MD Maite Torres-Salido

MD Josefina Cortés-Hernández

PhD Eva Balada

PhD Miquel Vilardell Tarrés

MD, PhD Josep Ordi-Ros

Systemic lupus erythematosus as a first presentation of common variable immunodeficiency associated with infrequent mannose-binding lectin gene polymorphisms

Keywords: Mannose-Binding Lectin, Common Variable Immunodeficiency, Systemic Lupus Erythematosus

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Introduction

CIVD is a heterogeneous syndrome characterized by failure of B cell differentiation associated to a defective immunoglobulin (Ig) production.

In addition to recurrent bacterial infections, up to 25% of patients with CVID can develop an autoimmune event [START_REF] Brant | Common variable immune deficiency and autoimmunity[END_REF], that in some instance, can be the initial presenting symptom of the disease. Association of CVID and SLE is uncommon, but approximately 1% of the patients with CVID will develop symptoms or laboratory abnormalities than can support the diagnosis of SLE or SLE-like syndrome [START_REF] Brant | Common variable immune deficiency and autoimmunity[END_REF][START_REF] Cunningham-Rundles | Common variable immunodeficiency: clinical and immunological features of 248 patients[END_REF][START_REF] Fernández-Castro | Common variable immunodeficiency in systemic lupus erythematosus[END_REF].

The underlying aetiology of increased autoimmunity and development of SLE in CVID is not completely understood. Genetic as well as persistent antigen exposure and ineffective antigen clearance in a background of lack of immunologic regulatory mechanisms seem all likely to contribute.

In recent years CVID has been significantly linked in a minority of patients to defects in genes encoding for molecules as ICOS (inducible co-stimulator) [START_REF] Grimbacher | Homozygous loss of ICOS is associated with adult-onset common variable immunodeficiency[END_REF], TACI (transmembrane activator and calcium-modulator and cytophillin ligand interactor) [START_REF] Salzer | Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans[END_REF], CD19 (B-lymphocyte antigen CD19) and BAFF-R (tumor necrosis factor superfamily member 13C) [START_REF] Van Zelm | An antibody-deficiency syndrome due to mutations in the CD19 gene[END_REF][START_REF] Yong | Common Variable Immunodeficiency: An update on etiology and management[END_REF], but in none of these patients SLE was observed [START_REF] Carneiro-Sampaio | Understanding systemic lupus erythematosus physiopathology in the light of primary immunodeficiencies[END_REF]. In addition, a specific MBL allele, a component of the innate immune response, has been strongly associated with overall autoimmune disease in patients with CVID by contributing to both susceptibility to infections and increased frequency of autoimmune and inflammatory manifestations [START_REF] Mulligham | Mannose binding lectin polymorphisms are associated with early age of disease onset and autoimmunity in common variable immunodeficiency[END_REF].

We describe 2 patients who developed SLE as an initial presentation of probable CVID.

Methods

Case presentations

Case 1

A 15 year-old girl was diagnosed of SLE in June 1994. She presented with malar rash, photosensitivity, polyarthritis, positive antinuclear antibodies (ANA) 1:1280 and raised anti-dsDNA antibodies (200 IU/mL; normal range < 40) for which she was started on oral steroids. Six months later, she was admitted to our hospital with a 2-week history of low grade temperature followed by gait disturbances and acute urinary retention. Physical examination on admission revealed horizontal and vertical nistagmus and left sided hemiparesis. The immunological profile showed positive ANA 1:1280 and anti-dsDNA antibodies (360 IU/mL; normal range < 40 IU/mL), reduced serum C3 level of 75 mg/dL (80-170 mg/dL) and normal immunoglobulin levels. CSF analysis showed PMN pleocytosis (WBC 370 with 89% PMN) and elevated protein content (1.55 g/l; normal range <0.5). Bacterial, fungal and mycobacterium cultures in both blood and CSF fluid were negative; brain and whole spine MRI showed high signal intensity regions on T2-weighted throughout the spinal cord suggestive of encephalomyelitis. Clinical improvement was achieved after pulses of methylprednisolone (1gx3day) followed by intravenous cyclophosphamide (1000 mg) and oral steroids. One week after discharge, the patient presented with a tonic-clonic seizure and was re-admitted with a status epilepticus.

Phenytoin infusion was started and the patient had to be transferred to intensive care for ventilatory support. On discharged she was on prednisolone (50 mg/day), azathioprine (75 mg/day) and carbamazepine (400 mg/day) with good control of her symptoms.

One year later, she developed respiratory symptoms with frequent episodes of productive cough and recurrent chest infections. Laboratory tests showed a new persistent panhypogammaglobulinemia with serum IgG levels of 320 mg/dL (850-1600 mg/dL), IgA levels of 7 mg/dL (75-350 mg/dL), and IgM levels of 20 mg/dL (58-265 mg/dL). ANA and anti-dsDNA antibodies were negative. No specific antibody response was detected after Pneumoccocus and tetanus vaccinations. A probable CVID diagnosis was made and intravenous gammaglobulin replacement therapy (0.5mg/kg/month) was initiated. Since then, she continues on replacement therapy and there has been neither clinical nor laboratory evidence of active SLE in the absence of immunosuppressive therapy.

Case 2

In October 2002, a 13-year-old girl was admitted to our hospital with fever, sore throat, mild bilateral cervical lymphadenopathy, polyarthritis, malar rash and a non-specific generalized maculo-papular rash. Laboratory tests revealed a haemoglobin value of 9.8 g/dL. Bacterial and viral screen were negative. The immunological profile showed positive ANA 1:2560, raised anti-dsDNA antibodies (926 IU/mL; normal range < 40 IU/mL) with normal serum immunoglobulin and complement levels. The thoracoabdominal CT showed minimal bilateral pleural effusions. During her admission, the patient developed a progressive jaundice, hepatosplenomegaly and reduced level of consciousness. Blood test confirmed the diagnosis of an acute hepatitis (ALT 2394 IU/l (< 30 IU/l), AST 848 UI/l (<34 IU/l), alkaline phosphatase 570 IU/l (< 90 IU/l), GGT 469 IU/l (< 40 IU/l), and LDH 11989 IU/l (< 330 IU/l). Hepatitis A, B, C, CMV and EBV viral serologies were negative as well as the liver autoantibodies. There was no history of hepatotoxic drugs. The abdominal ultrasound was normal and the liver biopsy showed changes compatible with autoimmune hepatitis. Treatment with pulses of methylprednisolone (1g x3 day) and oral azathioprine (1.5mg/kg) was started followed by clinical and serological improvement and resolution of her liver dysfunction. One month afterwards, azathioprine had to be stopped due to severe leucopoenia (WBC <1x10 9 /l (4.9-19.3x10 9 WBC/l)) that required treatment with granulocyte colony-stimulating factor. Patient was discharge on oral steroids and tacrolimus 1mg/12 hours as maintenance therapy. In 2004, due to persistent polyarthritis, methotrexate (15 mg/week) was introduced in addition to her previous medication. In April Replacement immunoglobulin therapy (0.5 g/Kg/month) was initiated. Until now, she remains on regular replacement therapy and asymptomatic of her SLE in the absence of immunosuppressive therapy.

MBL genotyping and measurement of MBL levels

Ethical approval was obtained by the local Ethical Committee. Genomic DNA was prepared from whole blood using the QIAamp DNA Blood Maxi Kit (QIAGEN GmbH, Hilden, Germany) and 272 bp-polymerase chain reaction (PCR) fragments were generated. Combinations of forward and reverse allelespecific primers (Table 1) were used to amplify fragments which contained the 5'-untranslated region +4 c/t (P/Q) polymorphisms and the exon 1 polymorphisms at codon 57 t/c (57Gly→Glu) as previously described [START_REF] Mulligham | Mannose binding lectin polymorphisms are associated with early age of disease onset and autoimmunity in common variable immunodeficiency[END_REF]. MBL levels were determined by ELISA (MBL Oligomer ELISA kit, BioPorto Diagnostics, Gentofte, Denmark). Case 1 had the heterozygous genotype 4P-57Glu/4Q-57Glu and case 2 had the homozygous genotype 4Q-57Glu (Figure 1). Serum MBL concentrations were 4,328 ng/mL and 58.4 ng/mL, respectively.

Discussion

Although CVID is a rare disease, the estimated prevalence in the western population is approximately of 1 in 25,000 according to the different studies.

Despite respiratory i nfections being the most common clinical manifestations of CVID, an increased frequency of autoimmune disorders, granulomatous inflammation and malignancy have been described [START_REF] Cunningham-Rundles | Common variable immunodeficiency: clinical and immunological features of 248 patients[END_REF][START_REF] Blanco-Quirós | Common variable immunodeficiency. Old questions are getting clearer[END_REF][START_REF] Giannouli | Autoimmune manifestations in common variable immunodeficiency[END_REF]. Association of CVID and SLE is uncommon. So far, including our two, a total of twenty patients with SLE and CVID have been described in the literature. In the majority of cases, the development of SLE preceded the development of CVID. At the time of the initial diagnosis, patients had normal sera immunoglobulins and it was later that hypogammaglobulinemia developed. While in the majority, the disease activity and immunological markers decreased after the development of CVID, in other patients the disease remained unchanged [START_REF] Fernández-Castro | Common variable immunodeficiency in systemic lupus erythematosus[END_REF].

Diagnosis of CVID may be masked in those patients presenting with SLE that receive immunosuppressive therapy. Cyclophosphamide, azathioprine, mycophenolate mofetil, rituximab, corticosteroids and other drugs including phenytoin have been reported to cause hypogammaglobulinemia [START_REF] Fernández-Castro | Common variable immunodeficiency in systemic lupus erythematosus[END_REF][START_REF] Blanco-Quirós | Common variable immunodeficiency. Old questions are getting clearer[END_REF][START_REF] Giannouli | Autoimmune manifestations in common variable immunodeficiency[END_REF][START_REF] Yong | Management of hypogammaglobulinaemia occurring in patients with systemic lupus erythematosus[END_REF].

Unlike CVID, drug-induced hypogammaglobulinaemia is reversible and resolves after drug withdrawal. Although our patients received several immunosuppressive agents and antiepileptic treatment during the course of their disease, it seems unlikely that they were the cause of hypogammaglobulinaemia since some agents were only administered during a short period of time and in both patients hypogammaglobulinaemia was severe and sustained despite being stopped.

