(CANCER RESEARCH 50, 3816-3825, July 1, 1990]
Special Lecture
Molecular Cytogenetics: Rosetta Stone for Understanding Cancer—Twenty-ninth
G. H. A. Clowes Memorial Award Lecture1
Janet D. Rowley
University of Chicago, Chicago, Illinois 60637
This article provides me with an occasion to review our
progress in a central area of cancer research, namely the genetic
changes that occur within the cancer cell that are critically
involved in the transformation of a normal to a malignant cell.
I think it is especially appropriate that three named awards
(Clowes, Rosenthal, and Rhoads) presented at the 1989 annual
meeting of the American Association for Cancer Research went
to investigators who have made contributions to our under
standing of the genetic changes in cancer cells. Clearly, to
concentrate on genes to the exclusion of cell biology would be
too narrow and short-sighted an approach. Nonetheless, I am
convinced that until we have isolated the genes that are centrally
involved in at least some of the malignant processes in different
cell types, we will be unable to answer the fundamental ques
tions about malignant transformation. More importantly, we
will be unable to answer the questions with precision. I will
limit my consideration to those changes that have been detected
by analyzing the karyotypic pattern of human cancer cells using
chromosome banding, and in particular to those found in leu
kemia.
We are living in a golden age of the biomedicai sciences.
Increasingly sophisticated instruments and creative scientific
strategies allow remarkably precise understanding of some as
pects of cancer biology. It is clear that during the course of the
last three decades, the scientific community's assessment of the
role of chromosome changes in the complex process of malig
nant transformation has changed from considering them to be
merely trivial epiphenomena to recognizing their fundamental
involvement for at least some tumors. This change in attitude
has occurred for at least two reasons. First, the demonstration
of specific recurring chromosome rearrangements, including
translocations and deletions, that were often uniquely associ
ated with a particular type of leukemia, lymphoma, or sarcoma
provided clear evidence that these rearrangements were criti
cally involved in malignant transformation (1-3). About 70
recurring translocations as well as many nonrandom deletions
and other structural abnormalities are listed in the chapter on
structural chromosome changes in neoplasia included in Hu
man Gene Mapping 10 (4). The evidence for the presence of
recurring chromosome abnormalities in a wide variety of human
neoplasms was the result of 30 years of painstaking chromo
some analysis by my cytogenetic colleagues around the world.
The honor attached to this Clowes award should be shared with
all of them.
A second, and I believe an even more powerful, force acting
within the general scientific community to reassess the role of
karyotypic alterations was the identification of the genes in
volved in some of the chromosome rearrangements and the
discovery that some of these genes were the human counterparts
Received 3/23/90.
1The research described has been supported by Department of Energy Contract
DE-FG02-86ER60408 and USPHS Grant CA 42557. Presented at the Eightieth
Annual Meeting of the American Association for Cancer Research, May 26,
1989, in San Francisco, CA.
of the viral oncogenes (5). In a sense, each group of investigators
gave the other scientific validity. The fact that oncogenes were
directly involved in chromosome translocations demonstrated
that both translocations and oncogenes were critically involved
in human cancer. The correlation of the chromosome location
of human protooncogenes as well as other cancer related genes
with recurring chromosome rearrangements is shown in Fig. 1.
Cytogeneticists thus progressed from being mere "stamp collec
tors" to being major contributors in cancer research, an im
provement in status that I welcome.
The genetic changes that occur in different types of malignant
cells are quite varied and clearly several different changes occur
in the same cell as it is altered from a normal to a fully
malignant cell. Cytogenetic analysis has been the key to defining
at least two major categories of rearrangements, namely recur
ring translocations and consistent deletions. One of the first
translocations, identified in 1972, was the 9;22 translocation in
chronic myeloid leukemia about which I will say more later (6).
There are now at least 70 recurring translocations that have
been detected in human malignant cells. But the identification
of consistent chromosome deletions has been equally important
because it has provided the absolutely essential information
regarding the chromosome location of the genes that are in
volved in cancer. I submit that the retinoblastoma gene would
not have been cloned, or at least not yet, if cytogeneticists had
not identified deletions of the long arm of chromosome 13, and
specifically of band 13ql4, in patients with constitutional chro
mosome abnormalities who had a high incidence of retinoblas
toma (7). This is not to detract from the careful and exciting
work of many scientists in actually cloning the gene, but at
least they knew where to look (8). This triumph has now been
joined by the recent cloning of the DCC (deleted in colorectal
carcinomas) gene on chromosome 18; the fact that a gene
important in the transformation of colorectal cells was located
on chromosome 18 was the result of cytogenetic analysis of
colon cancer cells that revealed that loss of chromosome 18
was a recurring abnormality (9, 10).
It has been a source of great disappointment to me that we
have progressed so slowly in cloning most of the genes located
at the breakpoints in the recurring translocations or inversions
in human leukemia. This emphasizes the fact that knowing the
location of the breakpoint is very helpful in selecting the genes
to use as probes for these rearrangements. However, a chro
mosome band contains at least 5 million base pairs, and the
likelihood that the DNA probe that you "pull off the shelf is
at the breakpoint and can detect a rearrangement on Southern
blot analysis is vanishingly small. The lymphoid leukemias and
lymphomas are the major exceptions to this slow progress,
because the immunoglobulin genes in B-cell tumors and the Tcell receptor genes in T-cell tumors have provided the essential
DNA probes to clone several dozen translocations (11-13).
