[CANCER RESEARCH 40. 3426-3429, October 1980]
0008-5472/80/0040-0000$02.00
Chromosome Markers and Evidence for Clone Formation in Lymphocytes of
a Patient with SézarySyndrome1
Jan C. Liang,2 Mary Esther Gaulden, and James H. Herndon, Jr.
Radiation Biology Section. Department of Radiology ¡J.C. L., M. E. G.I and Department
University of Texas Health Science Center at Dallas, Dallas, Texas 75235
ABSTRACT
Cytogenetic studies of 222 metaphase lymphocytes stimu
lated by phytohemagglutinin
were carried out on a patient
diagnosed clinically as having Sézarysyndrome. Twenty-two
cells (10%) contained 42 to 100 chromosomes. The remaining
200 cells contained 46 chromosomes and revealed evidence
of clone formation; 45 were apparently normal diploid cells, but
155 were pseudodiploid with at least one long submetacentric
marker in each cell. This marker was shown to have a consist
ent banding pattern from cell to cell. Of the 25 pseudodiploid
cells karyotyped, there were other types of markers present.
Normal chromosomes 2 and 17 were missing in all 25 karyotypes. There were seven sets of two cells, each with an identical
karyotype, suggesting subclonal formation. Many of the phytohemagglutinin-stimulated
nondividing white blood cells had
one or more nuclear protrusions. Cytogenetic examination of
peripheral lymphocytes may be of value in diagnosing and
following the course of this disease.
of Internal Medicine [J. H. H.¡.Southwestern
Medical School,
The
patient, some of whose Cytogenetic abnormalities resemble
those that have been described and some of which have not
previously been reported.
CASE HISTORY
The patient was a white male born in 1901. He began having
cutaneous symptoms in 1952 to 1953 and was treated with
unspecified doses of X-rays (60 kVp) to the right ear and
hairline and to both palms. In 1960, he developed a scaly
dermatitis on the extensor surfaces of the arms and legs; pain
and swelling developed in the small joints of the hands and
feet. The diagnosis of psoriasis complicated by psoriatic ar
thritis was made. After topical medications had given little relief,
systemic medication was prescribed, including methotrexate
(10 mg p.o. once a week for 4 weeks in April 1968) and
hydroxyurea (amounts and exact time of administration not
known).
In 1974, he developed marked cracking and splitting of his
palms and soles. In January 1975, his WBC rose to 17,300
INTRODUCTION
(normal range, 4,800 to 10,500) with 68% lymphocytes. An
internist tentatively diagnosed chronic lymphocytic leukemia
The Sézarysyndrome, first described by Sézaryand Boubut prescribed no treatment. In May 1976, he developed gen
vrain (25) in 1938, is a rare disease characterized by chronic
eralized redness with induration especially marked over the
erythroderma and the presence of so-called Sézarycells in the
legs. By this time, his WBC had risen to 36,500.
peripheral blood and affected skin. The morphological char
In September 1976, Dr. J. H. Herndon, Jr., examined the
acteristics of these cells have been described by Taswell and
patient and tentatively diagnosed mycosis fungoides. The pa
Winkelmann (28). Crossen ef al. (7) first showed that Sézary tient received 3 injections of methotrexate (25, 50, and 75 mg,
cells behave like lymphocytes rather than histiocytes or monrespectively) at approximately 2-week intervals. A skin biopsy
ocytes as suggested by other investigators (4, 24). The cells
at this time revealed a dense polymorphic dermal infiltrate
do not normally divide but can be stimulated to divide in vitro
including large histiocytes with atypical nuclei along with many
by the mitogen PHA.3 Other features provide strong evidence
small cells of mixed type. There were moderate exocytosis of
that Sézarycells represent abnormal but partially functional Tsmall round cells and several clumps of such cells within the
lower epidermis (Pautrier's microabscesses). In late October
lymphocytes (6).