Different mechanisms may account for the development of autoimmunity in those patients. The first one would be a possible breakdown in central and peripheral mechanisms of tolerance induction or maintance. The second contributing mechanism would be related to the existing variable degree of immune dysregulation. Thridly, the innate and adaptative immunity deficiencies would lead to persistent/recurrent infections and to a defective processing and clearing of external antigens that may result in a chronic inflammation and recurrent tissue damage secondary to end-organ deposition of immune complexes, formation of anti-tissue antibodies, and cross-reactivity between normal tissues and foreign antigens. Finally, there is a genetic predisposition that can also lead to autoimmunity [START_REF] Lopes-Da-Silva | Autoimmunity in common variable immunodeficiency[END_REF].

MBL is a protein belonging to the collectins family that is involved in several of the above mentioned mechanisms and altered levels of MBL seem to play a role contributing to both the development of CVID and autoimmunity [START_REF] Yong | Management of hypogammaglobulinaemia occurring in patients with systemic lupus erythematosus[END_REF]. MBL enhances opsonisation of different mannose -rich microorganisms and it takes part in complement activation pathway that is an important component of the innate immunity, the others important functions of MBL are recognition of altered self structures, and modulation of the inflammation [START_REF] Aghamohammadi | Mannose-binding lectin polymorphisms in common variable immunodeficiency[END_REF]. MBL has shown to bind and clear-out DNA molecules [START_REF] Palaniyar | Nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins A and D and mannose-binding lectin[END_REF], its deficiency appears to mildly increase susceptibility to SLE and associations between SLE and different MBL polymorphisms have been also reported [START_REF] Lee | The mannose-binding-lectin gene polymorphisms and systemic lupus erythematosus: two case-control studies and a meta-analysis[END_REF].

MBL also plays an important role in CVID. Low MBL levels have been significantly associated with an increased frequency of respiratory tract infections and brochiectasis [START_REF] Fevang | Common variable immunodeficiency and the complement system; low mannose-binding lectin levels are associated with bronchiectasis[END_REF]. Recently, low-producing coding alleles and promoter haplotypes for MBL have been correlated to an early age of CVID onset and variations in the +4Q allele have been associated with inc reased autoimmunity incidence [START_REF] Mulligham | Mannose binding lectin polymorphisms are associated with early age of disease onset and autoimmunity in common variable immunodeficiency[END_REF]. The exact mechanism by which MBL polymorphisms may play a role in the development of autoimmunity in patients with CVID is not fully understood, but similarly to what occurs in SLE, it is possible that low MBL levels associated with these variants result in defective pathogen clearance and immune complex formation that ultimately triggering the autoimmune response [START_REF] Seelen | A role for mannose-binding lectin dysfunction in generation of autoantibodies in systemic lupus erythematosus[END_REF].

In agreement with Mulligan et al., we also found that our two patients with concomitant CVID and SLE carried this allele. Although polymorphisms in the promoter and the coding region of the MBL gene are closely correlated with serum levels of MBL, the functional effects of the +4Q allele are poorly understood. Mullighan et al. [START_REF] Mulligham | Mannose binding lectin polymorphisms are associated with early age of disease onset and autoimmunity in common variable immunodeficiency[END_REF] also found that the MBL 4Q-57Gly haplotype was associated with autoimmune disease in CVID. However, none of our two patients showed this allele combination. Interestingly, case 2, who also had low levels of circulating MBL, presented the haplotype 4P-57Glu. To our knowledge, only a few of the theoretically possible MBL haplotypes have been described [START_REF] Garred | Mannose-binding lectin deficiency-revisited[END_REF] and the 4P-57Glu haplotype has never been detected. It is unclear whether the low MBL levels may really be due to this particular haplotype. Even though, we did not perform an analysis of the frequency of this haplotype in the Spanish population, Lozano et al [START_REF] Lozano | Novel MASP2 variants detected among North African and Sub-Saharan individuals[END_REF] could not find it among the 104 Spaniard blood donors controls. It is also worth mentioning that, although our patients had the 4Q-57Glu haplotype, the frequency of this allele combination in the general Spanish population is the lowest (1.92 %) among all the other haplotypes [START_REF] Lozano | Novel MASP2 variants detected among North African and Sub-Saharan individuals[END_REF].

Thus, the MBL haplotypes we have found in our patients may support the role of the innate immune system in the development of SLE and/or CVID. Due to the small sample size, these results should be interpreted with caution.

Conclusion

We have reported two patients with CVID that initially presented with manifestations of SLE. Although the association of both diseases is uncommon, it should be considered in any SLE patient with persistent hypogammaglobulinemia. Further genetic studies are needed to clarify the role of the MBL gene polymorphisms in the development of autoimmunity in CVID.

  2006,she was re-admitted with a 6-month history of diarrhoea, abdominal pain, malar rash, worsening polyarthritis, and mouth ulcers. Two days prior admission she also developed severe epigastric pain, nausea and vomiting. Raised ser um amylase (1403 IU/L (<350 IU/l) and the evidence of pancreatic oedema in the abdominal CT supported the diagnosis of acute pancreatitis. By exclusion, and by the possibility of a drug-induced pancreatitis, tacrolimus and methotrexate were stopped with clinical and biochemical improvement. At that time, that were normal, and a colonic biopsy that only showed non-specific colitis. The most outstanding blood test result was the presence of a new persistent panhypogammaglobulinaemia with serum IgG levels of 303 mg/dL (850-1600 mg/dL), IgA levels of 17 mg/dL (75-350 mg/dL) and IgM levels of 26 mg/dL (58-265 mg/dL). ANA and anti-dsDNA had become negative. The lack of immune response to pneumococcus and tetanus vaccination supported the diagnosis of probable CVID.

Figure 1 .

 1 Figure 1.

Figure 1 .

 1 Figure 1. Mannose binding lectin (MBL) haplotyping for case 1 (lanes 1-4) and

Table 1 .

 1

	Primers	Sense/antisense	Sequence (5' to 3')
	4P	sense	TAggACAgAgggCATgCTC
	4Q	sense	TAggACAgAgggCATgCTT
	57GLU	antisense	CCTggTTCCCCCTTTTCTC
	57GLY	antisense	CCTggTTCCCCCTTTTCTT

Table 1 .

 1 Allele specific primers used to amplify the sequence spanning the 5' untranslated region and the codon 57 of MBL exon 1.

Acknowledgements

Dr. Maite Torres-Salido is funded by the Instituto de Salud Carlos III (CM07/00143).


Georgia Metzgeroth

Eva-Maria Kanders

Philipp Erben

Wolf-Karsten Hofmann

Jan Hastka
email: jan.hastka@umm.de

The role of zinc protoporphyrin measurement in the differentiation between primary myelofibrosis and essential thrombocythaemia

Keywords: Zinc protoporphyrin, primary myelofibrosis, essential thrombocythaemia

The differentiation between primary myelofibrosis (PMF) and essential thrombocythaemia (ET) may be difficult especially in early stage disease. In PMF, increased levels of inflammatory cytokines induce impaired iron-utilization and disturbed erythropoiesis. In conditions with impaired iron support to erythropoiesis, zinc protoporphyrin (ZPP) is produced instead of heme. Here we investigate, whether ZPP concentration can be useful in the differentiation between PMF and ET.

Seventy newly diagnosed patients (PMF, n=24; ET, n=46) were analysed. Intraerythrocytic ZPP concentration (normal: <40 mol/mol heme) was measured by Aviv front-face hematofluorometer. In PMF, ZPP concentrations were significantly increased when compared to ET (9937 mol/mol heme vs. 3613 mol/mol heme, p<0.0001). There was also a significant difference between early stage PMF and advanced disease (77 ± 30 mol/mol heme vs. 122 ± 31 mol/mol heme, p=0.003). ZPP >76 mol/mol heme as observed in 71% of PMF-patients were not seen in ET. In PMF-patients responding to immunosuppressive treatment (n=4), the increase of hemoglobin was accompanied by declining ZPP. In summary, by detecting the disturbed iron metabolism common in PMF, ZPP may assist in the differentiation between PMF and ET. Concentrations >60 mol/mol heme are unlikely in ET if iron deficiency is excluded. ZPP determination is also useful for monitoring the effect of therapy in PMF.

Introduction

Primary myelofibrosis (PMF) is a clonal proliferative neoplasm of the hematopoietic stem cell which is clinically characterized by pancytopenia, splenomegaly and extramedullar hematopoiesis [START_REF] Rambaldi | From palliation to epigenetic therapy in myelofibrosis[END_REF][START_REF] Tefferi | Myelofibrosis with myeloid metaplasia[END_REF]. The diagnosis of PMF is based on clinical features, laboratory findings and bone marrow biopsy [START_REF] Tefferi | Classification and diagnosis of myeloproliferative neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic algorithms[END_REF]. The latter typically reveals megakaryocytic proliferation, accompanied by a reactive deposition of fibrous connective tissue which results in a characteristic picture in advanced disease. However, at early stages of the disease and especially in the prefibrotic phase, the accurate morphological differentiation between PMF and other chronic myeloproliferative neoplasms poses a diagnostic challenge [START_REF] Wilkins | Bone marrow pathology in essential thrombocythemia: interobserver reliability and utility for identifying disease subtypes[END_REF]. This is in particular the case for the essential thrombocythaemia (ET), as marked thrombocytosis is often the only finding in both entities and subtype-specific markers are absent. The histological examination of the bone marrow biopsy plays a pivotal role in distinguishing these two entities. The entire histological pattern, ranging from cellularity to granulopoiesis, erythropoiesis and especially the megakaryopoiesis, has to be considered in these cases. Apart from the new diagnostic criteria of the revised WHO classification, the precise differentiation of PMF and ET strongly depends on investigators experience and remains a problem in daily clinical practice.

The outstanding finding in PMF is the reticulin or collagen fibrosis, which is regularly present in more advanced disease. One major pathogenetic mechanism involved in the development of myelofibrosis represents the increase of inflammatory cytokines such as platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and transforming growth factor β-1 (TGFβ-1) [START_REF] Mc | Involvement of the fibrogenic cytokines, TGF-beta and bFGF, in the pathogenesis of idiopathic myelofibrosis[END_REF]. These cytokines mediate essential changes in the bone marrow homeostasis. These changes result in shortened survival of red blood cells, suppression of erythroid progenitor cells, inadequate erythropoietin production, impaired iron utilization and finally in increased production of connective tissue [START_REF] Mc | Involvement of the fibrogenic cytokines, TGF-beta and bFGF, in the pathogenesis of idiopathic myelofibrosis[END_REF][START_REF] Martyre | Critical review of pathogenetic mechanisms in myelofibrosis with myeloid metaplasia[END_REF][START_REF] Mc | Myelofibrosis: pathogenesis of myelofibrosis with myeloid metaplasia. French INSERM Research Network on Myelofibrosis with Myeloid Metaplasia[END_REF]. Therefore, the PMF displays changes typical for anaemia of chronic diseases (ACD). The latter develops in the course of chronic disorders and is also associated with impaired iron metabolism caused by an increase of inflammatory cytokines.

