Pathology of Leukemia in the Brain and Meninges: Postmortem

Studies of Patients with Acute Leukemia and of Mice Given

Inoculations ofLl2lO Leukemia

LOUIS B. THOMAS

Pathologic Anatomy Branch, National Cancer Institute, Bethesda, Maryland

SUMMARY

Data previously published were reviewed and compared with pathologic observations on more recently autopsied patients. Special consideration was given to the

frequency of leukemic cell involvement of the brain and meninges and the effects ofanti-leukemic chemotherapy on the amount and distribution of leukemic cells in thecentral nervous system at the time these patients died. The material from man wasalso compared with that from mice given inoculations of L1210 leukemia becausemeningeal leukemia of the mouse was found to be similar pathologically to meningealleukemia of man.

There was little change in the amount of leukemic cell infiltration in the meninges ofsubjects autopsied during 2 periods, 1953—58 and 1961—63. Intracerebral leukostasisand leukemic nodule formation occurred with equal frequency in the 2 series.

Pathologically there did not appear to be any relationship between intracerebralleukostasis and leukemic nodule formation and the degree of leukemic cell infiltrationill the meninges. Leukemic cell inifitration in the area postrema, the tuber cinereum,

and other “non-neural―areas was usually associated with leukemic cell infiltration ofthe arachnoid.

Many of the histologic features of leukemic cell infiltration in the meninges andnon-neural areas of the mouse after inoculations of L1210 leukemia were similar tothose in man. Intracerebral leukostasis and leukemic nodules, and massive dural andarachnoidal hemorrhages, were occasionally seen in the brains of patients with acuteleukemia; however, they were not observed in the central nervous system of miceinoculated with L1210 leukemia.

Diffuse meningeal leukemia will develop in mice given s.c. inoculations of L1210leukemia if their life is prolonged with methotrexate treatment. In mice given s.c.inoculations of L1210 leukemia and treated with methotrexate, leukemic cells enteredthe arachnoid by direct migration and growth through the perivascular and perineuraltissues of vessels and nerves which bridge the subdural space. Although direct observations about the route of spread of leukemic cells to the arachnoid cannot be madein the human, it was speculated after examination of the human material that directspread of leukemic cells from dura to the arachnoid may occur in man as it did in themouse. Pathologic observations in mice with diffuse meningeal leukemia confirmedthe fact that most of the commonly used anti-leukemic drugs, when given parenterally,failed to cross the blood-brain barrier in sufficient quantity to eradicate leukemic cellsfrom the central nervous system. One of the nitrosoureas, BCNU (given parenterally), did appear to be effective against meningeal leukemia cells in the arachnoid.

Leukemic involvement of the meninges, brain, and with acute leukemia who developed signs and symptoms ofspinal cord has been known for nearly 100 years, and many meningeal irritation and increased intracranial pressure.neurologic changes have been ascribed to the presence of This syndrome is now referred to as meningeal leukemia.the infiltrating leukemic cells. Since the advent of the Second, most of the anti-leukemic drugs do not cross theanti-leukemic chemotherapy era, interest in the central blood-brain barrier in sufficient quantities to eradicatenervous system in acute leukemia has increased. First, leukemic cells which are in the brain and meninges.there appeared to be an increasing number of patients More than 400 subjects dead of acute leukemia have

1555

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Series of casesNo. of cases―Degree ofinfiltrate%1953—58

1961—6369 421—2+ 1—2+35 1651381953—58

1961—6369 423—4+ 3—4+13 619141953—58

1961—6369 421—4+ 1—4+48 2270 52

Series of casesNo. of cases―Degree of infiltrateNo. withinfiltrate1953—58

1961—6339 391—2+ 1—2+21 1354331953—58

1961—6339 393-4+ 3—4+2 2551953—58

1961—6339 391—4+ 1-4+23 1559 43

1556 Cancer Research Vol.25,October 1965

been autopsied in the Department of Pathologic Anatomyduring the years 1953—63. The over-all frequency ofleukemic involvement of the central nervous system of the117 cases autopsied during the 5-year period from 1953 to1958 has been rej)orted (6). The pathologic findings inthese 117 cases are summarized and compared with thefindings in 100 consecutive cases in which death was due toacute leukemia and which were autopsied during a laterperiod, December, 1961, to June, 1963. In general, therewas greater clinical awareness of the problem of meningealleukemia during the period of treatment of the more recent series of l)atients, and a number of them receivedintrathecal chemotherapy for meningeal leukemia in addition to nrnltiple courses of systemic chemotherapy. Forthese reasons it proved interesting to compare the extentand degree of leukemic cell infiltration in the brain andmeninges of the I)atients in the 2 series.

The results of a number of animal experiments are alsoreviewed, because experimental meningeal leukemia ofmice given inoculations of L1210 leukemia mimics the human disease closely. Some of the observations in miceappear to have furnished answers to questions which theobservations in man did not answer.

Pathologic studies of the subjects dead of acute leukemiahave usually been conducted in association with Dr. EmilFrei and Dr. Emil J Freireich of the Medicine Branch ofthe National Cancer Institute. The experimental studiesof L1210 leukemia in mice have all been done in collaboration with Dr. @\IichaelChirigos of the Viral ChemotherapySection, Cancer Chemotherapy National Service Centerof the National Cancer Institute.

