1. No category

From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
1992 80: 1279-1283
Growth and spread of human malignant T lymphoblasts in
immunosuppressed nude mice: a model for meningeal leukemia
F Cavallo, M Forni, C Riccardi, A Soleti, F Di Pierro and G Forni
Updated information and services can be found at:
http://www.bloodjournal.org/content/80/5/1279.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
Growth and Spread of Human Malignant T Lymphoblasts in Immunosuppressed
Nude Mice: A Model for Meningeal Leukemia
By Federica Cavallo, Marco Forni, Carlo Riccardi, Antonio Soleti, Francesco Di Pierro, and Guido Forni
Previous work has shown that nude (nu/nu) mice additionally immunosuppressed by splenectomy, sublethal irradiation, and treatment with antiasialo GMI antiserum (SIAnu/nu mice) have no detectable natural killer activity and
allow the growth of human malignant lymphoblasts. We
show here that all SIA-nulnu mice engrafted intravenously
with 5 x 108 malignant lymphoblastsoriginally derived from
a child with a T-cell acute lymphoblastic leukemia (PF382)
and from a boy with a T-cell lymphoma (ST-4) develop lethal
meningealleukemia and die within 35 days. Histologicexam-
ination of moribund SIA-nu/nu mice showed that vertebral
and skull bone marrow was always replaced by proliferating
human T lymphoblasts.From the spinal canal, lymphoblasts
spread to the meninges, causing hind leg paralysis. Leaving
the skull, they permeatedthe meninges and then invaded the
nervous parenchyma. This efficient and reproducibleexperimental model may be suitable for experimental studies on
the pathogenesisof meningeal leukemia.
0 1992by The American Society of Hematology.
M
pathogenesis of meningeal leukemia, and in the development of experimental strategies designed to prevent it.
ENINGEAL LEUKEMIA emerged as an important
complication of acute lymphoid leukemia when
improved therapeutic strategies increased the remission
rate and prolonged survival.' Meningeal prophylaxis based
on skull irradiation, intrathecal chemotherapy, or both has
reduced the incidence of meningeal leukemia to less than
10%.2,3Morbidity and intellectual disabilities may be associated with these treatments. A better understanding of the
process by which leukemic cells enter the brain may lead to
an improved management of meningeal leukemia and,
potentially, reduce the risk of overtreatment and of secondary complications.
Numerous studies on h u m a d 6 and animals6-I1have
disclosed several features of meningeal leukemia, although
little is known about the sequence of events through which
the meninges are colonized by malignant lymphoblasts.12
Entry into the central nervous system (CNS) via the
systemic circ~lation~,~J2
or from the skull bone marr0w3,~J~
are the two main hypotheses.
The difficulty of setting up an appropriate experimental
model with human leukemic cells has represented a major
limiting factor. Heterotransplantation of human tumors
into athymic nude (nulnu) mice enables interesting models
to be built. However, hematopoietic malignancies have
consistently proved difficult to tran~p1ant.I~The severe
combined immunodeficiency (scidlscid)mouse can be used
to enable the growth and spread of childhood acute
lymphoblastic leukemias in many organs, including CNS.14
We have recently shown that nulnu mice additionally
immunosuppressed by splenectomy, sublethal irradiation,
and treatment with antiasialo GM1 antiserum (SIA-nulnu
mice) display no natural killer (NK) activity in the blood
and spleen, and allow the growth and systemic dissemination of several human malignant blasts, either directly
obtained from the patients or growing in vitro.15-'7
The aim of the present study was to reproduce in mice a
situation in which CNS involvement is the only or the most
serious clinical problem, and not just an occasional part of a
widespread disease with invasion of many organs, as commonly found in scidlscid mice challenged with malignant
1ymphobla~ts.l~
An intravenous (IV) challenge with human
malignant T lymphoblasts was capable of giving rise to a
selective meningeal leukemia with a consistent homing and
progression pattern in 100% of SIA-nulnu mice. This
model could help in investigating the factors involved in the
Blood, Vol80, No 5 (September 11,1992: pp 1279-1283
MATERIALS AND METHODS
Mice. Five-week-old female nulnu mice on Swiss background
were purchased from Charles River Laboratories (Calco, Italy) and
allowed to rest for 1week before any treatment. They were fed and
maintained under specific patogen-free conditions, and received
sterilized food pellets and tap water ad libitum. Mice were
splenectomized as previously described in detai1,'5-'7 and received
total body, sublethal irradiation of 4.5 Gy from a 137Cssource
providing a dose rate of 0.5 Gy/min 3 days before tumor challenge.
