epstein-barr virus in monitoring the response to therapy of acquired immunodeficiency...

Interferon-g Secretion byPeripheral Blood T-CellSubsets in Multiple Sclerosis:Correlation with DiseasePhase and Interferon-bTherapyBurkhard Becher, PhD, Paul S. Giacomini, BSc,Daniel Pelletier, MD, Ellie McCrea, BSc,Alexandre Prat, MD, and Jack P. Antel, MD

Interferon-g (IFN-g) is implicated as a participant in theimmune effector and regulatory mechanisms consideredto mediate the pathogenesis of multiple sclerosis (MS).We have used an intracellular cytokine staining tech-nique to demonstrate that the proportion of ex vivo pe-ripheral blood CD4 and CD8 T-cell subsets expressingIFN-g is increased in secondary progressing (SP) MSpatients, whereas the values in untreated relapsing-remitting (RR) MS patients are reduced compared withthose of controls. Patients treated with interferon-b(IFN-b) have an even more significant reduction in thepercentage of IFN-g–secreting cells. The finding that thenumber of IFN-g–expressing CD8 cells is increased inSPMS patients, a group with reduced functional suppres-sor activity, and is most significantly reduced by IFN-btherapy, which increases suppressor activity, indicatesthat IFN-g secretion by CD8 T cells and functional sup-pressor defects attributed to this cell subset in MS can bedissociated.

Becher B, Giacomini PS, Pelletier D, McCrea E,Prat A, Antel JP. Interferon-g secretion byperipheral blood T-cell subsets in multiple

sclerosis: correlation with disease phaseand interferon-b therapy.

Ann Neurol 1999;45:247–250

Multiple sclerosis (MS) is considered to be animmune-mediated disorder that results in multifocalsites of demyelination within the central nervous sys-tem. The disease most frequently manifests an initialrelapsing-remitting (RR) course which subsequentlyevolves into a progressive phase. In the animal model

of MS, experimental autoimmune encephalomyelitis(EAE), interferon-g (IFN-g)–producing Th1 myelin-reactive CD4 T cells are used to adoptively transfer thedisease, whereas Th2 cells are not encephalitogenic.1,2

IFN-g has been directly implicated as participating inthe MS disease process based on the observation thatwhen given to MS patients, systemic IFN-g increasesthe frequency of clinical relapse.3 In EAE, however,disease severity can be increased either by systemic ad-ministration of antagonistic IFN-g antibodies or by de-letion of the genes encoding IFN-g.4,5 These resultssuggest that IFN-g can also have a protective role inautoimmune disease. Th1 cells are now recognized tohave relevant autoimmunity properties other than cy-tokine (IFN-g) production, including those related tochemokine responses and migration.6,7 Thus, whetherthe disease-inducing capacity of Th1 CD4 T cells isdirectly related to IFN-g production remains to beresolved.

IFN-g can be produced by other lymphoid cells, in-cluding CD8 T cells and natural killer cells.8,9 As re-gards CD8 T cells, Balashov and colleagues10 reportedreduced production of IFN-g by CD8 T cells of pro-gressive MS patients in an autologous mixed lympho-cyte reaction assay system and correlated this findingwith reduced suppressor function. Suppressor functionmediated by activated mononuclear cells has also beenfound to be reduced in MS.11,16 We subsequently ob-served, however, that this functional defect could bedetected using CD8 T-cell lines from MS patients.12

Interferon-b (IFN-b), the initial approved therapy forMS, augments mitogen-induced suppressor function.13

The purpose of the current study was to combineintracellular cytokine staining with T-cell subset immu-nostaining to evaluate expression of IFN-g in bothCD4 T cells and CD8 T cells derived from peripheralblood of untreated MS patients with either RR or pro-gressive forms of disease or from MS patients treatedwith IFN-b.

Patients and MethodsPatientsMS patients were selected on the basis of having secondaryprogressing (SP) disease (10 women, 1 man; mean age, 47 69 years; mean disease duration, 11.0 6 2.4 years; meanEDSS, 5.5 6 0.4) or RR disease. The latter were furtherdivided into those who were untreated (11 women, 1 man;mean age, 40 6 4 years; mean disease duration, 10 6 4years; mean EDSS, 2.5 6 0.3) or were receiving IFNb-1b (4women, 1 man; mean age, 41 6 4 years; mean disease du-ration, 7 6 4 years; mean EDSS, 2.7 6 0.8). All had con-firmatory magnetic resonance imaging abnormalities consis-tent with MS. None of the patients were having acuterelapses, and none had received corticosteroids in the last 3months. The mean age of subjects in the healthy controlgroup was 29 years.

From the Neuroimmunology Unit, Montreal Neurological Institute,McGill University, Montreal, Quebec, Canada.

Received Jul 6, 1998, and in revised form Sep 1. Accepted for pub-lication Sep 1, 1998.

Address correspondence to Dr Becher, Neuroimmunology Unit,Montreal Neurological Institute, McGill University, 3801 Univer-sity Street, Montreal, Quebec H3A 2B4, Canada.

BRIEF COMMUNICATIONS

Copyright © 1999 by the American Neurological Association 247

MethodsPeripheral blood-derived mononuclear cells (PBMCs) weretreated for 4 hours with PMA (25 ng/ml), ionomycin (1 mg/ml), and Brefeldin A (10 mg/ml) (all from Sigma, Missis-sauga, Ontario, Canada). The cells were then extensivelywashed with phosphate-buffered saline and immunostainedfor 30 minutes with either anti-CD8 monoclonal antibody(mAb) conjugated to phycoerithrine (PE) or IgG1-IgG2a iso-type control conjugated to fluorosein-isothyocyanate (FITC)and PE, respectively (Becton Dickinson, Mississauga, On-tario, Canada). The cells were washed and fixed overnight in4% PFA. The cell membrane was permeabilized with HBSSbuffer containing 0.2% saponin (Sigma) and 2% fetal calfserum and was stained for 30 minutes with anti–IFN-gmAbs conjugated to FITC (Becton Dickinson). Cells wereanalyzed using a FacsScan and Lysis II software (BectonDickinson). The data were analyzed using WinMDI software(Scripps, La Jolla, CA). Statistical analysis comparing the dif-ferent MS groups with controls was by single-factor ANOVAand Dunnet posttest (PrismII).

ResultsExpression of IFN-g–Secreting T Cells in MSPatient-Derived LymphocytesIn initial experiments using ex vivo PBMCs, we foundthat virtually all cells gated as lymphocytes were eitherCD8 or CD4 cells but only rarely were natural killercells (CD561, CD161). Upon activation with PMAand ionomycin, the levels of CD4 expression de-creased, limiting the capacity to perform dual-color

flow cytometry using anti-CD4 mAbs. Figure 1 showsa representative FACS result of PBMCs derived froman SPMS patient when stimulated for 4 hours andstained for CD8 and IFN-g. We further documentedthat the intracellular cytokine staining technique coulddetect shifts in T-cell cytokine polarization by showingthat the proportion of IFN-g–secreting lymphocyteswas significantly increased when cultured for 18 hourswith anti-CD3 mAbs in the presence of recombinantinterleukin-12 (IL-12) or reduced if cultured withanti–IL-12 mAbs plus IL-4 (data not shown).

Figure 2 compares the MS patient subgroups andcontrols with regard to the percentage of IFN-g–secreting cells in the overall lymphocyte population.Values were increased in the untreated SPMS groupcompared with the control group and even more socompared with the RRMS group. The values in theRRMS group were reduced compared with the con-trols. The SPMS group has a wider range of valuesthan the other groups. The mean percentage ofIFN-g1 cells was significantly lower in the IFN-b–treated group compared with all other groups.

When results are analyzed in terms of T-cell subsets,there were no significant differences between groupswith regard to CD4:CD8 ratios (data not shown). Asshown in Figure 3, the mean percentage of CD4 Tcells expressing IFN-g is significantly less than in theCD8 population (p , 0.05). The proportion of CD4T cells expressing IFN-g was significantly increased inthe SPMS patients compared with the RRMS patients(p , 0.05). For CD8 T cells, the percentage of IFN-g–expressing cells was significantly increased in the

Fig 1. Interferon-g (IFN-g) production by CD8 T cells frommultiple sclerosis patients. Representative cytofluorometric anal-ysis of peripheral blood mononuclear cells that were activatedfor 4 hours in the presence of PMA (25 ng/ml), ionomycin(1 mg/ml), and Brefeldin A (10 mg/ml) and then werestained for CD8 (ordinate) and IFN-g (abscissa).

Fig 2. Comparison of percentage of interferon-g (IFN-g)–secreting T cells from multiple sclerosis (MS) patient subgroupsand controls. Cytofluorometric analysis of IFN-g secretingwithin the total lymphocyte population derived from the fol-lowing donor groups: ctrl 5 control, RR 5 nontreatedrelapsing-remitting (RR) MS, SP 5 secondary progressive MS,IFN-b 5 RRMS treated with interferon-b. Data indicateresults from individual donors plus the mean 6 SEM for eachsubgroup. #p , 0.05, *p , 0.01 (compared with controlvalues).

248 Annals of Neurology Vol 45 No 2 February 1999

SPMS patients compared with the control group (p ,0.01), whereas values in the RRMS group were de-creased compared with the control group (p , 0.05).In the IFN-b–treated group, the percentage of IFN-g1

cells was significantly reduced (p , 0.01) in bothT-cell subsets compared with both untreated patientsand controls.

DiscussionIn this study, we have used intracellular cytokine stain-ing to assess IFN-g cytokine secretion by CD4 andCD8 T-cell subsets contained in peripheral blood ofMS patients and have correlated the results with clini-cal disease phenotype and effects of IFN-b therapy.The intracellular cytokine staining technique used inthis study assessed the original ex vivo population inthat no significant cell proliferation is required and iswell suited to identify the cell source of the cytokine.

Our data indicating significant differences in IFN-gexpression in lymphocyte populations derived from MSpatients in the SP compared with the RR phase of thedisease could reflect on the potential contribution ofthis cytokine to the susceptibility and course of MS.The increased proportion of IFN-g–expressing lym-phocytes detected by intracellular cytokine staining inSPMS patients is consistent with reports of increasedIFN-g production by mitogen, and anti-CD3 antibod-ies stimulated PBMCs in vitro in this population.14,15

The increases could reflect the effects of the Th1 po-larizing cytokine IL-12, whose production by mono-

cytes is increased in this patient group.15 Whether theincreased production of IFN-g underlies the transitionof MS from the RR phase to the progressive phase re-mains speculative. The wider range of values found inthe SPMS group may reflect a heterogeneity of diseasewithin this group.

Our findings regarding reduced IFN-g expression inRRMS patients compared with healthy controls are inaccordance with previous studies using short-term acti-vation of MS PBMCs.16 A number of studies havenow reported reduced IFN-g in short-term cultures ofmitogen or anti-CD3–activated mononuclear cells de-rived from RRMS patients.17,18 Whether the reducedIFN-g levels in the RRMS patients reflect a suscepti-bility factor analogous to the effect of depleting sys-temic IFN-g in the EAE model remains unanswered.4,5

Among its multiple actions, IFN-g has been impli-cated as being the molecular mediator of CD8 T-cellfunctional suppressor activity.10 As regards SPMSpatients, functional suppressor defects have been demon-strated using multiple assay systems.10,12 Our current re-sults of increased IFN-g production by CD8 T cellsfrom such patients would not suggest a correlation be-tween these suppressor functions and IFN-g production.