The following pathologic conditions in the patients withacute leukemia and in the mice given inoculations ofL1210 leukemia are given special consideration:

I. Human material1. Frequency of leukemic iIl%olvement of the central

nervous system in patients dead of acute leukemia

a) Leukemic cell infiltration of the dura

b) Leukemic cell infiltration of the arachnoidc) Leukosta.sis and intracerebral leukemic nodules

associated with ‘‘blastic crisis―d) Diffuse intracerebral leukemic cell infiltratione) Leukemic cell infiltration in non-neural areas of

the brain1) Leukemic cell infiltration of the pineal gland,

pituitary gland, and choroid plexus2. Presence of leukemic cells in the cerebrospinal fluid

of Patients with acute leukemiaII. Experimental material

1. Leukemic cell infiltration of the central nervoussystem of mice given inoculations of L1210 leukemia

a) Development of meningeal leukemia in micegiven intracerebral inoculations

b) Development of meiiingeal leukemia iii micegiven 5. c. inoculations

c) Leukemic cell infiltration iii non-neural areas ofthe brains of mice

d) Failure of development of leukosta.sis and intracerebral leukemic nodules in mice

2. Effect of chemotherapy on meningeal leukemia inmice given intracerebral inoculations of L1210leukemia

I. HUMAN i@%lATERIAL

1. Frequency of leukemic involvement of the central nervoussyste@n in patients dying of acute leukemia.—The over-allfrequency of leukemic involvement of the central nervoussystem in 2 autopsy series is presented, and the distributionand degree of the leukemic cell infiltrations are compared.The 1953—58series included the first 117 patients withacute leukemia autopsied in this department. The 100patients included in the 1961-63 series represent the mostrecent consecutively autopsied patients with acute leukemia. In Tables 1—4the cases in the 2 series are groupedaccording to the degree of leukemic cell inifitration (6).The cases in the 2 series are comparable by age, sex, andtype of leukemia (Tables 6 and 7).

a) Leukemic cell inifitration of the dura : Infiltration of1 + or more was noted in the dura of 70 % of the cases withacute lymphocytic leukemia in the 1953—58series and in52 % of the cases in the 1961—63series (Table 1). Of thecases with acute myelogenous leukemia, 59 % of the 1953—58 series and 43 % of the 1961—63series had 1+ or moreleukemic cell infiltration (Table 2). The differences arenot statistically significant.

TABLE 1LEUKEMIC CELL INFILTRATION OF DURA IN ACUTE

LYMPHOCYTIC LEUKEMIA

a Sections of dura were not available for evaluation in 5 of the1953—58cases and in 9 of the 1961—63cases.

TABLE 2LEUKEMIC CELL INFILTRATION OF DIJRA IN ACUTE

MYELOGENOUS LEUKEMIA

a Sections of dura were not available for evaluation in 4 of the1953—58casesand in5ofthe1961—63cases.

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Series of casesNo. of casesaDegree of infiltrateN@O@W1@th

1716%

23321953—581961—6374 501—2+1—2+1953—58

1961—6374 503—4+ 3—4+11 615121953—58

1961—6374 501—4+ 1—4+28 2238 44

Series of casesNo. of casesaDegree of infiltrateNo. withinfiltrate°‘@°1953—58

1961—6343 421—2+ 1—2+4 59121953—58

1961—6343 423—4+ 3—4+3 2751953—58

1961—6343 421—4+ 1—4+7 716 17

PatientsAcute lymphocyticleukemiaAcute

myelogenousleukemiaAcute

leukemia, t@rpenotclassifiedTotalChildrenb

MaleFemale

AdultMaleFemRle41

2318

10829

45

3520153

350

2723

4823

15Total5144

@ 398

Series of casesNo. of casesLeukemicnodule%1953—5811714121961—63981010

PatientsAcute lymphocytic leukemiaAcute

myelogenousleukemiaTotalChildren'49554Male33336Female16218Adult253863Male142640Female111223Total7443117

THoMAS—Pathology of Leukemia in the Brain and Meninges 1557

tients in the 1961—63 series exhibited this lesion (Table 5).Four of the 10 l)atientS with the lesion had acute lymphocytic leukemia, 5 had acute myelogenous leukemia, and 1had an acute leukemia of type not classified. Thus in the2 groups of patients the frequency of leukosta.sis and intracerebral leukemic nodules at the time of death is nearlyidentical. This is remarkable, because greater effort wasmade during the time of treatment of the 1961—63seriesto control blastic crisis whenever it was clinically evident.

Pathologically there did not appear to be any relationship between leukostasis and intracerebral leukemic noduleformation, with or without fatal intracerebral hemorrhage,and the degree of leukemic cell infiltration in the meninges.None of the 10 patients with leukosta.sis and intracerebralleukemic nodules in the 1961—63series had a 4+ dural or a4+ arachnoidal infiltration. In fact there was 3+ infiltration in the dura of only 1 of these patients, and therewere no recognizable leukemic cells in the arachnoid of 2patients. The remaining 6 had either 1+ or 2+ leukemiccell infiltration of the dura and arachnoid.

d) Diffuse intracerebral leukemic cell infiltration : Except for the intracerebral leukemic nodules associated withblastic crisis described above, and infiltration of so-callednon-neural areas which will be described below, diffuseintracerebral infiltration by leukemic cells was seldomencountered in either the 1953—58or the 1961—63series of

TABLE 6PATIENTS WITH ACUTE LEUKEMIA INCLUDED IN 1961-63

POSTMORTEM STUDYa

TABLE 3LEUKEMIC CELL INFILTRATION OF ARACHNOID IN ACUTE

LYMPHOCYTICLEUKEMIA

a Sections of arachnoid were not available for evaluation in 1 ofthe 1961—63cases.

TABLE 4LEUKEMIC CELL INFILTRATION OF ARACHNOID IN ACUTE

MYELOGENOUS LEUKEMIA

a Sections of arachnoid were not available for evaluation in 2of the 1961—63cases.

TABLE 5

INTRACEREBRAL LETJKEMIC NODULES ASSOCIATED WITH

BLASTIC CRISIS

a Study includes 100 consecutive postmortem examinations,

but permission to examine brain was not given in 2 instances.b This group includes patients 15 years of age or less.

TABLE 7PATIENTS WITH ACUTE LEUKEMIA INCLUDED IN 1953—58

POSTMORTEM STUDY

b) Leukemic cell infiltration of the arachnoid : Infiltration of 1+ or more was noted in 44 % of the cases withacute lymphocytic leukemia and in 17 % of the cases withacute myelogenous leukemia i@ the 1961—63 series (Tables3 and 4), as compared with 38 and 16% of the 1953-58series. There is 110 significant difference between thesepercentages of arachnoidal infiltration.