Chromosome abnormalities in the peripheral lymphocytes of
1976, his WBC had risen to 89,000 with 86% lymphocytes, all
a Sézarypatient were first reported by Crossen et al. in 1971
of which were determined to be T-lymphocytes (40% rosetted
(7). Two types of abnormal cells were found: hypotetraploid
with sheep RBC, and the remainder were positive for the o
cells with a large metacentric chromosome (designated as "A"
surface antigen). In addition, electron microscopy of thin sec
tions of the patient's cell buffy coat showed that all lymphocytes
marker); and hypertetraploid cells with a large submetacentric
chromosome (designated as "B" marker). So far, only 26
contained the cerebriform nuclei characteristic of Sézarycells.
cases of Sézarysyndrome have been investigated cytogenetiIt was thus possible to make a positive diagnosis of Sézary
cally (1, 5-8, 15, 16, 31), and in 17 of them, chromosomes
syndrome.
strikingly similar to one or both of the A and B markers of the
After entering a program of intermittent leukapheresis in
patient of Crossen et al. have been the most frequently ob
February 1977, the patient improved clinically and the WBC
served markers.
remained low. He was treated twice weekly from February to
In the present paper, we present data on a Sézarysyndrome
April, once a week during May and June, and intermittently in
July. In August 1977, his skin condition again worsened in
1 This work was supported in part by NIH Grant HD06832.
spite of a WBC of 6000. At this time, nitrogen mustard was
2 To whom requests for reprints should be addressed.
applied
topically to the whole body (10 mg in 50 ml water) once
3 The abbreviations used are: PHA, phytohemagglutinin; AML, acute myelogdaily for approximately 3 weeks. In late April 1978, he was
enous leukemia.
given whole-body topical electron beam therapy (60 MeV, a
Received June 25, 1979; accepted June 13, 1980.
3426
CANCER
RESEARCH
VOL. 40
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Chromosome Markers in SézarySyndrome
total of 900 rad, all delivered
over a 3-week
period).
He
developed progressively severe edema and disability and died
of a myocardial infarction in January 1979.
MATERIALS
AND METHODS
Leukocyte-rich samples of blood obtained April 19 and June
28, 1977, immediately following leukapheresis were cultured
in A-1 chromosome medium (Difco Laboratories) containing
PHA for 72 hr. Colcemid (final concentration, 0.1 jug/ml) was
added to the cultures for the final 1.5 hr. Cells were then
harvested, treated with 0.075 M KCI, and fixed in absolute
methanoliglacial acetic acid (3:1). The slides were air dried
(12) and either uniformly stained with Giemsa or treated by
Seabright's trypsin method (23) for G-banding. Analyzed by
eye were 222 metaphases (113 from the first sample and 109
from the second sample). Karyotypes were prepared for 25
uniformly stained cells and one G-banded cell.
RESULTS
Because there were no significant differences in the cytogenetic observations in the 2 blood samples analyzed, the
results have been combined. Nuclear protrusions, which ac
cording to Atkin and Baker (3) are indicative of long marker
chromosomes, were frequently found on the nuclei of the PHAstimulated, nondividing, WBC of this patient (Fig. 1).
The distribution of cells with respect to chromosome number
is given in Table 1. Two hundred cells had 46 chromosomes,
and the remaining 22 cells were aneuploid to hypertetraploid,
with 42 to 100 chromosomes. Of the 200 with 46 chromo
somes, 45 (23%) were apparently normal diploid cells, and
155 (77%) were pseudodiploid with at least one long submetacentric chromosome marker (M1) appearing in each cell.
The M1 marker has a consistent banding pattern from cell to
cell, 3 of which are shown in Fig. 2, inset. One distinct band on
the short arm near the centromere and 3 prominent bands on
the long arm characterize this marker. The origin of M1 is not
obvious, but the fact that one normal chromosome 2 is missing
in all cells containing an M1 suggests that the latter may be
derived in part from a chromosome 2 (perhaps bands p12,
q22, and q24 of chromosome 2). Each of the 2 proximal q
bands of M1 can sometimes be seen to consist of double
bands, which conforms to the multiple banding patterns at the
q22 and q24 regions of chromosome 2 as shown by Francke
and Oliver (10) for extended chromosomes.
