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

Florentina Kosely

Jens Hillengass

Michael Hundemer

Stefan Schmitt

Kai Neben

Thomas Moehler

Ute Hegenbart

Anthony D Ho

Hartmut Goldschmidt

Jens Hillengaß

: U Hegenbart

Effective prophylaxis of thromboembolic complications with low molecular weight heparin in relapsed multiple myeloma patients

Keywords: IMiDs, Lenalidomide, Low molecular weight heparin, Multiple myeloma, Prophylaxis, Thrombosis

Introduction

Venous thromboembolism (VTE) is well known to be a common complication in cancer patients and represents a significant cause of morbidity and mortality [START_REF] Kakkar | Venous thrombosis in cancer patients: insights from the FRONT-LINE survey[END_REF]. Large studies of patients with hematological malignancies have demonstrated the increased risk of VTE in this population [START_REF] Baron | Venous thromboembolism and cancer[END_REF]. Of these patients, multiple myeloma (MM) patients have the highest risk of thromboembolic complications [START_REF] Blom | Malignancies, prothrombotic mutations, and the risk of venous thrombosis[END_REF]. The general incidence of VTE in newly diagnosed MM patients is higher than in relapsed/refractory patients [START_REF] Weber | Thalidomide alone or with dexamethasone for previously untreated multiple myeloma[END_REF][START_REF] Neben | Dose-dependent effect of thalidomide on overall survival in relapsed multiple myeloma[END_REF]. The risk of developing a VTE is highest during induction therapy while patients still have a high tumor burden [START_REF] Heit | Risk factors for deep vein thrombosis and pulmonary embolism: a population-based case-control study[END_REF][START_REF] Zangari | Increased risk of deep-vein thrombosis in patients with multiple myeloma receiving thalidomide and chemotherapy[END_REF]. Beside the myeloma-related risk factors in itself, the appearance of various other individual risk factors like age, obesity, central venous catheters, previous history of VTE, diabetes, chronic renal insufficiency, heart disease, and the kind of anti-myeloma treatment are known.

Thalidomide and lenalidomide have shown significant activity in the treatment of MM [START_REF] Neben | Dose-dependent effect of thalidomide on overall survival in relapsed multiple myeloma[END_REF][START_REF] Weber | Lenalidomide plus dexamethasone for relapsed multiple myeloma in North America[END_REF]. Nevertheless, the associated side effect of VTE is a severe complication in the treatment with these immunomodulatory drugs. Published data for lenalidomide/dexamethasone demonstrate high rates of VTE [START_REF] Knight | Lenalidomide and venous thrombosis in multiple myeloma[END_REF]. The risk significantly increases in analogy to the experience with thalidomide in case of combination with chemotherapy or erythropoietin [START_REF] Rajkumar | Lenalidomide and venous thrombosis in multiple myeloma[END_REF]. In two recently published phase III studies of lenalidomide/ dexamethasone combination therapy with no recommended anticoagulation, VTE occurred more often in the lenalidomide group compared to the placebo group with 14.7% vs. 3.4% (MM-009) and 11.4% vs. 4.6% (MM-010) [START_REF] Weber | Lenalidomide plus dexamethasone for relapsed multiple myeloma in North America[END_REF][START_REF] Dimopoulos | Lenalidomide plus dexamethasone for relapsed or refractory multiple myeloma[END_REF]. A randomized double-blind, placebo-controlled trial for previous untreated MM patients compared dexamethasone in combination with lenalidomide vs. high dose dexamethasone alone. A high initial rate of VTE was seen in newly diagnosed myeloma patients treated with lenalidomide/ dexamethasone, therefore acetysalicyl acid (ASA) 325 mg was mandated. Of 198 patients, 25 patients developed a VTE (12.6%) during initial therapy with lenalidomide/ dexamethasone or after crossover while seven patients (3.5%) had VTE in the high dose dexamethasone arm [START_REF] Zonder | Superiority of lenalidomide (Len) plus high-dose dexamethasone (HD) compared to HD alone as treatment of newly-diagnosed multiple myeloma (NDMM): results of the randomized, double-blinded, placebo-controlled SWOG Trial S0232[END_REF].

Analyzing published data for newly diagnosed patients, the risk is lower at the time of relapse with up to 15% than in newly diagnosed patients with a rate of more than 20% [START_REF] Palumbo | Enoxaparin or aspirin for the prevention of recurrent thromboembolism in newly diagnosed myeloma patients treated with melphalan and prednisone plus thalidomide or lenalidomide[END_REF][START_REF] Rajkumar | A randomized trial of lenalidomide plus high-dose dexamethasone (RD) versus lenalidomide plus low-dose dexamethasone (Rd) in newly diagnosed multiple myeloma (E4A03): a trial coordinated by the Eastern Cooperative Oncology Group[END_REF]. Nevertheless, the rate of 10-15% VTE complications is significantly high in relapsed MM patients. Reducing the incidence of VTE is critical to patient survival, as approximately one fourth of pulmonary embolism (PE) cases present as sudden death [START_REF] Heit | Predictors of survival after deep vein thrombosis and pulmonary embolism: a population-based, cohort study[END_REF].

The heterogeneity of published data does not allow a particular recommendation for VTE prophylaxis for patients receiving an immunomodulatory-based therapy. The optimal pharmacological VTE prophylaxis for patients treated with a combination regime with dexamethasone or chemotherapy and immunomodulatory drugs remains controversial. Recently presented preliminary data of a randomized phase III study of enoxaparin vs. aspirin vs. low-fixed dose of warfarin in newly diagnosed MM patients treated with thalidomide-based regimes revealed that ASA patients had a higher frequency of VTE than LMWH patients, but differences did not reach statistical significance [START_REF] Palumbo | randomized, phase III study of enoxaparin versus aspirin versus low-fixed-dose of warfarin in newly diagnosed myeloma patients treated with thalidomidecontaining regimens[END_REF]. For patients with VTE risk factors, ASA may not be an adequate prophylaxis as a retrospective analysis of the MD Anderson Cancer Centre has recently shown [START_REF] Wright | Characteristics of patients experiencing thromboembolic events during treatment for multiple myeloma: aspirin may not be adequate thromboprophylaxis in patients with multiple risk factors[END_REF].

Currently, a recommendation according to a risk assessment model based on various risk factors is discussed [START_REF] Palumbo | Prevention of thalidomide-and lenalidomide-associated thrombosis in myeloma[END_REF]. Especially in relapsed/refractory patients, limited data are available on the efficacy of thromboprophylaxis. We therefore performed a single center analysis regarding the use of LMWH for prevention of VTE in relapsed/refractory patients treated with lenalidomide/dexamethasone therapy.

Patients and methods

From July 2005 to January 2008, 45 patients with relapsed MM were treated with lenalidomide/dexamethasone in our department. The median age was 62 years (range 29-78 years), 21/45 patients (46.7%) were ≥65 years. Twentyeight patients were male and 17 patients were female. The patients were intensively pre-treated with a median of four previous treatment regimens (range 1-11). Thirty-seven patients (82.2%) underwent at least one autologous transplantation. Twenty-six patients (57.8%) had a thalidomidebased therapy before initiating lenalidomide medication; 29 patients (64.4%) were pre-treated with bortezomib. The median time from first diagnosis to lenalidomide/ dexamethasone therapy was 69 months (range 14-155 months). Twelve patients (26.7%) had a previous medical history of VTE; nine patients (20%) had a port catheter system during treatment with lenalidomide/dexamethasone. The distribution of other VTE risk factors like obesity, diabetes, renal insufficiency, and cardiac disease is scheduled in Table 1.

Treatment was administered with 25 mg lenalidomide days 1-21 and initially with 40 mg oral dexamethasone days 1-4, 9-12, and 17-20. After treatment of ten patients, the dosage of dexamethasone was regularly reduced to 20 mg days 1-4, 9-12, and 17-20 because of severe infectious complications in the preceding patients. The 45 patients received a total number of 192 cycles, respectively a median of three cycles per patient (range 1-17 cycles). The median dosage of dexamethasone was 240 mg per cycle. All of the 45 patients received prophylactic anticoagulation from the start of therapy with LMWH at least for the first three cycles. Also, 167 cycles (86.9%) were given with LMWH in which 152 cycles (79.2%) were administered with enoxaparin: 147 cycles with enoxaparin 0.4 ml daily subcutaneously, two cycles with enoxaparin 0.3 ml due to renal insufficiency, and three cycles with enoxaparin 1.0 ml because of previous severe and recurrent thrombosis of the vena cava superior. Ten cycles (5.2%) were administered with dalteparin. Of these ten cycles, nine were administered with a dosage of dalteparin 2,500 IE once daily and one cycle was given to a patient with the dosage of 5,000 IE once daily; three (1.6%) cycles were performed with nadoparin 7,600 IE once daily. Two cycles (1.0%) were given to one patient with a combination of ASA 100 mg and enoxaparin 0.2 ml. ASA had already been continuously administered due to coronary heart disease before initiating medication with lenalidomide. Twenty-five cycles (13.0%) were administered during medication with ASA 100 mg. All of these patients started with LMWH prophylaxis for at least the first three cycles but due to adverse reaction in one case and easier management of care in two other cases, prophylaxis was switched to ASA after four and five cycles, respectively. All patients were seen at least once every 2 weeks in our department and examined for clinical signs of thrombosis. At the time of analysis, 37 patients (82.2%) were alive, 24 patients (53.3%) were still on therapy with lenalidomide/dexamethasone while 21 patients (46.6%) had stopped due to progressive disease or severe side effects.