These figures indicate a similar degree of leukemic cellinfiltration of the meninges in the 2 series.

c) Leukostasis and intracerebral leukemic nodulesassociated with blastic crisis : The association of a distinctive type of intracerebral lesion characterized by leukostasis, intracerebral leukemic nodule formation, andacute intracerebral hemorrhage with a rapidly risingperipheral leukocyte count (blastic crisis) in patients withleukemia has been previously reported (4, 5). Fourteen(12 %) of the brains of the 1 17 patients included in the1953—58 series and 10 (10 %) of the brains of the 98 pa a This group includes patients 15 yeam of age or less.

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AGE (yr)DEGREE OFLEUKEMICCELLINFILTRATIONMaleFemaleArachnoidArea

postremaTubercinereum2mo+++4++++++++++4++++++4±06±±700078++

+++++88

89++

±0++++

0

009±01400160017±±28++++++340059++00

AGE (yr)DEGREE OF LEUKEMIC CELLINFILTRATIONMaleFemale

2.5ArachnoidArea

postremaTubercinereum0010++++17019+++++++++2031++

00039

4141485153±

0+0

±

+0

±0

±

+0

0

055±05658±

0±0++67000

1558 Cancer Research Vol.25,October 1965

cases. In the report of the earlier series it was noted thatperivascular “cuffing―by leukemic cells in the substanceof the brain or spinal cord was always associated with leukemic cell infiltration within the arachnoid (6). Thisassociation was also evident in the 1961—63series. It isworth while to note that diffuse leukemic cell infiltrationinto the substance of the brain or spinal cord usually didnot occur eveii in the presence of 3—4+ leukemic cell infiltration in the arachnoid and 3-4+ perivascular cuffing.Occasionally focal areas of intracerebral leukemic cell infiltration were found in or adjacent to areas of softeningand hemorrhage in patients with septicemia. However,intracerebral leukemic cell infiltration was not extensiveeven around these pyemic lesions.

e) Leukemic cell infiltration in non-neural areas of thebrain: Leukemic cell infiltration of the so-called non-neuralareas of the brain—i.e., area postrema, subfornical organ, ortuber cinereum—was first observed in mice given intracerebral inoculations of L1210 leukemia (9). Subsequently, leukemic cell infiltration was found in the nonneural areas iii the brain of some of the patients who haddied of acute leukemia. The similarity between the appearance of the infiltration in the non-neural areas of themouse and of the human is striking (Figs. 1—7). It wasalso observed in mice that infiltration of the non-neuralareas developed only in association with infiltration ofleukemic cells in the arachnoid. A marked associationbetween the degree of leukemic cell infiltration in thearachnoid and Hi the Ilon-neural areas was also found in thehuman (Tables 8, 9). The 35 patients from the 1961—63

series who are arranged by age, sex, and type of leukemiain these 2 tables were selected because routine histologicsections of the central nervous system included either thearea postrema or the tuber cinereum. In these patientsthe degree of leukemic cell infiltration seen microscopicallyin either of these non-neural areas could be compared withthe amount of leukemic cell infiltration seen in the arachnoid.

f) Leukemic cell infiltration of the pineal gland, pituitary gland, and choroid plexus : These structures are alsosometimes referred to as non-neural areas of the brain. Asystematic study of the association between the degree ofarachnoid infiltration and the degree of leukemic cell inifitration in each of these structures in each of the patientsincluded in the 1961—63series has not been made. However, routine sections of the central nervous system oftenincluded at least one of these structures. Therefore, thesections which included pituitary gland, pineal gland, andchoroid plexus have been reviewed, and the degree of leukemic cell infiltration has been noted.

No leukemic cell infiltration was seen in 12 of the 15pineal glands which had been sectioned. The arachnoidin 9 of these 12 was also free of leukemic cell infiltration,and in 3 others was 1+ . The 3 infiltrated pineal glandshad 1+, 2+, and 4+ degrees of leukemic cell infiltration,and the arachnoid in the same patients was 2+, ±, and1 +, respectively.

Leukemic cell infiltration was present in 8 of 35 pituitaryglands which were sectioned and suitable for evaluation.Seven of the pituitary glands exhibited 1+ infiltration, and1 had 2+ infiltration. In 6 of the 7 the degree of arach

TABLE 8COMPARISON OF LEUKEMIC CELL INFILTRATION IN AREA

POSTREMA AND TUBER CINEREUM WITH ARACHNOIDALINFILTRATION IN ACUTE LYMPHOCYTICLEUKEMIA

TABLE 9COMPARISON OF LEUKEMIC INFILTRATION IN AREA POSTREMA AND

TUBER CINEREUM WITH ARACHNOIDAL INFILTRATION INACUTE MYELOGENOUS LEUKEMIA

noidal infiltration was either 1+ or more than 1+ . Inonly 1 instance was the degree of pituitary infiltrationgreater than the degree of arachnoidal infiltration: 2+and ±,respectively.

The degree of infiltration in the choroid plexus wasevaluated in 26 patients. In 21 the choroid plexus had noleukemic cell infiltration, and in 17 of the 21 the arachnoidwas also free of leukemic cell infiltration. In the other 4the arachnoidal infiltration was 1+, 2+, 2+, and 4+.

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THoMAs—Pathology of Leukemia in the Brain and Meninges 1559

In 5 patients there was either 1+ or 2+ leukemic cell infiltration in the choroid plexus, and the arachnoid in eachof these 5 patients was also infiltrated, usually with anequal degree of leukemic cell infiltration.

