A historical outline of the development of cancer cytogenetics

A Historical Outline of the Development of Cancer Cytogenetics T. C. H s u

INTRODUCTION For decades, biologists have suspected that cancer m a y be initiated by a change in the genetic c o m p o s i t i o n of the somatic cell. In pathologic preparations, cancer tissues often exhibit a high frequency of abnormal cell division figures; so the chromosomes, hence, the genetic constitution, of the cancer cells must be abnormal. However, no one knew w h e t h e r the abnormalities were the cause or the consequence of neoplasia, and if the abnormalities were the cause of cancer, w h e t h e r or not they were specific. The earliest hypothesis regarding the origin of cancer in relation to cellular genetics was p r o p o s e d by Boveri in 1914 [1]. The Boveri hypothesis can be briefly p a r a p h r a s e d as follows. 1. Malignant cells are derived from normal tissue cells 2. The cause of the abnormal behavior lies within the t u m o r cell itself, and not in its e n v i r o n m e n t 3. The t u m o r cell is a defective cell in the sense that it has lost the properties of a normal cell 4. Typically, each tumor cell takes its origin from a single cell 5. The malignant cell is one with certain abnormal chromatin content Each process that brings about this variable chromatin constitution w o u l d result in the origin of a malignant tumor. During the ensuing four decades, this hypothesis was not tested because of technical difficulties in analyzing mutations and c h r o m o s o m e s of somatic cells of vertebrates. Cytogenetic investigations on vertebrates, especially mammals, gave such inaccurate results that until 1956 even the d i p l o i d c h r o m o s o m e n u m b e r of man was erroneously d e t e r m i n e d [2, 3]. In the early period, cytologic investigations of cancer concentrated on recording mitotic abnormalities, but such studies a d d e d little to our k n o w l e d g e of the genetic c o m p o s i t i o n and c h r o m o s o m e constitution of tumor cells. The field of cancer cytogenetics grew slowly. Even after several technical breakthroughs and conceptual advances, progress was not rapid until recent years, when other d i s c i p l i n e s of b i o m e d i c a l science interfed with cytogenetics. In chronicling the d e v e l o p m e n t of cancer cytogenetics, I do not intend to present in detail topics that belong to h u m a n and m a m m a l i a n cytogenetics in general, because these subjects have been reviewed in a n u m b e r of articles, including books. However, it is From the Section of Cellular Genetics, University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, TX.

Address requests f o r reprints to Dr. T. C. H s u , Section of Cellular Genetics, University of T e x a s M. D. Anderson Hospital and T u m o r Institute, H o u s t o n , TX 77030.

5 © 1987 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017

Cancer Genet Cytogenet 28:5-26(1987) 0165-4608/87/$03.50

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T . C . Hsu necessary to mention some of them because they made vital contributions to the d e v e l o p m e n t of cancer genetics. Readers may consult two references [3, 4] for more details. For convenience, I shall divide the studies on cancer cytogenetics into three overlapping periods: the p r e h y p o t o n i c period, the p o s t h y p o t o n i c period, and the p o s t b a n d i n g (modern) period. It can be seen that m u c h critical information was gathered after the invention of the b a n d i n g procedures during the early 1970s. Even so, activities in c h r o m o s o m e studies on cancer, particularly on solid tumors, have become intensive only during the last few years. The primary purpose of cancer cytogenetic investigations is to find evidence for the etiology of cancer (the Boveri concept), and that each type of cancer is initiated by a specific genetic change or changes. We can say that the explosive research activities in cancer cytogenetics have clarified a n u m b e r of vexing problems, but the field still has a long way to go. For a fossilized s p e c i m e n like myself who has experienced, through the years, frustration in dealing with this subject, it is i n d e e d gratifying to witness the advancement of late. By and large, m o d e r n - d a y information supports the basic concept of Boveri, but it offers more precise definitions and modifications.

THE PREHYPOTONIC PERIOD

Histological preparations of h u m a n and m a m m a l i a n tumors can u s u a l l y reveal that the tumor cells have larger chromatin masses than normal cells, both in mitotic stages and in interphase nuclei. In mitotic cells, abnormal division figures, including scattered c h r o m o s o m e s at metaphases, multipolarity, and abnormal anaphases, are more c o m m o n than in normal tissues. A p p l i c a t i o n of the squash technique did not significantly help investigations in m a m m a l i a n cytology. Although the squash method can force metaphase plates to flatten from a t h r e e - d i m e n s i o n a l orientation to a more or less t w o - d i m e n s i o n a l configuration, the chromosomes were still crowded at the equatorial plates, so that observations on i n d i v i d u a l chromosomes were still extremely difficult. One may scan h u n d r e d s of metaphase figures and observe one whose c h r o m o s o m e s are barely countable. Probably the earliest attempt to study the cytological p h e n o m e n a of tumors was m a d e by Biesele et al. [5], who used several transplantable tumors and methylchola n t h r e n e - i n d u c e d primary tumors of C3H mice. The preparations were made by the acetic carmine squash method. A few camera l u c i d a drawings of metaphases were presented in that article, but it was not possible to accurately count the chromosomes from the drawings (Fig. 1). The authors e m p h a s i z e d the variation in the nuclear volume and the large size of the chromosomes of the neoplastic cells, interpreting the latter p h e n o m e n o n as representing polytene chromosomes. A p p a r e n t l y such " p o l y t e n e c h r o m o s o m e s " represented the product of what is now k n o w n as e n d o r e d u p l i c a t e d c h r o m o s o m e s (see the last metaphase in Fig. 1). One of the difficulties of studying the cytology of solid tissues, including tumor tissues, is that the cells are not dissociated into a suspension. Even if the tissue is cut into small fragments prior to making squash preparations, the tissue fragments still contain t h o u s a n d s of cells in a mass. Inoculation of transplantable tumors into the peritoneal cavity of susceptible animals to obtain the ascites form of tumors greatly facilitated the p r o c u r e m e n t of s u s p e n d e d single or small c l u m p s of cells, w h i c h were more amenable to cytologic investigation. The earliest attempts at converting solid rat sarcomas to ascites tumors were carried out in Japan, and Makino et al. [6-9] studied the c h r o m o s o m e s of these tumors. These investigators found several significant cytologic characteristics in Yoshida sarcoma and several other rat tumors (MTK sarcomas, Hirosaki sarcomas): a) the c h r o m o s o m e constitution of

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Fig. 26. Fig. 27. Fig. 28. Fig. 29. Fig. 30. Fig. 31. Fig. 32. Fig. 33. Fig. 34.

r IG. 33. FIG. 34.. Metaphase of Fetal Liver. 41 (?) Chromosomes. Average Volume 0.46/z3. Metaphase of Healing Ear Wound. 40 Chromosomes. Average Volume 0.52 ~3. Metaphase in Muscle Cell in Methylcholanthrene Tumor. 40 Chromosomes. Average Volume 0.42 ~3. Metaphase in Methylcholanthrene Tumor. 38 Chromosomes. Average Volume 1.2 ~3. Metaphase in Methylcholanthrene Tumor. 86 Chromosomes. Average Volume 1.0 ~3. Metaphase in Transplant of Spontaneous Tumor. 138 Chromosomes. Average Volume 1.2/z3. Metaphase in Tumor XG. 40 Chromosomes. Average Volume 2.4#5. Metaphase in Transplant of Spontaneous Tumor. 44 Chromosomes. Average Volume 2.2 ~3. Metaphase in Methylcholanthrene Tumor. 39 Chromosomes. Average Volume 6.2 ~3

F i g u r e 1 C a m e r a l u c i d a d r a w i n g s of n o r m a l a n d n e o p l a s t i c c h r o m o s o m e s . Reproduced from Biesele et al. [51 with permission.

