Fatal JC-virus Granular Cerebellar Neuronopathy associated with Autoimmune Lymphoproliferative Syndrome and Common Variable Immunodeficiency


 Background: JC-virus (JCV) associated granular cerebellar neuronopathy (JCV-GCN), which causes severe ataxia, is a rare complication of severe immunodeficiency and caused by a variant of JCV with a VP1-deletion. JCV-GCN has not yet been reported in patients with autoimmune lymphoproliferative syndrome (ALPS-FAS). We report a 34-year old woman with autoimmune lymphoproliferative syndrome (ALPS-FAS) and common variable immunodeficiency (CVID) who developed JCV-GCN which progressed to fatal progressive multifocal leukoencephalopathy (PML).Methods: Extensive longitudinal virologic, genetic and immunologic characterisation by JCV CSF quantification and sequencing, peripheral blood flowcytometry and exome sequencing. Results: A diagnosis of JCV-GCN was established by findings of severe cerebellar vermis atrophy on MRI and a JCV variant with a VP1-deletion (JCV-VP1) in CSF. During a period of 18 months, at which treatment with high dose immunoglobulin, cidofovir, mirtazapine and high dose fucidic acid were given, cerebellar ataxia was relatively stable. In CSF a mixture of predominantly JCV-VP1 with only low levels of wildtype JCV was found. However, classic PML ensued, with rising JCV- CSF levels and a complete change to JCV-wildtype quasi species without JCV-VP1. Immunological findings were unusual, with disappearance of the original ALPS-FAS phenotype. Trio-exome sequencing identified a heterozygote de novo variant of unknown significance in RAG1. FAS mutations were not detected in the patient or in the parents, suggesting that her previously diagnosed ALPS-FAS-FAS was caused by somatic mosaicism. At the progression to PML, increased expression of T-cell PD-1 was demonstrated, suggesting T-cell exhaustion. Treatment with pembrolizumab, an inhibitor of PD-1, was attempted but followed by rapid and fatal PML progression. Conclusion: JCV-GCN may complicate ALPS and may progress to PML. In JCV-GCN, wild-type and JCV-VP-1 can co-exist in CSF. Quantification and sequencing of JCV in CSF is helpful to characterize and monitor disease and treatment. Progression of disease may be associated with development of T-cell exhaustion, but the benefit PD-1 inhibitor treatment is uncertain.

and fatal PML progression.
Conclusion: JCV-GCN may complicate ALPS and may progress to PML. In JCV-GCN, wild-type and JCV-VP-1 can co-exist in CSF. Quanti cation and sequencing of JCV in CSF is helpful to characterize and monitor disease and treatment. Progression of disease may be associated with development of T-cell exhaustion, but the bene t PD-1 inhibitor treatment is uncertain.

Background
In immunocompromised patients, the primary disease caused by reactivation of JCV is PML. JCV affects the white matter of the brain by productive and lytic infection of oligodendrocytes and astrocytes. However, in 2003 a rare syndrome caused by productive and selective infection of cerebellar granule cell neurons by a JCV variant with a small deletion in the VP1 capsid was described (1)(2)(3).

Autoimmune lymphoproliferative syndrome (ALPS-FAS)
Autoimmune lymphoproliferative syndrome (ALPS-FAS) is characterized by FAS gene mutations that leads to defective lymphocyte apoptosis with uncontrolled proliferation of lymphocytes accompanied by autoimmune cytopenia, splenomegaly, lymphadenopathy and increased risk of lymphoma (4). ALPS-FAS was rst characterized in the 1990'ies in patients with chronic lymphoproliferation and an increased number of a characteristic "double negative T cells (DNT)". Germ-line FAS mutations is the most common cause of ALPS-FAS, but it can also be caused by somatic FAS mutations (10%) (5). The disease has variable penetrance and severity. In the largest cohort study of ALPS-FAS, patients had relatively good prognosis (estimated survival by age 50, 85%) but a high risk of post-splenectomy sepsis and lymphoma. In this cohort opportunistic infection was rare and typically associated with immunosuppressive treatment of autoimmunity (6). A subset of ALPS-FAS patients with ALPS-FAS have associated CVID, which may be di cult to distinguish in clinical practice.

Methods
Extensive longitudinal virologic, genetic and immunologic characterisation by JCV CSF quanti cation and sequencing, peripheral blood owcytometry and exome sequencing.