Our observation that IFN-b therapy downregulatesIFN-g expression in both CD4 and CD8 T cells isconsistent with that of Crucian and co-workers,19 whoapplied a similar intracellular cytokine staining proto-col to unfractionated PBMCs derived from IFN-b–treated MS patients.19 Similar results have been foundin in vitro assays dependent on cell proliferation.20–22

Our finding that IFN-b reduced IFN-g expression inCD8 T cells together with the report that IFN-b ther-apy augmented mitogen-induced suppressor activity13

would further support the postulate that defects inIFN-g production by CD8 T cells and suppressorfunction are not directly linked. The combined intra-cellular cytokine/cell subset detection assay can be ex-tended to analysis of multiple cell subsets and cytokinesso as to permitmore detailed analysis of the apparentlycomplex immunoregulatory networks that participatein the MS disease process.

Dr Becher has a fellowship from the German Academic ExchangeService (DAAD-HSPIII). This work was supported by a grant fromthe Multiple Sclerosis Society of Canada and the MRC.

References1. Ando DG, Clayton J, Kono D, et al. Encephalitic T cells in the

B10.PL model of experimental allergic encephalomyelitis (EAE)are of the Th-1 lymphokine subtype. Cell Immunol 1989;124:132–143

2. Racke MK, Bonomo A, Scott DE, et al. Cytokine-induced im-mune deviation as a therapy for inflammatory autoimmune dis-ease. J Exp Med 1994;180:1961–1966

3. Panitch HS, Hirsch RL, Schindler J, Johnson KP. Treatment ofmultiple sclerosis with gamma interferon: exacerbations associ-

Fig 3. Comparison of percentage of interferon-g (IFN-g)–secreting CD8 and CD4 T cells within each subset. Cytoflu-orometric analysis of IFN-g–secreting CD8 and CD4 T cellsderived from the following donor groups: ctrl 5 control,RR 5 nontreated relapsing-remitting (RR) MS, SP 5secondary progressive MS, IFN-b 5 RRMS treated withinterferon-b. Patients are the same as in Figure 2. Data arepresented as mean 6 SEM percentage of either CD8 or CD4IFN-g–secreting T cells. #p , 0.05, *p , 0.01 (comparedwith control values).

Brief Communication: Becher et al: Altered IFN-g Secretion in MS 249

ated with activation of the immune system. Neurology 1987;37:1097–1102

4. Heremans H, Dillen C, Groenen M, et al. Chronic relapsingexperimental autoimmune encephalomyelitis (CREAE) in mice:enhancement by monoclonal antibodies against interferon-gamma. Eur J Immunol 1996;26:2393–2398

5. Krakowski M, Owens T. Interferon-gamma confers resistanceto experimental autoimmune encephalomyelitis. Eur J Immunol1996;26:1641–1646

6. Austrup F, Vestweber D, Borges E, et al. P- and E-selectin me-diate recruitment of T-helper-1 but not T-helper-2 cells intoinflamed tissues. Nature 1997;385:81–83

7. Bonecchi R, Bianchi G, Bordignon PP, et al. Fifferential ex-pression of chemokine receptors and chemotactic responsivenessof type 1 T helper cells (Th1s) and Th2s. J Exp Med 1998;187:129–134

8. Zhang B, Yamamura T, Kondo T, et al. Regulation of experi-mental autoimmune encephalomyelitis by natural killer (NK)cells. J Exp Med 1997;186:1677–1687

9. Koh DR, Fung-Leung WP, Ho A, et al. Less mortality butmore relapses in experimental autoimmune encephalomyelitis inCD81/2 mice. Science 1992;256:1210–1213

10. Balashov KE, Khoury SJ, Hafler DA, Weiner HL. Inhibition ofT cell responses by activated human CD81 T cells is mediatedby interferon-gamma and is defective in chronic progressivemultiple sclerosis. J Clin Invest 1995;95:2711–2719

11. Bania MB, Antel JP, Reder AT, et al. Suppressor and cytolyticcell function in multiple sclerosis: effects of cyclosporin A andinterleukin-2. J Clin Invest 1986;78:582–586

12. Antel JP, Bania MB, Reder A, Cashman N. Activated suppres-sor cell dysfunction in progressive multiple sclerosis. J Immunol1986;137:137–141

13. Noronha A, Toscas A, Jensen MA. Interferon beta augmentssuppressor cell function in multiple sclerosis. Ann Neurol 1990;27:207–210

14. Noronha A, Toscas A, Jensen MA. Interferon beta decreases Tcell activation and interferon gamma production in multiplesclerosis. J Neuroimmunol 1993;46:145–153

15. Balashov KE, Smith DR, Khoury SJ, et al. Increased interleukin12 production in progressive multiple sclerosis: induction byactivated CD41 T cells via CD40 ligand. Proc Natl Acad SciUSA 1997;94:599–603

16. Neighbour PA, Miller AE, Bloom BR. Absence of virus-induced lymphocyte suppression and interferon production inmultiple sclerosis. Neurology 1981;31:561–566

17. Crucian B, Dunne P, Friedman H, et al. Alterations in periph-eral blood mononuclear cell cytokine production in response tophytohemagglutinin in multiple sclerosis patients. Clin DiagnLab Immunol 1995;2:766–769

18. Brod SA, Khan M, Bright J, et al. Decreased CD3-mediatedinterferon-gamma production in relapsing-remitting multiplesclerosis. Ann Neurol 1995;37:546–549

19. Crucian B, Dunne P, Friedman H, et al. Detection of altered Thelper 1 and T helper 2 cytokine production by peripheral bloodmononuclear cells in patients with multiple sclerosis utilizing in-tracellular cytokine detection bu flow cytometry and surfacemarker analysis. Clin Diagn Lab Immunol 1996;3:411–416

20. Arnason BG. Interferon beta in multiple sclerosis. Clin Immu-nol Immunopathol 1996;81:1–11

21. Petereit HF, Bamborschke S, Esse AD, Heiss WD. Interferongamma–producing blood lymphocytes are decreased by inter-feron beta therapy in patients with multiple sclerosis. MultScler 1997;3:180–183

22. Bongioanni MR, Durelli L, Ferrero B, et al. Systemic high-doserecombinant alpha-2a-interferon therapy modulates lymphokineproduction in multiple sclerosis. J Neurol Sci 1996;143:91–99

An Association betweenAutosomal DominantCerebral Cavernomas and aDistinctive HyperkeratoticCutaneous VascularMalformation in 4 FamiliesPierre Labauge, MD,* Odile Enjolras, MD,†Jean-Jacques Bonerandi, MD,‡ Sophie Laberge, MD,*Michel Dandurand, MD,§ Jean-Marie Joujoux, MD,i

and Elisabeth Tournier-Lasserve, MD*¶

Cerebral cavernomas (CCMs) are vascular malformationsthat may be inherited as an autosomal dominant condi-tion for which a gene, CCM1, was mapped to chromo-some 7. Poorly defined cutaneous malformations weresometimes described in association with CCMs. During anational survey, 57 French CCM families were studied.Co-occurrence of CCMs and a distinctive cutaneous vas-cular malformation was observed in 4 families. Ten in-dividuals belonging to these families showed similar hy-perkeratotic cutaneous capillary venous malformations(HCCVMs). In 3 families, the histology showed ortho-keratosis and hyperkeratosis as well as dilated capillaries inthe dermis extending to the hypodermis and confirmed thediagnosis of HCCVM. Genetic analysis strongly supportslinkage of these families to the CCM1 locus on chromo-some 7. The HCCVM seems to be a peculiar cutaneousvascular malformation associated with CCMs. These datastrongly suggest that HCCVMs and CCMs in these fami-lies are due to the same genetic abnormality.

Labauge P, Enjolras O, Bonerandi J-J, Laberge S,Dandurand M, Joujoux J-M, Tournier-Lasserve E.An association between autosomal dominant cerebral

cavernomas and a distinctive hyperkeratoticcutaneous vascular malformation in 4 families.

Ann Neurol 1999;45:250–254

Cavernomas are vascular malformations mainly locatedin the central nervous system. They are characterizedby abnormally enlarged capillary cavities without inter-

From *INSERM U25 Faculte de Medecine Necker, Paris, †Consul-tation des Angiomes, Hôpital Lariboisiere, Paris, ‡Service de Der-matologie, CHU Timone, Marseille, and §Service de Dermatolo-gie and iService d’Anatomopathologie, CHU Caremeau, Nîmes,¶Hôpital Lariboisiere, Paris, France.

Received Jul 16, 1998, and in revised form Sep 14. Accepted forpublication Sep 16, 1998.

Address correspondence to Dr Tournier-Lasserve, INSERM U25Faculte de Medicine Necker, 156 Rue de Vaugirard, 75730 ParisCedex 15, France.

250 Copyright © 1999 by the American Neurological Association

vening brain parenchyma.1 Large autopsy and mag-netic resonance imaging (MRI) series evaluated theirfrequency at 0.5% in the general population.2 Theymay be inherited as an autosomal dominant conditiondesignated as familial cerebral cavernomas (CCMs).CCMs have been studied extensively in the Hispanicpopulation in the United States, in which more than50% of cavernomas are familial.3 An as yet unidenti-fied gene mapping on chromosome 7, CCM1 4, hasbeen shown to be involved in all CCM families be-longing to this ethnic group.5,6 Few non-Hispanicfamilies have been studied so far, with some of thembeing linked to chromosome 7 and some unlinked,demonstrating the implication of at least one othergene.7 In some rare sporadic and familial cases of

CCM, angiomatosis may affect other organs such asretina and/or skin.8

During a recent survey of 57 French familial cerebralcavernomas families, including 173 CCM patients,9 weidentified 10 cases of localized peculiar cutaneous hy-perkeratotic vascular malformations segregating in 4families, including at least 2 cases of these cutaneousvascular lesions in each family. Cerebral MRI per-formed in 8 of the patients with skin lesions showedCCMs in all cases. The cosegregation of these types ofcutaneous and neurological vascular malformations hasnot been reported before. It is unlikely to have oc-curred by chance, as these distinctive cutaneous lesionsare uncommon and, to our knowledge, not familial.Genetic linkage analysis with chromosome 7 markers

Fig 1. Pedigree structure of the 4 families. Affected individuals are represented by filled symbols, unaffected individuals by emptysymbols, and individuals having an unknown status by symbols with a question mark. Individuals designated by an asterisk bear ahyperkeratotic cutaneous capillary venous malformation (HCCVM). Alleles of the polymorphic markers spanning the CCM1 intervalon chromosome 7 (D7S2410, D7S2409, D7S1813, D7S1789, M65B, D7S646, D7S558, D7S689) that segregate within thefamilies are shown for each individual. Filled boxes indicate the affected haplotypes cosegregating with the disease. Empty boxes in-dicate haplotypes unlinked to the disease. All cerebral cavernoma (CCM) and HCCVM patients share the affected haplotype segre-gating with the cavernous angioma phenotype in their family. Two unaffected individuals (I-15, III-4) were carrying the affectedhaplotype, which is most likely a consequence of the established incomplete penetrance of CCMs.

Brief Communication: Labauge et al: Association between CCMs and HCCVMs 251

spanning the CCM1 interval strongly suggests that all 4families are linked to chromosome 7.

Materials and MethodsA total of 57 French CCM families were studied during asurvey conducted throughout all 28 French neurosurgerycenters from November 1996 to June 1997.9 The “affected”diagnosis was based on the presence of typical histological orMRI-detected lesions (n 5 173 subjects). The “unaffected”diagnosis was retained based on the normality of MRI (n 589). In addition to detailed neurological and neuroimaginginvestigations, specific inquiries on skin lesions were con-ducted. All skin lesions were examined by a certified derma-

tologist (O.E.), and pathological samples were examined by apathologist (J-J.B.). Eight polymorphic microsatellite markersspanning the interval containing the CCM1 locus were se-lected for linkage analysis.9 Two-point linkage analysis wasperformed (data not shown but available on request).

