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`
`Whole brain volume changes in patients
`with progressive MS treated with
`cladribine
`
`M. Filippi, MD; M. Rovaris, MD; G. Iannucci, MD; S. Mennea; M.P. Sormani, PhD; and G. Comi, MD
`
`Article abstract—Objective: To compare changes in whole brain volume measured using MRI scans in patients with
`progressive MS enrolled in a double-blind, placebo-controlled trial assessing the efficacy of two doses of cladribine (0.7 and
`2.1 mg/kg) and to assess the correlations between change in whole brain volume and change in other conventional MRI
`measures. Background: Measuring brain parenchymal volumes is an objective and reliable surrogate for the destructive
`pathologic process in MS. The dynamics and the mechanisms of tissue loss in progressive MS are unclear. Methods: Whole
`brain volumes were measured using postcontrast T1-weighted scans with 3 mm slice thickness from 159 patients with
`progressive MS (70% secondary progressive and 30% primary progressive) enrolled in a double-blind, placebo-controlled
`trial of 12-month duration. Results: Whole brain volumes were similar in the placebo and cladribine-treated patients on
`the baseline scans. A significant decrease of brain volume over time was observed both in the entire population of patients
`(p ⫽ 0.001) and in the placebo patients in isolation (p ⫽ 0.04). No significant treatment effect of either dose of cladribine
`on brain volume changes over time was found. In the 54 patients who received placebo, the change in brain volume was
`not significantly correlated with other MRI measures at baseline (enhancing lesion number and volume and T2-
`hyperintense and T1-hypointense lesion volumes) or at follow-up (cumulative number of enhancing lesions and absolute
`and percentage changes of enhancing T2- and T1-hypointense lesion volumes). Conclusions: This study shows in a large
`cohort of patients that brain parenchymal loss occurs, even over a short period of time, in progressive MS and that
`cladribine is not able to alter this process significantly. It also suggests that MRI-visible inflammation and new lesion
`formation has a marginal role in the development of brain atrophy in patients with progressive MS.
`NEUROLOGY 2000;55:1714 –1718
`
`Cladribine (2-chlorodeoxyadenosine; 2-CdA) is a pu-
`rine nucleoside analogue resistant to the action of
`adenosine deaminase, which results in preferential
`lymphocytotoxicity. In cells with a high ratio of de-
`oxycytidine kinase to deoxynucleotidase (e.g., lympho-
`cytes and monocytes), cladribine is phosphorylated into
`the active triphosphate deoxynucleotide, which dam-
`
`ages DNA and promotes cell death.1 Preliminary trials
`reported that the long-lasting lymphocytotoxic activity
`of cladribine has the potential for modifying the evolu-
`tion of progressive MS.2,3 In a recent multicenter, ran-
`domized, double-blind, placebo-controlled trial of
`patients with progressive MS,4,5 it was shown that
`cladribine had a dramatic effect on the volume and
`
`From the Neuroimaging Research Unit (Drs. Filippi, Rovaris, Iannucci, and Sormani, and S. Mennea) and Clinical Trials Unit (Dr. Comi), Department of
`Neuroscience, Scientific Institute Ospedale San Raffaele, University of Milan, Italy.
`Supported by The R.W. Johnson Pharmaceutical Corporation, Raritan, NJ.
`Received April 21, 2000. Accepted in final form August 24, 2000.
`Address correspondence and reprint requests to Dr. Massimo Filippi, Neuroimaging Research Unit, Department of Neuroscience, Scientific Institute
`Ospedale San Raffaele, Via Olgettina, 60, 20132 Milan, Italy; e-mail: m.filippi@hsr.it
`
`1714
`
`Copyright © 2000 by AAN Enterprises, Inc.
