.6 g/kg followed by 0.2 g/kg every 3 weeks in addition to cidofovir treatment at day 125, which successfully terminated the second hyperfibrinolytic syndrome. From day 131 on, i.e., 6 days after the first application of IVIG + cidofovir, the patient's clinical status rapidly improved; gastrointestinal bleeding resolved, diarrhea was less frequent, fever disappeared, and LDH elevation was reduced to 884 U/l at day 128 and to 236 U/l at day 138, respectively. When EBV DNA levels increased in parallel with LDH levels after further applications of cidofovir at days 131, 141, 148, and 155, antiviral therapy was switched to foscarnet (180 mg/kg per day) at day 156. LDH peaked at 356 U/l at day 159 but normalized with foscarnet treatment within 6 days. There was no worsening of PTLD symptoms. At day 169, antiviral therapy was stopped and maintenance immunosuppression was reduced to prednisolone 15 mg/day only. The patient was discharged from hospital and continued IVIG applications every 3 weeks on an outpatient basis. With a followup of more than 19 months, the patient stayed in complete response and is still in an excellent condition, although immunosuppression had to be increased to transplant rejection and the patient's underlying autoimmune disease (graft rejection at approximately day 190, activity index according to Banff 3 of 9; histologically proven inflammatory bowel disease without evidence of PTLD on day 243). EBV-PCR remained positive during follow-up, but quantitative EBV-PCR of whole blood and plasma confirmed lytic EBV replication only until day 332, when the initially Fig. 2 a Immunosuppressive therapy consisted of 3 mg/day FK506 and 7.5 mg/day prednisolone at diagnosis of PTLD, of 1 mg/day FK506 and 7.5 mg/day prednisolone from days -7 to +169, of 15 mg/day prednisolone from days +169 to +189, of high-dose methylprednisolone from days +190 to +195 (black bar), of 30 mg/day prednisolone and 2 g/day MMF from days +196 to +217, of 0.5 mg/day FK506 and 30 mg/day prednisolone from days +218 to +249, of 1.5 mg/d FK506, 7.5 mg prednisolone and 125 mg/day AZA from days +250 to +295, and of 1.5 mg/day FK506, 7. Due to an interval of more than 1 year between transplantation and PTLD in this formerly EBV-seronegative transplant recipient, we suspected community acquired primary infection 4 to 8 weeks prior to clinical presentation rather than a transplant-associated transmission. The first approach of treatment in PTLD usually is to reduce immunosuppression. For patients that do not respond to a reduction of immunosuppression, many will go on with rituximab monotherapy [START_REF] Oertel | Effect of anti-cd 20 antibody rituximab in patients with post-transplant lymphoproliferative disorder (ptld)[END_REF][START_REF] Choquet | Efficacy and safety of rituximab in b-cell post-transplant lymphoproliferative disorders: results of a prospective multicentre phase ii study[END_REF][START_REF] Gonzalez-Barca | Prospective phase ii trial of extended treatment with rituximab in patients with b-cell posttransplant lymphoproliferative disease[END_REF] and/or CHOP-like chemotherapy [START_REF] Trappe | Sequential treatment with rituximab and chop chemotherapy in b-cell ptld-a new standard in therapy[END_REF]. However, early treatment algorithms for PTLD also included antiviral therapy in an attempt to control EBV infection [START_REF] Hanto | Epstein-Barr virus-induced b-cell lymphoma after renal transplantation: acyclovir therapy and transition from polyclonal to monoclonal b-cell proliferation[END_REF][START_REF] Hanto | Classification of Epstein-Barr virus-associated posttransplant lymphoproliferative diseases: Implications for understanding their pathogenesis and developing rational treatment strategies[END_REF][START_REF] Rees | Disappearance of an Epstein-Barr virus-positive post-transplant plasmacytoma with reduction of immunosuppression[END_REF] and polymorphic and monomorphic PTLD with expression of ZEBRA-an early marker of EBV's lytic replication-do respond to foscarnet [START_REF] Oertel | Identification of early antigen bzlf1/zebra protein of Epstein-Barr virus can predict the effectiveness of antiviral treatment in patients with post-transplant lymphoproliferative disease[END_REF]. Thus, immunologic and antiviral therapy may be moderately successful for treating EBV-associated PTLD especially PTLDs with a significant lytic replication of EBV, which may have been a critical point to success in the patient described. Fever, cytopenia, coagulopathy, and high LDH levels combined with a considerable increase of serum ferritin levels (42 μg/l at day 21, 1,334 μg/l at day 110, 13,620 μg/l at day 128) further evokes macrophage activation syndrome (hemophagocytic syndrome; HPS) in this patient. HPS is classically associated with EBV primary infection [START_REF] Reisman | Virus-associated hemophagocytic syndrome due to Epstein-Barr virus[END_REF]. Although not present at diagnosis of PTLD, HPS may have been induced by lysis of EBVinfected lymphoma cells at day 88 when cidofovir was commenced and by progression of PTLD at day 124. Application of IVIG in combination with prolonged antiviral therapy (and IR) terminated these processes inducing a significant and long-lasting response, possibly by enabling EBV-specific T cell control. (EBV-specific T cell counts were not monitored during the patients clinical course). The resumption of cidofovir therapy alone was accompanied by a rapid decrease in viral load (Fig. 2d). This is compatible with recent data suggesting that the resumption of ganciclovir therapy in primary CMV viremia can induce CMV-specific CD4 T-cells that remain stable for a period extending beyond several years [START_REF] Sester | Dominance of virus-specific cd8 t cells in human primary cytomegalovirus infection[END_REF]. Conversely, another study that analyzed patients during and after primary CMV infection in the absence of antiviral treatment demonstrated an initial rise in specific CD4 T-cells but that after a subsequent, rapid decrease, CMV-specific CD4 T cell counts remained low or undetectable in the face of detectable viral load [START_REF] Rentenaar | Development of virus-specific cd4 (+) t cells during primary cytomegalovirus infection[END_REF]. Similarly, other groups have reported in HIV infections that even transient administration of highly active antiretroviral therapy prior to seroconversion can preserve HIV-specific immunity, enabling the host immune system to subsequently control viral replication, preserving HIV-specific CD8 T cell number and function while HIV-specific T cell help is sustained [START_REF] Oxenius | Early highly active antiretroviral therapy for acute hiv-1 infection preserves immune function of cd8+ and cd4+ t lymphocytes[END_REF].

While the mechanism of immunomodulation by IVIG (and/ or anti-CMV-specific IG) in PTLD still remains to be determined, recent reports suggest significant action in the prophylaxis of PTLD [START_REF] Opelz | Effect of cytomegalovirus prophylaxis with immunoglobulin or with antiviral drugs on post-transplant non-Hodgkin lymphoma: a multicentre retrospective analysis[END_REF] via increased humoral EBV immunity [START_REF] Abedi | Response of elevated Epstein-Barr virus DNA levels to therapeutic changes in pediatric liver transplant patients: 56-month follow up and outcome[END_REF] and IVIG may be active even in patients that do not respond to upfront antiviral therapy [15].

With respect to monitoring therapy, in our patient, progression of PTLD was not accompanied by increasing EBV loads and nor were increasing EBV loads, in response to intensified immunosuppression after day 190, associated with a PTLD relapse (Fig. 2a-c). These findings are in accordance with previous observations in EBV-associated PTLD with upfront rituximab treatment [START_REF] Oertel | Epstein-Barr viral load in whole blood of adults with posttransplant lymphoproliferative disorder after solid organ transplantation does not correlate with clinical course[END_REF].

Fig. 1

 1 Fig. 1 Polymorphic PTLD containing CD20-positive B-blasts as well as numerous cells showing plasmacellular differentiation with a polytypic expression pattern of the immunoglobulin light chains. The infiltrate contained several LMP-1-positive B-blasts, as well as ZEBRA-expressing cells in the absence of EBNA-2 expression. Molecular analysis revealed polyclonal immunoglobulin gene rearrangements. a The mucosa shows a dense infiltrate composed of blasts as well as of numerous cells with features of plasmacellular differentiation (H & E stain, insert represents higher magnification). b Immunostain for CD20 (monoclonal antibody, clone L26) reveals several labeled cells frequently with large nuclei. Labeling for

  Fig.2a Immunosuppressive therapy consisted of 3 mg/day FK506 and 7.5 mg/day prednisolone at diagnosis of PTLD, of 1 mg/day FK506 and 7.5 mg/day prednisolone from days -7 to +169, of 15 mg/day prednisolone from days +169 to +189, of high-dose methylprednisolone from days +190 to +195 (black bar), of 30 mg/day prednisolone and 2 g/day MMF from days +196 to +217, of 0.5 mg/day FK506 and 30 mg/day prednisolone from days +218 to +249, of 1.5 mg/d FK506, 7.5 mg prednisolone and 125 mg/day AZA from days +250 to +295, and of 1.5 mg/day FK506, 7.5 mg/day prednisolone and 75 mg/day AZA from days +296 to +767. b LDH values from start of therapy (day 1: first application of rituximab) to end of follow-up (day +750). Arrows indicate hyperfibrinolytic syndrome due to tumor lysis (first arrow) or due to PTLD progression (second arrow). LDH values below 245 U/l were regarded within the normal range. c Treatment: solid arrows indicate the application of rituximab (375 mg/m 2 ) at days +1, +7, +14, and +21. Dotted arrows indicate application of CHOP (750 mg/m 2 cyclophosphamide, 2 mg vincristine, 50 mg/m 2 doxorubicin, 100 mg prednisolone) at days +38, +55, and +72 (information on individual dose reductions given in the text). Smalldashed arrows indicate applications of cidofovir (5 mg/kg) at days +86, +94, +104, +125, +131, +141, +148, and +155. The largedashed arrow indicates the start of foscarnet therapy (180 mg/kg per day, days +156 to +169). The light gray bar indicates continuous application of IVIG 0.2 g/kg every 3 weeks starting at day +125, indicated by a light gray line. d EBV viral load (whole blood) in copies per milliliter in a logarithmic scale from start of therapy to end of follow-up

Acknowledgements R. Trappe, H. Riess, I. Anagnostopoulos, R. Neuhaus, B. C. Gärtner, S. Jonas, M. Papp-Vary and S. Oertel are member of the German Study Group on PTLD. The German Study Group on PTLD (DPTLDSG) is a member of the German Competence Network Malignant Lymphomas (KML). Editorial support was provided by Tracy McNally and Jude Manifold at Gardiner-Caldwell Communications; this support was funded by Roche Pharma AG.