It is apparent from these figures that there is a markedassociation between the degree of leukemic cell infiltrationin the arachnoid and the degree of leukemic cell infiltrationin the pineal gland, pituitary gland, and choroid plexus.Usually the leukemic cell infiltrations in the interstitialtissues of the pituitary gland and pineal gland were penvascular and multifocal. Their appearance suggesteddirect infiltration from the thin-walled vessels in theseglands. The leukemic cell infiltrations in the choroidplexus, on the other hand, did not appear to result fromdirect spread from the vessels of the choroid plexus.Rather, it appeared that leukemic cells migrated from thearachnoid through the loose interstitial tissues of the telachoroidea to reach the choroid l)lexus. In other words,they migrated to the choroid plexus along with the bloodvessels supplying the choroid plexus.

2. Presence of leukeinic cells in the cerebrospinal fluid ofpatients with acute leukemia.—Del Vecchio et al. have reported on the cytology of spinal fluid in 95 patients withacute lymphocytic leukemia and 15 patients with acutemyelocytic leukemia who were admitted to the LeukemiaService of the National Cancer Institute (3). Their observations were based on cytologic examination by the1\Iillij)Ore membrane technic of ap@)roximately 7 cerebrospinal fluid specimens from each of the patients with acutelymphocytic leukemia and of approximately 2 specimensfrom each patient with acute myelocytic leukemia. Immature lymphocytes were found in at least 1 specimen ofcerebrospinal fluid from 68 % of the patients with acutelymphocytic leukemia and from 47 % of the latients withacute myelocytic leukemia.

II. EXPERIMENTAL MATERIAL

1. Leukemic cell infiltration of the central nervous systemof mice given inoculations of L1210 leukemia.—For severalyears we have been studying the pathology of leukemiccell infiltration in the meninges and the central nervoussystem of susceptible strains of mice given inoculations ofL1210 leukemia. The technics used and the detaileddescriptions of some of these experiments have been published (2, 9). These experiments have increased ourunderstanding of the routes by which leukemic cells enterand leave the meninges, brain, and spinal cord of the mouseand have also been useful in testing the effectiveness ofchemotherapeutic agents against diffuse meningeal

leukemia. As mentioned earlier, the experimentally produced L1210 meningeal leukemia in mice has many pathologic features which are strikingly similar to those observed in patients dying of acute leukemia. For thisreason it is hoped that a number of the observations in theexperiments with mice will contribute to our understanding of the problem of meningeal leukemia in the human.

a) Development of meningeal leukemia in mice giveninoculations intracerebrally of L1210 leukemia: It wasfound that the easiest and most direct method of producing meningeal leukemia was by intracerebral inoculation. If an appropriate inoculum of L1210 leukemia was

injected intracerebrally into mice of a susceptible strain,diffuse leukemic cell infiltration of the arachnoid and duradeveloped, and the animals died in 10—12days. Diffuseinfiltration of leukemic cells through the tissues of thebrain and spinal cord did not occur, even though there was4+ infiltration in the meninges at the time the mice died.In fact, leukemic cells usually did not proliferate or groweven along the intraeerebral inoculation needle track.

The early fate of leukemic cells iiioeulated into thecerebral tissue of the mouse is not known because detailedpathologic studies have not been made immediately afterinoculation or during the next 2 days. However, it appeared from @)atholog1cobservations on the spread of leukemic cells during the period from the 3rd to the 6th dayafter intracerebral inoculation that the l)robable sequenceof events was as follows: (a) Most of the L1210 leukemiacells inoculated into the substance of the brain died ; (b) afew leukemic cells survived and migrated or were carriedby the flow of cerebrospinal fluid to the region of relativelylarge dural sinuses or veins or occasionally to the region of

arachnoidal veins; (c) some of the leukemic cells infiltrated the walls of the dural veins and entered the bloodstream to be carried to extracranial organs and tissues ; and(d) other leukemic cells started proliferating in the tissuesadjacent to the dural sinuses and eventually l)roduceddiffuse meningeal leukemia. Usually there were only 2 or3 microscopic foci of leukemic cells around dural sinuses onthe 3rd and 4th days after intracerebral inoculation, andmoderately diffuse meningeal leukemia was not established until the 6th day. Obviously these time relationships in the development of meningeal leukemia variedsomewhat according to the size and potency of the leukemic cell iiioculum, which was injected intracerebrally.It was because of these pathologic observations thatchemotherapy was not started until the 6th day afterintracerebral inoculation in experiments in which theeffectiveness of anti-leukemic chemotherapy was beingstudied.

b) Development of meningeal leukemia in mice givens.c. inoculations of L1210 leukemia: 1\Iice given inoculations subcutaneously of L1210 leukemia occasionally developed slight, focal infiltrations of leukemic cells in thearachnoid but did not develop diffuse leukemic cell inifitration of the arachnoid even though there were massiveleukemic cell infiltrations in the liver and spleen and largenumbers of leukemic cells circulating in the blood severaldays before death. Pathologically it appeared that thesmall focal deposits of leukemic cells which were presentin the arachnoid had reached the arachnoid directly fromthe dura by migrating through the adventitial tissueswhich surrounded blood vessels and nerves bridging thesubdural space (9). Leukemic cell infiltration of the duraappeared to be direct from dural veins and also from adjacent bone marrow. Both the circulatingbloodand thebone marrow of these mice contained many leukemic cells.

Because of these observations it was concluded thatdiffuse leukemic cell infiltration of the arachnoid would develop if mice inoculated s. c. were kept alive for a sufficientlength of time to allow large numbers of leukemic cells tomigrate from the dura to the arachnoid. The desired cxperimental conditions were obtained by giving mice s.c.

