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Cell Cycle 11:6, 1151-1166; March 15, 2012; © 2012 Landes Bioscience

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*Correspondence to: Peter Duesberg; Email: Duesberg@berkeley.eduSubmitted: 11/07/11; Revised: 02/01/12; Accepted: 02/02/12http://dx.doi.org/10.4161/cc.11.6.19580

Introduction

Metastases are colonies of cancers that have spread to distant sites in an organism. Since metastases severely complicate the diagno-sis and treatment of cancers, they have long been compared with primary cancers for clues of causative changes.1-18

Despite these efforts, which we analyze below, Molecular Biology of the Cell writes in its latest edition, “The changes in tumor cells that lead to metastasis are still largely a mystery” and “We have yet to identify mutations that specifically per-mit cells to invade surrounding tissues, spread through the body and form metastases.”19 If mutations would, however, “permit” cells to metastasize, normal cells should metasta-size just as much as cancer cells, since the mutation rates of cancers and normal cells are about the same.20-22 Also assum-ing mutations, a recent review finds it “puzzling” that cancers develop “metastasis virulence genes… at appreciable frequency” and that these genes “confer… advantages only within foreign microenvironments.”23

Conventional mutation theories do not explain (1) why the karyotypes of metastases are related to those of parental cancers but not to those of metastases of other cancers and (2) why cancers metastasize at rates that often far exceed those of conventional mutations. To answer these questions, we advance here the theory that metastases are autonomous subspecies of cancers, rather than mutations. Since cancers are species with intrinsically flexible karyotypes, they can generate new subspecies by spontaneous karyotypic rearrangements. This phylogenetic theory predicts that metastases are karyotypically related to parental cancers but not to others. Testing these predictions on metastases from two pancreatic cancers, we found: (1) Metastases had individual karyotypes and phenotypes. The karyotypes of metastases were related to, but different from, those of parental cancers in 11 out of 37 and 26 out of 49 parental chromosomal units. Chromosomal units are defined as intact chromosomes with cancer-specific copy numbers and marker chromosomes that are > 50% clonal. (2) Metastases from the two different cancers did not share chromosomal units. Testing the view that multi-chromosomal rearrangements occur simultaneously in cancers, as opposed to sequentially, we found spontaneous non-clonal rearrangements with as many new chromosomal units as in authentic metastases. We conclude that metastases are individual autonomous species differing from each other and parental cancers in species-specific karyotypes and phenotypes. They are generated from parental cancers by multiple simultaneous karyotypic rearrangements, much like new species. The species-specific individualities of metastases explain why so many searches for commonalities have been unsuccessful.

Origin of metastasesSubspecies of cancers generated by intrinsic karyotypic variations

Peter Duesberg,1,* Christine Iacobuzio-Donahue,2 Jacqueline A. Brosnan,2 Amanda McCormack,1 Daniele Mandrioli1 and Lewis Chen1

1Department of Molecular and Cell Biology; Donner Laboratory; University of California at Berkeley; Berkeley, CA USA; 2Departments of Pathology and Oncology; The Sol Goldman Pancreatic Cancer Research Center; Johns Hopkins Medical Institutions; Baltimore, MD USA

Key words: cancer autonomy, marker chromosomes, high rates of karyotypic variation, speciation, stochastic karyotypic variation, multi-chromosomal rearrangements, intrinsic instability of aneuploidy, stabilization of karyotype by selection for autonomy,

saltational evolution

In a new effort to find an alternative theory, which explains, why “mutations” have not yet solved the “mystery” of metasta-sis, we have first summarized here the known (1) karyotypic, (2) genetic and (3) kinetic connections between metastases and cancers and then tested a new theory.

Karyotypic relationships between metastases and cancers.Cytogenetic comparisons between cancers and metastases pub-lished since 1979 show that the karyotypes of metastases are related to those of parental cancers but distinct in various indi-vidual chromosomes.1-18 By contrast, the karyotypes of metasta-ses from different cancers are not related.3,8,13,16,24 In short, no common metastasis-specific chromosomes were found. Thus metastases are karyotypically related to parental cancers, but not to those of other cancers.