Adrian Tempescul
email: tempescul@hotmail.com

Johanna Feuerbach

Jean-Christophe Ianotto

Florence Dalbies

Veronique Marion

Marie-Josée Bris

Marc Braekeleer

Christian C Berthou

Jean-Christophe Ianotto

Marie-Josée Le Bris

Marc De Braekeleer

J.-C Ianotto

M.-J Le Bris

A combination therapy with fludarabine, mitoxantrone and rituximab induces complete immunophenotypical remission in B-cell prolymphocytic leukaemia

HAL is

Dear Editor,

We present here the cases of two patients with B-cell prolymphocytic leukaemia (B-PLL), both expressing a translocation t(8;14)(q24;q32) with rearrangement of cmyc gene found in FISH analysis. A treatment protocol that includes six cycles of Fludarabine and Mitoxantrone followed by four injections of Rituximab allows obtaining complete cytological and immunophenotypical remission.

B-PLL is a rare disease that accounts for less than 2% of chronic lymphatic leukaemia (CLL). The characteristics of the disease include high lymphocytosis, over 4 Gigas/L, with at least 55% cells described as prolymphocytes and splenomegaly. The prolymphocytes are medium-sized cells with prominent, round or oval nuclei and a single, central nucleolus [START_REF] Robak | Current treatment options in prolymphocytic leukaemia[END_REF]. Bone marrow aspirate shows a heavy infiltration by prolymphocytes with a variable degree of residual haematopoiesis. Immunophenotypically, there is a strong expression of the B-cell antigens CD19, CD20, CD21, CD79b and FMC7 and of surface IgM, whereas CD23 is negative and CD5 is variably expressed [START_REF] Robak | Current treatment options in prolymphocytic leukaemia[END_REF].

In cytogenetic studies, the implication of the 14q32 region of the chromosome 14 is found in 50% of the cases [START_REF] Robak | Current treatment options in prolymphocytic leukaemia[END_REF]. Translocation (11;14) is present in 25% of the cases, in particular t(11;14)(q13;q32) with a rearrangement of bcl1 protein. Other partners for the IgH gene of the 14q32 region of chromosome 14 could be genes from the regions 11q23 and 13q14 [START_REF] Robak | Current treatment options in prolymphocytic leukaemia[END_REF]. A translocation (8;14)(q24;q32) typically exists in type 3, B-cell acute lymphoblastic leukaemia and Burkitt lymphomas. This anomaly, which also involves cmyc, was found in only a few cases [START_REF] Kuriakose | Translocations (8;14)(q24;q32) as the sole cytogenetic abnormality in B-cell prolymphocytic leukaemia[END_REF][START_REF] Crisostomo | Complex caryotype including chromosomal translocation (8;14)(q24;q32) in one case with B-cell prolymphocutic leukaemia[END_REF]. B-PLL is not treated when asymptomatic or there are no signs of bone marrow deficiency. Treatment options are various, including combination chemotherapies with alkylating agents or purine nucleoside analogues as monotherapy. Monoclonal antibodies such as Rituximab and Alemtuzumab are also used.

A 63-year-old man was first seen in October 2006 with symptoms of asthenia and dyspnoea. A physical examination revealed a splenomegaly grade 3 and lymphadenopathy. The blood count showed a significant anaemia (Hb 5,4g/dL), thrombocytopenia (108 Gigas/L) and a hyperleukocytosis at 247Gigas/L with 92% abnormal circulating lymphocytes, which presented the typical morphology and phenotype of B-PLL. Genetic studies revealed a complex caryotype (Fig. 1), involving a translocation t(8;14)(q24;q32) with a rearrangement of c-myc, confirmed by fluorescence in situ hybridisation analysis (Fig. 2).

The leukaemia was resistant to initial treatment by Pentostatine and Dexamethasone association and to Burkittlymphoma standard therapy: COPADM (Methotrexate, Doxorubicine, Cyclophosphamid and Vincristin). A third-line treatment by Fludarabine (25mg/sqm D1-D5), Mitoxantrone (12ms/sqm at D1) was introduced, with complete cytological and immunophenotypical remission. At the end of treatment, the splenomegaly was at 1cm and we did not notice any lymphadenopathy. After six cycles of chemotherapy, the patient had a major lymphopenia at 0.03Gigas/L, without any circulating B-cell, and a normal bone marrow aspirate. The remission was consolidated by four weekly cycles of Rituximab (375mg/sqm). The patient is in complete cytological and immunophenotypical remission at 6months.

We followed the case of a 70-year-old woman presenting a hyperleukocytosis (13. analysis found the presence of a complex caryotype with translocation t(8;14)(q24;q32) with a rearrangement of cmyc gene confirmed in FISH analysis (Fig. 3).

Four years after the diagnosis, we decided to treat the patient because of the presence of signs of evolution: lymphocytosis at 85 Gigas/L with 89% B prolymphocytes, signs of medullar failure-anaemia at 11 g/dL and the presence of grade 2 splenomegaly. We used the same association: Fludarabine (25 mg/sqm D1-D5) and Mitoxantrone (12 mg/sqm at D1).

After three cycles of Fludarabine and Mitoxantrone, the patient achieved a complete cytological and immunophenotypical remission (CR). The grade 2 splenomegaly, noticed at the time of diagnosis, disappeared at course 3 of chemotherapy. The monoclonal antibody Rituximab was used as consolidation therapy. The patient is in complete remission at 5 months.

B-PLL is a rare disease that has a more critical prognosis than typical chronic lymphocytic leukaemia. A longer period of static disease occurs less frequently than in CLL. In this article, we described the cases of two patients with very different clinical presentations, one showing a sudden, more aggressive presentation of the disease while the other patient was asymptomatic for years. These findings correspond to an Israeli study that emphasised the heterogeneity of this disease [START_REF] Nguyen-Khac | Burkitt-type acute leukemia in a patient with B-prolymphocytic leukemia: evidence for a common origin[END_REF].

Translocation (8;14)(q24;q32), as found in both of our cases, is frequently found in B-cell acute lymphoblastic leukaemia, Burkitt lymphomas and other non-Hodgkin lymphomas. However, some rare cases of t(8;14)(q24;q32) in B-PLL are reported [START_REF] Kuriakose | Translocations (8;14)(q24;q32) as the sole cytogenetic abnormality in B-cell prolymphocytic leukaemia[END_REF][START_REF] Crisostomo | Complex caryotype including chromosomal translocation (8;14)(q24;q32) in one case with B-cell prolymphocutic leukaemia[END_REF]. One could consider that B-cell PLL typically presents this translocation, associated with splenomegaly and lymphocytosis. The presence of TP53 mutations in B-PLL has a peculiar prognosis; the outcome is marked by a tendency towards high-grade transformation [START_REF] Nguyen-Khac | Burkitt-type acute leukemia in a patient with B-prolymphocytic leukemia: evidence for a common origin[END_REF].

In a multicentric study of 41 patients, Hercher et al. found two groups of patients according to their clinical course: patients who died within the first year after the diagnosis and a second group, patients who had a prolonged survival, without any treatment in some cases. The differences between the two groups were the age of patients and the presence of anemia. They also noticed that the patients with p53 mutations had the worst prognosis [START_REF] Hercher | Groupe Français d'Hématologie Cellulaire-"A multicentric study of 41 cases of B-prolymphocytic leukemia: two evolutive forms[END_REF].

There is no consensus on how to treat this entity, which presents the translocation t(8;14)(q24;q32) of B-PLL. It is considered incurable and is usually not treated when asymptomatic. Combination chemotherapy with alkylating agents (COP, CHOP) allowed promising results in several studies. Nevertheless, CR was achieved in only a few cases [START_REF] Shvidel | B-cell prolymphocytic leukaemia: a survey of 35 patients emphasizing heterogeneity, prognostic factors and evidence for a group with an indolent course[END_REF][START_REF] Taylor | Prolymphocytic leukemia: Treatment with combination chemotherapy to include doxorubicin[END_REF][START_REF] Sibbald | Complete remission in prolymphocytic leukaemia with the combination chemotherapy CHOP[END_REF]. In different studies, the purine nucleoside analogues Fludarabine, Cladribine or Pentostatin, used alone, allowed achieving CR in 0% to 11% of the cases and PR in 11% to 60% [START_REF] Taylor | Prolymphocytic leukemia: Treatment with combination chemotherapy to include doxorubicin[END_REF][START_REF] Montillo | Phase II study of cladribine and cyclophosphamide in patients with chronic lymphocytic leukemia and prolymphocytic leukemia[END_REF]. CR has also been reported with the monoclonal antibodies Alemtuzumab and Rituximab [START_REF] Mourad | Successful treatment of B-cell prolymphocytic leukemia with monoclonal anti-CD20 antibody[END_REF][START_REF] Perz | Level of CD 20-expression and efficacy of rituximab treatment in patients with resistant or relapsing B-cell prolymphocytic leukemia and B-cell chronic lymphocytic leukemia[END_REF][START_REF] Chaar | Complete response to Alemtuzumab in a patient with B prolymphocytic leukaemia[END_REF]. We treated our patients according to a protocol including a combination of a purine analogue: Fludarabine and an anthracendione: Mitoxantrone followed by four consolidating injections of monoclonal antibody: Rituximab. This combination therapy allowed obtaining complete cytological and immunophenotypical remission. The tolerance of the treatment was very good; the patients did not require intercourse hospitalisation.

In this article, we reported about two cases of B-PLL, with very different clinical presentations. The cytogenetic analysis revealed in both cases a rare translocation: t(8;14) (q24;q32). In both cases, we could found, in FISH analysis, the rearrangement of the c-myc gene. The use of a combination chemotherapy including Fludarabine, Mitoxantrone and Rituximab allowed achieving cytological and immunophenotypical remission.

Even if the follow up of the patients is still short, our results encourage further investigations, suggesting that the treatment protocol associating Fludarabine, Mitoxantrone and Rituximab could be an alternative as first-line treatment of this entity.