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Cancer Research Vol.25,October 19651560

inoculations of L1210 leukemia and treating them withmethotrexate. The methotrexate treatment was startedon the 8th day after inoculation, when the mice were

nearly terminal, so that the mediati survival time was increased by only 1 day. However, some of the mice survived froiu 2 to 9 days beyond the median survival time,and each of these mice was found at autopsy to have developed diffuse infiltration and growth of leukemic cells inboth the dura and arachnoid (10).

c) Leukeniic cell infiltration in non-neural areas of thebrains of mice: As noted earlier there is a striking similarity between the appearance of leukemic cell infiltrationin the area postrema and tuber cinereum of the human andthe leukemic cell infiltrations in these 2 structures and thesubfornical organ of mice (Figs. 1—7). In animals givenintracerebral inoculations the degree of leukemic cell infiltration in these non-neural areas was always accom

panied by a similar degree of leukemic cell infiltrationelsewhere in the arachnoid. Similarly, in the experiments iii which mice were given s.c. inoculations andtreated with methotrexate, only those animals which haddeveloped extensive and widespread leukemic cell infiltration of the arachnoid exhibited leukemic cell infiltrationin the non-neural areas. The degree of leukemic cell infiltration H) the pineal gland and choroid l)lexus of the

mice given inoculations of L1210 leukemia, like that observed in the human, was also related to the degree ofleukemic cell infiltration in the arachnoid.

d) Failure of development of leukosta.sis and intracerebral leukemic nodules in mice given inoculations of L1210leukemia : Leukosta.sis and intracerebral leukemic noduleformation have not developed in any of the mice in theexl)erin)ents ill which L1210 leukemia was inoculatedeither intracerebrally or subcutaneously. Even thosemice treated with methotrexate which developed diffuseleukemic cell infiltrations in the meninges and non-neuralarea-s of the brain exhibited neither leukostasis nor intracerebral leukemic nodules. This is one of 2 principaldifferences between the pathologic appearance of meningeal leukemia observed in the human dying of acute leukemia and that produced by inoculating L1210 leukemiainto susceptible strains of niice. The other pathologicaldifference is the absence of intracerebral, subarachnoidal,or dural hemorrhage in the mouse. In the human, massiveintracerebral hemorrhage occurred in association with thedevelopment of intracerebral leukemic nodules, and subarachnoid and subdural hemorrhages occurred in association with thrombocytopenia (4, 5). Also, in the human,many of the leukemic cell infiltrates, whether in the dura,arachnoid, or the non-neural areas, were grossly and microscopically hemorrhagic. Occasionally leukemic cell infiltration in the non-neural areas of the mouse was observed to be hemorrhagic, but leukemic cell infiltrations inthe dura and arachnoid were characteristically free ofhemorrhage both grossly and microscopically. Massivehemorrhage in either the dura or the arachnoid ofthe mouse was not seen.

2. Effect of chemotherapyon meningealleukemia in micegiven inoculations intracerebrally of L1210 leukemia.—It isnot necessary for the purpose of this presentation to review the extensive literature on the numerous anti-leu

kemic drugs which have been shown to have some therapeutic effect on intracerebrally inoculated L1210 leukemia.It is appropriate, however, to summarize the pathologicchanges we have observed in several experiments in which

various chemotherapeutic agents were gi yen after menin

geal leukemia was established in mice by intracerebralinoculation. The drugs' used in these experiments wereCytoxan (cyclophosphamide), methotrexate (amethopterm), MeGAG (methylglyoxal-bis-guanylhydrazone), hydroxyurea, leurocristine (vincristine sulfate), 6-MP(6-mercaptopurine), sarcolysine [p-di (2-chloroethyl)aminophenylalanine], terephthalanilide (2-chloro-4', 4―-di2-imidazolin-2-ylterephthalanilide), TEM (triethylenemelamine), and BCNU [1 , 3-bis(2-chloroethyl)-l -nitrosourea] . Systemic chemotherapy was started on the 6thday after intracerebral inoculation of L1210 leukemia.By this time meningeal leukemia was usually well established, and leukemic cell infiltrations had also appeared inthe liver, spleen, bone marrow, and other extracranialtissues.

The pathologic observations in mice treated with cytoxan have been reported (9). Cytoxan destroyed all ofthe leukemic cells in the extracranial organs and the durabut had little effect on the leukemic cells infiltrating thearachnoid and the non-neural areas of the brain. Theinterpretation of these observations was that cytoxan is a

@articularly j)Otent chemotherapeutic agent against L1210leukemia but that an insufficient amount of the drug passedthrough the blood-brain and blood-cerebrospinal fluidbarriers to be effective against leukemic cells in the arachnoid. Cytoxan was capable of curing mice inoculateds.c. if it was given before any of the leukemic cells hadreached the arachnoid.

A detailed report on the pathologic and biologic findingsin the animals treated with the other chemotherapeuticagents will be published later. In general, none of thesedrugs except BCNU was capable of completely eradicatingL1210 leukemia from either the extracranial organs or themeninges and ceiitral nervous system. BCNU and othernitrosoureas have been reported to be the first class ofcompounds which “possess an encouraging degree of activity against intracerebrally inoculated L1210 leukemia―(8). In our experiments BCNU is the only chemotherapeutic drug which is capable of completely eradicatingL1210 leukemic cells both from the extracranial organsand tissues and from the dura, the arachnoid, and the nonneural areas of the brain. Within 2 days after the institution of parenteral therapy with BCNU, in mice giveninoculations intracerebrally 6 days previously, leukemiccells iii the arachnoid exhibited marked cytotoxic and cytolytic changes and by the 4th day of treatment either hadcompletely disappeared or were so degenerated that theywere difficult to recognize as leukemic cells microscopically(Fig. 8). Many arachnoidal vessels near foci of degencrating leukemic cells were ifiled with fresh thrombus, andsome acute hemorrhage and necrosis appeared in adjacentbrain tissue.

I Obtained through the Cancer Chemotherapy National Service

Center, National Cancer Institute.

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THOMAS—Pathology of Leukemia in the Brain and Meninges 1561

in the memnges to produce the syndrome of meningeal leukemia do not occur in all these patients.