In a previous study we have shown that zinc protoporphyrin (ZPP) reflects the extent of the impaired iron utilisation in ACD [START_REF] Hastka | Zinc protoporphyrin in anemia of chronic disorders[END_REF], being produced instead of heme when iron is not available. By qualitative and quantitative assessment of the impaired iron supplementation to the erythropoiesis, measurement of the ZPP concentration does not only support the diagnosis of ACD but also the supervision of the course of the disease [START_REF] Hastka | Zinc protoporphyrin in anemia of chronic disorders[END_REF].

Supposing similar effects on iron metabolism in PMF and ACD and the lack of iron disturbance in ET, we evaluated whether the ZPP measurement could be useful for the discrimination between PMF and ET and for the management of PMF.

Patients and Methods

The study was a single center analysis at III. All patients were diagnosed and regularly followed-up in our institution. Patients without stainable iron in bone marrow were not enrolled into this analysis.

The complete blood counts and tests of iron metabolism including ferritin, serum iron, transferrin, transferrin saturation, ZPP and the serum concentration of the soluble transferrin receptor (sTfR) were determined at baseline in all enrolled patients.

The measurements of hemoglobin and the other parameters of the blood count were performed by an automatic cell counter (Model CC-180, Sysmex, Kobe, Japan). Serum ferritin (normal value: 35-310 g/L for men, 25-210 g/L for women) was determined by the Access Ferritin immunoassay (Beckmann-Coulter, Krefeld, Germany). Serum iron, transferrin and transferrin saturation were determined by ELISA. ZPP concentration (normal <40 mol/mol heme) was measured with the Aviv front-face hematofluorometer (Aviv Biomedical Company, Lakewood, New Jersey, USA) on washed erythrocytes [START_REF] Hastka | Washing erythrocytes to remove interferents in measurements of zinc protoporphyrin by front-face hematofluorometry[END_REF].

For JAK2V617F mutation analysis the genomic DNA was extracted from the peripheral blood using QIAampDNA Kit (Qiagen, Hilden, Germany). The stored DNA samples were tested for the JAK2V617F mutation by tetra ARMS PCR Assay as described [START_REF] Jones | Widespread occurence of the JAK2V617F mutation in chronic myeloproliferative disorders[END_REF] in 55 patients.

Statistical analyses were performed using the GraphPad Prism statistical package for Windows (Version 4.0, GraphPad Software Inc., San Diego, CA, USA). The non-parametric two-tailed Mann-Whitney test was used to determine significances between groups. Correlation coefficients were determined by Pearson`s correlation coefficient test. Differences between frequencies were proved for significance by the Chi-square Test, a p-value less than 0.05 was considered as statistically significant.

The diagnostic accuracy was defined as the ratio of "true positives" + "true negatives" divided by the sum of "true positives" + "true negatives" + "false positives" + "false negatives".

Ethics:

The study was conducted in accordance with the Declaration of Helsinki 1975, as revised in 1983 and approved by the institutional review board. The study did not influence in any way the management of the patients, as all the tests used in the study including the bone marrow examinations were routinely done in all patients with ET and PMF at time of diagnosis. All patients gave written informed consent for testing and analysis of their data.

Results

Forty-six patients (19 males and 27 females, median age 62 years, range 25-78) were diagnosed as ET 2). The drop in ZPP concentration was accompanied by an improvement of hemoglobin levels. One case (UPN 2) showed a ZPP reduction from 136 mol/mol heme to normal values with a significant reduction of bone marrow fibrosis accompanied by a complete normalisation of the peripheral blood cell counts for now more than 4 years without further treatment.

In summary, 20 of 24 (83%) PMF patients and 3 of 46 (7%) ET patients had a ZPP concentration > 60 mol/mol heme at diagnosis of the disease. Accordingly, the diagnostic accuracy of a ZPP concentration > 60 mol/mol hemeto identify PMF in a PMF/ET patients group was 90%, with a sensitivity of 83% and a specificity of 93%.

Discussion

In the present study, we could show that the measurement of the ZPP concentration provides a useful contribution to differentiate PMF from ET. ZPP concentration further allows a monitoring of the therapeutic measures in PMF.

The intraerythrocytic ZPP concentration observed in our PMF patients at diagnosis was significantly higher when compared to the patients diagnosed with ET. Whereas 61% of the ET patients had normal ZPP, the concentrations were elevated in 92% of the PMF patients. The only two PMF patients with a normal ZPP concentration had markedly elevated platelet counts and normal hemoglobin levels. They were diagnosed as PMF due to bone marrow morphology in which clusters of atypical megakaryocytes were present. The marrow specimens, however, revealed no relevant marrow fibrosis and the clinical appearance of these patients corresponded better to ET.

In contrast to PMF, the elevation of the ZPP concentration observed in ET individuals was only minimal. ZPP concentrations > 60 mol/mol heme, as detected in 83% of PMF patients, were observed only in 7% of the ET individuals (p<0.0001; chi-square test). Therefore, the ZPP concentration of 60 mol/mol heme seems to be a suitable cut-off value to separate ET and PMF.

The diagnostic potential of the ZPP determination to separate PMF from ET patients is obviously based on the disturbance of iron utilization in PMF [START_REF] Martyre | Critical review of pathogenetic mechanisms in myelofibrosis with myeloid metaplasia[END_REF][START_REF] Mc | Cellular and molecular mechanisms underlying bone marrow and liver fibrosis: a review[END_REF], whereas iron deficient erythropoiesis is uncommon in ET. The impaired iron bioavailability observed in PMF seems to be a consequence of the overexpression of inflammatory cytokines such as interleukin-1 and transforming growth factorbeta (TGF-ß) which contributes to the underlying pathogenetic mechanism finally leading to fibrosis [START_REF] Rameshwar | Monocyte adhesion in patients with bone marrow fibrosis is required for the production of fibrinogenic cytokines. Potential role for interleukin-1 and TGF-beta[END_REF]. This aspect of the PMF pathogenesis strongly resembles features known from the anaemia of chronic diseases (ACD). In ACD there is also a cytokines-mediated impairment of iron bioavailability, mainly caused by tumor necrosis factor-α (TNF-α), interleukin-1 and interferon  [START_REF] Means | Recent developments in the anemia of chronic disease[END_REF]. The impaired iron supply to the bone marrow leads to an iron-deficient erythropoesis with decreased hemoglobin production finally resulting in anaemia [START_REF] Philippe | Iron and inflammation[END_REF][START_REF] Labbe | Iron assessment tests: transferrin receptor vis-a-vis zinc protoporphyrin[END_REF][START_REF] Braun | Erythrocyte zinc protoporphyrin[END_REF]. The impaired bioavailability in ACD is regarded to be an unspecific defence mechanism to suppress the inflammatory process [18;19]. It is characterized by a redistribution of iron to storage sites, which is expressed by an increase of ferritin and low levels of iron and saturation of transferrin in serum. In this condition, as soon as the iron concentration gets insufficient to satisfy the need of the erythropoiesis, zinc is increasingly incorporated into protoporphyrin IX and ZPP is produced [START_REF] Philippe | Iron and inflammation[END_REF][START_REF] Labbe | Iron assessment tests: transferrin receptor vis-a-vis zinc protoporphyrin[END_REF][START_REF] Braun | Erythrocyte zinc protoporphyrin[END_REF]. Similar to ACD, the sustained inflammatory process in patients with PMF obviously leads to a shifting of iron to storage sites, resulting in increase of stainable iron in bone marrow and serum ferritin, as also observed in our study. The bone marrow specimens of the PMF patients showed increased amounts of stainable iron and the serum ferritin concentration was significantly elevated when compared to the ET group. However, due to the wide range of serum ferritin levels in both disorders a reliable discrimination between ET and IMF by the determination of the serum ferritin concentration was not feasible.

The extent of the ZPP elevation depends on the severity and duration of the inflammatory process. Our results indicate that the increased inflammatory activity caused by overexpressed cytokines is obviously present already in the prefibrotic phase [START_REF] Bock | Bone morphogenetic proteins are overexpressed in the bone marrow of primary myelofibrosis and are apparently induced by fibrogenic cytokines[END_REF]. Despite the lack of significant marrow fibrosis, there was a significant increase of ZPP observed in patients with hypercellular prefibrotic stage PMF comparing to both normal population and ET group.

The ZPP concentration directly reflects the amount of impaired iron bioavailability, which depends directly on the inflammatory activity of the underlying disease [START_REF] Hastka | Central role of zinc protoporphyrin in staging iron deficiency[END_REF][START_REF] Blumberg | Hemoglobin determined in 15 microL of whole blood by "front-face" fluorometry[END_REF][START_REF] Mclaren | Erythrocyte protoporphyrin in the detection of iron deficiency[END_REF]. By quantitative assessment of the impaired iron bioavailability, follow-up measurements of ZPP allow a reliable monitoring of response of the underlying disease to therapy [START_REF] Hastka | Central role of zinc protoporphyrin in staging iron deficiency[END_REF][START_REF] Blumberg | Hemoglobin determined in 15 microL of whole blood by "front-face" fluorometry[END_REF][START_REF] Mclaren | Erythrocyte protoporphyrin in the detection of iron deficiency[END_REF]. Thus, the goal of the treatment in such a clinical situation must be the complete normalisation of this parameter, which verifies the positive effect of the therapy on the underlying pathophysiological process. The clinical value of the ZPP measurement for supervising the success of the therapy was clearly demonstrated in ACD [START_REF] Hastka | Central role of zinc protoporphyrin in staging iron deficiency[END_REF]. The four presented PMF patients who responded to immunsuppressive therapy illustrate that follow-up measurements of ZPP provide useful information for treatment monitoring also in PMF. The response, visible by the increase of the hemoglobin concentration was associated by a substantial decrease of ZPP (Figure 2). One of the patients (UPN 2) who presented with a hemoglobin value of 83 g/L and fibrotic marrow at diagnosis reached a complete normalisation of the peripheral blood counts and regression of the marrow fibrosis. The response was accompanied by a decrease of ZPP from 136 mol/mol heme, at the beginning of treatment to normal levels during normalisation of peripheral blood counts.