1 derFig. 3

 13 Fig. 2 FISH using LSI IGH (spectrum green)/MYC (spectrum orange), CEP8 (spectrum aqua) tri-colour, dual fusion translocation probes (Abbott, Rungis, France) showing the t(8;14)(q24;q32) in patient 1

Fig. 1

 1 Fig. 1 Conventional cytogenetics showing the 49,XY, +der(3)t (3;18)(p11;q11), +der(3)t(3;18) (p11;q11), +7,t(8;14)(q24;q32), der(9)t(1;9)(q22;q34) karyotype in patient 1


Dear Editor, Caspofungin belongs to standard drugs for empirical antifungal therapy (A1 recommendation) [1]. Breakthrough fungal infections are rare complications under treatment of neutropenic fever with Caspofungin [2]. However, emergence of candidiasis under treatment with Caspofungin was reported in poorly penetrated organs [3]. Drug concentrations of Caspofungin are sub-optimal in brain tissue [4]. Cerebral fungal infections are rare but life-threatening complications after allogeneic stem cell transplantation. Early diagnosis and critical monitoring of response to the chosen antifungal agents is critical for successful treatment. We report about the diagnosis and treatment of a 69-year-old female patient who underwent allogeneic peripheral blood stem cell transplant from a related donor against AML M2 developed out of MDS. Conditioning comprised of TBI 2Gy, Fludarabin 120 mg/m 2 , ATG 40 mg/kg, Cyclophosphamid 120 mg/kg. Immunosuppression was accomplished by Ciclosporin A and Mycophenolat Mofetil. The patient did not completely engraft. Six months after transplantation, the patient developed pneumonia. As she did not improve under treatment with piperacillin or combactam and teicoplanin, we added caspofungin after 72 h. Hereunder, she improved tremendously. After 4 weeks, she developed ataxia and dysarthria. The cranial magnetic resonance imaging (MRI) showed two lesions most likely to be of infectious origin in the left parietal lobe and in the cerebellum (Fig. 1). Candida polymerase chain reaction of the liquor was positive. A second liquor puncture confirmed this result. Treatment was initiated with liposomal Amphothericin B 3 mg/d/kg intravenous and Voriconazol 200 mg twice a day per os. Within 2 weeks, all clinical symptoms resolved and within 4 weeks, the control MRI showed remission. Treatment with Voriconazol was continued for 6 weeks.

In part, Candida breakthrough infections may be due to the changing scope of strains [5]. However, to our knowledge, only a few cases of candida breakthrough infections under Caspofungin are reported [3][4][5][6][7][8][9]. Krogh-Madson et al. isolated Caspofungin resistant C. glabrata from a critically ill liver transplant patient [6]. In another case C. parapsilosis infection emerged under co-administration of phenytoin maybe leading to decreased Caspofungin concentrations [6]. Mougdal et al. showed by molecular-genetic studies that one initial susceptible Candida glabrata strain acquired resistance to Caspofungin under therapy [9]. As penetration of caspofungin in brain tissue is low, we think Candida could resist treatment in this tissue in our case. This report indicates that cerebral fungal infections can occur during Caspofungin therapy and should be considered as differential diagnosis.

  LETTER TO THE EDITOR Successful treatment of Candida spp. encephalitis occurring under Caspofungin treatment after allogeneic stem cell transplantation G. Behre & M. Christopeit & T. Weber Received: 6 May 2008 / Accepted: 24 June 2008 / Published online: 31 July 2008 # Springer-Verlag 2008

Ann Hematol (2009) 88:93-94 DOI 10.1007/s00277-008-0551-3 G. Behre and T. Weber contributed equally. G. Behre (*) : M. Christopeit : T. Weber Department of Oncology and Hematology, University Hospital Halle, Halle, Germany e-mail: GerdBehre@aol.com Fig. 1 The cranial magnetic resonance imaging (MRI) showed two lesions most likely to be of infectious origin in the left parietal lobe and in the cerebellum


est destinée au dépôt

Reversible cardiac dysfunction without myocytolysis related to all-trans retinoic acid administration during induction therapy of acute promyelocytic leukemia

Annunziata Manna, Luigi Cadenotti, Andrea Motto, Piercarlo Ballo

To cite this version:

Annunziata Manna, Luigi Cadenotti, Andrea Motto, Piercarlo Ballo. Reversible cardiac dysfunction without myocytolysis related to all-trans retinoic acid administration during induction therapy of acute promyelocytic leukemia. On day 6 of ATRA treatment, echocardiography showed left ventricular enlargement (end-diastolic volume 160 ml, normal range: 67-155 ml; indexed end-diastolic volume 90 ml/m 2 , normal range 35-75 ml/m 2 ) with diffuse hypokinesia and reduced ejection fraction (45%, normal range: ≥55%; figure, left panels). Electrocardiographic findings were within normal limits. The patient became febrile and reported asthenia (NYHA II), dyspnea, and diffuse muscular pain. A pleural effusion and weight increase (+6 kg) were also observed. Repeated measurements of troponine I, myoglobin, and CK-MB were within the normal ranges, whereas brain natriuretic peptide was elevated (321 pg/ml, normal range: <100 pg/ml). White blood cell count showed leukocytosis (17.1×10 9 /l) with atypical granular blasts. A diagnosis of ATRA syndrome was made. Treatment with diuretics and full-dose prednisone was started while ATRA was discontinued. In the following days, fever and muscular pain disappeared. Three months later, echocardiography showed reduction of left ventricular size (end-diastolic volume, 124 ml; indexed end-diastolic volume, 72 ml/m 2 ) with normalization of ejection fraction (62%; figure, right panels).

The pathogenesis of myocarditis in patients under ATRA treatment depends on complex interaction between extravasation of acute promyelocitic leukemia cells, leukocyte infiltration, endothelial injury, and cytokine release, leading to myocardial cell lysis and release of cardiac biochemical markers into the blood [START_REF] Fabbiano | All-trans retinoic acid induced cardiac and skeletal myositis in induction therapy of acute promyelocytic leukaemia[END_REF]. To our knowledge, only three cases of ATRA-induced myocarditis-all with electrocardiographic modifications and increase in myocardial necrosis indices-have been reported [START_REF] Fabbiano | All-trans retinoic acid induced cardiac and skeletal myositis in induction therapy of acute promyelocytic leukaemia[END_REF][START_REF] Klein | Two cases of isolated symptomatic myocarditis induced by all-trans retinoic acid (ATRA)[END_REF]. In this case, clinical and echocardiographic evidence of left ventricular systolic dysfunction occurred without electrocardiographic changes and despite normality of myocardial biomarkers. Although causal relation between ATRA administration and cardiac

A. Manna : L. Cadenotti Department of Oncohaematology, Felettino Hospital, La Spezia, Italy A. Motto : P. Ballo Cardiology Operative Unit, S. Andrea Hospital, La Spezia, Italy P. Ballo (*) U.O. Cardiologia, Ospedale "S. Andrea", via Veneto 197, 19100 La Spezia, Italy e-mail: pcballo@tin.it dysfunction cannot be directly demonstrated, we found no evidence of other conditions associated with increased risk of myocarditis. Based on the absence of clinical or echocardiographic signs of concomitant endocarditis, a potential role of recent dental surgery also seems unlikely. Moreover, based on the Naranjo scale [START_REF] Naranjo | A method for estimating the probability of adverse drug reactions[END_REF]-an objective algorithm for assessing the probability of adverse drug reactions-the causal relation between ATRA administration and cardiac dysfunction was categorized as probable. This suggests the intriguing hypothesis that ATRA cardiac toxicity may lead to myocardial damage and reversible dysfunction even in the absence of significant myocytolysis.


Ricardo García-Muñoz

Paula Rodriguez-Otero

Carlota Pegenaute

Juana Merino

Juan Jakes-Okampo

Luis Llorente

Maurizio Bendandi

Carlos Panizo

Splenic marginal zone lymphoma with Evans' syndrome, autoimmunity, and peripheral gamma/delta T cells

Dear Editor, Several theories have tried to explain the occurrence of autoantibodies in lymphoid malignancies. Autoimmune phenomena like Evans' syndrome, lupus anticoagulants, autoimmune thrombocytopenia, and autoimmune hemolytic anemias are frequently associated with lymphomas [START_REF] Hauswirth | Autoimmune hemolytic anemias, Evans' syndromes, and pure red cell aplasia in non-Hodgkin lymphomas[END_REF][START_REF] Hauswirth | Autoimmune thrombocytopenia in non-Hodgkin's lymphomas[END_REF]. Recently, systemic lupus erythematosus (SLE) was associated with an increased risk of diffuse large B cell and marginal zone lymphomas [START_REF] Smedby | Autoimmune disorders and risk of non-Hodgkin lymphoma subtypes: a pooled analysis within the InterLymph Consortium[END_REF]. Splenic marginal zone lymphoma (SMZL) is an indolent B cell lymphoma, which generally presents splenomegaly and involvement of both bone marrow and peripheral blood. In this report, we describe a patient with autoimmunity and bone marrow infiltration that probably broke the mechanisms of central tolerance in bone marrow producing the emergency of new auto-reactive clones and autoantibodies.

A 60-year-old man had a 12-month history of B symptoms. Physical examination was unremarkable, except for the presence of massive splenomegaly. A computerized tomograhpy (CT) scan of the abdomen confirmed spleno-megaly and visualized enlarged lymph nodes in retroperitoneum (Fig. 1). Blood smear and peripheral blood immunophenotype suggest SMZL with villous lymphocytes.

The patient underwent laparotomy for splenectomy, lymph node, and bone marrow biopsies. Histology corresponds to SMZL. Follow-up studies detected the persistence of lymphoid neoplastic cells (1.4%) and a γδ T cell population (TCR γδ CD2+, CD3+, CD5 low+, CD7+, CD8 low+/-, CD16 -/+ CD56+, CD57 +/-) that represented 12% of the cells in peripheral blood. Five months after diagnosis, the patient presented with abdominal discomfort. On a CT scan, abdominal adenopatic progression was observed and the two populations (3.4% of SMZL and 4.9% of γδ T cells) persisted in peripheral blood. The patient received eight cycles of chemotherapy with cyclophosphamide-doxorubicin-vincristin-prednisone regime, resulting in radiological remission. At this moment, flow cytometry of peripheral blood revealed 0.0026% of tumor cells and 13.75% of γδ T cells.

Four months after chemotherapy, the patient was admitted to the hospital with a recurrence of SZML. He showed, besides, respiratory insufficiency and Evans' syndrome, associated with high titers of antiphospholipid antibodies, lupus anticoagulant and prolonged partial tromboplastin time, high levels of immune complexes, and low levels of both immunoglobulins and C3 and C4 fractions of complement. Antinuclear, antineutrophil cytoplasmic, anticardiolipin, antiplatelets, anti-HLA I, and anti-EPCR antibodies were also detected. CT scan ascertained the presence of pulmonary emphysema and hepatomegaly, without associated enlarged lymph nodes. Peripheral blood flow-cytometry indicated an increase in the neoplastic B cell population (50%) and a decrease of γδ T cells (5%). The bone marrow biopsy confirmed lymphoid infiltration (SMZL 35% and γδ T cells 4%) by immunophenotype.