Results

One of 45 patients developed a VTE (2.2%). The 74-year-old Caucasian male had a previously diagnosed thrombosis. It had developed several years before starting the treatment with lenalidomide/dexamethasone. After the first cycle of lenalidomide/dexamethasone, he underwent surgery for a double-sided inguinal hernia. The patient was taking 100 mg ASA regularly because of coronary heart disease. Therewith beside the myeloma-related risk, he had four additional VTE risk factors: age ≥65 years, previous history of VTE, surgery, and a chronic heart disease. He was intensively pre-treated with six prior treatment regimens including autologous transplantation but no previous therapy with thalidomide had been performed. Before starting anti-myeloma treatment with lenalidomide/dexamethasone, prophylaxis was extended with additional continuous enoxaparin 20 mg subcutaneously. Anti-myeloma treatment was administered as previously mentioned. The dosage of dexamethasone was reduced in the second cycle to 20 mg days 1-4. At day 20 of the second cycle, the patient developed deep vein thrombosis and pulmonary embolism (PE). Treatment with lenalidomide was stopped and changed to bendamustine. In January 2008, the patient was still alive. None of the other 44 patients had clinical signs of thrombosis or embolism and none of the patients experienced bleeding complications.

Discussion

Multiple myeloma remains incurable with conventional treatment but the development of new agents like bortezomib, thalidomide, and lenalidomide have changed the management of the disease essentially. All of these drugs have shown significant activity in the treatment of MM [START_REF] Weber | Lenalidomide plus dexamethasone for relapsed multiple myeloma in North America[END_REF][START_REF] Richardson | Extended follow-up of a phase 3 trial in relapsed multiple myeloma: final time-to-event results of the APEX trial[END_REF]. Nevertheless, they have side effects that partly entail the end of the therapy. Venous thromboembolism is such a severe complication in the treatment with the immunomodulatory drugs thalidomide or lenalidomide (see Table 2).

With the frequent use of these drugs, VTE have attained a new clinical dimension, but the optimal pharmacological prophylaxis remains controversial [START_REF] Durie | Deep vein thrombosis in myeloma: estimate of prevalence and recommendations for therapy based upon a survey of members of the International Myeloma Working Group (IMWG)[END_REF]. For the prophylaxis of VTE in cancer patients in general, LMWH is recommended as standard of care [START_REF] Geerts | Prevention of venous thromboembolism: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy[END_REF]. In the absence of randomized data, ASA became a candidate for VTE prophylaxis for patients receiving lenalidomide/dexamethasone. Earlier published data may suggest that ASA is a potential drug for prevention of VTE with many advantages compared to LMWH such as oral administration, low cost, and high availability [START_REF] Baz | The role of aspirin in the prevention of thrombotic complications of thalidomide and anthracycline-based chemotherapy for multiple myeloma[END_REF].

In a retrospective analysis of different trials of newly diagnosed patients, Palumbo et al. showed a rate of VTE of 2.1% for patients who were treated with melphalan/ lenalidomide/prednisone and who received a thromboprophylaxis with ASA. But it needs to be considered that dexamethasone has a much higher potency to provoke VTE than prednisone. Beside that, the dosage of dexamethasone in itself is much higher with 240 to 480 mg/cycle compared to 100 mg prednisone [START_REF] Palumbo | Enoxaparin or aspirin for the prevention of recurrent thromboembolism in newly diagnosed myeloma patients treated with melphalan and prednisone plus thalidomide or lenalidomide[END_REF]. For newly diagnosed patients who receive a dexamethasone-containing regime, ASA may not be an adequate prophylaxis as recently published data have shown [START_REF] Zonder | Superiority of lenalidomide (Len) plus high-dose dexamethasone (HD) compared to HD alone as treatment of newly-diagnosed multiple myeloma (NDMM): results of the randomized, double-blinded, placebo-controlled SWOG Trial S0232[END_REF]. The American Society of Clinical Oncology determined that a routine prophylaxis for ambulatory patients with anticoagulation is not recommended, with the exception of patients receiving thalidomide or lenalidomide. Until data of randomized studies are available, LMWH or low dose warfarin is recommended in myeloma patients who are receiving anti-angiogenic therapy in combination with chemotherapy or dexamethasone [START_REF] Lyman | American Society of Clinical Oncology guideline: recommendations for venous thromboembolism prophylaxis and treatment in patients with cancer[END_REF].

Likewise, for patients with additional risk factors beside the myeloma-related risk in itself, ASA seems to be insufficient. Taking this into account, a strategy according a risk assessment model was initiated. The panel recommends aspirin for patients with ≤1 risk factor for VTE and LMWH for those with ≥2 individual/myeloma-related risk factors [START_REF] Palumbo | Prevention of thalidomide-and lenalidomide-associated thrombosis in myeloma[END_REF]. Nevertheless, data of larger-sized trials are lacking.

We retrospectively analyzed refractory/relapsed MM patients who were treated in our center with lenalidomide/ dexamethasone. With a median of 240 mg/cycle, we administered an intermediate dosage of dexamethasone. All patients received prophylactic anticoagulation with LMWH from the start of treatment. Only one case of DVT and PE occurred. The 74-year-old male received aspirin before starting the treatment with lenalidomide/ dexamethasone because of coronary heart disease. Therefore, the dosage of enoxaparin was reduced from the common dosage of 0.4 to 0.2 ml. In consideration of the experience with no observed bleeding complication, we currently determine an additional prophylaxis with enoxaparin 0.4 ml in such patients.

Being aware of the small number of patients and a refractory/relapsed setting, the rate of 2.2% VTE remains lower than the incidence of VTE in the dexamethasone control arms of the two randomized phase III trials with 4.6% and 3.4%, respectively. Taking into account that 42.2% of patients had beside the myeloma-related risk, one more VTE-specific risk factor and an additional proportion of 44.4% even ≥2 risk factors LMWH prophylaxis seems to be very effective even in patients of higher risk. Patients with no additional risk factors may be protected adequate with the administration of aspirin. Regarding no complications or severe side effects caused by the use of LMWH, we propose prophylaxis with LMWH in patients being treated with lenalidomide/dexamethasone at least during the first 3 months of therapy until randomized trials have proven the equality of other pharmacological prophylaxis. In the future, analysis of randomized trials may sustain the model for the individualized probability of VTE risk [START_REF] Palumbo | Prevention of thalidomide-and lenalidomide-associated thrombosis in myeloma[END_REF].

Table 1

 1 Patient characteristics

	Risk factor	Number (%)
	Previous VTE, port catheter system, age
	≥65 years, obesity (BMI ≥30), high dose
	dex 480 mg/month, diabetes, cardiac disease,
	renal insufficiency Crea.-Cl. ≤40 ml/min
	Previous VTE	12 (26.7)
	Port catheter system	9 (20)
	Age ≥65 years	21 (46.7)
	obesity (BMI ≥30)	5 (11.1)
	High dose dexamethasone 480 mg/month	10 (22.2)
	Diabetes mellitus	2 (4.4)
	Cardiac disease	5 (11.1)
	Renal insufficiency with Crea.-Cl. ≤40 ml/min	7 (15.6)
	No risk factor	6 (13.3)
	1 risk factor	19 (42.2)
	≥2 risk factors	20 (44.4)

Table 2

 2 VTE rates in patients with multiple myeloma treated with thalidomide or lenalidomide and dexamethasone

	Study	N	Therapy	Prophylaxis	VTE rate (%)
	Dimopoulos et al. [11]	351	Len/high dose Dex vs. Placebo/Dex	None	11.4 vs. 4.6
	Weber et. al [8]	353	Len/high dose Dex vs. Placebo/Dex	None	14.7 vs. 3.4
	Zonder et al. [12]	198	Len/high dose Dex vs. high dose Dex	ASA (not initially)	12.6 vs. 3.5
	Rajkumar et al. [14]	445	Len/high dose Dex vs. Len/Dex low dose	ASA	25 vs. 9
	Palumbo et al. [16]	200 (interim analysis)	Thal-based therapy	ASA vs. LMWH vs.	9
				low-fixed dose warfarin	3
					3
	Minnema et al. [24]	211	Thal/Adriamycin/Dex high dose (TAD)	LMWH	8
	Klein et al. (this study)	45	Len/intermediate Dex	LMWH	2.2

Acknowledgement We thank Frank Müller for carefully collecting data.