Genetic relationships between metastases and cancers. Complete genome sequencing methods developed recently show that metastases share some clonal mutations with parental can-cers,24-28 but these mutations are not “metastasis-specific muta-tions per se.”29 Instead they are only shared by parental cancers

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steps.”36,37 Several investigators have since confirmed Foulds’ rules: (1) Based on metastatic spreading probabilities of melano-mas that range from 1 in 5 x 102 to 1 in 108 cells, Michaelson et al. conclude “that the spread of human breast cancer and melanoma cells is unlikely to occur by a mechanism requiring mutation at the time of spread.”38 (2) Kuukasjarvi et al. conclude, because of “early stemline evolutions” of breast cancers, “a linear progres-sion model is unlikely to account for the progression of primary tumors to metastases.”8 (3) Wang et al. report that “all the metas-tases were derived from the same primary tumor [melanoma], although they were each probably not derived from the most recent previous metastasis in a sequential manner.”39 (4) Sabatino et al. conclude that metastasis of melanomas “did not follow a strict temporal progression but stemmed independently at vari-ous time points.”15 (5) Torres et al. state, “Metastases may occur relatively early during breast carcinogenesis,” because all metasta-ses differed from primary cancers in many karyotypic elements.14 (6) Khalique et al. find that “The data support a model in which primary ovarian cancers have a common clonal origin… with different clones at both early and late stages of genetic divergence acquiring the ability to progress to metastasis.”40 (7) Studying Barrett’s carcinoma, Walch et al. concluded, “clonal evolution is more complex than would be predicted by linear models.”41 (8) Studying breast cancers, Navin et al. advanced a phylogenetic theory that “tumors grow by punctuated clonal expansions—bor-rowing a term from species evolution to explain gaps in the fossil record,” because metastasis could not be reconciled with the low rates predicted by “a linear progression model.”24 Animal studies have also shown “evolutions” of metastases42 at “rapid”43 or “high rates”44 that exceed those of conventional mutations by orders of magnitude.44-46 All these observations, including Foulds’ rules, are hard to reconcile with the conventional view that metastases are end products of multiple steps of sequential mutations.19,23,28

Thus conventional mutation theories cannot explain (1) why metastases are karyotypically related to parental cancers but not to those of other cancers and (2) why cancers metastasize at rates that often exceed those of conventional mutations by several orders.

Phylogenetic theory of metastasis. In an effort to answer these questions, we advance here the theory that metastases are subspecies of cancers, rather than mutations. Since cancers are species with flexible karyotypes, they can generate new subspe-cies, such as metastases, by spontaneous karyotypic rearrange-ments, independent of mutation.17,47-52 This flexibility of cancer karyotypes is a consequence of an equilibrium between two com-peting forces: (1) inherited imbalances of normal mitosis genes destabilizing cancer karyotypes. These imbalances reflect the very karyotypic rearrangements that generated cancers from nor-mal cells.17 (2) Selection for cancer-specific autonomy stabilizing the flexible karyotypes of cancers within cancer-specific margins of autonomy.17,51 Accordingly, cancers can generate new autono-mous subspecies with new species-specific karyotypes by intrin-sic chromosomal rearrangements, as, for example, metastases and drug-resistant subspecies17,22,51,53 (Fig. 1). The rates and ranges of these subspeciations would depend on the karyotypic flexibility of a cancer. This flexibility is proportional to the extent of can-cer-specific aneuploidy, in other words, the differences between

and their metastases.27,29-33 Some of the pancreatic cancers and metastases studied here have also been sequenced recently in ref-erences 29 and 30 (see Materials and Methods) and were found to share a “few” mutations,25 including Kras.29 Since Kras muta-tions are, however, shared by metastases and parental cancers in our case, they cannot be metastasis-specific. Moreover, since Kras mutations are in no more than 70% of human pancreatic can-cers,17,34,35 this mutation is also not cancer-specific.

