Relationship of Urinary Myelin Basic Protein–Like Material With Cranial Magnetic Resonance Imaging in Advanced Multiple Sclerosis FREE

MANY IMPORTANT advances have been made in recent years in the clinical management of patients with multiple sclerosis (MS). Among those are the increased accuracy and certainty of diagnosis,^{1 - 2} recognition of clinical subtypes,³ and the introduction of 2 types of immunomodulatory agents, type 1 interferons and glatiramer acetate, which have been shown to improve the natural history of MS by reducing the number of relapses^{4 - 6} and slowing progression⁶ of relapsing-remitting (RR) MS and slowing progression of secondary progressive (SP) MS.⁷ Numerous trials with a number of agents are now in progress or being planned. This new stage in managing MS has increased the awareness of the need to be able to conduct clinical trials more rapidly and reliably and to monitor patients with MS to determine treatment failure. The latter is particularly important, since RR MS changes to SP MS and the patient becomes increasingly disabled.

A surrogate marker is defined as a nonclinical assessment that may predict ultimate clinical change.⁸ Among the various procedures that might be used as such a marker in MS, cranial magnetic resonance imaging (MRI) has been firmly established as a noninvasive means to aid in the diagnosis and gauge the dynamic changes occurring in MS.⁹ Although gadolinium-enhancing T1 lesions on cranial MRI imply active phases with disruption of the blood-brain barrier with perivenular inflammatory collections,¹⁰ correlations with progression of disease have been more difficult to ascertain. For example, spatial mapping of T2 and gadolinium-enhancing T1 lesion volumes does not appear directly linked and suggests that progressive gliosis and wallerian degeneration may occur without an inflammatory disruption of the blood-brain barrier.¹¹ Attempts to measure a variety of changes on cranial MRI and loss of central nervous system tissue volume accompanying atrophy are addressing the onset and progression of disability in MS.^{12 - 13} The presence of decreased signals, commonly referred to as black holes,¹⁴ on T1-weighted images and cervical spinal cord atrophy in the upper segments¹⁵ appear to be the best neuroimaging correlates of disability, and inferentially progression, of MS. Diminished N-acetylaspartate detected by magnetic resonance spectroscopy relates directly to disease disability and progression^{16 - 18} and correlates with the severity of the hypointense T1 lesions.^{19 - 20}

Urinary myelin basic protein–like material (MBPLM) also correlates with disease progression.^{21 - 24} Urinary MBPLM has been used to designate an immunoreactive substance(s) detected by antibodies reactive with MBP. The major chemical component of urinary MBPLM has recently been identified as p-cresol sulfate.²⁵ It is known that MBPLM in urine represents material that (1) cross-reacts with a cryptic epitope in MBP peptide 83-89²² ; (2) is normally present in low levels in neonates that rise above adult levels in childhood²⁶ ; (3) is normal in RR MS but elevated in SP MS and, to a lesser degree, in primary progressive MS^{23 ,27} ; (4) does not correlate with disease activity in MS²² ; (5) when elevated, correlates with a transition to the SP phase of MS from RR MS^{23 - 24} ; and (6) when elevated, correlates with lesion number and volume of T2-weighted central nervous system lesions manually identified and quantitated on 0.15-T cranial MRI.²³

The predictive value of the level of urinary MBPLM in a large prospective trial was planned as an "add-on" study in the multicenter Linomide (roquinimex) trial on RR and SP MS.²⁸ In this article, the results at enrollment of that investigation are described with evidence presented to demonstrate that urinary MBPLM, alone or after adjustment for creatinine (MBPLM/Cr), correlates well with black hole volume (BHL VOL) detected on T1-weighted cranial MRI, especially in SP MS and, more specifically, in those without relapses and with more advanced disease.