ResultsOf the overall 57 identified families, 4 were character-ized by the association of CCMs and cutaneous lesions(Fig 1). Within these 4 families, 8 of 21 individualswho had CCMs detected by MRI (Fig 2) also had cu-taneous lesions (Table). Two additional family mem-bers (Patients I-23 and IV-9) in whom MRI was notperformed because of young age (Patient I-23) and re-fusal (Patient IV-9), respectively, had cutaneouschanges. These lesions were congenital, single, and lo-calized on the lower limbs in all patients, except 1 inwhom the lesion was located on the left arm (PatientIII-6). No extension was noticed since childhood. Sur-gical removal was performed in 3 patients (PatientsI-18, I-23, and IV-9) followed by local recurrence in 1(Patient IV-9). Lesions were crimson-colored tiny pap-ules (3–10 mm) or larger macules (20–65 mm) with ahyperkeratotic epidermis. Bluish discoloration of theskin or several tortuous vessels radiating outward fromthe superficial lesion led us to consider clinically thediagnosis of associated subcutaneous venous malforma-tion (Fig 3). Histological confirmation was available in4 patients (Patients I-18, I-23, II-4, IV-9) belonging to3 families (Families I, II, IV). The histology revealedorthokeratosis and hyperkeratosis, abundant dilatedcapillaries, and blood-filled spaces in papillary and re-ticular dermis extending to the hypodermis in all 4 pa-tients (Fig 4). These vascular lesions can be describedas hyperkeratotic cutaneous capillary venous malforma-tions (HCCVMs). None of the 89 clinically healthyindividuals having a normal MRI scan had anyHCCVMs.

Haplotypes obtained with eight microsatellite mark-ers spanning the CCM1 locus on chromosome 7 are

Table. Cutaneous Findings of HCCVMs and CCMs

Patients Age (yr) CCM MRIHCCVMLocation Size (mm)

Skin PathologicalConfirmation

I-4 60 1 Left calf 10 3 5 2I-12 29 1 Left calf 8 3 5 2I-18 34 1 Buttock 3 3 4 1I-23 7 NA Left calf 10 3 10 1II-4 60 1 Right thigh 65 3 60 1II-11 34 1 Left thigh 20 3 10 2III-3 26 1 Left leg 5 3 4 2III-6 31 1 Left arm 4 3 2 2IV-2 78 1 Left thigh 20 3 15 2IV-9 28 NA Right thigh 10 3 5 1

CCM 5 cerebral cavernoma; HCCVM 5 hyperkeratotic cutaneous capillary-venous malformation; NA 5 not available.

Fig 2. Axial T2-weighted magnetic resonance image of thebrain of an affected patient (Patient II-4). Mixed hypersignalsand hyposignals surrounded by a rim of hyposignal locatedin the right external capsule are strongly suggestive of acavernoma.


shown in Figure 1. Detailed linkage analysis and LODscore values of these families are not shown but areavailable on request. In each family, a distinct haplo-type was shared by all CCM patients, and the 8 indi-viduals bearing HCCVMs were carrying the CCMhaplotype segregating in their family. Patient I-23, whodid not undergo MRI, shared the CCM haplotype seg-regating in his affected relatives. The affected haplo-types segregating with the CCM and HCCVM pheno-types were distinct in the 4 families. Two-point and

multipoint linkage as well as HOMOG analysisshowed that Families I, II, and IV had a greater than90% probability of being linked. No definite conclu-sion could be made for Family III (58% probability)due to its small size.

DiscussionSkin lesions in our patients consisted of more or lessprominent, plaque-like, irregularly shaped, crimson-colored vascular malformations. These circumscribedlesions were strikingly similar in all patients. Accordingto the current classification of superficial vascular ab-normalities accepted by the International Society forthe Study of Vascular Anomalies,10 they can be definedas capillary and venous malformations. All the cutane-ous lesions of our patients were vascular malformationsof predominantly capillary type with a venular compo-nent in association with an overlying hyperkeratoticepidermis. No cellular hyperplasia was present, and thevascular proliferation invaded not only the dermis butalso the hypodermis.

Several arguments strongly suggest that the occur-rence of HCCVMs and CCMs in several members ofthe families reported here is not fortuitous, includingthe facts that (1) HCCVM is a rare vascular lesion that

Fig 3. Aspect of skin lesion of the right thigh (Patient II-4).Note the underlying tortuous bluish vessels. The white areawas left by a previous partial treatment (radiotherapy andcryotherapy).

Fig 4. Cutaneous biopsy findings (Patient II-4). Hyperkeratosis and acanthosis associated with dilated vessels in the upper dermis(A) and capillaries and veins extending into the hypodermis (B) (hematoxylin-eosin and safran; magnification 3 100 before 47%reduction).

Brief Communication: Labauge et al: Association between CCMs and HCCVMs 253

was observed in 8 of 173 individuals with CCMs andin none of the 89 healthy family members, and (2) ineach case, an HCCVM was observed in at least 2members of the affected families, with 1 family (FamilyIV) including 4 cases.

There are few reports about the association betweenCCMs and cutaneous lesions. The types of these cuta-neous lesions are quite heterogeneous and were mainlydescribed as bluish nodules,11,12 cherry angiomas,13

and capillary vascular anomalies.14 Filling-Katz andcolleagues15 reported 1 family suffering from terminaltransverse limb defect, CCMs, and skin lesions calledcavernous “angiomas.” In 1 patient, several CCMswere associated with eruptive multiple angiokerato-mas.16 Histological data are available in only one ofthese case and family reports.16 All of these reportsconcerned patients bearing multiple CCMs11,12,16 orbelonging to a family with multiple cases of caverno-mas.13–15 To our knowledge, CCMs have never beenassociated with HCCVMs. In addition, this is the firstreport on familial cases of this hyperkeratotic vascularmalformation.

Interestingly, all 9 genetically studied HCCVM pa-tients share the affected haplotype present in theirCCM-affected relatives. These data strongly suggestthat the occurrence of the vascular cutaneous lesions inthese patients is due to the same genetic abnormalitycausing CCMs, although with a lower penetrance. Aswe did observe these HCCVMs only in a subset of theFCC families linked to chromosome 7, one may sug-gest that these 4 families may harbor a specific CCM1mutation. This fact will be tested only when CCM1gene identification is achieved.

In conclusion, we focus on HCCVMs, a cutaneousvascular hallmark for the development of CCMs in at-risk family members.

Dr Laberge is the recipient of a grant from the Fonds de Rechercheen Sante du Quebec (FRSQ). Dr Labauge benefitted from a posted’accueil INSERM during the initial part of the study and is nowsupported by the College des Enseignants de Neurologie. This workwas supported by INSERM, Ministere de l’Enseignement Superieuret de la Recherche (MESR, ACCSV 1995).

We are indebted to the families included in this study for theirparticipation; to Drs A. L. Benabid, A. Carriere, P. Freger, B.George, J. Guillemette, D. Hache, J.-P. Lejeune, J.-P. Machayeki, J.Petit, B. Silhouette, and A. Turcarelli for referring medical observa-tions; and to Dr M. Wassef and Prof J.-F. Pellissier for pathologicaladvice. We also thank Profs J.-C. Piette and E. Roullet for excellentcritical reading of this manuscript.

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10. Enjolras O, Mulliken JB. Vascular tumors and vascular malfor-mations. Adv Dermatol 1998;13:375–423

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15. Filling-Katz MR, Levin SW, Patronas NJ, Katz NNK. Termi-nal transverse limb defects associated with familial cavernousangiomatosis. Am J Med Genet 1992;42:346–351

16. Ostlere L, Hart Y, Misch KJ. Cutaneous and cerebral haeman-giomas associated with eruptive angiokeratomas. Br J Dermatol1996;135:98–101


Epilepsia Partialis Continua:A New Manifestation ofAnti-Hu–AssociatedParaneoplasticEncephalomyelitisYael Bellaıche Shavit, MD,* Francesc Graus, MD,‡Alphonse Probst, MD,† Ramon Rene, MD,§and Andreas J. Steck, MD*

We report on 3 anti-Hu–positive patients who presentedwith clinical and electroencephalographic (EEG) featuresof epilepsia partialis continua (EPC). Two of the patientshad an associated small cell carcinoma. Magnetic reso-nance imaging (MRI) disclosed a hyperintense nonen-hancing focal lesion in T2-weighted images in the senso-rimotor area in 2 patients. Histopathological analysis ofthe lesion revealed inflammatory infiltrates and neuronalcell loss. In the patient who had a postmortem study,these neuropathological changes were not observed inother areas of the nervous system. This study emphasizesthat the possibility of an anti-Hu–associated paraneoplas-tic disorder must be considered in patients with corticalencephalitis presenting with EPC when a brain tumorcan be excluded.

Shavit YB, Graus F, Probst A, Rene R, Steck AJ.Epilepsia partialis continua: a new manifestation of

anti-Hu–associated paraneoplastic encephalomyelitis.Ann Neurol 1999;45:255–258

Anti-Hu–associated paraneoplastic encephalomyelitisand sensory neuronopathy (PEM/SN) are two oftenconcomitant syndromes associated with small cell lungcarcinoma (SCLC) in nearly 80% of patients. Neuro-logical dysfunction is usually multifocal, including sen-sory neuronopathy, motor neuron dysfunction, brain-stem encephalopathy, limbic encephalitis, autonomicneuropathy, and cerebellar degeneration.1–3 Althoughthe inflammatory infiltrates and neuronal loss charac-teristic of PEM/SN may be observed in multiple areasof the cerebral cortex in postmortem studies, no symp-toms suggestive of a particular area of the cerebral cor-tex other than limbic encephalitis have been reported

in patients with Hu-associated PEM/SN. Seizures arerarely the presenting symptom of PEM/SN and occuralmost always in the setting of limbic encephalitis.2

We describe 3 patients with focal sensorimotor en-cephalitis presenting with epilepsia partialis continua(EPC), which represents a new clinical picture in thespectrum of anti-Hu–associated neurological disorders.

Case ReportsPatient 1A 56-year-old woman with a history of SCLC diagnosed inJune 1995 went into complete remission after chemotherapy.In September 1996, she presented with dysesthesia of the lefttrunk from T6 to T10 and involuntary clonic musculartwitching of the left leg. A neurological examination showedleft facial weakness, increased tendon reflexes on the left side,negative Babinski reflex, slight paresis, and distal hypesthesiaof the left extremities. A stimulus-sensitive myoclonic move-ment disorder of the left arm and leg could be observed.Magnetic resonance imaging (MRI) of the head showed anonenhancing lesion in the right postcentral area in T2-weighted images (Fig 1A). The electroencephalogram (EEG)demonstrated periodic epileptiform discharges in the rightparietal area, which were sometimes synchronous with an ob-served myoclonus of the left hand. A cerebrospinal fluid(CSF) examination, including oligoclonal IgG bands, wasnegative. There was no evidence for recurrence of her previ-ous cancer on chest computed tomography (CT), includingliver and adrenal glands, or on spinal and pelvic radiography.Tumor markers were normal, except for b2-microglobulin,which was slightly elevated (3.7 mg/L).

The patient underwent a stereotactic biopsy of the rightpostcentral lesion. A histopathological examination revealed asubacute cortical encephalitis with a few perivascular B-cellinfiltrates, a larger number of T lymphocytes diffusely infil-trating the cortex, and neuronal cell loss. There was evidencefor microglia activation and proliferation as well as for astro-cytic gliosis (Fig 2). Intracytoplasmic IgG reactivity wasfound in some neurons (not shown). Despite intravenousimmunoglobulin treatment and plasmapheresis, she experi-enced a relentless progression of her left hemiparesis until shebecame unable to walk. The motor seizures involving the leftface and the left upper and lower extremities became almostconstant but were finally partially controlled with a combi-nation of anti-epileptic drugs. On MRI 3 months later, thepatient showed an enlargement of the nonenhancing lesionto the right precentral area (see Fig 1B). She subsequentlyreceived cyclophosphamide under which the lesion regressed,the EPC disappeared, and the left hemiparesis notably im-proved. Nevertheless, at her last follow-up examination 2years after the onset of neurological symptoms, the hemipa-resis had again increased with subsequent extension of thelesion, although the SCLC is still in remission.