Fortunately, the rapid progress being made in mapping the
human genome, coupled with major advances in working effec-
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Fig. 1. Map of the chromosome location of protooncogenes or of genes that appear to be important in malignant transformation and the breakpoints observed in
recurring chromosome abnormalities in human leukemia, lymphoma, and solid tumors. Known protooncogenes are indicated in bold italics and other cancer related
genes are listed in standard type. The protooncogenes and their locations are placed to the left of the appropriate chromosome band or region (indicated by a bracket).
The breakpoints in recurring translocations, inversions, etc., are indicated with an arrowhead to the right of the affected chromosome band. The solid vertical lines on
the right indicate regions frequently present in triplicate; the dashed lines indicate recurring deletions. Recurring viral integration sites and cloned translocation
breakpoints with no identified transcripts are indicated to the right of the appropriate band. Genes or recurring breakpoints that have been cloned are identified by #.
The locations of the cancer specific breakpoints are based on the Report of the Committee on Structural Chromosome Changes in Neoplasia, Human Gene Mapping
10 (4). [Author's note: Any map of this sort involves selection as to the genes that should or should not be included: I have been relatively conservative. Also for
recurring breakpoints and deletions, I have included only those listed as Status I or II in HGM10.
This figure was prepared by Michelle S. Rebelsky.)
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MOLECULAR CYTOGENETICS
lively with large pieces of DNA, should lead to the successful
cloning of most of the recurring translocations in the acute
leukemias and sarcomas in the next 5 years.
From the beginning of the cytogenetic analysis of human
malignant disease it has been clear that virtually all solid
tumors, including the non-Hodgkin's lymphomas, have an ab
normal karyotype and that some of these abnormalities are
limited to a given tumor. With regard to the leukemias, it
appeared from studies in the 1960s and early 1970s that only
about 50% had an abnormal karyotype. With improved culture
techniques and with the development of processing methods
that resulted in longer chromosomes with a larger number of
more clearly defined bands, Yunis et al. (14) have provided
evidence that a karyotypic abnormality can be detected in
virtually all leukemias as well. Some malignant diseases, such
as Hodgkin's disease or multiple myeloma, continue to show a
chromosome, No. 21 or 22, was involved and was the Ph' really
the result of a chromosome deletion as had been assumed in
the 1960s? With the use of the banding, the Ph' chromosome
was shown to involve chromosome 22, and in 1972 I could
show that it occurred as the result of a translocation rather
than a deletion. This was one of two translocations that I
discovered in 1972; the other one was actually identified about
6 months earlier than the Ph' translocation. It involved chro
mosomes 8 and 21 and occurred in patients with acute myeloblastic leukemia [t(8;21)(q22;q22)] (17). The Ph' translocation
high frequency of normal karyotypes. These diseases are char
acterized by malignant cells with a low mitotic index; therefore
it is likely that the dividing cells studied do not represent the
malignant cells.
One of the major reasons to concentrate on cloning the genes
involved in rearrangements is that the consistent chromosome
changes pinpoint the location of the genes with functions that
are critical in the growth potential of that cell type. The chro
mosome changes that we concentrate on are present in all of
the malignant cells; thus they are not random events affecting
one or a few cells in the involved tissue. Moreover, they are
clonal in origin and are derived from a single cell in which the
initial chromosome change occurred. These changes are so
matic mutations in individuals who otherwise virtually always
have a normal karyotype in their uninvolved cells. These obser
vations provide the evidence that cancer is a genetic disease.
This notion seems self-evident today, but it was not generally
accepted several decades ago when many of us began working
in cytogenetics. Clearly, I am using genetic in a special way,
referring to changes in genes within the affected cell, not in the
more usual sense of a constitutional genetic disease such as
hemophilia or color blindness.
Cytogenetic studies of CML2 are important because they
provided a number of firsts. The first consistent chromosome
change in any human cancer was identified in CML by Nowell
and Hungerford (15) in 1960 in Philadelphia, thus giving this
morphologically distinct chromosome its special name, Phila
delphia or Ph1 chromosome. Investigation of many patients
involved chromosomes 9 and 22 [t(9;22)(q34;qll)] (6). They
were the first consistent translocations to be discovered in any
human or animal tumor, the first of a great many (1, 2).
Many of the translocations we discovered in the acute myeloid leukemias were shown to be relatively specifically associ
ated with particular morphological subtypes, and in the acute
lymphoid leukemias they were associated with specific immunological phenotypes (Tables 1 and 2). Equally important was
the observation in the acute myeloid leukemias that two differ
ent chromosome bands were consistently affected in each translocation and therefore that the critical change involved the
abnormal juxtaposition of two very specific genes. These obser
vations gave rise to the idea that the genes involved in these
translocations had restricted functions that were actively ex
pressed only in the appropriate cell types or stage of differen
tiation (18). Cloning of a number of translocations in the
lymphoid leukemias and lymphomas has validated this concept.