It has to be pointed out that the use of ZPP in patients with regular red blood cell transfusions is limited, as it is not the iron metabolism of the patient but rather the iron metabolism of the blood donor which is reflected by the ZPP determination. This is especially true for patients who have received numerous blood transfusions within 4 weeks prior to testing, as the life span of transfused red cells is supposed to be about 30 days [START_REF] Bard | The life span of erythrocytes transfused to preterm infants[END_REF].

In summary, while further studies are needed, based on the impaired iron bioavailability common in PMF, the ZPP measurement may provide valuable additional information which aids in the differential diagnosis between PMF and ET. A ZPP concentration of 60 mol/mol heme seems to be the appropriate cut-off value for this purpose. ZPP concentrations > 60 mol/mol heme are uncommon in ET if iron deficiency is excluded. Patients with PMF were subdivided in prefibrotic and fibrotic stage PMF. The values are mean ± standard deviation (and range). Values in square brackets are reference values. Abbreviations were notified as PMF, primary myelofibrosis; ET, essential thrombocythaemia; Hb, hemoglobin; sTfR, soluble transferrin receptor; WBC, white blood cell; ZPP, zinc protoporphyrin. 1 The differences between prefibrotic and fibrotic PMF group were statistically significant. Increased ZPP concentrations in PMF decreased successively under the treatment allowing a monitoring to therapy efficacy. The decline of ZPP was accompanied by an improvement of hemoglobin level. In one case (UPN 2, dashed line, follow-up for more than 4 years) bone marrow examination showed also a significant reduction of fibrosis.

Legends

Figure 1 ZPP

 1 Figure 1

Figure 2

 2 Figure 2

Figure 1 Individual

 1 Figure 1Individual ZPP concentrations and median ZPP concentration ( __ ) of all ET and PMF patients at

Figure 2 Individual

 2 Figure 2

Table 1 )

 1 . The mean ferritin concentration was 111 + 80 g/L (range 25-452) and the transferrin tested positive for the JAK2V617F mutation. The clinical course was much more comparable to the course of ET.When subdividing the PMF patient in prefibrotic stage and fibrotic stage according to the bone marrow examination, 12 PMF patients were in the hypercellular prefibrotic stage and 12 in the fibrotic phase at the time of the diagnosis. Comparing these groups, mean ZPP concentrations were significantly higher in fibrotic stage of the disease (122 ± 31 mol/mol heme, range 65-178) than in early stage PMF (77 ± 30 mol/mol heme, range 29-127; p=0.003). In fibrotic stage, hemoglobin values (96 ± 11 g/L, range 79-130) were lower than in the prefibrotic phase (121 ± 26, range 75-160; p = 0.02). Patients with fibrosis showed higher erythropoietin levels (84 ± 80 U/L, range 14-245) than patients in prefibrotic stage of PMF (19 ±23 U/L, range 3-79; p=0.01). There were no significant differences observed in mean WBC count, platelet count, transferrin saturation, sTfR and ferritin.

	saturation 26 + 7  (range 18-45). The mean ZPP concentration at diagnosis was 36 ± 13 mol/mol
	heme (range 11-76) (Figure 1). Twenty-eight of the 46 patients (61%) with ET had a ZPP
	concentration below the upper normal value of 40 mol/mol heme. Eighteen ET patients (39%)
	showed an increased ZPP, the concentration was, however, only marginally elevated. Forty ET
	patients (87%) had a ZPP concentration < 50 mol/mol heme, 43 patients (93%) < 60 mol/mol heme.

Only three patients revealed higher ZPP concentrations (61, 62 and 76 mol/mol heme, respectively).

ZPP concentrations > 76 mol/mol heme were not observed in ET.

Twenty-four patients (15 males and 9 females, median age 71 years, range 52-86) were classified to have PMF (Table

1

). The mean ferritin concentration (480 + 653 g/L, range 36-1792) was significantly higher (p=0.0004) compared to ET. There was no difference in transferrin saturation between PMF and ET patients observed.

The mean ZPP concentration at the diagnosis of PMF was 99 ± 37 mol/mol heme (range 29-178), which was significantly higher compared to patients diagnosed with ET (p<0.0001). The individual ZPP concentrations were above the upper normal value of 40 mol/mol heme in 92% (22/24) of cases.

ZPP concentrations > 50 mol/mol heme were exceeded by 21 of the 24 PMF patients (88%),

The two PMF patients with normal ZPP concentrations (29 and 40 mol/mol heme) showed remarkable high platelet counts (1,700 x10 9 /L and 808 x10 9 /L), an enlarged spleen (22 cm and 12 cm) and were

The JAK2V617F mutation was found in 26 of all patients analysed (26/55, 47%). Seventeen of the ET patients (17/38, 45%) and 9 of the PMF patients (9/17, 53%) were JAK2V617F positive. The mean ZPP, ferritin and sTfR concentration did not differ in both entities between patients with mutated and non-mutated JAK2. In ET with non-mutated JAK2 the mean platelet count was significantly higher (1027  333 x 10 9 /L; range 547-1740), than in JAK2-mutated ET (761  460, range 460-1370; p=0.008).

Table 1 Characteristics of all patients.

 1

			PMF		ET	P-values
		total	prefibrotic stage	fibrotic stage		ET vs. PMF
	N	24	12	12	46
	male / female	15 / 9	8 / 4	7 / 5	19 / 27
	ZPP	99 ± 37	1 77 ± 30	122 ± 31	36 ± 13	< 0.0001
	<40 mol/mol heme	(29 -178)	(29 -127)	(65 -178)	(11 -76)
	Ferritin	480 ± 653	324 ± 366	654 ± 848	111 ± 80	0.0004
	[male: 35 -310 g/L]	(36 -1792)	(80 -1369)	(36 -1792)	(25 -452)
	[female: 25 -210 g/L]
	sTfR	2.67  1.50	2.15  2.79	2.27  1.08	1.43 ± 0.42	< 0.0001
	0.81 -1.75 mg/L	(0.96 -6.80)	(0.96 -6.80)	(1.03 -5.17)	(0.82 -2.67)
	Transferrin saturation	33  18	33  22	33  13	26 ± 7	n.s.
	[16 -45%]	(17 -72)	(17 -72)	(20 -57)	(18 -45)
	Platelet count	363  276	468  317	259  189	941 ± 327	< 0.0001
	[male: 145-348 x 10 9 /L]	(31 -1700)	(106 -1700)	(31 -675)	(460 -1956)
	[women: 165-387 x 10 9 /L]
	WBC [4.2-10.2 x 10 9 /L]	(9.9-60.4) 13.0  14.5	(18.0-48.4) 15.6  12.2	(9.9-60.4) 10.3  16.9	(4.0 -17.1) 8.8 ± 3.4	n.s.
	Hb	109 ± 23	1 121 ± 26	96 ± 11	114 ± 14	< 0.0001
	male: 130 -170 g/L	(75 -160)	(75 -160)	(79 -130)	(111 -168)
	female:120 -160 g/L
	Erythropoetin	45 ± 60	1 19 ± 23	84 ± 80	12 ± 12	0.02
	6 -25 U/L	(3 -245)	(3 -79)	(14 -245)	(2 -67)
	JAK2V617F positive (N, %)	9/17	4/8	5/9	17/38	n.s.
		53%	50%	56%	45%


Ulrike Klein
email: ulrike.klein2@med.uni-heidelberg.de

Kai Neben

Thomas Hielscher

Christiane Heiß

Anthony D Ho

Hartmut Goldschmidt

Lenalidomide in combination with dexamethasone: Effective regimen in patients with relapsed or refractory Multiple Myeloma complicated by renal impairment

Keywords: Multiple Myeloma, Lenalidomide, renal insufficiency, pre-treatment

Over the last decade, treatment options for patients with Multiple Myeloma (MM) have improved substantially, resulting in better response rates and prolonged overall survival. Nevertheless, MM remains a challenging disease, especially if renal insufficiency (RI) or extensive pre-treatment aggravates the assignment of the optimal treatment schedule. In this retrospective study, we analyzed the outcome of lenalidomide + dexamethasone in 167 patients with relapsed or refractory MM with focus on RI. The baseline creatinine clearance (CLCr) was normal in 94 patients (CLCr ≥ 80 mL/min), while a RI was observed in 73 patients, including 40 patients with mild (50 ≤ CLCr < 80 mL/min) and 33 patients with moderate or severe RI (CLCr < 50 mL/min). Response rates declined depending on the severity of RI, being 67% among patients with normal kidney function, 60% among patients with mild RI and 49% among patients with moderate or severe RI. Time to progression (TTP) was significantly reduced in patients with severe RI and in case of >2 previous treatment lines. Overall survival (OS) was not significantly different between patients with normal and impaired renal function. In contrast, the number of previous treatment lines (2 versus <2) and the use of novel agents like bortezomib or thalidomide prior to lenalidomide + dexamethasone therapy had a more adverse effect on OS. In conclusion, lenalidomide + dexamethasone is an effective regimen for relapsed or refractory patients with MM complicated by RI with manageable toxicity.

Introduction

Considerable effort has been put into understanding the pathomechanism of Multiple Myeloma (MM), optimizing diagnostic procedures and therapeutic strategies, leading to substantial advances in treatment options and survival duration in the last decade, mostly due to new therapeutic agents ].

However, MM remains a challenging hematological neoplasm. Lenalidomide is an agent with proven efficacy and tolerability in relapsed and newly diagnosed patients. Its targets are not only the MM cell, but also cell-host interactions, cytokine-secretion and the BM milieu ]. Management of MM patients is often complicated by the fact that up to 20 to 40% of patients suffer from renal insufficiency (RI) ]. The main reason for this is the accumulation and precipitation of light chains forming casts, resulting in renal tubules obstruction. They also seem to alter renal function by direct toxic effects on the proximal renal tubules; hypercalcemia and dehydratation, with nephrotoxic medication enhancing this renal damage ].