Conflict of Interest Disclosure

The authors declare no competing financial interests.


Thomas Elter
email: thomas.elter@uk-koeln.de

Janne J Vehreschild

John Gribben

Oliver A Cornely

Andreas Engert

Michael Hallek

: A Engert

Management of infections in patients with chronic lymphocytic leukemia treated with alemtuzumab

Keywords: Alemtuzumab, Campath, CLL, Infection, Management

Infection is a significant cause of morbidity and death in patients with chronic lymphocytic leukemia (CLL). Increased infectious events may arise from the multiple courses of immunosuppressive therapy and progressive deterioration of a patient's immune system over the course of disease. The humanized, anti-CD52 monoclonal antibody alemtuzumab (Campath or Campath-1H) has shown notable activity for both untreated and fludarabinerefractory CLL. The antibody not only targets malignant cells but also affects normal, healthy immune cells. The cumulative effects of the malignancy and successive courses of treatments adversely impinge on a patient's defense response to certain bacterial, fungal, and viral infections. In this review article, we provide an overview of common infectious events associated with alemtuzumab therapy in CLL. We also discuss recommendations for effectively monitoring and managing infections in CLL patients.

Background

As one of the most common types of leukemia, B-cell chronic lymphocytic leukemia (CLL) afflicts approximately <1 to 5.5 per 100,000 people and accounts for 22% to 30% of leukemia cases worldwide [START_REF] Jemal | Cancer statistics, 2007[END_REF][START_REF] Redaelli | The clinical and epidemiological burden of chronic lymphocytic leukaemia[END_REF]. Although the disease remains incurable without allogeneic transplantation at present, the development of a number of chemotherapeutic (alkylating agents, nucleoside analogues, and novel drugs) and targeted immunotherapeutic agents (monoclonal antibodies) has improved clinical outcomes in patients with CLL [START_REF] Nabhan | Purine nucleoside analogues and combination therapies in B-cell chronic lymphocytic leukemia: dawn of a new era[END_REF][START_REF] Robak | Monoclonal antibodies in the treatment of chronic lymphoid leukemias[END_REF][START_REF] Wierda | A retrospective comparison of three sequential groups of patients with recurrent/ refractory chronic lymphocytic leukemia treated with fludarabinebased regimens[END_REF].

Alemtuzumab is a humanized monoclonal antibody that binds to the CD52 antigen, which is present on an array of mature hematopoietic cells, including lymphocytes, monocytes, macrophages, and eosinophils, but not on progenitor cells [START_REF] Gilleece | Effect of Campath-1H antibody on human hematopoietic progenitors in vitro[END_REF]. Its mechanism of action includes complementdependent cytotoxicity and antibody-dependent cellmediated cytotoxicity, and probably apoptosis [START_REF] Nuckel | Alemtuzumab induces enhanced apoptosis in vitro in B-cells from patients with chronic lymphocytic leukemia by antibody-dependent cellular cytotoxicity[END_REF][START_REF] Ravandi | Alemtuzumab[END_REF][START_REF] Stanglmaier | Rituximab and alemtuzumab induce a nonclassic, caspase-independent apoptotic pathway in B-lymphoid cell lines and in chronic lymphocytic leukemia cells[END_REF][START_REF] Zent | Alemtuzumab (CAMPATH 1H) does not kill chronic lymphocytic leukemia cells in serum free medium[END_REF]. Single-agent alemtuzumab is highly effective as both first-line treatment for CLL and treatment for relapsed/ fludarabine-refractory CLL, and has been approved for both indications [START_REF] Hillmen | Alemtuzumab compared with chlorambucil as first-line therapy for chronic lymphocytic leukemia[END_REF][START_REF] Keating | Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study[END_REF]. Alemtuzumab is also being evaluated intensively in the settings of combination therapy [START_REF] Elter | Fludarabine in combination with alemtuzumab is effective and feasible in patients with relapsed or refractory B-cell chronic lymphocytic leukemia: results of a phase II trial[END_REF], consolidation therapy [START_REF] Fischer | Doseescalation study to evaluate dose limiting toxicity (DLT), maximum tolerated dose (MTD) and safety of alemtuzumab for consolidation therapy in patients with chronic lymphocytic leukemia: phase I/II trial of the German CLL Study Group (GCLLSG)[END_REF][START_REF] Lin | Consolidation therapy with subcutaneous (SC) alemtuzumab results in severe infectious toxicity in previously untreated CLL patients who achieve a complete response (CR) after fludarabine and rituximab (FR) induction therapy: interim safety analysis of the CALGB Study 10101 [abstract 755[END_REF][START_REF] Montillo | Alemtuzumab as consolidation after a response to fludarabine is effective in purging residual disease in patients with chronic lymphocytic leukemia[END_REF][START_REF] O'brien | Updated guidelines on the management of cytomegalovirus reactivation in patients with chronic lymphocytic leukemia treated with alemtuzumab[END_REF][START_REF] Wendtner | Consolidation with alemtuzumab in patients with chronic lymphocytic leukemia (CLL) in first remission-experience on safety and efficacy within a randomized multicenter phase III trial of the German CLL Study Group (GCLLSG)[END_REF], and as part of the conditioning regimen for patients undergoing autologous [START_REF] Montillo | Alemtuzumab as consolidation after a response to fludarabine is effective in purging residual disease in patients with chronic lymphocytic leukemia[END_REF] or allogeneic stem cell transplantation (SCT) [START_REF] Delgado | Results of alemtuzumab-based reducedintensity allogeneic transplantation for chronic lymphocytic leukemia: a British Society of Blood and Marrow Transplantation Study[END_REF].

Despite its remarkable efficacy, alemtuzumab therapy can frequently be accompanied by serious, sometimes even lifethreatening complications, including cytopenia and subsequent infections. Careful monitoring and management of infectious events will be required in order to fully harness the benefit of alemtuzumab therapy and improve the clinical outcome in CLL patients. The overview and management recommendations provided in this article aim to help physicians better understand and manage alemtuzumabassociated infections in routine clinical practice setting.

Patient predisposition to infections

Patients with CLL may have immune dysfunctions inherent to the disease process that predispose them to infections. Microarray analysis revealed differential gene expression patterns in CD4 + and CD8 + T cells isolated from CLL patients and age-matched healthy donors, suggesting that the leukemic cells may have direct effects on normal T cells. Specifically, changes were noted in genes involved in Th1-induced differentiation (CD4 + cells), as well as vesicle trafficking, cytoskeletal formation, and cytolytic enzymes (CD8 + cells) [START_REF] Gorgun | Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells[END_REF]. Co-culture of CLL and normal cells showed that cell-to-cell interaction and cross-talk could cause adverse effects on the gene expression of normal cells, alluding again to the potential negative impact CLL cells may have on surrounding healthy cells in vivo [START_REF] Gorgun | Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells[END_REF].

In patients with CLL, there is an expansion of a subset of CD8 + cytotoxic T cells with a CD45RA + CD27 -phenotype that is often found in immunocompromised or cytomegalovirus (CMV)-seropositive individuals [START_REF] Gamadia | Differentiation of cytomegalovirus-specific CD8(+) T cells in healthy and immunosuppressed virus carriers[END_REF][START_REF] Hooper | Cytomegalovirus seropositivity is associated with the expansion of CD4+[END_REF] and thought to be involved in regulating CMV infection [START_REF] Briggs | T cells and CD45R expression in B-chronic lymphocytic leukemia[END_REF][START_REF] Mackus | Expansion of CMV-specific CD8+CD45RA+[END_REF]. Thus, in patients with CLL who are undergoing lymphocyte-depleting treatment regimens, the loss of CD8 + cytotoxic T cells may provide a basis for the frequent episodes of CMV reactivation. There is also an increase in T regulatory cells in CLL, which may contribute to impaired T-cell function [START_REF] Beyer | Reduced frequencies and suppressive function of CD4+ CD25hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine[END_REF].

