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1 1077 The Effect of Potent Antiretroviral Therapy and JC Virus Load in Cerebrospinal Fluid on Clinical Outcome of Patients with AIDS-Associated Progressive Multifocal Leukoencephalopathy Andrea De Luca, 1 Maria Letizia Giancola, 1,a Adriana Ammassari, 1 Susanna Grisetti, 2 Maria Grazia Paglia, 2 Marco Gentile, 2 Antonella Cingolani, 1 Rita Murri, 1 Giuseppina Liuzzi, 2 Antonella D Arminio Monforte, 3 and Andrea Antinori 2 1 Istituto di Clinica delle Malattie Infettive, Università Cattolica, and 2 Istituto di Ricovero e Cura a Carattere Scientifico per le Malattie Infettive Lazzaro Spallanzani, Rome, and 3 Istituto di Malattie Infettive e Tropicali, Università di Milano, Milan, Italy A multicenter analysis of 57 consecutive human immunodeficiency virus positive patients with progressive multifocal leukoencephalopathy (PML) was performed, to identify correlates of longer survival. JC virus (JCV) DNA was quantified in the cerebrospinal fluid (CSF) by polymerase chain reaction. Two months after therapy, 4% of the patients without highly active antiretroviral therapy (HAART) and 26% with HAART showed neurologic improvement or stability ( P p.03), and 8% and 57%, respectively, reached undetectable JCV DNA levels in the CSF ( P p.04). One-year probability of survival was.04 without HAART and.46 with HAART. HAART and lack of neurologic progression 2 months after diagnosis were independently associated with longer survival. Among HAART-treated patients, a baseline JCV DNA!4.7 log, and reaching undetectable levels after therapy predicted longer survival. Survival of AIDS-related PML is improved by HAART when JCV replication is controlled. Progressive multifocal leukoencephalopathy (PML), the demyelinating brain disease caused by JC virus (JCV), is an important cause of morbidity and mortality in patients with AIDS. The introduction of highly active antiretroviral therapy (HAART) into clinical practice has led to fewer opportunistic infections during human immunodeficiency virus (HIV) disease [1], but PML has been reduced less than other opportunistic diseases [2, 3]. Although potent antiretroviral therapies significantly prolong survival of patients affected by AIDS-related PML, even in large cohort studies [4 7], not all HIV-infected patients with this neurologic disorder receive significant benefit from HAART. Indeed, some case series have shown very poor prognosis, despite aggressive antiretroviral treatments [8], and cases of PML occurring after the start of potent antiretroviral therapy have been reported [9, 10]. Thus, it is important to establish which prognostic factors are related to better responses to HAART in terms of neurologic responses and survival. Preliminary data in patients treated with and without HAART have shown some association between JCV load in cerebrospinal fluid (CSF) and clinical findings [11 15]. To analyze the therapeutic, clinical, and virologic variables associated with the prognosis of AIDS-related PML, we performed an observation analysis of HIV-seropositive patients affected by histologically and/or virologically proven PML. These subjects had CSF samples collected serially in 3 large Italian AIDS treatment facilities. Received 13 March 2000; revised 19 June 2000; electronically published 31 August Presented in part: 7th European Conference on Clinical Aspects and Treatment of HIV Infection, Lisbon, October 1999 (abstract 727); 7th Conference on Retroviruses and Opportunistic Infections, San Francisco, February2000 (abstract 299). Informed consent was obtained from patients, and human experimentation guidelines of local institutional ethics committees were followed in the conduct of the research. Financial support: Istituto Superiore di Sanità, Italy; Programma Nazionale sull AIDS Progetto Infezioni Opportunistiche e TBC derivanti dall AIDS. a Present affiliation: Istituto di Ricovero e Cura a Carattere Scientifico per le Malattie Infettive L. Spallanzani, Rome, Italy. Reprints or correspondence: Dr. Andrea De Luca, Istituto di Clinica delle Malattie Infettive, Università Cattolica del S. Cuore, L.go A. Gemelli 8, Rome, Italy (andeluc@tin.it). The Journal of Infectious Diseases 2000;182: by the Infectious Diseases Society of America. All rights reserved /2000/ $02.00 Patients and Methods Patients and clinical data collection. Patients were recruited from 3 large tertiary infectious diseases clinics in Italy. Only patients with a confirmed diagnosis of PML were selected. The diagnosis was based on either the concomitant presence of a compatible clinical and neuroradiologic picture and the detection of JCV DNA in CSF by polymerase chain reaction (PCR) or by the presence of characteristic histopathologic features in brain tissue. Patients were divided into 2 main groups, according to the type of treatment they had received. These groups reflected the time of observation. Patients treated before the era of HAART received several treatments, including cytosine arabinoside, with or without a concomitant antiretroviral monotherapy or dual therapy, and were classified as group A; patients treated with HAART, with or without concomitant cidofovir, were classified as group B. Clinical data were collected retrospectively from clinical records.

