Another approach is the two-arm randomized cross-over trial. Each patient serves as his or her own control, and the concurrent parallel arm allows comparison of placebo versus treatment in the population. For this design to be effective, the wash-out interval must be sufficient relative to the duration of the pharmacologic effect. Unfortunately, this interval is rarely known at the time of the study, and it can be surprisingly long.[130]
Nevertheless, this design has considerable strength related to its inherent property of allowing statistical comparison between individual patients and between groups.
The third classical trial design is the parallel-group trial, which is also used in the setting of the phase III trials. One important modification of the parallel-group design is the use of additional baseline studies, a run-in series before randomization, which has the beneficial effect of reducing sample size.[60]
Fig. 6.6 Enhancing lesion profile in an individual patient over 104 months.
Contrast-enhancing lesions in a relapsing—remitting MS patient who was entered into three separate baseline-versus-treatment trials at the National Institutes of Health in the USA. The patient was initially entered into a study evaluating cyclosporine A (CSA), which had some effect on decreasing the frequency of enhancing lesions. Following a wash-out period, the patient returned to his natural history baseline enhancing lesion frequency of 11 lesions per month. At month 35, the patient was started on interferon (IFN) beta-1b and
responded to therapy for 18 months, at which time he developed severe
depression and suicidal ideation, and IFN beta-1b was terminated.
Approximately 6–8 months elapsed before the patient returned to his
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natural history baseline lesion
frequency. The patient entered into an early phase II trial of altered peptide ligand and after 2 months on therapy experienced a severe exacerbation accompanied by an increase in number of enhancing lesions, from 20 lesions to more than 90 lesions, which exceeded the predetermined stopping rule for this phase II trial. The patient was treated with intravenous
corticosteroids and started on standard therapy. From Frank and McFarland,
[120]with permission.
active treatment (Fig. 6.6). One strategy is to compare the enhancing lesion profile of individual patients based on their monthly MRI scans to look for greater than expected increases in activity as an indication of an adverse effect. An increase in an individual patient’s count of enhancing lesions could also be compared with enhancing lesion activity in an untreated cohort or with historical data. This is a practical approach within a trial setting because of the relative ease and speed in which counts of enhancing lesions can be performed by experienced imagers.
Using enhancing lesions to select for greater disease activity
A common strategy in phase II clinical trials is to use enhanced MRI to select patients with greater disease activity or ‘active disease’, based on one, two or three MRI scans.
The advantage of such preselection is that it minimizes potentially uninformative scans generated in studies from patients who are likely to show no new disease activity. Ideally, enhancing lesions on consecutive studies (e.g. three of three) or on a majority of consecutive studies (e.g. two of three) would provide the most informative appraisal;[68]
however, one common strategy is to permit trial entry after a single positive MRI scan in consecutive MRI evaluations over monthly intervals. In addition to minimizing uninformative scans, recruitment of only active patients may have a secondary advantage of reducing intra-patient variability. Unfortunately, preselection has the undesirable effect of making the results less generalizable to potential treatment recipients with less disease activity. This may not be a critical problem, since positive phase II trials would be followed by definitive phase III studies using a clinical outcome. Another disadvantage of preselection is the increased chance of regression to the mean, which can confound interpretation of the results, depending on the study design.
Multiple studies have established that enhancing lesions are the strongest predictors of clinically definite MS in patients with a clinically isolated syndrome.[95–98] The conversion to clinically definite MS is probably the consequence of greater activity in the population with enhancing lesions; consequently, a strategy can be developed for
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selecting a more active patient group based on the presence of enhancing lesions after a clinically isolated syndrome.
Statistical considerations
Several reports have addressed optimal sample size for MS trials using enhancing lesions as a marker, and guidelines for sample size have been published for each of the major trial designs.[60,61,68,121,123–125,134]
Conservative sample size estimates have been recommended, since the predictive analyses have been based on relatively small data sets, which may include a mixture of patients at various stages of disease. The optimal statistical methodology for analysing enhancing lesions has been considered.[135] Because new enhancing lesion counts in MS are not normally distributed, nonparametric methods have been used in phase II studies, whereas standard approaches based on normal approximations have been used in larger phase III studies. Specific parametric models for new enhancing lesion counts in MS have recently been shown to be applicable to MS trials.[134,136]
MRI as a post-selection, pre-randomization strategy
The number of enhancing lesions can be used in post-selection, pre-randomization procedures to insure balanced stratification of study arms.[137] Ensuring balance in treatment groups can be important because the small sample sizes in phase II trials may not result in equivalent treatment arms. This can significantly complicate study analysis.