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T.C. Hsu the tumor cells was distinctly n o n d i p l o i d (in the Norwegian rat, the d i p l o i d number is 42), and the c h r o m o s o m e n u m b e r m a y vary within a population, fluctuating around a m o d a l number. The m o d a l c h r o m o s o m e numbers of m a n y tumors were subdiploid, e.g., 39-41 for the Yoshida sarcoma, 37-39 for the MTK sarcoma, and 38-40 for the Hirosaki sarcoma. Other tumors had modal c h r o m o s o m e numbers between d i p l o i d y and tetraploidy, e.g., Usubuchi sarcoma (70-76) and Takeda sarcoma (84). b) The karyotype of the normal rat does not contain large metacentric chromosomes, but in the t u m o r cells, at least one large metacentric was found. Such abnormal chromosomes (now k n o w n as marker chromosomes) were apparently formed by Robertsonian fusions or m u l t i p l e translocations or both. In the United States, Hauschka and Levan [10-12], working on the chromosomes of several ascites tumors of the laboratory mouse, observed similar phenomena, i.e., each t u m o r had its characteristic c h r o m o s o m e constitution that differed from that of the normal mouse. I n d e p e n d e n t l y and almost simultaneously, Makino and Hauschka and Levan p r o p o s e d the stemline concept of cancer, i.e., the chromosome constitutions of cancer cells are not normal, and each tumor has its own characteristic cytologic features, a p p a r e n t l y selected as the most adaptive genome from a cell population. In other words, the stemline represents a clone from w h i c h more genetic variations m a y be generated for further selection. Even if one had never seen the original cancer cell, the data a p p e a r e d to i m p l y that it too may be a genetically aberrant cell.

THE POSTHYPOTONIC A N D PREBANDING PERIOD

Use of a h y p o t o n i c solution pretreatment m e t h o d [13, 14] has greatly facilitated studies on h u m a n and m a m m a l i a n cytogenetics because metaphase chromosomes can n o w be separated, and the gross m o r p h o l o g y of each chromosome can be critically delineated. Advances in tissue culture techniques also significantly contributed to investigations of cytogenetics, both normal and neoplastic. During this period, several important discoveries were m a d e as the foundation on w h i c h cancer cytogenetics was built. The most important discovery was the correction of the d i p l o i d n u m b e r of h u m a n s by Tjio and Levan in 1956 [15]. Lejeune's finding [16] that Down's s y n d r o m e was associated with a congenital aneup l o i d y stimulated a flurry of research activities on h u m a n syndromes to establish the relationship b e t w e e n d e v e l o p m e n t a l errors and a n e u p l o i d y in humans. Of course finding a n e u p l o i d i n d i v i d u a l s was not new. Plant cytogeneticists had described the effects of a n e u p l o i d y on growth and m o r p h o l o g y in several species; but a p p l i c a t i o n of chromosomal abnormalities to m e d i c i n e represented a new approach to h u m a n genetics. Another very important contribution relating to h u m a n cytogenetics was made by Nowell [17], who found that p h y t o h e m a g g l u t i n i n (PHA) could stimulate lymphocytes of p e r i p h e r a l blood to revert to the blast stage in short-term cultures. This finding was quickly a d o p t e d by Moorhead et al. [18] specifically for h u m a n cytogenetics work, and it has become an i n d i s p e n s a b l e tool in any laboratory involved in studies on h u m a n chromosomes. The field of cancer cytogenetics suffered some controversy in the late 1950s as claims were made that c o m p l e t e l y n o r m a l karyotypes were recorded for a variety of primary tumors in humans, mouse, cattle, and chicken. Many cytogeneticists (myself included) were skeptical about such a conclusion, as merely counting the chromosome number could lead to serious mistakes because m a n y subtle c h r o m o s o m e aberrations could not be positively detected by the methodology existing at that time. In a review on cell populations, I presented two photographs of a normal mouse metaphase, one of w h i c h had artificial deletions on three chromosomes cre-

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ated by darkroom manipulations, and the other photograph was the intact original [19]. O n l y by critical c o m p a r i s o n of the two could one tell the difference. This was perhaps a dramatic demonstration of the i n a d e q u a c y of using crude observations to determine the n o r m a l c y of a karyotype, especially in animal species whose chromosomes are similar in m o r p h o l o g y such as those of the cattle, the dog, and the mouse. Nevertheless, the question r e m a i n e d whether or not chromosome abnormalities in neoplastic cells had any meaning in relation to the origin of cancer. It seemed unreasonable, even at that time, that any r a n d o m change, numerical or structural, can cause a cell to become neoplastic, yet the available methodology was not able to detect c h r o m o s o m e aberrations with accuracy. The discovery by Nowell and Hungerford [20] of a shortened G group chromosome in h u m a n chronic myelogenous leukemia (CML) became a milestone in the chronicle of cancer cytogenetics because it pointed out, for the first time, that a specific c h r o m o s o m e change was associated with a specific neoplasm. Confirmation quickly came from several countries, and this particular marker chromosome became k n o w n as the P h i l a d e l p h i a (Ph) chromosome. Because there was no method for differentiating the two pairs of G group chromosomes (#21 and #22), the Ph c h r o m o s o m e was thought to be chromosome #21 with a terminal deletion. The reason that CML became a m o d e l case in cancer cytogenetics was the fact that CML cells were p r e d o m i n a n t l y diploid, and the Ph c h r o m o s o m e was, in the great majority of cases, the only anomaly. Because of this clear-cut p h e n o m e n o n , the "deletion" was regarded by m a n y cytogeneticists as the strongest evidence to link genetic alteration and the etiology of cancer. However, at the early stage of studies on the Ph chromosome, there was still some confusion, viz., whether every leukemic cell of CML contained this anomaly. Using cultured buffy coat stimulated by PHA, only a certain proportion of metaphases was found to exhibit the Ph c h r o m o s o m e in 3-day cultures. After experimenting with 2-day cultures and cultures without PHA, all mitoses showed the Ph chromosome [21]. These results indicated that PHA stimulated the growth of lymphocytes that had a normal c h r o m o s o m e complement, but it usually took about 3 days in culture for the stimulated l y m p h o c y t e s to enter mitosis. The CML cells were able to grow in culture without PHA and enter mitosis after 2 days in culture. These experiments not only clarified the confusion but also suggested that the anomaly occurred only in the myelocytic series, not the l y m p h a t i c series. In a d d i t i o n to CML, some other neoplasms were also explored by cytogeneticists during this era. Specific c h r o m o s o m e abnormalities were found in meningiomas [22] in w h i c h one G group c h r o m o s o m e was absent, and in neurogenic tumors [23, 24] in w h i c h there were n u m e r o u s round, paired chromatin bodies among seemingly normal metaphases. These particles have been k n o w n as double minutes (DM), but their nature and function were not known. In other leukemias and lymphomas, the cytogenetic pictures were not as clear as that of CML, but various other chromosome anomalies, instead of the Ph chromosome, were found. THE POST-BANDING (MODERN) PERIOD Without the b a n d i n g techniques d e v e l o p e d in the late 1960s and early 1970s, human and m a m m a l i a n cytogenetics, i n c l u d i n g cancer cytogenetics, w o u l d have rem a i n e d in the dark ages. In fact, without advances in cytogenetics, d e v e l o p m e n t in m a n y disciplines of b i o m e d i c a l science w o u l d have slowed down also. Of course the benefit has been mutual; cytogenetics, particularly cancer cytogenetics, has used extensively the information collected in the fields of biochemistry, molecular biology, cell biology, and m a n y branches of m o d e r n h u m a n genetics. There are a n u m b e r of b a n d i n g procedures in use, but the most valuable ones

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are still the original four listed in the Paris Conference [25]: Q-band, C-band, Gband, and R-band. In the field of cancer cytogenetics, c h r o m o s o m e banding, particularly the high-resolution b a n d i n g procedure advocated by Yunis [26], has rendered i n d i s p e n s i b l e assistance in solving a number of nagging problems and has helped in the formulation of new concepts in the genetic etiology of cancer.