It provides the hope that sustains us in our thus far fruitless
search for the genes involved in the translocation breakpoints
in acute myeloid leukemia. When we have successfully cloned
these breakpoints we will have discovered many new genes that
Table 1 Nonrandom chromosome abnormalities in malignant myeloid diseases
Disease"CMLCML
abnormalityt(9;22)(q34;qll)t(9;22)(q34;ql patients-100-70-2060-100100
of
phaseAML-M2"APL-M3,
blast
M3VAMMoL-M4EoChromosome
or t(16;16)(pl3;q22)%
(25% of M4)
AMMoL-M4 or M5
with CML revealed that marrow cells from the vast majority of
them had the Ph' chromosome. This cytogenetic analysis be
came important clinically because, paradoxically, patients
whose cells were Ph' positive appeared to have a longer survival
than these whose cells were Ph' negative (16). The reasons for
this finding will be considered later. In our naive enthusiasm
then, we assumed that consistent chromosome changes would
be found in most malignant cells. This hope was quickly dis
pelled in the 1960s before the development of chromosome
banding, because some leukemias showed a bewildering array
of chromosome changes whereas others had an apparently
normal karyotype. This variability led to the view I mentioned
earlier, that chromosome changes were merely epiphenomena.
The situation changed dramatically with the development of
chromosome banding in 1970. It became possible to identify
each human chromosome, and parts of chromosomes, precisely.
Thus it became possible to answer a number of questions
regarding the Ph' chromosome, namely which small G group
2The abbreviations used are: CML, chronic myeloid leukemia; ALL, acute
lymphoblastic leukemia.
1) with +8, +Ph',
ori(17q)t(8;21)(q22;q22)t(15;17)(q22;qll-12)inv(16)(pl3q22)
+ 19,
t(l;ll)(q21;q23)
t(2;ll)(p21;q23)
t(6;ll)(q27;q23)
t(10;ll)(pll-pl5;q23)
ins(10;ll)(pll;q23q24)
t(ll;17)(q23;q25)
t(Il;19)(q23;P13)
t(llql3orq23),
del(ll)(q23)
AMoL-M5
t(9;l I)(p22;q23), t(l Iql3 or q23)
AML
+8
-7 or del(7q)
-5 or del(5q)
t(6;9)(p23;q34)
t(3;3)(q21;q26) or inv(3)(q21q26)
del(20q)
t(12p)ordel(12p)
t(3;21)(q26;q22)
Therapy-related AML
-7 or del(7q) and/or -5 or del(Sq)
-35
-30
13
9
6
2
2
5
2
1
90
l(9;ll)(P22;q23)
<1
" AML, acute myeloblastic leukemia; AML-M2, AML with maturation;
AMMoL, acute myelomonocytic leukemia; AMMoL-M4Eo, acute myelomonocytic leukemia with abnormal eosinophils; AMoL, acute monoblastic leukemia;
APL-M3, M3V, hypergranular (M3) and microgranular (M3V) acute promyelocytic leukemia.
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MOLECULAR CYTOGENETICS
Table 2 Cytogenetic-immunophenotypic-genomic
correlations in malignant lymphoid diseases
Involved genes
Rearrangement
Phenotypc
Acute
leukemiaPre-BB(SIg+)°B
lymphoblastic
or B-myeloidt(l;19)(q23;pl3)I(8;14)(q24;q32)t(2;8)(pll-12;q24)t(8;22)(q24;qll)t(5;14)(q31;q32)dic(9;!2)(pll;pl2)t(9;22)(q34;qll)I(4;ll)(q21;q23)PRLMYC1GKMYCIL3ABLE2AI
Other
hyperdiploidy (50-60 chromosomes)
del(9p), t(9p)
del(12p), t(12p)
t(8;l7)(q24;q22)
IFNA/B
MYC
BCL3"
TALI"/TTGC
TCL2'
TCRD
TCRD
TCRA
TCRA
TCRA
TCRD
TCRD
t(8;14)(q24;qll)
inv(14)(qllq32.3)
inv(14)(qllq32.1)/t(14;14)(qll;q32)
t(10;14)(q24;qll)
t(l;14)(p32;qll)
t(7;9)(q35;q32)
t(7;9)(q35;q34)
t(7;7)(pl5;qll)
t(7;14)(pl5;qll)
t(7;14)(q35;qll)
t(7;19)(q35;pl3)
MYC
t(8;14)(q24;q32)
t(2;8)(pll-12;q24)
t(8;22)(q24;qll)
t(14;18)(q32;q21)
t(ll;14)(ql3;q32)
MYC
IGH
IGK
MYC
MYC
IGL
BCL2
IGH
IGH
TCLÌ
TCL3"-C
TALl"/SCL
TCRB
TCRB
TCRG
TCRG
TCRB
TCL3"
LYL1
Non-Hodgkin's lymphoma
B
IGH
BCLl'
see T-cell ALL
Chronic lymphocytic leukemia
B
Multiple myeloma
B
Adult T-cell leukemia
t(ll;14)(ql3;q32)
t(14;19)(q32;ql3)
t(2;14)(pl3;q32)
t(18;22)(q21;qll)
t(14q32)
+ 12
BCLl'
IGH
IGH
BCL2
IGH
BCL3"
t(8;14)(q24;qll)
inv(14)(qll;q32)
MYC
TCRA
TCLl
t(ll;14)(ql3;q32)
t(14q32)
BCLl
IGH
TCRA
IGL
IGH
t(14;14)(qll;q32)
inv(14)(qllq32)
+3
°SIg, surface immunoglobulin.
