In the last 4 years we have established five long-term cultures
from tumor material of Hodgkin's disease. The in vitro cells have
malignant characteristics and represent the in vivo Hodgkin- and
Sternberg-Reed-cells as shown by the identity of multiple properties.
Common immunological, functional, and morphological assays did not
characterize the in vitro cells as a known cell type of lymphoid,
myeloid, or monocytoid tissue. The in vitro Hodgkin's disease cells
are biologically active by producing factors involved in regulation
and promotion of immunological response and granulopoiesis. The
relevance of the findings for pathogenesis and clinical appearance
of Hodgkin's disease is discussed.
Hodgkin's disease is still one of the most challenging entities
in hematooncology. Of patients with this histopathological diagnosis,
60%- 70% can possibly be cured [3, II] and 30%-40% do not respond
with complete remission upon first treatment; of this group 15%-20%
will achieve remission upon secondary treatment, but 15%-20% will
die within 4-18 months in spite of intensive treatment strategies
(Fig. I). Secondary neoplasias are the most hazardous consequence
of intensive combined treatment modalities (radiochemotherapy) at
the moment and amount to 5%-10% of all initially diagnosed Hodgkin's
disease patients [ I, 2, 16]. The incidence of acute myeloid or
myelomonocytoid leukemia is 130 times higher in Hodgkin's disease
patients than in normal individuals . Attempts to investigate
the nature of the pathognomonic Hodgkin- (H) and Sternberg-Reed
(SR)-cells have been hampered by the fact that these cells constitute
only a small minority in the primary biopsy and seem to be utterly
growth restricted in the hitherto available culture systems .
Since 1978 we have established five cell lines from tumor-involved
specimens derived from HD patients [5, 6, 12]. All histologies were
confirmed by four independent hematopathologists.
C. Patients, Material, and Methods
All five lines were grown from HD specimens of four patients with
nodular sclerosing-type histologies, clinical stage IVB. The patients
had been submitted to intensive combination chemoradiotherapy. The
sources for the culture material were pleural effusions in three
cases and bone marrow and peripheral blood in one case. Two identical
cultures were established from the two different sources of this
particular patient. Four cell lines continously proliferate in vitro,
while on line stopped growing for unknown reasons after 7 months
in culture (L 439).
Fig. I. Hodgkin's disease: clinical course and secondary
The cell lines share an identical (L 428) or partially identical
phenotype (L 538/540, L 591) with in vivo H- and SR-cells and represent
a cell type previously unknown according to the methods used for
cellular discrimination . The neoplastic nature of the five
HO tumor cell lines is indicated by aneuploidy, except in one line
(L 591), and multiple structural and numerical chromosome abnormalities
associated with a monoclonal pattern of multiple marker chromosomes.
A comparison of the characteristic features of the in vitro cultured
HO cells with H- and SR-cells from freshly obtained biopsies is
shown in Table I. All cultured cells lacked surface- or cytoplasmic-Igs.
IgG present in fresh biopsy H- and SRcells was not found in vitro.
Ia-like antigens, receptors for human T cells, acid phosphatase,
and acid naphthol acetate esterase were present in all cultured
lines. EBV specific receptors were found in two out of two tested
lines, EBV genomes and EBV -induced antigens, however, only in one
line (L 591). All HO cell lines as well as fresh biopsy H- and SR-cells
are devoid of HTLA receptors for C3b, C3d, IgG-Fc, mouse-E, or sheep-E
and of lysozyme, peroxidase, and chloracetate esterase (Table I).
The identity of the in vivo and in vitro H- and SR-cells was shown
by congruent morphological, functional, and immunological markers.
The strongest proof for the derivation of the cultured cells from
Hand SR-cells in vivo was the demonstration of cross-reacting surface
and cytoplasmatic constituents on the in vivo and in vitro cells
by means of absorbed polyclonal (rabbit anti L 428 cells) (Table
I) and mouse monoclonal (anti L 428 cells) antibodies (Ki I, Ki
24, Ki 27) (Table 2). Furthermore, monoclonal antibodies directed
against .granulopoietic cell determinants (3C4, TU 9) were also
present on biopsy HO cells and the cell lines L 428 KS and L 540,
but were absent on the L 591 cells. In an attempt to determine the
origin and nature of the cultured H- and SRcells a multitude of
monoclonal antibodies directed against human lymphoid and hematopoietic
differentiation markers were tested against these cells. Table 3
summarizes the results by showing the most commonly accepted markers
as specific attributes of the different cell types. As demonstrated
in this table, the reactive pattern of the cell line L 428 and partially
of the cell line L 540 was identical
Table 1. Properties of in vivo and in
vitro H- and SR-cells
with that of the H- and SR-biopsy cells, , lacking markers characterizing
B cells, T cells, monocytes, dendritic reticulum cells, interdigitating
reticulum cells, and Null cells. They carried, however, granulopoietic
cell determinants, as shown by the reactivity of antibodies 3C4,
TÜ 9, and Vim 05, but they were unreactive for the peroxidase and
chloracetate esterase cytochemical staining. The L 540 cells reacted
like the L 428 cells, but were positive with the monocyte 1 antibody.