A number of factors have been identified that contribute to increased risk of infection in patients with CLL, including age, type and/or number of prior treatments, and immune cell status [START_REF] Hensel | Disease activity and pretreatment, rather than hypogammaglobulinaemia, are major risk factors for infectious complications in patients with chronic lymphocytic leukaemia[END_REF][START_REF] Itala | Infections and serum IgG levels in patients with chronic lymphocytic leukemia[END_REF][START_REF] Molteni | Multiple lines of chemotherapy are the main risk factor for severe infections in patients with chronic lymphocytic leukemia with febrile episodes[END_REF]. A retrospective analysis of 187 patients with CLL demonstrated that the number of chemotherapy (p<0.001), purine analogue (p<0.001), and monoclonal antibody treatments (p=0.019) significantly increased the risk for major infections [START_REF] Hensel | Disease activity and pretreatment, rather than hypogammaglobulinaemia, are major risk factors for infectious complications in patients with chronic lymphocytic leukaemia[END_REF]. Due to the chronic nature of CLL and subsequent disease relapses, most patients receive more than one treatment over the course of the malignancy. The cytotoxic effects of therapeutic regimens on normal immune cells result in greater patient susceptibility to infectious complications, and such risk increases as patients undergo each additional line of therapy. Patients may ultimately succumb to infections rather than malignancy. Patients with prior fludarabine treatment may be more susceptible to opportunistic infections due to the long-lasting effects of fludarabine on T cells, and up to 89% of these patients are at risk of developing severe infections [START_REF] Perkins | Frequency and type of serious infections in fludarabine-refractory B-cell chronic lymphocytic leukemia and small lymphocytic lymphoma: implications for clinical trials in this patient population[END_REF].

Patients receiving first-line therapy for CLL are generally at a much lower risk of developing infections compared with those who have received multiple courses of prior therapy. Nevertheless, previously untreated patients may also be susceptible to infections due to hypogammaglobulinemia and the underlying malignancy [START_REF] Tsiodras | Infection and immunity in chronic lymphocytic leukemia[END_REF]. Infections are less of a concern in younger patients, as shown in a recent study with frontline fludarabine-based treatment in which antibiotics or antiviral prophylaxes were not routinely used [START_REF] Eichhorst | Due to low infection rates no routine antiinfective prophylaxis is required in younger patients with chronic lymphocytic leukaemia during fludarabine-based first line therapy[END_REF].

Recent studies have reported on the potential association between high-risk prognostic markers (e.g., elevated thymidine kinase, CD38 + status, unmutated IgV H status, and presence of genomic abnormalities) and an increased risk of developing infectious complications in patients with CLL [START_REF] Eichhorst | Due to low infection rates no routine antiinfective prophylaxis is required in younger patients with chronic lymphocytic leukaemia during fludarabine-based first line therapy[END_REF][START_REF] Francis | The effect of immunoglobulin VH gene mutation status and other prognostic factors on the incidence of major infections in patients with chronic lymphocytic leukemia[END_REF]. The utility of these biomarkers in clinical practice remains to be established.

Effect of alemtuzumab on immune cells

A rapid decrease in normal lymphoid and malignant CLL cells is readily apparent within a few weeks of alemtuzumab treatment. In the pivotal study of alemtuzumab in patients with relapsed/refractory disease, CD19 + /CD5 + B cells and CD3 + /CD4 + and CD3 + /CD8 + T cells were significantly depressed after 4 weeks of treatment [START_REF] Keating | Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study[END_REF]; these findings were confirmed by another study that also noted a decrease in CD16 + natural killer (NK) cells [START_REF] Rai | Alemtuzumab in previously treated chronic lymphocytic leukemia patients who also had received fludarabine[END_REF]. The T-cell population gradually recovered to baseline levels by 36 weeks after therapy initiation [START_REF] Keating | Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study[END_REF]. Similar results on the recovery of CD4 + cells in alemtuzumab combination therapy were reported [START_REF] Elter | Fludarabine in combination with alemtuzumab is effective and feasible in patients with relapsed or refractory B-cell chronic lymphocytic leukemia: results of a phase II trial[END_REF]. Importantly, several studies have shown that infectious events typically occur around the time of T-cell and neutrophil nadirs (Fig. 1) [START_REF] Ferrajoli | Phase II study of alemtuzumab in chronic lymphoproliferative disorders[END_REF][START_REF] Keating | Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study[END_REF][START_REF] Moreton | Eradication of minimal residual disease in B-cell chronic lymphocytic leukemia after alemtuzumab therapy is associated with prolonged survival[END_REF][START_REF] Nguyen | Cytomegalovirus viremia during Campath-1H therapy for relapsed and refractory chronic lymphocytic leukemia and prolymphocytic leukemia[END_REF][START_REF] Rai | Alemtuzumab in previously treated chronic lymphocytic leukemia patients who also had received fludarabine[END_REF].

An array of immune cell populations was examined by flow cytometry at baseline and through 18 months following first-line alemtuzumab treatment [START_REF] Lundin | Cellular immune reconstitution after subcutaneous alemtuzumab (anti-CD52 monoclonal antibody, CAMPATH-1H) treatment as first-line therapy for B-cell chronic lymphocytic leukaemia[END_REF]. CD4 + and CD8 + T cells, CD3 -/56 + NK cells, CD3 + /56 + NK T cells, and CD19 + /5 -B cells were all profoundly decreased to <25% baseline through 9 months after treatment. Alemtuzumab also significantly depleted granulocytes and monocytes. The study showed that even lower cumulative doses of alemtuzumab could result in a sharp decrease in cell numbers for an extended period of time, although the cumulative dose level was not significantly associated with the extent or duration of lymphocyte depletion. There was, however, a trend toward lower levels of CD4 + and CD8 + T cells at the end of therapy in patients who received greater than median cumulative doses of alemtuzumab, although the actual bloodstream concentrations of alemtuzumab were not measured [START_REF] Lundin | Cellular immune reconstitution after subcutaneous alemtuzumab (anti-CD52 monoclonal antibody, CAMPATH-1H) treatment as first-line therapy for B-cell chronic lymphocytic leukaemia[END_REF]. Interestingly, no late-occurring opportunistic infections or major infections were reported during unmaintained, long-term follow-up; this may partially be explained by the patients' treatment-naive status prior to alemtuzumab therapy.

Alemtuzumab therapy is also known to cause transient neutropenia, anemia, and thrombocytopenia that resolve in most patients by a 2-month follow-up [START_REF] Keating | Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study[END_REF]. The effect of alemtuzumab on other blood cells such as macrophages has not been reported.

Infectious events associated with alemtuzumab therapy

Single-agent alemtuzumab therapy in first-line treatment of CLL In general, infections associated with alemtuzumab therapy are less frequent in previously untreated patients than in patients with relapsed/refractory diseases. The infectious events in two studies of first-line alemtuzumab therapy are summarized in Table 1 [START_REF] Hillmen | Alemtuzumab compared with chlorambucil as first-line therapy for chronic lymphocytic leukemia[END_REF][START_REF] Lundin | Phase II trial of subcutaneous anti-CD52 monoclonal antibody alemtuzumab (Campath-1H) as first-line treatment for patients with B-cell chronic lymphocytic leukemia (B-CLL)[END_REF].

The recent phase III randomized CAM307 study compared first-line alemtuzumab administered intravenously versus chlorambucil administered orally [START_REF] Hillmen | Alemtuzumab compared with chlorambucil as first-line therapy for chronic lymphocytic leukemia[END_REF]. Although grade 3/4 neutropenia was more common in the alemtuzumab arm, the incidence of grade 3/4 non-CMV infectious events was not significantly different between the two treatment arms (4.8% with alemtuzumab versus 2.7% with chlorambucil for febrile neutropenia; 3% and 1.4%, respectively, for bacteremia/sepsis). CMV events were more frequent in the alemtuzumab arm than in the chlorambucil arm. During treatment, 52.4% of patients receiving alemtuzumab had an asymptomatic positive CMV polymerase chain reaction (PCR), compared with 7.5% of patients receiving chlorambucil. No patient receiving chlorambucil had symptomatic CMV infection, whereas 15.6% of patients receiving alemtuzumab had symptomatic CMV infection without end organ involvement [START_REF] Hillmen | Alemtuzumab compared with chlorambucil as first-line therapy for chronic lymphocytic leukemia[END_REF].

