Long-term prognosis and overall mortality in patients with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a rare but fatal opportunistic infection and mainly occurs in patients with immunosuppressive conditions. Despite the increasing number of patients receiving immunosuppressive treatments, studies on PML are still lacking due to its low prevalence and incidence. We retrospectively reviewed patients diagnosed with PML in two tertiary hospitals in South Korea from 1999 to 2021. Total of 47 PML patients were included. Of 27 patients (57.4%) were diagnosed with human immunodeficiency virus (HIV). Median last follow-up modified Rankin Scale (mRS) score was higher in the non-HIV PML group than that in the HIV group (5 vs. 4, p = 0.020). Median survival duration was lower in the non-HIV group (184 vs. 1,564 days). The 1-year and overall mortality rates of PML patients were significantly higher in the non-HIV group than that in HIV group (60.0% vs. 25.9%, p = 0.019; 80.0% vs. 40.7%, p = 0.007). Initial mRS score (HR 1.685, p = 0.038) and highly active antiretroviral therapy (HAART) in HIV patients (HR 0.374, p = 0.013) had a significant effect on overall mortality. Our findings suggest that early detection of PML with low mRS score and early initiation of HAART in patients with HIV may improve prognosis.

www.nature.com/scientificreports/Therefore, this study aimed to investigate the long-term prognosis and prognostic factors for overall mortality of patients with PML through an extended observation period of over 21 years.

Results
Characteristics of patients with PML.Out of 68 patients with the PML diagnosis code, 47 patients meeting the American Academy of Neurology (AAN) diagnostic criteria were enrolled (Fig. 1) 19 .The median age was 46 years (interquartile range [IQR], 37-57), and 74.5% were male patients (Table 1).According to the AAN criteria, definite PML was observed in 44.7% of patients (21/47), while possible PML was observed in 55.3% (26/47).Of the 21 patients with a definite diagnosis of PML, eight had neuropathological demonstration of the typical histopathologic triad.The median initial mRS score was 4 (IQR, 3-4); 46.8% of patients had a mRS score of 4, and 8.5% had a mRS score of 5. Patients diagnosed with PML had a variety of immunosuppressive conditions; 27 (57.4%)were HIV positive, 7 (14.9%)had hematologic malignancies, 11 (23.4%) had solid organ cancer, 3 (6.4%)were diagnosed with rheumatologic disease, and 3 (6.4%)with solid organ transplant.Further review of concomitant medications revealed that 19 (40.4%) patients were using immunosuppressive drugs, and 10 (21.3%) had a history of chemotherapy.