Forty-eight and 24 hours before this challenge, they were additionally injected IV with 0.2 mL of a %O dilution in phosphate-buffered
saline (PBS) of antiasialo GM1 antiserum (Wako Chemicals
GmbH, Dusseldorf, Germany; batch PDG9536) and referred to as
SIA-nulnu mice.
Malignant lymphoblasts. PF382 lymphoblasts were originally
derived from the pleural exudate of a 6-year-old boy with T-cell
lymphoblastic leukemia in relapse, and maintained as an in vitro
cell line.I8 ST-4 lymphoblasts were from a lymph node biopsy of a
12-year-old boy with a malignant T-cell lymphoma (convoluted
type) currently alive and in prolonged complete remis~ion.'~
Lymphoblast engraflment. PF382 or ST-4 malignant T lymphoblasts (5 X lo6) in 0.3 mL of Hanks' Balanced Salt Solution
(HBSS) were injected 1V into the tail vein of SIA-nulnu mice,
which were then inspected daily for signs of systemic involvement
and spinal cord symptoms. Moribund mice were killed in extremis.
Each experiment was performed independently four times using
groups consisting of five mice. Because they gave homogeneous
From the Institute of Microbiology, University of Turin, Turin; the
Regina Margherita Children's Hospital, Turin; the Institute of Pharmacologx University of Perugia, Perugia; and the Immunogenetics and
Histocompatibility Center, CNR, Turin, Italy.
Submitted September 16, 1991; accepted May 7,1992.
Supported by research grants j?om the Italian Association for
Cancer Research (AIRC), the Ministry of Education (40 & 60%),
Regione Piemonte, and PF ACRO.
Address reprint request to Guido Forni, MD, the Immunogenetics
and Histocompatibiliv Center CNR, Via Santena 19, 10126 Turin,
Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1992 by TheAmerican Society of Hematology.
0006-497119218005-0002$3.00/0
1279
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
CAVALLO ET AL
1280
results, as determined by the Snedecor and Irvin test," the data
were cumulated.
Blood smears. At weekly intervals after challenge and when
mice displayed initial neurologic signs, peripheral blood smears of
all challenged SIA-nulnu mice were performed through punctures
of the tail lateral vein and routinely stained for differential count.
At least 300 leukocytes were counted for each mouse.
Hivtology. Thoracic and abdominal organs of all mice were
inspected, extracted, fixed in buffered formalin, and routinely
processed for histologic e~aminati0n.l~
To maintain the anatomical
connection among bones and meninges, the head was skinned,
fixed for 48 hours, completely decalcified in Osteodec (Farben;
Bio-Optica, Milan, Italy) for 24 to 48 hours, and then sliced with
frontal cuts in six to seven parallel slices 2 to 3 mm thick, including
cranial bones and cerebrum. A transversal section was cut from the
dorso-lumbar region of the decalcified spines, whereas the two
remaining halves were cut in half sagittally. The tissues were then
embedded in paraffin, sectioned, and stained with hematoxylin and
eosin. Some tissues were also snap-frozen and immunostained with
mouse monoclonal antibody (MoAb) to HLA A, B, C antigens
(TEC-HLA ABC; Sorin, Saluggia, Italy) using an avidin-biotin
procedure (ABC, Vectastain; Vector, Burlingame, CA), as previously described.15
Recovery of malignant T-blasts. Fragments of skull bones were
placed in complete medium in 24-well plates (Nunc, Roskilde,
Denmark). The lymphoblasts recovered were stained with fluorescein isothiocyanate (F1TC)-conjugated anti-HLA A, B, C, CD1
and CD7 murine MoAb (Sorin) and analyzed with a FACScan flow
cytometer (Beckton Dickinson, Milan, Italy). Karyotype analysis
was performed as previously des~ribed.'~,'~
RESULTS
Clinical course. By the day 25 to 30 after IV challenge
with malignant human lymphoblasts, SIA-nulnu mice presented anorexia, hunched posture, lethargy, and weight
loss. A gait disturbance and signs of hind limb weakness
become evident first in mice challenged with ST-4 lymphoblasts and later in those challenged with PF382 lymphoblasts. A marked leukopenia with relative neutrophilia but
no leukemic blasts was found on differential cell count of
smears of peripheral blood taken at weekly intervals after
challenge from asymptomatic mice. Occasional leukemic
blasts (1% or less) were found when mice displayed initial
neurologic signs. About 48 hours after the appearance of
overt neurologic symptoms, mice become moribund and
were killed. No neoplastic foci were ever found on histologic examination of lung and kidney from all of these
moribund mice. Liver infiltration by PF382 cells was found
in only one case (Fig 1A). By contrast, extensive bone
marrow and CNS infiltration was always evident (Table 1).
