[CANCER RESEARCH 37, 2950-2956, August 1977]
Chromosome Markers and Progression in Bladder Cancer1
Avery A. Sandberg
Roswell Park Memorial Institute, Buffalo, New York 14263
Summary
The chromosome constitution of 62 papillary tumors and
75 invasive transitional cell carcinomas of the bladder has
been examined. Of the 62 papillary tumors, 12 were benign
and were found to be preponderantly diploid; 2 of these
tumors recurred, however, and in each a few karyotypically
abnormal cells were present on the original examination.
The modal chromosome number in the 50 papillary cancers
ranged in the dipboid area (44 to 49 chromosomes) with the
karyotypic picture being different from tumor to tumor. Of
the 50 papillary cancers, 32 had 1 to 2 marker chromo
somes. Twelve of the papillary tumors recurred; only 1 did
not have a marker. Thus, the recurrence of the 11 papillary
tumors with markers indicates that their presence may be of
value
in predicting
the behavior
of papillary
cancers
of the
bladder. The chromosomal picture in the recurrent papillary
tumors did not differ materially from the original one, al
though a slight modification in the chromosome number
may occur. On the other hand, invasive transitional cell
cancer of the bladder was accompanied by a large number
of chromosomes and a relatively large number of marker
chromosomes with many complicated karyotypic pictures.
The presence of marker chromosomes in papillary cancers
may be indicative of the likelihood of recurrence and/or
progression of such tumors and should attract the attention
of those involved in the care of patients with these cancers.
Introduction
Chromosome studies in cancer of the bladder have had as
one of their major aims the establishment of cytogenetic
parameters that may characterize certain tumor types
(based on the anatomy, histology, and clinical behavior of
the tumors) and may possibly be of value in predicting the
progression of the tumor. Although a number of cytoge
netic studies on cancer of the bladder have appeared in the
past and have indicated a difference in the karyotypic find
ings, primarily in the chromosome number, between inva
sive and noninvasive tumors of the bladder (1, 9, 13), the
role of abnormal chromosomes (markers) remained un
clear, primarily since the exact incidence could not be
ascertained with the techniques then used. The introduc
tion of chromosome banding techniques, which afford rig
orous identification of all normal chromosomes and, thus,
also of abnormal chromosomes, has led to a more precise
evaluation of the role of such markers in human cancer (2,
, Presented
at the National
Bladder
Cancer
Conference,
November
28 to
December 1, 1976, Miami Beach, Fla. Supported in part by Grant CA-i 4555
from the National Cancer Institute through the National Bladder Cancer
Project.
2950
5—8,
10, 11). This is particularly true of those tumors with a
chromosome number in the dipboid range, which character
izes a substantial number of bladder cancers, particularly of
the noninvasive papillary type (1, 9). The present study was
undertaken to establish with various banding techniques a
correlation between (a) the chromosomab findings of early
bladder lesions, particularly papillary tumors, with the cyto
logical picture and clinical course, and (b) the karyotypic
picture in advanced carcinoma of the bladder with the re
sponse to therapy and clinical course, e. g., sensitivity to X
ray and/or chemotherapy, metastatic spread, recurrences,
etc. This presentation deals primarily with the former as
pectsofthisstudy.
Materialsand Methods
Specimens of tissues were obtained by means of cysto
scopic procedures or following cystectomy. In some cases,
specimens from different areas of the tumor were proc
essed for analysis; in others, samples from multiple lesions
present in the bladder were examined, and in still others
specimens were obtained on more than 1 occasion. In
cluded in the present study are those patients who had been
followed for a period of at most 3 years and for no less than
6 months.
The specimens were processed immediately after being
obtained, and chromosome preparations were performed
by 2 approaches. In one, the tissue was prepared by a direct
technique previously described (5—8,12), and in the other
the minced tissue was incubated overnight in the presence
of Colcemid. A few details will be given. The tissue was
minced on a watch glass into small segments with a small
scalpel, and the material was mixed with a hypotonic glu
cose solution [0.6% glucose in 0.7% sodium chloride:0.44%
sodium citrate (1:1)], pipetted into a centrifuge tube, and
allowed to stand for 2 to 5 mm. The supemnatant was re
moved and was incubated for 20 to 30 mm at 37°.These
procedures were performed in the presence of Colcemid, 10
ppm, in order to obtain well-contracted metaphases. After
incubation, the cells were collected by centrifugation and
fixed with 50% cold acetic acid (stored at 4°),and chromo
some preparations were made. In the 2nd procedure, utiliz
ing short-term culture (about 12 hr), the cells were incu
bated in Roswell Park Memorial Institute Tissue Culture
Medium 1640 and then were harvested. Such a procedure
yields a high number of metaphases suitable for analysis
that do not appear
to differ
significantly
from
obtained in the tissues by a direct technique.