Kinetics of the origins of metastases from cancers. In 1949 Leslie Foulds first described the peculiar rules under which metastases and other “progressions” arise from cancers, hence-forth called “Foulds’ rules:” “Progression is independent of the size or clinical duration of a tumor. Progression occurs indepen-dently in different characters in the same tumor. Progression is continuous or discontinuous, by gradual change or by abrupt

Figure 1. Ranges of karyotypic and phenotypic variations among subspecies of cancers generated by multi-chromosomal rearrange-ments. The figure shows a hypothetical cancer in the center, which is surrounded by four karyotypically and phenotypically distinct, but re-lated, potential subspecies. Since cancers are species with quasi-flexible karyotypes, they can generate new subspecies such as metastases by karyotypic rearrangements independent of mutation.17,52 This flexibility of cancer karyotypes is a consequence of an equilibrium between two competing forces: (1) Inherited imbalances of normal mitosis genes destabilizing cancer karyotypes. These imbalances reflect the very karyotypic rearrangements that generated cancers from normal cells.17 (2) Selection for cancer-specific autonomy stabilizing the flexible karyotypes of cancers within cancer-specific margins of autonomy.17,51 Accordingly cancers can generate new autonomous subspecies with new species-specific karyotypes and phenotypes by intrinsic chromo-somal rearrangements, as for example metastases and drug-resistant subspecies17,22,51,53 (Fig. 1). The rates and ranges of these subspeciations would depend on the karyotypic flexibility of a cancer. This flexibility is proportional to the extent of cancer-specific aneuploidy, in other words the differences between the karyotype of the cancer and that of the normal cell from which it evolved.17,51,54-57 The ranges of subspeciations are, however, limited by the conservation of certain as yet undefined karyotypic elements that maintain the autonomy of the paternal cancer and its subspecies.17 Figure 1 shows graphically the apparent borders set by these undefined conserved karyotypic elements that limit the autonomy of the parental cancer and its subspecies. The specia-tion theory thus predicts that metastases are individual species with species-specific karyotypes and phenotypes.

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Karyographs. To test the karyotypic relationships between A13A, A13B and A13D, we have compared their karyographs.17,50 Karyographs are three-dimensional tables that list the numbers of intact chromosomes and number designations of marker chro-mosomes on the x-axis, the cancer-specific copy numbers of each chromosome on the y-axis, and the number of about 20 meta-phases arrayed per table on the z-axis. By comparing 20 karyo-grams at once, karyographs immediately distinguish clonal from non-clonal chromosomes. Such karyographs can thus be used as photographic images of clonal cancer-specific individuality, which is primarily defined by the clonal cancer karyotypes, and secondarily by the “clonal heterogeneity” that results from cancer-specific karyotypic flexibility (See Introduction, “Phylogenetic theory of metastasis.”). Direct comparisons of the karyographs of A13A, A13B and A13D are shown in Figure 3.

The karyographs shown in Figure 3 below were typically con-structed from 20 karyograms obtained by microscopic analyses of metaphase-chromosome spreads. These metaphase spreads were prepared from cancer cells treated for 4 to 8 h with colcemid in cell culture (Materials and Methods). To facilitate karyotyp-ing, the metaphase chromosomes were hybridized to chromo-some-specific color-coded DNA probes from MetaSystems, as described in Materials and Methods. Typical karyograms of the primary pancreatic A13B and of the metastasis A13D are shown in Figure 4A and B, respectively.

Comparisons of cancers based on clonal chromosomal units. To calculate the karyotypic differences that set apart the cancers and metastases studied here, we introduced “chromosomal units.” Chromosomal units are defined here as intact chromosomes with cancer-specific copy numbers and marker chromosomes that are > 50% clonal. We also include in this definition “diploid sets” of chromosomes (i.e., with copy number 2) as cancer-specific chromosomal units, because a diploid set of two intact or normal chromosomes does not represent a normal gene balance in the context of an aneuploid cancer karyotype.

Karyotypic commonalities. Table 1 shows that A13B has 49, A13A has 48 and A13D has 42 such clonal chromosomal units. Table 1 further shows that all three A13-cancers share 18 highly

the karyotype of the cancer and that of the normal cell from which it evolved.17,51,54-57 The ranges of subspeciations are, however, limited by the conservation of certain as-yet-undefined karyotypic elements that maintain the autonomy of the parental cancer and its subspecies.17 Figure 1 shows graphically the ranges of subspeciations within the apparent borders set by these conserved karyotypic elements that maintain the autonomy of the parental cancer and its subspecies. The spe-ciation theory thus predicts that metastases are individual species with species-specific karyotypes and phenotypes.