* Same name used for different translocation breakpoints.
c No expressed gene identified at present.
d Breakpoint not named; source of translocation for cloning listed.
are of critical importance to these cells in their particular stage
of differentiation.
Having emphasized the challenge for the future, let me return
to the success of the present. As noted earlier, the Ph' translo
1036 (92%) (1). Variant translocations have been discovered,
however, in addition to the typical t(9;22). Until recently these
were thought to be of two kinds: one appeared to be a simple
translocation involving No. 22 and some chromosome other
cation involved chromosome 9 with a break in the last band in than No. 9 (about 4%); and the other was a complex translo
the long arm (9q34) and chromosome 22 with a break in the cation involving three or more different chromosomes, two of
long arm, band 22qll. It was clear with banding that a large which were No. 9 and No. 22 (about 4%). Recent data clearly
portion of No. 22 was on No. 9 (Fig. 2). Although we assumed
demonstrate that No. 9 is affected in the simple as well as the
that this was a reciprocal translocation, it was not until 1982 complex translocations and that its involvement had been over
that we had proof that this notion was correct (19). Other
looked (20). Virtually all chromosomes have been involved in
studies with fluorescent markers or chromosome polymor
these variant translocation, but No. 17 is affected more often
phisms showed that, in a particular patient, the same No. 9 and than are other chromosomes (21).
No. 22 were involved in each cell. The karyotypes of many Ph1
In 1972, we lacked the insights and the tools to discern the
positive patients with CML have been examined with banding
meaning of these translocations. However, the fact that virtually
techniques by a number of investigators; in a review of 1129 all of the translocation regularly involved the same two chro
Ph' positive patients, the 9;22 translocation was identified in mosomes indicated that two essential genes were involved in
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MOLECULAR CYTOGENETICS
K
Fig. 2. Karyotype of a CML cell, with chro
mosomes stained to show the Giemsa banding
pattern. The material missing from the Ph' chro
mosome has been translocated to the end of the
second chromosome No. 9, which has additional
pale material at the end of the long arm. The two
abnormal chromosomes are marked by arrows.
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the transforming event. The discovery that the Ph1 chromosome
was a translocation led to two questions: (a) was it reciprocal
and (b) what were the genes at the breakpoints? The evidence
that it was a reciprocal translocation was obtained only in 1982
and was directly related to the subsequent cloning of the break
point (19). Our efforts to clone chromosome translocation
breakpoints received remarkable assistance from the tumor
virologists. These scientists, beginning with Peyton Rous in
1911, had isolated a number of viruses from spontaneous
cancers and leukemia/lymphomas
in various animal species.
The particular segment of the RNA genome of these viruses
that was associated with its ability to induce transformation
could be isolated with molecular techniques. These segments
were called oncogenes. It was natural to ask where these oncogenes had come from, but the answer was something of a
surprise. It turned out that they were homologous to highly
conserved genes in normal cells. These cellular counterparts
were referred to as protooncogenes. These conclusions were
based on the research of many scientists who carried out these
studies in the early 1970s. The critical paper that reported these
results has been recognized recently by the selection of J.
Michael Bishop and Harold E. Varmus as 1989 recipients of
the Nobel Prize in Physiology or Medicine (22). The analysis
of these viral oncogenes and the protooncogenes has led to an
increasingly rich understanding regarding the aberrant function
of these genes in malignant cells, and in some ways more
importantly, their usual function in normal cells as essential
mediators of cell growth and differentiation (5).
Many of us were struck in the early 1980s by the remarkable
coincidence of the chromosome location of these protoonco
genes and the cancer/leukemia specific breakpoints (23). In
fact, molecular analysis of the first translocation junction in
1982 was based, in part, on the use of one of these oncogenes,
namely the MYC protooncogene, the normal human homo
logue of the \-myc oncogene from the avian myelocytomatosis
virus, which is located at the breakpoint in Burkitt's lymphoma
(24, 25). The breakpoint junction in CML was the next one to
be cloned and the gene that was the key to cloning the 9;22
translocation was the Abelson (ABL) protooncogene. The hu
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man ABL gene was first identified because of its homology to
the viral oncogene (\-abl) isolated from a mouse pre-B-cell
leukemia. It was shown in 1982 that ABL was located at the
end of chromosome 9 (26). The rapid discovery that ABL was
translocated to the Ph1 chromosome was the result of the
foresight of Professor Bootsma in Rotterdam and his colleague
Geurts van Kessel, who constructed somatic cell hybrids from
CML cells and isolated some hybrid cells that contained only
the Ph' chromosome. Using these hybrids and the probe for
ABL, it was shown that ABL was translocated to the Ph1
chromosome (19). In fact, it was the first gene on chromosome
9 that could be shown to be translocated to the Ph' chromo
some, thus providing clear evidence that this was a reciprocal
translocation. Using the v-abl probe to obtain human ABL
sequences in 1983, Heisterkamp et al. (27) walked across the
translocation junction and identified DNA sequences that were
derived from chromosome 22. Using this probe from chromo
some 22, they examined DNA from 17 CML patients and
found that the breakpoints on No. 22 all occurred in a 5.8kilobase segment which they called the breakpoint cluster re
gion, or ber (28). The gene in which this cluster is located has
also been cloned and, at least temporarily until its function is
defined, it too is called BCR. As you can imagine, at least in
verbal communication, it may be confusing as to whether one
is referring to the region ber, written in lower case, or to the
gene BCR written in capitals and italics. It is a large gene of
130 kilobases that contains many exons (29). The great majority
of the breakpoints in CML are located in the middle of the
gene. The breaks usually occur within either of two introns,
between exons 2 and 3 or between exons 3 and 4 (using the
original nomenclature of Heisterkamp et al. (29). The exact
location can be determined with the use of the polymerase chain
reaction. There are conflicting data regarding whether the pre
cise location of the breakpoint has prognostic significance.
