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Short Communications |
From the Departments of Anatomical and Cellular Pathology*
and Clinical Oncology,
Prince of Wales
Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The low proliferative rate of the tumor cells in this malignancy has
hampered the conventional cytogenetic analysis. An abnormal karyotype,
often a complex mixture of numeric and structural changes, is found in
~50% of patients.2
Recent data have revealed that 55%
of MM patients had three or more trisomies involving chromosomes 3, 5,
7, 9, 11, 15, 19, and 21.3
The significance of these
chromosomal aberrations in MM pathogenesis remains obscure. As a large
number of proliferating or nondividing cells can be examined, use of
fluorescence in situ hybridization (FISH) has improved the
detection of cytogenetic abnormalities in MM. In
addition, by combination with morphological assessment of the cells
studied, the lineage of cells that are involved in the neoplastic
transformation can be elucidated. Using FISH and 10 
-satellite DNA
probes, Drach et al4
showed that 88.9% of MMs were
aneuploid for at least one chromosome examined and 66% had aberrations
in three or more chromosomes. In the same study, mature myeloid cells
evaluated showed no abnormality.4
After FISH procedure,
cytomorphological details are so obscured that it would be very
difficult to ascertain the cell types being assessed. Using combined
morphological and FISH (May-Grunwald-Giemsa (MGG)/FISH) technique, more
reliable information on the cytogenetics of MM can be obtained,
irrespective of the percentages of the plasma cell infiltration.
Moreover, myeloid and lymphoid cells can be evaluated in the primary MM
samples for involvement of chromosomal aberration. To gain insight into
the real incidence of the numeric chromosomal aberrations and the
nature of the MM progenitor cell, we examined 18 Chinese MM patients by
MGG/FISH using three DNA centromeric probes specific for chromosomes 3,
7, and 9, which frequently showed aberrations in MM.3-6
In
addition, in our experience, these probes work out very well with
strong and discrete signals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bone marrow (BM) aspirate samples collected in EDTA bottles from 18 Chinese patients diagnosed to have MM in Prince of Wales Hospital, the Chinese University of Hong Kong, from June 1995 to January 1997 were recruited for the study. The diagnosis and staging were made according to the criteria of Durie and Salmon.7,8 Peripheral blood (PB) samples from five healthy volunteer staff and BM aspirates from four healthy BM donors were analyzed for determination of the cutoffs (mean + 3.291 SD).
MGG/FISH
Mononuclear cells from nine patient EDTA BM aspirate and five normal control PB samples were separated by Lymphoprep (Nycomed Pharma, Oslo, Norway) according to the manufacturer's protocol. The cytospin slides prepared were air dried thoroughly overnight and then stored at -70°C until use. All of the BM or PB cytospins and smears from the normal control and patients were fixed in absolute methanol and stained with MGG by standard procedure. The stained slides were then air dried and analyzed by FISH within 1 week after staining. The MGG-stained cytospins and BM smears were viewed under light microscope (POTIPHOT-2, Nikon, Tokyo, Japan) at x400 magnification. Fields of interest on the slides were photographed with Kodak Gold III 100 (GA 135-36 Eastman Kodak, Rochester, NY) films. The coordinates on the stage of the microscope were recorded for every shot taken.
Interphase FISH was performed using digoxigenin-labeled -satellite
DNA probes (Oncor, Gaithersburg, MD) specific for chromosomes 3 and 7
and digoxigenin-labeled Classical Satellite DNA probe (Oncor) specific
for chromosome 9. BM cytospins or smears were destained in Carnoy's
fixative (methanol/glacial acetic acid, 3:1, v/v), and procedures
followed then were according to Oncor's FISH protocol with minor
modification on post-hybridization wash (the slides were washed in
0.25x SSC (pH 7.0) for 8 minutes at 68°C without agitation).
Finally, the slides were put up for detection of hybridization
sig-nals. The cells were evaluated under the fluorescence microscope
(OPTIPHOT-2, Nikon) equipped with an oil immersion 100x objective and
an fluorescein isothiocyanate filter. One MGG-stained control PB
cytospin was hybridized together with each set of sample slides
subjected to the FISH procedure. A total of 600 nuclei were analyzed
each time under the fluorescence microscope as described previously.
Hybridization results of the samples were considered satisfactory if
the hybridization signals in the control slide were reasonably strong
and the frequency of nuclei showing numeric aberrations was
statistically insignificant. All sets of samples were satisfactorily
hybridized by these criteria, and the sample slides were then scored.
Fields previously photographed were relocated on the stage of the
microscope using the reference coordinates recorded. The same cells,
plasma cells (PCs), myeloid cells (MCs), and lymphoid cells (LCs),
identified by morphological assessment previously, were re-evaluated
under the fluorescence microscope for the number of fluorescence
signal(s) per nucleus, which was recorded on the LM microphotographs.