With the diagnosis of SMZL relapse, chemotherapy with fludarabin-mitoxantrone-dexamentasone and rituximab (FMD-R) was started. Surprisingly, after one cycle of FMD-R, all the autoimmune tests became negative, C3 and C4 fractions of complement reached normal levels, and immune complexes were found to be negative. After three cycles, a complete remission was achieved. Flow-cytometry of peripheral blood did not detect any B cells, although the γδ T cells persisted, representing 2.5% of all peripheral blood cells and 64% of the circulating lymphocytes.

It is generally accepted that most auto-reactive B cells are eliminated from the normal repertoire. Within the bone marrow, B cells that rearrange and express self-reactive receptors at the immature stage are either edited or eliminated by apoptosis. Defects in the retention of selfantigen could result in the loss of efficient negative selection and escape of self-reactive B cells to the periphery. Complement-binding in immune complexes or in autoantigens could protect from autoimmunity by enhancing presentation of antigens to self-reactive B cells at the immature stage or in the T1-T2 transitional stage [1,[START_REF] Carroll | The complement system in regulation of adaptive immunity[END_REF]. Interestingly, marginal zone B cells have been shown to initiate immune responses by transporting IgM antigenimmune complexes into the follicle and may play an important role in the removal of senescent cells, apoptotic debris, and immune complexes. The fact that removal of the spleen appears to alter the disease distribution in some patients with SMZL, resulting in an increase in the bone marrow infiltration, could contribute towards the migration of neoplastic SMZL with immune-complexes and autoantigens (e.g., apoptotic debris evoked by chemotherapy) to the bone marrow and alter central tolerance. Abnormally expanded γδ T cells could provide increased amounts of help to B cells to produce autoantibodies. Importantly, several autoimmune diseases show an expansion of altered γδ T cells like in Behcet's disease, rheumatoid arthritis, and SLE.


Carlo Visco
email: carlovisco@hotmail.com

Enrico Marchioni

Fabrizio Pomponi

Pasquale Ferrante

Serena Delbue

Gianpietro Pellizzer

Francesco Rodeghiero

: S Delbue

Progressive multifocal leuconcephalopathy and autoimmune haemolytic anemia in chronic lymphocytic leukaemia: more than a fortuitous combination?

LETTER TO THE EDITOR

Progressive multifocal leuconcephalopathy and autoimmune haemolytic anemia in chronic lymphocytic leukaemia: more than a fortuitous combination? Dear Editor, Progressive multifocal leucoencephalopathy (PML) is a subacute demyelinating disorder of the central nervous system usually affecting immunocompromised individuals [START_REF] Walker | The epidemiology of human polyomaviruses[END_REF], caused by JC polyomavirus (JCV) reactivation. Apart from being a relevant cause of morbidity in human immunodeficiency virus-seropositive (HIV+) patients, PML may be observed during lymphoproliferative disorders, rheumatic disease or bone marrow transplantation. The JCV remains lifelong latent in the kidneys and in the peripheral blood mononuclear cells (PBMC) [START_REF] Lafon | JC virus remains latent in peripheral blood B lymphocytes but replicates actively in urine from AIDS patients[END_REF] of infected patients unless immunosuppression occurs, allowing infected PBMC to migrate to the brain, where astrocytes and oligodendrocytes support JCV replication resulting in neurologic damage [START_REF] Sabath | Traffic of JC virus from sites of initial infection to the brain: the path to progressive multifocal leukoencephalopathy[END_REF]. Normal T-cell lymphocytes seem to play a crucial role in this setting, preventing proliferation and migration of infected PBMC. Low numbers of CD4+ T-lymphocytes have been related to PML development, while functional specific CD8+ T-cells are involved in early control of PML [START_REF] Pasquier | A prospective study demonstrates an association between JC virus-specific cytotoxic T lymphocytes and the early control of progressive multifocal leukoencephalopathy[END_REF]. Thus, immunosuppressive and immunomodulating treatments may enhance the risk for reactivation of latent JCV. This is the case of natalizumab, a humanised monoclonal antibody directed against α4 integrins, which has been associated to PML occurrence in patients treated for multiple sclerosis and Crohn's disease [START_REF] Boren | The emergence of progressive multifocal leukoencephalopathy (PML) in rheumatic diseases[END_REF]. Rituximab, a monoclonal antibody against B-cell lymphocytes, and fludarabine, a purine analogue that impairs the function of both T-and B-lymphocytes, have been associated with the occurrence of PML in haematologic malignancies and rheumatic disease [START_REF] García-Suárez | Changes in the natural history of progressive multifocal leukoencephalopathy in HIV-negative lymphoproliferative disorders: impact of novel therapies[END_REF], but a causal relation between these drugs and PML seems yet speculative.

We report a case of a 63-year-old HIV-negative male patient with a 10-year history of chronic lymphocytic leukaemia (CLL). Assessment of prognostic variables at presentation revealed a mutated pattern with VH3-21 gene usage and low expression of Zap-70 and CD38 by tumor cells, with a 13q deletion. The patient had been previously treated with two courses of chlorambucile and two courses of fludarabine-based chemotherapy. One month after the last chemotherapy, while the patient was still rituximab naïve, he presented to our Department because of an isolated progressively worsening visual loss. Anamnesis revealed a recent episode of transient diplopia. Brain computed tomography and magnetic resonance imaging (MRI) revealed aspecific non-enhancing bilateral occipital lesions. Cerebrospinal fluid (CSF) cell count and biochemical analysis was within normal limits. Haematologic assessment showed increasing lymphocytosis (from 130 to 170×10 3 /μL), accompanied by a deep fall in the haemoglobin level (from 12.4 to 6.7 g/dL) and by swelling of the spleen and lymphoadenopathies. Serum immunoglobulin levels were low (IgG 513 mg/dL; IgA 10 mg/dL; IgM 11 mg/dL), and immunophenotypic analysis revealed a striking reduction of CD4+, and CD8+ T-lymphocytes on peripheral blood (0.2 and 0.05×103/μL, respectively). A Coomb's positive autoimmune haemolytic anemia (AHA) by IgG warm antibodies was diagnosed. The patient was then started with a regimen including rituximab, cyclophosphamide, vincristine and prednisone (R-COP), completing three cycles and obtaining a rapid and complete regression of lymphoadenopaties, splenomegaly, lymphocytosis, and of the AHA. The visual defect remained stable during the 2 months of immunochemotherapy. However, 2 months after the end of treatment, the patient was hospitalised because of acute dyspnoea and persistent fever. Cytomegalovirus reactivation was detected and therapy with intra-venous gancyclovir and antibiotics was started, with improvement of respiratory symptoms. A new MRI of the brain showed bilateral non-enhancing foci of white matter abnormalities in the occipital regions, compatible with a diagnosis of PML. Biochemical analysis and cell count of the CSF was within normal limits, but JCV search was positive in both blood and CSF by real time PCR (3.58×10 4 copies/mL and 1×10 8 copies/mL, respectively). Cidofovir therapy was started, but neurologic symptoms progressed to hemiparesis, and then to a cognitive impairment in 1 month. The patient died 3 months later.

Visual loss, together with motor weakness, is the most frequent initial deficit in patients affected by PML. To the best of our knowledge, this is the first report describing the concomitant occurrence of neurologic symptoms attributable to PML and AHA in a patient with CLL. However, this association seems not surprising. CLL is typically characterised by profound immunosuppression, since primary antibody response declines early during the disease. Furthermore, T-cell response is impaired by pathological regulatory cytokines and by interactions between normal T-and Blymphocytes and CLL B-cells. In this setting, the altered function of regulatory T-cells (CD4+CD25+FOXP3+), which are also hampered by immunosuppressive treatments, allows the emergence of normally suppressed B-cell clones producing autoantibodies, giving rise to autoimmune phenomena. AHA is the most frequent autoimmune complication in the course of CLL, and its frequency significantly increases in heavily pre-treated patients with progressive disease, espe-cially after fludarabine monotherapy [START_REF] Dearden | The prognostic significance of a positive direct antiglobulin test in chronic lymphocytic[END_REF]8]. Only about 2% of patients with CLL develop haemolysis after one line of therapy, compared with about 5% of patients who have received more than one previous treatment and 20% of heavily treated patients [9]. Reduction of T-cell count, hypogamma-globulinemia and the immunosuppressive effect of treatment represent the pathogenetic link between occurrence of AHA and infectious complications as PML. As outlined before, at onset of neurologic symptoms, both CD4+, and CD8+ T-cell count and immunoglobulin levels were strikingly reduced in our patient and never recovered during the subsequent controls.

Although many cases of PML have been reported outside the HIV epidemic in patients with CLL treated with fludarabine [10], current and new immunosuppressive treatments may increase the risk of reactivation of latent infections. In our patient, neurologic symptoms preceded administration of rituximab, then remained stable for several months after the first dose, but worsened thereafter. Thus, while a causal role for this drug in the development of PML seems not likely in our patient, immunotherapy may have contributed to the subsequent progression to hemiparesis and cognitive impairment. Our case shows that PML must be considered in patients experiencing acute onset of neurologic symptoms in the CLL setting, especially when heavily pre-treated with agents impairing T-cell function like fludarabine. leukemia: a beneficial effect of the combination of fludarabine and cyclophosphamide on the incidence of hemolytic anemia. Blood 111:1820-1826 doi:10.

  Carlo Visco & Enrico Marchioni & Fabrizio Pomponi & Pasquale Ferrante & Serena Delbue & Gianpietro Pellizzer & Francesco Rodeghiero Received: 25 April 2008 / Accepted: 17 July 2008 / Published online: 12 August 2008 # Springer-Verlag 2008

  1182/blood-2007-07-101303 8. Visco C, Ruggeri M, Evangelista ML, Stasi R, Zanotti R, Giaretta I et al (2008) Impact of immune thrombocytopenia on the clinical course of chronic lymphocytic leukemia. Blood 111:1110-1116 doi:10.1182/blood-2007-09-111492 9. Hamblin TJ (2006) Autoimmune complications of chronic lymphocytic leukemia. Semin Oncol 33:230-239 doi:10.1053/j. seminoncol.2006.01.011 10. Kiewe P, Seyfert S, Körper S, Rieger K, Thiel E, Knauf W (2003) Progressive multifocal leukoencephalopathy with detection of JC virus in a patient with chronic lymphocytic leukemia parallel to onset of fludarabine therapy. Leuk Lymphoma 44:1815-181 doi:10.1080/1042819031000116625


M D Diamantidis
email: diamantidis79@yahoo.gr

P Klonizakis

S Haralambidou

E Vlachaki

I Klonizakis

I Venizelos

teaching and research institutions in France or abroad, or from public or private research centers.