Testing the theory that metastases are subspecies of cancers. To test the theory that metastases are subspecies of cancers, we compared the karyotypes of several metasta-ses to those of two parental pancreatic cancers that were recently isolated in the labs of Iacobuzio-Donahue and Vogelstein at Johns Hopkins University.29 We found (1) metastases and paren-tal cancers have closely related karyotypes that differ in multiple individual chromosomal units (defined in Abstract and Results, “Karyotypic and phenotypic relationships between the pancreatic cancer A13B and two metastases, A13A and A13D”); (2) metas-tases from two different pancreatic cancers are not karyotypi-cally related, and (3) multi-chromosomal rearrangements with as many new chromosomal units as in authentic metastases occur spontaneously in the cancer cells studied here. These results sup-port the theory that metastases are subspecies of cancers, and that simultaneous multi-chromosomal rearrangements may generate metastases stochastically, much like new species.

Results

To test the relationships between several metastases and two pri-mary pancreatic cancers predicted by our theory, we have com-pared here their karyotypes and phenotypes, such as cellular morphologies and growth rates.

Karyotypic and phenotypic relationships between the pan-creatic cancer A13B and two metastases, A13A and A13D. Phenotypes. Cell cultures of the concurrent pancreatic cancers, A13B and A13A and the lung metastasis, A13D are shown in Figure 2. Among the two pancreatic cancers, A13A is thought to be a metastasis of the much larger primary cancer A13B (Materials and Methods). It can be seen in this figure that the cells of the pancreatic cancer and those of the metastases are morpho-logically closely related. The A13A and A13D cells are, however, more homogeneous than those of A13B. In addition, the A13A and A13D cells grew about two times faster than the A13B cells. Metastasis would, indeed, typically select for the fastest-growing variants from a heterogeneous parental population of cancer cells, since it is a form of clonal selection. In view of this, we hypothesize A13B is the parental cancer and A13A is a pancreatic metastasis (see below). This hypothesis would also explain why A13B is more heterogeneous than the resulting metastases, A13A and A13D.

Figure 2. The morphology of cells of the pancreatic cancer A13B, a pancreas metastasis A13A and a lung metastasis A13D. The cells were grown in vitro as described in Materials and Meth-ods and photographed at 120x magnification with a phase contrast microscope.

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Figure 3. Karyographs of the pan-creatic cancer A13B, a corresponding pancreatic metastasis A13A and a lung metastasis A13D. Karyographs are 3-dimensional tables showing the chromosome numbers of about 20 cells on the x-axis, the chromo-some copy numbers on the y-axis and the numbers of metaphases arrayed for comparison to each other on the z-axis. By comparing 20 karyograms at once, karyographs immediately distinguish clonal from non-clonal chromosomes. The karyographs and the corresponding cytogenetic analyses (Tables 1 and 2) show that all three cancers were closely related, as predicted by our theory that metastases are subspecies of cancers. Moreover, the karyographs and tables show that the two metastases and the parental cancer each contained chromosomes with individual clonal copy numbers and clonal marker chromosomes. All three variant cancer species also contained cells with individual, non-clonal marker chromosomes (see Tables 1 and 2).

(80 to 100%) clonal A13 group-specific chromosomal units, which include 10 intact chromosomes with A13 group-specific copy numbers and eight A13 group-specific marker chromosomes (see also Fig. 3). Similar pair-wise com-parisons show that A13B shares 22 chromosomal units with A13A and 29 chromosomal units with A13D.

These karyotypic relationships confirm and extend preliminary cytogenetic analyses performed by one of us (C.I.-D.), which identi-fied nearly the same numbers of chromosomal units per cancer as in Table 1. Moreover, the preliminary study confirmed the A13-group-specific nullisomy 8, monosomy 13, trisomy 16, polysomy 20 and monosomy 22 shown in Table 1. Further, it identified seven of the eight A13 group-specific markers named here, der(1;5), der(1;11), der(7;18), der(8;17), der(9), der(13;8) and der (15) in Table 1.

Differences between the karyo-types of metastases and parental cancer. In the following we have quantitated the karyotypic dif-ferences between the metastases and the parental pancreatic cancer

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Figure 4. For figure legend, see page 1156.