Specific Chromosome Alterations in Specific Neoplasms The most important a c c o m p l i s h m e n t in cancer cytogenetics in the postbanding period should be the establishment of evidence for the concept of specific chromosome alterations in specific neoplasms. If a specific cancer has no specific genetic changes, then the entire field of cancer cytogenetics has no great value and is not worth pursuing with vigor. Fortunately, cancer investigators have found, in several types of neoplasms, not only cytogenetic specificity, but also explanations for the specificity. Because of the relatively simple chromosomal alterations in hematologic neoplasms and a few solid tumors, concepts of genetic etiology of cancer became clearer, but a p p l i c a t i o n of these concepts to the vast majority of solid tumors was still h a n d i c a p p e d by several factors, particularly the c o m p l e x cytogenetic changes in practically all samples observed. Although a considerable amount of information regarding cytogenetics of solid tumors was available in the 1970s, and although c o m p r e h e n s i v e collection and cataloging of the data were made through the efforts of Sandberg [27] and Mitelman [28, 29], the information still represented a confusing mess. During recent years, however, cytogeneticists began to search, through kaleidoscopic variations of c h r o m o s o m a l alterations, for c h r o m o s o m e abnormalities shared by tumor specimens of the same pathologic type (or the "colnmon cause"), and began to realize that tumors classified by pathologists as one type may actually represent several different genetic origins. This was learned some time ago in studies on CML that less than 10% of CML cases did not exhibit the Ph chromosome. The clinic behavior of these "aberrant" CML cases suggested that CML identified by hematologic criteria may actually contain more than one genetic origin. Solid tumors m a y be just as complex, if not more so. A n o t h e r realization, also arrived at in studies on hematologic neoplasms, is that a crucial rearrangement, such as a translocation, does not necessarily involve the same two chromosomes. For example, the majority of Burkitt's l y m p h o m a s exhibited t(8;14), but t(2;8) and t(8;22) were also found. This c o m p o u n d s the task of cytogeneticists who study solid tumors, but such a realization helps to avoid frustration because disturbance of one critical gene may be vital whereas the partner chromosome involved in exchanges may be either immaterial or have secondary importance. One of the excellent examples for the specificity of c h r o m o s o m e alterations in specific tumors was found in the T-cell l y m p h o m a s of the mouse. During the nonb a n d i n g days, Stich [30] i n d u c e d leukemia in low-leukemic Swiss mice by injecting 7,12-dimethylbenz(a)anthracene (DMBA) into newborns. Of the 16 mice that developed thymic l y m p h o s a r c o m a s , 15 showed a stemline of 41 (2n + 1) chromosomes. Unfortunately, at that time there was no way to determine if the trisomy was of the same chromosome. Subsequent b a n d i n g studies by several groups of investigators showed that all m u r i n e T-cell leukemias, spontaneous as well as i n d u c e d by various carcinogens, had a c h r o m o s o m e constitution of trisomy 15 or partial trisomy 15 [31-34]. In animals with a Robertsonian fusion between chromosomes #1 and #15 or between chromosomes # 6 and #15, the leukemic cells again showed trisomy 15, but they also had trisomy 1 or 6, respectively [35]. Chromosomes #3 and # 6 were not suspected of being etiologically related to the T-cell function; they

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were trisomic merely because they were fused with #15. These data emphasized the importance of chromosome #15 in maintaining the function of normal T-cell lymphocytes. An additional #15 in such cells upsets the genetic balance of murine T-cells such that the cells become neoplastic. Of special interest was the discovery [36] that, instead of trisomy 15, mouse plasmacytomas exhibited specific translocations, t(15;6) or t(15;12). Genetic studies have located the heavy chain of the murine immunoglobulin gene on chromosome #12, and the light chain gene on chromosome #6. These nonrandom chromosome alterations in mouse plasmacytoma constituted an excellent testimony for the genetic etiology of cancer because they confirmed that disruption of genes vital to the function of the cell type causes malfunction of the cell. In human neoplasms, several examples emphatically support the generalization that specific chromosomal alterations are associated with specific cancers. The best case is the Burkitt lymphoma in which an exchange of the terminal segments between chromosomes #8 and #14 was first described by Zech et al. [37]. Because the exchanged segments are similar in length, this translocation can be unequivocally identified only by chromosome banding. The important lesson we learned from Burkitt's lymphoma is that chromosome rearrangements (in this case, a translocation) must be specific, not only with regard to the chromosomes involved, but also to the breakpoints. The breakpoint in chromosome #14 is at the gene coded for the heavy chain of immunoglobulin and that in #8 is at the locus of the c - m y c oncogenel Apparently chromosome breakage at these gene loci cause disturbances in both genes and the functions of these genes become altered, resulting in malignancy. Subsequent studies by various investigators confirmed the finding, but added that the translocation was not limited to t(8;14). Cases of Burkitt lymphoma with t(8;2) and t(8;22) also have been found. All of these three chromosomes, #2, #14, and #22, are involved in the production of immunoglobulin. The chromosome aberrations in Burkitt lymphoma also implied that the same aberration, when occurred in cells of tissues other than the B cells, would cause no malignant changes. Cytogenetic data collected thus far suggest that, in human neoplasms at least, specific chromosome rearrangements may be more prevalent than trisomy. In CML, for example, soon after the banding technique became available, the Ph chromosome was found to be chromosome #22, but instead of the deletion, the bulk of 22q was translocated to chromosome #9 [38]. In both Burkitt lymphoma and CML, the genetic expression should be regarded as dominant, because in each somatic cell there should be at least two gene copies, one in each homologous chromosome. The genetic alteration affects only one of the two alleles. There has been no evidence that any mutational event occurred in the homologous allele.

The Two-Step Mutation Hypothesis and Its Cytogenetic Evidence Retinoblastoma can be hereditary or nonhereditary. The hereditary type may develop more than one tumor (bilateral), and the average age at onset is much earlier than that for the nonhereditary type, which usually has a unilateral development. From statistical analyses of family and clinical data, Knudson [39] hypothesized that mutation at a specific gene locus is responsible for developing a specific tumor and that two mutations are required to accomplish the process of oncogenesis. The hereditary type has one mutation transmitted germinally, so only one additional mutation in the homologous allele is required to develop retinoblastoma, resulting in earlier onset of the disease and multiple tumor loci. The nonhereditary type requires two mutational events, one in each target gene, to occur postzygotically. In other words, a gene, presumably vital to the normal development of the retina, has

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two copies in each diploid somatic cell. When one mutated gene is transmitted prezygotically, it is not enough to change the physiology because the normal allele in the homologous chromosome still functions. However, the hereditary mutation accomplishes the first step, i.e., only one postnatal mutational event in the normal allele is sufficient to transform a retinoblast into a malignant one. In the nonhereditary type, both genes are normal at birth. It will require two separate mutations, one on each chromosome, to cause malignancy. Therefore, the onset of the nonhereditary type of retinoblastoma is considerably delayed. According to the classic definition of inheritance, the hereditary type of retinoblastoma has a d o m i n a n t mode of inheritance. However, if K n u d s o n ' s two-step mutation hypothesis is valid, development of retinoblastoma is in reality recessive because both genes must be mutated [40]. This, of course, is only a semantic point. The significance of this hypothesis is that the same principle may be applicable to other heritable (as well as nonheritable) cancers as a generalized theory. Indeed, the second case, the Wilms' tumor, was found to almost completely mimic the retinoblastoma story [41]. Familial and bilateral Wilms' tumor have an early average age of diagnosis and a distribution pattern suggestive of a single-event process, whereas, unilateral and unselected cases do not. Association of hereditary Wilms' tumor with other phenotypes, especially aniridia, is of particular value because these other phenotypes support the mutation hypothesis [42]. K n u d s o n and Strong [41] offered two alternative explanations: a) the mutation causing aniridia could be an u n u s u a l aniridia allele that also results in Wilms' tumor, or b) the mutation could be a chromosomal abnormality involving more than one genetic locus. The discovery of the 13q interstitial deletion in retinoblastoma [43] and the l l p interstitial deletion in aniridia Wilms' tumor [44] gave strong support to K n u d s o n ' s hypothesis. An interstitial deletion of a chromosome segment that contains a gene vital to the normal function of a tissue creates a haploid condition, leaving only one allele in the homologous chromosome to perform its function. When that allele mutates, neoplasm of that tissue results. Of course, deletion is not a prerequisite for these cancers. Mutations occurring in both alleles can yield the same result (and, indeed, many retinoblastoma cases were found without the deletion), but finding this specific deletion in a specific cancer definitely added credence to the basic concept that cancer is initiated via a genetic change in a somatic cell. Molecular genetic studies on the loss of heterozygosity of genes located in the deleted segment(s) have confirmed such a supposition. All available evidence appears to endorse the Boveri hypothesis but with much more precise definitions. In a way, these deletions were lucky breaks for cancer geneticists because chromosomal abnormalities are at least easier to detect and to verify than gene mutations. From cases of retinoblastoma and Wilms' tumor with the deletion, one can infer that a mutation occurring at the critical gene locus is equivalent to a deletion, because that gene no longer performs its function. But if a chromosome aberration repeatedly occurs at a particular locus in one type of tumor cells, even if this aberration is not observed in all cases, there is a strong likelihood that this locus contains a gene responsible for the initiation of that neoplasm. Intensive research activities in cancer genetics have yielded preliminary findings suggesting that other neoplasms may also have specific genetic lesions [45]. In the not too distant future, characteristic genetic and/or cytogenetic lesions will be established for solid tumors other than retinoblastoma and Wilms' tumor.