Results
A 34-year old woman was diagnosed at early childhood with a primary immunode ciency presenting with autoimmune hemolytic anemia (AIHA), hypogammaglobulinemia, lymphadenopathy, recurrent upper respiratory infections and meningitis. After splenectomy for AIHA, she continuously received intravenous immunoglobulin substitution, during which infections were relatively infrequent. At the age of 13 years owcytometry revealed an increased fraction of CD4/CD8 double negative T-cells (DNT), comprising between 30-50% of the TCRab CD3 positive T-cells. Based on these ndings a defect in FAS induced apoptosis was suspected. At the age of 14 a missense Ile243-Arg FAS mutation (laboratory of Rieux-Laucat, Paris) was veri ed in peripheral blood cells. Based on these ndings and reduced mutated fraction of somatic hypermutation of expressed immunoglobulin kappa light chain (examined by restriction enzyme based hot-spot mutation assay (IgκREHMA)) in B-cell, a de nitive diagnosis of ALPS-FAS with CVID was made (7). At age of 27 years, she was treated for recurrent genital HPV condylomata. Two years later, progressive extensive bluish nodular skin lesions developed Fig. 1. Cutaneous lymphoma was suspected, but skin biopsies demonstrated a polyclonal T-cell lymphoproliferation and lymph node biopsy and PET-CT were without lymphoma. Treatment with alitretinoin followed by 6 months of peg-interferon had no effect on the skin lesions.
Early 2017, at age 34, she developed slowly progressive gait disturbance and vertigo, which during the next 6 months progressed to severe truncal ataxia, dyskinesia and nystagmus. Cognition was normal.
Severe cerebellar vermis atrophy was found on MRI. Figure 2, A and B. CSF was acellular with normal protein. Extensive investigations for infectious, autoimmune and paraneoplastic conditions were done on CSF and blood including gastrointestinal biopsies and whole-body PET-CT, which were all negative. High dose high dose iv methylprednisolone was given on suspicion of an auto-in ammatory aetiology but stopped when CSF was found positive for JCV DNA. A diagnosis of JCV-GCN was made.
Treatment IVIG treatment doses were increased to keep IgG at a minimum level of 12 g/l. Mirtazepin, which she already received for depression, was continued.
Cidofovir treatment was initially given 7 times for three months (September to November 2017), during which cerebellar symptoms and MRI changes were stable and CSF-JCV levels decreased moderately- Fig. 3. Three months later, however, progression was suspected because of slight worsening in ataxia and rising JCV-CSF levels, at which time cidofovir treatment was repeated once, but was terminated because of uveitis, possibly related to cidofovir (8) .
Fusidic acid, commonly used in Denmark for treatment of staphylococcal infections, has possible in vitro e cacy against JCV with a single case report describing effect against JCV associated nephropathy(9,10). In standard dosage, fusidic acid has relative low CSF penetration, but better into brain tissue (11)(12)(13). Since progression of ataxia was suspected and CSF-JCV-levels remained (Fig. 2), treatment with high dose fusidic acid (1500 mg x 2) was attempted. After 1 month at which a relatively high level of CSF-fucidic acid (2.5 mg/L) was measured JCV-CFS levels had decreased to 71 from 478 copies/ml. Because of gastrointestinal side-effects, the dosage was then reduced to 500 mg x 3 after which CSF-fucidic acid levels decreased to 0.4 mg/L, after which time treatment was stopped, Fig. 3. During the next 7 months, the ataxia, MRI and skin changes remained unchanged. JCV was detected twice in blood.
However, over a 2-week period in November 2018, she suddenly developed progressive severe blindness and reduced lower leg power. On MRI non-enhancing parietal PML lesions was found bilaterally- Fig. 2.
PML is associated with increased programmed cell death-1 (PD-1) expression, a marker of cellular immune exhaustion with increased JCV-speci c T-cell immune responses following PD-1 inhibition (14).

Virology
During illness, a total of 11 lumbar punctures were obtained, in which CSF remained acellular with normal CSF-protein, glucose and lactate- Fig. 1. Ten of the CSF samples were available for JCV quanti cation. DNA was isolated from CSF using QiaCube isolation and the JCV copy number was determined by quantitative PCR using a JCV standard with known copy number (15). Sequencing of the C terminus of the VP1 gene was performed after ampli cation of CSF samples using primer pair JC_F2469 (ACAGAGGAGCTTCCAGGGGA) and JC_R2629 (AAACCAAAGACCCCTCCCCC). Sequencing was carried out using the Nextera XT However, at the progression to fatal PML, CSF-JCV levels increased 10-fold to an average of 2000 copies/ml at which point only wildtype-JCV without JCV-GCN was detected (Fig. 3).