`
`Downloaded from https://www.neurology.org by Sally Charin on 29 January 2024
`
`Merck 2065
`TWi v Merck
`IPR2023-00049
`
`

`

`Table Mean (SE) volumes of the whole brain at study entry, month 6, and month 12 in patients treated with placebo,
`cladribine 0.7 mg/kg, and cladribine 2.1 mg/kg
`
`Brain volume, mL
`
`Treatment
`
`Entry scan Month 6
`
`Mean absolute
`change (SE) (mL),
`month 6 versus
`entry
`
`Mean
`percentage
`change (SE) (%),
`month 6 versus
`entry
`
`Brain volume,
`mL, month 12
`
`Mean absolute
`change (SE)
`(mL), month 12
`versus entry
`
`Mean
`percentage
`change (SE) (%),
`month 12 versus
`entry
`
`1042 (18.8) 1040 (19.0)
`Placebo
`Cladribine 0.7 mg/kg 1053 (18.1) 1051 (18.5)
`Cladribine 2.1 mg/kg 1082 (19.9) 1074 (19.1)
`
`⫺2.1 (5.5)
`⫺2.3 (4.6)
`⫺7.8 (8.4)
`
`⫺0.2 (0.5)
`⫺0.2 (0.4)
`⫺0.6 (0.7)
`
`1037 (18.3)
`1049 (18.3)
`1067 (20.6)
`
`⫺5.0 (5.9)
`⫺4.1 (3.6)
`⫺16.3 (5.8)
`
`⫺0.4 (0.6)
`⫺0.4 (0.3)
`⫺1.5 (0.5)
`
`T1-weighted images obtained at study entry, month 6, and
`at month 12 (end of the double-blind phase) to measure the
`volumes of the whole brain tissue.
`Brain volumes were measured by a single observer (un-
`aware of the acquisition order of the scans and to whom
`the scans belonged) in a three-step process, using a semi-
`automated segmentation technique based on local thresh-
`olding.16 The first step consists of the segmentation of the
`brain and the intraventricular CSF from all the other ex-
`tracerebral tissues. To this end, the observer chooses a
`point on the brain surface boundary, using a mouse-
`controlled cursor, and the algorithm starts contouring fol-
`lowing from the strongest edge point in the neighborhood
`of the user-selected point. This strongest edge point is
`found by searching over a 5 ⫻ 5 pixel square area with the
`manually selected point in its center. From the starting
`point found by the algorithm, the program finds the direc-
`tion of next contour point by searching north, east, south,
`and west and choosing the strongest direction among
`them. The next contour point must have at least as strong
`a gradient as the starting point. The program then traces a
`contour from the most recent point following the same
`principle above described; the contour is complete when it
`traces back to the starting point. Manual outlining may be
`required to modify part of the boundary of poorly defined
`areas. The observer uses the same local thresholding tech-
`nique to contour the ventricle borders (again, manual out-
`lining might be sometimes necessary in case of poorly
`defined areas). The resulting portions of the brain paren-
`chyma and intraventricular CSF are recorded in a file as
`regions of interest (ROI) and superimposed on each image
`slice. The second step consists of removal of intraventricu-
`lar CSF to obtain the brain parenchyma in isolation. The
`intraventricular CSF ROI are masked on a slice by slice
`basis to segment ROI containing only pixels belonging to
`the brain parenchyma. The third step consists of the auto-
`matic calculation of the brain volume. This is obtained by
`multiplying the number of pixels included in the ROI re-
`sulting from the two previous steps by the voxel size. In-
`traobserver measurement error was
`calculated by
`determining the coefficient of variation (COV) as a per-
`centage (SD/mean) for two measurements made at a test–
`retest interval of at least 1 month by a single observer in
`20 randomly selected cases.17 The mean COV was 2.2%.
`Hyperintense lesions on the dual-echo scans and en-
`hancing and hypointense lesions on postcontrast T1-
`weighted scans were also identified at baseline and month
`12, as previously described.4,5 The corresponding lesion vol-
`umes were then calculated using the same segmentation
`December (1 of 2) 2000 NEUROLOGY 55 1715
`
`For further details and statistical analysis, see the text.