Single-agent alemtuzumab therapy in relapsed/refractory CLL Infectious events in four published studies are summarized in Table 1. In an earlier study by Osterborg et al., patients who developed Pneumocystis carinii pneumonia (PCP) or septicemia tended to be more heavily pretreated or resistant to fludarabine treatment [START_REF] Osterborg | Phase II multicenter study of human CD52 antibody in previously treated chronic lymphocytic leukemia. European Study Group of CAMPATH-1H Treatment in Chronic Lymphocytic Leukemia[END_REF]. In another study in patients with relapsed or fludarabine-refractory CLL, infections were reported in 42% of patients, predominantly in the non-responder group. Seven of eight non-responders developed major infections during treatment and within 35 days following treatment completion, and two of them died from infections [START_REF] Rai | Alemtuzumab in previously treated chronic lymphocytic leukemia patients who also had received fludarabine[END_REF]. Similarly, in the pivotal study of 93 relapsed/refractory CLL patients, 25 patients (27%) experienced grade 3/4 infectious events (27%), which occurred more frequently in non-responders than in responders (36% versus 9.7%) [START_REF] Keating | Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study[END_REF]. In a recent study, 91 patients with relapsed/refractory CLL were treated with alemtuzumab until maximum response, which included achievement of minimal residual disease (MRD)-negative remission [START_REF] Moreton | Eradication of minimal residual disease in B-cell chronic lymphocytic leukemia after alemtuzumab therapy is associated with prolonged survival[END_REF]. There were four infectious episodes in 18 MRD-negative patients, compared with 27 episodes in 73 patients who did not achieve MRD negativity. Collectively, these studies suggest a trend that infectious complications are more frequent in patients who are heavily pretreated or fludarabine-refractory and who do not respond or only partially respond to alemtuzumab therapy.

Alemtuzumab in combination therapy

In light of the promising single-agent activity of purine analogues and monoclonal antibodies in CLL, various chemoimmunotherapy combination regimens are currently under investigation in an effort to further improve patient outcomes. In a recent study, 36 patients with relapsed/ refractory CLL were treated with fludarabine in combination with alemtuzumab (FluCam regimen: alemtuzumab 30 mg and fludarabine 30 mg/m 2 on three consecutive days administered every 28 days for up to 6 cycles) [START_REF] Elter | Fludarabine in combination with alemtuzumab is effective and feasible in patients with relapsed or refractory B-cell chronic lymphocytic leukemia: results of a phase II trial[END_REF]. Most infections occurred within the first 3 months of treatment, when CD4 + cells were at the lowest levels (Table 2). It is noteworthy that only two patients developed grade 3 subclinical CMV reactivation, which resolved with ganciclovir administered intravenously, although 80% of patients were CMV seropositive at baseline, suggesting that the rate of CMV reactivation might be lower with this 4-weekly combination regimen. Overall, patients who developed severe infectious complications were those who experienced progressive disease after FluCam therapy. The combination of alemtuzumab and steroid methylprednisolone was also investigated in patients with relapsed/refractory CLL [START_REF] Pettitt | Alemtuzumab in combination with high-dose methylprednisolone is a logical, feasible and highly active therapeutic regimen in chronic lymphocytic leukaemia patients with p53 defects[END_REF]. This regimen consists of methylprednisolone 1.0 g/m 2 administered intravenously on days 1-5 and alemtuzumab 30 mg thrice weekly in a 28day cycle for up to 4 cycles. Five patients with p53 defects were treated with this regimen. All five patients experienced infections that included CMV reactivation, suspected pulmonary aspergillosis, bacterial chest infection, and bacterial sinusitis, but all infections were successfully treated. The high infection rate was not unexpected, because four of the five patients were already at high risk of infection prior to therapy.

Alemtuzumab in consolidation therapy

Several studies have evaluated alemtuzumab as consolidation therapy after initial response to induction chemotherapy (Table 2). The rationale is that reduction in residual disease by alemtuzumab may improve patient response and survival. Indeed, an impressive improvement in progressionfree survival was demonstrated with alemtuzumab consolidation [START_REF] Schweighofer | Consolidation with alemtuzumab improves progression-free survival in patients with chronic lymphocytic leukemia (CLL) in first remission: long-term follow-up of a randomized phase III trial of the German CLL study group (GCLLSG)[END_REF][START_REF] Wendtner | Consolidation with alemtuzumab in patients with chronic lymphocytic leukemia (CLL) in first remission-experience on safety and efficacy within a randomized multicenter phase III trial of the German CLL Study Group (GCLLSG)[END_REF]. However, elevated infectious complications have also been noted in some studies [START_REF] Lin | Consolidation therapy with subcutaneous (SC) alemtuzumab results in severe infectious toxicity in previously untreated CLL patients who achieve a complete response (CR) after fludarabine and rituximab (FR) induction therapy: interim safety analysis of the CALGB Study 10101 [abstract 755[END_REF][START_REF] Wendtner | Consolidation with alemtuzumab in patients with chronic lymphocytic leukemia (CLL) in first remission-experience on safety and efficacy within a randomized multicenter phase III trial of the German CLL Study Group (GCLLSG)[END_REF]. For example, the earlier CLL4B trial of the German CLL Study Group had to be stopped due to severe infection in seven of 11 patients [START_REF] Wendtner | Consolidation with alemtuzumab in patients with chronic lymphocytic leukemia (CLL) in first remission-experience on safety and efficacy within a randomized multicenter phase III trial of the German CLL Study Group (GCLLSG)[END_REF]. In particular, the CALGB 10101 study reported six infection-related deaths in patients receiving alemtuzumab consolidation after fludarabine/rituximab induction therapy [START_REF] Lin | Consolidation therapy with subcutaneous (SC) alemtuzumab results in severe infectious toxicity in previously untreated CLL patients who achieve a complete response (CR) after fludarabine and rituximab (FR) induction therapy: interim safety analysis of the CALGB Study 10101 [abstract 755[END_REF]. It is not immediately clear why infection-related mortality is higher in this trial than in other consolidation trials [START_REF] Montillo | Alemtuzumab as consolidation after a response to fludarabine is effective in purging residual disease in patients with chronic lymphocytic leukemia[END_REF][START_REF] O'brien | Updated guidelines on the management of cytomegalovirus reactivation in patients with chronic lymphocytic leukemia treated with alemtuzumab[END_REF][START_REF] Wendtner | Consolidation with alemtuzumab in patients with chronic lymphocytic leukemia (CLL) in first remission-experience on safety and efficacy within a randomized multicenter phase III trial of the German CLL Study Group (GCLLSG)[END_REF]]. An open issue concerning alemtuzumab consolidation therapy is whether the alemtuzumab dosage should be adjusted according to the level of residual disease after induction therapy. If a patient is already in MRD-negative remission, it is probably not desirable to administer alemtuzumab at the full dose of 30 mg three times a week. We speculate that the treatment regimen in these trials may have been too intense and/or administered too early following induction chemotherapy, therefore resulting in excessive immunosuppression and increased infection.

To establish a dosing schedule that would maintain clinical efficacy of consolidation therapy while minimizing infectious complications, the German CLL Study Group recently conducted a phase I/II dose-escalation trial (CLL2I) to determine the maximum tolerated dose (MTD) of alemtuzumab consolidation in patients after second-line chemotherapy. The MTD of alemtuzumab consolidation administered intravenously was determined to be 10 mg once weekly, which was well tolerated, with no severe toxicity, no severe infections, and no clinically symptomatic CMV reactivation. Four patients had converted from PR to CR after consolidation and nine of 12 patients had a reduction in their MRD levels, suggesting that lower-dose alemtuzumab consolidation therapy is both safe and effective [START_REF] Fischer | Doseescalation study to evaluate dose limiting toxicity (DLT), maximum tolerated dose (MTD) and safety of alemtuzumab for consolidation therapy in patients with chronic lymphocytic leukemia: phase I/II trial of the German CLL Study Group (GCLLSG)[END_REF].

Alemtuzumab in SCT

Patients undergoing SCT are at a high risk of developing infections throughout different procedural stagespretransplantation conditioning through posttransplantation follow-up [START_REF] Kruger | Antimicrobial prophylaxis in allogeneic bone marrow transplantation. Guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Haematology and Oncology[END_REF]. This susceptibility may be influenced by a patient's underlying disease, conditioning therapy, presence of graft-versus-host disease (GvHD), or conformity between the donor and recipient's human leukocyte antigen. During the conditioning phase, patients are likely to develop treatment-related neutropenia, which adds to the high risk of developing infections within the first 30 days of treatment, including herpes simplex virus (HSV), CMV, Gram-negative bacteria, Staphylococcus spp., Enterococcus spp., Candida spp., and Aspergillus spp. During the postengraftment period, patients are susceptible to CMV reactivation, P. jiroveci, and Toxoplasma gondii at 30 to 100 days and varicella zoster virus (VZV) and encapsulated bacteria at >100 days. Respiratory and enteritic viruses may be present throughout all phases.