The North American Linomide Study was a phase 3 trial of patients with RR and SP MS conducted in 27 centers in a randomized, double-blind, placebo-controlled and multidose fashion.^{28 - 29} Of 718 patients entering the trial, 24-hour urine specimens were obtained from 662 patients at enrollment (Table 1). The study population was 95.2% white and 4.8% nonwhite, with a female-male ratio of 1.9:1. Of the 662 patients, 86 had RR MS, and 576 had SP MS.³ Of the group with SP MS, 317 were noted to have accompanying relapses and 259 to have no relapses. All patients were scored as to disability on the Expanded Disability Status Scale (EDSS).³⁰

Cranial MRIs were performed at the time of enrollment. All patients underwent imaging (Signa 1.5-T scanner; General Electric, Milwaukee, Wis) using version 5.4 or higher operating system, according to a defined protocol.²⁹ Sequences were applied to obtain T2-weighted, fluid-attenuated inversion recovery and magnetization transfer, T1-weighted with gadolinium, magnetic resonance angiography, and postgadolinium T1-weighted images.²⁹ Automated segmentation analysis was performed, and a composite score was derived.³¹ Low-signal intensity lesions, designated as "black holes," were determined by expert identification and local thresholding on postgadolinium T1-weighted magnetization transfer images.²⁹ Black holes measure a heterogeneous population of lesions. They were segmented based on having an intensity less than that of the normal-appearing white matter and greater than that of cerebrospinal fluid on the post–T1-weighted images. Black holes appear to include the most permanent and least reversible tissue destruction, especially when gadolinium-enhanced tissue is excluded as was done in this study.²⁹ As noted,²⁹ determination of BHL VOL was not part of the original study design, and these determinations on the MRI data obtained were not initiated until well after the study was begun.

In an attempt to compile the various abnormalities detected on cranial MRI, an MRI composite score, designated as the composite Z4, was determined.²⁹ The Z4 was derived from the volume of enhanced tissue, the normalized plaque volume, the normalized cerebrospinal fluid volume, and the BHL VOL. The more positive the Z4 number, the worse the subject is on MRI relative to his/her peers.

At the time of preenrollment, a 3-L plastic bottle was issued to the patient, who, during the 24 hours before the enrollment or second study visit, collected his/her urine. The total volume and duration of collection were determined, and, after thorough mixing, an aliquot of 25 mL of urine was placed in a plastic container (Boritex; Fisher, Suwanee, Ga) with 1 boric acid tablet. The vial was sent to the central laboratory of Quintiles, the contact research organization for the trial, stored frozen at −20°C, and subsequently shipped frozen to the laboratory of the primary author (J.N.W.) for analysis. Studies were conducted (data not shown) to demonstrate that this processing of urine did not alter the quantitative results of urine MBPLM.

Urinary MBPLM was determined by a double-antibody radioimmunoassay in which radiolabeled human MBP peptide 69-89 served as the radioligand, rabbit (R110) anti-MBP served as primary antibody, and human MBP peptide 83-89 served as assay standard.^{21 - 23} The performance, validation, and variation of this radioimmunoassay have been described elsewhere.^{22 - 23} Urinary MBPLM was expressed as nanograms per milliliter of unprocessed urine or as nanograms per milligram of creatinine measured by standard methods.²¹ The statement of MBPLM in relationship to creatinine was to use creatinine as an adjustment for renal function and dilution of urine. Since 24-hour urine collections were made, 24-hour values of MBPLM, designated as total MBPLM, were also calculated.

All correlations were performed on the comprehensive clinical and MRI data set available from the entire study²⁹ and the measurement of urinary MBPLM. Because of the early termination of the trial due to cardiac toxic effects, only a cross-sectional study was performed. Statistics mentioned for the cranial MRI results have been previously reported.²⁹ For the analyses and correlations of the urinary MBPLM data, group and subgroup comparisons were made primarily by using the general linear model approaches of analysis of variance with post hoc comparisons. Correlations were performed using Pearson or Spearman rank procedures where appropriate.

In this cross-sectional study, urinary MBPLM and MBPLM/Cr showed no differences in the population of patients with an EDSS score of 5.0 or lower or an EDSS score of 5.5 or higher, whereas BHL VOL was greater (P = .02) in the group with the higher EDSS scores (≥5.5).