Spine and medulla. In mice challenged with ST-4 cells,
the vertebral bone marrow was replaced by rapidly proliferating lymphoblasts (Fig 1B). When a high local concentration was reached, lymphoblasts broke through the nutrient
foramen and colonized muscles and/or meninges, and
eroded the trabeculae of the vertebrae (Fig 1C and D). The
meningeal infiltration was epidural and only later subdural,
without lesions of the pia mater. Therefore, the spinal cord
was never directly infiltrated, and hind leg paralysis was due
to compression. Whereas ST-4 uniformly infiltrated the
vertebral bodies, PF382 lymphoblasts were mainly located
in the lumbar area. This distinct infiltration pattern ex-
plains why mice injected with ST-4 lymphoblasts became
paralyzed earlier.
Encephalic area. In the initial experiments, the brains of
SIA-nulnu challenged mice were extracted before being
processed for histologic observation. However, it became
evident that this maneuver hampers correct evaluation,
because it alters the meninges structure and brain-skull
anatomical connections, and may lead to the loss of
lymphoblast clusters attached to the brain surface (Fig 2).
When the whole cranium was decalcified and processed
for histology, it was evident that lymphoblast infiltration
primarily affects the skull bone marrow. Infiltration was not
uniform, with the temporal bone being most frequently and
massively infiltrated, followed by the sphenoid. Meningeal
infiltration and extension of the leukemic infiltrates into the
brain parenchyma follow direct transit of skull-infiltrating
lymphoblasts. These lymphoblasts progressively erode the
junction of the squama with the petrosa portion of temporal
bone (Fig 2A and B) and the sphenoid (Fig 2C), invade the
dura mater and the subarachnoid spaces (Fig 2C) with
permeation of the pia mater, and infiltrate the VirchowRobin spaces and the nervous parenchyma late (Fig 2D, E,
and F).
This progression pattern was almost identical after both
ST-4 and PF382 lymphoblast challenge. Minor differences
were noted in the distribution and extension of bone
marrow infiltration, and meningeal spreading as PF382
lymphoblasts showed more extensive cerebral infiltration
with areas of necrosis.
Malignant lymphoblastsfromSZA-nulnumice. The immunoperoxidase technique was used to stain spinal cord and
encephalic area sections with an antihuman HLA A, B, C
MoAb, as previously described.15The large infiltrating cells
were always positive. Flow cytometry and cytogenetic analysis on lymphoblasts recovered from bone marrow and
meninges cultures displayed no significant modulation of
surface and karyotype markers when compared with ST-4
and PF382 lymphoblasts before engraftmentlsJ9 (data not
shown).
DISCUSSION
Splenectomy, irradiation, and injection of antiasialo GM1
antiserum required for SIA-nulnu mice are simple procedures that can be easily standardized and quickly performed. The resulting SIA-nulnu mice are healthy, longliving animals that display no detectable NK activity in the
blood and the spleen, clear human malignant lymphoblasts
very slowly, and allow their local growth and systemic
dissemination.l5-I7
The pattern of dissemination of malignant human T
lymphoblasts injected IV into SIA-nulnu mice enables a
model of selective leukemic invasion of the CNS to be
created. The effectiveness and reproducibility of CNS
invasion makes it a suitable model for experimental studies
on meningeal leukemia and on its pathogenesis.
In SIA-nulnu mice, both PF382 and ST-4 human lymphoblasts injected IV localize first in the bone marrow and then
disseminate to the surrounding tissues, and to the CNS in
particular, whereas a hematologic dissemination was never
found.
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
1281
A MODEL FOR MENINGEAL LEUKEMIA
Fig 1. Growth of PF382 and ST-4 human lymphoblasts in the liver and spine. (A) Liver: Evident lymphoblast foci were found in only 1/20 mice
challengedwith PF382 lymphoblasts. (B.C. and D) Spine: Infiltration by ST-4 lymphoblasts that colonize vertebral bone marrow (b) and meningeal
spaces. (B)The epidural infiltrate is evident at vertebral body level, whereas it disappears at the vertebraldisk level (arrow). (C) Fromthe vertebral
bone marrow lymphoblasts break through the nutrient foramen of the bone (arrow)to the meningeal spaces (arrow), whereas the medulla (m) is
not invaded. (D) Erosion of the vertebral bone table and spilling of the lymphoblasts into the epidural spaces (black arrow) and, on the opposite
site, invasion of skeletal muscles (white arrow). Original magnification: A and B, x10; C and D, ~ 2 5 .