Discussion
With the recent increase in organ transplantation and immunosuppressive drug usage, PML has been reported in various disease groups, including not only HIV infection but also lymphoproliferative disorders, post-organ transplantation, and autoimmune diseases 1,20 .In our study, we described the long-term prognosis and the factors influencing long-term overall mortality of patients with PML under the introduction of HAART and various immunosuppressive conditions.
In previous studies, the median survival duration increased up to 66 months after the introduction of HAART in the HIV population; however, the overall mortality rate was 74.5-78.0% 1,9.In the non-HIV population, the median survival duration of two months was reported, and the case-fatality rate was 90% 10 .In our study, median survival duration was 1,564 days (IQR 254-3444) in the HIV group and 184 days (IQR 74-1566) in the non-HIV group.Additionally, the overall mortality rate of patients with PML during the long observational period was 40.7% in the HIV group and 80.0% in the non-HIV group.Although advances in treatment options have been considered for only a few patients, the prognosis of PML has improved compared to the previous decade 4,8 .As the introduction of HAART had a significant effect on the median survival of PML patients in HIV population, it is likely that changes in HAART initiation regardless of CD4 count will further improve the prognosis of PML.
Various studies have attempted to elucidate predictors of prognosis and mortality in patients with PML.Khanna et al. showed that baseline CD4 + T cell count (HR 0.52, p = 0.010) and HAART (HR 0.37, p = 0.006) www.nature.com/scientificreports/were associated with overall mortality of patients with PML 2 .In a similar report, a higher baseline CD4 + T cell count (HR 0.33) and CSF inflammatory profile (HR 0.12) were significantly related to the long-term survival of patients with PML 21 .Another report by Koralnik et al. showed an association between JCV-specific cytotoxic T-lymphocytes (CTLs) and HIV-positive PML survivors and HIV-negative PML patients with improving clinical status 22 .In a comparative study, Marzocchetti reported that JCV-specific CTLs were associated with a trend toward more prolonged survival in patients with PML 23 .Our study showed that higher initial mRS score was a significant risk factor for overall mortality in a long-term follow up period.In addition, the introduction of HAART was a predictor of long-term survival in HIV-positive PML patients.The first stage of JCV infection is an asymptomatic, persistent infection of the kidneys by the nonpathogenic JCV that occurs in the majority of the general population 24 .During this stage, the virus may establish a latent infection in other secondary sites, including lymphoid tissues, bone marrow, and possibly the brain 24 .These  www.nature.com/scientificreports/asymptomatic latent infections can lead to PML by impairment of cellular immunity due to a particular underlying disease and immunosuppressive drug usage 24 .As a result, PML treatment proceeds in two directions: immune reconstitution or antiviral therapy.Drugs such as mefloquine and cidofovir, which have a direct mechanism of action against JCV infection and replication, have not shown clinically meaningful results; therefore, PML treatment mainly involves reconstitution of the immune system 16,17,25 .Immune reconstitution can be achieved through HAART initiation in patients with HIV; however, it is difficult to achieve in other immunosuppressive conditions.Hematologic malignancy is often pathophysiologically related to immune disruption and is accompanied by transplantation or high-dose chemotherapy 10 .In addition, immunosuppressive reduction therapy is a significant burden when immunosuppressive agents are required to modulate disease activity, such as in solid organ transplantation or rheumatic disease.HAART is the mainstream treatment for HIV-positive patients with PML.Various studies have reported a decrease in the incidence and mortality of PML in HIV-positive patients after the introduction of HAART.In the Eurosida cohort, the incidence of PML decreased from 0.7 to 0.07 per 100 cases per year (PY) after the introduction of HAART 26 .Similarly, Engsig et al. showed that the median survival of HIV-positive patients with PML increased from 0.4 to 1.8 years after the introduction of HAART 9 .In another study, the 1-year mortality due to PML in patients with HIV decreased from 82.3 per 100 cases PY in the pre-HAART era to 37.6 per 100 cases PY in the HAART era 2 .In a multivariable model of 186 patients with PML, the overall mortality was associated with HAART in patients with HIV (HR 0.37, p = 0.006) 2 .In our study, patients with HIV with HAART had a 63% reduction in overall mortality in PML compared to non-HIV patients.Therefore, HAART (HR 0.374, p = 0.013) in HIV patients was associated with decreasing overall mortality or long-term survival of PML patients.
PML may rarely occur in HIV patients receiving continuous HAART 27 .However, in this study, 29.6% (8 of 27) of HIV patients were diagnosed with PML during HAART.The 1-year mortality rate in the group receiving HAART was lower than that in the group newly starting HAART (12.5% vs. 31.6%,p = 0.311); however, no statistical difference was observed (Supplementary Table 1).Additionally, the median survival duration of the group receiving HAART was longer (1921 days [799-4995] vs. 1,221 days [126-2786], p = 0.243).This suggests that the prognosis may be better in HIV patients receiving HAART (who recovered some immune response) than in naive HIV patients.However, the factors contributing to the disease course of PML in HIV patients receiving HAART are unknown.Genetic risk factors, such as mutations in VP1 or polymorphisms in the tumor suppressor protein p53, may affect the pathogenesis of PML 28,29 .In this regard, a prospective study is required to analyze and compare the immunological characteristics of the two groups.
Our study also showed that the initial mRS score is an important prognosis factor, and in particular, the long-term prognosis is poor in the case of mRS score of 4 or higher through the Cox regression model.PML was dominated by motor weakness (51.1%), gait instability (46.8%), speech abnormalities (46.8%), and apraxia (42.6%), but various symptoms may appear depending on the site of involvement.Since PML can occur not only in the case of hematologic malignancy (14.9%) and HIV infection (57.4%) but also in various disease groups taking immune-modulating agents, its diagnosis is difficult and time-consuming.Early detection contributes to a better prognosis of PML with limited disease progression and rapid and effective immune reconstitution 24 .PML pathophysiology involves oligodendrocyte destruction and subsequent demyelination of the CNS 1 .Currently, treatments that can reverse CNS destruction and subsequent demyelination are unavailable, so early recognition of possible PML and achieving immune reconstitution are crucial for improving prognosis.
In this study, the median lymphocyte count was 785 cells/microliter, which was not as severe as expected.This may be due to the fact that HIV-negative patients had a much lower baseline lymphocyte rate of 3.5% (3.3-5.7%), while HIV-positive patients had a baseline lymphocyte rate of 26.3% (15.4-29.9%)and 830 (580-1530).PML was also observed in patients with already recovered lymphocytes, with eight patients taking HAART and nine patients suffering from immune reconstitution inflammatory syndrome (IRIS).Lymphopenia is a risk factor for PML, but monitoring of absolute lymphocyte counts alone is not an accurate predictor of risk because it does not take into account the complexity and diversity of the immune system 30 .The fact that dimethyl fumarateassociated PML typically occurs in the context of severe lymphopenia, whereas abnormal production of IL-10, expression of PD-1, and reduced expression of CD49d by JCV-specific T cells are observed in natalizumabinduced PML, suggests that not only the number of lymphocytes but also their composition is important in the pathogenesis of PML [30][31][32] .In addition, chronic infection with persistent antigens can lead to immune exhaustion, eventually rendering T cells unable to respond effectively to persistent antigens 33 .CD8 + T cells are specialized for intracellular pathogen clearance, but a combination of decreased effector function, inhibitory receptor expression, and cytokine hyporesponsiveness can lead to CD8 + T cell exhaustion in chronic viral infections 34,35 .CD4 + T cells are essential for the immune response to chronic viral infection, but due to decreased production of IL-2, TNFα, and IFNγ and increased expression of the suppressive cytokine IL-10, CD4 + T cells exhibit altered function during chronic infection, which is considered a form of functional exhaustion [35][36][37] .
Our study had several strengths.The long-term prognosis was evaluated while fully reflecting the introduction of HAART and the recommendation of antiretroviral treatment regardless of CD4 cell count in randomized controlled trials such as Temprano ANRS 12,136 and START 38,39 .In addition, the long-term prognosis was evaluated by reflecting the overall PML occurring under various immunosuppressive conditions through increased use of immunosuppressive agents.For patients with hopeless discharge or loss of follow-up, accurate mortality data could be obtained from the Ministry of the Interior and Safety of South Korea.This allowed us to accurately assess mortality or long-term prognosis, the primary or secondary endpoint of our study.
Our study has certain limitations.Due to the retrospective nature of this study, PML diagnosis or treatment may have physician-dependent selection bias.Furthermore, there were some cases where brain biopsy could not be performed depending on the patient's condition; the diagnosis was made based on clinical and radiographic findings.In addition, due to the low prevalence of PML, the sample size was small despite the long observation