A series of correlations were made for BHL VOL (Table 1) and the MRI composite Z4 score (Table 2) with urinary MBPLM. A weak direct correlation existed between the level of urinary MBPLM and BHL VOL in all patients with MS (Table 1). The fact that the correlations with MBPLM were not evident or as strong with MBPLM/Cr or total MBPLM presumably reflects the lack of linearity of the measurement of MBPLM using a standard of MBP peptide 83-89.^{21 - 22} When analyzed among MS subtypes, this correlation was restricted to those with SP MS, specifically those without relapses, and most significantly when the EDSS score was 5.5 or higher. The later correlation was highly significant regardless of the manner in which urinary MBPLM was expressed (Table 1). The greatest significance was for urinary MBPLM/Cr. The group of patients with SP MS with relapses showed no differences in urinary MBPLM related to EDSS scores of 5.5 or higher or less than 5.5 in regard to T1-weighted BHL VOL.

The population of patients studied was predominantly white, but no racial differences in the correlations were noted (data not shown). For correlations of MBPLM or total MBPLM, there were no differences between sexes; however, for MBPLM/Cr, in the group of patients with SP MS (92 females and 46 males), both females and males showed the same correlations, which were more significant for females. This is presumably related to the known lower body mass and urinary creatinine in females.³²

Although not as strong, similar correlations were noted between the expressions of urinary MBPLM and the MRI composite Z4 score (Table 2). No racial or sex differences were noted. Since BHL VOL is included in the Z4 composite score and since there was no significant correlation of urinary MBPLM and any of the other cranial MRI measurements (data not shown), this weaker relationship of the Z4 score and urinary MBPLM is presumably due to the impact of the BHL VOL component on the composite score. The composite Z4 score (Table 2) but not the BHL VOL measurement (Table 1) showed a weak correlation with MBPLM in patients with SP MS. This significant relationship of Z4 was present only in those with an EDSS score of 5.5 or higher. The correlation with the Z4 score and not with BHL VOL implies an impact of another component, not yet identified, on the composite Z4 score.

No correlations for urinary MBPLM and any of the cranial MRI measurements were detected for the RR MS group.

This investigation revealed that in patients with more advanced MS, that is, those with an EDSS score of 5.5 or higher and without relapses, urinary levels of MBPLM are significantly correlated with BHL VOL hypointense areas on T1-weighted images on cranial MRI. The slightly less strong correlation of urinary MBPLM with the MRI composite Z4 score is most likely the result of the inclusion of other MRI methods, along with BHL VOL, in that composite. These findings, an extension of previous studies relating increased levels of urinary MBPLM to the progressive phase of MS,^{22 - 24 ,27} provide additional evidence for the possible utility of urinary MBPLM to predict or reflect the more disabling form of MS. The recent identification of p-cresol sulfate as the major component of urinary MBPLM should facilitate the development of improved detection methods for MBPLM and other clinical or neuroimaging correlations.

Restriction of a significant correlation between urinary MBPLM and BHL VOL to patients with SP MS with an EDSS score of 5.5 or higher to those without relapses and not to patients with SP MS with relapses implies a pathological difference between the clinical expressions of relapses and progression. The varying combinations of the temporal events of relapses, remissions, and progression constitute the basis for the clinical subtypes of MS currently recognized. Cranial or spinal MRI features of these subtypes have not yet been clarified. The distinction of the relationship of T1-weighted BHL VOL and urinary MBPLM between SP MS patients with and without relapses adds to other unexplained observations of the varied histopathological tissue alterations in MS³³ and the apparent separate processes for T2 and gadolinium-enhancing T1 lesions on cranial MRI.

The multicenter, phase 3 trial of Linomide was well designed to detect beneficial treatment changes in RR MS and SP MS effected by Linomide. Unfortunately, cardiac toxic effects led to termination of the trial shortly after full enrollment.²⁸ Although there was a trend for improvement, especially at the medium dose of Linomide of 2.5 mg/d, persuasive effectiveness of treatment could not be demonstrated with the brevity of the trial.²⁸ Nevertheless, important experience was gained from that trial. As reported elsewhere,²⁹ there was evidence of slowing of progressive accumulation of lesion burden and of gadolinium positivity in patients who were treated with Linomide compared with placebo. In addition, the MRI measurement of BHL VOL also showed an effect of treatment. After 3 months of treatment in this trial, the proportion of lesions categorized as black holes was reduced by treatment, with the effect most prominent for patients with MS taking higher doses of drug (P<.05 for active treatment and P<.03 overall).²⁹