Advanced age of MM patients can also be a cofactor for renal insufficiency, as it might be associated with an increased incidence of cardiovascular or endocrine disorders. Data from retrospective analyses and sub-analyses of prospective trials for conventional chemotherapy reveal that TTP and OS are reduced in patients with renal insufficiency ]. Therefore, effective treatment modalities having no or little renal toxicity and the potential ability to improve renal impairment are preferred. In the literature, bortezomib has been identified to be the best choice for these patients, particularly as no dose adjustment is needed for any degree of renal insufficiency and response and improvenment of renal function take place rapidly . In case of impaired renal function, therapy with lenalidomide requires adjustment to be feasible. Oral absorption, protein binding, or non-renal elimination of lenalidomide are not altered by RI. However, renal clearance of lenalidomide decreases drastically in patients with severe renal dysfunction, whereas drug exposure and half-life time are increased. Therefore, dose adjustments are recommended for patients with a creatinine clearance CLCr of less than 50 ml/min ]. Prospective data from clinicals trials on the application of lenalidomide in patients with MM and renal impairment, however, are not available.

As data from randomized studies are lacking, the question of when to best apply salvage therapy in relapsed or refractory disease remains to be discussed as controversially as the correct implementation of new therapeutic agents, particulary since these agents took place in various upfront strategies. It is well known that the probability of treatment response decreases with increasing numbers of previous therapies ]. In addition, the time to progression is abbreviated with each salvage therapy applied.

The current investigation is a retrospective analysis to evaluate the influence of renal impairment and previous therapies in patients with relapsed MM who have been treated with lenalidomide at a single center.

Patients and Methods

Between July 2005 and June 2009, 167 patients with refractory or relapsed MM were treated at our center with a combination of lenalidomide and dexamethasone. Treatment consisted of lenalidomide (25 mg on day 1 to 21) and dexamethasone (cycle duration 28 days) according to the two phase III studies, wich lead to the approvel of lenalidomide in relapsed and refractory MM patients [18;19]. The first 13 pts (7.8%) received high-dose dexamethasone with 40mg day 1-4, 9-12, 17-20 but due to severe infectious complications we decided in our center to reduce the dosage for all patients about 50% to 20mg day 1-4, 9-12 and 17-20. One-hundred-thirty-two patients (79.0%) received this schema. With the emerging data of the low-dose dexamethasone schedule of Rajkumar et al. 12 patients (7.2%) received the reduced dosage of dexamethasone according to the ECOG E4A03 study with 40 mg on days 1, 8, 15 and 22 because of expected severe side effects of dexamethasone due to their personal risk profile . Of the 20 patients who received lenalidomide/dexamethsone after allogenic transplantation and due to the concomitant immunosuppressive therapy, 8 patients (4.8%) got a further reduced dexamethasone dosage (2 pts. 0mg dexamethasone, 4 pts. 80mg per cycle, 1 pt 96mg per cycle and 1 pt 144mg per cycle). The remaining 2 patients received 80mg and 120mg per cycle respectively as an individualized treatment decision. Dose adjustments for patients with RI were made in case of a baseline CLcr ≤50 mL/min or worse.

Patients with a moderate RI (30 ≤ CLCr ≤50 mL/min) received 10mg Lenalidomide daily; patients with a severe RI (CLCr < 30 mL/min) received 15mg every other day, whereby patients requiring dialysis received lenalidomide 15 mg after dialysis. However, 11 pts with moderate RI received due to their borderline CLCr and the fact that their serum creatinin was below 1.7mg/dl the full dosage with 25mg lenalidomide day 1-21. One pt with a baseline CrCl of 25ml/min received 25mg as well, because serum creatinine was normal and collectin volume of urine was only 1.200ml/24h. Treatment was administered until disease progression or unacceptable toxicity occured. Lenalidomide and dexamethasone were administerd as re-induction therapy in 41 patients, followed by high dose chemotherapy and autologous stem cell transplantation (ABSCT). At the time of this analysis, 40 patients have undergone ABSCT. In the statistical analysis ABSCT was computed as a competing risk. For the analysis of RI, patients were classified into the following RI subgroups based on their baseline cratinine clearance (CLcr) as determined in a 24-hour urine sample: no RI CLCr > 80 mL/min, mild RI 50 ≤ CLCr ≤ 80 mL/min, moderate RI 30 ≤ CLCr ≤50 mL/min or severe RI CLCr < 30 mL/min. As the dosage of lenalidomid is linked to the classification of the GFR (80-50ml/min, 50-30ml/min, <30ml/min without dialysis and dialysis dependency) we decided to classify the patients as well in these categories, even if nephrologists consider normal renal function as GFR >90 ml/min. Information on CLCr at the time of treatment initiation was available for 165 patients (98.8%). For two patients, CLCr at start therapy was not available; therefore the MDRD-formula (MDRD = Modification of Diet in Renal Disease) was used instead. Both patients had in previous measurements always normal CLCr and at the time of beginning normal values for creatinine and urea, so they were counted as normal kidney function. Due to the relatively small number of patients with moderate to severe RI, these groups were combined for the statistical analysis. The numbers of pre-treatment were recorded for all patients. Every treatment schedule which was necessitative due to relapse or refractory disease was counted as one therapy.

Induction, mobilization, autologous transplantation and maintenance therapy were counted as one therapy. Response to treatment was assessed according to the European Group for Blood and Marrow Transplantation criteria for complete remission (CR), partial response (PR), minimal response (MR), stable disease (SD) and progressive disease 21]. The criteria were complemented by the criteria of the International Myeloma Working Group for very good partial response (VGPR) and near complete remission (nCR) 2]. CR was defined as: no M-protein detectable by electrophoresis and immunofixation in the serum and urine, disappearance of any soft tissue plasmacytomas, and ≤ 5% plasma cells in the bone marrow. nCR was defined as complete disappearance of the monoclonal protein, except for a positive immunofixation. VGPR was defined as at least 90% reduction in serum M-protein and urine Mprotein level below 100 mg/24 hours. PR was defined as at least 50% reduction of serum M-protein and reduction in 24-hour urinary sample M-protein by at least 90% or less than 200 mg/24 hours. MR was defined as a 25 to 49% reduction in serum M-protein and reduction in 24-hour urinary sample M-protein by 50 to 89%. Progressive disease was defined by any of the following: an increase of at least 25% from baseline serum or urinary M-protein, equalling at least an absolute increase of at least 500 mg/dL in serum or 200 mg/24 hours in urine; newly occured or increased size of bone lesions or soft-tissue plasmacytomas, or development of hypercalcemia. Efficacy was measured by overall response rate (ORR), which is defined as CR+VGPR+PR rate. ORR was assessed as best response during treatment with lenalidomide and dexamethasone. Time to progression (TTP) was evaluated as time from start of lenalidomide/dexamethasone to first assessment of disease progression. Patients without evidence of progression were censored at their last information. Overall survival (OS) was defined as start of lenalidomide/dexamethasone until death from any cause or last follow-up visit, whichever occurred first.

Follow-up data on OS were obtained up to June 2009, for a median follow-up duration of 15.7 months.

The review board of our institution approved of the retrospective analysis and patients provided written informed consent.

Statistical analysis

Estimation of OS distribution was performed by the method of Kaplan and Meier. For comparisons of OS curves, the log-rank test was used. In addition, Cox PH regression analysis was used to evaluate the prognostic impact in a univariate model as well as in a multivariate model together with prognostically relevant clinical factors. TTP was evaluated in a competing risk analysis with ABSCT being the competing risk. TTP was analysed fitting a subdistributional hazard regression model [32]. This model directly assesses the effect of a prognostic factor on TTP in a competing risk setting and allows incorporating additional covariates. TTP distribution was estimated using cummulative incidence rates.

Post-baseline factors were included as time-dependent covariates. Response rates were compared with Fisher's exact test. The result of a test was always judged as statistically significant when the corresponding two-sided p-value was below 0.05. The prognostic value of clinical factors was assessed by their estimated hazard ratios including 95% confidence intervals. All statistical computations were performed with the statistical software environment R, version 2.9.0 using the R package cmprsk version 2.2-0 using the R add-on packages cmprsk and kmi [33].

A propensity score analysis was performed to account for imbalances at baseline between patients with severe and without renal impairment, adjusting for age, sex, number of previous therapies, previous treatment with velcade, thalidomide or both, time between start of first-line treatment and start of lenalidomide, and time between last multiple myeloma treatment and start of lenalidomide. Adjusted Kaplan-Meier curves for OS were computed using the propensity scores in an inverse probability weighting scheme 3].

Results

Patients

From July 2005 to June 2009, 167 patients with relapsed or refractory MM were treated with lenalidomide/dexamethasone in our center, forming the base of this analysis. Normal renal function with no RI (CLCr ≥ 80 mL/min) was observed in 94 patients, 40 (23.9%) patients had mild RI (50 ≤ CLCr < 80 mL/min), 21 (12.6%) had moderate (30 ≤ CLCr < 50 mL/min) and 12 patients (7.2%) had severe RI (CLCr < 30 mL/min), of whom 5 patients (3%) required hemodialysis. Further baseline characteristics are presented in table 1. When compared to patients without RI, patients with RI were more likely to be female and patients with normal renal function were more likely to have a lower median number of previous therapies (2 versus 3 in mild RI and 3 in severe RI) and are less likely to have received bortezomib and/or thalidomide prior to lenalidomide/dexamethasone when compared to patients with mild or severe RI (22.3% vs. 45.0% vs. 36.4%).

Efficacy

Among the entire group, the overall response rate (≥ PR) was 61.7%, while the CR rate was 11.4%.

Overall response rates and CR rates declined depending on the severity of renal impairement, being 67.0% among patients with normal kidney function (CR 14.9%), 60.0% among patients with mild RI (CR 10.0%) and 48.5% among patients with moderate or severe RI (CR 3.0%) (ORR adj. p-value 0.66).

Efficacy was also strongly influenced by the type of pre-treatment and the occurence of relapse from remission or primary refractory disease. A detailed description is listed in table 2a.

Time to progression (TTP)

TTP was defined as time from start of RD therapy until disease progression. During the follow-up of 15.7 month, a total of 101 events occurred. The median TTP based on the cumulative incidence function for the overall population is 9.02 months. TTP is significantly shortened in case of mild (p = 0.007) and ≥moderate RI (p = 0.005) versus no renal impairment (Fig. 2a). In order to identify the effect of more intense pre-treatment in patients with renal impairment, we performed a multivariate analysis.