Unhybridized nuclei were not counted and did not contribute to our
data. Only regions with more than 90% of cells showing hybridization
signals were analyzed. Only those well separated signals with similar
intensity, size, and shape were counted. Cells with ambiguous
morphology precluding accurate identification of cell types were
excluded from analysis. The fluorescence images were photographed with
Kodak Ektapress Plus 1600 color film (5PJC 135-36, Eastman Kodak).
Cutoff Levels
The MGG-stained PB cytospins and BM smears from control
donors were subjected to interphase FISH using the probes for
chromosomes 3, 7, and 9. A total of 600 per nuclei were analyzed and
scored in each slide. The mean percentage of trisomic/monosomic cells
in these control samples for the chromosomes assessed was as follows:
chromosome 3, 0.9 ± 0.7/5.4 ± 2.7 (cytospin (C)) and
0.8 ± 0.4/7.1 ± 2.5 (smear data (S)); chromosome 7,
0.4 ± 0.3/6.3 ± 2.2 (C) and 0.4 ± 0.5/5.8 ± 1.5
(S); chromosome 9, 1.0 ± 0.6/6.9 ± 2.4 (C) and 0.4 ±
0.3/8.2 ± 2.5 (S). The cutoff levels for the significant numeric
changes in the patient samples were defined as any values higher than
the mean percentages of aneuploid nuclei plus 3.291SD
(P = 0.001 by 
2
test) for the
corresponding chromosome in the control samples. As not a single cell
from 600 cells studied in the normal controls showed four or more
signals per nucleus, it had been determined that the cutoff level for
significant tetra- or polysomies were at 
0.3% of cells showing four
or more fluorescence signals per nucleus. As only single-probe
interphase FISH was performed, monosomies, which had not been verified
by dual-probe FISH analysis, were not presented in this paper.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Table 1
shows the clinical and FISH
data of the 18 Chinese MM patients (12 pretreated and 6 previously
treated) studied. The male-to-female ratio was 1:1, with a median age
of 62.5 years. It is noteworthy that one patient (patient 5) was
diagnosed at 25 years of age, which is extremely unusual for MM. Our
patient group included five patients with IgG
, five with IgG
, two
with IgA, four with IgA, one with BJP, and one with BJP MM.
Fifteen patients were at stage III and three at stage II disease.
Patient 14 was diagnosed as a relapse disease. The median follow-up
duration was 12 months from the first consultation at Prince of Wales
Hospital to the time of data analysis. The median treatment duration
before the FISH evaluation for the previously treated cases was 3
months. The complete blood counts revealed a mean hemoglobin level of
9.0 g/dl (range, 6.9 to 11.9 g/dl), white blood cell count of 5.8
x 109/L (range, 2.9 x 109
to 7.1 x
109/L), and platelet count of 224 x 109/L
(range, 52 x 109
to 432 x 109/L).
Six patients died, and eleven were alive at the time of data analysis.
One patient (patient 10) was lost to follow-up (Table 1)
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Eighteen BM aspirates were analyzed using DNA probes for
chromosomes 3, 7, and 9. Except for one patient (patient 8), the
results were considered acceptable for documentation of chromosomal
abnormalities, as arbitrarily defined, when the number of cells counted
was >100 for PCs, >50 for MCs, and >15 for LCs. This could be
achieved in most of the patient samples as indicated in Table 1
. The
number of cells studied was 266 ± 110 for PCs (range, 104 to
628), 215 ± 131 for MCs (range, 54 to 546), and 54 ± 46 for
LCs (range, 18 to 271).
Plasma Cells
FISH data were available for all except one patient (patient 11)
for the analysis of chromosome 3. Aneuploidies from one or more
chromosomes were found in all of the 18 patients. Multiple polysomies
of all three chromosomes were observed in 14 patients. The cytogenetic
aberration was also complex with multiple aneuploids (Table 1)
. Table 2
shows the frequency, range, and mean percentage (clone size) of cells
involved in the different chromosomal aberrations. Polysomies 3
(82.4%), 7 (83.3%), and 9 (83.3%) and trisomies 3 (64.7%), 7
(66.7%), and 9 (88.9%) were observed in the PCs. Trisomy 9 was the
most common aberration seen, in fact in all cell series. Incidences of
the various combinations of chromosomal aberrations in the three cell
series are illustrated in Figure 1
.