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chromosomal units, we compared the two liver metastases of an unknown primary pancreatic cancer, A2.1 and A2.2 (Materials and Methods).

Phenotypes. As can be seen in Figure 5, the cells of both A2-metastases have similar epithelial morphologies. If compared as populations, the A2.2 cells grew about two times faster than the A2.1 cells.

Karyotypic relationships between metastases A2.1 and A2.2. The karyographs of the two pancreas-derived liver metastases, A2.1 and A2.2, and the corresponding cytogenetic data show that the two metastases are closely related (see Fig. 6 and Table 2). For example, the total numbers of highly (>50%) clonal chromo-somal units of A2.1 were 37 and those A2.2 were 35 (Table 2). Moreover, the two metastases shared 25 chromosomal units. These 25 units included 16 intact chromosomes with highly clonal (over 85%) A2 group-specific copy numbers and nine A2 group-specific marker chromosomes. The preliminary Johns Hopkins-study had found seven of these nine marker chromo-somes, termed der(6;15), der (1;10), der(18;3), der(18;17), der (19;12), der(5) and i(7p) (Table 2).

Karyotypic individualities. Table 2 and Figure 6 also show that the two A2-metastases each contained seven individual intact chromosomes with highly clonal (> 50%), metastasis-specific copy numbers. Individually, A2.1 also contained four highly clonal A2.1-specific marker chromosomes, whereas A2.2 contained three such A2.2-specific markers (Table 2). The Johns Hopkins study had found previously two of the four A2.1-specific marker chromosomes, termed here der(14;1;19) and der(15), and two of the three A2.2-specific markers, termed here der(4) and der(15;3) in Table 2.

Based on these data, we deduce that A2.1 differs from A2.2 in 11 of 37 or 30% of the highly clonal chromosomal units of A2.1. The 11 A2.2-specific chromosomal units that set apart A2.1 from A2.2 were either differentially inherited from the unknown parental primary cancer or were generated during the evolution of A2.2 from the parental cancer or both. In short, A2.2 can be seen as derived from A2.1 through an unknown parent by replacing 11 of 37, or 30%, of the chromosomal units of A2.1 by A2.2-specific counterparts.

This result confirms and extends the above observation (Results, “Karyotypic and phenotypic relationships between the pancreatic cancer A13B and two metastases, A13A and A13D”) that relatively high percentages of the chromosomal units of a cancer species can be changed in the evolution of metastatic subspecies, while both species retain basic phenotypes, such as cell morphology, malignancy and autonomy. To understand the impact of this relatively high numerical discrepancy, it is necessary to consider that all of the chromosomal units that set apart either cancers from metastases or metastases from other metastases only

based on the number of parental chromosomal units replaced by metastasis-specific counterparts. On this basis we see in Table 1 that the metastasis A13A was generated from the parental cancer A13B by replacing 26 of its 49 chromosomal units either by new A13A-specific ones or by deletions (Table 1). Thus 26 of 49, or 53%, of the chromosomal units of A13B were changed to gen-erate the metastasis A13A. Similarly, the metastasis A13D was generated by replacing 23 of the 49, or 47%, of the chromosomal units of the parental cancer A13B either by new A13D-specific ones or by deletions.

We conclude that multi-chromosomal rearrangements gener-ated the metastases A13A and A13D from the parental cancer A13B. With the help of these karyotypic data we then tested the hypothesis advanced above that A13B is the parental cancer of both A13A and A13D.

Phylogenetic tree of the metastases A13A and A13D orig-inating from the primary pancreatic cancer A13B. Sharing a marker (hybrid) chromosome is nearly unambiguous evidence for a phylogenetic relationship between two cells because of the very low probability of forming the same hybrid chromosome twice by random karyotypic recombinations. Based on this principle, we asked whether differentially inherited marker chromosomes could confirm the above-mentioned hypothesis, that the distinct pancreatic cancer A13A was a metastasis of the apparent parental pancreatic cancer A13B. In our case the parental cancer A13B would share some specific marker chromosomes only with A13A and others only with the A13D. In other words, descendants could have selectively inherited parental marker chromosomes that they do not share with each other.