Imbalance in enhancing lesion activity between treatment arms can even have an impact in large populations in phase III trials, since studies demonstrate that an important factor in the development of new T2-lesions, new enhancing lesions, and T2-lesion volume is the number of enhancing lesions at trial study entry.[75] While a post hoc factor analysis may be applied, this is less satisfactory than achieving a balance before randomization.
The effect of enhancing lesions on subsequent activity can be dramatic. In one large prospective trial, untreated patients presenting with enhancing lesions showed a median 2-year change in T2-hyperintense lesion volume of 2.98 ml compared with only 0.67 ml for patients with no lesions on their initial MRI study, and the enhancing lesion rate was about five-fold greater for untreated patients with enhancing lesions on their baseline MRI study.[75] Consequently, imbalance at entry may mask detection of a significant treatment effect.
Equivalence of counts of enhancing lesions and counts of T2 lesions
Given the relationship between acute enhancing lesions and acute T2-hyperintense lesions, there is overlap in using these two outcomes for determining disease activity in trials. The correlation between new T2 lesions and new enhancing lesions is strong in series in which MRI is conducted weekly,[24] monthly,[127,138] and every second month.[72]
In theory, counts of T2 lesions could replace counts of enhancing lesions in studies with monthly MRI, after baseline inflammatory activity has been established with enhancing lesion analyses, however with some cost due to decreased new lesion detection. In one series, 15% of new (T2) lesions were missed because of their small size and another 5%
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were missed because of their periventricular location.[127] The converse situation, in which a new lesion is detected on a T2-weighted image but not detected as an enhancing lesion on monthly studies, is less likely, but may occur in primary progressive MS, which is characterized by a lower degree of focal inflammatory activity.[139] The benefits of replacing counts of enhancing lesions with counts of T2 lesions are the cost savings and the avoidance of intravenous injections. However, more experienced, and optimally expert readers are required for counting new T2 lesions compared with those needed for counting enhancing lesions, although the latter requires careful training and standardization as well. A strategy of determining new lesions by T2 lesion counts rather than enhancing lesions will be more successful in early MS, in which lesions rarely overlap anatomically, and less successful in more advanced relapsing MS, in which confluent and closely spaced T2 lesions make counts of T2 lesions less reliable. For trials in which the scan interval is 6 months or longer, counts of new T2 lesions provide a good index of interval activity, whereas counts of enhancing lesions would provide an index of inflammatory activity only around the time of the MRI scan.
IMAGING METHODOLOGY FOR ENHANCING LESIONS
Many MRI acquisition methodologies suited to displaying enhancing lesions are available, and most of these techniques can be readily accomplished (Table 6.1). As described above, the magnetic resonance contrast agents used to assess the blood-brain barrier are gadolinium chelates, which are visualized by MRI in the CNS on the basis of their paramagnetic effect. The gadolinium chelates affect the T1 relaxation time of the innumerable water molecules in their vicinity, shortening it from seconds to several hundred milliseconds, and this provides a mechanism for separating and observing their anatomic
Table 6.1 Summary of imaging methodologies for enhancing lesions
Imaging Technique Advantages Disadvantages Standard T1-weighted Gold standard
Minimal cost Maximal compliance
Sensitivity not optimized
Standard T1-weighted plus magnetization transfer
Increase in number of lesions detected
False-positive lesions (blood flow-induced motion artifacts)
Need pre-magnetization transfer image
Variation in technique among magnetic resonance instruments
Standard T1-weighted plus delayed imaging
Increase in number of lesions detected Fewer false-positive vascular
enhancements
Increased imaging time (and therefore cost)
Edges of lesions may be ill-defined since contrast diffuses outwards Measures of gadolinium enhancement in multiple sclerosis 121
Standard T1-weighted plus triple-dose contrast
Combination of above (optimized protocol)
Increase in number of lesions detected Optimal increase in number of lesions detected
Direct purchase cost Potential for increase in false-positive lesions Cost
Imaging time
Potential increase in false-positive scans
May not have a meaningful impact on sample size
distribution. To observe this distribution, images are created with a set of MRI pulse parameters biased towards display of these short T1 relaxation time water molecules (T1-weighted images). Short T1 fractions appear bright or hyperintense relative to other water fractions.[140]