The biological relationship of BHL VOL on T1-weighted MRI appears to be that of axonal loss and central nervous system tissue atrophy.¹⁴ Axonal damage, previously demonstrated in MS but inferred to be less obvious or occurring later in the course of MS, has more recently been shown to be an early accompaniment of inflammatory demyelination.³⁴ The amount of atrophy that must exist in central nervous system tissue to be detected by either a decline in N-acetylaspartate by magnetic resonance spectroscopy^{16 - 19} or by increase in hypointensity on T1-weighted cranial MRI is unknown.¹⁴ It is clear that in normal-appearing white matter the N-acetylaspartate is diminished when no abnormalities are detected on MRI.³⁵ Thus, among the measurements that can be readily made in patients with MS to include magnetic resonance spectroscopy, cranial MRI, and body fluid collections and subsequent measures, there may be a useful and feasible means to monitor changes at different times in the temporal profile of the early, middle, and late phases of MS and to acquire an earlier indication of beneficial or nonbeneficial results of new treatments.

Accepted for publication May 5, 2000.

This investigation was supported by the research program of the Veterans Administration, Washington, DC, and also by Pharmacia and Upjohn, Kalamazoo, Mich.

Preliminary results of this study were presented at the 51st Annual Meeting of the American Academy of Neurology, Toronto, Ontario, April 21, 1999.

Jeanine Goodwin provided excellent technical assistance, and Linda Brent and Denise Ball furnished excellent assistance in the preparation of the manuscript.

MRI–Analysis Center Assistants

MRI–Analysis Center, University of Texas Health Science Center, Houston: Jonathan Carlson, Jennifer Chambers, Brian Decuir, Lucy Mendia, Skipp Slattenow, Tom Thomas.