Figure 1 .
Figure 1.Flow chart depicting study population with progressive multifocal encephalopathy.ICD International Classification of Diseases, PML Progressive multifocal leukoencephalopathy, HIV Human immunodeficiency virus, DLBCL Diffuse large B cell lymphoma, NSCLC Non-small cell lung cancer, CNS Central nervous system, AQP Aquaporin.

Figure 2 .
Figure 2. Kaplan-Meier curve for overall mortality in patients with progressive multifocal leukoencephalopathy according to (A) HIV infection and (B) mRS score.HIV human immunodeficiency virus, mRS modified Rankin Scale, OS overall survival.

Table 1 .
Baseline characteristics of patients with progressive multifocal leukoencephalopathy.AAN American Academy of Neurology, mRS modified Rankin Scale.a Other diseases include hypogammaglobulinemia, autoimmune cholangiopathy, psoriasis with monoclonal antibody treatment, and disseminated tuberculosis.

Other underlying medication treatments include ustekinumab, total body irradiation, and radiation therapy, in combination with an immunosuppressive or chemotherapeutic agent. Total (N = 47) %
vival was longer in the HIV patient group than in the non-HIV patient group (1564 days [IQR, 254-3444] vs.

Table 2 .
Clinical manifestation, imaging and laboratory findings of patients with progressive multifocal leukoencephalopathy.Significant values are in bold.HIV Human immunodeficiency virus, RNA Ribonucleic acid, PCR Polymerase chain reaction, CSF Cerebrospinal fluid, PML Progressive multifocal leukoencephalopathy, MRI Magnetic resonance imaging.

Table 3 .
Treatment and survival outcomes of patients with progressive multifocal leukoencephalopathy.Significant values are in bold.HIV Human immunodeficiency virus, HAART Highly active antiretroviral therapy, IRIS Immune reconstitution inflammatory syndrome, mRS modified Rankin Scale.

Table 4 .
Univariable and multivariable analyses of overall mortality in patient with progressive multifocal leukoencephalopathy by Cox Proportional-Hazards Model.Significant values are in bold.mRS modified Rankin Scale, HIV Human immunodeficiency virus, HAART Highly active antiretroviral therapy, IRIS Immune reconstitution inflammatory syndrome.