used in our laboratory for the preparation of
materials, photography, the presentation
data, and the performance of G, Q, and C
been described previously (5-8, 12).
the karyotypes
The methods
chromosomal
of karyotypic
banding have
CANCER RESEARCH VOL. 37
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Chromosome
Results
Markers and Progression
28
Chromosomesof Normal BladderTissues. Examination
of the chromosome counts of 75 specimens (52 male, 23
24
female) of normal bladder mucosa revealed the preponder
ant number (more than 90%) of the 757 metaphases ob
served to have 46 chromosomes with a diploid karyotype.
20
The number of metaphases available for analysis varied
C,)
from as few as 2 to as many as 50 following short-term
Li
(1)
incubation (6 to 8 hr) in the presence of Colcemid. The cells
with less than 46 chromosomes, constituting no more than
16
5 to 8% of the metaphases of any single bladder specimen,
were due to random loss of chromosomes.
Metaphases with a polypboid number of chromosomes,
12
e.g., tetraploid cells with 92 chromosomes, were not ob
served in any of the specimens examined. Since it is possi
:D
ble that polypboid cells may not undergo mitosis and,
8
hence, that metaphases with 92 or more chromosomes may
not be available for observation, we examined interphase
cells in the bladder specimens of the male patients using a
quinacrine fluorescent technique (5). This method leads to
4
high fluorescence of the V chromosome, which can be
readily identified as a single V body in dipboid mnterphase
cells. No cells with 2 such fluorescent bodies were ob
0
served, which would be expected if polypboid cells were
44 45 46 47 48 49
present in the male bladder specimens. Thus, these obser
CHROMOSOMEMODE
vations lend further support to the lack of polyploid cells in
Chart 1. Distribution of the modal chromosome numbers in 62 papillary
human bladder mucosa.
of the bladder, 12 benign and 50 cancerous. The 12 benign tumors
In contrast to the observations on elderly males, of whom tumors
had a diploid number of 46 chromosomes (cross-hatched area). The papillary
a high percentage have a missing Y chromosome in their cancers had a chromosome number ranging from 44 to 49, with markers
marrow cells (11), we could not find any significant number being present in 32 tumors (hatched areas) and absent in 18 (clear areas).
of cells with a missing V chromosome in the bladder, in
(more than 300 metaphases) of dividing cells for reliable
cluding 22 specimens from males over the age of 65.
NoninvasivePapillaryTumorsof the Bladder.A total of kamyotypic examination, with the average number of meta
62 noninvasive papillary tumors of the bladder has been phases being about 50. Six papillary cancers were found to
examined. Of these, 12 were thought to be benign lesions have a chromosome mode of 44 (3 with markers), 13 tumors
had a mode of 45 (8 with markers), 16 had a mode of 46
(Chart 1). The number of metaphases available for exam ma
tion in these 12 tumors was rather small (2 to 19 metaphases chromosomes but with a pseudodipboid karyotypic picture
per tumor), a characteristic previously observed in other (11 with markers), 9 had a mode of 47 chromosomes (6 with
benign tumors (11); in the 107 metaphases examined, a markers), 4 had a mode of 48 chromosomes (2 with
preponderantly
dipboid chromosome constitution
was markers), and 2 had a mode of 49 chromosomes (2 with
found except for 2 cases in which some abnormal meta
markers). Thus, of the 50 papillary tumors examined, 32 had
phases were present. In 1 case, 3 metaphases of 12 exam
markers in a few (5%) to a large proportion (nearly 100%) of
med had a chromosomal number of 45. This tumor recurred the cells examined. It appears, then, that marker chromo
and became invasive 11 months after the original observa
somes may appear frequently in papillary bladder cancers
tion, at which time all the cells had a chromosomal picture regardless of the chromosome count.
similar to that observed in the aneupboid cells of the original
Although most of the papillary tumors had a definite
papillary tumor. In the 2nd case, 2 metaphases out of 14 modal number of chromosomes, the distribution of the
were aneupboid, i. e., contained 47 chromosomes; the tu
number of chromosomes about the mode was decidedly
mom recurred about 1.5 years later, was noninvasive, and different from that observed in benign tumors or normal
wascompletelyresectabbe.