`
`number of active lesions (ⱖ90% reduction) seen on en-
`hanced MRI scans of the brain, a modest effect on the
`accumulation of T2 lesion volume, and no effect on the
`accumulation of disability and T1-hypointense lesions.
`Recently, several studies have shown that mea-
`suring brain volume changes over time is an objec-
`tive and reliable surrogate for the destructive
`pathologic processes in MS.6-15 In detail, these stud-
`ies have demonstrated that 1) brain volume mea-
`surements are sensitive to MS-related changes over
`time since the earliest phases of the disease9,13;
`2) brain volume and disability changes are correlated
`even over relatively short periods of time9,11,13,14 and
`the magnitude of the correlation is particularly
`strong in patients with secondary progressive
`MS11,14,15; and 3) there are treatments able to modify
`favorably the progressive loss of cerebral tissue in
`MS.12 In this study, we investigated the effect of two
`doses of cladribine (0.7 mg/kg and 2.1 mg/kg) on the
`changes in brain volume in a cohort of patients with
`progressive MS enrolled in a double-blind, parallel-
`group, placebo-controlled trial with cladribine4 to
`provide additional information about the role of this
`drug in the treatment of progressive MS. We also
`assessed the magnitude of the correlation between
`change of whole brain volume and change of other
`conventional MRI measures to elucidate the mecha-
`nisms leading to tissue loss in progressive MS.
`
`Patients and methods. A total of 159 patients with
`progressive MS (70% with secondary progressive and 30%
`with primary progressive MS) were enrolled in a random-
`ized, double-blind, parallel-group, placebo-controlled study
`to assess the safety and efficacy of 0.7 mg/kg and 2.1 mg/kg
`of cladribine administered by subcutaneous injection.4 The
`study included a 4-week screening phase, a 1-year double-
`blind phase, and a 6-year open label phase. Patients were
`assigned to one of three parallel treatment groups (2.1
`mg/kg cladribine, 0.7 mg/kg cladribine, or placebo). Fur-
`ther details about study population and design have been
`reported previously.4
`At study entry and at months 6, 12, 18, and 24, dual-
`echo (proton density and T2-weighted) and enhanced T1-
`weighted scans (5 to 10 minutes after the injection of 0.1
`mmol/kg gadolinium-DTPA) were obtained from all pa-
`tients. For T1-weighted images, slices were axial, contigu-
`ous, 3 mm thick, with a matrix size of 256 ⫻ 256 mm and a
`field of view of 250 ⫻ 250 mm. We used the 40 central
`
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`
`

`

`demonstration that the magnitude of the correlation
`between brain parenchymal loss and new lesion for-
`mation and inflammation is poor in patients with
`progressive MS.
`Previous studies using several different measure-
`ment strategies showed that the brain volume of pa-
`tients
`with
`relapsing-remitting,
`secondary
`progressive, and primary progressive MS is reduced
`compared to that of sex- and age-matched healthy
`controls,6-14,18 and that significant changes in brain
`volume can be detected over a 1- to 2-year period
`early in the course of MS.9,13 Only two studies, how-
`ever, assessed the dynamics of brain parenchymal
`loss in patients with progressive MS.15,19 These stud-
`ies consisted of only nine secondary progressive15 and
`16 secondary or primary progressive19 MS patients
`and showed very different annual rates of brain pa-
`renchymal loss, which in one of the studies15 corre-
`lated strongly with changes in disability. Our annual
`rate of brain parenchymal loss is similar to that re-
`ported by Fox et al.19 and to that found for relapsing-
`remitting MS.12 Whereas these studies are difficult
`to compare because different methodologies to mea-
`sure brain volumes were used, it is important that
`our study of 159 patients with progressive MS
`achieved the same conclusion as the two previous
`preliminary studies15,19 that tissue loss continues to oc-
`cur in the most advanced phases of the disease. This
`agrees with the findings of previous magnetization
`transfer (MT) imaging20,21 and MR spectroscopic22-24
`studies showing that patients with primary and sec-
`ondary progressive MS have reduced MT ratios (MTR)
`and N-acetylaspartate levels both in T2-visible lesions
`and in normal-appearing white matter (NAWM). This
`also fits well with the progressive increase of T1-
`hypointense lesion volumes25 and the progressive de-
`crease of lesion and NAWM MTR26 shown to occur in
`patients with secondary progressive MS.