2 1078 De Luca et al. JID 2000;182 (October) In the 3 clinical centers, patients routinely receive a standardized complete neurologic assessment at each hospitalization. During follow-up, neurologic examination was done mostly at monthly intervals, but in every case after and after months. For analysis, we used the results of baseline, month 2, and month 6 neurologic examinations. The following score system was used according to the severity of the neurologic picture: 1, presence of 0 or 1 focal signs; 2, 2 4 focal signs; 3, 14 focal signs; and 4, alteration of consciousness. Follow-up neurologic examination was classified in comparison with baseline as follows: progressed, worsening of previously detected focal signs and/or appearance of new focal signs and/or alteration of consciousness; improved, improvement or resolution of previously detected neurologic signs; and stable, lack of improvement or progression. Semiquantitative JCV DNA assay in CSF. We used a semiquantitative assay with a slight modification of a PCR technique described elsewhere [14, 16], with nested primers from the large T antigen region. In brief, an external standard of a known concentration of recombinant JCV DNA of the Mad-1 strain was serially 2-fold diluted in parallel with the study sample. Samples were extracted on resin columns in accordance with manufacturer s instructions (QIAamp; Qiagen, Hilden, Germany), amplified, and run on ethidium bromide stained agarose gel electrophoresis, as described elsewhere [16]. Gels were analyzed by the Gel Doc 2000 documentation technique (Biorad, Segrate, Italy). The last positive dilution of the clinical sample was multiplied by the lowest detectable concentration of the standard 100, to give the final concentration per milliliter of CSF. In preliminary experiments, 2000 DNA copies/ml of the standard were detected 100% of the time (20/20 experiments), whereas 1000 copies/ml were detected 40% of the time (8/20 experiments). By use of the Poisson distribution, the detection limit of the assay was 1600 copies/ml. Therefore, for statistical reasons, an assay in which no JCV DNA was detected was computed as 1599 JCV DNA copies/ml. HIV RNA measurements. Plasma HIV RNA was measured using a branched DNA assay with a detection limit of 500 copies/ ml (Quantiplex 2.0; Chiron, Emeryville, CA). HIV RNA levels in CSF were quantified either by an ultrasensitive reverse-transcriptase (RT) PCR, with a detection limit of copies/ml (Roche, Branchburg, NJ) or by nucleic acid sequence based amplification (NASBA) with a detection limit of 80 copies/ml (Organon Teknika, Baxtel, The Netherlands). For statistical reasons, plasma HIV RNA values below the limit of detection of the bdna assay were computed as 499 copies/ml; CSF values below the limit of detection of either RT-PCR or NASBA were computed as 79 copies/ ml. Statistical analysis. Viral copy concentrations in body fluids were transformed in log units/ml before calculations. Differences between proportions were analyzed by Fisher s exact test, and differences between continuous variables by Student s t or Mann- Whitney U tests, as