In SIA-nulnu mice, the spread and progression pattern
of PF382 and ST-4 cells is remarkably consistent. However,
in untreated nulnu mice, as well as in those only splenectomized or sublethally irradiated, ST-4 and PF382 lymphoblasts either do not grow or they vary to a great extent their
growth and dissemination pattern.16J7 Moreover, when
PF382 lymphoblasts are injected in SIA-nulnu mice treated
with a concentration of antiasialo GM1 antiserum, only half
of that used in this study, their growth pattern is markedly
different. They first invade the liver and kidney, whereas
CNS localization is an occasional and late event.17Scidlscid
mice have T- and B-lymphocyte defects, but a normal NK
activity. They allow the growth and spread of many childhood acute lymphobastic leukemias, but the CNS is never
primarily affected.14 In these mice, PF382 and ST-4 lymphoblasts form a tumor when injected subcutaneously, but do
not spread after an IV challenge (data not shown). As a
whole, these findings show that the addition of antiasialo
GM1 treatment to other major immunodeficiency statuses
as well as the dose used are important in determining the
spread of malignant lymphoblasts and getting a selective
CNS invasion.
One of the most evident effects of antiasialo GM1
antiserum is the abolition of the NK activity of recipient
Table 1. Features of Clinical Course and Lymphoblast Localization 111
SIA-nulnu Mice ChallengedWith PF382 and ST-4 Human
T-Malignant Lymphoblasts
Mice
Challenged
IV With
Meningeal
Leukemia
Takes/Total
Challenged
Mice
PF382
20/20
ST-4
19/19
Spread of
Malignant Lymphoblasts
Organ
% of
Mice
Lung*
Liver*
Kidney*
Bone marrow*
Meninges*
BIood(>l/lOO)t
Lung'
Liver*
Kidney"
Bone marrow*
Meninges*
Blood (>1/100)t
0
5
0
100
100
0
0
0
0
100
100
0
Days When
Mice Becomt
Moribund
26-32
23-27
*Presence of histologically detectable foci of malignant lymphoblasts.
tLymphoblasts/peripharal blood leukocytes.
Fig 2. Meningeal invasion of the encephallc area by PF382 and
ST-4 lymphoblasts. (A and B) Bone marrow infiltration of the temporal
bone by PF382 lymphoblasts. Slight (A) (arrows) and massive (B)
infiltration of the meningeal spaces by lymphoblasts coming from the
bone marrow. (C) ST-4 lymphoblasts coming out from the sphenoid
bone(s) form a large meningeal deposit in the base of the skull.
Interestingly, the biconvex shape of the infiltrate is reminiscent of a
subdural hemorrhage. Pie mater and cortex (c) are not yet infiltrated.
(D, E, and F) Progressive phases of brain parenchyma invasion by
PF382 lymphoblasts. (D) Initially, the meningeal leukemic infiltration
is confined to arachnoidal spaces. No violation of pia mater is yet
present. Virchow-Robin spaces are not invaded by lymphoblasts
(whiie arrow). (E) Initial permeation of pia mater. Few lymphoblasts
are invading the superficial cortex. Virchow-Robin spaces are both
noninvaded (white arrow) and invaded (black arrow). (F) Diffuse
infiltration of the superficial cortex and invasion of Virchow-Robin
spaces (black arrows). Original magnification: A, B, and C, ~ 2 5 D,
; E,
and F, x100.
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
CAVALLO ET AL
1282
mi~e.17,2~,~~
This produces a major delay in the clearance of
human and murine lymphoblasts from nu/nu mice and
increases the growth and dissemination of many tum o r ~ . ~Thus,
~ , it~ is~ likely
, ~ ~that NK cells play a direct role
in the spread of malignant lymphoblasts and the appearance of meningeal leukemia. However, in addition to NK
cells, other asialo GM1+ cells responsible for natural
resistance to malignant lymphoblasts can be affected by the
amounts of antiserum used.% The role of natural immune
reactivity mechanisms in the pathogenesis of meningeal
leukemia is a subject for future research.