Discussion
We here report the rst a case of ALPS-FAS/CVID associated JCV-GN. During the rst part of illness, repeated CSF samples demonstrated a genotype mixture with almost complete predominance of a JCV variant, with a 16 bp deletion in the VP1-gene, associated with JCV-GN, together with low levels of wild type JCV (17). For 18 months, at which treatment with high-dose IVIG, intermittent cidofovir and high-dose fucidic acid were given, the severe ataxia remained stable and CSF-JCV levels decreased. However, a sudden switch to classic PML was observed, with progressive blindness, increasing CSF-JCV levels with a complete change from JCV-VP1 to wildtype JCV. Treatment with the PD-1 inhibitor pembrolizumab was unsuccessful and associated with rapid progressive fatal encephalopathy.
JCV associated disease primarily manifests as PML, through lytic infection of oligodendroglial cells with progressive demyelinating white matter lesions. Rarely, JCV may also selectively affect granule cell neurons, with resulting neuronal loss, gliosis and a clinical cerebellar syndrome.
PML is in frequent in patients with primary immunode ciency, In a literature case study review, a total of 26 cases of PML complicating primary immunode ciency disease (PID) was described of which 7 cases were associated with CVID and combined immunode ciency (18). A recent French study, reported 11 patients with PID and PML, of which two patients had CVID, several cases were seen in connection with immunosuppressive treatments (19). CVID associated Granule Cell Neuronopathy has only been reported once, in a recent case (20). Her ALPS-FAS was probably caused by a somatic mutation leading to mosaicism, since parents had no germline FAS mutations. The persisting immunode ciency including CVID lead to a comprehensive genetic screening (EXOM-sequencing) revealing a heterozygous RAG1 mutation and no FAS mutation in accordance with the "disappearance" of the ALPS-FAS phenotype. A heterozygous RAG1 mutation has previously been described in a patient with adult-onset manifestation of idiopathic CD4 T-cell lymphopenia and B-cell dysfunction (23). We speculate that the heterozygous RAG1 mutation in our patient may have contributed to the T-cell as well as B-cell dysfunction, which might have been an additional risk factor in combination with the somatic FAS mutation.
Treatment options for PML are limited. JCV CSF quanti cation as a surrogate marker for treatment response is debated, and PCR methods are not standardized. However, overall there seems to be reasonable correlation between CSF-JCV levels and clinical treatment response (24,25).
In our patient, decreasing JCV-CSF levels and stable disease was observed in relation to cidofovir treatment. Whether this re ects treatment effect or merely could be a result of complete depletion of target cerebellar granula cells is however uncertain. Cidofovir treatment of PML remains controversial, as a pooled analysis of several small observational studies failed to show evidence of e cacy against AIDS-associated PML (26). However, several case reports in non-HIV associated PML have observed a possible bene t of Cidofovir (27)(28)(29)(30)(31) After the death of our patient, 5 publications from 2019 describing a total of 12 patients, have reported bene cial effects of PD-1 inhibitor treatment in 9 PML patients, with increased JCV-speci c T-cell immune responses (24,(32)(33)(34)(35). In the NIH study, 5 of the 8 patients had clinical improvement or stabilization, with MRI showing stabilization or reduction in lesion burden and viral load reductions in CSF (24). The remaining 3 patients had no clinical bene t. Immune reconstitution in ammatory syndrome (IRIS), was suspected in one patient, who had worsening shortly after infusion, but in which MRI did not show contrast enhancement, which was interpreted that IRIS was unlikely.
In our patient a rapid worsening with encephalopathy was observed shortly after pembrolizumab infusion without MRI enhancement. Despite the lack of contrast enhancement on MRI, we are concerned that the progression may have been provoked by the PD1-inhibitor treatment. Although the observed e cacy ofPD-1 inhibitor treatment is promising, cases of PML have been reported after treatment with nivolumab or pembrolizumab, why the use of this treatment should be cautious (36).

Conclusion
JCV-GCN may complicate ALPS and may progress to PML. In JCV-GCN, wild-type and JCV-VP-1 can coexist in CSF. Quanti cation and sequencing of JCV in CSF is helpful to characterize and monitor disease and treatment. Progression of disease may be associated with development of T-cell exhaustion, but the bene t PD-1 inhibitor treatment is uncertain. Abbreviations