Theoretically, circulating leukemic cells might enter thebrain or meninges by 5 different routes: They might passthrough the thin walls of the capillaries of the brain andenter the brain tissues directly. There was no pathologicevidence either in the mice or in the humans that this occurred. Periva.scular cuffing of intracerebral and intraspinal vessels by leukemic cells in the absence of leukemiccell infiltration in the arachnoid did not occur. Rather, itappeared that leukemic cell cuffing of cerebral vessels wasdue to migration of leukemic cells from the arachnoid tissues on the surface of the brain. A 2nd route of spreadinto the central nervous system might be by migration ofleukemic cells through the capillaries and specializedependymal cells which form the choroid plexus. This didnot seem to be a very important route of spread, even if itoccurred at all, because isolated leukemic cell infiltrationin the choroid plexus was never seen in the mice and onlyseldom seen in the human material. Choroid plexus infiltration was nearly always associated with leukemic cellinfiltration in adjacent arachnoidal or pial tissues, and inmany histologic sections it appeared that leukemic cellswere migrating from the arachnoid into the choroid plexus.The capillaries in the brain are thought to be the anatomicsite of the blood-brain barrier, and the choroid plexus isthought to be the anatomic site of the blood-cerebrospinalfluid barrier. It is interesting to note that these 2 barriers,which are remarkably impermeable to many drugs andcompounds, also seem to be effective barriers against circulating leukemic cells. A 3rd route by which leukemiccells could enter the central nervous system is directlythrough the walls of vessels which lie in the arachnoid.Leukemic cells could reach the subarachnoid space directlyby this route and be distributed widely by the flow ofcerebrospinal fluid.

If leukemic cells did enter by this route and were carriedaway immediately by the spinal fluid there would, ofcourse, be nothing for the pathologist to observe whichwould indicate that leukemic cells had entered by thisroute. It is more probable, however, that if leukemic cellsentered by this route some would remain and form localinfiltrates in the vicinity of the arachnoidal vessels. Because isolated perivascular arachnoidal infiltrations of

leukemic cells were never seen in the mice given s.c. inoculations of L1210 leukemia, and were only seldom encountred in the human material, it is believed that this route ofspread is also relatively unimportant. A 4th route ofspread could be through the capillaries of the non-neuralareas. From the animal experiments it appeared that thisroute of spread was also relatively unimportant, becausenone of the mice that received s.c. inoculations was foundto have leukemic cell infiltrates in the non-neural areas ofthe brain. In both the animal and the human material,leukemic cell infiltration in these areas was almost alwaysassociated with the development of widespread leukemiccell infiltration in the arachnoid. The 5th route by whichleukeinic cells could enter the meninges and brain is bydirect migration and growth through perivascular andperineural tissues of vessels and nerves which cross thesubdural space. In the animal experiments this appeared

DISCUSSION

The pathologic studies on patients with acute leukemiaand on mice given inoculations of L1210 leukemia havebeen presented in some detail, because it is hoped thatthey will answer some of the principal questions about thecentral nervous system manifestations of acute leukemia.The most important questions that arise may be dividedinto 2 groups. (a) The first group is concerned with thebasic mechanisms of the spread of leukemia in the meningesand tissues of the central nervous system. What are theroutes by which leukemic cells reach the dura? How dothey reach the arachnoid? Do they infiltrate and proliferate in the cerebral and spinal cord tissues? Whatqualitative or quantitative features of the leukemic cellinfiltration of the central nervous system can be correlatedwith the meningeal syndrome? What is the mechanismby which leukemic cell inifitration of the meninges andcentral nervous system produces increased intracranialpressure and hydrocephalus? Do all patients with acuteleukemia have leukemic cells in the cerebrospinal fluid atsome time during the course of the disease? (b) The seeond group of questions is concerned with the effects produced by anti-leukemic chemotherapy on leukemic cellinifitrations in the meninges and central nervous system.The principal and foremost question in this group iswhether all leukemic cells are eradicated from the centralnervous system by any particular drug. In other words, isany drug completely effective, and, if not, what are thetherapeutic effects of each particular drug, and are theseeffects histologically distinctive? Inasmuch as the nonneural areas of the brain are reported to have a decrease orabsence of the blood-brain barrier, does systemic chemo.therapy produce any effect on leukemic cells infiltratingthese non-neural areas? Do any of the chemotherapeutic

agents used parenterally or intrathecally produce pathologic lesions in the central nervous system? There are nodefinitive answers to any of these questions. However, itis interesting to consider some of the partial answerswhich have come from pathologic study of the human andanimal material.

Study of the pathology of L1210 leukemia in mice hasgiven considerable information about the actual physicalroutes by which leukemic cells enter the brain andmeninges. Each mouse or patient with circulating leukemic cells had millions or even billions of these cells passing through the blood vessels of the brain and meningesevery few minutes. The remarkable observations werethat only about@ of the patients developed signs and symptoms of meningeal leukemia and that none of the micegiven s.c. inoculations of L1210 leukemia exhibited diffuseleukemic cell infiltration in the arachnoid unless their lifewas prolonged by methotrexate chemotherapy. Thecytologic studies revealed that a considerable proportionof patients had leukemic cells in the cerebrospinal fluid atsome time during the course of the disease : 68 % in acutelymphocytic leukemia and 47 % in acute myelocytic leukemia. There were no comparable studies on the cerebrospinal fluid in mke given inoculations of L1210 leukemia.These observations suggest that a few leukemic cells mayenter the cerebrospinal fluid in a majority of patients butthat sufficient proliferation and growth of leukemic cells

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Cancer Research Vol. 25, October 19651562

to be the principal route by which leukemic cells spread tothe arachnoid. In most experiments the mice that hadreceived s.c. inoculations died before diffuse arachnoidalleukemia had developed. If the life of the leukemic micewas prolonged by methotrexate, however, the number ofleukemic cells spreading from dura to arachnoid via thenerves and vessels increased greatly, and diffuse arachnoidal leukemia developed. There is no pathologic cvidence in the human that leukemic cells spread by this sameroute directly along vessels and nerves from the dura tothe arachnoid. However, it is possible that this is also theprincipal route in the human. If so, it might help to cxplain 2 clinical and pathologic observations : (a) the factthat all patients with acute leukemia have not developedarachnoidal infiltration by the time they die, and (b) anapparent increase in meningeal leukemia during the antileukemic chemotherapy era. Additional informationabout these 2 observations could be obtained by a systematic review of the gross and microscopic pathology ofthe brains of patients with acute leukemia who died priorto the anti-leukemic chemotherapy era.