appropriate. Relationships between continuous variables were identified by Spearman s test. Survival analysis was performed using Kaplan-Meier curves and univariate Cox model with odds ratios and 95% confidence intervals. Cox proportional hazards regression was used to detect the independent association of variables with survival. P.05 (2-sided) was considered statistically significant. All analyses were performed using the Statistica 5.0 software package (Statsoft, Padova, Italy). Results Baseline characteristics of patients and treatments. We identified 57 patients with PML. All were observed between January 1993 and June Diagnoses relied on clinical and magnetic resonance imaging findings. Fifty-five patients had CSF-positive JCV DNA; these findings were histologically confirmed in 10 cases (3 biopsies and 7 autopsies). By treatment, 25 patients were classified as group A, and 32 were classified as group B. Of the 25 group A patients (the controls of this study), 23 were previously included in a study on clinical and virologic responses to cytarabine [14]. The initial survival of 10 group B patients was described in a preliminary work on responses to HAART [8]. Baseline epidemiologic, demographic, clinical, and viroimmunologic characteristics are summarized in table 1. All patients were white. There was no significant baseline difference between groups. Group A patients were treated with intrathecal cytosine arabinoside (23 patients), antiretroviral monotherapy (17 patients), or dual therapy (2 patients). Group B subjects were treated as follows: 31 patients, 3 antiretroviral drugs (2 nucleoside analogues and 1 protease inhibitor); 1 patient, 4 antiretrovirals (2 nucleoside analogues and 2 protease inhibitors); and 14 patients, concomitant intravenous treatment with cidofovir cycles. Neurologic outcome. After 2 months, 9 (16.1%) of 56 patients showed neurologic improvement or stabilization; all others progressed or died. One of 25 in group A and 8 (25.8%) of 31 in group B showed no progression at 2 months ( P p.03). After 6 months, of the 15 surviving patients (all but 1 in the HAART group), 9 were stable, 4 progressed, and 2 were improved, compared with baseline. Table 1. Characteristic Baseline characteristics of patients, by treatment group. Group A a (n p 25) Group B b (n p 32) Median age, years Sex, men/women 21/4 23/9 Transmission category MSM, % 16 3 Intravenous drug user, % Heterosexual, % 8 13 Median days from disease onset to diagnosis (IQR) 62 (28 89) 41 (27 88) Median neurologic score 2 2 Median Karnofsky score (range) 50 (30 80) 50 (20 80) Previous AIDS, % Median CD4 cell count, 10 6 cells/l (IQR) 28 (10 81) 38 (15 77) Median plasma HIV RNA, log 10 copies/ml (IQR) 4.79 ( ) Median CSF HIV RNA, log 10 copies/ml (IQR) 3.22 ( ) 3.23 ( ) Median CSF JCV DNA, log 10 copies/ml (IQR) 3.60 ( ) 3.30 ( ) NOTE. Variables showed no significant difference between groups. MSM, men who have sex with men; IQR, interquartile range; HIV, human immunodeficiency virus; CSF, cerebrospinal fluid; JCV, JC virus. a Group A, no highly active antiretroviral therapy (HAART). b Group B, HAART.