The following parameters were considered in addition to kidney function and previous treatment schedules within a multivariate Cox PH model: age, previous therapies (1, 2,>2), previous new agents (bortezomib, thalidomide, both), time between first myelomaspecific therapy and RD therapy, time between last treatment and RD therapy, progressive disease during treatment with new agents, prior allogeneic transplantation and quality of response (≥ PR). Multivariate analysis revealed that the quality of response (p = 0.03), severe renal impairment (p = 0.04), more than two previous treatment lines (p = 0.03) and time between last treatment and RD therapy (p = 0.02) were significantly related to TTP. The median TTP lies between 13.02 months among patients with no RI, 7.67 months among patients with mild RI and 6.00 months among patients with severe kidney dysfunction. The TTP according to the kind of pre-treatment revealed the following results: median TTP not reached for patient with no history of novel agents, 11.34 months for patients who only received previous thalidomide, 8.92 months among patients who were prior exposed to bortezomib only and 5.57 months among patients who received both new agents before the treatment with lenalidomide/dexamethasone (Fig. 2c). Based on the hazard ratio we calculated a forest plot to illustrate the impact of the different parameters on TTP (Fig. 1a).

Overall survival

Overall survival was defined as time from start of lenalidomide/dexamethasone therapy until death from any cause. After a median follow-up of 15.7 months, death occurred in 62 patients (29 among patients with normal kidney function, 17 among patients with mild RI and 16 among patients with moderate/severe RI), with a median time to death of 25.8 months. With regards to RI, no significance could be detected in the differentiation between normal kidney function and all severity codes of renal impairment (p = 0.233) (Fig. 2b). There was a trend towards shorter duration of survival (p = 0.107) among patients with severe RI (CLCr < 50 mL/min) when compared to patients with normal kidney function. This trend, however, was not significiant. Patients who had received bortezomib or thalidomide prior to lenalidomide/dexamethasone had a significant shorter duration of survival when compared to patients treated with schedules without new agents (p = 0.018), with bortezomib (p = 0.011) having a stronger influence than thalidomide (p = 0.103). In case both new agents were applied prior to lenalidomide/dexamethasone, overall survival was negatively impacted (p < 0.001) (Fig. 2d). Similar results were seen when regarding the number of previous treatment lines: patients with 2 (p = 0.028) or more than 2 pre-treatment schemes (p < 0.001) had a significant shorter duration of overall survival. A detailed description is shown in table 2b. Results are illustrated as forest plot as well (Fig. 1b). In order to verify the influence of other variables we performed a multivariate analysis. The following parameters were considered within a multivariate Cox PH model: age, previous therapies (1, 2,>2), previous new agents (bortezomib, thalidomide, both), time between first myelomaspecific therapy and RD therapy, time between last treatment and RD therapy, progressive disease during treatment with new agents, stem cell transplantation after RD therapy (time dependent co-variate), prior allogeneic transplantation and quality of response (≥ PR). In multivariate analysis the time between first myelomaspecific therapy and RD therapy (p = 0.03) and the quality of response (p = 0.02) were significant. Additionally a propensity score analysis was performed to account for imbalances at baseline. The following baseline covariates are used to model the probability of having a severe renal impairment compared to having no renal impairment: age, sex, number of previous therapies, previous treatment with velcade, thalidomide or both, time between first MM treatment and start of RD, and time between last MM treatment and start of RD therapy (Fig. 3).

Toxicity

Due to the retrospective nature of the study, complete toxicity data are not available for all patients. We therefore performed a subgroup analysis of 108 patients with available toxicity information referring to haematological toxicity, infectious complication and thromboembolic events. Patients with renal insufficiency had a higher incidence of neutropenia than patients with normal kidney function (p = 0.05).

RI was also associated with a higher incidence of infections (p = 0.03) especially with pneumonia (p = 0.03). Thrombocytopenia was not significantly different. Anemia naturally occurred more frequently in the group of patients with renal impairment due to additional renal caused anemia. Thromboembolic events did not differ in the subgroups.

The tolerance of therapy with lenalidomide/dexamethasone was additionally measured by the frequency of dose reduction or discontinuation of therapy. These data are available for all patients. In 58 of 167 patients (34.7%) either therapy interruption or dose reduction was necessary. While patients with severe RI more often required interruption of treatment, for patients with moderate RI more frequently a dose reduction was performed (24.2 vs. 12.5% and 24.2 vs. 30.0%). In the Fisher exact test the reduction of therapy reveals significance (p = 0.02) in case of RI, whereby in the testing for discontinuation of treatment, data do not reach significance (p = 0.67). Further information on toxicity can be found in tables 3a and 3b.

Alterations of renal impairment during lenalidomide/dexamethasone treatment Among 167 patients, 152 were evaluable for follow-up CLCr, of which 64 patients suffered from RI at baseline. Principally it needs to be taken into consideration that the pathogenesis of renal impairment remains unclear in the main proportion of patients. Beside myeloma specific alterations, RI is probably related to other than myeloma associated factors as well.

No alteration in the degree of renal impairment was seen in 123 of 152 patients (80.9%). When regarding patients with renal insufficiency at the beginning of therapy 17 of 64 (26.6%) showed an improvement of at least one level of the previously described subclasses. On the contrary, CLCr had deteriorated in 8 of 64 patients (12.5%). However, 2 of these 8 patients had suffered from progressive disease at the time of deterioration of their renal function. We subsequently looked at the relationship between the improvement of renal function and the response to RD treatment. Patients who experienced an improvement or a stabilization of their kidney function noticeably obtained a higher frequency of at least a partial response than patients who experienced a worsening of their renal function. Further information is displayed in table 4.

Discussion

Our data demonstrate that lenalidomide is highly active in relapsed and refractory MM patients. Even with impaired renal function or previous treatment schedules with new agents a remarkable overall response rate of about 62% and a CR rate of 11.4% can be reached. Nevertheless, response rates decreased with the extent of renal impairment from 67% to 48.5%. It needs to be taken into account that this is not mainly an effect of the severity of renal impairment but the impact of more prior therapies in the two groups with RI and a higher percentage of bortezomib containing regimes. This imbalance applies as well for the TTP which is significantly shortened in univariate analysis in case of mild and ≥moderate RI. To account for these heterogenities we performed a multivariate analysis regarding TTP and OS. Multivariate analysis was only significant in patients with severe kidney dysfunction according the TTP. This finding is mainly caused by the significantly higher frequency of dose reduction in patients with renal insufficiency due to more toxic side effects. Patients with RI had a higher incidence of neutropenia and infections. Especially the occurrence of pneumonia was more frequent than in patients with normal kidney function. Due to these adverse events treatment schedules could not be realized as planned. Delay of treatment, dose reduction and interruption entailed not only the deterioration of response rates but also explain the shortened TTP. Rajkumar et al could demonstrate that the dosereduction of dexamethasone in the combination therapy with len/dex may result in a significantly worse ORR, but due of the reduction of toxic side effects and the nessessity of less dose adjustments or dose interruptions a prolonged OS was obtained [20]. Recently, a dose adjustment protocol for patients with renal impairment was approved for use in the US and Europe. It suggests that lenalidomide 5 mg daily be used in patients requiring dialysis. This dosage has been chosen in order to decrease the fluctuation of lenalidomide plasma-levels and the individual body-load of each single dosage. This adjustment will most likely reduce toxic side effects and improve the compliance. Due to our single-centre experience we reduced the dosage of dexamethasone per cycle about 50%, from 480mg to 240mg. A further reduction in analogy to the ECOG study to 160mg might additionally reduce toxic side effects.

A main goal in the treatment of MM patients with renal insufficiency is the improvement of kidney function. Our data demonstrated that among the 152 evaluable patients 140 pts. (92.1%) had an unchanged renal status or an improvment of their renal impairment. Focussing on 64 patients with a CLCr of 50 ml/min or less at baseline, 17 (26.6%) had an improvement in renal function. Patients who experienced an improvement or a stabilization of their kidney function noticeably obtained a higher frequency of at least a PR than patients who experienced a worsening of their renal function. The effect of disease control through the lenalidomide/dexamethasone treatment seems to have a major impact on the course of renal function. Yet it has to bear in mind that for the main proportion of patients in our study population the pathogenesis of renal impairment remains unclear. Focussing on patients with proven solely myeloma associated renal dysfunction, would more likely result in an even higher percentage of renal function improvement.

As repeatedly mentioned lies great relevance regarding the efficacy of lenalidomide in the type, duration and sequence of previous myelomaspecific treatments. The effect of decreasing OS and TTP with each additional treatment line is a general feature for all kinds of myeloma-specific chemotherapy, reflecting the biology of the disease mainly by an increasing resistence-feature of the plasma cell. Recently published data from a subset analysis of two phase III studies with lenalidomide and dexamethasone suggested the greatest benefit in terms of TTP in early use ]. In our analysis, ORR diminished with increasing numbers of previous new agents from 64.3% to 52.9%. In relation to intensity and type of pre-treatment, OS in a univariate setting was significantly shortend after two and more previous treatments. The application of one of the two novel agents (thalidomide or bortezomib) resulted in a significant reduction of OS. While the use of thalidomide had no significant impact, previous therapy with bortezomib was significant. Applying both novel agents significantly decreased OS (p < 0.001). In accordance to OS, the type of previous treatment schemes affected TTP. While the influence of thalidomide showed only a trend towards a shorter duration of TTP (p = 0.067), the impact of bortezomib was highly significant (p = 0.002). The application of both agents prior to lenalidomide was associated with a significant reduction of TTP (p < 0.001). With regards to mechanism of action, it can not to be anticipated that the influence of the closely related drug thalidomide is less favorable in relation to response rate and TTP than previous therapy with bortezomib 31]. In our analysis, the use of thalidomide seemed to be partly negligible, with OS and TTP being significantly influenced by the previous application of bortezomib.

The comparison of TTP and OS among different studies is limited because of varying inclusion and exclusion criteria. However, bortezomib seems to achieve a TTP duration of about 5 to 7 months in relapsed MM patients 7]. Data on the efficacy and safety of bortezomib in patients with RI from the subanalysis of the APEX study revealed that TTP was less than 4.9 months for patients with CLCr <50ml/min and 6.2 months for patients with moderate RI 4]. Comparable patient characteristics with 1-3 previous therapies can be found in an other phase III study which investigated the use of pegylated liposomal doxorubicin plus bortezomib versus bortezomib alone with 199 days for the bortezomib mono group and 190 days among patients with normal kidney function 8]. In the phase II CREST study, two doses of bortezomib alone or in combination with dexamethasone were evaluated as second-line therapy in MM. With the addition of dexamethasone the 1.3mg/m² cohort achieved a TTP of 11 month [29]. Patient characteristics that correlate better to our heavily pretreated cohort can be found in the phase II study in which 202 patients were enrolled. These mainly refractory patients reached an ORR (≥PR) of 27% and a median TTP of 7 month [30]. Compared to the above mentioned studies the duration of response is noticeably longer for patients receiving len/dex than bortezomib monotherapy.

