Dear Editor,

Progressive multifocal leukoencephalopathy (PML) results from productive infection of glial cells by John Cunningham polyomavirus (JCV), facilitated by immune deficiency [1]. We report three cases of PML in HIV-negative patients who had been treated with rituximab (their main clinical features are summarized in Table 1).

Table 1 Summary of patients' clinical features
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FormalPara Case 1

A 45-year-old male patient with mantle cell lymphoma was treated with four courses of R-hyperCVAD (October 2000–August 2001), followed by mitoxantrone and melphalan supported by autologous hematopoietic stem cell transplantation (HSCT; October 2001). Complete remission was maintained with four courses of rituximab 375 mg/m2 (May–June 2002). On November 2005, a bone relapse required four courses of R-DHAP (ended in February 2006) and BEAM-conditioned allogeneic HSCT (March 2006). Graft-versus-host disease prophylaxis was carried out with methotrexate 15 mg/m2 on day +1 and 10 mg/m2 on days +3, +6, and +11, and cyclosporine A titrated to keep serum levels between 200 and 400 pg/ml until October 2006. Five courses of maintenance rituximab (375 mg/m2) were then administered (January 2007–April 2008). On April 2008, while in complete remission, he developed bilateral hemianopsia. Magnetic resonance imaging (MRI) displayed subcortical lesions of the white matter. A stereotactic brain biopsy (June 2008) revealed the presence of JCV DNA by polymerase chain reaction (PCR), and histomorphology confirmed PML. Despite treatments with mirtazapine 30–45 mg/day orally, cidofovir 5 mg/kg intravenously (four courses), and risperidone 4 mg/bid orally, follow-up MRIs (June–September 2008) showed progression of demyelinating lesions. The patient died from respiratory failure on September 2008.

FormalPara Case 2

A 68-year-old male patient received nine courses of intravenous cladribine for splenic marginal zone lymphoma (May–December 2002), achieving complete remission. Because of relapsing disease, he received six courses of R-CVP (November 2006–April 2007). Since a partial response was achieved, he was treated with ten courses of maintenance rituximab (375 mg/m2; June–October 2007). On November 2007, disease progression prompted a treatment with intravenous bendamustine 90 mg/m2 (five courses). On December 2007, the patient complained of dysmnesia and visual disturbances, and MRI revealed demyelinating lesions suggestive of PML (Fig. 1a–d). Due to lymphoma progression, the patient started lenalidomide 25 mg/day (May–June 2008). Histomorphology and a positive JCV PCR on a stereotactic brain biopsy confirmed PML (June 2008). Treatment with mirtazapine 30 mg/day was then started. On September 2008, a follow-up MRI showed new areas of demyelination, despite a moderate improvement in the original demyelinated area (Fig. 1e–f). The patient then underwent four courses of COMP chemotherapy to treat lymphoma progression. On December 2008, a new MRI evaluation showed further worsening of the disease (Fig. 1g–h). The patient finally died from leukostasis in March 2009.

Fig. 1
figure 1

MRI in case 2. The first MRI examination (December 2007) revealed a left frontal homogeneous hyperintense lesion of the centrum semiovalis on axial fluid-attenuated inversion recovery (FLAIR) images (a) corresponding to an hypointense (c) nonenhancing (d) area on T1-weighted images. Diffusion-weighted imaging image (b) of the same lesion depicted a central core of hypointensity surrounded by a rim of high signal intensity suggesting a demyelinating lesion probably due to PML. Nine months later, the left frontal lesion showed a slight size reduction with atrophic feature of involved cerebral gyri (e). Nevertheless, similar white matter lesions involving also the subcortical “U” fibers with “scalloped” appearance developed in the left temporal and parietal lobes (f). White matter lesions progressively became multiple and confluent, affecting asymmetrically both cerebral hemispheres as shown in axial FLAIR images obtained 12 months later (g–h)

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FormalPara Case 3

A 66-year-old male patient with leukemic mantle cell lymphoma received two doses of rituximab (375 mg/m2) within CHOP regimen (January–February 2004). Due to occurrence of infusion reactions (fever, chills, and lumbar pain), the last two doses were not administered. As only a partial response was achieved, the patient received eight courses of P-VMyBEC (September–October 2004). On December 2004, the patient developed paraplegia. Brain MRI and computed tomography showed multiple areas of subcortical demyelination. Despite JCV DNA testing negative in cerebrospinal fluid, due to the low sensitivity of this assay in HIV-negative patients [2], he was finally diagnosed as probable PML, according to consensus terminology [3]. Unfortunately, the patient died in December 2004 before a stereotactic brain biopsy could be performed, and no necropsy was carried out.

PML occurs uncommonly in HIV-negative patients with lymphoprolipherative disorders, and it has been rarely reported during rituximab therapy [4–6]. The causal involvement of antiblastic therapies (i.e., purine analogs) in PML is not conclusive [1], and the possible contribution of drugs other than rituximab to PML occurrence in our cases cannot be ruled out. However, case 1 developed PML while in complete remission and free from drug treatments (other than rituximab) since 18 months, making causality highly suspected.

Although immunodeficiency is recognized as a necessary condition for PML development, the mechanism by which JCV crosses the blood–brain barrier (BBB) remains uncertain. Serotonin receptor 2A (5-HT2AR) seems to be involved in JCV internalization into glial cells and some serotonin receptor antagonists (i.e., risperidone, mirtazapine) have been proposed as agents potentially blocking JCV entry [7, 8]. Two of the present cases received mirtazapine and/or risperidone as a tentative salvage therapy, but these drugs did not provide appreciable benefits. Of note, case 2 showed a delay in the progression of the primary lesion, but new focal lesions developed concomitantly. Consistently with recent investigations [9, 10], the unfavorable outcome in our patients suggests that JCV might use alternative gates, besides 5-HT2AR, to cross the BBB.

In conclusion, the present cases support the hypothesis that exposure to rituximab may be an independent risk factor for the development of PML. Moreover, 5-HT2AR antagonists were ineffective in two of our cases, thus pointing out the need for more suitable therapeutic options.