The Campath series of antibodies was initially developed to deplete T cells, and early studies demonstrated its ability to provide durable engraftment and to reduce GvHD [START_REF] Hale | Removal of T cells from bone marrow for transplantation: a monoclonal antilymphocyte antibody that fixes human complement[END_REF][START_REF] Hale | T cell depletion with CAMPATH-1 in allogeneic bone marrow transplantation[END_REF][START_REF] Waldmann | Elimination of graft-versus-host disease by in-vitro depletion of alloreactive lymphocytes with a monoclonal rat anti-human lymphocyte antibody (CAMPATH-1)[END_REF]. Recent studies confirmed that the incorporation of alemtuzumab into non-myeloablative conditioning regimens reduced the occurrence of GvHD and associated mortality in patients undergoing allogeneic SCT [START_REF] Chakrabarti | T-cell depletion with Campath-1H "in the bag" for matched related allogeneic peripheral blood stem cell transplantation is associated with reduced graft-versus-host disease, rapid immune constitution and improved survival[END_REF][START_REF] Chakraverty | Limiting transplantation-related mortality following unrelated donor stem cell transplantation by using a nonmyeloablative conditioning regimen[END_REF]. Alemtuzumab-based conditioning (with fludarabine and melphalan) prior to allogeneic transplantation in 41 young patients (<70 years of age) with advanced CLL showed reduced acute and chronic GvHD, with a 10% GvHDrelated mortality rate [START_REF] Delgado | Results of alemtuzumab-based reducedintensity allogeneic transplantation for chronic lymphocytic leukemia: a British Society of Blood and Marrow Transplantation Study[END_REF]. Patients received antifungal (fluconazole or itraconazole), acyclovir, and trimethoprim and sulfamethoxazole (TMS) (or alternatively, dapsone or subcutaneously has yet to be determined pentamidine) prophylaxis. Treatment-or GvHD-related mortality from infectious complications was caused by pulmonary aspergillosis (two patients), bacterial pneumonia (two patients), neutropenic sepsis (two patients), respiratory syncytial virus (RSV) pneumonia (one patient), EBVrelated posttransplantation lymphoproliferative disease (one patient), and fungal cerebral abscess (one patient). Non-lethal infections were mainly viral and fungal, with 68% of patients at risk for CMV reactivation, two of whom were treated for CMV. Alemtuzumab therapy may not be beneficial in combination with total body irradiation in the autologous SCT setting. In the CLL3C trial, patients received high-dose alemtuzumab and total body irradiation prior to autologous SCT [START_REF] Zenz | Autologous graft-versus-host disease-like syndrome after an alemtuzumab-containing conditioning regimen and autologous stem cell transplantation for chronic lymphocytic leukemia[END_REF]. Compared with the CLL3 trial, in which a similar treatment regimen was given without alemtuzumab, the CLL3C trial reported a high incidence of GvHD-like syndrome, high non-relapse-related mortality and a wide array of infectious complications (including CMV, herpes zoster, VZV, and pneumonia), despite improved disease control at the molecular level.

Management of infectious events associated with alemtuzumab therapy

In this section, we discuss some well-established practical recommendations that are based on previous clinical experiences.

Timeline of alemtuzumab-associated infections

A qualitative timeline (Fig. 1) [START_REF] Keating | Management guidelines for use of alemtuzumab in B-cell chronic lymphocytic leukemia[END_REF][START_REF] Keating | Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study[END_REF] shows that bacterial infections appear mainly during the first weeks of alemtuzumab treatment and become less frequent during subsequent weeks of treatment; viral infections, particularly CMV reactivation, were evident beginning in week 3 and generally tapered off over time; and fungal infections were mainly evident during the posttreatment phase of alemtuzumab. Unknown (or unclassified) infectious episodes were seen mainly during and just after treatment and may include fever of unknown origin (FUO).

Monitoring, prophylaxis, and treatment of infection

Before initiating alemtuzumab treatment, baseline status should be evaluated through examination of overall physical characteristics; computed tomography (CT) or ultrasound scan of enlarged and potentially palpable lymph nodes, liver, or spleen (where indicated); assessment of comorbid disease; serological status of hepatitis B, hepatitis C, CMV, HSV, VZV, EBV, and HIV; and complete blood counts [START_REF] Oscier | Guidelines on the diagnosis and management of chronic lymphocytic leukaemia[END_REF]. The strategy of infection monitoring and prophylaxis must always consider these baseline findings and other risk factors from the patient's medical history, such as age, comorbid diseases, and earlier infections.

The following prophylactic practices were recommended for patients receiving alemtuzumab: TMS (160 mg trimethoprim, 800 mg sulfamethoxazole) BID (or alternatively, aerosolized pentamidine, dapsone administered orally, and atovaquone given orally) for prevention of PCP; and acyclovir, famciclovir, or valacyclovir as antiviral prophylaxis for at least 2 months after completion of therapy (or more conservatively, 4 months after therapy or until CD4 + counts recover to at least 250/μL; Table 3) [START_REF] Hughes | Guidelines for the use of antimicrobial agents in neutropenic patients with cancer[END_REF][START_REF] Keating | Management guidelines for use of alemtuzumab in B-cell chronic lymphocytic leukemia[END_REF]. Prophylaxis with valganciclovir has been shown to be effective in preventing the onset of symptomatic CMV reactivation in a randomized trial [START_REF] O'brien | Alemtuzumab as treatment for residual disease after chemotherapy in patients with chronic lymphocytic leukemia[END_REF] and should be considered at the discretion of attending physicians.

Broad-spectrum antibacterial prophylaxis should be avoided because it offers no improvement in mortality, has the potential to select for resistant bacterial strains, and frequently causes adverse events such as hypersensitivity or Clostridium difficile-associated diarrhea [START_REF] Akova | A European organization for research and treatment of cancer-International Antimicrobial Therapy Group Study of secondary infections in febrile, neutropenic patients with cancer[END_REF]. Prophylactic TMS may provide some antibacterial activity, although it primarily targets PCP.

Routine antifungal prophylaxis is also not recommended. The results of evidence-based trials suggest the use of fluconazole 400 mg/day administered orally as antifungal prophylaxis only in allogeneic SCT, but not in conventional chemo-or immunotherapy. Posaconazole was proven effective in preventing fungal infections and death in long-term neutropenic patients and in patients with GvHD after allogeneic SCT [START_REF] Cornely | Posaconazole vs. fluconazole or itraconazole prophylaxis in patients with neutropenia[END_REF][START_REF] Ullmann | Posaconazole or fluconazole for prophylaxis in severe graft-versus-host disease[END_REF]; however, the dosage needed to treat patients receiving alemtuzumab remains unknown and the substance has not yet been tested in this setting. Similarly, little evidence is available to support the prophylactic use of itraconazole or liposomal amphotericin [START_REF] Cornely | Prophylaxis of invasive fungal infections in patients with hematological malignancies and solid tumors-guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Oncology (DGHO)[END_REF]. It is important to note that agents such as fluconazole will not protect against filamentous fungi (i.e., Aspergillus spp.). In patients experiencing prolonged FUO (>72 h) in whom CMV reactivation has been ruled out as a cause of fever, pulmonary high-resolution or thoracic CT should be performed [START_REF] Caillot | Increasing volume and changing characteristics of invasive pulmonary aspergillosis on sequential thoracic computed tomography scans in patients with neutropenia[END_REF][START_REF] Heussel | Pneumonia in febrile neutropenic patients and in bone marrow and blood stem-cell transplant recipients: use of high-resolution computed tomography[END_REF]. Early treatment with caspofungin (70 mg loading dose followed by 50 mg QD) or liposomal amphotericin B (3 mg/kg QD) may be clinically warranted [START_REF] Walsh | Caspofungin versus liposomal amphotericin B for empirical antifungal therapy in patients with persistent fever and neutropenia[END_REF]. In the event of probable (measured by two positive serum galactomannan samples or halo signs in pulmonary CT scan per modified European Organization for Research and Treatment of Cancer [EORTC]/Mycoses Study Group [MSG] criteria) or proven invasive aspergillosis, the treatment of choice is voriconazole 6 mg/kg loading dose followed by 4 mg/kg BID, as recommended by the Infectious Disease Society of America (IDSA) [START_REF] Herbrecht | Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis[END_REF].