Several recent papers suggest that a break between exons 3 and
4 may be associated with a shorter time before blast transfor
mation (30-32).
In contrast to the breaks in ber, the breaks in ABL on No. 9
occur over an incredible distance of more than 200 kilobases,
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MOLECULAR CYTOGENETICS
but again, they always occur in an intron. Pulsed field gel
electrophoresis was used by my colleagues, Drs. Charles Rubin
and Carol Westbrook at the University of Chicago to great
advantage in the study of the ABL protooncogene. Southern
blotting with standard gel electrophoresis leads to separation
of DNA fragments in the size range of 2 to about 25 kilobases.
Since the ABL gene is larger than 200 kilobases, mapping it in
10- to 20-kilobase pieces is a formidable task. In contrast, by
using pulsed field gel electrophoresis one can separate frag
ments larger than 1000 kilobases, and this technique is also
very effective in the 100- to 600-kilobase range. As I mentioned
earlier a normal chromosome band contains roughly 5,000 to
10,000 kilobases and thus, several very large, overlapping frag
ments could encompass a single band. Using many probes for
ABL provided by various investigators, Rubin and Westbrook
in collaboration with Bernards and Baltimore constructed a
partial map of the normal ABL gene (33).
This is a very complex gene that normally uses one of two
alternative beginnings, exon la or Ib. During transcription,
either of these exons can be spliced at the same point to the
remainder of the gene, which is called the common splice
acceptor site of exon II. One of their first discoveries was that
the type Ib exon mapped more than 200 kilobases upstream
from exon II. Thus, in normal cells, a very large segment of the
RNA transcript was removed or spliced out to form the mature
mRNA. This was a remarkable feat, not identified before in
biological systems. The sizes of exons la and Ib are different
and therefore the size of the mature mRNA is different. If exon
Ib is used, the mRNA is 7.0 kilobases whereas it is 6.0 kilobases
if exon la is used. In CML, breaks can occur anywhere within
the introns of the ABL gene 5' of exon II. As a result of the
translocation, the ABL gene from exon II to its 3' terminus is
moved next to the midportion of the BCR gene forming a fusion
or chimeric gene (Fig. J>A)(34). In essence, the normal ABL
exon la or Ib is replaced by a part of the BCR gene. Despite
the great variability of the breakpoints in the 5' region oÃ-ABL,
the same ABL fusion mRNA is formed because the 5' region
of the BCR gene is spliced into the same ABL common splice
acceptor site of exon II. This fusion mRNA is 8.5 kilobases,
which is much larger than the two normal ABL mRNAs of 6.0
and 7.0 kilobases.
Cloning the translocation breakpoint has helped to clarify
several problems. One of these relates to patients discussed
earlier, who appear to have CML, although their leukemic cells
lack the Ph1 chromosome. Many such patients can be shown
with careful analysis of the morphology of their bone marrow
cells not to have CML but to have some other myelodysplastic
Fig. 3. A, map of the BCR-ABL fusion gene in CML and in some adult ALL
patients. In this example, the breakpoint has occurred between the third and
fourth exons included in the ber region, which are equivalent to exons 11 and 12
in the BCR gene. The chimeric mRNA is diagrammed below the gene. B, map of
the fusion gene in some ALL patients showing the breakpoint in the first intron
of BCR. The breakpoint in ABL is identical with that in CML in this example.
Only one BCR exon is included so that the mRNA is much smaller than in CML.
syndrome (35, 36). It is no wonder that they do not have a Ph1
chromosome because they do not have CML. But a very inter
esting group of patients, possibly up to 5%, have clinically
typical CML. Using the ber probe, many of these patients have
the same DNA rearrangements that are identified in the Ph1
positive CML. Moreover, with special techniques including in
situ chromosome hybridization, the ABL gene can be identified
on one of the apparently normal No. 22 chromosomes (37, 38).
Therefore the ABL gene has been cut out of one No. 9 and
inserted into a No. 22 adjacent to the BCR gene resulting in
the same fusion gene that is present in the typical Ph' chro
mosome. Therefore it seems valid, at present, to consider this
BCR-ABL fusion gene as the sine qua non of CML.
We faced another dilemma in the study of the Ph' chromo
some. I have emphasized the specificity of chromosome translocations, and yet the Ph1 chromosome appeared to be an
exception. As well as its association with CML, an apparently
identical translocation involving chromosomes 9 and 22 was
seen in patients with ALL which is a very different disease. The
cloning of the translocation breakpoints in Ph' positive ALL
has shed a little light on this confusing issue.