The North American Linomide Investigators

University Hospital (University of Alabama, Birmingham): John N. Whitaker, MD (clinical principal investigator), Galen W. Mitchell, MD, Christopher C. LaGanke, MD, Beverly Layton, RN, University of Alabama, Birmingham, MR Imaging, Taher El-Gammal, MD (imaging principal investigator), Cleve Crews, Wladyslaw T. Sobol, PhD; Arizona Health Sciences Center, Tucson : William A. Sibley, MD (clinical principal investigator), Scott Sherman, MD, Barbara Geisser, MD, Jean Kunkel-Thomas, MD, Janet Mar, RN, Todd McGregor, University Medical Center MRI, Joachim Seeger, MD (imaging principal investigator), Joseph Berg, Arthur Gmitro, PhD, Bill Ahern; The Bowman Gray School of Medicine, Salem, NC: Douglas R. Jeffrey, MD (clinical principal investigator), B. Todd Troost, MD, D. Leftkowitz, MD, William McKinney, MD, Lorraine Harris, RN, MRI Center, Department of Radiology, Allen Elster, MD (imaging principal investigator), Lisa Smith, Elaine James; Buffalo General Hospital, Buffalo, NY: Lawrence Jacobs, MD (clinical principal investigator), Reza Pordell, MD, Frederick E. Munschauer III, MD, Elizabeth Doherty, MD, Steven J. Greenberg, MD, Susan Krantz, RN, Roswell Park MRI, Henry Z. Wang, MD, PhD, Wendy Zimmer, MD (imaging principal investigators), Carol Kaminski, Richard Mazurchek, PhD, Mark Smerka; The University of Calgary: Luanne Metz, MD (clinical principal investigator), David Patry, MD, Robert Bell, MD, W. F. Murphy, MD, Amanda Pitts, RN, Sandra McGuinness, MN, Magnetic Resonance Imaging Centre, Foothills Hospital, Carla Wallace, MD (imaging principal investigator), Pierre LaForge, RTNM, Ken Bott; The University of California, Davis Medical Center, Davis: Mark A. Agius, MD (clinical principal investigator), David Richman, MD, N. Vijayan, MD, Lee Eun Kyu, MD, Janelle Adams, RN, University of California, Davis Medical Center, MRI Center, Michael Buonocore, MD, PhD (imaging principal investigator), Stephen Hecht, MD, Cindy DuPreeThompson, Lisa Wall, Jose Gacayan, John Tinker, John Ryan, Danna Whitfield, David A. Weber, PhD, Jim Deal; The University of California at Los Angeles: Lawrence Myers, MD (clinical principal investigator), Joanna Girard, MD, Robert Baumhefner, MD, Louis Rosner, MD, Sharon Craig, RN, UCLA MR Imaging Center, John R. Bentson, MD (imaging principal investigator), Valerie Gausche, BRST, Mary Ann Burns, CRT, Angela Wallace, CRT, Shantanu Sinha, PhD; The University of Chicago, Chicago, Ill: Anthony Reder, MD (clinical principal investigator), Avertano Noronha, MD, Barry Arnason, MD, Gwen Jacobs, RN, The University of Chicago Hospital Department of Radiology, Daniel Huddle, DO (imaging principal investigator), Vence Edmonds, Robert Meyers; Georgetown University Medical Center, Washington, DC: John Richert, MD (clinical principal investigator), Carlo Tornatore, MD, Kiren Kresa Reahl, MD, Jorge Kattah, MD, Andrew Pachner, MD, Tara Gustafson, Shady Grove MRI, Robert Isaacs, MD (imaging principal investigator), Joe Previte, Kevin Quinn; University Hospital, London, Ontario: George Rice, MD (clinical principal investigator), George Ebers, MD, Pejjx Wilma Koopman, University Hospital Imaging, Donald Lee, MD (imaging principal investigator), Karen Kennedy, RTNM, Brian Rutt, PhD; Maimonides Medical Center, New York: Aaron Miller, MD (clinical principal investigator), Marshall Keilson, MD, Kersti Bruining, MD, Ellen Drexler, MD, Linda Sciarra, RN, MSc; The New York Hospital–Cornell Medical Center, New York: Brian Apatoff, MD (clinical principal investigator), Barry Singer, MD, Justine Wheatley, RN, Priscilla Periconi, MPA, The New York Hospital–Cornell Medical Center Imaging, Michael D. F. Deck, MD (imaging principal investigator), John A. Markisz, MD, PhD, Michael Aquilia, RT; The University of Maryland Hospital, Baltimore: Christopher Bever, Jr, MD (clinical principal investigator), Kenneth P. Johnson, MD, Omar Khan, MD, Hillel Panitch, MD, Suhayl Jalbut, MD, Eleanor Katz, RN, Cathy Conway, RN, Anna Gudusky MRI Center, Michael Rothman, MD (imaging principal investigator), Erma Owens, Moriel Nessaiver, PhD, Steve Crum; MCP Hahnemann University: Fred D. Lubin, MD (clinical principal investigator), Flo Trantas, RN, Leith Kelly, RN, PhD, Thomas Jefferson University, Philadelphia, Pa: Robert Knobler, MD, Jefferson Imaging–Bala, Carlos Gonzalez, MD (imaging principal investigator), Lynn Adinolfi, BSRT(R), Simon Viniski, PhD, Keith Kodash; Mayo