Inherited Chromosomal Alterations and Cancer Predisposition The existance of familial breast and colon cancers, for example, has been recognized for a long time. Without k n o w i n g the basic genetic lesions of these cancers, however, finding the target gene at the molecular level is a laborious and perhaps

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u n r e w a r d i n g endeavor. If a specific cytogenetic lesion can be found in a specific neoplasm, such as the one in retinoblastoma, then analysis of constitutional lesions within a family becomes feasible. In fact, some c h i l d r e n with retinoblastoma have a congenital 13q deletion [46]. Such a deletion can be inherited from a parent with a c h r o m o s o m a l rearrangement, e.g., an insertion of the 13q segment into another c h r o m o s o m e [47]. The unfortunate zygote (the proband) received the defective #13 but not the c h r o m o s o m e with the insertion, thus, p r e d i s p o s i n g the child with the lesion to develop retinoblastoma. Cytogenetic studies of familial cancers in w h i c h chromosomal aberrations are present m a y be extremely useful for throwing some light on the genetic lesions associated with a particular neoplasm. Perhaps the best example is still the congenital t(3;8) found in cultured l y m p h o c y t e s of family members with hereditary renal cell carcinoma [48]. This congenital translocation was found in all members of the family with renal cell carcinoma and in three n o n s y m p t o m a t i c members at the time of cytogenetic analysis. These three persons, considered to be the at-risk i n d i v i d u als, subsequently d e v e l o p e d the same tumor. This particular incident stimulated a search of the c h r o m o s o m e constitutions of renal cell carcinomas because there were now specific changes to look for in these particular chromosomes to determine if one or both chromosomes might be responsible for the etiology of that cancer or if the aberrations found in that family were not related to the n e o p l a s m in question. Indeed, cytogenetic analyses of a n u m b e r of renal cell carcinomas [49, 50] revealed that all tumor cells had a 3p aberration and that the breakpoint was the same (3p14), whereas, the translocation partner was not limited to c h r o m o s o m e #8. This finding p i n p o i n t e d a gene or genes, located in the 3p14 band region as vital to renal cell function. A translocation with a breakpoint in this band, w h e t h e r transmitted from the parental generation or occurring s p o n t a n e o u s l y in the renal cells, once again, causes a h a p l o i d constitution as far as that gene is concerned. The a p p r o a c h of examining c h r o m o s o m e rearrangements of both the tumor cells and the normal cells of the same patients is useful, as long as one recognizes the fact that constitutional c h r o m o s o m e aberrations are not always present.

Gene Mapping In classic h u m a n genetics, statistical analyses of family pedigrees with special phenotypes constituted the p r e d o m i n a n t m e t h o d for studies on h u m a n heredity. Cytogenetic investigations on congenital s y n d r o m e s c o n d u c t e d in the late 1950s and the 1960s represented an entirely n e w approach. However, without knowing the genes responsible for each p h e n o t y p i c expression, the findings (e.g., trisomy 21 for Down's syndrome) were still empirical. Thus, the ability of locating genes in chromosomes of a species should revolutionize m a m m a l i a n , particularly human, genetics. The discovery that somatic cells can fuse into a heterokaryon [51], with subsequent methodologic improvements, quickly became a powerful tool for investigations on somatic cell genetics [52]. After cell fusion, the two nuclei may undergo mitosis synchronously, and each of the resulting daughter nuclei will contain both genomes in one nucleus to become true somatic cell hybrids. One of the important p h e n o m e n a in somatic cell hybrids is that most chromosomes of one genome are selectively eliminated [53]. Biologists capitalized on this p h e n o m e n o n to locate genes on particular chromosomes. One of the first such attempts was made in human/mouse cell hybrids in w h i c h the mouse cells lacked the gene t h y m i d i n e kinase ( T k ) and the h u m a n c h r o m o s o m e s were eliminated. Under a given selection condition [54], the mouse cells could not survive unless a t h y m i d i n e kinase gene was i n t r o d u c e d into its genome. Thus, the h u m a n c h r o m o s o m e that carries the Tk gene

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must be retained. From a n u m b e r of such hybrid colonies, one can identify the h u m a n c h r o m o s o m e that was invariably present in the p r e d o m i n a n t l y m o u se genome. This h u m a n c h r o m o s o m e was found to be an E group c h r o m o s o m e [55, 56], and subsequently was identified as c h r o m o s o m e #17 [57]. Thus, the h u m a n Tk gene must be located on c h r o m o s o m e #17. When I was preparing the book on the history of h u m a n and m a m m a l i a n cytogenetics [3], I asked a n u m b e r of investigators, w h o had made significant contributions to the d e v e l o p m e n t of the field, for background stories surrounding their discoveries. I have an opportunity here to add two scientists, Frank H. Ruddle and James L. German, w h o have been leaders in research areas of utter importance through their desire for p l o w in g virgin lands, their imagination, and their relentless pursuit of perfection. Ruddle has made n u m e r o u s contributions to genetics and cytogenetics; but if one must single out one area as his major acco m p l i sh m en t , it should be gene mapping. Frank was interested in c h r o m o s o m e s w h e n he was an undergraduate student at Wayne State University in Detroit, Michigan. He continued research in this area but d e v e l o p e d interest in many related (and perhaps more exciting) subjects, i n c lu d i n g parasexual systems in the late 1950s and early 1960s. His idea was to use parasexual systems of cultured cells for the purpose of extracting genetic information on higher eukaryotes. It was necessary to have a good genetic system in somatic cell populations, and he selected isozyme variants as genetic markers. The earliest work on isozyme markers was to establish their genetic basis, and this was done w i t h inbred m o use populations in vivo. One of his first reports along this line was coauthored by Thomas Roderick [58]. The link between these studies and somatic cell gene m a p p i n g was the demonstration that genetically distinct isozymes in mice could be expressed in somatic cells. The first time that this was done was in 1968 [59]. Earlier studies had been carried out on esterases, w h i c h were not particularly well expressed in tissue cultured cells; so it took several years to find the appropriate isozymes that were strongly expressed in cells in vitro. The first actual m a p p i n g report was published in 1969 [60] with Charlotte Boone, then Ruddle's graduate student. The absence of linkage was demonstrated between a n u m b e r of segregated genes, such as TK, LDH, and IDH, but it really showed the feasibility of gene mapping. The definitive report, assigning LDH-A to c h r o m o s o m e #11, TK to #17, and IDH to #20, [61] was published in 1972. In the meantime, Ruddle worked with another group of investigators in his laboratory to demonstrate the absence of linkage b et w een h u m a n lactate dehydrogenase A and B and linkage b e tw e e n LDH-B and peptidase B [62]. Recalls Frank, This paper is one of the most satisfying papers I have ever written. 1 remember having all the data with me on a trip to Denver; I stayed in my motel an extra day, wrote the paper, and then sent it off when I returned to New Haven. It is the only paper that has been accepted by N a t u r e by return telegram. They published it without page proof, which bothered me a little bit, but everything came out well in the end. Another paper [63] that I was very pleased with was one describing the assignment of peptidase C to chromosome 1. That paper was interesting because it provided the first assignment of an autosomal marker without recourse to any kind of selection technique. It also allowed the assignment of a linked marker, in this case the Rh linkage group, to chromosome 1. I guess some day it would be useful to write down an account of those early days of linkage studies. They certainly were interesting because no matter where one turned in the laboratory, a new result of interest emerged. One can see h o w ideas e v o l v e d and h o w logically the research proceeded. With refined technology, not only m a n y genes can be located in a particular chromosome, but can be p i n p o i n t e d to a band region. Combining gene m a p p i n g with several other

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new research areas, such as the in situ h y b r i d i z a t i o n techniques, and oncogenes studies, h u m a n genetics and genetic oncology have made t r e m e n d o u s advances during recent years. However, it w o u l d be i m p o s s i b l e to cover all these vast fields in this brief review, w h i c h is p r i m a r i l y concerned with cancer cytogenetics. But it suffices to say that h u m a n genetics has changed its countenance during the last two to three decades to such a degree that I believe if a classic h u m a n geneticist who died 30 years ago, s u d d e n l y comes out of his grave, attends a few seminars and reads several papers on m o d e r n h u m a n genetics, he probably w o u l d make a hasty retreat to his resting place.