Numeric chromosomal changes were commonly derived from a spectrum of
variable copy numbers of the three chromosomes in different cells,
particularly in PCs (Figure 2, A–H)
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MCs recruited for FISH analysis included erythroblasts and
intermediate and mature granulocytic cells. FISH data on MCs were
obtained for all except one patient (patient 11) for the analysis of
chromosomes 3 and 7. Again, all of the MM patients demonstrated
multiple chromosomal aberrations in MCs, which exhibited similar
patterns as observed in PCs (Table 1)
. Almost all aneuploids found in
MCs could be demonstrated in PCs except in the two cases (patients 11
and 18), in which trisomies 9 and 3 were observed, respectively, in MCs
but not PCs. However, in contrast to PCs, several exceptional features
were noted. First, MCs did not show any polysomies beyond tetrasomies
(more than four signals/nucleus) (Figure 2, I–L)
. Second, as shown in
Table 2
, the trisomic/tetrasomic clones,
if found, were also very small (0.5% to 5.5%). Third, numeric
aberrations found in PCs were observed less frequently in MCs. Fourth,
MCs showed a smaller number of aneuploids than PCs in each MM case
(Table 1)
.
Lymphoid Cells
Analysis of the lymphoid cells was frequently hampered by
inadequate samples, which occurred in nine cases (five patients with
chromosome 3, two with chromosome 7, and two with both chromosomes).
Multiple cytogenetic aberrations were found in LCs in all except one
(patient 3) of the MM patients studied (Table 1)
. Like MCs, LCs did not
demonstrate polysomies beyond tetrasomies, and the trisomic/tetrasomic
clones (2.6% to 8.4%), although slightly larger than those of MCs,
were also much smaller than those of PCs (Table 2)
. In three patients
(11, 13, and 18), trisomies were found in LCs but not PCs.
Similarities and Differences of Aneuploidies among PCs, MCs, and LCs
For all three cell series, FISH data on chromosomes 9, 7,
and 3 were assessed for 18, 14, and 11 patients, respectively.
Identical aneuploids with chromosomes 9 (12/18), 7 (6/14), or 3 (4/11)
were commonly seen across the three cell series. FISH data on
chromosomes 9, 7, and 3 for all three cell series were evaluated in
nine patients. Two (patients 4 and 6) of these nine patients showed
gains in the same copy numbers of all three chromosomes in PCs, MCs,
and LCs (Table 1)
. Trisomies or tetrasomies found in MCs or LCs could
almost always be demonstrated in PCs with few exceptions. The trend of
expanded clones of increased polyploidization (multiple polysomies of
chromosomes 3, 7, and 9) with multiple gains of various high (three to
six) copy numbers of different chromosomes in PCs was in striking
contrast to the much smaller trisomic/tetrasomic clones in
LCs and MCs.
Heterogeneity of Aneuploidies
The myeloma cells (PCs) displayed a wide spectrum of the number of
hybridization signals (range, 1 to 11, but mostly 2 to 6) (Figure 2
, A–D, G, and H). In a few cases where multinucleated myeloma cells were
present, the progeny nuclei also displayed different combination of
variable numbers of hybridization signals (Figure 2, K and L)
. As
illustrated in Table 3
, the percentage of
myeloma cells affected by each chromosomal aberration was variable in
the same MM cases, suggesting that there were probably multiple
subclones carrying different numeric cytogenetic abnormalities. For
example, in case 4, the percentages of trisomic 3 and 7 cells were,
respectively, 17.1%, and 11.1%, whereas trisomic 9 was 62.8%. It is
possible that there was a triploid clone making up for ~11% of MM
cells whereas ~46% of MM cells may harbor trisomy 9 alone, with 6%
showing trisomies 3 and 9. There is much less cellular heterogeneity
among myeloid and lymphoid cells in terms of polysomies.
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The patterns of chromosomal aberrations were not significantly
different between the previously treated and untreated groups. Numeric
changes of chromosomes were not found significantly correlated with any
of the clinical parameters as listed in Table 1
. The only case with
gain in chromosome 3 as the sole numeric abnormality was found in a
treated IgA MM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The MCs in MM have been assumed or found to be cytogenetically normal previously. In the FISH study on MM by Drach et al, only mature myeloid cells with distinct nuclear outlines were analyzed and reported to have no chromosomal changes.4 In their other FISH study on monoclonal gammopathy of undetermined significance (MGUS) patients, abnormality of MCs was not observed.9 Our finding thus represents the first description of involvement of chromosomal aberration of MCs in MM.
The MM clonogenic cell has been a myth despite recent advances in MM research. The simultaneous occurrence of MM and another hematopoietic disorder in the same patient has been well known in MM. Early sporadic case reports of simultaneous occurrence of acute myeloid leukemia and MM may be the first evidence suggesting that there might be a potential link in the pathogenesis of the two disease entities.10-12 In the study by Rosner et al,13 among 58 acute-leukemia-associated MM patients, 11 cases had either simultaneous development of the two diseases or within several months of each other. Thus, it raised the possibility that acute leukemia may occur as part of the natural history of MM.13 In the last two decades, phenotypic and cell culture studies in MM cells have demonstrated a high frequency of expression of multiple-lineage-associated surface antigens (eg, myelomonocytic), similar in vitro growth requirements for interleukin (IL)-3 and IL-6 as early hematopoietic progenitors, and the presence of a typical Philadelphia chromosome in short-term culture and a cell line from cases of disseminated MM or plasma cell leukemia.14-18 These findings provide strong evidence that malignant transformation of MM may start early in the hematopoietic development.