Patient 2A 54-year-old man was admitted to the emergency room dueto a generalized tonic-clonic seizure. The patient recoveredcompletely within a few hours. Routine laboratory analysisand a CT scan of the head were normal. Over the ensuing 3days, the patient noticed progressive difficulty in evoking

From the Departments of *Neurology and †Pathology, UniversityHospital, Basel, Switzerland, ‡Department of Neurology, HospitalClınic i Provincial, University of Barcelona, and §Department ofNeurology, C.S.U. Bellvitge, L’Hospetalet, Barcelona, Spain.

Received May 21, 1998, and in revised form Sep 28, 1998. Ac-cepted for publication Sep 28, 1998.

Address correspondence to Prof Steck, Neurologische Klinik, Kan-tonsspital Basel, Petersgraben 4, CH-4031 Basel, Switzerland.


names, with dysarthria and frequent focal-clonic seizures in-volving the muscles of the right side of the face.

General physical and neurological examinations were nor-mal, except for severe dysarthria, mild facial paresis of centralorigin, increased deep tendon reflexes, and extensor plantar re-sponse in the right side. A second CT scan of the head, repeatMRI; three CSF examinations; routine laboratory analysis; se-rologies for herpesvirus, human immunodeficiency virus, bor-relia, and syphilis; testing of somatosensory evoked potentials;and four-vessel angiography were all normal or negative. Sev-eral EEGs demonstrated periodic lateralized epileptiform dis-charges in the left parasagittal frontoparietal region.

The clinical course of the patient was characterized by fo-cal clonic seizures involving the right side of the face and

right hand, which became almost continuous and unrespon-sive to treatment. The patient remained collaborative andoriented throughout the clinical course. He died due to as-piration pneumonia 30 days after the onset of the neurolog-ical disorder.

The autopsy study did not show any tumor. The macro-scopic examination of the brain was normal. The micro-scopic study of multiple areas of the brain was normal, exceptfor the presence of perivascular and intraparenchymatousinflammatory infiltrates, gliosis, and neuronal cell loss in theleft motor strip. Immunohistochemical studies demonstratedmicroglia proliferation. Most of the inflammatory cells wereT cells, with occasional perivascular B-cell lymphocytes.

Patient 3A 43-year-old man noticed involuntary repetitive movementsof the tongue that interfered with his speech and deglutitionin April 1997. The movements were almost continuous andpersisted during sleep. The patient was evaluated elsewhere,and head MRI and a CSF examination were normal. He wasdischarged with a diagnosis of lingual myoclonus and treatedwith clonazepam, which reduced the movements. Over theensuing months, the patient noticed additional left perioraltwitching and occasional clonic movements of the first twofingers of the left hand.

The patient was admitted for a severe hematemesis in No-vember 1997. A general physical examination was unreveal-ing. A neurological examination was normal, except for fre-quent twitching of the tongue and platysma of both sides,which was more prominent on the left side. There were oc-casional clonic jerks of the left side of the face. The twitchinginterfered with voluntary movements of the tongue, whichwere slowed. Deep tendon reflexes were abolished in the legs.Pallesthesia was diminished in the left foot. Routine labora-tory analysis and a CSF examination were normal. The EEGdemonstrated periodic epileptiform discharges in the rightfrontoparietal area, and MRI of the head showed a nonen-hancing lesion in the inferior portion of the right Rolandicarea (Fig 3). The electromyographic examination was nor-mal, except for absent sensory potential in the left sural andsuperficial peroneal nerves. Upper gastrointestinal endoscopyand a CT scan of the abdomen disclosed a large tumor in thecardia of the stomach with enlarged perigastric lymph nodes.A biopsy of the tumor confirmed a small cell tumor. Afterthree cycles with carboplatin and etopiside, the tumorshowed partial remission. The patient’s movements greatlyimproved, and MRI revealed a complete remission of thebrain lesion.

Immunological StudiesSerum and CSF anti-Hu antibodies were detected on frozensections of normal human cortex and confirmed by immu-noblotting of human neuronal nuclei extracts and recombi-nant HuD as described in detail previously.4 Anti-Hu anti-bodies were detected in the serum of the 3 patients and inthe two available CSF samples. Anti-Hu antibodies remainedpositive at serum dilutions greater than 1:10,000, a featurethat is almost always associated with paraneoplastic neurolog-ical disorders.4

Fig 1. (A) T2-weighted axial magnetic resonance imaging(MRI) performed during the initial course of the disease dis-closed high signals located in the right postcentral area (TR 52,500). (B) Head MRI performed 9 months later. T2-weighted images show an extension of the hyperintense lesionto the precentral area.


DiscussionThese 3 patients demonstrate a previously unreportedclinical presentation of PEM/SN associated withanti-Hu antibodies. The clinical, EEG, and histopatho-logical findings in our patients fulfill the criteria of theEPC syndrome.5 In addition, MRI disclosed a focalnonenhancing lesion in the sensorimotor area as de-scribed in patients with EPC of other etiologies.6 EPCis usually caused by structural focal lesions (cerebrovas-cular disorders, trauma, or tumors), metabolic disor-ders, or presumed encephalitis, but in some patients,the cause is unknown.5,6 The present study shows thatthe possibility of a paraneoplastic origin must be in-cluded in the differential diagnosis. The absence of a tu-mor in the autopsy of Patient 2 does not rule out thispossibility. The autopsy was done without the clinicaldiagnosis of a paraneoplastic syndrome, and a small tu-mor can easily be missed in the postmortem analysis if adirect search for an occult tumor is not considered.7 Thepossibility of a brain metastasis was reasonably excludedby the brain biopsy in Patient 1 and by a complete au-topsy in Patient 2, which did not disclose malignantcells in the brain. Furthermore, the clinical course ofPatient 1, who survived for 2 years after the onset ofmanifestations, is not typical for a brain metastasis.

Fig 2. Biopsy of the cerebral cortex of Patient 1. (a) The cerebral cortex is diffusely infiltrated by lymphocytes. The cell infiltratessurrounding the vessel mainly consist of B lymphocytes. (b) The cerebral cortex shows hypertrophic astrocytes (arrowheads), loose neu-ropil texture, and loss of neurons. (c) The patient’s biopsy was incubated with pan–T-cell antibody CD3. The cortex is diffuselyinfiltrated by T cells, with most of them bearing CD8 surface antigen (not shown). (d) Immunostaining with antibody CD68shows diffuse infiltration of the cerebral cortex by activated microglia/macrophages. (All hematoxylin and eosin; original magnifica-tion: a 3 40, b 3 200, c 3 80, d 3 100.).

Fig 3. T2-weighted transverse magnetic resonance imaging ofPatient 3. The arrow shows the hyperintense lesion in theright frontoparietal area.

Brief Communication: Shavit et al: EPC in Anti-Hu–Associated PEM 257

Pathological and immunohistochemical abnormali-ties in the brain tissue of patients with PEM/SN areusually widespread, with certain areas being more af-fected than others.8 This may be explained by theubiquitous expression of Hu antigens in the nervoussystem. The remarkable findings in our patients are theexquisitely focal clinical, paraclinical, and, to a certainextent, pathological features. Why some patients withPEM/SN and anti-Hu antibodies demonstrate a focalneurological disorder throughout the clinical course ofthe disease is an unsettled issue. Patients with anti-Hu–associated PEM/SN sometimes build an immuneresponse against other neural antigens, and they maydemonstrate other autoantibodies such as anti-CV29 orantiamphiphysin.10 Our patients could have otherautoantibodies that would be relevant to the seizuredisorder. Patients with Rasmussen’s encephalitis, a pro-gressive disorder characterized by intractable focal sei-zures and inflammatory neuropathology, demonstrateantibodies against subunit 3 of the glutamic receptor(GLuR3). Animals immunized with GLuR3 developanti-GLuR3 antibodies and clinical and pathologicalfeatures similar to those of Rasmussen’s encephalitis.11

Recently, antibodies against GLuR1, GLuR4, andGluR5/6 were reported in 6 of 7 patients with para-neoplastic cerebellar degeneration.12 Although we didnot detect GLuR antibodies in our 3 patients, no an-tibodies against any subunit of GLuR were found in alarge number of patients with paraneoplastic neurolog-ical disorders, including anti-Hu–associated PEM/SN(J. Dalmau, personal communication, 1997).13

Whether the tissue distribution of the products ofthe different genes of the Hu family correlates with thediverse neurological syndromes of PEM/SN is still amatter requiring further research.14 Studies by Manleyand colleagues15 could not find any correlation be-tween antibody reactivity with any of the Hu antigensand the type of neurological symptoms. Furthermore,analysis of IgG subclass distribution did not reveal anycorrelation between anti-Hu isotype and specific brainarea or clinical features.16 Finally, the focal nature ofthe symptoms could be explained by the circumscribedexpression of major histocompatibility complex pro-teins, which eventually leads to the demonstration ofHu antigens. This could induce a T-cell–mediated pro-cess either as a direct cytotoxic reaction with neuronalcells or as the activation of T and B cells.17

In conclusion, the present study extends our knowl-edge of the clinical spectrum of PEM/SN and demon-strates that the possibility of a paraneoplastic origin mustbe considered in the differential diagnosis of EPC.

We thank Prof E. W. Radu for providing the MR images of Patient1 and Dr H. R. Stockli for referring Patient 1. We are grateful to V.Bruce for revising the manuscript.

References1. Graus F, Ribalta T, Campo E, et al. Immunohistochemical

analysis of the immune reaction in the nervous system in para-neoplastic encephalomyelitis. Neurology 1990;40:219–222

2. Dalmau J, Graus F, Rosenblum MK, Posner JB. Anti-Hu–associated paraneoplastic encephalomyelitis/sensory neuronopa-thy. A clinical study of 71 patients. Medicine 1992;71:59–72

3. Henson RA, Urich H. Encephalomyelitis with carcinoma. In:Cancer of the nervous system. Oxford, UK: Henson BlackwellScientific Publications, 1982:314–345

4. Graus F, Dalmau J, Rene R, et al. Anti-Hu antibodies in pa-tients with small cell lung cancer: association with complete re-sponse to therapy and improved survival. J Clin Oncol 1997;15:2866–2872

5. Thomas JE, Reagan TJ, Klass DW. Epilepsia partialis continua.A review of 32 cases. Arch Neurol 1977;34:266–275

6. Singh BM, Strobos RJ. Epilepsia partialis continua associatedwith nonketotic hyperglycemia: clinical and biochemical profileof 21 patients. Ann Neurol 1980;8:155–160

7. Anderson NE, Budde-Steffen C, Wiley RG, et al. A variant ofthe anti-Purkinje cell antibody in a patient with paraneoplasticcerebellar degeneration. Neurology 1988;38:1018–1026

8. Dalmau J, Furneaux HM, Rosenblum MK, et al. Detection ofthe anti-Hu antibody in specific regions of the nervous systemand tumor from patients with paraneoplastic encephalomyelitis/sensory neuronopathy. Neurology 1991;41:1757–1764

9. Honnorat J, Antoine JC, Derrington E, et al. Antibodies to asubpopulation of glial cells and a 66-kDa developmental pro-tein in patients with paraneoplastic neurological syndromes.J Neurol Neurosurg Psychiatry 1996;61:270–278