3 JID 2000;182 (October) HAART, JCV Load, and PML in AIDS Patients 1079 Concentration of JCV DNA in CSF. For 53 of 57 patients (25 group A and 28 group B), there was a baseline (time of diagnosis) CSF sample available for the semiquantitative JCV DNA assay. For 38 patients (21 group A and 17 group B), 1 follow-up sample was available for analysis. Fifteen group A patient baseline samples reported elsewhere [14] were reanalyzed for this study. The median baseline concentration of JCV in CSF was similar in the 2 groups (see table 1). However, beginning at 2 months of follow-up, the proportion of subjects with undetectable JCV load was significantly higher in subjects treated with HAART (see figure 1). After we excluded patients who received cidofovir concomitantly with HAART, the proportion with undetectable JCV DNA was still significantly higher in the HAART group at 2 months ( P p.027); at 3 6 months, the difference was not significant because of the low number of subjects ( P p.11), but all HAART-treated subjects with available samples at this time point had undetectable JCV in CSF (not shown). HIV RNA load in plasma and CSF. Plasma HIV-1 load data were available for patients treated with HAART only. In this group, the median plasma HIV RNA change after therapy was 1.10 log 10 copies/ml. The proportion of patients ever reaching undetectable (!500 copies/ml) plasma HIV RNA levels during follow-up was 76%. CSF HIV RNA was analyzed retrospectively in both treatment groups. At baseline, median CSF HIV-1 load was similar in both groups (see table 1). The median changes of HIV RNA in CSF 2 months after therapy were 0.68 and 0.31 log 10 /ml in groups A and B, respectively (P, not significant [NS]). The proportions of subjects ever reaching undetectable (!80 copies/ml) HIV RNA levels in CSF were 20% and 35% in groups A and B, respectively ( P p NS). Survival analysis. Clinical follow-up was censored at 31 December At that time, 41 deaths had occurred: 25 of 25 group A and 16 of 32 group B patients. All but 3 deaths (in group A subjects) were PML related. Those 3 deaths were excluded from the survival analysis. Findings that were associated with significantly longer survival (table 2) included a baseline CSF JCV load of!4.7 log 10 copies/ml, undetectable JCV DNA levels during follow-up, HAART versus no HAART before and after the diagnosis of PML, and no neurologic progression from baseline until after 2 months of follow-up. Baseline and follow-up HIV RNA levels in plasma and CSF, sex, age, HIV transmission categories, a previous diagnosis of AIDS, and baseline neurologic score were not associated with survival. Persons with 1100 CD4 cells/ml at baseline had a trend toward a longer survival (table 2). Baseline CD4 cell counts were inversely correlated with JCV load ( R p 0.45; P p.0004). All variables significantly associated with survival were included in the multivariate model. Cox regression analysis showed that only HAART and lack of neurologic progression at 2 months were independently associated with longer survival (table 2). Group A patients survived a median of 66 days after Figure 1. Proportion of patients with progressive multifocal leukoencephalopathy with undetectable JC virus (JCV) DNA (!1600 copies/ ml) in cerebrospinal fluid at diagnosis and after therapy. Dashed line, group A patients (no highly active retroviral therapy [HAART]); solid line, group B patients (HAART). Months are approximate 0.5 month. Differences of proportions: at 2 months, P p.038; at 3 6 months, P p.04. diagnosis, whereas group B patients had a median survival of 245 days. The cumulative proportion of HAART-treated patients surviving 11 year was versus for patients who did not undergo HAART. All patients who died despite HAART did so during the first months (median time from diagnosis to death, 7 weeks; range, 1 35 weeks). HAART was associated with longer survival, even after exclusion of the 14 patients who were concomitantly treated with cidofovir (log rank P p.02). When HAART-treated patients were considered alone, baseline JCV DNA levels!4.7 log U/mL, 2-month neurologic stability, and 1100 CD4 cells/mm 3 during follow-up were associated with longer survival; undetectable JCV DNA during follow-up showed a trend toward longer survival (table 2). In a multivariate model that excluded the 2-month neurologic outcome, a variable too strong for this smaller subset of patients, JCV DNA!4.7 log at baseline and undetectable during followup were independently predictive of longer survival in HAARTtreated patients (table 2). Kaplan-Meier curves of variables independently predictive of a longer survival are shown in figure 2. PML in patients already on HAART. Of the 32 group B patients, 9 developed PML a median of 9 weeks (range, 3 28 weeks) after beginning HAART. As mentioned, this variable was significantly correlated with prolonged survival by univariate but not multivariate analysis (table 2). The mean baseline JCV DNA load was significantly lower than in patients not taking HAART before the diagnosis of PML (mean, 3.26 vs log U/mL; P p.003). The number of CD4 cells was significantly higher in persons with PML onset after beginning HAART (mean, 127 vs. 56 cells/ml; P p.003). Five of 9 patients showed no neurologic progression after 2 months (vs. 3