The amount of hydrocephalus in the brains of the patients in the 1961—63 series and the types of treatment

which these j)atients received have not yet been studied.In the 1953—58series of patients, a marked association wasnoted between arachnoidal infiltration and hydrocephalusin the patients who had the meningeal leukemia syndrome.It wa.s postulated that leukemic cells infiltrating the arachnoid interfered with cerebrospinal fluid flow to produce increased intracranial pressure and hydrocephalus. Leukemic cell infiltrations in the dura might augment the development of the syndrome, but the principal cause ofincreased intracranial pressure was considered to be thearachnoidal infiltration. At the time of the pathologicreview of the 1953—58series of patients, leukemic cell infiltrations in the non-neural areas, particularly the areapostrema and the tuber cinereum, had not been observed.

Since then infiltration of these areas was observed in themouse and human brains and was found to be associatedwith arachnoidal infiltraticn. The function of these nonneural areas is unknown. The area postrema in both themouse and man has a close anatomic relationship to thetela choroidea of the choroid plexus of the 4th ventricle,and in mice there is a close relationship between the veinsdraining the choroid plexus of the 3rd ventricle and thesubfornical organ. Because of these anatomic relationships it is possible to speculate that these non-neural structures have some influence on the rate of production ofcerebrospinal fluid by the choroid plexus. If this were so,leukemie cell infiltration of the non-neural structures mightplay some role in the development of increased intracranial pressure and hydrocephalus. A number of micewith infiltrations in the arachnoid and non-neural areashave developed hydrocephalus. Unfortunately, it hasproved difficult to make a systematic study of hydrocephalus in these animals given inoculations of L1210 leukemia because of the shrinkage artifact which was usuallyproduced by fixation of the mouse head and brain.

The pathologic data presented for the patients in the1953—58 and 1961—63 postmortem series indicated thatleukemic cell infiltration of the meninges is present in anumber of patients at the time of death in spite of vigorousanti-leukemic chemotherapy and increasing clinical awareness of the problem of meningeal leukemia. Even thoughintrathecal chemotherapy produced remission of meningealleukemia in a number of the patients (7), leukemic cellinfiltrations were found in the meninges at the time ofdeath. Thus, it appears that none of the therapeuticdrugs or regimens of therapy used in these patients wascapable of permanently eradicating leukemic cells from thecentral nervous system. In experiments with mice givenintracerebral inoculations of L1210 leukemia, systemicchemotherapy with cytoxan, methotrexate, MeGAG, hydroxyurea, leurocristine, 6-MP, sarcolysine, terephth

FIG. 1._Upper medulla and rostral portion of spinal canal ofmouse. The area postrema is situated in the dorsal portion ofthe medulla and has a 3+ infiltration of leukemic cells. The infiltrating leukemic cells are grouped around small blood vesselsof the area postrema and form a diffuse infiltrate in the overlyingarachnoidal tissues. Sections of arachnoid and dura elsewherealso exhibited 3—4+ leukemic cell infiltrations. 1)iffuse infiltration of leukemic cells into the medulla or perivascular cuffing ofintramedtillary vessels was not observed. The mouse was killed16 days after s.c. inoculation of L1210 leukemia. It was treatedwith daily s.c. injections of methotrexate, 0.75 mg/kg, from the8th to the 15th day after inoculation. H & E, X 108.

FIG. 2.—(A56-206). Upper medulla and rostral portion ofspinal canal of a 12-year-old girl with acute lymphocytic leukemia.The appearance of leukemic cells infiltrating the area postrema isremarkably similar to the infiltration of the area postrema of themouse shown in Fig. 1. There was also 3—4+ leukemic cell infil

tration in the dura and arachnoid over the surface of the brainand spinal cord. Other sites of the central nervous system infiltrated by leukemic cells were both lobes of the pituitary gland,4+ ; choroid plexus, 4+ ; Pacchionian granulations, 1+ ; tubercinereum and adjacent wall of the third ventricle, 4+; and intracerebral perivascular cuffing, 4+. There was no hydrocephalus.The patient had meningeal leukemia 3-4 months before death andhad a partial remission following intrathecal methotrexate therapy, which was given about 2 months before death. She had systemic relapse for the last 2 months of life. H & E, X 20.

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FIG. 3.—Brain at level of tuber cinereum and supraoptic recessof the 3rd ventricle of a mouse. The tuber cinereum has a discrete3—4+infiltration of leukemic cells. Elsewhere there was 1—4+leukemic cell infiltration in the arachnoid and dura, 3+ infiltration in the area postrema, subfornical body, and choroid plexus,and 2+ infiltration in the pineal gland. Except for these nonneural areas there was no leukemic cell infiltration into the substance of the brain or spinal cord. The mouse was killed on the9th day after intracerebral inoculation of L1210 leukemia. H &E, X 150.

FIG. 4.—(A61-132). Floor of the third ventricle and hypothalamus at level of tuber cinereum and stalk of pituitary gland of a16-year-old boy who died of acute lymphocytic leukemia. Theoptic tracts can be seen laterally. There is a 3—4+leukemic cellinfiltration into the tuber cinereum and pituitary stalk. Thepatient had meningeal leukemia clinically and had some improvement 2-3 months before death following intrathecal treatmentwith aminopterin and ‘@Aubut was in relapse for several weeksbefore death. Note the similarity between this leukemic cell infiltration and the infiltration in the area postrema shown in Fig.2. Leukemic cell infiltration was seen in other sections of thebrain and meninges, 1+ in the pineal gland and arachnoid, 1—2+in the dura, 3—4+perivaseular cuffing of 2 vessels in 1 of the cerebral peduncles, and 0-± in the area postrema. There was no hydrocephalus. H & E, X 12.