10. Dropcho EJ. Anti-amphiphysin antibodies with small-cell lungcarcinoma and paraneoplastic encephalomyelitis. Ann Neurol1996;39:659–667

11. Rogers SW, Andrews PI, Gahring LC, et al. Autoantibodies toglutamate receptor GLuR3 in Rasmussen’s encephalitis. Science1994;265:648–652

12. Gahring LC, Twyman RE, Greenlee JE, Rogers SW. Autoan-tibodies to neuronal glutamate receptors in patients with para-neoplastic neurodegenerative syndrome enhance receptor activa-tion. Molecular Medicine 1995;1:245–253

13. Degenhardt A, Hoard R, Duvoisin RM, et al. Glutamate re-ceptor antibodies and paraneoplastic neurologic disorders. Neu-rology 1996;46:A410 (Abstract)

14. Darnell RB. Onconeural antigens and the paraneoplastic neu-rologic disorders: at the intersection of cancer, immunity, andthe brain. Proc Natl Acad Sci USA 1996;93:4529–4536

15. Manley GT, Smitt PS, Dalmau J, Posner JB. Hu antigens: re-activity with Hu antibodies, tumor expression, and major im-munogenic sites. Ann Neurol 1995;38:102–110

16. Jean WC, Dalmau J, Ho A, Posner JB. Analysis of the IgGsubclass distribution and inflammatory infiltrates in patientswith anti-Hu–associated paraneoplastic encephalomyelitis. Neu-rology 1994;44:140–147

17. Dalmau J, Graus F, Cheung NV, et al. Major histocompatibil-ity proteins, anti-Hu antibodies, and paraneoplastic encephalo-myelitis in neuroblastoma and small cell lung cancer. Cancer1995;75:99–109


Epstein-Barr Virus inMonitoring the Responseto Therapy of AcquiredImmunodeficiencySyndrome–Related PrimaryCentral NervousSystem LymphomaAndrea Antinori, MD,* Antonella Cingolani, MD,*Andrea De Luca, MD,* Gianluca Gaidano, MD, PhD,‡Adriana Ammassari, MD,* Luigi M. Larocca, MD,†and Luigi Ortona, MD*

To evaluate the value of Epstein-Barr virus DNA (EBV-DNA) assay in cerebrospinal fluid (CSF) for monitoringthe response to treatment in acquired immunodeficiencysyndrome–related primary central nervous system lym-phoma (AIDS-PCNSL), 9 human immunodeficiencyvirus–infected patients with biopsy-proven AIDS-PCNSLwho underwent multimodal therapy were investigated forEBV-DNA detection in CSF by semiquantitative nestedpolymerase chain reaction (PCR). Tumoral tissue expres-sion of bcl-6 oncogene protein and of EBV-encoded la-tent membrane protein (LMP-1) was also investigated.The 2 patients who had a response to chemotherapyshowed a reduction of mean EBV-DNA concentrationvalues after chemotherapy and displayed a large non-cleaved morphology and a BCL-61/LMP-12 phenotype.Conversely, the 4 patients with progressive disease afterchemotherapy showed increasing mean values of EBV-DNA and displayed an immunoblastic morphology and aBCL-6 2/LMP-11 phenotype. No significant changeswere observed for patients with stable disease. EBV-DNAburden reduction was significantly associated with pro-longed survival. These results suggest that EBV-DNAmonitoring might be helpful in predicting response tochemotherapy and in segregating distinct biological andprognostic categories of AIDS-PCNSL.

Antinori A, Cingolani A, De Luca A, Gaidano G,Ammassari A, Larocca LM, Ortona L. Epstein-

Barr virus in monitoring the response to therapyof acquired immunodeficiency syndrome–related

primary central nervous system lymphoma.Ann Neurol 1999;5:259–261

From the Departments of *Infectious Diseases and †Pathology,Catholic University, Rome, and ‡Division of Internal Medicine,Department of Medical Sciences, University of Torino at Novara,Novara, Italy.

Received Aug 18, 1998, and in revised form Oct 5, 1998. Acceptedfor publication Oct 5, 1998.

Address correspondence to Dr Antinori, Department of InfectiousDiseases, Catholic University, L.go A. Gemelli 8—00168 Rome,Italy.

Epstein-Barr virus (EBV) infection is associated withvirtually all acquired immunodeficiency syndrome–related primary central nervous system lymphoma(AIDS-PCNSL),1 and EBV-DNA is detectable in thecerebrospinal fluid (CSF) of most patients with the dis-ease.2–4 For these reasons, EBV-DNA detection inCSF has been proposed as a valid tool for the rapidselection of patients to undergo brain biopsy.5 Re-cently, our group reported that detection of EBV-DNAin CSF may allow a minimally invasive diagnosis ofAIDS-PCNSL.6,7 At present, it is noteworthy to assessthe value of this assay for monitoring tumor responseto treatment.7 Here, we report the results of a pilotstudy performed to assess the relationship betweenEBV load in CSF, response to therapy, and survival ofpatients with AIDS-PCNSL who received multimodaltherapy.

Materials and MethodsNine consecutive patients with biopsy-proven AIDS-PCNSLwho underwent multimodal therapy between January 1995and April 1998 were included in the study. All pathologicalspecimens were classified according to the working formu-lation for non-Hodgkin’s lymphoma8 and to a revisedEuropean-American classification of lymphoid neoplasms.9

EBV-encoded small RNAs were detected by in situ hybrid-ization. All cases were investigated for protein expression ofthe BCL-6 proto-oncogene and of EBV-encoded latentmembrane protein-1 (LMP-1). The BCL-6 protein was de-tected by the PG-B6 monoclonal antibody directed againstthe amino terminal portion of the human bcl-6 gene product(obtained from B. Falini, Institute of Hematology, Univer-sity of Perugia, Perugia, Italy). The LMP-1 antigen was de-tected using a pool of four anti-LMP-1 monoclonal antibod-ies (CS-1–4, Dakopatts, Glostrup, Denmark). Subsequently,after incubation with primary antibodies, an avidin-biotinperoxidase complex method was employed using tyramideamplification (Dako Catalyzed Amplification System, Dako-patts) and diaminobenzidine as a chromogen. A positive re-sult was defined as 10% or greater positive neoplastic cells.10

Eight patients received multimodal therapy with high-doseintravenous methotrexate (MTX), oral procarbazine, and in-trathecal MTX followed by whole-brain irradiation to 30Gy. The last patient (Patient 9 in Table) was treated with acombination of high-dose intravenous MTX, high-dosezidovudine, and intrathecal MTX followed by whole-brainirradiation to 50 Gy. The response to chemotherapy as wellas that to radiation therapy was determined by computedtomographic scan or magnetic resonance imaging accordingto reported criteria.11

EBV-DNA amplification was performed in CSF samplesat baseline and after the end of the chemotherapy phase us-ing a nested polymerase chain reaction (PCR) technique ex-ploiting primers derived from the EBNA1 gene.3 The detec-tion limit of the nested PCR assay was 1,000 EBV-DNAcopies per milliliter as defined by serial dilution experiments.EBV-DNA load in CSF was measured semiquantitatively byserial testing of twofold dilutions of each sample. Sample di-lutions were prepared and run in triplicate; the assay was


considered positive only if at least two of the triplicate PCRtests for each dilution scored positive. Changes of the EBV-DNA concentration in CSF between the baseline value andthe value after chemotherapy were expressed as log copies permilliliter. Differences between mean values of EBV-DNAconcentration at baseline in different response groups wereanalyzed by one-way ANOVA. Mean values of EBV-DNAconcentration at baseline and after chemotherapy were com-pared by paired t test. Survival probability was estimated bythe Kaplan-Meier method, and differences between groupsaccording to the reduction of EBV-DNA log copies were cal-culated using the Mantel-Cox test.

ResultsThe Table summarizes the host’s CD4 count, tumorhistology, and expression of BCL-6, EBV-encodedsmall RNAs, and LMP-1 as well as the viral load inCSF and the response to therapy of all patients in-cluded inthe study.

Changes of EBV-DNA burden correlated with re-sponse to chemotherapy. In fact, in patients who re-sponded to chemotherapy (Cases 1 and 2), the meanvalue of the EBV-DNA load was 4.85 log copies permilliliter at baseline (range, 4.70–5.0) and 3.50 logcopies per milliliter after therapy (range, 3.3–3.7) (p 50.022, paired t test). In patients who progressed (Cases6–9), the mean values were 3.75 (range, 3.0–4.7) and4.82 (range, 4.0–5.7) log copies per milliliter, respec-tively (p 5 0.0049). In patients with stable disease(Cases 3–5), the mean values were 4.10 (range, 3.3–5.3) and 4.43 (range, 3.3–5.3) log copies per milliliter,respectively (p 5 0.60) (Fig). Six of 9 patients had afinal response to multimodal therapy, but only thosepatients whose EBV-DNA concentration in CSF wasreduced after chemotherapy showed a response to the

chemotherapy phase as well. A final assessment ofEBV-DNA burden after radiotherapy was not per-formed in most patients (see Table).

Unlike longitudinally observed changes, absolute val-ues of EBV-DNA at baseline did not predict a responseto therapy. In fact, no significant differences for meanEBV-DNA concentrations were detected at baseline inthe three response groups of patients (4.85, 4.10, and3.75 log copies per milliliter for responders, stable pa-tients, and progressive patients, respectively; F value atANOVA 5 1.205; p 5 0.36).

The correlation with response to chemotherapy re-sulted in an effect on patients’ survival. In fact, themedian length of survival was 243 days in those with a

Table. Host’s CD4 Count, Tumor Histology, and Expression of BCL-6, EBV-Encoded Small RNAs, and LMP-1 as Well asViral Load in CSF and Response to Therapy of Patients Included in the Study

CaseCD4(per mL) REAL WF bcl-6a

EBV-EncodedSmall RNAb LMP-1a

BaselineEBV-DNA atDiagnosisc

Changes inEBV-DNA afterChemotherapyc

Response toChemotherapy

Response toMultimodalTherapy

1 5 DLCL LNCCL 1 1 2 5.0 21.3 PR CR2 5 DLCL LNCCL 1 1 2 4.7 21.4 PR PR3 20 DLCL LNCCL/IBPL 1 1 1 5.3 0.0 SD PR4 4 DLCL IBPL 2 1 1 3.3 1.4 SD PR5 4 DLCL IBPL 2 1 1 3.7 20.4 SD PR6 13 DLCL IBPL 2 1 1 3.0 1.3 PD PD7 8 DLCL IBPL 2 1 1 3.3 0.7 PD SD8 29 DLCL IBPL 2 1 1 4.0 1.3 PD NE9 41 DLCL IBPL 2 1 1 4.7 1.0 PD PR

aAnalyzed by immunohistochemistry.bAnalyzed by RNA in situ hybridization.cEpstein-Barr virus DNA is expressed as log copies per milliliter in cerebrospinal fluid.

LMP-1 5 latent membrane protein; REAL 5 revised European-American lymphoma classification; WF 5 working formulation for non-Hodgkin’slymphoma; DLCL 5 diffuse large B-cell lymphoma; LNCCL 5 large noncleaved-cell lymphoma; IBPL 5 immunoblastic plasmacytoid lymphoma;LNCCL/IBPL 5 diffuse large-cell lymphoma with an admixture of centroblasts and immunoblasts; CR 5 complete response; PR 5 partial response;SD 5 stable disease; PD 5 progressive disease; NE 5 not evaluable due to early death before starting radiotherapy.