As can be seen in Table 1 (top right column), A13B does indeed share one highly clonal and one sub-clonal marker solely with A13A, namely, der(1;12) and der(5;20). Further down, the Table shows that A13B shares four highly clonal markers only with A13D, namely der(14;8p), i(12p), i(10p) and i(2p). The Johns Hopkins group mentioned above has previously identified two of these, i(12p) and i(10p). By contrast A13D and A13A do not share any of these parental markers with each other. It follows that A13A and A13D are independent metastatic descendents of the parental cancer A13B.

The evidence that A13B has a few more chromosomal units than A13A and A13D and that its chromosomal units are per-centage-wise less clonal than those of A13A and A13D lends fur-ther support to our evidence that A13B is the parental cancer (see Table 1). So we conclude that A13B is the parental cancer of the A13-series.

Karyotypic and phenotypic relationships between two liver metastases of an unknown pancreatic cancer, A2.1 and A2.2. As a further test of our theory that metastases from the same cancer are related subspecies with common and individual

Figure 4 (See previous page). Karyograms of a typical cell of the pancreatic cancer A13B (A) and of a corresponding lung metastasis A13D (B). The cells of the metastasis A13D and the parental cancer A13B (Materials and Methods) were treated for 4 to 8 h with colcemid in cell culture before metaphase chromosomes were prepared as described in Materials and Methods. The metaphase chromosomes were spread on glass microscope slides by conventional methods (Materials and Methods). After incubation overnight at room temperature, the chromosomes were hybridized on the slides with chromosome-specific, color-coded DNA probes to facilitate karyotyping following published procedures that are described in Materials and Methods. As one can see by comparing these karyograms with the population of 20 such karyograms listed in Table 1, each cancer cell contained clonal and individual non-clonal copy numbers of intact chromosomes and marker chromosomes.

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Table 1. Numbers of clonal intact and marker chromosomes of pancreas cancer A13B, and metastases, A13A and A13D

Cancer Clones A13A A13B A13D

A13A A13B A13D Chromosome no. Copy number (% clonal)

Clonal chromosome numbers der (1;12) 1(95) 1(90) 0

48 49 42 der(5;20) 1(15) 1(5) 0

Chromosome no. Copy number (% clonal) der(14;8p) 0 1(95) 1(95)

1 1(75) 2(55) 2(80) i(12p) 0 1(95) 1(100)

2 2(85) 1(80) 2(90) i(10p) 0 1(85) 1(70)

3 2(100) 2(80) 2(100) i(2q) 0 1(80) 1(95)