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FIG. 5.—Brain of mouse at the level of the subfornical organ.On each side of the corpus callosum are portions of the lateralventricle which communicate with the 3rd ventricle through theforamen of Monroe. Portions of the choroid plexus extend intothe 3rd ventricle and into the lateral ventricles. The subfornicalorgan, a flattened structure on the undersurface of the corpuscallosum, protrudes slightly into the 3rd ventricle. The histologicfeatures of this organ can be seen better in Fig. 6. The mousewas killed on the 6th day after intracerebral inoculation of L1210leukemia. There was only a minimal take. Except for 1 microscopic focus of leukemic cells in the tentorium, the arachnoid,dura, brain, choroid plexus, and subfornical body appeared normal. H&E,X43.

FIG. 6.—Brain of mouse. The subfornical organ is the distinctstructure situated in the midline on the ventricular surface of thecorpus callosum. It is covered by ependymal cells of the 3rd yentricle and laterally is adjacent to the branches of the internalcerebral veins which drain the choroid plexus of the 3rd ventricleand lateral ventricles. Akert et at. (1) have studied the anatomyand histology of this organ in several species and point out “thatit can be found consistently in the rostral wall of the third yentricle at- the point in the midline to which the choroid plexuses ofthe lateral and third ventricles converge.― H & E, X 210.

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FIG. 7.—Brain of mouse. The subfornical organ has a 4+ infiltration of L1210 leukemia cells. A few leukemic cells are present around each choroidal vein which can be seen on each side ofthe subfornical organ. Laterally, portions of each choroid plexuscan be seen and these also have a 4+ leukemic cell infiltration.A few leukemic cells are detached and are lying on the ependymalsurface of the 3rd ventricle. There are no leukemic cells infiltrating the corpus callosum. Leukemic cell infiltrates were also seenin other sections of the central nervous system: 4+ in the tubercinereum and pineal gland, 3+ in the dura, arachnoid, and areapostrema. The mouse was killed on the 11th day after intracerebral inoculation of L1210 leukemia. Sarcolysine, 7 mg/kg/day:was started on Day 6. H & E, X 150.

FIG. 8.—Brain of mouse. Centrally, the thickened papillaryprocesses of the choroid plexus almost completely fill the thirdventricle. Large numbers of degenerating pyknotic and fragmented nuclei of L1210 leukemic cells are present in the interstitial tissues of the choroid plexus. A fresh thrombus fills thechoroidal vein, which is situated just above the choroid plexus.Other sections of the brain and spinal cord also revealed largenumbers of necrotic leukemic cells in the arachnoid, tuber cinereum, and dura. Several other veins in the arachnoid were occluded by fresh thrombus. Focal areas of encephalomalacia andacute hemorrhage were seen in the hippocampus. The mousewas killed 8 days after intracerebral inoculation of L1210 leukemia. BCNU, 12.5 mg/kg, was injected subcutaneously on Days6and7. H&E,X 160.

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THOMAS—Pathology of Leukemia in the Brain and Meninges 1571

alanilide, and TEM also failed to eradicate leukemic cellsinfiltrating the meninges. Only 1 drug, BCNU, appearedto be capable of completely eradicating L1210 leukemiccells from the arachnoid and central nervous system.

REFERENCES

1. Akert, K., Potter, D., and Anderson, J. W. The SubfornicalOrgan in Mammals. I. Comparative and Topographical Anatomy. J. Comp. Neurol., 116: 1—14,1961.

2. Chirigos, M. A., Thomas, L. B., Humphreys, S. R., andGoldin, A. Intracerebral Growth and Treatment of LeukemiaL1210. Proc. Soc. Exptl. Biol. Med., 104: 643-45, 1960.

3. Del Vecchio, P. R., Chu, E. W., Malmgren, R. A. and Freireich, E. J Cytology of Spinal Fluid in Leukemia. Abstr.American Society of Cytology, Inc., Eleventh Annual Meeting, 1963.

4. Freireich, E. J, Thomas, L. B., Frei, E., Fritz, R. D., andForkner, C. E., Jr. A Distinctive Type of Intracerebral Hemorrhage Associated with “BlasticCrisis― in Patients withLeukemia. Cancer, 13: 146—54,1960.

5. Fritz, R. D., Forkner, C. E., Jr., Freireich, E. J, Frei, E.,

and Thomas, L. B. The Association of Fatal IntracranialHemorrhage and “Blastic Crisis― in Patients with Acute Leukemia. New Engl. J. Med., 261: 59—64,1959.

6. Moore, E. W., Thomas, L. B., Shaw, R. K., and Freireich,E. J A Postmortem Study of 117 Consecutive Cases, withParticular Reference to Hemorrhages , Leukemic Infiltrations,and the Syndrome of Meningeal Leukemia, the Central Nervous System in Acute Leukemia. A. M. A. Arch. Internal Med.,105: 451—68,1963.

7.Rieselbach,R. E.,Morse,E. E.,Rall,D. P.,Frei,E.,andFreireich, E. J Intrathecal Aminopterin Therapy of Meningeal Leukemia. Ibid., 111 : 620—30,1963.

8. Schabel, F. M., Jr., Johnston, T. P., McCaleb, G. S., Montgomery, J. A., Laster, W. R., and Skipper, H. E. ExperimentalEvaluation of Potential Anticancer Agents. Cancer Res., 25:725—33,1963.

9. Thomas, L. B., Chirigos, M. A., Humphreys, S. R.,and Goldin, A. Pathology of the Spread of L1210 Leukemiain the Central Nervous System of Mice and Effect of Treatment with Cytoxan. J. Natl. Cancer Inst., 28: 1355—89,1962.

10.@— . Development of Meningeal Leukemia (L1210) duringTreatment of Subcutaneously Inoculated Mice with Methotrexate. Cancer, 17: 352-60, 1964.

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1965;25:1555-1571. Cancer Res   Louis B. Thomas  Inoculations of L1210 LeukemiaStudies of Patients with Acute Leukemia and of Mice Given Pathology of Leukemia in the Brain and Meninges: Postmortem

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