Fatal chylous ascites, pericarditis and extensive venous thrombosis, due to an aggressive T cell non-Hodgkin lymphoma

Dear Editor, Chylous ascites or chyloperitoneum is the extravasation of milky peritoneal fluid rich in triglycerides in the abdominal cavity, because of lymphatic obstruction or trauma. Cirrhosis, malignancies and infectious agents are frequent etiologies, whereas congenital, inflammatory, post-operative and traumatic causes have been described [START_REF] Almakdisi | Lymphomas and chylous ascites: review of the literature[END_REF]. The incidence of chylous ascites is between 1 in 50,000 and 1 in 187,000 hospital admissions. Neoplasms are mainly responsible for chyloperitoneum, with lymphomas predominating among malignant causes in young patients [START_REF] Bachmeyer | Chylous ascitis revealing a non-Hodgkin lymphoma[END_REF]. We report a case of a young patient presenting with chylous ascites, chylothorax, pericarditis and venous thrombosis in several anatomic regions, due to an aggressive T cell non-Hodgkin lymphoma (T-NHL). To the best of our knowledge, this is the first case of such co-existing pathologic features, revealing a T-NHL. The need for effective treatment is essential in such medical incidents, especially when young patients suffer from aggressive neoplasms. However, rarity of such cases and lack of guidelines concerning therapeutic options impede the task of haematologists. Therefore, reporting lymphomas combined with chyloperitoneum facilitates the choice of appropriate treatment and the outcome comparison between similar incidents.

A 38-year-old man with 3-month recurrent effusive right pleuritis, after being initially treated for presumed tuberculosis, was referred to our centre. The patient had increasing abdominal distention and shortness of breath. Physical examination revealed ascites, left axillary lymphadenopathy and upper limb oedema. Lymph node biopsy was diagnostic of peripheral T-NHL, unspecified. There was complete effacement of the nodal architecture, along with infiltration by neoplasmatic, lymphoid cells with abundant mitotic figures. They were medium-sized or enlarged with slightly eosinophilic cytoplasm and irregular-shaped nuclei. Immunohistochemically, tumour cells were strongly positive for CD5, mildly positive for CD3 and negative for CD20, CD79a, CD30, CD56, CD23 and cyclin D1 (Fig. 1). Peripheral, unspecified T-NHLs derive from post-thymic T cells at various differentiative stages. Computed tomography (CT) scan revealed ascites, ambilateral pleuritic fluid causing mediastinal compression, pericarditis and excessive left axillary, mediastinal, mesenteric and retroperitoneal lymphadenopathy (Fig. 2). Enlarged lymph nodes surrounding the thymus were traced. The presence of pericardial fluid was confirmed by an echocardiogram. Paracentesis elicited chylous-effusive pleuritic fluid and creamy white ascetic fluid with elevated triglycerides (1,212 mg/dl), low protein (2.6 g/dl) and cell count (550 cells per decilitre) with lymphocytic predominance.

Moreover, thrombosis was detected in the left brachiocephalic, subclavian and internal jugular veins, as a result of the CT scan. Simultaneously, after inflammation of the right thigh skin was traced, color-flow Doppler ultrasonography revealed extensive thrombosis of the right femoral and popliteal veins (Fig. 3). D-dimers (382.6 ng/ml) and fibrinogen levels (791.6 mg/dl) were elevated. Protein S, protein C, antithrombin III and homocysteine levels were within normal range. No anti-phospholipoid antibodies or factor V Leiden mutation was present. Therefore, severe thrombosis was attributed to aggressive T-NHL.

The patient underwent six cycles of bi-weekly combination chemotherapy with cyclophosphamide (1,200 mg/m 2 ), doxorubicin (60 mg/m 2 ), vincristine (2 mg) and prednisone (500 mg) plus etoposide (600 mg/m 2 ; CHOEP) regimen. Treatment duration was 80 days. Granulocyte colonystimulating factor (300 μg subcutaneously [s.c.]/week) and pegfilgrastim were also applied. Extensive thrombosis was successfully treated by fondaparinux sodium (7.5 mg s.c./ day). Ascites receded for 5 months, before a new relapse. Albumin and diuretics were unsuccessfully used to treat recurrent chyloperitoneum. After another CHOEP cycle, therapeutic paracentesis was still necessary to overcome ascetic symptoms. Two cycles of dexamethasone (40 mg/ m 2 ), mesna (400 mg/m 2 ), ifosfamide (1,500 mg/m 2 ), cisplatin (40 mg/m 2 ) and etoposide (180 mg/m 2 ) regimen at highest dosage were then applied, without success. Alemtuzumab (Campath ® ; 30 mg, three times weekly, for 6 weeks) was the final therapeutic approach [START_REF] Gallamini | Alemtuzumab (Campath-1H) and CHOP chemotherapy as first-line treatment of peripheral T-cell lymphoma: results of a GITIL (Gruppo Italiano Terapie Innovative nei Linfomi) prospective multicenter trial[END_REF]. Eighteen months after diagnosis, the patient died, due to thrombopaenia, resulting in fatal gastrorrhagia. Despite rarity of lymphoma-caused chyloperitoneum, some cases have been reported. A case of Hodgkin's disease presenting with chylous ascites and cavernous transformation of the portal vein has been described [START_REF] Gonen | Hodgkin's disease presenting with chylous ascites and cavernous transformation of the portal vein[END_REF]. Moreover, symptomatic enterocele as an unusual presentation of chylous ascites and follicular lymphoma has been reported recently [START_REF] Ward | Symptomatic enterocele. An unusual presentation of chylous ascites and lymphoma[END_REF]. There is a lack of data regarding appropriate treatment of T-NHLs, which develop the complication of chylous ascites. The prognosis is dismal. The majority of patients die within a few months, regardless of chemotherapy [START_REF] Almakdisi | Lymphomas and chylous ascites: review of the literature[END_REF].

Despite fatal outcome, our patient survived 18 months, which is much longer than the expected 3 to 6 months, and became free of ascites for 5 months. This rare case shows the efficacy of CHOEP regimen, even in aggressive NHLs of young patients, which present with systemic complications and are characterized by bad prognosis. Furthermore, our patient received full average relative chemotherapy dose intensity (ARDI), as the administered dosage of all regimens was exactly equivalent to the dosage planned by our initial protocol. Thus, ARDI in our patient was 100%, which was an important goal achieved, regardless of the patient's death. When ARDI is less than 90%, NHL patients are at risk of reduced survival [START_REF] Pettengell | Association of reduced relative dose intensity and survival in lymphoma patients receiving CHOP-21 chemotherapy[END_REF]. In conclusion, more clinical trials testing new chemotherapy combinations and novel drugs are needed for increasing treatment options in aggressive T-NHLs of young patients. The latter will lead to better dosing schemes and therapeutic results.

  Ioannidou-Papagiannaki, Michael D. Diamantidis, Ioannis Livanis, Philippos Klonizakis, Styliani Haralambidou-Vranitsa, Efthymia Vlachaki, Ioannis Venizelos, Ioannis Klonizakis To cite this version: Elizabeth Ioannidou-Papagiannaki, Michael D. Diamantidis, Ioannis Livanis, Philippos Klonizakis, Styliani Haralambidou-Vranitsa, et al.. Fatal chylous ascites, pericarditis and extensive venous thrombosis, due to an aggressive T cell non-Hodgkin lymphoma. Annals of Hematology, 2008, 88 (4), pp.371-373. 10.1007/s00277-008-0570-0. hal-00486523 LETTER TO THE EDITOR

Fig. 1 aFig. 2

 12 Fig. 1 a Large and medium-sized lymphoma cells (haematoxylin and eosin stain, ×400). b Positive immunohistochemical stain for CD3


J.-R Eveillard

C Berthou

V Marion

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Dear Editor, We reported an exceptional case of a tri-lineage disease arising in a 67-year-old man. The patient was followed for IgG lambda monoclonal gammopathy of undetermined signification (MGUS) for 10 years. He developed painful symptomatic multiple myeloma (14% marrow mature plasmocytes IgG lambda of 28 g/l, osteolytic lesions of humerus, skull and femur) and his ISS score was 2 (β2microglobuline at 4.5 mg/l and albumin at 40 g/l). Conventional karyotype showed loss of chromosome Y, and in-situ fluorescence hybridisation revealed del(13q). Interestingly, the same marrow aspirate revealed 5% blasts and 31% erythroblasts despite the haemoglobin level being 9.5 g/dl. Refractory anaemia with ringed sideroblasts was identified by Perls' staining, which showed 53% ringed sideroblasts (Fig. 1).

The patient was treated using the VAD (Vincristin, adriamycin and dexamethasone) standard protocol and EPO. After three courses of chemotherapy, the response was evaluated to be at 20% only on the heavy chain compound in the plasma. Before the fourth course of treatment, he developed a dyspnoea. Pulmonary computed tomography showed four tumours ranging in size from 2 to 7 cm. A transparietal biopsy was performed, and immunohistochemistry revealed the presence of CD5-/CD10-/ CD20+ diffuse large B-cell non-Hodgkin's lymphoma (All B-cells appeared positive for lambda light chain (Fig. 2). Unfortunately, the patient died 5 days after the biopsy. This is the first case reporting the concomitant appearance of refractory anaemia with ringed sideroblasts, multiple myeloma and B-cell non-Hodgkin's lymphoma in the same patient. Furthermore, the lambda light chain was identified in the plasmocytic and lymphoid tissues. Association of the first two diseases has been published before [START_REF] Economopoulos | Myelopathies during the course of multiple myeloma[END_REF][START_REF] Itoh | Sideroblastic anemia associated with multiple myeloma in Turner's syndrome[END_REF][3][4]. However, only four concomitant cases with a predominant IgA isotype have been reported. Secondary cases of ringed sideroblasts following treatment of myeloma, with odd-ratio of 100, have been described as well [5]. The association between a non-Hodgkin's lymphoma (T type) and multiple myeloma has been reported only in one case [6].

Our brief report confirmed that a tri-lineage haematological disease may appear in the same patient. We suggest that the multiple myeloma and DLCBL may originate from the same clone because both presented the same light chain.

The cumulative haematological diseases were fatal to the patient.