Our current data support that both lymphoid-plasma cell and myeloid lineages carrying similar chromosomal aberrations were possibly pathogenetically related and affected by a common source of genetic defect(s). However, we could not rule out the possibility that these chromosomal abnormalities arose from pathogenetically unrelated causes. One of the possibilities of the former scenario is that the MM precursor cell may be derived from the pluripotent progenitor cell capable of differentiation into myeloid and lymphoid cell lineages. Thus, this may lend further support to the postulation that the neoplastic transformation may occur early in the hematopoietic development.
An alternative explanation for the presence of chromosomal abnormalities in MCs may be that they were acquired genetic damages from aging unrelated to MM pathogenesis. To test this possibility, we evaluated the myeloid and lymphoid cells in the peripheral blood of two male and one female healthy elderly (70 to 80 years old) subjects for chromosomal aberrations. Abnormalities found in the disease group were not observed. Thus, we believe that these abnormal micro-populations of MCs were genuine abnormalities, which were probably pathogenetically related to the MM development.
Reactive plasma cells (found usually in 2% to 5% in normal subjects),
shown to be disomic by previous studies, were not distinguished
morphologically from MM tumor cells in this investigation. As the
number of the cytogenetically abnormal PCs was much smaller than the
total plasma cell population, it is reasonable to assume that many of
the MM cells were disomic (Figure 2, I and J)
. However, the highly
heterogeneous and complex pattern of numeric chromosomal aberrations
involving mainly subclones in MM suggest that these may be secondary
events resulting from genomic instability (GIN), which may occur when
there is error in cell cycle control and DNA repair mechanism. However,
alterations of the p53 gene, which are associated with GIN,
are infrequent in MM, and a late event in the disease.19,20
This discrepancy may be explained by the recent finding in MM of the
presence of overexpression of mdm-2, an inhibitor of p53
protein.21
Furthermore, the presence of tumor cells with
gain of a wide variety of different copy numbers of the same chromosome
(Figure 2)
may imply a loss of control of chromosome numbers, which is
possibly a result from an underlying defect of chromosomal segregation
during cell division. A similar phenomenon has also been observed in
colorectal cancers.22
This is also supported by our
observation on chromosomal aberrations in binucleated PCs in which one
nucleus showed a higher number of the same chromosome than the other.
However, the problem looks more complex because in some situations, the
sum of the chromosome copy numbers from the progeny nuclei was not an
even number (Figure 2, K and L)
. This may imply that other mechanisms,
such as defects in DNA replication or translocation, may be involved to
produce this unbalanced chromosomal status. Consistent with our
observation, a similar postulation of an incapacity of PCs to control
their number of chromosomes was made by Zandecki et al23
in
their recent study on MGUS where they identified heterogeneous BMPC
clones differing only in the number of chromosomal abnormalities
exemplified.
Although the pathogenesis of MM remains unclear, it can be postulated that MM transformation is a multistep process24 with cryptic mutations, including those associated with GIN, possibly occurring early in the hematopoietic development. This abnormal early progenitor cell may subsequently differentiate into the myeloid and lymphoid precursors through clonal evolution with additional mutations. Abnormal lymphoid cells with increased polysomies or polyploidies might have significant growth advantages for clonal expansion and development into the myeloma cell pool. On the other hand, tiny subpopulations of the very early myeloid precursors, with further mutations, may enter the aberrant pathway to produce tumor cells retaining the myeloid antigen expression. This may create a mechanism for the continuous expansion and development of the neoplastic clone(s), which becomes more heterogeneous both genotypically and phenotypically as the disease progresses. Other genetically unstable myeloid precursors may develop into acute leukemia with other critical mutations particularly driven by chemotherapy.
Previous studies have shown that gain of chromosome 3 was significantly correlated with IgA paraprotein and decrease in renal function.4,25 Our observation of trisomy 3 as the sole numeric chromosomal abnormality in one treated IgA MM patient may also suggest its biological significance in this MM subtype, which needs to be confirmed by a larger study.
The results presented may possibly imply that one of the earliest genetic lesions in MM may occur before the commitment to myeloid lineage development. The abnormality may be potentially cryptic and manifested as chromosomal instability. However, this hypothesis remains speculative and needs to be confirmed by further study of clonality of different lineages in MM.
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Acknowledgements |
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Footnotes |
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Accepted for publication September 22, 1998.
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References |
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