The banding pattern of the B marker of Crossen ef al. (7)
has not been previously described. Because the M1 chromo
some of our patient appeared to resemble the unhanded B
marker, the 2 marker chromosomes were compared for arm
ratios (p:q, short:long) and length relative to those of the normal
chromosome 1. Both the arm ratios, 0.37 for the B marker and
0.39 for the M1, and the relative lengths, 1.13 for the B marker
and 1.11 for the M1 chromosome, are remarkably similar.
Other types of markers were found among the 25 pseudo
diploid karyotypes. Repeated attempts at banding all of these
markers with a number of slides were unsuccessful in that only
one cell showed good banding patterns in most of the chro
mosomes. In this cell (Fig. 2), one each of chromosomes 2, 10,
12, 15, and 17 and both chromosomes 3 were missing, and
band deletions were noticed in one of chromosomes 6 and 8.
Table 1
Chromosome complements in 222 lymphocytes
of a Sézarysyndrome patient
Cells
No. of chromosomes
200(90)
7 (3)
4 (2)
11 (5)
46
42-45
47-48
60-100
Six markers in addition to the M1 were observed in this cell. It
should be noted that clear banding was obtained in a normal
subject's lymphocytes processed at the same time. There were
some repetitive patterns of chromosome loss; e.g., the absence
of one each of normal chromosomes 2 and 17 was noted in all
25 karyotypes. Also, either a chromosome 1 or 3 or both were
absent; in 3 cells, both of the normal chromosomes 3 were
absent. The presence of 7 sets of 2 cells with an identical
karyotype suggests subclonal formation. Cells with ring chro
mosomes, dicentrics, and minutes that have been reported in
some Sézarypatients (5, 16, 31) were not observed in our
patient.
DISCUSSION
This paper reports the first case in which nuclear protrusions
have been observed in a Sézarysyndrome patient. Since
Ruddle (21) first described nuclear protrusions on interphase
nuclei of an established line of pig kidney cells, they have been
observed on nuclei of both tumor and nontumorous cells by
other investigators (2, 11, 14, 27). It is believed that nuclear
protrusions are indicative of the presence of long marker
chromosomes (3). This proved to be true for the present case.
Most cases of Sézarysyndrome previously reported were of
the large-cell type, characterized by a near-tetraploid chro
mosome complement (7, 16, 31). A small-cell type with pseu
dodiploid or hyperdiploid chromosome number
scribed by Lutzner et al. (16). Since the majority
our patient have a diploid-range chromosome
patient's disease appears to be of the small-cell
was first de
of the cells in
number, our
type.
The B marker of Crossen era/. (7) is by far the most frequent
one that has been found in Sézarypatients (7, 16, 31). As
noted above, it is quite similar to the M1 chromosome in our
patient. The presence of the M1 always accompanied the
absence of one normal chromosome 2, which suggests that
the marker may be derived from a chromosome 2. Its exact
origin can be determined only by comparing a number of cells
with clear banding patterns.
The difficulty we experienced in obtaining good banding of
the chromosomes of Sézarycells has also been reported in 15
of the 19 patients to whose chromosomes banding methods
have been applied (1, 5, 8, 16, 31). In some patients, clear
bands could be obtained on chromosomes of the apparently
normal cells but not on those of the heterodiploid cells contain
ing markers (1, 8, 16). Although the reason for the refractori
ness of the chromosomes of some Sézarycells to banding is
not obvious, the fact that 6 groups of investigators, including
us, have observed this unusual phenomenon suggests that it
may be real. We were not able to explore it further with our
patient because additional blood samples could not be ob
tained.