No recurrenceshavebeenseen tissues, in which the cells with 46 chromosomes constitute
in the other 10 benign tumors, with the time of follow-up
more than 90% of the metaphases (Chart 2) (11). Thus, in
ranging from 8 months to 3 years.
papillary tumors the metaphases with the modal number did
The 50 noninvasive papillary cancers had modal chromo
not exceed 35% of the total cells and were as bow as 19% in
some numbers in the diploid mange(5 = 44 to 49), but all 1 of the tumors. Although the papillary tumors had modal
were abnormal cytogenetically, either containing numerical numbers around the dipboid range, no similarity in the chro
deviations from the dipboid number of 46 chromosomes,
mosomal picture among the various papillary cancers was
i.e. , aneupboidy, and/or morphobogically abnormal chromo
observed, with the karyotype varying from one case to an
somes (markers) (Figs. 1 to 4; Chart 1). In contrast to the other regardless of the chromosome count. Six of the papib
benign papillary tumors, the cancerous lesions usually lary cancers (3 with markers) had multiple foci in the blad
yielded from a sufficient (18 metaphases) to a large number der; the karyotypic picture was very similar in the foci of
z
AUGUST 1977
2951
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A. A. Sandberg
each tumor.
The application of various banding techniques in the
present study has afforded a rigorous identification of not
only the normal chromosomes in the tumors but also the
genesis of markers. As stated above, the latter were present
in 32 of the 50 papillary cancers examined, such markers
being seen in 5% to nearly 100% of the metaphases ana
lyzed in the tumors. In no case were more than 2 markers
observed in a cell. This differs from the usual picture of a
large number of markers seen in the bulk of the more
advanced forms of cancer of the bladder (2, 4) (see below).
Banding analysis revealed that the most common markers
originated from chromosome 1 or involved a translocation
of chromosomes 14, 17, and some of those in the C group.
Eleven of the papillary cancers recurred; 10 of these had
markers, and 1 did not. The latter tumor had a chromosome
mode of 48 and, when it recurred, the chromosome consti
tution differed only slightly from the original one, i. e., the
chromosome mode that was 48 persisted but some cells
with a near-tetmapboidnumber of chromosomes were pres
ent. The recurrence of the tumor without markers occurred
locally, whereas all the recurrent tumors with markers be
came invasive. In the 11 cases with markers and in whom
the papillary cancers recurred, the karyotypic picture did
not change materially in 6 cases; whereas in 5 cases near
tetrapboid cells with more than 2 markers appeared, ab
though the original chromosome constitution was observed
in most of the cells. When deviations from the original
picture in these tumors were seen, the chromosomal pic
ture that emerged appeared to be a variation on the chro
mosomal picture of the original examination. Recurrence of
the tumors took place within 6 months of the initial chromo
some examination in 8 of the 11 cases with markers.
30
20
0
Li
l0
4
x
LU
U)
-J
-J
LU
0
30
TransitionalCell Cancerof the Bladder.Forcomparison
z
with the findings in papillary tumors, data are presented on
75 patients with invasive bladder cancer studied cytogeneti
cabby(Figs. 5 and 6). Karyotypic examination revealed, gen
erally, a picture similar to that already published in the
literature; i.e. , relatively well-differentiated tumors with only
local invasiveness were shown to have a neam-dipboidrange
of chromosomes, with the more advanced cancers having a
neam-triploid or tetraploid number of chromosomes (Chart
3) (2, 9). Of more importance, perhaps, was the appearance
of an increased number of marker chromosomes with pro
gression of the cancer, with 2 barge markers of metacentric
morphology being most common. These large markers
have been shown with banding techniques to be probably of
common origin (chromosome 1?) in most of the tumors
investigated (Fig. 4). Thus, it appears that the evolution and
progression of marker chromosomes may be a more impor
tant cytogenetic parameter to evaluate than either the chro
mosomal number or the numerical progression of the tu
mor. In a preliminary evaluation, the impression was gained
that the response to radiation therapy was more readily ob
tamed in tumors that had a relatively large number of
marker chromosomes, usually 4 to 9 markers, than in those
C-)
20
10
(]@@-@@1_
38
40
42
44
46
48
50
52
CHROMOSOME
NUMBER
Chart 2. Distribution of the chromosome numbers in 2 papillary cancers
of the bladder. In each case there was considerable variation in the counts
about the modal number, which was 46 but karyotypically pseudodiploid with
a marker in one case (lower) and 48 chromosomes without markers in the
other (upper).