Clinic, Rochester, Minn: John H. Noseworthy, MD (clinical principal investigator), Claudia Lucchinetti, MD, Brian Weinshenker, MD, Moses Rodriguez, MD, Andrea Adams, MD, Mindy Arneson, RN, Mayo Clinic MR Imaging, Bradley J. Erickson, MD, PhD (imaging principal investigator), John Rasmusson, Joel P. Felmlee, PhD, Richard Westlund; Mayo Clinic, Scottsdale, Ariz: Jonathan L. Carter, MD (clinical principal investigator), Richard Caselli, MD, Kathryn J. Hirschorn, MD, Timothy J. Ingall, MD, Alycia Metcalf, RN, Carrie Meshulam, CA, MRI Center, Kent D. Nelson, MD (imaging principal investigator), Kay Dinoncourt, Dan Peterson; The Mellon MS Center–Cleveland Clinic, Cleveland, Ohio: Jeffrey Cohen, MD (clinical principal investigator), Thomas Masaryk, MD, Bianca Guttman, MD, Revere P. Kinkel, MD, Richard Rudick, MD, Patricia Adler, RN, MSN, Lakewood MRI Center, Jeffrey S. Ross, MD (imaging principal investigator), Judy Wilms, RT, Jean Tkach, PhD, Steve Bowers; The University of Minnesota, Minneapolis: Gary Birnbaum, MD (clinical principal investigator), Randall Shapiro, MD, David Knopman, MD, Crispin See, MD, Rosemary Nelson, RN, Midwest MRI, David Kispert, MD (imaging principal investigator), Kimberly Carley, Pat Miller, John Gaughan; Montreal Neurological Institute: Gordon Francis, MD (clinical principal investigator), William Barkas, MD, Yves Lapierre, MD, Rozie Arnaoutelis, Montreal General Hospital, Raquel Del Carpio-O'Donovan, MD (imaging principal investigator), Laurian Rohoman, Christopher Henri, Gennare Durante; UMD New Jersey Medical School, Newark: Stuart Cook, MD (clinical principal investigator), Shalini Bansil, MD, Mary Ann Picone, MD, Annette Jotkowitz, James Quinless, Department of Radiology, Leo J. Wolansky, MD (imaging principal investigator), Janice Comiskey, Wen Ching Liu, PhD; The University of Rochester Medical Center, Rochester, NY: Andrew Goodman, MD (clinical principal investigator), David H. Mattson, MD, PhD, Steven R. Schwid, MD, Eileen Scheid, RN, Department of Radiology, David Shrier, MD (imaging principal investigator), Constance H. White, BSRT, Edmund Wing-Chi Kwok; Rush-Presbyterian-St Luke's Medical Center, Chicago, Ill: Dusan Stefoski, MD (clinical principal investigator), Floyd A. Davis, MD, Karyn Karlin, MD, Jean Rush, RN, Greg Podraza, RN, ARSC–Circle Imaging Center, William Greenlee, MD (imaging principal investigator), Ginny Flynn, RT, Jin-Zhao Wang, PhD, Brad Phillips; St Michael's Hospital, Toronto, Ontario: Paul W. O'Connor, MD (clinical principal investigator), Trevor Gray, MD, Paul Marchetti, MD, Julie Hall, Sunnybrook Health Science Center MRI Centre, Gordon Cheung, MD (imaging principal investigator), Pauline Houston; University Medical Center SUNY at Stony Brook: Patricia K. Coyle, MD (clinical principal investigator), Lauren Krupp, MD, O. Gerber, MD, Carol Doscher, NP, Department of Radiology, Robert G. Peyster, MD (imaging principal investigator), Robert Day, Haifang Li, PhD, Christopher Runz; The University of Texas–Houston, Health Science Center: J. William Lindsey, MD (clinical principal investigator), Staley Brod, MD, Mazen Dimachkie, MD, Emily Cerreta, RN, MSN, Hermann Hospital MRI Department, Larry Kramer, MD (imaging principal investigator), June Garcia, RT, Scot Duncil, RT; Vanderbilt University Medical Center, Nashville, Tenn: Jane E. Howard, MD (clinical principal investigator), Subramanian Sriram, MD, Howard Kirshner, MD, Renee Browning, RN, Vanderbilt University Magnetic Resonance Imaging, Robert Kessler, MD (imaging principal investigator), Richard Paulsen, MD, Ric Andal, Joe Knuutila, Ronald R. Price, PhD, Dan West; Wayne State University School of Medicine, Detroit, Mich: Robert P. Lisak, MD (clinical principal investigator), Alex C. Tselis, MD, PhD, John Kamholtz, MD, PhD, James Garbern MD, PhD, Richard Lewis, MD, Linda Tvardek, RN, Children's Hospital of Michigan, Cristie J. Becker, MD (imaging principal investigator), Barbara Peters, Greg Moore.

External Data Safety Monitoring Committee

Henry McFarland, MD, Chair; Walter H. Carter, Jr, PhD, Charles Flexnor, MD, Stephen L. Hauser, MD, John Petkau, PhD, Stephen Reingold, PhD.

Pharmacia and Upjohn

Per Gjörstrup, MD, Director of Clinical Research; Anders Linde, MB, Study Director; Herman Sullivan, MD, Clinical Program Leader.

Reprints: John N. Whitaker, MD, Department of Neurology, University of Alabama at Birmingham, 625 19th St S, Birmingham, AL 35233-7340 (e-mail: jnwhit@uab.edu).