Consistent loss of one chromosome 17 was noted in the
aberrant cells of our patient as well as in 3 other Sézary
OCTOBER 1980
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3427
J. C, Liang et al.
patients (16, 31 ) and in 7 mycosis fungoides patients (9). Both
of these lymphoproliferative syndromes are now regarded as
cutaneous T-cell lymphomas. Frequent loss or gain of one or
more E-group chromosomes has been reported in patients with
various kinds of lymphomas (17, 26). It has been hypothesized
by Spiers and Baikie (26) that chromosome 17 abnormalities
play a special role in the genesis and/or evolution of neoplasia,
particularly in lymphoid and reticuloendothelial tissues. In this
connection, it is of interest that the gene for thymidine kinase,
which regulates nucleic acid metabolism, is located on the
chromosome 17 (20). Aberrations in chromosome 17, either in
structure or number, may provide a selective growth advantage
which promotes neoplasia (17, 18).
Aneuploidy has been reported in all seven patients with
established small-cell Sézarysyndrome who have been ex
amined cytogenetically (16, 31). The relationship between
aneuploidy and prognosis of AML has been investigated by
Sakurai and Sandberg (22) in 88 patients. The mean survival
time of those patients with only aneuploid metaphases in their
bone marrow was 1.2 months, that of those with both diploid
and aneuploid metaphases was 7.2 months, and that of those
with only diploid metaphases was 9.1 months. The results of
our study and those of others show that all the Sézarypatients
have at least some fraction of cells with an apparently normal
karyotype. The long benign course of this disease may be due
to normal functioning of these cells. All types of aneuploidy
have been found in the Sézarypatients studied thus far: pseudodiploidy (our study and Ref. 16); hyperdiploidy (16, 31 ); and
hypodiploidy (8). In a study of 170 adults with acute leukemia
(including 143 with AML), Trujillo er al. (29) found that the
mean survival time varied with the type of ploidy. For hyperdiploid cases, it was 49 weeks; for the pseudodiploids, it was 36
weeks; and for the hypodiploids, it was only 13 weeks. Whether
the aneuploidy found in the Sézarysyndrome will have the
same prognostic significance as that in AML still remains to be
determined when additional data are available.
Evidence for clone formation has been reported in only 4 out
of 26 Sézarypatients who have previously been examined
cytogenetically (1, 7, 16, 31). In our patient, the fact that all
155 pseudodiploid cells had the same marker, M1, is evidence
for but not proof of their derivation from an initial clone with
this abnormal chromosome. The 7 sets of 2 cells each with an
identical karyotype suggest subclone formation in this patient.
Methotrexate, hydroxyurea and X-rays, which were given to
the patient prior to our study, have been reported to produce
chromosome breaks and other structural abnormalities (13,
19, 30). Although we cannot rule out the possibility that these
mutagens may have produced in stem cells all of the chromo
somal abnormalities reported in this paper, the fact that some
strikingly similar marker chromosomes have also been found
in untreated patients (16, 31) renders this possibility less
probable. Banded chromosome studies on additional Sézary
patients prior to treatment are needed to help clarify this
point.
ACKNOWLEDGMENTS
The
help in
Pathak
Hallford
3428
authors thank Dr. Howard R. Toben and Dr. Amanallah Khan for their
identifying the T-lymphocytes; Dr. V. G. Dev, Dr. T. C. Hsu. and Dr. S.
lor help with interpretation of banded marker chromosomes; and Dianna
for technical help.
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Chromosome Markers in SézarySyndrome
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i ,
••
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MARKERS
Fig. 1. Some nuclear protrusions seen on the lymphocytes of Sézarysyndrome patient, x 3750.
Fig. 2. A G-banded karyotype of a Sézarycell. Inset, 3 M1 chromosomes from 3 other pseudodiploid
cells, x 9700.
OCTOBER 1980
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3429
Chromosome Markers and Evidence for Clone Formation in
Lymphocytes of a Patient with Sézary Syndrome
Jan C. Liang, Mary Esther Gaulden and James H. Herndon, Jr.
Cancer Res 1980;40:3426-3429.
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