The sequence of events behind leukemic infiltration of
the meninges and brain is debated.3-12 Our histologic
findings strongly suggest that the primary event is lymphoblast infiltration of the bone marrow, whereas meningeal
and brain invasion comes later. Confirmation of this succession is provided by the observation of a high blast proportion in the meninges right beside the sites where the
passage from the bone compartment to the surrounding
areas is evident. However, this passage must not be regarded as a sign of tropism towards the CNS on the part of
leukemic blasts, because they equally spread from the
marrow towards the muscles. In fact on reaching the brain
from the skull malignant blasts proliferate to the point
where the pressure they build up drives them first through
the nutrient foramen and then through holes they themselves erode in the bone table. The escaping cells accumulate in the epidural space and are again driven by compression to permeate the dura mater and infiltrate the
leptomeninges, but do not occupy the Virchow-Robin
spaces. When this infiltration becomes massive, they traverse and disrupt the pia mater to reach the brain parenchyma. Virchow-Robin space invasion becomes evident at
this stage only. Therefore, infiltration of the meninges is not
the direct outcome of invasion of the walls of their veins.
Systemic circulation of malignant blasts serves only as the
means whereby they reach the marrow. The lack of massive
blood invasion by lymphoblasts and the pattern of progression of the CNS invasion with the late invasion of VirchowRobin spaces make it unlikely that a major entry of
malignant lymphoblasts into the (=NS significantly occurs
via the systemic circulation. It would not have been possible
to establish this sequence of events had the brain been
removed from the skull, and in the absence of in toto
sections.
Because only the PF382 and ST-4 lymphoblasts have
been used in these experiments, no general conclusions can
be drawn. However, their spreading in SIA-nulnu mice may
be representative of malignant T lymphoblasts, because
PF382 and ST-4 cells display a similar spreading pattern,
but differ in membrane markers and stage of maturation
along the T-cell differentiation lineage, and were obtained
from children with a very different clinical c0urse.18,~~
Admittedly, injection of human lymphoblasts into immunodeficient and further experimentally immunosuppressed
xenogeneic hosts results in an artificial model. The extent to
which the data obtained mimic the situation occurring in
humans needs to be carefully considered.= The possibility
that the spreading pattern observed is the result of the
peculiar interactions between human and murine homing
receptors cannot be ruled out. Nevertheless, our data fit in
well with the findings of Thomas6 and Bleyer3 concerning
the direct spread of both syngeneic murine and human
leukemic cells from the skull bone marrow to the brain and
nervous system in meningeal leukemia. If human leukemic
cells primarily infiltrate the CNS by direct spread from cranial bone marrow, craniospinal radiotherapy, which eradicates malignant lymphoblasts from cranial and vertebral
bone marrow, may be a more effective way of preventing
such infiltration than systemic ~hemotherapy.~
Unfortunately, it is associated with significant adverse neuropsychologic and neuroendocrine effects, including impairment of
intellectual function.26However, in SIA-nulnu mice, malignant lymphoblast infiltration shows remarkable differences
between skull bones. If the SIA-nu/nu data truly delineate
some aspects of human meningeal leukemia, the identification of bones more at risk than others in children with acute
leukemia could lead to the establishment of a maximally
effective and minimally damaging form of prophylaxis.
ACKNOWLEDGMENT
We are indebted to Dr M. Geuna for skilful cytofluorimetric
analysis, and to L. Miserendino and A. Sassi for histologic
assistance. We also wish to thank Prof R. FOBand Dr J. Iliffe for
careful review of this report.
REFERENCES
1. Evans A, Gilbert E, Zandstra R: The increasing incidence of
central nervous system leukemia in children. Cancer 26:404,1970
2. Frei E 111: Acute leukemia in children. Cancer 53:2013, 1984
3. Bleyer AW: Biology and pathogenesis of CNS leukemia. Am J
Pediatr Hematol Oncol11:57,1989
4. Price RA, Johnson WW: The central nervous system in
childhood leukemia: I. The arachnoid. Cancer 31:520,1973
5. Evans EA Central nervous system involvement in children
with acute leukemia. A study of 921 patients. Cancer 17:256,1964
6. Thomas LB: Pathology of leukemia in the brain and meninges: Postmortem studies of patients with acute leukemia and of
mice given inoculations of L1210 leukemia. Cancer Res 25:1555,
1965
7. Chirigos MA, Thomas LB, Humphreys SR, Goldin A Intrac-
erebral growth and treatment of leukemia L1210. Proc SOCExp
Biol Med 104:643,1960
Torgersen JA, Chirigos M A An animal
8. Perk K, Pearson JW,
model for meningeal leukemia. Int J Cancer 13:863,1974
9. Peterson RDA, Varakis JN, Cheshire LB: New experimental
model of meningeal leukemia. Cancer Res 403130,1980
10. Kase CS, Wilborn WH, Varakis JN, Cheschire LB, Peterson
R D A Pathology of an experimental extradural spinal T cell tumor.