4 2(90) 2(90) 2(100) der(14;19?;16q) 1(100) 0 0

5 1(75) 1(15) 1(90) der (3;9) 1(100) 0 0

6 3(95) 3(80) 3(95) der (8;5) 1(95) 0 0

7 3(85) 3(55) 3(95) der (8) 1(95) 0 0

8 1(5) 0 0 der (12;16) 1(95) 0 0

9 1(95) 1(80) 1(95) der(17;19) 1(95) 0 0

10 1(80) 2(65) 1(80) der (17p;19) 1(85) 0 0

11 1(95) 2(75) 1(80) der (2;10) 1(80) 0 0

12 2(95) 2(70) 2(95) der (2;19) 1(80) 0 0

13 1(90) 1(70) 1(95) der (10;2) 1(75) 0 0

14 1(90) 1(90) 1(90) der (10;3) 1(70) 0 0

15 2(90) 1(90) 1(90) der (1;18) 1(50) 0 0

16 2(85) 3(80) 3(100) der(3;1) 1(40) 0 0

17 1(90) 2(85) 2(95) der (1;3) 1(25) 0 0

18 2(85) 1(80) 2(95) i(10q) 1(10) 0 0

19 1(50) 2(80) 2(80) der(5;12) 0 1(100) 0

20 5(45) 4(75) 4(100) der(14;8q) 0 1(100) 0

21 2(95) 3(70) 2(90) der(10;12) 0 1(95) 0

22 1(100) 1(95) 1(100) dmin(18) 0 1(95) 0

X 1(100) 1(95) 1(100) der(1;2) 0 1(95) 0

Y 1(100) 1(90) 0 i(19;12;19) 0 1(85) 0

der (1;5) 1(100) 1(90) 1(90) der(2;5) 0 1(85) 0

der (1;11) 1(100) 1(90) 1(100) der(5;3) 0 1(80) 0

der (14;16p) 1(100) 1(5) 1(90) der(4;15) 0 1(75) 0

der (7;18) 1(95) 1(80) 1(100) i(18q) 0 2(65) 0

i(11p) 1(95) 1(5) 1(75) i(11q) 0 1(60) 0

der (8;17) 1(95) 1(85) 1(95) der(11) 0 1(20) 0

der(9) 1(95) 1(90) 1(95) der(19;21) 0 1(10) 0

i(15q) 1(95) 1(90) 1(80) der (8;10) 0 0 1(100)

i(5q) 1(95) 1(90) 1(5) der (12;22;19) 0 0 1(95)

i(5p) 1(85) 1(10) 1(80) der(1;5q) 0 0 1(90)

der (13;8) 1(85) 1(85) 1(95) der (15;3) 0 0 1(90)

der(15) 1(85) 1(90) 1(85) i(12q) 0 0 1(90)

der (19;12;19) 1(10) 1(80) 1(80) der(10q) 0 0 1(45)

der(1) 1(5) 1(20) 1(85) der(10;15) 0 0 1(25)

der(20;19) 0 0 1(15)

der(10;19) 0 0 1(25)

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alter the copy numbers of shared chromosomal regions or genes quantitatively rather than qualitatively (see Tables 1–4). Furthermore, the comparisons between the related cancers and metastases studied here also show that there is an upper border of chromosomal varia-tions allowed for sub-speciation, which limits the range of exchanges of chromosomal units to about 50% (see also Fig. 1).

How are the new multi-chromosomal rearrangements of metastases generated in cancer cells? The high 30 to 53 percent-ages of chromosomal units of cancers that are replaced in the generation of metastases raise the question whether the underlying chromo-somal rearrangements happen sequentially or simultaneously.

Sequential accumulations of multiple chro-mosomal alterations would depend on mul-tiple intermediates consisting of non-clonal karyotypes, namely intermediates between the clonal and stable karyotypes of the parental cancers and the resulting metastases. Non-clonal karyotypic variants of clonal cancers are, however, unstable.17 The instability of non-clonal karyotypic variants of cancers has been shown here in Tables 1 and 2 and, specif-ically, in Tables 3 and 4 (below) as well as pre-viously by others and us in references 17, 48, 51, 53, 55, 58–60. Thus sequential multi-step accumulations of non-clonal karyotypic vari-ants of cancer cells would be highly unlikely as a mechanism to generate metastases differ-ing from parental cancers in multiple chromo-somal units.

The alternative, simultaneous mechanism of metastasis proposes a saltational origin of multiple unstable chromosomal units, in which certain stochastic combinations of non-clonal chromosomal units would be stabilized by the new autonomy of a new metastasis or other subspecies. This view is compatible with the observation that the origins of metastases are “discontinuous,” “unpredictable,” “nonlin-ear,” “abrupt,” “punctuated” or “stochastic,” as described in the Introduction.

In contrast to sequential karyotypic varia-tions, this view predicts simultaneous multi-chromosomal rearrangements. To test this prediction, we set out to determine the fre-quency of new non-clonal and multi-chromo-somal rearrangements in the cancers studied here. As a basis for the detection of such non-clonal rearrangements, we first determined the stability of the clonal karyotypes of these can-cers over extended cell generations.

Figure 5. The morphology of the cells of two pancreatic liver metastases, A2.1 and A 2.2 of an unidentified parental cancer. The cells were grown in vitro as described for Figure 2, and photographed at 120x magnification with a phase contrast microscope.

Figure 6. Karyographs of two liver metastases, A2.1 and A2.2, of an unidentified parental cancer. The karyographs and the corresponding cytogenetic analyses listed in Tables 3 and 4 show that the karyotypes of both metastatic cancers were clonal and flexible, much like those of other cancer species.17 The karyographs and tables further show that the karyo-types of the two metastases were closely related to the parental cancer and to each other, as predicted by our theory that metastases are subspecies of cancers. At the same time, both metastases and the parental cancer each had individual clonal chromosome copy numbers and clonal marker chromosomes. Moreover, all three variant cancer species contained cell-specific non-clonal marker chromosomes in most of their cells (see text).