The L 591 cells showed a very peculiar pattern insofar as they lacked
Table 2. Reactivity of HO and SR cells
in biopsies and in vitro (lines)
with monoclonal L 428- and granulopoietic cell antibodies
Table 3. Differential characteristics of HD and SR cells
and HD cell lines
Table 4. Biological activities of HD line supernatants
B-cell markers like Ig production or Ig surface reactivity (anti
IgM, anti IgD), but carried C3b and C3d receptors, as well as EBV
receptors and EBV antigens. Furthermore, they reacted positively
with T -cell antisera like OKT -11 and Lyt 13, but lacked monocyte.
granulopoietic cell, dendritic reticulum cell, and interdigitating
reticulum cell, as well as Null cell markers. The cell lines produced
a variety of sub stances which are known to be mediators of response
and gran ulopoiesis
1. L 428 respresents in vitro the in vivo H- and SR-cell population.
The other reported lines exhibit most but not all markers of in
vivo H- and SR-cells. The non-LCL character of L 591 is still the
subject of discussion. 2. The HO cells (in vivo and in vitro) represent
no known cell type or any cell class so far identified by common
immunological, functional, and morphological tests. 3. The in vitro
HO cells exhibit biological activities regulating and/ or promoting
immune response and granulopoiesis.
F. Hypothetic Pathogenesis of Hodgkin's Disease
Figure 2 summarizes all the data obtained on the described HO-derived
cell lines in an attempt to propose a hypothesis for some of the
pathogenetic mechanisms involved in Hodgkin's disease: The origin
of H- and SR-cells in unknown. It is possible that the Ki 1 antibody
not only recognizes H-SR-specific determinants, but also depicts
Fig.2. Hypothetic pathogenesis of Hodgkin's disease
undefined cell in normal tissue [ 13, 15], which could be the normal
counterpart of the "malignant" H- and SR-cells (see Stein, this
volume). The pathogenetic mechanisms involved in the transformation
of a normal cell, possibly playing some role in immune and hematopoietic
regulation, is unknown. Endogenous (genetic?) and exogenous (viruses,
chemical agents, both?) might induce a gradual "evoIution" from
a primarily nonproliferating, biologically active cell, which by
its products (CSF, II 1) might create the clinically not very aggressive
"Hodgkin's lymphoma ", to a genetically altered (Fonatsch et al.
unpublished results) more malignant cell, embedded in the histological
entity of a "Hodgkin sarcoma." Radiochemotherapy might act as a
cofactor in this process of gradual malignization. Of the HD patients,
however, 60%-90% are cured by radio- and/ or chemotherapy in the
early stages of this process before genetically altered cells have
chance to commence rapid proliferation and possibly exert resistance
to cytoreductive therapy. The variance in the histological presentation
of Hodgkin's disease could reflect this gradual malignization process:
Paragranuloma and/ or lymphocytic predominance and lymphocyte-enriched
nodular sclerosis would identify a stage of "low risk", with a high
functional activity of the H- and SR-cells, producing mediators
like CSF, Interleukin 1, but still restricted in cellular proliferation.
If cytoreductive therapy is carried out at this stage, cure is possible
in up to 90% of cases ([ 10], Schellong, personal communication).
If the HD cells withstand therapy by either genetically inherent
or resistance mechanisms acquired during treatment, the patient
will present a picture of a more malignant Hodgkin's sarcoma with
a higher number of rapidly proliferating H- and SR-cells. These
cells could still have retained their biological mediator production,
but the balance might be toward more production of immune suppressive
and EBV transformation enhancing factor. The fact that many Hodgkin's
disease patients develop high antibody serum titers against EBV
antigens and give rise to EBVinduced lymphoblastoid cell cultures
significantly more than normal individuals  could be explained
not only by T -cell immunosuppression but also by a direct influence
of an EBV transformation enhancing factor. The resulting polyclonal
lymphoblastoid transformation could "feed" or protect the tumor
cell, possibly under a concomitant protection of the rosetting OKT-4-positive
T -helper cells, attaching to the H- and SR-cells. These protection
mechanisms might enable an a priori "lowgrade malignant" HD cell
to "sneak through" to a higher malignant proliferating tumor cell,
which in 15%-20% of the clinical outcome could eventually kill the
patient. Most Hodkgin's disease patients, however, do not die of
tumor cell proliferation, but of biological side effects of immune
deficiency and hematological complications, possibly due to some
of the descri bed factors.
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