For clinical trials, T1-weighted imaging is most commonly accomplished using a classic two-dimensional spin-echo technique with short repetition time and short echo delay time. Imaging is based on slices that are 3 mm or 5 mm thick, without gaps and with a pixel size (plane resolution) of 1 mm×1 mm or less. Good signal-to-noise ratios are achieved using this approach, particularly with increased averaging, since the signal-to-noise ratio in two-dimensional imaging is proportional to the square root of the number of excitations. An alternative method is the use of a three-dimensional T1-weighted acquisition, based on a spoiled gradient echo methodology or magnetization-prepared gradient echoes, both of which allow thinner slices (or, in three-dimensional terminology, partitions).[141,142] With a three-dimensional acquisition, partitions are typically 1–2 mm with no gaps, with a good signal-to-noise ratio since signal-to-noise in a three-dimensional acquisition is proportional to the square root number of slices, which is relatively high. An advantage of three-dimensional acquisition is that it allows nearly seamless post hoc image reconstruction in any scan plane, which may help in image evaluation. A disadvantage is the greater transmission of motion-related noise through the image.
Other MRI factors can be introduced into the imaging paradigm to increase the conspicuity of enhancing lesions. One common approach is the use of a magnetization transfer pulse.[143,144] Here, an additional radiofrequency pulse, which is offset from the resonance frequency of free water and does not directly affect the free water, is added to the standard magnetic resonance pulse sequence to influence the bound water fraction (i.e. water interacting with protein, lipid, and membranes in general). The effect of this pulse when seen by bound water fractions is a transfer of magnetization to the free water fractions and a subsequent decrease in the measurable water signal, which is most extreme in intact CNS tissue. As a result, areas of disruption to the blood-brain barrier that ‘leak’ and are enriched for gadolinium chelates, will show relatively greater signal intensity than normal tissues and their surrounding tissue-containing lesions. This strategy can be used to increase sensitivity to abnormal contrast enhancement. There are disadvantages to using magnetization transfer pulses as a strategy to increase the detection of enhancing lesions. Because the magnetization transfer pulse causes relatively increased signal in some regions of normal brain (e.g. basal ganglia) and abnormal brain (e.g. chronic lesions), it is critical that the postcontrast images are always compared to precontrast images. In addition, the magnetization transfer images are sensitive to normal
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blood pools (intravascular blood) and as a result flow-related ghost artifacts may be strong and may confound interpretation. Magnetization transfer pulse sequences vary between magnetic resonance instruments, more so than conventional sequences, and there is evidence of increased interobserver error using magnetization transfer pulse sequences with, for example, single-dose contrast as compared with conventional imaging combined with triple-dose MR contrast.[145] Nevertheless, contrast-enhanced imaging based on magnetization transfer pulses are occasionally used today in MS clinical trials and, with attention to the potential problems, they can be a reasonable trials methodology.
An increased dose of contrast also increases the relative signal intensity of enhancing lesions, since dose and signal are nearly linearly related for normal and abnormal tissues in the brain. Unfortunately, dose and cost are linearly related as well. Triple-dose contrast increases the number of enhancing lesions in relapsing and secondary progressive MS, of the order of 25–75% in most studies. Triple-dose contrast increases lesion contrast, and to a lesser extent the fraction of patients with enhancing lesions.[146,147] In primary progressive MS, where lesion detection is generally poor with conventional approaches, triple-dose contrast can be especially beneficial,[148] although this has not been so in all studies.[106] There is a good safety profile for high-dose (triple-dose) contrast,[149] as there is for multiple repeated single doses of MR contrast.[150]
A third approach to improving detection of contrast-enhanced MS lesions is the use of delayed imaging after injection. Here, rather than obtaining an image immediately after injection, which is the relatively poor standard adopted from clinical practice, the T1-weighted image acquisition is delayed for 5–6 minutes or longer beyond the completion of the intravenous injection. There is a positive relationship between signal intensity and time for enhancing MS lesions, with signal intensity increasing for as long as 20–60 minutes in many lesions. Apart from the expense associated with increased imaging time, the benefits of long delays may be offset by blurring of the edges of lesions caused by diffusion of contrast agents away from lesions.