Gene Amplification W h e n the p l o i d y of a cell remains u n c h a n g e d but one or more genes become multiplied out of proportion, this p h e n o m e n o n is k n o w n as gene amplification. In m a m m a l i a n cell systems, gene amplification was first suspected in neurogenic tumors and in methotrexate-resistant cell lines. Biochemical investigations demonstrated an elevated level of the enzyme dihydrofolate reductase in the resistant cells, but the m e c h a n i s m for the overproduction of this enzyme was not known. Biedler and Spengler [64] discovered a cytologic abnormality in these resistant lines: segments of c h r o m o s o m e s that s h o w e d homogeneous staining in G-banded preparations. This type of a n o m a l o u s c h r o m o s o m e segments has been k n o w n as the h o m o g e n e o u s l y staining regions (HSR). These investigators speculated that HSR represented amplification of the genes coding for dihydrofolate reductase, but they had no supporting evidence. According to Biedler's interpretation, the methotrexate-resistant cells m a y have survived via the o v e r p r o d u c t i o n (amplification) of a particular gene that can synthesize a large amount of the target product (dihydrofolate reductase) to overcome the toxicity of methotrexate. There are two types of methotrexate-resistant cell lines, namely, the stable type and the unstable type. In the absence of selection pressure, the stable type retained its methotrexate-resistant p h e n o t y p e [65], whereas, the unstable type gradually lost its resistance [66]. Cytogenetic studies on resistant cell lines revealed that cells of the stable p h e n o t y p e contained HSR that has become portions of the existing chromosomes [67], whereas, cells with the unstable p h e n o t y p e contained double minutes (DM) [68]. The DM s h o w e d a m o r p h o l o g y similar to those in neurogenic tumors originally described by Cox et al. [23] and Lubs and Salmon [24]. Studies by Barker and Hsu [69], Levan and Levan [70], and Barker a n d Stubblefield [711 s h o w e d that DM are chromatin bodies without centromeres. They do not divide during mitosis in the same m a n n e r as chromosomes. They are distributed to daughter cells at random. Therefore, one daughter cell may receive more DM than the other. Biochemical and in situ h y b r i d i z a t i o n investigations c o n c l u d e d that HSR and DM, indeed, do represent the amplified genes [67, 68, 72]. Cytogenetic investigations of cancer ceils and cancer cell lines revealed that DM are not limited to neurogenic tumors [73, 74]. Of particular interest is the report by Levan et al. [75] on the m u r i n e tumor cell line SEWA in w h i c h new chromosomes a p p e a r e d with HSR characteristics. The n e w l y formed HSR chromosomes (called the C-M chromosomes) were not incorporated as HSR segments in existing chromosomes that already had centromeres. These new metacentric chromosomes had their own centromeres and telomeres. Where did the centromeres come from? The classic concept regarding centromeres considers that centromeres can be acquired (or borrowed) only from existing c h r o m o s o m e s via translocation and they can be removed only via deletions. Both m e c h a n i s m s require chromosome breakages and restitutions. Because all mouse c h r o m o s o m e s have paracentric heterochromatin, acquiring a centromere from an existing c h r o m o s o m e is expected to have some C-

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T.C. Hsu

band material a c c o m p a n y i n g the centromeric loan. Yet the C-M chromosomes of the SEWA line showed no trace of heterochromatin. Moreover, w h e n these chromosomes first a p p e a r e d in the cell population, they did not behave like normal chromosomes. At metaphase, their sister chromatids were sometimes slightly separated at the primary constriction, and they did not always divide n o r m a l l y in anaphase (Fig. 2). Such abnormal behavior suggests defective centromeres. As cell propagation continued, the abnormalities d i s a p p e a r e d (Fig. 3). We speculate that the C-M c h r o m o s o m e s were formed originally without a functional centromere. They behaved in mitosis similar to DM. We have learned, at least in the yeast, that there are genes coding for centromeres [76]. It is conceivable that similar centromere genes exist in m a m m a l i a n cells. The p82H sequence of the a l p h o i d repeated DNA family, w h i c h is localized in the centromere region of all h u m a n chromosomes [77], may not represent the centromere gene, but we believe that there are genes coding for centromeres in higher organisms. The C-M chromosomes, in order to survive, might have " e x p e r i m e n t e d , " by mutating the DNA sequences coding for different products, to create a centromere gene. W h e n the C-M chromosomes were first discovered, their "centromere gene" had not been perfected, so that their centromeres a p p e a r e d defective. In otherwords, we may have witnessed the genesis of centromeres. If the foregoing speculation has some validity, then one may view the p h e n o m enon described by Merry et al. [78] as the other side of the same coin. These investigators, using antikinetochore serum from a scleroderma CREST patient, demonstrated that a c h r o m o s o m e resulting from a t a n d e m translocation between h u m a n chromosomes # 9 and #11, exhibited a n o r m a l l y fluorescent (chromosome #11) kinetochore and a w e a k l y fluorescent (chromosome #9) kinetochore. It is quite possible that in order to survive, this dicentric c h r o m o s o m e must inactivate one of the two centromeres. Mutations in the DNA sequence coding for the centromere of one of the c o m p o n e n t c h r o m o s o m e s (in this case, c h r o m o s o m e #9) may change its code to eliminate its activity of synthesizing a kinetochore but the process was not yet completed. Therefore, the centromere gene of c h r o m o s o m e # 9 could still synthesize a "defective" kinetochore. Perhaps given sufficient time, the C-M chromosomes are able to develop a fully functional centromere, without borrowing an existing centromere, while the dicentric c h r o m o s o m e may c o m p l e t e l y inactivate a centromere without losing its centromere DNA. Perhaps in both cases, we are witnessing the intermediate stages of two d y n a m i c processes in opposite directions. Indeed, using m o n o c l o n a l antibodies against kinetochore proteins, Earnshaw and Migeon [79] failed to observe a second kinetochore in a c o m p o u n d X chromosome.

Chromosome Instability and Mutagen Susceptibility The two-step m u t a t i o n hypothesis of carcinogenesis suggests that even if one mutation is inherited, initiation of cancer still requires a mutational process in the normal allele. Considering the early onset and bilateral d e v e l o p m e n t of retinoblastoma and Wilms' tumor, it appears that mutation rates in m a m m a l i a n somatic cells are alarmingly high. The discoveries of the c h r o m o s o m e breakage s y n d r o m e s (Bloom's syndrome, F a n c o n i ' s anemia, ataxia telangiectasia), whose normal cells (fibroblasts, lymphocytes) exhibit an extraordinarily high rate of c h r o m o s o m e aberrations, indicated that some h u m a n genomes are more unstable than others. Because chromosome breakage represents only the m i c r o s c o p i c a l l y detectable fraction of the total mutation load, mutation rates in somatic cells of these i n d i v i d u a l s must be higher still. It is not surprising, therefore, to find that i n d i v i d u a l s with these s y n d r o m e s are more cancer-prone than normal persons.

Q

F i g u r e 2 A C-banded metaphase of the mouse tumor line SEWA showing 5 small C-band negative chromosomes (C-M chromosomes) with precocious separation of sister chromatids. Courtesy of Professors A. Levan and G. Levan.

V

,,,]

18

Figure 3a F i g u r e 3 Duplicate photographs of a mouse tumor cell (SEWA line) with 5 C-band ~legativ~ metacentric and 1 acrocentric chromosomes (C-M chromosomes): (a) regular Giemsa staining: (b) C-band preparation. The chromatids of the C-M chromosomes do not exhibit precocious centroinere separation. Courtesy of Professors A. Levan and G. Levan.

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Figure 3b Although numerous investigators contributed to the progress of this area of study, credit should go to James German who tirelessly brought all the information together through the years and led the field of genetic instability in the human population and its relationship with neoplasia. German received his medical education in The University of Texas Medical School in Galveston, Texas. During this period, he was exposed to tissue culture and cell studies in the laboratory of