-....-
12
@
WELL
AND
MODERATELY
WELL DIFFERENTIATED
D
10
POORLY AND VERY
POORLY DIFFERENTIATED
C/)
w
(08
4
@
C.)
Chart 3. Distribution of the modal chromosome numbers in
72 invasive transitional cell cancers of the bladder.
U-
6@
I
0
In
z
44
46
48
50
66
68
70
72
84
86
88
MODAL CHROMOSOMENUMBER
2952
CANCER RESEARCH VOL. 37
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Chromosome Markers and Progression
in which 0 to 2 markers were present. A correlation with endoreduplication and/or nondisjunction in the original tu
chemo- and/or radiation therapy is somewhat premature at mor cells or to some other processes leading to the com
this time, since many of the patients are still on various plicated cytogenetic pictures. In any case, it would appear
that the presence of marker chromosomes points to a defi
courses of such therapy. We hope to have such a correla
niteby more serious lesion than their absence. Thus, it be
tion within 2 to 3 years of this writing.
In 9 cases, a recurrence and spread of the disease took hooves clinicians to pay particular attention to the cytoge
netic picture of papillary cancers, since the presence of
place, and in each case it was shown that the chromosomal
picture did not deviate materially from the previous one and marker chromosomes may point to a more complicated
essentially represented a variation on the karyotypic theme lesion requiring a more careful follow-up, with particular
of the original chromosomal pattern. All 9 tumors had 5 to 7 attention being paid to recurrence of such lesions. Re
marker chromosomes. However, all 9 of these cancers of cently, others have also pointed out that marker chromo
the bladder were very poorly differentiated and had already somes have potential value as a prognostic aid in early
cancer of the bladder and, furthermore, that the triad of
shown rather advanced disease when studied initially.
tetrapboidy, markers, and submucosal, invasive, moderately
well-differentiated carcinoma appears to carry such a lethal
Discussion
prognosis as to militate for early radical resection (3). Thus,
it appears that the chromosomab
findings
in cancer of the
Although cytogenetic studies in cancer of the bladder bladder, whether early or late, may prove to be of consider
have not revealed a characteristic or specific chromosomal
able value to those engaged in the evaluation, diagnosis,
change, akin to the Ph' chromosome in chronic myebocytic and therapy of these complicated lesions.
leukemia (10, 11), the present findings, as well as those
published in the recent literature (1-4), may prove to be of
considerable value in the evaluation of cancer of the blad
der, particularly of papillary lesions. Thus, the presence of References
marker chromosomes appears to endow papillary tumors
1. Falor, W. H. Chromosomes in Non-invasive Papillary Carcinoma of the
with more likelihood of recurrence and/or invasiveness
Bladder. J. Am. Med. Assoc., 216: 791-794, 1971.
than those tumors that do not contain such markers. In the
2. Falor, W. H., and Ward, R. M. DNA Banding Patterns in Carcinoma of the
Bladder. J. Am. Med. Assoc., 226: 1322-1327, 1973.
present study it was shown that only 1 of the 18 papillary
3. Falor, W. H., and Ward, R. M. Cytogenetic Analysis: A Potential Index of
cancers without markers recurred, whereas 11 of the 32
Recurrence of Early Carcinoma of the Bladder. J. Urol., 115: 49—52,
papillary cancers with markers recurred and became inva
1976.
4. Falor, W. H., and Ward, R. M. Prognosis in Well-differentiated Non
sive.
Invasive Carcinoma of the Bladder Based on Chromosomal Analysis.
Similar findings and conclusions have been reached in
Surg. Gynecol. Obstet., 144: 515—518,
1977.
another study (1, 4). In a recent publication the cytogenetic
5. Kakati, S., Hayata, I., Oshimura, M., and Sandberg, A. A. Chromosomes
and Causation of Human Cancer and Leukemia. x. Banding Patterns in
data are presented on 27 well-differentiated noninvasive
Cancerous Effusions. Cancer, 36: 1729—1738,
1975.
cancers of the bladder (4). Almost all the patients who had
6. Kakati, S. , Hayata, I., and Sandberg, A. A. Chromosomes and Causation
of
Human
Cancer
and
Leukemia.
xlv.