`We also showed that the rate of brain parenchy-
`mal loss in patients with progressive MS is not influ-
`enced favorably by treating such patients with
`cladribine. On the contrary, atrophy progression ap-
`peared to be worse, albeit not significantly, in pa-
`tients treated with the higher dose of cladribine.
`Although this may just be a chance finding, it sug-
`gests that high doses of cladribine may affect MS
`evolution negatively. The results of the current study
`agree with the demonstration that cladribine does
`not have any impact on disability and T1-
`hypointense lesion accumulation4,5 and limits the
`value of the encouraging results of two pilot studies
`of cladribine in progressive and relapsing-remitting
`MS2,3 and the observation that cladribine dramati-
`cally reduces the number of enhancing lesions and
`has a moderate, but statistically significant, effect on
`the accumulation of T2 lesion burden.4 The discrep-
`ancy among the effects of cladribine on different
`MRI-derived measures is likely due to the inability
`of the drug to modify the mechanisms leading to
`tissue destruction in MS. MRI enhancing lesions just
`reflect the transiently increased blood– brain barrier
`
`technique described above. Further details regarding scan
`acquisition and post-processing have been reported
`previously.4,5
`The difference in brain volumes among the three pa-
`tient groups and the effect of the two doses of cladribine on
`the absolute and percentage brain volume changes over
`the follow-up period were assessed using the test of
`Kruskal–Wallis. This analysis was also performed consid-
`ering patients with primary and secondary progressive MS
`separately. The correlations between brain volume
`changes and other MRI-derived variables were tested us-
`ing the Spearman rank correlation coefficient.
`
`Results. Demographic and baseline characteristics of the
`patients studied as well as the effect of the two doses of
`cladribine on disability, enhancing lesion number and vol-
`ume, and T2-hyperintense and T1-hypointense lesion vol-
`umes have been reported previously.4,5 For the whole
`population studied, the average brain volumes were 1059
`mL (SE 11 mL) on the entry scans, 1055 mL (SE 12 mL) on
`the scans obtained at month 6 (mean absolute change ⫽
`⫺4.6 mL [SE 4 mL], mean percentage change ⫽ ⫺0.3%
`[SE 0.3%]), and 1050 mL (SE 11 mL) on the scans obtained
`at month 12 (mean absolute change compared to baseline
`⫽ ⫺8.4 mL [SE 3 mL], mean percentage change ⫽ ⫺0.7%
`[SE 0.3%], p ⫽ 0.001). This significant decrease in brain
`volume over time was also observed when only placebo
`patients were considered (p ⫽ 0.04). There was no signifi-
`cant correlation between brain volume at entry and
`changes of brain volume over time. The average brain vol-
`umes and the absolute and percentage volume differences
`between months 6 and 12 and at study entry for the three
`treatment groups are reported in the table. Whole brain
`lesion volumes were similar in placebo and cladribine-
`treated patients on the baseline scans (p ⫽ 0.24). No sig-
`nificant treatment effect of either dose of cladribine on
`brain volume changes over the entire follow-up was ob-
`served (p ⫽ 0.33 for the absolute brain volume change and
`p ⫽ 0.34 for the percentage brain volume change). The
`same was true when treatment effect was assessed over
`the first or the second 6-month periods, or when primary
`and secondary progressive MS patients were considered
`separately (data not shown). In the 54 patients who re-
`ceived placebo, absolute and percentage changes of brain
`volume were not correlated with other MRI measures at
`baseline (enhancing lesion number and volume and T2-
`hyperintense and T1-hypointense lesion volumes) or at
`follow-up (cumulative number of enhancing lesions seen at
`baseline, month 6, and month 12, and absolute and per-
`centage changes of enhancing, T2-hyperintense, and T1-
`hypointense lesion volumes). In addition, no correlation
`was found between the change in brain volume during the
`second 6-month period and either the cumulative number
`of enhancing lesions seen at baseline and month 6 or the
`percentage change of T2 lesion volume during the first
`6-month period. The r values for all these correlations
`were always lower than 0.16.