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T . C . Hsu Charles M. Pomerat (who was also m y mentor, some years later), and was always fascinated with that line of study. Subsequently, he worked at The National Institutes of Health on l u p u s erythematosus. This e p i s o d e actually led h i m to his career, although he had not w o r k e d on lupus since. At that time Henry Kunkel was to set up a laboratory of h u m a n genetics at the Rockefeller Institute. Because of his background in lupus, he was invited to join Kunkel's laboratory, w h i c h was p r i m a r i l y engaged in b i o c h e m i c a l research. The discoveries on abnormal c h r o m o s o m e constitutions in several h u m a n d e v e l o p m e n t a l defects came at that time, and German talked Kunkel into setting up a pilot tissue culture laboratory in 1959. He was told to show his ability in c h r o m o s o m e study by demonstrating the chromosomes of the mouse as a p r e c o n d i t i o n for this n e w venture. He successfully d i d so in bone marrow preparations with drawings of two mouse metaphases showing 40 chromosomes. Immediately, he d i s c o n t i n u e d the b i o c h e m i c a l studies, and with i m m e n s e pleasure returned to cell studies and for the first time h a n d l e d chromosomes. He was then o v e r w h e l m e d by requests from the New York City area for analyzing chromosome constitutions of congenital syndromes. One of these requests was from a dermatologist, David Bloom, who had described a s y n d r o m e consisting of stunted growth and a lupus-like lesion of the face, then called "congenital telangiectatic erythema resembling lupus erythematosis in dwarfs" [80]. Dr. Bloom w a n t e d to have a cytogenetic study to rule out a n e u p l o i d y in " h i s " syndrome, now k n o w n as the Bloom's syndrome. Dr. Peyton Rous, the r e n o w n e d virologist at Rockefeller and a friend of Bloom's, suggested German. James' previous interest in lupus and his respect for Rous p r o v i d e d a suitable incentive to accept the request. During that early period of h u m a n cytogenetics, p e o p l e were oriented to look for constitutional numerical or structural c h r o m o s o m e abnormalities in various syndromes. The l y m p h o c y t e s of the first patient with Bloom's s y n d r o m e s h o w e d structurally abnormal c h r o m o s o m e s in a high p r o p o r t i o n of cells, but no two cells had the same abnormality! The basic karyotype was normal. James was not p r e p a r e d to discover genetically d e t e r m i n e d genomic instability, nor was the general public. In 1963, w h e n he first described his observations on the cytogenetic p h e n o m e n a of the Bloom's s y n d r o m e at the A m e r i c a n Society of H u m a n Genetics meeting, the session chairman Klaus Patau thought that the results may be caused by dirty glassware or unidentified m e d i u m components. Perhaps Patau's criticisms also d a m p e n e d the enthusiasm of the audience. Nevertheless, James c o n t i n u e d his studies in the Bloom's s y n d r o m e and found the same results in several patients. He told me that during that p e r i o d he felt very lonely. I am quite certain that all pioneers felt lonely at one time or another. James jotted d o w n his thoughts in his notebook that the t e n d e n c y to extensive chromosome breakage and rearrangements conceivably could have importance in relation to a u t o i m m u n i t y or cancer. In fact the first Bloom's s y n d r o m e patient he studied at age 10 d i e d from acute l e u k e m i a at age 13. The hematologists who cared for this child informed German that a dwarf w o m a n with features reminiscent of this child had died of acute l e u k e m i a in Mexico City. Bloom and German p r o m p t l y flew to Mexico to get as m u c h information as possible about this deceased patient, including interviewing the family members using a combination of Yiddish, Hebrew, Polish, and Russian. They were c o n v i n c e d that the woman, in fact, had the Bloom's syndrome. Since then he set up a w o r l d - w i d e surveillance program to locate and to monitor persons afflicted with this syndrome. Confirmation on hereditary c h r o m o s o m e instability and their p r e d i s p o s i t i o n to neoplasia came from studies on other congenital diseases, i n c l u d i n g F a n c o n i ' s anemia [81, 82] and ataxia telangiectasia [83]. Victor McKusick urged German to pull

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the ideas together and call these disorders "chromosome breakage syndromes" [84]. One of the ideas cherished by James was the possibility of somatic gene recombination, an interpretation resulted from observations of chromatid exchange figures so frequently occuring in lymphocytes of patients with Bloom's syndrome. German called these exchange figures quadiradials because they are reminiscent of meiotic configurations of organisms heterozygous for a translocation. His interpretation was that these exchanges represented cytologic evidence for crossing-over in mammalian somatic cells. Such somatic crossing-over can create a clone with a genome that differs from the parent cell, as well as from other cells in the cell population. The new clone may lack certain genes, yet requires no mutations. The original lymphoid tissue may become foreign to this new clone of cells, which may have antigen losses, and consequently this new clone may develop into neoplasm [85]. The fact that in the human population genetic instability can be found in a number of hereditary disorders is now well-documented [86]. The chromosome breakage syndromes probably represent the extreme end of a distributional spectrum in the human population in terms of genetic stability, which spans from very stable to very unstable. However, it is difficult to use genetic and/or cytogenetic methods to identify moderately unstable genomes, which may be next in line regarding cancer proneness. The important fact we learned from studies on chromosome instability syndromes is that different syndromes may show differential sensitivities to different environmental mutagens. It is a well known fact that our environment is loaded with carcinogens and mutagens, each of which has its mechanism of action. Studies on mutagen sensitivity of the various chromosome breakage syndromes revealed that one syndrome may be hypersensitive to one mutagen but not to another, and another syndrome may be susceptible in the reverse fashion. For example, cells of persons with xeroderma pigmentosum are highly susceptible to ultraviolet light, resulting in a higher frequency of mutations [87] and chromosome damage [88] than cells of normal persons similarly exposed. Biochemical data showed that these patients are deficient in the initial step of excision repair [89, 90]. Subsequent studies showed that cells of xeroderma pigmentosum patients are not hypersensitive to ionizing radiations. Conversely, patients with ataxia telangiectasia are hypersensitive to ionizing radiations and to bleomycin but are not hypersensitive to UV light [91, 92]. Thus, genetic sensitivity to environmental mutagens may depend on the agents encountered. Most biologists consider environmental carcinogenesis as the result of defective, probably genetically determined DNA repair mechanisms [93, 94]. From the data on chromosome instability syndromes, it appears that spontaneous chromosome instability and mutagen sensitivity are two separate phenomena. Thus far, cells of Bloom's syndrome are not known to be hypersensitive to a particular agent, whereas, xeroderma pigmentosum cells do not exhibit a significantly high rate of spontaneous chromosome aberrations. A person may be perfectly stable in terms of spontaneous mutagenesis rate and chromosome breakage rate (thus, not be classified into chromosome breakage syndrome), but may be sensitive to one mutagen or another. If, within the human population, some individuals are moderately sensitive to the UV light, then such individuals may be more liable to acquire skin cancers but not necessarily neoplasms of internal organs. On the other hand, persons moderately sensitive to ionizing radiations and radiomimetic chemicals (e.g., bleomycin) may develop other cancers more easily than resistant individuals when equal amounts of exposure are received. Indirect evidence, indeed, shows that a higher proportion of patients with lung and colon carcinomas are sensitive to bleomycin than normal persons similarly tested [95].

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OUTLOOK

I am confident that intensive collaborative investigations among molecular biologists, biochemists, geneticists, and cytogeneticists will eventually find specific genetic changes in each type of neoplasm, m a n y of w h i c h are cytogenetically demonstrable. With the wealth of information a c c u m u l a t e d in genetics, molecular genetics, and cytogenetics of humans, concerted efforts should n o w focus on h u m a n cancers. A l t h o u g h the resolution of cytogenetics eventually will reach a limit, and m o l e c u l a r geneticists must take over from there, cytogenetics plays a vital role in cancer etiology because it will be utterly impractical or even fruitless to walk thousands of genes in the hope to find one or a few that are responsible for the initiation of a particular cancer. Cytogeneticists can eventually locate the breakpoints, the deleted or d u p l i c a t e d bands, or other suspected c h r o m o s o m e regions responsible for cancer initiation. From this point molecular geneticists can place their efforts e c o n o m i c a l l y as they have done with Burkitt's l y m p h o m a , retinoblastoma, Wilms' tumor, and other neoplasms. In a d d i t i o n to f u n d a m e n t a l contributions to the origin of cancer and gene action, results of cytogenetic investigations should help pathologists in cancer diagnosis and classification, and clinicians in cancer management. A growing b o d y of information has shown cytogenetic p r e d i s p o s i t i o n to cancer, both in terms of specific congenital anomalies and mutagen susceptibility. Extensive research in this direction should be most useful in identifying genetically atrisk individuals, who s h o u l d be p e r i o d i c a l l y monitored and counselled for possible signs of danger. It should be e m p h a s i z e d that a n u m b e r of c h r o m o s o m e anomalies have been shared by a variety of tumors. Very probably these c o m m o n aberrations found in different tumors do not represent cancer initiation, but rather represent genetic changes for cancer promotion, progression, and metastasis. At present, no concrete data are available regarding these subjects, but this area deserves more scrutiny in the future. Technically, the time for using long-term cell lines derived from neoplasms is about over for cytogeneticists who desire to find basic changes of cancer. Methods must be d e v e l o p e d to obtain acceptable cytogenetics preparations directly from biopsy s p e c i m e n s or from short-term cultures of b i o p s y specimens. Because m a n y tumor cells show h y p e r c o n d e n s e d c h r o m o s o m e s that are not amenable to detailed cytogenetic analysis, i m p r o v e m e n t s in m e t h o d o l o g y should be systematically developed to achieve a high degree of success with b i o p s y specimens. The field of cancer genetics, as a whole, has evolved from the nearly hopeless situation of yesteryear to the highly optimistic and exciting stage of the present. The future is unquestionably bright. Supported in part by a research grant from the John S. Dunn Foundation of Houston.