Origin
of a Large Number of
markers in their tumors (14 of 15) developed recurrences of
Markers in a Cancer. Cancer, 37: 776-782, 1976.
the cancer, whereas only 1 of the 12 patients without
7. Kakati, S., Oshimura, M., and Sandberg, A. A. The Chromosomes and
markers in their tumors developed such recurrence. Eleven
Causation of Human Cancer and Leukemia. xlx. Common Markers in
Various Tumors. Cancer, 38: 770-777, 1976.
of the latter 12 patients have been recurrence free for 8
8. Kakati, S., Song, S. V., and Sandberg, A. A. The Chromosomes and
years. The authors believed that in early lesions of the
Causation of Human Cancer and Leukemia. XXII. Karyotypic changes in
bladder the presence of markers is “a
highly accurate prog
Malignant Melanoma. Cancer, in press.
9. Lamb, D. Correlation of Chromosome Counts with Histological Appear
nostic aid―(4).
ances and Prognosis in Transitional-Cell Carcinoma of Bladder. Brit.
It is possible during the genesis and progression of papil
Med.J., 1:273-277,1967.
10. Sandberg, A. A. Chromosomes in Cancer and Leukemia. J. Reprod.
lary cancer of the bladder that the initial chromosomal
Med.,17:21-24,1976.
change is merely a deviation from the dipboid number of ii. Sandberg,
A. A., and Hossfeld, D. K. Chromosomes in the Pathogenesis
chromosomes, followed by the appearance of abnormal
of Human Cancer and Leukemia. In: J. F. Holland and E. Frei (eds.),
Cancer Medicine, pp. 151-177. Philadelphia: Lea & Febiger, 1973.
marker chromosomes and, ultimately, by the development
12. Sonta, S-I., Oshimura, M., Evans, J. T., and Sandberg, A. A. Chromo
of a totally abnormal kamyotypic picture consisting of chro
somes and Causation of Human Cancer and Leukemia. xx. Banding
mosome numbers above the dipboid range and the presence
Patterns of Primary Tumors. J. NatI. Cancer Inst., 58: 49-59, 1977.
of a barge number of marker chromosomes. It is possible 13. Spooner, M. E., and Cooper, E. H. Chromosome Constitution of Transi
tional Cell Carcinoma of the Urinary Bladder. Cancer, 29: 1401-1412,
that the genesis of this latter picture may be related to
1972.
Fig. 1. Karyotype of a C-banded metaphase with 45 chromosomes from a papillary tumor of the bladder showing the presence of an unusual marker (M)
with 3 C bands. Missing chromosomes are shown in Groups 5 and 8.
Fig. 2. Pseudodiploid karyotype with 46 chromosomes from a papillary cancer of the bladder, which recurred about 1 year after the 1st examination. Note
the large marker chromosome (Ml); missing chromosome in Groups *2, C, D, and E17; and extra ones in Groups E18 and F.
Fig. 3. 0-banded karyotype of a papillary cancer of the bladder containing 45 chromosomes with a missing chromosome in Groups 7 and 14 and an extra
one in Group 8. No marker chromosomes were present.
Fig. 4. Karyotype of a 0-banded metaphase with 48 chromosomes from a papillary cancer of the bladder with a marker (M). The derivation of the latter
could not be established with certainty. Note missing chromosome in Group 7 and extra ones in Groups 15 and 17. This tumor recurred within 9 months.
Fig. 5. Karyotype with 82 chromosomes from a poorly differentiated transitional cell cancer of the bladder with 2 small marker chromosomes (M). Note
increased numbers of chromosomes in all of the groups, including 2 Y chromosomes.
Fig. 6. Karyotype of a polyploid cell from a transitional cell cancer of the bladder containing 67 chromosomes with 11 markers, 6 with origins that could be
identified with G banding: M = iso-iq; M2= t(1 p, 13q); M3= 2p—;M4= iso-17q. The origin of the other 5 markers could not be established with certainty.
AUGUST 1977
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CANCER RESEARCHVOL. 37
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Chromosome Markers and Progression in Bladder Cancer
Avery A. Sandberg
Cancer Res 1977;37:2950-2956.
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