Ph' positive ALL is common in adults and accounts for at
least 20% of adult ALL patients (39). In fact, this is the single
most frequent abnormality in adult ALL. It has been recognized
for some time that certain chromosome abnormalities have
prognostic importance. One of the most important of these is
the presence of the Ph1 chromosome which is associated with
a very poor prognosis in both children and adults. A recent
prospective study using ber probes and pulsed field gel analysis
indicates that a rearrangement of the BCR gene can be identified
in 32% of adult ALL patients (40). Earlier analysis of a large
number of ALL patients showed that about one-half of adults
had the CML-type breakpoint in the BCR gene and one-half
did not (41). In contrast, the data from the molecular study
showed that only 2 of 11 Ph1 positive adult ALL patients had
a breakpoint in the CML region (40). Although Ph1 positive
ALL accounts for only about 5% of childhood ALL, the great
majority appear to have a non-CML breakpoint.
A number of investigators have cloned the non-CML break
point and have shown that it is 5' of ber, the CML breakpoint,
and that it is in the first large (70-kilobase) intron of the BCR
gene (Fig. 3Ä)(42). Moreover, the breakpoints appear to be
clustered in the 3' 20-kilobase segment of this intron (43). The
breakpoint in ABL is similar to CML, in that it can occur
anywhere in the two introns 5' of exon II. The translocation
results in a chimeric BCR-ABL mRNA which is 7.0 kilobases
(Fig. 3B). This is smaller than that seen in CML, because a
much smaller portion of the BCR gene is included. In fact, this
chimeric message is the same size as one of the two normal
ABL mRNAS. This fusion mRNA is translated, in turn, into a
fusion or chimeric protein. The normal ABL protein is 145
kDa; its precise function in normal cells is not well understood
(Fig. 4). The CML fusion protein is larger, 210 kDa, and it has
somewhat increased tyrosine kinase activity (44). The fusion
protein seen in some Ph' positive ALL patients is smaller,
about 190 kDa (45). The tyrosine kinase activity of this protein
is greater than that seen in CML and the increased kinase
activity of the BCR-ABL fusion protein in ALL probably leads
to a more aggressive leukemia (46). Fortunately, a mouse model
for CML appears to have been developed recently in which the
myeloid cells express the BCR-ABL 210-kDa fusion protein
(47). In addition, a transgenic mouse model for Ph' positive
acute leukemia, either lymphoid or myeloid, has also been
developed (48). The BCR-ABL transgene contains only BCR
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MOLECULAR CYTOGENETICS
Normal
CML
Fig. 4. Diagram of the Abelson protein in normal cells (left), in cells infected
with the abl virus (second), in Ph1 positive ALL (third), and in CML cells (right).
In the ALL and CML cells, the protein is larger because some of it is derived
from the BCR gene and some from the ABL gene. The molecular weight of the
protein is given below each one.
exon 1 and the leukemic cells express the BCR-ABL 190-kDa
fusion protein. The mice die of leukemia within the first 2
months of life. These models will provide the experimental
systems that are necessary to understand the pathophysiology
of Ph1 positive leukemia. Although we do not yet fully under
stand how the different fusion proteins are related to malignant
transformation in either CML or ALL, we at least have the
tools to ask sensible questions. You can appreciate how complex
the study of what appeared to be a simple translocation has
become.
As well as raising a number of questions regarding the func
tion of the BCR and ABL proteins in normal cells and of the
fusion protein in leukemic cells, cloning the translocation has
provided hematologists and oncologists with an important di
agnostic tool. One can use the DNA probes for the breakpoint
cluster region and can diagnose CML on the basis of DNA
rearrangements detected with this probe (30-32, 40). With the
use of amplification of the translocation junction in both CML
and ALL with the polymerase chain reaction (PCR) technique,
one can monitor the clinical status of patients and can detect
the very few abnormal cells in early relapse samples (49).
Moreover, as indicated earlier, it is possible to identify the
precise region of the breakpoint in the BCR gene and to
correlate this information with a variety of clinical indicators
to improve our ability to distinguish patients who will not
respond or who will have only a very short response to standard
treatment.
Specificity of Translocations
Let me return again to the specificity of chromosome translocations which is, I believe, a very significant observation
despite the quandry posed by the apparent identity of the 9;22
translocation in CML and in some Ph1 positive ALL patients.
The data obtained from the analysis of the lymphoid leukemias
and lymphomas have provided insights which I assume will be
applicable to the other recurring translocations. The mechanism
or mechanisms by which the specific translocation occur are
unknown. Evidence in some lymphoid tumors suggests that the
recombinase involved in normal immunoglobulin and T-cell
receptor gene rearrangements may play a role (13, 50, 51).
Sequencing of the normal homologues of some of the chromo
somes involved in the translocation in both B- and T-cell
neoplasms has revealed the presence of the heptamer-nonamer
sequences that are involved in recombination. This has led to
the proposal that, in at least some instances, the translocation
has occurred because of an error in the normal gene re
arrangement mechanism in lymphoid cells. In some T-cell
tumors, only the heptamer sequences that are 5' of the other
gene, e.g., MYC in the t(8;14)(q24;ql 1) in the SKW3 cell line,
have been identified. In other situations there is no evidence
that the recombinase system is involved at all (13). However,
these aberrant rearrangements would not be seen as a clonal
proliferation if they did not provide the involved cells with a
selective proliferative advantage vis-Ã -visunaffected cells in the
same tissue. Possibly some chromosome rearrangements occur
by chance in a cell that has some other genetic change leading
to a malignant phenotype. It is difficult to imagine that these
chance events would occur repeatedly in a number of independ
ent translocations. However, the recent studies of Tycko et al.