REFERENCES

1. Boveri T (1914): Zur Frage der Entstehung maligner Tumoren. Jena Gustav Fischer. 2. Kottler MJ (1974): From 48 to 46: Cytological technique, preconception, and the counting of the human chromosomes. Bull Hist Med 48:465 502. 3. Hsu TC (1979): Human and Mammalian Cytogenetics: A Historical Perspective. SpringerVerlag, New York. 4. Therman E (1980): Human Chromosomes: Structure, Behavior, Effects. Springer Verlag, New York.

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5. Biesele JJ, Poyner H, Painter TS (1942): Nuclear p h e n o m e n a in mouse cancer. University of Texas Publication 4243, pp. 7-68. 6. Makino S (1951): Some observations on the chromosomes in the Yoshida sarcoma cells based on the homoplastic and heteroplastic transplantations. Gann 42:87-90. 7. Makino S (1952): A cytological study of the Yoshida sarcoma, an ascities tumor of white rats. Chromosoma 4:649-674. 8. Makino S (1952): Cytological studies on cancer. III. The characteristics and individuality of chromosomes in tumor cells of the Yoshida sarcoma which contribute to the growth of the tumor. Gann 43:17-34. 9. Makino S (1957): The chromosome cytology of the ascites tumors of rats, with special reference to the concept of the stemline cell. Intl Rev Cytol 6:25-84. 10. Levan A, Hauschka TS (1952): Chromosome numbers of three mouse ascites tumors. Hereditas 38:251-255. 11. Hauschka TS (1953): Cell population studies on mouse ascites tumors. Trans NY Acad Sci 16:64-73. 12. Levan A (1959): Relation of chromosome status to the origin and progression of tumors: The evidence of chromosome numbers. In: Genetics and Cancer. University of Texas Press, Austin, TX, pp. 151-182. 13. Hsu TC (1952): Mammalian chromosomes in vitro. I. The karyotype of man. J Hered 43:167-172. 14. Makino S, Nishimura I (1952): Water-pretreatment squash technique: A new and simple practical method for the chromosome study of animals. Stain Tech 27:1-7. 15. Tjio JH, Levan A (1956): The chromosome n u m b e r of man. Hereditas 42:1-6. 16. Lejeune JC, Gautier M, Turpin R (1959): Etude des chromosomes somatiques de neuf enfant mongoliens. Comp Rend 248:1721-1722. 17. Nowell PC (1960): Phytohemagglutinin: An initiator of mitosis in cultures of normal human leukocytes. Cancer Res 20:462-466. 18. Moorhead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA (1960): Chromosome preparations of leukocytes cultured from h u m a n peripheral blood. Exp Cell Res 20:613616. 19. Hsu TC (1961): Chromosomal evolution in cell populations. Intl Rev Cytol 12:69-161. 20. Nowell PC, Hungerford DA (1960): A minute chromosome in h u m a n chronic granulocytic leukemia. Science 132:1497. 21. Nowell PC, Hungerford DA (1961): Chromosome studies in h u m a n leukemia. II. Chronic granulocytic leukemia. J Natl Cancer Inst 27:1013-1035. 22. Zang KD, Singer H (1967): Chromosome constitution of meningiomas. Nature 216:84-85. 23. Cox D, Yuncken C, Spriggs AI (1965): Minute chromatin bodies in malignant tumors of childhood. Lancet i:55-58. 24. Lubs HA, Salmon JH (1965): The chromosomal complement of h u m a n solid tumors. II. Karyotypes of glial tumors. J Neurosurg 22:160-168. 25. Paris Conference (1971): Standardization in h u m a n cytogenetics. Birth Defects Article Series 8. The National F o u n d a t i o n - - M a r c h of Dimes, New York, pp. 1-46. 26. Yunis JJ (1976): High resolution of h u m a n chromosomes. Science 191:1268-1270. 27. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier, New York. 28. Mitelman F, Levan G (1981): Clustering of aberrations to specific chromosomes in h u m a n neoplasms. IV. A survey of 1871 cases. Hereditas 95:79-139. 29. Mitelman F (1983): Catalogue of chromosome aberrations in cancer. Cytogenet Cell Genet 36:1-515. 30. Stich HF (1960): Chromosomes of tumor cells. I. Murine leukemias induced by one or two injections of 7, 12-dimethylbenz(a)anthracene. J Natl Cancer Inst 25:649-661. 31. Dofoku R, Biedler JL, Spengler BA, Old LJ (1975): Trisomy of chromosome 15 in spontaneous leukemia of AKR mice. Proc Natl Acad Sci USA 72:1515-1517. 32. Friedman JM, Fialkow PJ, Bryant J, Reddy AL, Salo AC (1978): Neoplastic behavior of chromosomally abnormal clones in New Zealand black mice. Intl J Cancer 22:458-464.

24

T.C. Hsu

33. Chang TD, Biedler JL, Stockert E, Old LJ (1977): Trisomy 15 in X-ray induced mouse leukemia. Proc Am Assoc Cancer Res 18:225. 34. Wiener F, Spira S, Ohno S, Haran-Ghera N, Klein G (1978): Chromosome changes (trisomy 15) in murine T-cell leukemia induced by 7, 12, dimethylbenz(a)anthracene (DMBA). Intl J Cancer 22:447-453. 35. Spira J, Wiener F, Ohno S, Klein G (1979): Is trisomy cause or consequence of murine Tcell leukemia development? Studies of Robertsonian translocation mice. Proc Natl Acad Sci USA 76:6619-6621. 36. Ohno S, Babonits M, Wiener F, Spira J, Klein G, Potter M (1979): Nonrandom chromosome changes involving the Ig gene-carrying chromosomes 12 and 6 in pristane induced mouse plasmacytomas. Cell 18:1001-1007. 37. Zech L, Haglund U, Nilsson K, Klein G (1976): Characteristic chromosomal abnormalities in biopsies and lymphoid-cell lines from patients with Burkitt and non-Burkitt lymphomas. lntl J Cancer 17:47-56. 38. Rowley JD (1973): A new consistent chromosome abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining. Nature 243:290-293. 39. Knudson AG (1971): Mutation and Cancer: Statistical study of retinoblastoma. Proc Natl Acad Sci 68:820-823. 40. Benedict WF, Murphree AL, Banerjee A (1983): Patient with 13 chromosome deletion: Evidence that the retinoblastoma gene is a recessive cancer gene. Science 219:973-975. 41. Knudson AG, Strong LC (1972): Mutation and Cancer: A model for Wilms' tumor of the kidney. J Natl Cancer Inst 48:313-324. 42. Miller RW, Franmeni JF Jr, Manning MD (1964): Association of Wilms' tumor with aniridia, hemihypertrophy and other congenital malformations. N Engl J Med 270:922-927. 43. Wilson MG, Melnyk J, Towner JW (1969): Retinoblastoma and deleted D(14) syndrome. ] Med Genet 6:322-327. 44. Riccardi VM, Sujansky E, Smith AC, Franke U {1978): Chromosomal imbalance in the aniridia-Wilms' tumor association: 11p interstitial deletion. Pediatrics 61:604-610. 45. Pathak S, Goodacre A (1986): Specific chromosome anomalies and predisposition to human breast, renal cell, and colorectal carcinoma. Cancer Genet Cytogenet 19:29-36. 46. Francke U (1983): Specific chromosome changes in the h u m a n heritable tumors retinoblastoma and nephroblastoma. In: Chromosomes and Cancer, Rowley JD, Ultmann JE, eds. Academic Press, New York, pp. 99-115. 47. Strong LC, Riccardi VM, Ferrell RE (1981): Familial retinoblastoma and chromosome 13 deletion transmitted via an insertional translocation. Science 213:1501-1503. 48. Cohen AJ, Li FP, Berg S, Marchetto DJ, Tsai S, Jacobs SE, Brown RS (1979): Hereditary renal-cell carcinoma associated with a chromosomal translocation. N Engl J Med 301:592595. 49. Pathak S, Strong LC, Ferrell RE, Trindade A (19821: Familial renal cell carcinoma with a 3;11 chromosome translocation limited to tumor cells. Science 217:939-941. 50. Pathak S (1986): Cytogenetics of solid tumors. In: The Human Oncogenic Viruses, Lederer AA, Weetall HH, eds. The Humana Press, Clifton, NJ, pp. 43-87. 51. Barski G, Sorieul S, Cornefert F (1960): Production dans des cultures in vitro de deux souches cellulaires en association, de cellules de caractere "hybride". CR Hebd Seances Acad Sci 251:1825-1827. 52. Ringertz N, Savage RE (1976): Cell Hybrids. Academic Press, New York. 53. Wiess MC, Green H (1967): H u m a n - m o u s e hybrid cell lines containing partial complements of h u m a n chromosomes and functioning h u m a n genes. Proc Natl Acad Sci USA 58:1104-1111. 54. Littlefield JW (1964): Selection of hybrids from matings of fibroblasts in vitro and their presumed recombination. Science 145:709-710. 55. Matsuya Y, Green H, Basilico C (1968): Properties and use of h u m a n - m o u s e hybrid cell lines. Nature 220:1199-1202. 56. Migeon BR, Miller CS (1968): Human-mouse somatic cell hybrids with single h u m a n chromosome (group E): Link with thymidine kinase activity. Science 162:1005-1006. 57. Miller OJ, Allderdice PW, Miller DA, Breg WR, Migeon BR (1971): Assignment of human