Combined approaches to optimizing enhancing lesion counts
It is clear that higher doses of magnetic resonance contrast, magnetization transfer pulses, or delayed imaging may all individually increase the yield of contrast-enhancing lesions in clinical trials. While not necessarily fully additive, in combination these techniques consistently improve lesion conspicuity and increase the number of enhancing lesions and the percentage of patients with enhancing lesions.[143,151] Recently, Silver et al.
evaluated conventional imaging with a standard magnetic resonance contrast dose compared with a modified protocol based on delayed imaging (40 minutes), the addition of a magnetization transfer pulse, and triple-dose contrast.[143] In a study of eight patients with relapsing MS and eight patients with secondary progressive MS, the modified protocol increased the total number of brain lesions by a factor of 2.17 and the number of new enhancing lesions by a factor of 1.6. However, there was only a 7% increase in the proportion of active scans. Enhancing spinal cord lesions were observed in 22% of cases with the modified protocol, compared with 16% with the standard protocol. Despite the increased yield by these methodologies, the effect on sample size in clinical trials may be inconsequential. Silver et al. found with their modified, optimized protocol that, although the sample size could be decreased in a cross-over study, the benefit was not apparent for
Measures of gadolinium enhancement in multiple sclerosis 123
a parallel-group design.[143] Finally, any method that increases lesion yield may increase the false-positive rate, particularly for a less experienced imager. This problem is mitigated when using delayed imaging, because enhancement of vascular structures is less problematic.
Accuracy of counts of enhancing lesions
One of the major benefits of counts of enhancing lesions in MS clinical trials is the ease with which these counts can be made by an experienced imager. However, few studies have addressed the issues of accuracy, inter- and intraobserver error, and reproducibility over time. In one detailed study by Barkhof et al., there was 100% agreement between observers for scans with no activity.[152] For scans with one or more lesions, there was agreement as to presence of lesions in 96% of the observations. Agreement on the number of lesions decreased with increasing numbers of lesions, however, the agreement rate was 80% for scans with five lesions or fewer.
Enhancing lesion volume is also used as a trial measure. In practice, enhancing lesion number and volume are highly correlated but, in theory, treatment may have a differential effect on these two outcomes. Enhancing lesion volume can be determined by relatively simple image processing methodologies, which can be semiautomated.[56,57,153]
As for T2 lesion volume measures, training and predetermined rules are important.[154]
Counts of enhancing lesions based on imaging of the spinal cord and optic nerve
The enhancement patterns in the spinal cord are similar to those in the brain, the key difference being a reduced number of lesions compared with the brain, proportional to the volume of these structures.[143,155,156]
Imaging the spinal cord for enhancing lesions is complicated by its small size and by artifacts induced by adjacent cerebrospinal fluid and cardiac or respiratory motion transmitted over the cord. The approaches that have been applied to brain imaging to increase the yield of enhancing lesions also increase the yield in cord imaging.[143,157] However, the small gain derived from adding spinal cord imaging to MS trials may not justify its inclusion, even with optimized methodology, because there is no significant impact on sample size when lesion number is the outcome.
Optic nerve imaging is useful for understanding the pathophysiology of MS and for correlative electrophysiologic, clinical, and imaging studies, but it has not been used in a formal trial setting.[158] The enhancement pattern in the optic nerve is similar to that in the brain and spinal cord, although enhancement may affect the full thickness of the nerve or be more perineural than central.[159]
EFFECT OF TREATMENT ON ENHANCING LESIONS
The results of clinical trials with multiple pharmaceutical agents are discussed in detail elsewhere in this book. The use of enhancing lesions, apart from being relatively simple, reproducible measures of disease activity, has become the standard in phase II and phase III trials, in part as a result of the success of several interventions that include
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inflammatory properties. While the list of agents showing this effect is increasing, the next generation of neuroprotective agents may have less of an effect on the inflammatory aspects of the disease, and their effect on enhancing lesions may be inconsequential in some cases despite important impact downstream of the inflammatory events in MS.