  Tri-lineage disease involving sideroblastic anaemia, multiple myeloma and B-cell non-Hodgkin's lymphoma in the same patient Jean-Christophe Ianotto, Adrian Tempescul, Jean-Richard Eveillard, Véronique Marion, Isabelle Quintin-Roué, Christian C. Berthou To cite this version: Jean-Christophe Ianotto, Adrian Tempescul, Jean-Richard Eveillard, Véronique Marion, Isabelle Quintin-Roué, et al.. Tri-lineage disease involving sideroblastic anaemia, multiple myeloma and Bcell non-Hodgkin's lymphoma in the same patient. Annals of Hematology, 2008, 88 (3), pp.273-274. 10.1007/s00277-008-0586-5. hal-00486527 LETTER TO THE EDITOR

Fig. 1

 1 Fig. 1 Cytological aspect of bone marrow aspirates with mature plasmocytes and erythroblasts. Perls coloration confirmed the presence of ringed sideroblasts


M Griva

N I Georgiakaki

Anagnostopoulos

A Galanopoulos

S I Papadhimitriou

M Kritikou-Griva

N I Anagnostopoulos

E Kritikou-Griva

M Georgiakaki

Μultiple myeloma developing after imatinib mesylate therapy for chronic myeloid leukemia

M ultiple myeloma developing after imatinib mesylate therapy for chronic myeloid leukemia

Dear Editor, The coexistence of chronic myelogenous leukemia (CML) and multiple myeloma (MM) is an extremely rare event [START_REF] Nitta | Multiple myeloma preceding the development of chronic myelogenous leukemia[END_REF]. Imatinib mesylate (IM) has become the mainstay first line treatment for CML [START_REF] Peggs | Imatinib mesylate-the new gold standard for treatment of chronic myeloid leukemia[END_REF]. We report the case of a patient who was diagnosed with CML and developed MM after 10 months of treatment with IM. To our knowledge, our patient is the third case of CML preceding the occurrence of MM and the second case of occurrence of MM post IM treatment for CML [START_REF] Garipidou | Development of multiple myeloma in a patient with chronic myeloid leukemia after treatment with imatinib mesylate[END_REF].

A 76-year-old white male was diagnosed with CML in May 2003. Cytogenetic analysis revealed the presence of Philadelphia (Ph 1 ) chromosome: 46 XY, t(9;22) (q34;q11). BCR/ABL mRNA transcript was detected by reverse transcription polymerase chain reaction (RT-PCR). Interphase fluorescence in-situ hybridization (iFISH) in bone marrow smears revealed deletion of the 5′ ABL on the derivative chromosome 9 in 62% of the cells. The patient received interferon-alpha. Treatment with interferon was switched to IM (400 mg once daily) after 4 months because of severe side effects as whole body worsening erythema and pruritus related to interferon. Bone marrow aspirate, 10 months after the initiation of imatinib treatment, showed normal cellularity, white and red lineages decreased and a plasma cell count of 60%. Cytogenetic analysis showed no cytogenetic response. A new iFISH in bone marrow revealed again the deletion of the 5′ ABL on the derivative chromosome 9 in 28% of the cells and trisomy 9 in 49% of the cells. Laboratory tests showed anemia, but a skeletal survey showed no lytic lesions. Serum immunofixation demonstrated an IgA κ-type chain M-protein (19.5 g/l) without depression of the other immunoglobulins and k light chains 1,700 mg/dl. Bone marrow biopsy disclosed diffuse bone marrow infiltration by plasma cells, which were k light chain positive with immunoperoxidase staining. iFISH on plasma cells showed no rearrangement involving IGH gene, 13q-or p53 but trisomy of one, three and seven chromosomes in all plasma cells, indicating a hyperdiploid MM. The patient's clinical condition deteriorated and he was dependent on red blood cell transfusion. He was treated with melphalan and prednisolone; imatinib was continued. Six months later, a bone marrow aspirate showed normal findings with a plasma cell count of 3%. No M-protein was detectable on serum immunofixation. iFISH study showed 97% of the marrow cells to be BCR/ABLpositive but it failed to reveal any aneuploid cells. Six months later, the patient progressed to blast crisis and died because of massive bleeding from the upper gastrointestinal tract.

Our patient developed MM 10 months post IM treatment, but it is difficult to prove whether the development of MM was random or accelerated by IM. It is reported that IM has inhibited the proliferation of MM cells by arresting cell cycle progression and furthermore inhibited the proliferation of MM cells resistant to dexamethasone or melphalan and had an additive effect when combined with dexamethasone. Nevertheless, IM had a small stimulatory effect on the proliferation of MM cells through activation of the Erk1 and Erk2 mitogen-activated protein kinases (MAPKs) route [START_REF] Schwarzmeier | Simultaneous occurrence of chronic myeloid leukemia and multiple myeloma: evaluation by FISH analysis and in vitro expansion of bone marrow cells[END_REF]. In the literature, only six patients among 189 CML patients treated with IM have developed a second malignancy [START_REF] Pandiella | Imatinib mesylate (STI571) inhibits multiple myeloma cell proliferation and potentiates the effect of common antimyeloma agents[END_REF].

The coexistence of CML and MM cannot be explained by the hypothesis that both disorders arise from malignant transformation of the common pluripotent stem cell because in our case only marrow granulocytic and not myeloma cells were positive for BCR-ABL fusion signal by FISH analysis.

Correlation of IM in the development of MM cannot be ruled out. Further long-term analyses are necessary in patients receiving IM, to conclude about the long-term side effects of this, so widely administered in many diseases, tyrosine kinase inhibitor.


M Christina Cox

Massimo Marignani

Barbara Veggia

Stefano Angeletti

Francesco Leone

Paola Cipriani

Sara Gallina

Gianfranco Delle Fave

M Antonietta Aloe-Spiriti

Bruno Monarca

Gianfranco Delle

M Antonietta

Is management with lamivudine

Dear Editor, We read with interest the article by Yi-Fu H. et al. [START_REF] Yi-Fu | The effectiveness of lamivudine in preventing hepatitis B viral reactivation in rituximab-containing regimen for lymphoma[END_REF] about the effectiveness of Lamivudine (LAM) prophylaxis in preventing hepatitis B viral (HBV) reactivation in Rituximab containing regimens (so called immunochemotheraphy, ICT) for lymphoma and would like to make a contribution to this emerging topic. All patients diagnosed with Non-Hodgkin Lymphoma (NHL) from January 2005 to March 2008, at our Institution, were screened for HBV serology: HBsAg, anti-HBs, anti-HBc, HBeAg, anti-HBe. Patients were also tested for HCV antibodies (HIV-positive patients were not included): 18 out of 159 patients (11.3%) had serological signs of HBV exposure. 12 out of 159 (8.1%) patients were HCV-infected. Seven out of 18 HBV carriers were coinfected with HCV (38.8%). In 11 out of 18 patients (61%), HBsAg were negative [START_REF] Raimondo | Occult hepatitis B virus in liver tissue of individuals without hepatic disease[END_REF], anti-HBc (nine patients), anti-HBs (one patient, no history of previous vaccination), anti-HBc/anti-HBe/anti-HBs (one patient) being the only positive markers of previous HBV exposure.

Three patients were known to be affected by mild HBV chronic active hepatitis at the time of lymphoma diagnosis and were treated with LAM to contain the disease process before and after ICT. The remaining four inactive HBsAg carriers and the 11 HBsAg-negative/antiHBc-positive patients were started on LAM as a preemptive treatment to prevent possible disease reactivation [START_REF] Marzano | Italian association for the study of the liver. Prophylaxis and treatment of hepatitis B in immunocompromised patients[END_REF].

Patients began treatment with LAM 100 mg/day po on average 3-4 weeks before embarking on chemotherapy, after signing informed consent. All 159 patients were given R-CHOP or R-CHOP-like regimens [START_REF] Coiffier | CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma[END_REF]. LAM treatment was extended to 18 months after that the new indications from the dedicated commission of the Italian Study of the Liver became widely available in 2007 [START_REF] Marzano | Italian association for the study of the liver. Prophylaxis and treatment of hepatitis B in immunocompromised patients[END_REF]. During and after ICT discontinuation, patients were tested every 2 months for HBsAg, quantitative-HBV-DNA, ALT, and AST. Median follow-up time was 7 months (range 3-43). Five of the seven HBsAg-positive patients (71.4%) became HBV-DNA positive during follow-up at 25, 18, 18, 10, and 7 months after commencing LAM. In two patients, emergence of HBV-DNA positivity was associated with an increase of serum transaminases (up to five times the upper normal limit). In these five patients, Adefovir-Dipivoxyl, 10 mg/day po, was combined with LAM, resulting in a disappearance of HBV-DNA and regression of the biochemical signs of hepatitis. None of these patients had to discontinue ICT because of HBV reactivation. No liver-related deaths occurred. None of the HBcAbpositive, anti-HBs-positive/HBsAg-negative patients (potential silent carriers) developed HBV reactivation during the period of observation. No side effect related either to LAM or ADV was registered. Four out of seven of these patients are now undergoing new treatments because of lymphoma progression.

The prevalence of HBV infection is high among patients diagnosed with NHL as compared to less than 2% of the Italian adult population [START_REF] Villa | Impact of hepatitis B vaccination in a higly endemic area of south Italy and long-term duration of anti-HBs ancibody in two cohorts of vaccinated individuals[END_REF]. Reactivation of HBV infection can present a serious threat to these patients and to the completion of their chemotherapeutic protocol. Thus, before proceeding to ICT, it is widely accepted that HBsAg active carriers should be treated, and preventative measures should be given to the silent carriers. In addition, HBsAgnegative or anti-HBc+patients (potential occult carriers) with onco-hematological malignancies can also develop severe hepatitis flare when treated with immunosuppressive regimens such as ICT. We, therefore, following the suggestions of the consensus conference [START_REF] Marzano | Italian association for the study of the liver. Prophylaxis and treatment of hepatitis B in immunocompromised patients[END_REF], also administered LAM to our HBV-occult carriers. In our series, the incidence of reactivation in the HBsAg-positive cases (both active and inactive carriers) was high (71.4%). Yu-FE et al. described a very low incidence of reactivation in HBsAgpositive, possibly due to the fact that in their series, patients were monitored by transaminase screening and as a result, the true incidence of virus reactivation might have been underestimated. Nevertheless, existing data on the effectiveness of LAM prophylaxis for patients treated with ICT can vary widely depending on the sample size.