H i s t o r i c a l D e v e l o p m e n t of C a n c e r C y t o g e n e t i c s

25

thymidine kinase gene locus to chromosome 17 by identification of its distinctive quinacrine fluorescence in a m a n - m o u s e somatic hybrid. Science 173:244-245. 58. Ruddle FH, Roderick TH (1966): The genetic control of two types of esterases in inbred strains of the mouse. Genetics 54:191-202. 59. Ruddle FH (1968): lsozymic variants a genetic markers in somatic cell populations in vitro. Natl Cancer Inst Monogr 29:9-13. 60. Boone CM, Ruddle FH (1969): Interspecific hybridization between human and mouse somatic cells: Enzyme and linkage studies. Biochem Genet 3:119-136. 61. Boone CM, Chen TR, Ruddle FH (1972): Assignment of three human genes to chromosomes (LDH-A to 11, TK to 17, and IDH to 20) and evidence for translocation between human and mouse chromosomes in somatic cell hybrids. Proc Natl Acad Sci 69:510-514. 62. Ruddle FH, Chapman VM, Chen TR, Klebe RJ (1970): Linkage between human lactate dehydrogenase A and B and peptidase B. Nature 227:251-257. 63. Ruddle FH, Riccinti F, McMorris FA, Tischfield J, Creagan R, Darlington G, Chen TR (1972): Somatic cell genetic assignment of peptidase C and the Rh linkage group to chromosome A-1 in man. Science 176:1629-1631. 64. Biedler JL, Spengler A (1976): Metaphase chromosome anomaly: Association with drug resistance and cell specific products. Science 191:185-187. 65. Fischer GA (1961): Increased levels of folic acid reductase as a mechanism of resistance to amethopterin in leukemic cells. Biochem Pharm 7:75-80. 66. Courtney VP, Robbins AB (1972): Loss of resistance to methotrexate in L5178Y mouse leukemia in vitro. J Natl Cancer Inst 49:45-53. 67. Nunberg JH, Kaufman RJ, Schimke RT, Urlaub G, Chasin LA (1978): Amplified dihydrofolate reductase genes are localized to a homogeneously staining region of a single chromosome in a methotrexate-resistant Chinese hamster ovary cell line. Proc Natl Acad Sci USA 75:5553-5556. 68. Kaufman RJ, Brown PC, Schimke RT (1979): Amplified dkyhdrofolate reductase genes in unstably methotrexate-resistant cells are associated with double minute chromosomes. Proc Natl Acad Sci USA 76:5669-5673. 69. Barker PE, Hsu TC (1978): Are double minutes chromosomes? Exp Cell Res 113:456-458. 70. Levan A, Levan G (1978): Have double minutes functioning centromeres? Hereditas 88:81-92. 71. Barker PE, Stubblefield E (1979): Ultrastructure of double minutes from a human carcinoma cell line. J Cell Biol 83:663-666. 72. Melera PW, Lewis JA, Biedler JL, Hession G (1980): Antifolate-resistant Chinese hamster cells. Evidence for dihydrofolate reductase gene amplification among independently derived sublines overproducing dihydrofolate reductases. J Biol Chem 255:7024-7028. 73. Barker PE, Hsu TC (1979): Double minutes in human carcinoma cell lines, with special reference to breast tumors. J Natl Cancer Inst 62:263-271. 74. Quinn LA, Moore GE, Morgan RJ, Woods LK (1979): Cell lines from human colon carcinoma with unusual cell products, double minutes and homogeneously staining regions. Cancer Res 39:4914-4924. 75. Levan A, Levan G, Mandahl N (1978): A new chromosome type replacing the double minutes in a mouse tumor. Cytogenet Cell Genet 20:12-23. 76. Yeh E, Bloom K (1985): Characterization of a tightly centromere-linked gene essential for mitosis in the yeast Saccharomyces cerevisiae. In: Aneuploidy: Etiology and Mechanisms, Dellarco VL, Voytek PE, Hollaender A, eds., Plenum Press. New York, pp. 231-242. 77. Mitchell AR, Gosden JR, Miller DA (1985): A clonal sequence, p82H, of the alphoid repeated DNA family found at the centromeres of all human chromosomes. Chromosoma 92:369-377. 78. Merry DE, Pathak S, Hsu TC, Brinkley BR (1985): Anti-kinetochore antibodies: Use as probes for inactive centromeres. Am J Hum Genet 37:425-430. 79. Earnshaw WC, Migeon BR (1985): Three related centromere proteins are absent from the inactive centromere of a stable isodicentric chromosome. Chromosoma 92:290-296. 80. Bloom D (1954): Congenital telangiectatic erythema resembling lupus erythematosis in dwarfs. Am J Dis Child 88:754-758.

26

T.C. Hsu

81. Schroeder TM, Anschutz F, Knopp A (1964): Spontane chromosomenaberrationen bei familiarer Panmyelopathie. Humangenetick 1:194-196. 82. German J, Archibald R, Bloom D (1965): Chromosomal breakage in a rare and probably genetically determined syndrome of man. Science 148:506-507. 83. Hecht F, Koler RD, Rigas DA, Dahnke GS, Case MP, Tisdate V, Miller RW (1966): Leukemia and lymphocytes in ataxia-telangiectasia. Lancet ii:1193. 84. German J (1969): Chromosome breakage syndromes. Birth Defects: Original Article Series 5:117 131. 85. German ] (1964): Cytological evidence for crossing-over in vitro in h u m a n lymphoid cells. Science 144:298-301. 86. German J (1983): Chromosome Mutation and Neoplasia. Alan R. Liss, New York. 87. Maher VM, Ouellette LM, Curren RD, McCormick JJ (1976): Frequency of ultraviolet light induced mutation is higher in xeroderma pigmentosum variant cells. Nature 261:593-595. 88. Marshall RR, Scott D (1976): The relationship between chromosmne damage and cell killing in UV-irradiated normal and xeroderma pigmentosum cells. Mutat Res 36:397-400. 89. Setlow RB, Regan JD, German ], Carrier WL (1969): Evidence that xeroderma pigmentosum ceils do not perform the first step in the repair of ultraviolet damage to their DNA. Proc Natl Acad Sci 64:1035-1041. 90. Cleaver JE (1968): Defective repair replications of DNA in xeroderma pigmentosum. Nature 218:652-656. 91. Taylor AMR, Metcalfe JA, Oxford JM, Harnden DG (1976): Is chromatid-type damage in ataxia telangiectasia after irradiation at Go a consequence of defective repair? Nature 260:441-443. 92. Taylor AMR, Rosney CM, Campbell JB (1979): Unusual sensitivity of ataxia telangiectasia cells to bleomycin. Cancer Res 39:1046-1050. 93. Setlow RB (1978): Repair deficient h u m a n disorders and cancer. Nature 271:713-717. 94. Glickman BW (1980): DNA repair and its relationship to the origins of h u m a n cancer. In: Genetic Origin of Tumor Cells, Cleton FJ, Simons JWIM, eds. Martinus Nijhoff, The Hague, pp. 25-51. 95. Hsu TC (1986): Genetic susceptibility to carcinogenesis. Cancer Bull 38:125-128.