The three major interventions to date with predominantly anti-inflammatory effects, the so-called ABC drugs, have been the subject of extensive study, yet their specific mechanisms of action (including their interaction at the level of the blood-brain barrier) are not well understood. The beta-interferon products show a consistent effect in decreasing enhancing lesion frequency and the percentage of positive scans,[75,160] evident in monthly MRI studies,[127,161] and effects that can be seen within weeks after initiating therapy.[162] The biological wash-out period has been evaluated in a pilot study using monthly MRI scans, and it appears to be on the order of about 6–10 months, since baseline MRI activity was restored over this interval in a study of two patients using interferon beta-1b.[130] Suggestions of wash-out effects have also been seen in phase II studies of alpha interferon.[163,164] Glatiramer acetate also suppresses enhancing lesions, but with a different pattern than that of the beta interferons. The effect demonstrated using monthly MRI is delayed for several months, then increases to maximum benefit after an interval of about 4–6 months.[79] The beta interferon products are believed to influence the integrity of the blood-brain barrier and indirectly enhancing lesions by way of mechanisms that include peripheral regulation of T-cell activation, down-regulation of adhesion molecule expression on endothelial cells, and inhibition of proinflammatory cytokine and matrix metalloproteinase secretion.[127,162,165,166]
Many of these effects may be similar to those of corticosteroids.[30]
Other immune-suppressing agents may have strong and sustained effects on enhancing lesions, and counts of enhancing lesions provide a convenient measure of the magnitude and duration of effect.[167,168] A note of caution. In theory, with profound immune cell suppression it is possible that the typical imaging features of enhancement may be masked, despite continued injury. Consequently, reliance on enhancing lesion rates alone as a measure of efficacy may fail to provide a reliable measure of disease activity.
The corticosteroids penetrate the blood-brain barrier and achieve high concentrations in the cerebrospinal fluid within about 6 hours.[169] The response to corticosteroids alone may occur within minutes to hours of administration. In one study, the blood-brain barrier was shown to be restored within 8 hours of treatment.[170] Consequently, the interval between corticosteroid administration and acquisition of an enhanced MRI scan is an important factor in MS trials and one that can influence results. The effects of corticosteroids on enhancing lesions can be sustained over weeks and months although the effect on individual lesions may be transient, over days.[171] Interestingly, while corticosteroids either alone or in combination with other therapy have transiently decreased enhancing lesions, they do not prevent some of the natural sequelae of enhancing lesions such as their T2 footprints.[172,173] However, it can be shown using magnetization transfer ratio measurements that corticosteroids reduce tissue damage and promote lesion recovery.[30] As with the beta interferon preparations, corticosteroids may close the blood-brain barrier by down-regulating adhesion molecule expression and inhibiting proinflammatory cytokine and matrix metalloproteinase secretion.[30,174–176]
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THE FUTURE
Counts of enhancing lesions are now well-established, verified outcome measures for MS clinical trials. Their success has been in part a fortuitous result of a prominent impact on enhancement by established MS treatments, trials of which have demonstrated that counts of enhancing lesions can provide direct, quantitative, and objective evidence for pharmacologic reduction in the inflammatory activity in the CNS. Enhancing lesion measures are likely to remain important in screening studies and definitive phase III trials, and they may become standard safety measures in phase I and phase II trials. It is important to recall, however, that absence of a significant effect on enhancement does not provide evidence that a pharmacologic intervention is ineffective in MS. This scenario would be anticipated in trials of neuroprotective agents, for example. Equally important, a significant effect on enhancement alone does not establish efficacy, although to date discordance between effect on enhancing lesions and other indicators of benefit have been rare. In the future, more sophisticated imaging techniques that provide improved imaging of the inflammatory processes in MS may prove complementary and will provide incremental information relative to the simple enhanced lesion methodologies described here. In order for us to benefit from these emerging technologies, studies are needed that will address specific pathophysiologic mechanisms that underlie enhancement, the details of the blood-brain barrier at cellular and molecular levels, and factors bridging these events that link the inflammatory enhancing lesion and the ultimate clinical outcome.
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