The TTP in our group among patients with severe RI (6.0 months) is nearly as long as for patients without renal disorder who are treated with bortezomib in the APEX study (6.3 months) or in the phase III study which investigated the use of pegylated liposomal doxorubicin plus bortezomib 8].

Considering that our investigated cohort was heavily pre-treated a median OS of 25.8 month is considerable. Although our data indicate a trend towards a shorter OS in patients with severe renal impairment compared to those with normal renal function, findings were not significant. Additionally a propensity score analysis was performed to account for imbalances at baseline, mainly the differences in the kind and intensity of previous therapies (Fig. 3). The approach of the curves is obvious and reflects the indeed sequence of events. We can demonstrate that OS is not significantly shortened in patients with severe renal insufficiency.

In conclusion, our data endorse the potency of this immunomodulatory drug even in heavily pre-treated MM patients with various degrees of renal impairment. This analysis substantiates previous data that the effectiveness is higher in its early use. However, the most favourable sequence of various new agents can not be deducted from our data. At this point we want to elude the limitations of our study due to its retrospective nature. Nevertheless reflects this study population the daily routine of hematologists in a university hospital. Instead, prospective trials are needed to answer the question on how to effectively sequence myelomaspecific therapy regimens. Considering the ongoing discussion of effective initial therapy with a combination of different kinds of new agents, the strategy of salvage therapy should be reconsidered as well. Lenalidomide is able to induce the longest TTP in relapsed and refractory MM patients compared to other standardized conventional myeloma therapies and achieves even in heavily pre-treated patients an improvement of renal function in a significant number of patients (25-30%). Side effects needs to be recognized but are manageable for versed hematologists and oncologists. The new medication schedule for patients with moderate and severe renal impairment will in all likelihood reduce the problems of adverse effects, dose reduction and treatment interruption.

There is no reason to generally avoid or shift the potent treatment option with lenalidomide for patients with renal insufficiency at a later time.

Fig. 1 Fig. 2

 12 Fig. 1 Forestplot of univariate prognostic effects displaying the hazard ratio including 95% confidence levels for Time to Progression (a) and Overall Survival (b) Fig. 2 Cumulative incidence functions for Time to Progression (a, c) and Kaplan-Meier estimates for Overall Survival (b, d) according to renal impairment and pre-treatment Fig. 3 Kaplan-Meier estimates for Overall Survival by renal impairment adjusted for additional covariates according to propensity score analysis

Table legends Tab . 1

 legends. Patient characteristics and disease features Tab. 2a Overall response rate and time to progression duration Tab 2b Prognostic value of various parameters on Overall Survival Patient characteristics and disease features : RI, renal insufficiency; CR, complete remission, VGPR, very good partial remission, ORR overall response rate, PR, partial response, TTP time to progression Tab. 2b: Prognostic value of various parameters on Overall Survival Tab. 3a CTC grade 3/4 toxicity according to renal function

	a and initiation of len/dex treatment 0 5 10 15 20 0.0 0.2 0.4 0.6 0.8 1.0 months since start of RD therapy 25 estimated cummulative incidence no RI moderate RI severe RI Time to progression according renal impairment 49.2 0.4 month Time between first line therapy OS Probability 0.6 0.8 1.0 Tab. 2a Overall response rate and time to progression duration b 79.4 OS Probability 0 0.0 0.2 0.4 0.6 0.8 1.0 no RI Severe RI Adjusted Kaplan-Meier plot for overall survival 6 Overall survival according renal impairment months since start of RD therapy 37.0 12 18 24 30 36 no RI Moderate RI Severe RI Tab. 1 Normal renal function % Mild RI 50 ≤ CLCr < 80 mL/min % ≥ moderate RI % CR (%) ≥VGPR ORR (%) (≥PR) Median TTP (month) Entire group n = 167 19 (11.4) 43 (25.7) 103 (61.7) Event All n = 108 No RI n = 59 ≥ Moderate RI Fisher's exact Mild RI n = 31 Test n = 18 (grade 3/4 combined) 9.02 Normal vs. mild/ CLCr < 50 mL/min Number of pts. 94 40 33 Sex (male) 67/94 71.3 14/40 35.0 12/33 36.4 Age median [range] 62 [30 -78] 66 [43 -77] 66 [29 -80] Normal kidney function n = 94 14 (14.9) 26 (27.7) 63 (67.0) 13.02 Mild RI n = 40 4 (10.0) 12 (30.0) 24 (60.0) Grade 3 Grade 4 Grade 3 Grade 4 Grade 3 Grade 4 Grade 3 moderate/ severe Grade 4 RI combined 7.67 Moderate + severe RI n = 33 1 (3.0) 4 (12.1) 16 (48.5) Hematologic 6.00 disorder No bortezomib or thalidomide as previous Neutropenia 29 (26.7) 2 (1.9) 11 (18.6) 1 (1.7) 11 (35.5) 1 (3.2) 7 (38.9) 0 (0.0) 0.05 Monoclonal Protein IgG IgA BJ Other 60 17 8 9 63.8 18.1 8.5 9.6 23 10 5 2 57.5 25.0 12.5 5.0 15 8 9 1 45.5 24.2 27.3 3.0 Time between first diagnosis and initiation of len/dex treatment in 54.2 88.7 38.0 therapy n = 42 8 (19.0) 16 (38.1) 27 (64.3) Thrombopenia 11 (10.2) 5 (4.6) 7 (11.9) 1 (1.7) 2 (6.5) 3 (9.7) 2 (11.1) 1 (5.6) 0.79 NA bortezomib or thalidomide as previous therapy n = 74 8 (10.8) 18 (24.3) 49 (66.2) Anemia 16 (14.8) 1 (0.9) 6 (10.2) 0 (0.0) 5 (16.1) 0 (0.0) 5 (27.8) 1 (5.6) 0.11 9.28 only thalidomide n = 27 2 (7.4) 6 (22.2) 19 (70.4) Infection 11.34 only bortezomib n = 47 6 (12.7) 12 (25.5) 30 (63.8) bortezomib and thalidomide as previous Other 8 (7.4) 0 (0.0) 4 (6.8) therapy n = 51 3 (5.9) 9 (17.6) 27 (55.1) FUO 8 (7.4) 1 (0.9) 3 (5.1) 5.57 Pneumonia 15 (13.9) 0 (0.0) 4 (6.8) 8.92 All kinds 31 (28.7) 1 (0.9) 11 (18.6) 1 (1.
	in month
	Tab. 3a CTC grade 3/4 toxicity according to renal function Tab. 3b Frequency of dose reduction and interruption according to renal function Tab. 4 Shift in renal function during treatment with lenalidomide/dexamethasone c 0.2 0.4 0.6 0.8 1.0 estimated cummulative incidence none new agent Time to progression according pre-treatment Overall survival according pre-treatment 0.2 Dosage of dexamethasone/cycle 480mg 8 5 0 d 240mg 76 31 25 1.0 160mg 5 3 4 Vel or Thal Vel+Thal OS Probability 0.2 0.4 0.6 <160mg 5 1 4 0.0 No. pre-treatment median [range] 2 [1 -10] 3 [1 -8] 3 [1 -9] Hazard ratio .95 lower CI .95 upper CI p-value 0.8 months since start of RD therapy 0 6 12 18 24 30 36 Bortezomib 46/94 48.9 30/40 75.0 23/33 RI yes vs. No 1.4 0.82 2.24 0.233 69.7 Thalidomide 39/94 41.5 22/40 55.0 17/33 Moderate RI vs. No 1.2 0.63 2.11 0.637 51.5 Bortezomib and Thalidomide 21/94 22.3 18/40 45.0 12/33 Severe RI vs. No 1.7 0.90 3.05 0.107 36.4 Autologous Transplantation 79/94 84.0 36/40 90.0 25/33 bortezomib or thalidomide 3.2 1.22 8.37 0.018 75.8 No. cycles median [range] 3 [1 -25] 5 [1 -30] Only thalidomide 2.5 0.83 7.76 0.103 3 [1 -21] Only bortezomib 3.7 1.35 9.91 0.011 none Vel or Thal Vel+Thal Fig. 3 bortezomib and thalidomide as previous therapy 6.4 2.50 16.55 <0.001
	0.0					0.0
	0	5	10	15	20	25	0	6	12	18	24	30	36
		months since start of RD therapy			months since start of RD therapy
												Fig. 2, a-d

Abbreviations: RI, renal insufficiency; CLCr, Creatinine Clearance; pts, patients; No, number AbbreviationsAbbreviations: RI, renal insufficiency; CI, confidence interval

  Abbreviations: FUO, fever of unknown origin; DVT, deep vein thrombosis; PE, pulmonary embolism, RI, renal insufficiency Tab. 3b Frequency of dose reduction and interruption according to renal function

				0.37
				1
	Dose reduction			%
	Entire group	n = 167	31/167	18.6
	Normal kidney function	n = 94	11/94	11.7
	Mild RI	n = 40	12/40	30.0
	Moderate + severe RI	n = 33	8/33	24.2
	Dose interruption
	Entire group	n = 167	27/167	16.2
	Normal kidney function	n = 94	14/94	14.9
	Mild RI	n = 40	5/40	12.5
	Moderate + severe RI	n = 33	8/33	24.2

Abbreviations: RI, renal insufficiency

Acknowledgement

The authors would like to thank Frank Müller for carefully collecting data and Fabienne McClanahan for spell checking. This work was supportet in part by the Dietmar-Hopp-Stiftung GmbH, Walldorf, Germany 32.

Fine JP, Gray RJ (1999) A proportional hazards model for the subdistribution of a competing risk. J Am Stat Assoc 94:496-509 33. R Development Core Team (2009). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.Rproject.org.

Fig. 1,a


Stefano Licci
email: stefano.licci@hotmail.it

Luca Morelli

Renato Covello

Primary mantle cell lymphoma of the testis

Dear Editor,

A 45 -year-old man presented wit h a 3-week histor y of right lo wer quadrant pain radiati n g to the right ingu i n a l area and testis. Physical exa m i n a t i o n revealed an enlarged right testis. Ultraso u n d exa m i n a t i o n of the right testicle sho we d a hypoec h o i c mass inv o l v i n g alm ost the entire organ and consiste nt wit h a neoplastic lesion. Biochemical assays for alpha fetoprotein and beta human chorionic gonadotrophin were within normal limits. A right orchi f u n i c u l e c t o m y was perfor m e d and histol o g i c a l exa m i n a t i o n revealed the neoplastic mass was represented by a diff use proli fe rati o n of pred o m i n a n t l y medium-sized monotonous lymphoid cells resembling lymphoblasts, with dispersed chromatin, scant cytoplasm and a high mitotic rate (25 per 10 high power field) (Fig. 1A-B).