Fig. Epstein-Barr virus DNA log copies per milliliter expressedas mean and range values at baseline and after chemotherapyin patients with acquired immunodeficiency syndrome–relatedprimary central nervous system lymphoma according to thegrade of response to therapy. Statistical significance was as-sessed by paired t test.


log EBV-DNA reduction and 121 days in those with-out a reduction. The 6-month probabilities of survivalwere 0.67 and 0.20, respectively ( p 5 0.048, Mantel-Cox test).

DiscussionThe results of this study suggest a prognostic signifi-cance of the changes in EBV-DNA viral burden inCSF before and after chemotherapy for AIDS-PCNSL.In particular, an increasing burden of EBV-DNA inCSF may reflect an active proliferation of an EBV-positive tumor cell population which is totally or par-tially resistant to therapy. Conversely, a significantdecrease of viral load after chemotherapy may be asso-ciated with response to therapy and prolonged survival.

AIDS-PCNSL cases showing a response to therapyand a decrease in EBV-DNA viral load appear to dis-play biological peculiarities when compared with casesthat are resistant to therapy and show no decrease inEBV-DNA viral load (see Table). Previously, it hasbeen shown that the expression of BCL-6 and LMP-1is mutually exclusive in AIDS-PCNSL and segregatestwo distinct phenotypical categories of the disease, thatis, BCL-6 1/LMP-12 and BCL-6 2/LMP-11.10 AIDS-PCNSL showing the BCL-61/LMP-12 phenotype re-flects a germinal center stage of B-cell differentiation,tends to display a large noncleaved cell morphology,and as suggested by the present study, appears to bemore susceptible to currently available therapeuticstrategies (see Table).10 Conversely, BCL-6 2/LMP-11

AIDS-PCNSL reflects a postgerminal center stage ofB-cell differentiation, tends to display an immunoblas-tic morphology, and appears to be more resistant totherapy (see Table).10

This pilot study includes a limited number of closelymonitored patients. Nevertheless, this is one of thelargest series of AIDS-PCNSL cases treated with mul-timodal therapy published to date. Overall, our resultsprompt large-scale investigation aimed at definingwhether molecular monitoring of EBV-DNA viral loadin CSF may be a reliable tool for predicting the re-sponse of AIDS-PCNSL to therapy and identifyingsubgroups of patients with different clinical outcomesand lengths of survival.

This work was supported by the Programma Nazionale di Ricercasull’AIDS—1997. Istituto Superiore di Sanita (Rome, Italy) grantno. 30A.0.47.

Special thanks to Emanuela Vaccher from Aviano, Italy, for the ma-jor contribution to the zidovudine/MTX chemotherapy protocoland to Laura Gillini for invaluable technical assistance.

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7. Yarchoan R, Jaffe ES, Little R. Diagnosing central nervous sys-tem lymphoma in the setting of AIDS: a step forward. J NatlCancer Inst 1998;90:346–347

8. Non-Hodgkin’s Lymphoma Pathologic Classification Project.National Cancer Institute sponsored study of classification ofnon-Hodgkin’s lymphomas: summary and description of aworking formulation for clinical usage. Cancer 1982;49:2112–2135

9. Harris NL, Jaffem ES, Stain H, et al. A revised European-American classification of lymphoid neoplasms: a proposal fromthe International Lymphoma Study Group. Blood 1994;84:1361–1392

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Brief Communication: Antinori et al: EBV-DNA Monitoring and Cerebral Lymphoma 261

Genetic Locus Heterogeneityin Lafora’s ProgressiveMyoclonus EpilepsyBerge A. Minassian, MD,*†‡§ Jesus Sainz, PhD,*†Jose M. Serratosa, MD, PhD,*†i Manyee Gee, PhD,*†Lise M. Sakamoto,*† Saeed Bohlega, MD,¶Guy Geoffroy, MD,** Cathy Barr, PhD,§††Steve W. Scherer, PhD,§ Uwamie Tomiyasu, MD,‡‡Stirling Carpenter, MD,§§ Karen Wigg,††§§A. V. Sanghvi,* and Antonio V. Delgado-Escueta, MD*†ii

In 1995, we mapped a gene for Lafora’s progressive my-oclonus epilepsy in chromosome 6q23-25. In 1997 and1998, we reduced the size of the locus to 300 kb, and aninternational collaboration identified mutations in theprotein tyrosine phosphatase gene. Here, we examine forheterogeneity through the admixture test in 22 familiesand estimate the proportion of linked families to be 75 to85%. Extremely low posterior probabilities of linkage(Wi), exclusionary LOD scores, and haplotypes identify 4families unlikely to be linked to chromosome 6q24.

Minassian BA, Sainz J, Serratosa JM, Gee M,Sakamoto LM, Bohlega S, Geoffroy G, Barr C,

Scherer SW, Tomiyasu U, Carpenter S, Wigg K,Sanghvi AV, Delgado-Escueta AV. Genetic locus

heterogeneity in Lafora’s progressive myoclonusepilepsy. Ann Neurol 1999;5:262–265

In 1911, Lafora and Glueck1,2 showed characteristiclarge basophilic periodic acid-Schiff (PAS)–positive in-clusion bodies in neuronal perikarya of the central ner-vous system of a young adult with a fatal form of pro-gressive myoclonus epilepsy (PME). Almost half acentury later, Harriman and Millar3 and Schwarz andYanoff 4 showed that PAS-positive granular depositscan be found in liver,5 heart, retina, peripheral nerve,

and skeletal muscle, and others showed similar inclu-sion bodies in sweat glands and apocrine myoepithelialcells.6–9 In 1965, Schwarz and Yanoff4 described elec-troencephalographic abnormalities consisting of back-ground slowing and diffuse spike wave and polyspikewave bursts. In 1977, Schwarz10 contended that thecharacteristic clinical features of stimuli-sensitive myoc-lonus (ie, absences, tonic-clonic seizures, rapid deterio-ration in neurological function ending in dementia anddeath by 30 years of age, and typical electroencephalo-gram and pathology) together with its autosomal reces-sive mode of inheritance warranted its separation fromother PMEs and its designation as a single homoge-neous entity called Lafora’s disease (LD).

In 1995, our laboratories localized a gene for LD toa 17-cM span of chromosome 6q23-25 betweenD6S292 and D6S420.11 In 1997, we reduced the LDregion to less than 3 cM (1.2 Mb of physical distance)between D6S1003 and D6S311.12 In 1998, homozy-gosities and recombinations in 30 families further re-duced the region to 300 kb, and an international col-laborative effort isolated the LD gene and identifiedmutation(s) in a gene encoding for a protein tyrosinephosphatase.13,14

Here, we present the results of the HOMOG admix-ture test in 22 LD families, their posterior probabilityof linkage, and the exclusionary LOD scores and hap-lotypes of 4 families. The present data suggest that ge-netic locus heterogeneity exists even within this rarebut clinically homogeneous entity.

MethodsFamily MaterialWe studied 39 patients with biopsy-proven LD who be-longed to 26 unrelated families, 14 of which were inbred.Families LD3, LD4, LD6, LD9, LD12, LD27, LD28 andLD33 have 2 or more affected offspring. These eight multi-plex pedigrees and the 14 consanguineous families were usedfor linkage analyses. This study was approved by the HumanSubjects Protection Committees at the UCLA School ofMedicine and the West Los Angeles DVA Medical Center.Each participating patient, or the responsible adult on behalfof minors or deceased relatives, signed an informed consentform before a blood sample was drawn.

DNA Analyses and GenotypingDNA was extracted from 200 ml of peripheral blood usingthe QIAamp Blood kit (Qiagen, Inc, Valencia, CA), andhighly polymorphic short tandem repeats or microsatelliteswere typed using the method of Weber and May.15 We used30 microsatellites in 6q23-25, including D6S314, D6S471,D6S453, D6S308, D6S409, D6S1003, D6S1010,D6S1049, D6S1703, D6S1042, D6S311, and D6S420. Theorder of the markers had been firmly established in our po-sitional cloning of the LD gene using high-resolution yeastartificial chromosome and P1-derived artificial chromosomemapping.13,14

From the *Comprehensive Epilepsy Program, Department of Neu-rology, and iiBrain Research Institute, University of California, LosAngeles School of Medicine, and †Neurology and Research and‡‡Pathology Services, West Los Angeles DVA Medical Center, LosAngeles, CA; ‡Division of Neurology, Department of Pediatrics,Bloorview Epilepsy Program, §Department of Genetics, Hospital forSick Children, and Departments of ††Psychiatry and §§Pathology,The Toronto Hospital, University of Toronto, Toronto, Ontario,and **Department of Neurology, Ste-Justine Hospital, University ofMontreal, Montreal, Canada; iEpilepsy Unit, Jimenez Diaz Foun-dation, Madrid, Spain; and ¶Department of Medicine, King FaisalSpecialist Hospital, Riyadh, Saudi Arabia.

Received May 29, 1998, and in revised form Oct 8, 1998. Acceptedfor publication Oct 9, 1998.

Address correspondence to Dr Delgado-Escueta, ComprehensiveEpilepsy Program, UCLA and West Los Angeles DVA MedicalCenter, Building 500, Room 3405, 11301 Wilshire Boulevard, LosAngeles, CA 90073.

262 Copyright © 1999 by the American Neurological Association

Linkage AnalysesModel-dependent pairwise linkage analyses were performedusing the MLINK portion of the LINKAGE program pack-age16 and GENEHUNTER,17 assuming a single autosomalrecessive gene with gene frequency 0.001 and 100% pen-etrance.12 Unaffected family members under 20 years ofage were classified as unknown. The admixture test in theHOMOG program was used to test for genetic heterogene-ity. Haldane’s mapping function was used to convert recom-bination fractions to map distances.18

ResultsWe observed extended areas of homozygosities, rangingfrom 2.7 to 31 cM, in 15 offspring belonging to 10families. Of these 10 families, 6 with homozygositieswere consanguineous, but 4 families (LD15, LD16,LD17, LD20) with homozygosities did not reportconsanguinity.

Of the 14 families reporting consanguinity, 4 (LD7,LD27, LD28 @Quebec, Canada#, and LD25 @SaudiArabia#) did not show evidence of homozygositiesamong the chromosome 6q23-25 markers. This madeus suspect heterogeneity. As a result, we performedpairwise linkage analyses between LD in 22 consan-guineous or multiplex families and chromosome 6qmarkers D6S471, D6S310, and D6S311 under the as-sumption of heterogeneity. LOD scores excluded link-age to chromosome 6q23-25 markers in Families LD7,LD25, LD27, and LD28. LOD scores for FamiliesLD6 and LD12 were in the positive range but werenot significant.

Next, we tested for homogeneity in the 22 multiplexor consanguineous families using pairwise LOD scoresfor D6S471, D6S310, and D6S311 (Table). H2 versusH1 showed significance (p 5 0.0009 for D6S311 andp 5 0.004 for D6S310), supporting the hypothesis oflinkage with heterogeneity, and provided an estimateda of 0.75 (95% CI, 0.05–0.99) for D6S311, an esti-mated a of 0.70 for D6S310, and an estimated a of0.85 for D6S471 for the proportion of chromosome6q–linked families among these 22 families. Theposterior probability of linkage (Wi) for D6S471,

D6S310, and D6S311 favored a chromosome 6q locusfor all 22 families, except Families LD7, LD25, LD27,and LD28. The Lafora progressive myoclonus epilepsytraits of these latter 4 families are unlikely to be linkedto chromosome 6q24 because of their posterior proba-bility of linkage; for example, the probability of linkagefor D6S311 was 0.0002 for Family LD7, 0.0002 forFamily LD25, and 0.0000 for Family LD27.