Lab Invest 46:535,1982
11. Varakis JN. Kase CS. Wilbom WH, Cheshire LB, Peterson
RDA: Pathogenesis of an experimental meningeal leukemia model.
Clin Immunol Immunopathol23:400,1982
12. West RJ, Graham-Pole J, Hardisty RM, Pike MC: Factors in
pathogenesis of central-nervous-system leukemia. Br Med J 3:311,
1972
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
A MODEL FOR MENINGEAL LEUKEMIA
13. Fogh J, Giovanella B C The Nude Mouse in Experimental
and Clinical Research, Vol 1. New York, NY,Academic, 1982
14. Cesano A, OConnor R, Lange B, Finan J, Rovera G, Santoli
D: Homing and progression pattems of childhood acute lymphoblastic leukemias in SCID mice. Blood 77:2463,1991
15. Roncella S, Ramarli D, Cooper MK, Forni M, Caretto P,
Rowe M, Francia di Celle P, Sessarego M, FOB R, Forni G,
Burrows P, Pistoia V, Ferrarini M: Establishment of an EBVpositive lymphoblastoid cell line that grows as a lymphoma in nude
mice and express membrane CD2 molecules. Int J Cancer 45:299,
1991
16. FOB R, Caretto P, Fierro MT, Bonferroni M, Cadorna S,
Guarini A, Lista P, Pegoraro L, Mandelli F, Forni G, Gavosto F:
Interleukin-2 does not promote the in vitro and in vivo proliferation and growth of human acute leukaemia cells of myeloid and
lymphoid origin. Br J Haematol75:34,1990
17. Cavallo F, Riccardi C, Forni M, Pericle F, Giovarelli M,
Bosco MC, Soleti A, Forni G: Growth and dissemination of human
malignant lymphoblasts in immunosuppressed nu/nu mice. Nat
Immun Cell Growth RegullO256,1991
18. Martinotti MG, Arione R, FOB R, Pegoraro L, Jemma C,
Forni G: Serial transplantation of a human acute T lymphoblastic
leukemia into nude mice. Tumori 72553,1986
19. Arione R, Jemma C, Forni M, Marchese C, Benetton G,
Giubellino C, Modesti A, Martinotti MG, Cordero di Monteze-
1283
molo L, Musso T, Forni G: A new childhood T-cell lymphoma
established in nude mice and in vitro. Cancer Res 48:1312,1988
20. Snedecor GW: Statistical Methods Applied to Agriculture
and Biology. Ames, IA, Collegiate, 1946
21. Forni G, Giovarelli M, Santoni A Lymphokine activated
tumor inhibition in vivo. I. The local administration of interleukin 2
triggers nonreactive lymphocytes from tumor bearing mice to
inhibit tumor growth. J Immunol134:1305,1985
22. Riccardi C, Puccetti P, Santoni A, Herberman RB: Rapid in
vivo assay of mouse natural killer activity. J Natl Cancer Inst 4773,
1979
23. Barlozzari T, Leonhardt J, Wiltrout R, Herberman RB,
Reynolds C: Direct evidence for the role of LGL in the inhibition
of experimental tumor metastases. J Immunol134:2783,1985
24. Solomon FR, Higgins TJ: A mAb with reactivity to AsialoGM1 and murine natural killer cells. Mol Immunol2457,1987
25. Amadori A, De Rossi A, Belardelli F, Cavallo F, Jemma C,
Comaglia Ferraris P, Doria G, Forni G, Forni M, Giavazzi R,
Lollini PL, Nanni P, Lusso P, Mezzanzanica D, Parmiani G, Semi
ML, Roncella S, Scala G: Mind the mouse! A consensus view on
the use of immunodeficient mice in immunology and oncology. J
Immunol Res 41,1992
26. Pochedly C Meningeal leukemia: Current concepts in biology and treatment. Am J Pediatr Hematol Oncol11:55,1989