Antiviral prophylaxis is generally not recommended in the SCT setting if the donor and recipient are seronegative for viral titers. Prophylaxis for HSV/VZV is not recommended unless the patient is seropositive and has additional risk factors including low CD4 count (<50/μL), previous corticosteroid or fludarabine treatment, a background of recurring herpes infections, or total body irradiation during conditioning [START_REF] Sandherr | Antiviral prophylaxis in patients with haematological malignancies and solid tumours: Guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society for Hematology and Oncology (DGHO)[END_REF]. Acyclovir 400 mg QD or three times per day (TID) is recommended if the donor or recipient is HSV seropositive. Acyclovir or valacyclovir both have similar prophylactic efficacy for patients with hematological malignancies [START_REF] Warkentin | Valacyclovir versus acyclovir for HSV prophylaxisin neutropenic patients[END_REF]. For patients who are seropositive for hepatitis B virus, lamivudine 100 mg/day is recommended as prophylaxis, although it has the potential to enhance the risk of developing hepatitis B-resistant viral strains [START_REF] Sandherr | Antiviral prophylaxis in patients with haematological malignancies and solid tumours: Guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society for Hematology and Oncology (DGHO)[END_REF].

Treatment of infections

In instances of fever, the patient needs to be examined for bacterial, fungal, or viral infections using monitoring techniques outlined in Table 4. Early blood cultures and start of empirical antibiotic treatment are especially imperative in severely immunosuppressed patients. Efforts to identify the source of infection should always be undertaken.

The timing of serious infections may be anticipated using the timeline discussed above (Fig. 1). Upon examining the types of bacteria present in alemtuzumab-treated patients, it was found that 60% to 70% of bacterial infections are caused by Gram-positive bacteria [START_REF] Perkins | Frequency and type of serious infections in fludarabine-refractory B-cell chronic lymphocytic leukemia and small lymphocytic lymphoma: implications for clinical trials in this patient population[END_REF]. In immunocompromised patients, empiric antibiotic treatment should be started immediately after the onset of infection to treat Gram-positive and Gram-negative bacteria (including Pseudomonas spp.). Empirical treatment should consider frequently isolated pathogens of the hospital environment and their resistances. In patients experiencing prolonged FUO (>48 h), pulmonary high-resolution or spiral CT should be performed [START_REF] Caillot | Increasing volume and changing characteristics of invasive pulmonary aspergillosis on sequential thoracic computed tomography scans in patients with neutropenia[END_REF][START_REF] Heussel | Pneumonia in febrile neutropenic patients and in bone marrow and blood stem-cell transplant recipients: use of high-resolution computed tomography[END_REF]. Early treatment with caspofungin (70 mg loading dose followed by 50 mg QD) or liposomal amphotericin B (3 mg/kg QD) may be clinically warranted in high-risk patients [START_REF] Walsh | Caspofungin versus liposomal amphotericin B for empirical antifungal therapy in patients with persistent fever and neutropenia[END_REF]. In the event of probable (measured by two positive serum galactomannan samples or halo signs in pulmonary CT scan per modified Data from [START_REF] Cornely | Prophylaxis of invasive fungal infections in patients with hematological malignancies and solid tumors-guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Oncology (DGHO)[END_REF][START_REF] Keating | Management guidelines for use of alemtuzumab in B-cell chronic lymphocytic leukemia[END_REF][START_REF] Sandherr | Antiviral prophylaxis in patients with haematological malignancies and solid tumours: Guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society for Hematology and Oncology (DGHO)[END_REF] BID Twice daily, DS double strength, PO oral, QD once daily, TID three times daily a Prophylaxis begins at onset of alemtuzumab therapy through ≥6 months posttherapy or >0.25×10 9 CD4 + cells/L. b Dose and schedule determined according to local standards (i.e., double-strength BID); alternative prophylaxis is dapsone 100 mg 5 days/week PO, nebulized pentamidine 300 mg/month, or atovaquone 750 mg BID PO. c Initiated following an increase of viremia as determined by viral antigen or polymerase chain reaction. d Prophylaxis begins 1 week prior to 2 months posttherapy. e Only if donor or recipient is seropositive. f Only if recipient is HBs-Ag-or anti-HBc-positive.

EORTC/MSG criteria) or proven invasive aspergillosis, the treatment of choice is voriconazole 6 mg/kg loading dose followed by 4 mg/kg BID, as recommended by IDSA [START_REF] Herbrecht | Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis[END_REF]. Another well-established treatment choice is liposomal amphotericin B 3 mg/kg QD [START_REF] Cornely | Liposomal amphotericin B as initial therapy for invasive mold infection: a randomized trial comparing a high-loading dose regimen with standard dosing (AmBiLoad trial)[END_REF].

Management of CMV reactivation and disease

CMV reactivation is the most common opportunistic infection observed in alemtuzumab-treated patients, although CMV-related organ disease or death is rare. Early detection of symptomatic reactivation, along with increased awareness of monitoring techniques and the use of preemptive therapy, has largely improved the management of CMV reactivation. This is evident in the recent report by Montillo and colleagues, in which active CMV infection was prevented with preemptive ganciclovir therapy (or by spontaneous resolution) in patients who developed CMV reactivation [START_REF] Montillo | Alemtuzumab as consolidation after a response to fludarabine is effective in purging residual disease in patients with chronic lymphocytic leukemia[END_REF].

CMV reactivation typically occurs between weeks 3 and 8 of alemtuzumab treatment, around the time of or shortly after T-cell nadir [START_REF] Keating | Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study[END_REF][START_REF] Moreton | Eradication of minimal residual disease in B-cell chronic lymphocytic leukemia after alemtuzumab therapy is associated with prolonged survival[END_REF][START_REF] Nguyen | Cytomegalovirus viremia during Campath-1H therapy for relapsed and refractory chronic lymphocytic leukemia and prolymphocytic leukemia[END_REF]. Routine CMV-specific prophylaxis is not recommended due to lack of data from large randomized studies. Patients should be closely monitored for CMV reactivation using antigenemia testing (pp65 and pp67) and/or PCR throughout the treatment and follow-up periods, particularly in those who are CMV seropositive at baseline (Table 5) [START_REF] Keating | Management guidelines for use of alemtuzumab in B-cell chronic lymphocytic leukemia[END_REF]. Intervals between CMV testing may be increased after the second month of therapy, but patients presenting with FUO should be tested promptly, because FUO (≥38°C or 101°F) may signal CMV reactivation (even in a patient who is CMV negative at baseline). A recently updated guideline on the management of CMV recommends that alemtuzumab therapy should be held only when patients have persistent, symptomatic CMV reactivation [START_REF] O'brien | Valganciclovir prevents CMV reactivation in patients receiving alemtuzumab based therapy[END_REF]. If a patient is CMV positive for two consecutive tests but remains asymptomatic, ganciclovir to be administered orally is recommended and alemtuzumab treatment should be continued. In symptomatic cases, ganciclovir (5 mg/kg BID) should be administered as first-line treatment for 14 to 21 days and alemtuzumab should be paused until negative CMV status is achieved. Foscarnet (60 mg/kg TID) may be added in the event of clinical CMV disease or substituted for ganciclovir if patients remain unresponsive or minimally responsive to ganciclovir treatment [START_REF] Nguyen | Cytomegalovirus viremia during Campath-1H therapy for relapsed and refractory chronic lymphocytic leukemia and prolymphocytic leukemia[END_REF][START_REF] O'brien | Valganciclovir prevents CMV reactivation in patients receiving alemtuzumab based therapy[END_REF].

Conclusions

The broad umbrella of infections present in patients with CLL reaffirms the need to closely monitor and manage infectious events beginning at treatment initiation. Various factors contribute to the development of infectious events in patients with CLL, including increased susceptibility due to immune defects inherent to the disease process itself, as well as the immunosuppressive effects of therapeutic regimens. Because of its promising efficacy, both as a single agent and in combination therapy, alemtuzumab is emerging as an important component of therapeutic regimens for CLL. A major complication of alemtuzumab therapy is infection that is predictable and manageable but also potentially severe and life-threatening if left undetected or untreated. As this agent is being used more and more widely, it is critical that infectious complications be managed with vigilance to minimize infection-related mortality and maximize therapeutic benefit.