In addition, we believe that it has to be taken into account that a substantial proportion of lymphoma patients will unfortunately suffer a relapse and will, therefore, require additional cycles of chemotherapy, prolonging their state of immunodepression. On the basis of our observations, we suggest LAM as a preemptive treatment in onco-hematologic HBsAg-positive patients with a high probability of cure after first line treatment and as a preventative measure in HBV occult carriers undergoing ICT. While new drugs, such as Entecavir [START_REF] Lai | Globe Study Group (2007) Telbivudine versus lamivudine in patients with chronic hepatitis B[END_REF], are more powerful and faster acting, they might be more useful when longer lasting or repeated treatments are required as they provide protection against the development of drug resistance. Whichever antiviral drug is administered, an extended follow-up term, for HBV reactivation, is recommended after ICT discontinuation. What is remarkable in our series is that 38.8% of HBV carriers were co-infected with HCV. Since HCV and HBV carriers are supposed to be more prone to the development of lymphoproliferative diseases [START_REF] Gaeta | Epidemiological and clinical burden of chronic hepatitis B virus/hepatitis C virus infection. A multicenter Italian study[END_REF], it should not be ruled out that a double viral infection determines an even greater risk of lymphoma occurrence [START_REF] Chen | High prevalence of occult hepatitis B virus infection in patients with B-cell non-Hodgkin's lymphoma[END_REF].


Dries Deeren

Deferasirox in pyruvate kinase deficiency

Dear Editor, In a recent article in Annals of Hematology, Metzgeroth and colleagues reported the effects of oral iron chelation therapy with deferasirox in myelodysplasia patients with transfusion-related iron overload [START_REF] Metzgeroth | Deferasirox in MDS patients with transfusion-caused iron overload-a phase-II study[END_REF].

Patients with other blood disorders with complications of iron overload may also benefit from deferasirox.

We report the case of a 40-year-old woman with pyruvate kinase deficiency who was referred because of fatigue. She had a history of severe haemolysis requiring frequent red cell transfusions. Twenty years earlier, she underwent cholecystectomy and splenectomy after which the transfusion requirement was abolished and the haemoglobin level was stable at 9.5 g/dl. Now, the ferritin level was 4,500 μg/l, aspartate aminotransferase 169 U/l, alanine aminotransferase 147 U/l and gamma glutamyltransferase 96 U/l. The black hypointense liver on magnetic resonance imaging confirmed iron overload. There were no cardiac abnormalities. She was heterozygous for the His63Asp mutation of the HFE gene.

Deferasirox was started on January 21st 2007 with a dose of 20 mg/kg. Side effects were mild and included anorexia, nausea, abdominal cramps, diarrhoea and weight loss. On July 28th, the ferritin level was decreased to 507 μg/l, and the liver enzyme levels had normalised. Fatigue was no longer present. Treatment with deferasirox was stopped, and the ferritin level and liver enzymes are regularly being monitored.

The prevalence of pyruvate kinase deficiency is 1/20,000. The degree of haemolysis is variable, but iron overload is common, even in non-transfused patients [START_REF] Zanella | Pyruvate kinase deficiency[END_REF]. The observation of effective iron removal and improvement of liver function tests in our patient suggests that iron chelation with deferasirox may be considered in patients with pyruvate kinase deficiency and severe iron overload.

Funding and conflicts of interest None to declare.


D Deeren
email: ddeeren@hhr.be

B-lymphoblastic leukaemia presenting as autoimmune haemolytic anaemia

Dear Editor, In the March 2008 issue of Annals of Hematology, Niscola and colleagues reported a case of B-lymphoblastic leukaemia 9 months after the diagnosis of autoimmune haemolytic anaemia (AIHA) [START_REF] Niscola | Acute lymphoblastic leukemia subsequent to autoimmune hemolytic anemia: a case report[END_REF].

The preceding AIHA was in remission and its treatment was stopped, so nor the preceding AIHA nor its treatment influenced the leukaemia treatment. However, when Blymphoblastic leukaemia presents immediately as AIHA, treatment should be directed against both leukaemia and haemolysis, and toxicity should be limited.

Recently, a 61-year-old man was admitted to the emergency department because of dyspnoea. The Hb count was 6.7 g/dl, WBC count 1.2•10 9 /l, 5.8% blasts, neutrophil count 0.3•10 9 /l, platelet count 84•10 9 /l. Despite a normal reticulocyte count, there was evidence of relevant haemolysis suggested by increased lactate dehydrogenase and indirect bilirubin, and decreased haptoglobin. The peripheral blood smear showed no schistocytes. The direct antiglobulin (Coombs') test was positive (3+) with polyvalent antibodies and anti-C3d, and negative with anti-IgG. Testing for cold agglutinins was negative. Bone marrow examination showed a common acute lymphoblastic leukaemia (ALL) with 97.5% marrow blasts and a normal karyotype.

Because he had a history of myocardial ischemia, packed cells were transfused. This caused a very modest Hb increase. Because of neutropenia and the expectation of several more weeks of neutropenia and mucositis, we wanted to reduce the amount of corticosteroids. We chose to start preinduction treatment for ALL with cytarabine, etoposide and methotrexate. After 1 month, the Coombs' test was only slightly positive, and laboratory evidence of haemolysis had disappeared. Induction therapy for ALL was started, this time with corticosteroids (dexamethasone), vincristine and adriamycin. One month later, the Coombs' test was negative. After the induction courses, the Hb started to increase. A complete remission of ALL was achieved. After consolidation with cytarabine and asparaginase, the patient was admitted for allogeneic stem cell transplantation.

Cases of ALL with a positive Coombs' test at the time of diagnosis have been reported [START_REF] Solal-Celigny | Positive Coombs test in acute leukaemia[END_REF]. All had a positive Coombs' with anti-Cd3 but not with anti-IgG, and cold agglutinins could not be found. In these patients, the Coombs' test became negative when complete remission was achieved.

Even though cytostatic drug are immunosuppressive, corticosteroids are most often used as first-line therapy for haemolytic anaemia caused by warm antibodies. Our results suggest that corticosteroids may not be necessary in the initial treatment of AIHA caused by ALL, as long as effective treatment directed against ALL is started.

Received: 3

 3 September 2008 / Accepted: 23 September 2008 / Published online: 7 October 2008 # Springer-Verlag 2008


MBChB Colin Andrew Hutchison
email: c.a.hutchison@bham.ac.uk

Reduction of serum free light chains predict renal recovery

Keywords: Multiple myeloma, renal failure, free light chains Multiple myeloma, renal failure, free light chains

Monoclonal free light chains are the principal cause of severe renal failure in patients with multiple myeloma. Increasing evidence suggests that targeting early reductions in serum concentrations of free light chains is associated with improved clinical outcomes. Further work is required to determine whether direct removal of free light chains from the serum provides any additional benefit to effective chemotherapy alone.

Reduction of serum free light chains predict renal recovery

Reduction of serum free light chains predict renal recovery

Dear editor, in a recent issue of Annals of Hematology Matsue et al [START_REF] Matsue | Reversal of dialysis-dependent renal failure in patients with advanced multiple myeloma: single institutional experience over 8 years[END_REF] report the outcomes of a population of patients with severe renal failure secondary to multiple myeloma. Of particular note, the authors highlight that where serum free light chains (FLC) were measured (five patients) an early reduction in their concentrations was associated with renal recovery. This data is supported by two larger case series which report 40 and 20 patients with severe renal failure and myeloma [START_REF] Leung | Improvement of cast nephropathy with plasma exchange depends on the diagnosis and on reduction of serum free light chains[END_REF][START_REF] Hutchison | Treatment of acute renal failure secondary to multiple myeloma with chemotherapy and extended high cut-off hemodialysis[END_REF]. These series independently demonstrated renal recovery to be associated with an early reduction in serum FLCs of greater than 50%.

How this reduction in serum FLCs is achieved, and whether direct removal of FLCs from the serum has a role, remain to be determined. Undoubtedly effective chemotherapy is essential, without which direct removal can have little impact on circulating FLC levels [START_REF] Clark | Plasma exchange when myeloma presents as acute renal failure: a randomized, controlled trial[END_REF]. Historically, plasma exchange has been used to aid this reduction [5], more recently however a novel method for FLC removal, high cut-off haemodialysis, has been demonstrated to have an increased capacity [START_REF] Clark | Plasma exchange when myeloma presents as acute renal failure: a randomized, controlled trial[END_REF]. The potential benefit of FLC removal in this setting, relates to the extended serum half lives of FLCs in renal failure, which result serum concentrations remaining high even when chemotherapy has been effective. This is likely to translate to the time to renal recovery. In line with other series which used novel chemotherapy agents in this setting [6,7], Matsue et al [START_REF] Matsue | Reversal of dialysis-dependent renal failure in patients with advanced multiple myeloma: single institutional experience over 8 years[END_REF] report a high rate of independence of dialysis. However, the median time to independence of dialysis, 2 months, is uncharacteristically long for an acute kidney injury. Potentially if serum FLCs were reduced more rapidly, using direct removal to overcome the prolonged serum halflives, the duration of dialysis dependence would be shorter. This hypothesis is currently being evaluated by the European Trial of Free Light Chain Removal by Extended Haemodialysis in Cast Nephropathy (EuLITE) [8], a multi-centre randomised controlled trial based in the United Kingdom and Germany.

Currently we can conclude that recent use of novel chemotherapy regimes in patients with renal failure secondary to multiple myeloma has resulted in improved outcomes for this population. Further work in now required to determine whether therapeutic strategies for directly removing FLCs from the serum can provide an additional benefit.

  Hutchison CA, Cockwell P, Reid S, et al (2007). Efficient removal of immunoglobulin free light chains by hemodialysis for multiple myeloma: in vitro and in vivo studies. J Am Soc Nephrol 18(3):886-95. 6. Kastritis E, Anagnostopoulos A, Roussou M, et al (2007). Reversibility of renal failure in newly diagnosed multiple myeloma patients treated with high dose dexamethasone-containing regimens and the impact of novel agents. Haematologica 92(4):546-9. 7. Ludwig H, Drach J, Graf H, Lang A, Meran JG (2007). Reversal of acute renal failure by bortezomib-based chemotherapy in patients with multiple myeloma. Haematologica 92(10):1411-4. 8. Hutchison CA, Cook M, Heyne N, Weisel K, Billingham L, Bradwell A, Cockwell P (2008). European trial of free light chain removal by extended haemodialysis in cast nephropathy (EuLITE): A randomised control trial. Trials 9:55.