4 1080 De Luca et al. JID 2000;182 (October) Table 2. Variables (Cox regression) associated with longer survival in all patients and in the subset of patients treated only with highly active antiretroviral therapy (HAART). Variables Unadjusted HR All patients Adjusted HR Unadjusted HR HAART only Adjusted HR HAART after PML onset 0.30 ( ) a 0.37 ( ) b ND ND HAART before PML onset 0.16 ( ) b NE 0.27 ( ) ND 2-Month neurologic stability 0.05 ( ) c 0.05 ( ) c NC a NC, NE Baseline JCV DNA in CSF!4.7 log 0.46 ( ) b NE 0.22 ( ) c 0.13 ( ) b Undetectable JCV in CSF after therapy 0.36 ( ) b NE 0.27 ( ) 0.17 ( ) b Plasma HIV RNA!500 after therapy ND ND 2.0 ( ) ND CSF HIV RNA!80 after therapy 0.81 ( ) ND 0.71 ( ) ND Baseline CD4 cells 1100/mm ( ) ND 0.38 ( ) ND Follow-up CD4 cells 1100/mm ( ) ND 0.12 ( ) b NE Male sex 1.52 ( ) ND 1.86 ( ) ND No previous AIDS 1.31 ( ) ND 1.06 ( ) ND Age!35 years 0.80 ( ) ND 0.66 ( ) ND Neurologic score! ( ) ND 1.00 ( ) ND NOTE. HR, hazard ratio; CI, confidence interval; PML, progressive multifocal leukoencephalopathy; ND, not done; NE, did not enter in multivariate model; NC, could not be calculated (no uncensored events); JCV, JC virus; CSF, cerebrospinal fluid; HIV, human immunodeficiency virus. a P!.001. b P!.05. c P!.01. of 21 other group B patients; P p.03): 1 was improved, and 4 were stable. Discussion Our results confirm that potent combination antiretroviral regimens have a significant effect on the survival of persons with AIDS-associated PML. Indeed, in our series with a historical control group of patients treated, in most of those given antiretroviral monotherapy, HAART was independently associated with longer survival with, 46% cumulative survival after 1 year. This observation is consistent with several recent reports [4 7]. Our series is one of the largest with histologically or virologically confirmed PML. Our initial observation of a lack of benefit from HAART in a series of 10 subjects (all included in the present study) [8] can now be interpreted as a proportion of persons representing the lowest quartile of survival (i.e., rapid progressors) and underscores the importance of large observations for a disorder that is characterized by marked prognostic variability. We show for the first time in the HAART era that the lack of clinical progression during the first 2 months after diagnosis is independently predictive of longer survival and that the persons who benefit most from treatment have a naturally slower progressing disease. This confirms and reinforces a previous analysis of patients from the pre-haart era that showed that neurologic improvement after PML diagnosis is a strong predictor of long-term survival [17]. In this series, most AIDS-related PML patients had CSF samples collected at baseline and longitudinally after therapy. The JCV burden in the CSF both before and after therapy was predictive of survival. Multivariate analysis did not confirm the independent predictive value of JCV load at baseline and during follow-up on survival, probably because of the strong effect of HAART on the outcome. Nevertheless, when only CSF samples from HAART-treated patients were analyzed, having a lower baseline JCV burden (!50,000 copies/ml) and reaching undetectable JCV load during follow-up (!1600 copies/ml) were independently associated with longer survival. Furthermore, a significantly higher proportion of patients treated with HAART than of control patients achieved undetectable levels of JCV during follow-up. JCV DNA clearance was also associated with a lack of neurologic progression during the first 2 months of therapy (not shown). Moreover, JCV load at baseline was negatively correlated with CD4 cell counts. These findings confirm and substantially reinforce preliminary observations of the prognostic value of JCV load at PML diagnosis [11 13] and show for the first time in a larger series that the clearance of JCV DNA from the CSF during follow-up is associated