Table 1

 1 Infectious events in single-agent alemtuzumab therapy

	Fig. 1 Qualitative timeline of
	infections in conjunction with immune cell nadir in patients	Bacterial Bacterial
	with relapsed/refractory CLL receiving alemtuzumab	Fungal Fungal
		Viral Viral
		Unknown Unknown
		Week 1 Week 1	Week 2 Week 2	Week 3 Week 3	Week 4 Week 4
			Month 1 Month 1	Month 2 Month 2	Month 3 Months 4 Month 3 Months 4
		Alemtuzumab Alemtuzumab	T-cell nadir T-cell nadir	Neutrophil nadir Neutrophil nadir	Lowest cell counts: ALC Lowest cell counts: ALC
					T lymphocytes T lymphocytes
					CD3 + /CD4 + CD3 + /CD4 +
					CD3 + /CD8 + CD3 + /CD8 +
					Malignant cells Malignant cells
					CD19 + /CD5 + CD19 + /CD5 +

Table 2

 2 Infectious events in alemtuzumab combination and consolidation therapy

	Additional notes		Grade 3/4 infection was reported	in 4 patients (11%), and 2 of the	3 patients with grade 4	infections were refractory to	prior therapy and to FluCam				One case each of pneumonia of	unknown origin and sinusitis.	One patient with CMV	reactivation also had	concomitant EBV and large cell	lymphoma and died			Trial had to be stopped due to	sever infection in 7 of 11	patients. No death was reported									1 hemorrhagic cystitis									2 DLT at 20 mg IV: 1 FUO, 1	exacerbated erythema	exsudativum multiforme. MTD	of alemtuzumab administered	intravenously was determined	as 10 mg once weekly. MTD of	alemtuzumab administered
	Viral infection (other	than CMV)	Not reported								Viral myocarditis (1),	influenza A (1)							HSV/HHV-6 (1),	herpes zoster (1)				Not reported						1 viral meningitis	(fatal) in patients	with CR, 1 EBV	lymphoproliferative	disorder (fatal)					No severe infection	observed
	CMV infection Fungal Bacterial	infection infection	2 grade 3 infections Aspergillus Escherichia	pneumonia coli sepsis	(2, both in (after CMV	patients infection,	with PD) fatal),	Pseudomonas	aeruginosa	(1)	9 symptomatic CMV Not reported Septicemia (1),	reactivation (1 fatal Listeria (1)	due to CMV	hepatitis). None of 4	patients with	ganciclovir	prophylaxis develop	CMV reactivation	4 cases of grade 3 Grade 4 Reactivation of	infection pulmonary pulmonary	aspergillosis tuberculosis (1)	(1)		CMV reactivation in No No documented	18 patients documented bacterial	fungal infection	infection			3 cases in 34 patients 4 cases in Cryptococcus	with PR, 3 cases in patients (1), Listeria	17 patients with CR with PR, 1 meningitis (1,	(1 fatal) PCP in fatal) and	patients Legionella	with CR pneumonia (1,	(fatal) fatal) in	patients with	CR	2 subclinical CMV No severe No severe	reactivation infection infection	observed observed
	No. of patients Neutropenia		36 Grade 1-2 in	25% patients,	grade 3-4 in	26% of	patients				41 Grade 3-4 in	71% at	30 mg level	versus 17%	at 10 mg	level			11 Grade 3 in 4	patients,	Grade 4 in 3	patients		34 No	hematologic	toxicity	observed			51 Not reported									6 at 10 mg IV, Not reported	4 at 20 mg	IV, 2 at	10 mg SC
	Reference Alemtuzumab therapy Anti-infective	prophylaxis	Elter et 30 mg IV in TMS,	al. [12] combination with valacyclovir	fludarabine 30 mg/	m 2 /day on the first	3 days of a 28 day	cycle, in patients	with relapse/	refractory CLL	O'Brien 10 or 30 mg IV 3 TMS,	et al. times a week for valacyclovir	[42] 4 weeks, in patients	with relapse/	refractory CLL				Wendtner 30 mg IV 3 times a Cotrimoxazole,	et al. week after a median famciclovir	[61] of 67 days following	first-line F or FC	therapy	Montillo 10 mg SC 3 times a Cotrimoxazole,	et al. week for 6 weeks acyclovir	[37] after a median of	16 weeks following	fludarabine-based	first-line therapy	Lin et al. 30 mg SC 3 times a Standard PCP	[32] week for 6 weeks and varicella	after 4 months zoster	following first-line prophylaxis	FR therapy					Fischer et Dose escalation trial, Trimethoprim/	al. [14] IV or SC once cotrimoxazole	weekly for 8 weeks Valacyclovir	after 90-150 days	following second-	line F/FC/FCR	therapy (CLL2I	protocol of	GCLLSG)
			Combination	therapy							Consolidation	therapy

Table 3

 3 Prophylaxis strategies for common infections associated with alemtuzumab therapy

	Infection types	CLL	Allogeneic transplantation
	Bacterial
	Staphylococcus aureus	Broad-spectrum antibacterial prophylaxis	Broad-spectrum antibacterial prophylaxis
	Other Gram-positive bacteria	not recommended	not recommended
	Mycobacterium tuberculosis
	Septicemia
	Fungal
	Aspergillosis	General antifungal prophylaxis not recommended	Fluconazole 400 mg QD
	Cryptococcus	PCP a : trimethoprim/sulfamethoxazole b
	Pneumocystis jiroveci (formerly
	P. carinii) pneumonia (PCP)
	Systemic candidiasis	Prophylaxis for high-risk patients: fluconazole
		50 mg QD or itraconazole 5 mg/kg QD PO
	Viral
	Cytomegalovirus (CMV)	CMV prophylaxis in early development	CMV prophylaxis not recommended
			Preemptive therapy c : ganciclovir 5 mg/kg daily
			or ID or foscarnet 60 mg/kg TID or valacyclovir
			250-500 mg BID PO
	Herpes simplex virus (HSV)	Acyclovir 200-400 mg TID PO or famciclovir	Acyclovir 400 mg QD or TID (HSV) e
	Varicella zoster virus (VZV)	500 mg BID or valacyclovir 250-500 mg BID PO d
	Epstein-Barr virus (EBV)	Prophylaxis for EBV or HBV not recommended	Lamivudine 100 mg (HBV) f
	Hepatitis B virus (HBV)

Table 5

 5 CMV negative, asymptomatic Monitor CMV status by weekly or biweekly PCR/antigenemia testing CMV negative, pulmonary infection Perform bronchoscopy and lavage to test for other pathogens If unresponsive to antibiotics (or test is negative for other pathogens), treat with ganciclovir for 14-21 days CMV positive, asymptomatic Monitor for symptoms If CMV positive for 2 consecutive tests, begin preemptive therapy with oral ganciclovir or equivalent for 7-14 days Continue alemtuzumab therapy CMV positive, symptomatics Treat with i.v. ganciclovir or equivalent for 14-21 days If persistently symptomatic, hold alemtuzumab until asymptomatic and CMV negative Perform bronchoscopy and lavage if symptomatic for pulmonary infection

	Guidelines for
	monitoring CMV status during	Patient status	Recommendation
	alemtuzumab therapy in CMV
	IgG-positive patients	FUO	Test for CMV via PCR/antigenemia (in both CMV positive
	at baseline		and negative patients)
		Persistent FUO, no testing	Treat empirically with ganciclovir
		available
	CMV Cytomegalovirus, FUO
	fever of unknown origin, PCR
	polymerase chain reaction

Table 4

 4 Patient monitoring techniques Bacterial Chest X-ray a Microbiologic cultures a Bronchoalveolar lavage b Fungal Sputum swab/nasal culture a Serum galactomannan a CT scan of chest/sinuses c Bronchoalveolar lavage (galactomannan, culture) b Viral Serologic baseline screen (CMV IgM & IgG, hepatitis B and C, HIV, HSV, VZV) d CMV IgG positive: frequent PCR or antigenemia tests e CMV Cytomegalovirus, CT computed tomography, HIV human immunodeficiency virus, HSV herpes simplex virus, IgG or IgM immunoglobulin G or M, PCR polymerase chain reaction, VZV varicella zoster virus a In case of fever/infection b If deemed necessary c For patients with previous history of fungal infection or fever of unknown origin >72 to 96 h and in case of lung infiltrates d At baseline e In patients with positive CMV IgG at baseline or any patient with fever

Acknowledgments This manuscript was supported in part by the German Competence Network on Malignant Lymphoma (KML) of the BMB-F. Editorial assistance for this manuscript was provided by Meniscus Health Care Communications, with the financial support of Schering AG. JG is a speaker for Roche, Genentech and Schering AG/ Berlex. OAC is supported by the German Federal Ministry of Research and Education (BMBF grant 01KN0706); has received research grants from Astellas, Basilea, Bayer, Genzyme, Gilead, Pfizer, Merck, Schering-Plough, and Vicuron; is a consultant to Astellas, Basilea, F2G, Gilead, Pfizer, Merck, Mölnlycke, Nektar, Schering-Plough, and Zeneus; and served at the speakers' bureau of Astellas, Gilead, Merck, Schering-Plough, SpePharm, and United Medical.

Disclosures JJV received Travel grants from Astellas, Gilead, Merck, Pfizer, Schering-Plough, and Viropharma. TE, AE, MH received travel, speaker and research grants from BayerSchering.