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Dr Kalliopi N Manola
email: pmanola@ipta.demokritos.gr

Despina Pantelidou

Maria Papaioannou

Yours Sincerely

Keywords: CML, plasmablastic crisis, Philadelphia chromosome

We report for the first time a rapidly fatal case of a Plasma Cell Leukemia crisis in a young patient with Ph chromosome positive CML. The patient, a 43-year-old male suffered from non diagnosed CML and presented at the time of blast crisis, which turned to be a plasmablastic leukemia. This hypothesis relies on the cytogenetic and immunophenotypic analysis of the leukemic cells. Two related clones were detected. The first clone was characterized only by t(9;22)(q34;q11.2). The evolutionary clone showed severe hypodiploidy, including monosomy 13 and chromosome aberrations different from that of the blast crisis of CML, in addition to t(9;22)(q34;q11.2). Severe hypodiploid karyotype and monosomy 13 are common cytogenetic features of plasma cell leukemia. Positivity for CD38 and cytoplasmic kappa light chain revealed by immunophenotypic analysis of the leukemic cells, confirmed the plasmacytic origin of blasts. Therefore, the most likely diagnosis of our patient is that of Plasmablastic crisis of Philadelphia chromosome positive CML.

Response to Reviewers: Dear Sirs,

The manuscript was revised according to the Reviewer's comments as follows: 1) "The pictures relating to karyotypes should be deleted, the information is already in the text ".

-The pictures relating to karyotypes were deleted.

2) "Instead, a clear peripheral blood microphotograph (600-1000x) should document the presence of plasmablastic-type cells".

-A clear peripheral blood microphotograph (600x) was added to document the presence of plasmablastic-type cells.

3) "The immunophenotype study should report in detail the antisera used with respective percentages of positivity; this is especially important for CD45, CD20, CD56, CD30 and cytoplasmic/surface IgG and IgA (not reported in text)".

-The immunophenotype study is now reported in detail [CD45+ (79%), CD38+(77%), CD34+ (56%), surface κ(-), surface λ(-), cytoplasmic κ +(90%), cytoplasmic λ( -), CD20 (-)]. 4) "Was kidney failure related to any degree of hypercalcemia or proteinuria? Were serum and urine free light chains quantified?" -Neither hypercalcemia nor proteinuria was observed. Serum and urine free light chains were not quantified as the assay was not available.

Thank you very much for your suggestions. The manuscript was revised according to the Reviewer's comments as follows:

1) "The pictures relating to karyotypes should be deleted, the information is already in the text ".

-The pictures relating to karyotypes were deleted.

2) "Instead, a clear peripheral blood microphotograph (600-1000x) should document the presence of plasmablastic-type cells".

-A clear peripheral blood microphotograph (600x) was added to document the presence of plasmablastic-type cells.

3) "The immunophenotype study should report in detail the antisera used with respective percentages of positivity; this is especially important for CD45, CD20, CD56, CD30 and cytoplasmic/surface IgG and IgA (not reported in text)".

-The immunophenotype study is now reported in detail [CD45+ (79%), CD38+(77%), CD34+ (56%), surface κ(-), surface λ(-), cytoplasmic κ +(90%), cytoplasmic λ( -), CD20 (-)].

4) "Was kidney failure related to any degree of hypercalcemia or proteinuria? Were serum and urine free light chains quantified?" -Neither hypercalcemia nor proteinuria was observed. Serum and urine free light chains were not quantified as the assay was not available.

Plasmablastic crisis of Philadelphia chromosome positive Chronic Myeloid

Leukemia

The cytogenetic hallmark of Chronic Myeloid Leukemia (CML) is the Philadelphia chromosome (Ph), the consequence of the reciprocal translocation t (9;22)(q34;q11.2) that results in the BCR-ABL1 fusion gene, located in the Ph chromosome. 1 The natural history of untreated CML is an initial indolent chronic phase which is followed by a blast phase either suddenly or through an accelerated phase. Blast crisis may be diagnosed when blast cells are ≥20% of the peripheral blood WBC or the nucleated cells of the bone marrow or when there is an extramedullary blast proliferation. 1,2 In approximately 70% of cases the blast lineage is myeloid while in 20-30% of cases the blasts are lymphoblasts. 1,2 Progression is usually associated with clonal evolution, and at the time of transformation to blast crisis, 80% of patients demonstrate cytogenetic changes in addition to the Ph chromosome such as an extra Ph chromosome, +8, i(17q) +19, t(3;21)(q26;q22), and very rarely t(7;11)(p15;p15). 3 Plasma Cell Leukemia (PCL) is an aggressive and rare hematological malignancy which is characterized by circulating plasma cells >2x10 9 /l in peripheral blood or by a plasmatocytosis >20% of blood leukocytes and confers a poor prognosis. It originates as a primary disease or as a secondary leukemic transformation of multiple myeloma. 4 The most frequent features at diagnosis are asthenia, severe anemia, thrombocytopenia, splenomegaly or hepatomegaly while CD38 and CD138 antigens are excellent plasma cell markers. 5,6 In the majority of the cases karyotypes are complex and hypodiploid or pseudodiploid. chromosomes 13 and X (in women), trisomy 18, amplification of 1q21, and MYC rearrangements. [4][5][6][7] Here, we report for the first time a rapidly fatal case of a Plasma Cell Leukemia crisis in a young patient with Ph chromosome positive CML .

The patient, a 43-year-old male with a 2-month history of persistent fatigue, weakness and loss of weight was admitted to our department. On physical examination spleen was remarkably enlarged and routine complete blood count revealed leukocytosis with a WBC of 22x10 9 /l, anemia with a Hb of 7 gr/dl, and thrombocytopenia with platelet count (PLT) of 30x10 9 /l. Neither hypercalcemia nor proteinuria was observed.

Blood smear showed 90% blasts with the appearance of plasma blasts (Figure 1).

Immunophenotyping of blast cells by flow cytometry showed CD45+ (79%), CD38+(77%), CD34+ (56%), surface κ(-), surface λ(-), cytoplasmic κ +(90%), cytoplasmic λ(-), and CD20(-). The cytogenetic analysis of G-banded chromosomes in peripheral blood cells revealed two evolutionary clones, which were positive for Ph chromosome. The karyotype was described as: 46,XY,t(9;22)(q34;q11.2)[4]/ 32,XY,-2,-3,-4,-5,t(9;22)(q34;q11.2), -10,-11,-12,-13,-15,-16,-17,-18,-20,-22[16)].

Unfortunately, the patient developed acute renal failure on the second day after admission and died two days later.

Ph chromosome and/or BCR-ABL1 fusion gene which are detected in 90-95% of cases of CML is used as a major criterion for diagnosis of CML.

Legend to figure

Figure 1 .

 1 Figure 1. A peripheral blood microphotograph (600x) showing the presence of

Figure

  Figure Click here to download high resolution image

  Please accept the revised manuscript, entitled "Plasmablastic crisis of Philadelphia chromosome positive Chronic Myeloid Leukemia" by K.N. Manola et al., for consideration for publication as Letter in Annals of Hematology, containing the corrections according to the reviewer's recommendations.

	To: Tiziano Barbui
	Associate Editor
	Annals of Hematology	October 6, 2009
	Dear Dr Tiziano Barbui,

Manuscript Click here to download Manuscript: K. Manola et.al_revised manuscript.doc Click here to view linked References

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	53 54	Common chromosome abnormalities are
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	56	14q32 translocations such as t(11;14)(q13;q32), t(4;14)(p16.3;q32), t(14;16)(q32;q23),
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	58 59	deletions of the chromosomal regions 17p13, 13q, and 1p21, monosomies of
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  1,2 Ph chromosome, marked splenic enlargement, thrombocytopenia, and the high percentage of blasts are compatible with the diagnosis of blast crisis of CML. It seems possible that our patient suffered from non diagnosed CML and presented at the time of blast crisis, which turned to be a plasmablastic leukemia. This hypothesis relies on the cytogenetic and immunophenotypic analysis of the leukemic cells. Two related clones were Primary plasma cell leukaemia: a report of 18 cases. LeukRes. 2001;25:103-7. 6 García-Sanz R, Orfão A, González M, Tabernero MD, Bladé J, Moro MJ, et al.

		detected in our patient. The first clone was characterized only by t(9;22)(q34;q11.2), ES, Pileri SA, Stein H, Thiele J, et al. WHO Classification of Tumours of
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	2 3 2 3 2 3	indicating that Ph chromosome was the primary aberration, possibly created in the Haematopoietic and Lymphoid Tissues. 4th Edition 2008;32-7.
	4 5 6 4 4 5 6 5 6	chronic phase of CML. The evolutionary clone showed severe hypodiploidy, 2 Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, et
	7 8 7 8 7 8	including monosomy 13 and chromosome aberrations different from that of the blast al. The 2008 revision of the WHO classification of myeloid neoplasms and
	9 10 11 9 9 10 11 10 11	crisis of CML, in addition to t(9;22)(q34;q11.2). Severe hypodiploid karyotype and acute leukemia: rationale and important changes. Blood 2009; e-pub ahead of
	12 13 12 13 12 13	monosomy 13 are common cytogenetic features of plasma cell leukemia. Positivity print 8 Apr 2009;doi:10.1182/blood-2009-03-209262.
	14 15 16 14 14 15 16 15 16	for CD38 and cytoplasmic kappa light chain revealed by immunophenotypic analysis 3 Calabretta B, Perrotti D. The biology of CML blast crisis. Blood.
	17 18 17 17 18 18	of the leukemic cells, confirmed the plasmacytic origin of blasts. Therefore, the most 2004;103:4010-22.
	19 20 21 19 19 20 21 20 21	likely diagnosis of our patient is that of Plasmablastic crisis of Philadelphia 4 Tiedemann RE, Gonzalez-Paz N, Kyle RA, Santana-Davila R, Price-Troska T,
	22 22 22	chromosome positive CML. To the best of our knowledge this case presents the first
	23 23 23	Van Wier SA, et al. Genetic aberrations and survival in plasma cell leukemia.
	24 25 26 24 24 25 26 25 26	reported case of Plasmablastic crisis of Ph chromosome positive CML. Leukemia 2008;22:1044-52.
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	31 32 31 31 32 32	Kalliopi N Manola 1* , Despina Pantelidou 2 , Maria Papaioannou 2
	33 34 33 33 34 34	1 Laboratory of Cytogenetics, National Center for Scientific Research (NCSR)
	35 35 35
	36 37 36 36 37 37	Primary plasma cell leukemia: clinical, immunophenotypic, DNA ploidy, and "Demokritos", 15310 Aghia Paraskevi, Athens, Greece
	38 39 38 39 38 39	2 1st Department of Internal Medicine, Hematology department, Medical School cytogenetic characteristics. Blood. 1999;93:1032-7.
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	41 42 41 41 42 42	7 Chang H, Qi X, Yeung J, Reece D, Xu W, Patterson B. Genetic aberrations AUTH, Thessaloniki, Greece
	43 44 43 44 43 44	*E-mail: pmanola@ipta.demokritos.gr including chromosome 1 abnormalities and clinical features of plasma cell
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