with a better neurologic outcome and longer survival. Unlike a recent study of 25 HAART-treated subjects in whom undetectable plasma HIV RNA levels during therapy were predictive of longer survival [5], in our study, HIV RNA levels in plasma and CSF at baseline and during follow-up were not associated with survival. In our study, 16 (76%) of 21 patients reached undetectable plasma virus load levels while on HAART. The discrepancy between the study results might in part be explained by the lower baseline CD4 cell counts in our series (median CD4 cells/mm 3, 38 vs. 104). In fact, the inhibition of HIV replication might be a necessary but not sufficient condition, if a consistent immune recovery does not occur. In our analysis of HAART-treated subjects, those with 1100/mm 3 CD4 cells while on therapy survived longer. Together, these findings support the hypothesis that the benefit of potent antiretroviral

5 JID 2000;182 (October) HAART, JCV Load, and PML in AIDS Patients 1081 Figure 2. Kaplan-Meier curves of patient survival, by variables shown to be independent predictors of survival by multivariate model. Observations:, complete;, censored. In all patients, significance (log rank) was as follows: effect of highly active antiretroviral therapy (HAART), P p.001; effect of lack of neurologic progression 2 months after diagnosis, P! In HAART subgroup, significance (log rank) was as follows: effect of baseline cerebrospinal fluid (CSF) JC virus DNA concentration!4.7 log 10 copies/ml CSF, P p.011; effect of reaching undetectable levels (!1600 copies/ml) of JCV DNA in CSF during follow-up, P p.06. therapies on the prognosis of PML is a consequence of immune reconstitution (through HIV control) and of the eventual control of the replication of JCV. The relationship with baseline CD4 cell counts seems to indicate an immune control of JCV replication even before therapy. Nevertheless, baseline CD4 cell counts were a weaker predictive value of survival than was JCV load. Our knowledge of the correlates of JCV-specific immunity is preliminary and requires more investigation before moredefined conclusions on the mechanisms of JCV replication control can be drawn [18, 19]. Our findings have several implications. First, both baseline quantification and follow-up qualitative determination of JCV DNA in CSF are significant prognostic indications and are important tools for monitoring AIDS-associated PML, particularly in the HAART era. The quantitative assay is relatively simple and inexpensive and can be performed on the diagnostic CSF sample. Other groups have reported techniques for JCV quantitation in CSF [11 13, 20]. Follow-up lumbar puncture is minimally invasive and can be performed after 2 months, in conjunction with a new neurologic assessment. Second, even though HAART has profoundly changed the prognosis of PML, this effect is limited. Most patients with long-term survival in this and previous series showed neurologic stabilization [17, 21]. Therefore, a prompt diagnosis and immediate therapy aimed at limiting the neurologic damage is mandatory, to improve survival and to reduce chronic physical and neurocognitive disabilities. Furthermore, subjects with higher replication of JCV in the CSF seem to have significantly shorter survival, despite potent antiretroviral therapy. It remains to be established whether these persons would benefit by the addition of agents that have a direct inhibitory effect on JCV, such as cidofovir [22]. There have been anecdotal reports of a beneficial effect of cidofovir therapy in AIDS-related PML [23 25]. In our series, follow-up of patients treated with

6 1082 De Luca et al. JID 2000;182 (October) cidofovir was too short for a comparative analysis (not shown). To check for the potential influence of the coadministration of cidofovir in some patients from the HAART group, we repeated survival analyses, by excluding cidofovir-treated subjects. Despite the substantial loss of statistical power, this analysis confirmed that HAART-treated subjects survived significantly longer than historical controls. In our series, there were 9 PML episodes in patients after beginning HAART. Most occurred a few weeks after the start of antiretroviral therapy. Subjects showed lower JCV loads and higher CD4 cell counts at diagnosis and had better neurologic outcomes and survival than did patients without prior HAART. The lower JCV DNA concentration in the CSF at diagnosis might be a reason for a decreased diagnostic yield of JCV PCR in the HAART era, indicating that more sensitive assays might be required. 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