`
`Discussion. The three main results of this study
`are 1) the confirmation in a large cohort of patients
`that significant brain parenchymal loss occurs even
`over a short period of time in progressive MS, 2) the
`demonstration that cladribine at 0.7 and 2.1 mg/kg is
`not able to alter this process significantly, and 3) the
`1716 NEUROLOGY 55 December (1 of 2) 2000
`
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`

`

`results of a recent study with interferon beta-1a in
`relapsing-remitting MS12 give support to this hypoth-
`esis. Despite the immediate effect of interferon
`beta-1a in reducing enhancement,36 a significant
`treatment effect on brain parenchymal loss was seen
`only during the second year of treatment. Finally,
`enhancing lesions might be an MS epiphenomenon,
`which can be suppressed by the lymphocytotoxic ac-
`tivity of cladribine without interfering with more
`fundamental mechanisms of the disease.
`Previous studies of relapsing-remitting MS11,15
`also did not find significant correlations between
`brain volume and T2 lesion volume changes. The
`current study confirmed the absence of such a corre-
`lation in patients with progressive MS and also
`showed that the change in brain volume does not
`correlate with change of T1 lesion volume. The T2
`lesion load represents the total amount of MRI-
`visible lesions and the T1 lesion load provides a mea-
`sure of the severity of the intrinsic tissue damage
`within such lesions.37 All of this indicates that tissue
`loss in progressive MS is, at least partially, indepen-
`dent from the amount of macroscopic lesions and
`suggests that the pathology occurring in the NAWM
`might be relevant in this regard. There is a large
`body of evidence emerging from MT imaging and
`MRS studies suggesting that NAWM changes are
`prominent in the progressive forms of the disease20-23
`and one study showed that the average MTR of the
`entire brain tissue appearing normal on conventional
`scans is strongly correlated with the size of the
`brain.20
`The results of this study have implications for de-
`signing and monitoring clinical trials in MS that go
`beyond the demonstration that cladribine does not
`modify the rate of tissue loss in progressive MS. The
`lesson to be taken from this study is that the ability
`of a drug to reduce the amount of enhancing lesions
`and the accumulation of T2 lesions in MS is not
`necessarily translated into a beneficial effect on the
`mechanisms leading to tissue loss and, ultimately, to
`irreversible neurologic disability. At present, virtu-
`ally all treatment trials in MS are monitored with
`dual-echo and post-contrast T1-weighted scans. Our
`results call for the incorporation in MS clinical trials
`of other MRI measures with the potential to monitor
`the most severe aspects of the disease (i.e., irrevers-
`ible demyelination and axonal loss). This seems to be
`particularly important in trials of patients with pro-
`gressive MS, where this study showed that there is a
`dissociation between the development of brain atro-
`phy and MRI-visible disease activity.
`
`Acknowledgment
`The authors thank the investigators of the Cladribine Clinical
`Study Group (the complete list can be found in reference 4) who
`acquired the MRI scans used for the current analysis.
`
`References
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`
`December (1 of 2) 2000 NEUROLOGY 55 1717
`
`permeability and inflammation and T2-weighted im-
`aging provides nonspecific information about the
`pathologic substrate of MS lesions, whereas progres-
`sive brain volume reduction is conceivably related to
`loss of neurons, axons, oligodendrocytes, and myelin
`sheaths. Admittedly, histopathologic studies27,28 dem-
`onstrated large numbers of transected axons at the
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`transected at the site of inflammation and when a
`measurable amount of tissue is actually lost. The
`
`Downloaded from https://www.neurology.org by Sally Charin on 29 January 2024
`
`

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