In addition, LOD scores (#2) were exclusionary formany of the chromosome 6q23-25 microsatellites(D6S314–D6S420) in Families LD7, LD25, andLD27. The fourth family (LD28) showed positiveLOD scores of 1.03 at u 5 0 for D6S420 and 1.54 atu 5 0 for D6S1042. Nevertheless, because of the lowconditional probability of linkage and because a chainof homozygous markers is not seen in the affectedmembers as shown by their haplotypes, it is unlikelythat the LD gene in this consanguineous family(LD28) is located in the chromosome 6q23-25 region.

Haplotype AnalysesIn almost all cases, consanguineous unions of first-degree cousins resulting in rare autosomal recessive dis-orders are due to transmission by the carrier parents oftwo copies of the same mutation that they inheritedfrom their common grandparent.19 Similarly, geno-types of microsatellite markers in the vicinity of thegene are expected to be the same on both chromo-somes carrying the mutated gene.

In Families LD7 and LD28 from Quebec and Fam-ily LD25 from Saudi Arabia, the parents of the affectedindividual are first-degree cousins. There is no chain ofhomozygous markers in the 6q23-25 LD gene region.Moreover, the affected individuals have the same hap-lotypes for the LD region as unaffected siblings who arepast the age of possible onset of LD (.26 years old).

In Family LD27 from Quebec (Fig 1), the parentsare first-degree cousins (LD27-2, LD27-3, LD27-4,LD27-5). There is no chain of homozygous markers inthe living affected individual (LD27-6). He and hishealthy 16-year-old sister (LD27-7) have the same hap-

Table. Tests of Linkage Homogeneity

Marker Test (df )

D6S471 D6S310 D6S311

x2 p x2 p x2 p

H2 versus H1 (1) 1.526 0.2167 8.115 0.004 11.092 0.0009H1 versus H0 (1) 36.123 ,0.0001 38.725 ,0.0001 38.683 ,0.0001H2 versus H0 (2) 37.649 ,0.0001 46.840 ,0.0001 49.775 ,0.0001

The HOMOG test generates the likelihood of the data under each hypothesis, allowing the use of the likelihood ratio test. Theresulting 22 Ln (likelihood) is approximately distributed as a x2 with df equal to the difference in the number of parametersestimated. Heterogeneity is suspected if the H1 versus H2 test is significant.

H0 5 the hypothesis of no linkage in any families; H1 5 the hypothesis of linkage in all families; H2 5 the hypothesis oflinkage in only a subset of families.

Brief Communication: Minassian et al: Lafora’s Disease 263

lotypes. Although LD27-2 and his brother, LD27-4,both have sons with LD, the haplotype they share(boxed haplotype, Fig 1) is not transmitted to LD27-2’s son, LD27-6. Similarly, the haplotype common toSisters LD27-3 and LD27-5 (shaded haplotypes, seeFig 1) is not transmitted to LD27-6.

BiopsiesThe biopsy slides of LD patients belonging to FamiliesLD7, LD25, LD27, and LD28 were carefully reana-lyzed by two senior neuropathologists (U.T. and S.C.),who reconfirmed the presence of Lafora inclusions.Figure 2 shows an example of a skin biopsy of an af-

fected individual from Family LD25 that revealedprominent PAS-positive oval inclusions in peripheralcells of eccrine ducts. Hepatocytes in liver biopsiesfrom the affected individuals of Family LD27 and au-topsy brain tissue from a deceased member of FamilyLD28 revealed numerous large Lafora bodies with adark core and paler peripheral zone.

DiscussionThree of the four families who do not show linkage tochromosome 6q23-25 are from the French-Canadian“isolate” of Quebec (LD7, LD27, LD28). Noneshowed homozygous 6q23-25 microsatellites, despitebeing products of first-cousin marriages. Recombina-tions could have eliminated homozygosities fromaround the gene in affected individuals. Chances forsuch strategically placed recombinations resulting inelimination of homozygosities are more likely to occurin families where the relationship is distant, however.Recombinations eliminating homozygosities are leastlikely to occur in offspring of first-degree cousins, be-cause the number of generations separating the com-mon grandparent from the affected individuals is only 2.

Nevertheless, the absence of homozygosities in first-degree consanguineous families such as LD7, LD25,LD27, and LD28 is not of itself absolute proof of ex-clusion of the LD gene from the 6q23-25 region. It isstill possible, although highly unlikely, that strategic re-combinations within the narrow LD gene region mighthave occurred on either side of the gene even withinthe 2 generations separating the founder haplotypefrom the one seen in the affected individuals. This

Fig 1. Haplotypes of Family LD27 suggest alocus outside chromosome 6q23-25. An opensquare means an unaffected male individual,an open circle means an unaffected femaleindividual, and a filled symbol means anaffected individual. A double line indicatesa consanguineous union. A diagonal linethrough a filled symbol signifies a deceasedindividual. The vertical columns of numbersindicate genotypes for each of the microsatel-lites arranged in proper physical order fromcentromere to telomere, according to a yeastartificial chromosome and P1-derived artifi-cial chromosome contig of chromosome6q23-25.

Fig 2. Example of Lafora inclusion body in a family member(LD25-9) afflicted with the clinical disease but whose family(LD25) does not genetically link to chromosome 6q24. Ovalinclusions in peripheral cells of an eccrine duct stain stronglypositive with periodic acid-Schiff–hematoxylin. Bar 520 mm.


could offer a possible explanation for the positive LODscores in Family LD28.

Taking all the data into account, including an a es-timate of 75 to 85% of LD families linked to chromo-some 6q23-25, low conditional probability of linkagein 4 consanguineous families who lack homozygosities,sharing of 6q23-25 haplotypes by affected and unaf-fected individuals in these 4 families, and exclusionaryLOD scores in 3 of these 4 families, one has to acceptthe possibility of another genetic locus in LD.

This study was supported by NIH grant NS21908 to Dr Delgado-Escueta, the DVA West Los Angeles Medical Center, The Hospitalfor Sick Children, Bloorview Children’s Hospital Foundation, andthe Lafora’s Disease Associations of Quebec (Odette Malenfant) andSweden (Vera Faludi).

We thank Dr Jacques Thibeault for providing the biopsies on Fam-ily LD28.

References1. Lafora GR. Uber das vorkommen amyloider korperchen im in-

nern der ganglienzellen; zugleich ein beitrag zum studium deramyloiden substanz im nervensystem. Virchows Arch A PatholAnat Histopathol 1911;205:295–303

2. Lafora GR, Glueck B. Contribution to the histopathology andpathogenesis of myoclonic epilepsy. Bull Gov Hosp Insane1911;3:96

3. Harriman DGF, Millar JHD. Progressive familial myoclonicepilepsy in three families: its clinical features and pathologicalbasis. Brain 1955;78:325–349

4. Schwarz GA, Yanoff M. Lafora’s disease. Distinct clinicopatho-logic form of Unverricht’s syndrome. Arch Neurol 1965;12:172–188

5. Nishimura RN, Ishak KG, Reddick R, et al. Lafora disease:diagnosis by liver biopsy. Ann Neurol 1980;8:409–415

6. Carpenter S, Karpati G. Sweat gland duct cells in Laforadisease: diagnosis by skin biopsy. Neurology 1981;31:1564–1568

7. Busard HLSM, Gobreels-Festen AAWM, Renih WU, et al. Ax-illa skin biopsy; a reliable test for the diagnosis of Lafora’s dis-ease. Ann Neurol 1987;21:599–601

8. Van Heycop Ten Ham MW. Lafora disease, a form of progres-sive myoclonus epilepsy. Handbook of Clinical Neurology1974;15:382–422

9. Roger J, Pellissier JF, Bureau M, et al. Le diagnostic precoce dela maladie de Lafora. Importance des manifestations paroxys-tiques visuelles et interet de la biopsie cutanee. Rev Neurol(Paris) 1983;139:115–124

10. Schwarz GA. Lafora’s disease: a disorder of carbohydrate me-tabolism. In: Goldensohn ES, Appel SH, eds. Scientific ap-proaches to clinical neurology. Philadelphia: Lea & Febiger,1977:148–159

11. Serratosa JM, Delgado-Escueta AV, Posada I, et al. The genefor progressive myoclonus epilepsy of the Lafora type maps tochromosome 6q. Hum Mol Genet 1995;4:1657–1663

12. Sainz J, Minassian BA, Serratosa JM, et al. Lafora’s progressivemyoclonus epilepsy: narrowing the 6q24 locus by recombinationsand homozygosities. Am J Hum Genet 1997;61:1205–1209

13. Minassian BA, Lee JR, Hherbrick JA, et al. Mutations in a geneencoding a novel protein tyrosine phosphatase cause progressivemyoclonus epilepsy, Lafora type. Nat Genet 1998;20:171–174

14. Serratosa JM, Gomez-Garre P, Anta B, et al. A novel protein ty-

rosine phosphatase gene is mutated in progressive myodonus epi-lepsy of the Lafora type (EPM2). Hum Mol Genet 1999,8 (Inpress)

15. Weber JL, May PE. Abundant class of human DNA polymor-phisms which can be typed using the polymerase chain reac-tion. Am J Hum Genet 1989;44:388–396

16. Lathrop GM, Lalouel JM, Julier C, Ott J. Multilocus linkageanalysis in humans: detection of linkage and estimation of re-combination. Am J Hum Genet 1985;37:482–498

17. Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES. Parametricand nonparametric linkage analysis: a unified multipoint ap-proach. Am J Hum Genet 1996;58:1347–1363

18. Haldane JBS. The combination of linkage values and the cal-culation of distances between the loci of linked factors. J Genet1919;8:299–309

19. Lander ES, Botstein D. Homozygosity mapping: a way to maphuman recessive traits with the DNA of inbred children. Sci-ence 1987;236:1567–1570

Three-Dimensional Trackingof Axonal Projections inthe Brain by MagneticResonance ImagingSusumu Mori, PhD,* Barbara J. Crain, MD, PhD,†V. P. Chacko, PhD,* and Peter C. M. van Zijl, PhD*

The relationship between brain structure and complexbehavior is governed by large-scale neurocognitive net-works. The availability of a noninvasive technique thatcan visualize the neuronal projections connecting thefunctional centers should therefore provide new keys tothe understanding of brain function. By using high-resolution three-dimensional diffusion magnetic reso-nance imaging and a newly designed tracking approach,we show that neuronal pathways in the rat brain can beprobed in situ. The results are validated through compar-ison with known anatomical locations of such fibers.

Mori S, Crain BJ, Chacko VP, van Zijl PCM.Three-dimensional tracking of axonal projections

in the brain by magnetic resonance imaging.Ann Neurol 1999;45:265–269

The white matter fibers in the brain are essential inlinking functional regions. These neuronal projectionshave been traced in experimental animals, using inva-sive in vivo experiments,1 but comparable human data

From the *Department of Radiology, Division of MRI Research,and †Department of Pathology, Johns Hopkins Medical School,Baltimore, MD.

Received Aug 17, 1998, and in revised form Oct 21. Accepted forpublication Oct 23, 1998.

Address correspondence to Dr Mori, 217 Traylor, 720 Rutland Ave,Baltimore, MD 21205.


are necessarily much more limited. The availability of anoninvasive method for fiber tracking therefore wouldhave a tremendous impact on our understanding ofnormal and abnormal brain function. Diffusion-weighted magnetic resonance imaging (MRI) allows invivo mapping of the diffusional properties of brain wa-ter, and has revealed a high degree of diffusional an-isotropy (directionality) in white matter.2–11 Despitemany studies of this anisotropy and the production ofvector pictures showing fiber orientation within thevolume elements (voxels) of image planes, the actualreconstruction of neuronal projections by tracking ofthese vectors has never been accomplished. In thepresent study, we demonstrate an approach called fiberassignment by continuous tracking (FACT), which isable to achieve such high-resolution three-dimensional(3D) tracking of axonal projections. The results aresubsequently validated by in situ tracking of known fi-ber tracts in a rat brain.