(52) show that chimeric rearrangements involving the V and J
segments of the TCRD and TCRG receptors can be detected in
normal individuals using PCR. These data confirm cytogenetic
observations made years before (1975) that translocations in
volving 14ql 1 and either 7pl5 or 7q34-35 are a recurring albeit
rare event (10~3 to 10~4/cell) in normal individuals (53-55).
Three groups reported a total of 12 unrelated individuals who
had a 7; 14 translocation in phytohemagglutinin
stimulated
lymphocyte cultures (53-55). All 12 had a break in 14qll; 6
had a 7q34-35 breakpoint, and 6 had a break identified as 7pl3,
which is more likely to be 7pl5. Thus in these individuals, it
would appear that there was nonhomologous recombination
involving TCRA and either TCRB or TCRG. These same
changes occur with much higher frequency in patients with
ataxia-telangiectasia (56, 57).
The question related to the interaction of the two genes
involved in chromosome translocations can be answered by an
analysis of the two different translocations involving the MYC
protooncogene in B-cell and T-cell tumors. The first translo
cation, cloned in 1982 independently by Leder and Croce and
their colleagues, was the 8; 14 translocation in Burkitt's lymphoma which involved the immunoglobulin heavy chain gene
at 14q32 and MYC at 8q24 (24, 25). Breakpoints in the variant
Burkitt's translocations involving the immunoglobulin light
chain genes, K at 2pl2 and X at 22ql 1, and MYC were also
cloned (58, 59). More recently, we and others have cloned an
analogous translocation in T-cell leukemias (50, 60-62). This
is also an 8; 14 translocation with the break in chromosome 8
near the MYC gene; the break in No. 14 is in the proximal
region in band 14ql 1 which is the location of the gene encoding
the a/5 chain of the T-cell receptor (TCRA/TCRD). In this
situation, as in the variant Burkitt's translocations, MYC re
mains on No. 8 and the J region of TCRA is translocated to
the 3' end of the MYC gene and is a variable distance 3' of the
3rd exon of MYC. With very rare exceptions, the MYC-immunoglobulin rearrangements are seen in B-cell tumors and the
MYC-TCRA rearrangements are seen in T-cell tumors. We
recently described an unusual patient with a B-cell lymphoma,
with an 8; 14 translocation involving 14ql 1 and 8q24 in whom
the breakpoint in chromosome 14 was in the J region of TCRA
(63). As far as is known at present, the MYC alterations are
similar if not identical in both B- and T-cell tumors. Therefore,
the specificity of the rearrangements is associated with the gene
that is specifically active in the particular cell type, clearly
immunoglobulins in B-cells and T-cell receptor genes in T-cells.
A reasonable paradigm is that translocations bring together, in
an aberrant relationship, a gene intimately involved in regulat
ing growth (a protooncogene in the examples defined to date)
adjacent to an active cell specific gene. The active cell specific
gene in turn causes the inappropriate expression of the growth
regulating gene.
A number of laboratories are actively investigating the many
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factors related to gene expression that interact with each other
as well as with positive and negative regulatory sequences in
the 5' region of genes. Some of the factors that activate genes
show relatively ubiquitous expression; others have an extremely
restricted expression (64).
It has been rather puzzling that some recurring transloca
tions, identified primarily in B-cells, do not appear to involve
an expressed gene. The first example of this was the BCL1
putative oncogene identified on chromosome 11 in the 11;14
translocation [t(l I;14)(ql3;q32)] in B-cell chronic lymphatic
leukemia (65). Extensive searches for an expressed gene on
I Iql3 have been unsuccessful. One possible explanation is that
the gene has a very limited range of expression either in a
restricted cell type or stage in differentiation. Another expla
nation is related to chromosome structure. Rabbitts and his
colleagues have recently analyzed a different breakpoint on
I1 p 13, called TCL2 in leukemias with a t( 11;14)(p 13;q 11), and
found that the breaks in all 10 cases occurred within a range of
less than 25 kilobases in band Ilpl3 and that 8 of them
clustered within 6.7 kilobases (66). DNA sequence analysis of
both 1Ipl3 and 1Iql3 breakpoints revealed alternating purinepyrimidine sequences suggestive of potential Z-DN A. In 11p 13,
the purine-pyrimidine region consists of a run of 62 alternating
T-G residues; it is 160 base pairs from the breakpoint in one
tumor cell line and within 900 base pairs in other tumors. The
region near the Ilql3 breakpoint seems to be larger and has
an unusual repeating series of alternating T-G residues (67). It
is proposed that the location of potential regions of Z-DNA
near to translocation breakpoints could make the chromatin
accessible to site specific recombinases in the absence of tran
scription of nearby genes. How this rearrangement would pro
vide a growth potential to the affected cell is unclear.