Semeniferous tubules appeared entrapped in the lymphomatous tissue, were immature and lacking spermatogenesis (Fig 1A-B). On immunohistochemistry, the neoplastic cells expressed CD45, CD20 (Fig. 1C), CD5, cyclin D1 (Fig. 1D), bcl-2, and, focally, CD43, with no expression of CD3, CD10, bcl-6, CD23, TdT, CD34, MUM1/IRF4 and CD138. The proliferation index measured by immunostaining with monoclonal antibody MIB1 (anti-Ki-67 nuclear antigen) was high with more than 60% of the cells staining positive. Morphological and immunohistochemical features were consistent with a mantle cell lymphoma (MCL), blastoid variant. A fluorescence in situ hybridization (FISH) revealed that more than 75% of the cells contained t(11;14). Biochemical and hematological tests were normal, and computed tomography scans of the head and neck, thorax, abdomen, and pelvis were unremarkable.

A bilateral bone marrow biopsy revealed no infiltration by lymphoma.

On the basis of all these findings, a diagnosis of primary MCL of the testis, blastoid variant, was made.

The patient underwent localized radiotherapy and six cycles of CHOP chemotherapy (doxorubicin, cyclophosphamide, vincristine, prednisone). Currently, 10 months after completion of therapy, he continues to be healthy with no apparent manifestation of the disease by physical examination and total body computed tomography.

MCL is a B cell non Hodgkin lymphoma (NHL) of older adults, with a male preponderance. Most patients present with disseminated disease, with lymphadenopathy, often with splenomegaly, and frequent involvement of the bone marrow. A minority of patients present with extranodal disease, most often involving the gastrointestinal tract 1 and occasionally Waldeyer ring 2 , but rarely involving other sites, such as the skin [START_REF] Sen | Mantle cell lymphoma involving skin: cutaneous lesions may be the first manifestation of disease and tumors often have blastoid cytologic features[END_REF] or the ocular adnexa [START_REF] Ferry | Lymphoma of the ocular adnexa: a study of 353 cases[END_REF] . Nearly all patients with extranodal presentation of MCL will be found to have lymphadenopathy or more widespread disease on staging.

However, cases of primary extranodal mantle cell lymphoma have been described. The most frequent site is represented by the gastrointestinal tract [START_REF] Meral | Diffuse gastrointestinal involvement of mantle cell lymphoma[END_REF] , with the characteristic clinico-pathological presentation of a multiple lymphomatous polyposis. Other primary MCLs have been reported in the skin [START_REF] Sen | Mantle cell lymphoma involving skin: cutaneous lesions may be the first manifestation of disease and tumors often have blastoid cytologic features[END_REF][START_REF] Estrozi | Primary cutaneous blastoid mantle cell lymphoma-case report[END_REF] , the oral cavity [START_REF] Guggisberg | Mantle cell lymphoma of the oral cavity: case series and comprehensive review of the literature[END_REF] and the conjunctiva 8 , but they can be considered extremely rare.

Primary testicular lymphoma represents 1-2% of all NHLs 9 . This entity is chiefly a disease of the elderly, with 85% of the cases occurring in patients older than 60 among whom it is the most common testicular tumour [START_REF] Zucca | Primary extranodal non-Hodgkin's lymphomas. Part 1: Gastrointestinal, cutaneous and genitourinary lymphomas[END_REF] . Almost all cases are diffuse large B cell lymphoma (DLBCL). Few cases of primary testis follicular lymphoma (FL) have been reported, mainly in children [START_REF] Bacon | Primary follicular lymphoma of the testis and epididymis in adults[END_REF] , even though adults also rarely develop testicular FL [START_REF] Bacon | Primary follicular lymphoma of the testis and epididymis in adults[END_REF] . A case of MCL has been previously described in the testis [START_REF] Iwuanyanwu | Mantle cell lymphoma with a rare involvement of the testicle[END_REF] , but it represented a rare involvement of this organ in a patient with disseminated disease.

As far as we are aware, our case represents the first report in the English literature of primary MCL of the testis.

In cases of primary testis lymphoma which are not morphologically consistent with DLBCL, it is recommended to make a correct differential diagnosis among small to medium-sized B cell NHL.

The distinction is important for therapeutic and prognostic implications, since MCL has a significantly worse outcome than other lymphomas, in particular primary testis FL, whose spread beyond the primary site is very unusual, with a consequent very good prognosis [START_REF] Bacon | Primary follicular lymphoma of the testis and epididymis in adults[END_REF] .

Figure legend

Fig. 1

 1 Fig. 1 The testicular neoplastic lesion was composed of a diffuse proliferation of medium-sized lymphoid cells resembling lymphoblasts, with dispersed chromatin and scant cytoplasm. A semeniferous tubule is entrapped in the lymphomatous tissue, and shows only immature intraluminal germinal cells (A, hematoxylin-eosin, original magnification 200x; B, hematoxylineosin, original magnification 630x). Neoplastic cells show a diffuse cytoplasmic positivity for CD20 (C, original magnification 200x) and a strong nuclear positivity for cyclin D1 (D, original magnification 200x).


Konstantinos Mr

MD Liapis

M.D.; Konstantinos Liapis
email: kosliapis@hotmail.com

M.D.; Evangelia Charitaki

M.D. Susan Delimpasi

M.D. Arnold Ganser

Keywords:

Dear Editor,

We read with interest the article by Mehta et al. regarding stomatocytosis as a heralding sign of acute Wilsonian crisis [START_REF] Mehta | Stomatocytosis heralding a case of acute Wilsonian crisis[END_REF]. Stomatocyte formation is frequently observed in liver disease irrespective of the underlying etiology and has been attributed to an increase of lysolecithin in the inner layer of the red cell membrane. The degree of stomatocytosis is analogous to the severity of liver disease [START_REF] Bain | Blood cells: a practical guide[END_REF]. Stomatocytosis per se is not characteristic of Wilson's disease, but as the authors indicate, evidence of liver dysfunction in a young patient should raise suspicions for the disease. However, it is important to note that Wilson's disease can cause both acute hemolytic anemia with no or non-specific morphologic abnormalities and a distinctive hemolytic crisis with the characteristic features of oxidant-induced erythrocyte damage [START_REF] Bain | Blood cells: a practical guide[END_REF]. In our letter, we comment on an adolescent who manifested brisk oxidative hemolysis as the presenting symptom of Wilson's disease.

A 17-year-old girl, previously in good health, was admitted with a 5-day history of weakness and jaundice. The patient denied any exposure to drugs or chemicals and there was no family history of anemia or jaundice. On examination, she was alert, temperature was 37.5 0 C and the liver span enlarged at 15 cm. Spleen was not palpable and no neurological abnormalities were detected. The hemoglobin level was 6.7 g/dL, mean corpuscular volume 100 fL, the leucocyte count 22000/μL and platelets 186000/μL. Serum level of total bilirubin was 37 mg/dL (conjugated bilirubin 22 mg/dL), ALT 25 U/L, AST 133 U/L, LDH 550 U/L and alkaline phosphatase 10 U/L. Serum creatinine level was normal and the prothrombin time ratio was prolonged at 1.6. The reticulocyte count was 12% and serum haptoglobulin <7 mg/dL. The blood film showed polychromasia, basophilic stippling, many irregularly contracted cells and occasional blister cells (Fig. 1). A Heinz-body preparation with methyl violet at 1-hour incubation was positive, confirming thus the oxidative nature of hemolysis. Coomb's test and screening for hemoglobin instability and G-6-PD deficiency gave negative results. Wilson's disease was suspected because of the patient's liver dysfunction and oxidantinduced hemolysis in the absence of any drug intake; this was confirmed by the detection of Kayser-Fleischer rings on slit-lamp examination and the copper studies: ceruloplasmin 4.7 (normal 22-61) mg/dL, serum copper level 362 (normal 78-131) μg/dL. The patient was stabilized with transfusions and chelation therapy and she was referred for evaluation to a liver transplantation center.

Erythrocyte damage in Wilson's disease is believed to be caused directly by the oxidant effects of copper. Central to the pathophysiology is the impaired biliary excretion, due to the mutated transporter protein ATP7B, and accumulation of copper within the hepatocytes [START_REF] Pfeiffer | Wilson's Disease[END_REF]. Unbound inorganic copper spills out of the damaged liver into the bloodstream and interacts with the erythrocytes generating free oxygen radicals and hydrogen peroxides via the Fenton's reaction [START_REF] Pfeiffer | Wilson's Disease[END_REF][START_REF] Goldman | Wilson's disease presenting as Heinz-body hemolytic anemia[END_REF][START_REF] Lehr | Haemolytic anaemia as initial manifestation of Wilson's disease[END_REF]. Various lines of evidence suggest that high levels of free serum copper have a direct effect on either oxidation of hemoglobin, direct damage to the membrane phospholipids, or inhibition of glycolytic enzymes [START_REF] Forman | Hemolytic anemia in Wilson disease: clinical findings and biochemical mechanisms[END_REF]. The oxidative damage of the membrane accounts for the irregularly contracted cells and the precipitation of denaturated hemoglobin for the Heinz-bodies. The subsequent destruction of the red cells occurs both intravascularly and extravascularly.

As mentioned above, Wilson's disease can cause both acute hemolytic anemia with no specific morphologic abnormality and an acute Heinz-body hemolytic anemia. However, the mechanism of red cell destruction is the same in both instances and subsequent on the copper-mediated oxidation of erythrocytic components. Factors that may account for the morphologic differences include the speed and amplitude of copper flux, the rate of splenic clearance and the diverse effects of the metal in many parts of the erythrocyte. The shape changes commonly observed in hepatic disease, such as the stomatocytosis reported by Mehta et al., may contribute to the hemolysis by enhancing extravascular destruction.

The hemolytic crisis may rarely be the initial manifestation of Wilson's disease and antedate symptomatic liver or neurologic disease by months. The presence of oxidant-induced hemolysis in a young patient in the absence of G6PD deficiency, unstable hemoglobin or a history of drug or chemical exposure should be indicative of Wilson's disease.

Figure 1 .

 1 Figure 1. Peripheral blood film photomicrograph showing the presence of

Figure 1

 1 Figure 1 Click here to download high resolution image