Materials and MethodsThe principle of water-diffusion anisotropy is shown in Fig-ure 1a. For a region where axons are aligned, water is re-stricted in the direction perpendicular to the axons and dif-fuses preferentially in a direction parallel to them. Thissituation can be represented mathematically by a so-calleddiffusion ellipsoid5–11 (see Fig 1b), characterized by diffusionconstants l1, l2, and l3 along its three orthogonal directionsand the (vector) direction of the longest axis (l1). For exam-ple, l1 .. l2 5 l3 (anisotropic diffusion) suggests the ex-istence of cylindrical structures preferentially aligned alongl1, whereas l1 5 l2 5 l3 (isotropic diffusion) suggestssparse or unaligned axons. Although diffusion anisotropieshave been detected previously in white matter, until now ithas not been possible to translate these into neuronal trajec-tories. This problem is related to difficulties in the postpro-cessing reconstruction of 3D fiber structures from diffusiontensor MRI data, where decisions have to be made concern-ing the connections between voxels of the image. This is il-lustrated in Figure 1c, where the fibers (long curved arrows)are assumed to be confined to the two-dimensional (2D)plane and the fiber direction within each voxel is indicatedby a straight open arrow. Starting from the voxel with anasterisk, tracking should follow the bold curved arrow. Themost intuitive way to perform this tracking is by connectingeach voxel to the adjacent one toward which the fiber direc-tion is pointing. However, when using this approach, thetracking (indicated by the dotted voxels) often deviates fromthe true fiber orientation, because the choice of direction islimited to only eight angle ranges (26 in the case of 3D) (seeFig 1c). This problem is avoided when tracking a continuousrather than a discrete vector field (see Fig 1d). Here, trackingis initiated from the center of a voxel and proceeds accordingto the vector direction. At the point where the track leavesthe voxel and enters the next, its direction is changed to thatof the neighbor. Due to the presence of continuous inter-cepts, this tracking now connects the correct voxels and theactual fiber (bold straight arrows in Fig 1d) can be assigned.We therefore dubbed this approach FACT. The end point of

the projection is judged based on the occurrence of suddentransitions in the fiber orientation (see Fig 1e and f). Theseverity of such a transition is quantified through a parame-ter R, presenting the summation of the inner products ofnearby data points:

R 5 Oi

s Oj

s

abs~nl1i z nl1j!/s~s 2 1! (1)

where nl1 is the unit vector representing the longest princi-pal diffusion axis (l1) and s is the number of data points

Fig 1. Principle of fiber tracking. (a) Schematic view of re-stricted water diffusion (solid sphere) in an environment withstrongly aligned fibers (depicted by bars). The diffusion proper-ties can be fully described by an ellipsoid (b) with three prin-cipal axes (l1, l2, and l3 ) of which the orientation of themain axis represents the average fiber direction. Once thisdirection is determined in each voxel, tracking can be per-formed by using either a discrete (c) or a continuous (d) num-ber field. The actual fibers are indicated by curved arrows,and the average fiber directions in the voxel are displayed asopen arrows. The connected voxels resulting from tracking areshaded, using dots. The discrete approach leads to deviationfrom the actual fiber to be tracked (c), whereas the continuousapproach succeeds as indicated by the train of solid arrows(d). A three-dimensional axonal projection can be tracked aslong as nearby vectors are strongly aligned (e). When vectororientation becomes random, as judged quantitatively fromsummation of the inner products of these vectors (Eq 1), thetracking is ended ( f ).


referenced. When adjacent fibers are aligned strongly (see Fig1e), the R value is large, while it becomes small (see Fig 1f)in regions without continuity in fiber direction. Theoreti-cally, there are many ways to modify this criterion. In thepresent study, we calculated R values by using the fourneighboring voxels closest with respect to the continuoustracking position, and a fiber was judged discontinued forR , 0.8. The procedure of mapping the neuronal connec-tions is started through the input of an arbitrary point in 3Dspace, after which the extent of the axonal projections intofunctional regions is traced in both the orthograde (forward)and the retrograde (backward) directions.

Studies were performed on a 400 MHz GE Omega (9.4T), using a field of view of 32 3 16 3 16 mm and 128 364 3 64 data points zero-filled to a final resolution of256 3 128 3 128. Diffusion images were recorded by usinga spin-echo Stejskal-Tanner sequence (echo time [TE] 5 44msec) and 10 gradient orientations. Gradient strength andlength were 16 G/cm and 5 msec, with a separation of 16msec. For an additional image with low diffusion weighting,a strength of 5 G/cm was used in the xyz direction. Diffusiontensor elements at each voxel were determined by multivari-ant least-square fitting weighted by signal-to-noise ratio.12

The tensors were diagonalized to obtain l1, l2, and l3

and the direction of l1. The brain sample was obtained froman adult Sprague–Dawley rat and fixed on 5% formalin for2 weeks.

ResultsFigure 2a shows a 2D representation of the water-diffusion measurements. Although existence of axonalprojections can be clearly appreciated from this 2Dvector picture, tracing of neuronal projections throughthe brain requires a complete 3D analysis. After ex-tending the diffusion MRI into 3D, we applied theFACT analysis to track 10 different pathways in aformalin-fixed rat brain (Fig 3; see Fig 2b). It can beseen that our method successfully reconstructs the well-known structure of these projections. To further illus-trate the validity of our approach, the tracking of theanterior commissure is also shown in a series of 2Dplanes and compared with the corresponding rat brainatlas from Paxinos and Watson13 (see Fig 3d–f). The Ushape of this structure can be easily appreciated fromthe atlas and it can be clearly seen that our diffusionresult (dark blue) accurately traces this projection.These 2D slices also show part of the tracking of ol-factory tract (ol), middle forebrain bundle (mfb), andfornix (f).

One interesting aspect of Figure 2a is the demon-stration of aligned fibers in most areas of gray matter.Such anisotropy of gray matter has been reported pre-viously, using diffusion measurements in two spatialorientations,14 and our results extend on this earlierfinding by showing the ordered fiber structures thatcause the anisotropy.

DiscussionOur results from using the FACT approach show thatit is now possible to track the 3D structure of axonalprojections by using the diffusion MRI technique. Al-though this is very promising, some limitations shouldbe pointed out. First, MRI can only give informationon the average axonal orientation within a voxel.Therefore, it is resolution dependent and it cannot dis-tinguish among several very small projections that areimmediately adjacent to each other. Second, the track-ing method is unable to distinguish between afferentand efferent fibers. Further problems may occur if por-tions of two pathways actually touch, because the pro-gram may inadvertently switch pathways. For example,the initial tracking of the optic tract was complicatedwhen reaching the level of the dorsal lateral geniculate,where the algorithm began a partial tracking of adja-cent fimbrial axons. Other limitations are functions ofthe current algorithm. For example, one of the mostchallenging problems is the tracking of branched axonsor axons leaving major pathways as they approach theirtargets. Our program traces only one of the branchesin such a situation. An example is the tracking of thelateral olfactory tract (see Fig 3) for which the anteriorportion is quite faithfully followed (see arrow in Fig 3dand e), but where the fibers that are actually identifiedin Figure 3f are small branches into olfactory and piri-form cortex (indicated by dotted lines in the atlases ofFig 3d–f). As a result, the lateral olfactory tract itself,the location of which is still visible in the T2-weightedMRI scan (see arrow in Fig 3f), is not labeled at thislevel. Possible solutions to this problem depend on thespecific question. For instance, if the goal is to followvarious components of a pathway to their end points,one might simply identify multiple points in a largefiber bundle and track each component separately.

In summary, we introduced a 3D tracking methodfor diffusion MRI that allows the successful identifica-tion of axonal projections. Potential applications of thismethod include the study of neuronal developmentand of diseases affecting neuronal integrity. In thepresent study, a total of 12 hours were needed for thecompletion of the study. This time period can be easilyshortened by a factor of 8 to 32 by using multiple-echo acquisition techniques.15–17 Combined with re-cent methodological advances for the suppression ofmotion-related artifacts,18–20 we expect clinical appli-cation of this fiber-reconstruction technique to be fea-sible in the near future.

This research was funded in part by a grant from Johns HopkinsSchool of Medicine, the American Federation of Aging Research,and the Whitaker Foundation.

Brief Communication: Mori et al: 3D Brain Fiber Tracking 267

Fig 2. Two-dimensional (2D) and three-dimensional (3D) track-ing of prominent axonal projections. (a) 2D vector field presenta-tion in the parietal lobe of a rat brain as localized from a sectionof the T2-weighted magnetic resonance imaging scan. Becauseaxonal orientations are projected onto the 2D plane, axons per-pendicular to the plane appear as dots. Rough boundaries forregions of prominent fiber bundles are indicated by color lines(yellow 5 corpus callosum [cc] and external capsule [ec];green 5 fimbria [ fi]; and red 5 internal capsule [ ic]. Noticethe presence of preferential axonal orientation in the gray matter.(b) 3D presentation of the fibers. Color coding is the same as in(a) except for the blue color that shows axons tracked from thesplenium of corpus callosum into the external capsule. Some axonswithin the fimbria are tracked into ventral hippocampal commis-sure, and axons within the internal capsule are tracked into thecorpus callosum.

Fig 3. Three-dimensional (3D) projections andtwo-dimensional (2D) validation of eight fiberbundles in the rat brain. The results of the track-ing are superimposed on 3D volume images, usingan oblique angle (a) or three orthogonal angles (band c). The vertical lines between (b) and (c)indicate the positions of the 2D axial slices ofT2-weighted images on which the position of thetracking is superimposed (d–f ). Images from therat brain atlas13 corresponding to the three slicesin (d–f ) are shown in the bottom row. Colorcodes are as follows: green 5 fimbria; darkblue 5 anterior commissure (ac); light blue 5medial forebrain bundle (mfb); yellow 5 fornix(f); white 5 stria terminalis; pink 5 stria med-ullaris; red 5 optic tract; peach 5 lateral olfac-tory tract (ol).


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CorrectionThe abstract by Yamasaki and colleagues that appearedin the September issue (Ann Neurol 1998;44:490–491) and the program of the American NeurologicalAssociation’s 123rd Annual Meeting (pp 76–77)contained an error of copy editing that distortedits meaning. The full text of the corrected abstractappears here:

T193. A Protein Critical for Immune-MediatedDemyelinating DiseaseKenji Yamasaki, G. D. Ghadge, and R. P. Roos; Chicago, IL

DA and other members of the TO subgroup of Theiler’smurine encephalomyelitis virus persistently infect mice andcause a chronic demyelinating disease that resembles multiplesclerosis. Both diseases have a similar pathology, and in boththe immune system plays a role in pathogenesis. In contrast,members of a GDVII subgroup produce an acute fatal en-cephalomyelitis and do not persist. We previously reportedthat demyelinating (but not nondemyelinating) strains havean initiation codon for translation for a protein (L*) out offrame with the viral polyprotein; mutant DA virus that doesnot have the L* initiation codon fails to induce a persistentinfection or demyelination. These data suggest that L* is crit-ically important for viral persistence and demyelination. Tofurther test the role of L* in demyelinating disease, we havenow engineered the GDVII viral genome to contain the L*initiation codon and to synthesize L*. The efficiency of ex-pression of L* in mutant GDVII depends on particular nu-cleotides in an RNA stem loop structure near the L* initia-tion codon. Mutant GDVII virus that synthesizes L* is nolonger neurovirulent. The presence of demyelination and vi-rus persistence in survivors is under study.

Study supported by the National Multiple Sclerosis Society.

The publisher apologizes for the error.

Brief Communication: Mori et al: 3D Brain Fiber Tracking 269

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