It should be emphasized that many of the protooncogenes
were identified in viruses that cause tumors. However, these
genes have not been conserved through evolution from yeast
and Drosophila to the chicken, mouse, and humans to cause
cancer. Where we have any insight into the function of these
genes in normal cells, they are growth factors, growth factor
receptors, or DNA binding proteins. It is not unexpected that
the genes which a virus might coopt if it developed into a tumor
producing virus would be genes that control proliferation, genes
which under viral regulation would function abnormally with
regard to cell growth. It is unfortunate that these genes were
first identified because their mutant forms were associated with
cancer. The term "oncogene" is too short and easy for it to be
discarded, but it really refers to respectable genes for regulating
cell growth.
Up to now, I have extolled the importance of chromosome
aberrations as powerful tools to locate genes that are central to
the process of malignant transformation. Although I have con
centrated on hematological malignant diseases, it is clear from
the remarkable progress that has been made in cloning genes
in solid tumors, both the RBI gene in retinoblastoma and a
series of genes in colon cancer, that knowing the location of
cytogenetic abnormalities has been central to this progress.
I will conclude with some comments regarding the longerterm potential impact of discovering new genes via chromosome
rearrangements. Once these genes are identified, many previ
ously unknown, BCR, e.g., they become the focus of very active
investigation. Scientists try to find answers regarding the func
tion of these genes in normal cells; how are they altered by the
chromosome rearrangements, and how does this relate to ma
lignant transformation? The questions are endless. The answers
will provide insights into cell biology that have very profound
implications. Within the next decade or two, we should be able
to define the major genetic abnormalities in many types of
cancer and to identify the specific changes in the tumor cells of
many patients. For most leukemias, lymphomas, and sarcomas,
unique chromosome changes often are associated with a partic
ular subtype of these neoplasms.
Cloning of the genes involved in these chromosome changes
will provide specific DNA markers that will have diagnostic
importance. For some solid tumors, on the other hand, current
evidence suggests that deletions of the same chromosome region
may occur in different types of tumors, such as the deletions of
13q in retinoblastoma, osteosarcoma (not secondary to radia
tion for retinoblastoma), breast cancer, and lung cancer. (Re
viewed in Ref. 68.) The deletion of the same region does not
necessarily imply that the same gene is involved or that the
change within the gene is identical; witness the fact that two
different translocations in band 22ql 1 involve different genes,
namely, the X light chain gene in the 8;22 translocation in
Burkitt's lymphoma and the BCR gene in CML; furthermore,
the breakpoints within BCR in Ph1 positive leukemia are also
somewhat variable.
The multistep process of malignant transformation is com
plex. In the leukemias and lymphomas, we often see specific
chromosome translocations combined with loss or gain of par
ticular chromosome segments. Some combination of alterations
in dominant!) acting protooncogenes and in recessively acting
tumor suppressor genes certainly acts synergistically to enhance
the malignant phenotype.
In the future, the precise definition of the genetic changes in
the malignant cells of a patient will be used to select the most
appropriate treatment for cells with these genetic defects. This
treatment will be less toxic for the normal cells in the patient.
Moreover, this genetic profile may allow monitoring of the
patient's course and early detection of relapse. These same
genetic markers may be used to detect the involvement of other
tissues such as bone marrow, spleen, or lymph nodes, for
example. These changes in treatment strategies will clearly
benefit the patient. Of more general scientific importance,
however, will be the identification of dozens of genes, many
hitherto unknown, that can be used to study the complex
process of the regulation of cell growth and differentiation. This
development may be the most significant result of our success
in understanding the genetic changes that occur in cancer cells.
I would like to conclude with a more personal note based on
my continuing amazement at the interrelatedness of the
biomédicalsciences. This should be no surprise to me, but it is.
Many investigators have found that a successful system for
carrying out some function in primitive organisms has evolved
and then this system is used repeatedly with varying modifica
tions as the organisms become more complex. As a cytogeneticist, I had to learn something about the cell cycle and DNA
replication, about chromosome structure, and about various
agents that can alter both of these. More recently, I have had
to become an amateur tumor virologist at least with regard to
the action of viral oncogenes and their cellular counterparts,
the protooncogenes. With the cloning of translocations, espe
cially some of the recent ones, a knowledgeable cancer cytogeneticist must understand cell cycle control genes in yeast (cdclO
and SW14 and 6) and developmentally regulated genes in Dro
sophila (Notch for example) and nematodes (lin 10, gip 1). My
colleague, Timothy McKeithan, has recently cloned the BCL3
gene that is involved in the 14; 19 translocation in B-cell leu
kemias (69). If I am to understand the possible roles this gene
plays in B-cell transformation, I must understand how its
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MOLECULAR CYTOGENETICS
homology to portions of the cdclO and Notch genes might
provides clues as to its functions in both normal and malignant
cells. As more genes involved in translocations and deletions
are defined, many of us in cancer research will continue to have
to "go back to school" to be able to incorporate the knowledge
provided by molecular geneticists and cell biologists into our
concepts of carcinogenesis. The golden ages in biology and in
medicine are nourishing one another as never before in history.
Acknowledgments
My colleagues, Drs. Manual Diaz, Michelle Le Beau, and Timothy
McKeithan, have been generous with their review of the manuscript
and with advice on the construction of Fig. 1. I acknowledge the expert
secretarial assistance of Felecia Stokes.
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Molecular Cytogenetics: Rosetta Stone for Understanding
Cancer−−Twenty-ninth G. H. A. Clowes Memorial Award Lecture
Janet D. Rowley
Cancer Res 1990;50:3816-3825.
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