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Karyotypic stability within clonal margins. The karyotypic sta-bility of the pancreatic cancers studied here can be traced back 30, even 60, generations from their known clinical history as fol-lows. Since these cancers and their metastases were each over 1 g by the time they were isolated, we deduce that their individual stem cells had each undergone at least 30 cell generations in vivo by the time they were isolated. This follows, because 1 g of tis-sue corresponds to about 109 cells or 230 doublings or 30 genera-tions of the stem cell.61 In the specific case of A13B, which was of “grapefruit size” or about 250 g by the time it was isolated, the clonal age corresponds to about 38 cell generations.

After these 30 to 38 independent generations in vivo, the clon-alities of the many chromosomal units shared by the two metas-tases and the parental A13-cancer were exactly the same, namely, between 70 to 100% (Table 1 and Fig. 3). The same argument of clonal stability also applies to the many common, cancer-specific chromosomal units of the two A2-metastases (Table 2 and Fig. 6). Considering that the clonalities of the shared chromosomal units of the metastases and their parental cancers were practically iden-tical (Tables 1 and 2), we can say that the shared chromosomal units have in fact been stable for over 60 generations.

This means that all shared chromosomal units and thus prob-ably the whole clonal karyotypes of these cancers were highly stable over 60 generations. It should thus be possible to detect new, non-clonal chromosomes arising spontaneously in these cancer cells.

Accordingly, we asked next whether the pancreatic cancers studied here are sufficiently flexible to generate simultaneously new multi-chromosomal rearrangements with as many new chro-mosomal units as those that set apart the metastases from their parental cancers.

Karyotypic variability from without clonal margins. To estimate the range and frequency of spontaneous multi-chromosomal vari-ations in the cancers studied here, we have listed all non-clonal chromosomal alterations associated with 20 individual cells of the metastatic pancreatic cancers A13D and A2.1 in Tables 3 and 4.

As can be seen in Table 3, 55% of 20 A13D cells each con-tained between 1 and 6 (!) new, non-clonal or cell-specific marker chromosomes. Similarly, it can be seen in Table 4 that 25% of 20 A2.1 cells each contained between 1 and 4 new, non-clonal or cell-specific marker chromosomes. (In a previous study of a highly aneuploid bladder cancer, we even found a metastatic cell with eight new, non-clonal marker chromosomes).17 None of these new, non-clonal markers were found in subsequent or par-allel analyses of these cancers here and previously in reference 51. The non-clonality thus indicates that these karyotypic variants are unstable and consequently lost in subsequent generations. It would follow that single and multiple marker chromosomes arise de novo in mitoses of 25 to 60% of the pancreatic cancer cells studied here.

Among the cells with new non-clonal chromosomal units studied here, metaphase 19 of A13D holds a record of newly acquired complexity (Table 3). Metaphase 19 of A13D includes as many 24 new, cell-specific chromosomal units, including six new non-clonal markers and 18 intact chromosomes with non-clonal copy numbers. Metaphase 19 thus differs from the clonal

Table 2. Numbers of clonal intact and marker chromosomes of two liver metastases of an unknown pancreas cancer, A2.1 and A2.2

Cancer clones

A 2.1 A 2.2

Clonal chromosome numbers

37 35

Chromosome no. Copy number (% clonal)

1 1(85) 1(100)

2 3(95) 2(100)

3 2(100) 1(100)

4 2(100) 2(95)

5 3(80) 2(100)

6 2(100) 2(100)

7 2(70) 2(85)

8 3(80) 3(95)

9 2(85) 2(100)

10 2(100) 2(90)

11 3(95) 3(95)

12 2(80) 2(100)

13 4(55) 3(90)

14 2(60) 2(100)

15 1(100) 1(90)

16 2(90) 2(65)

17 2(85) 2(95)

18 2(90) 1(95)

19 1(90) 1(90)

20 3(85) 4(95)

21 2(65) 1(90)

22 2(80) 2(100)

X 2(100) 2(100)

Y 0 0

der(6;15) 1(100) 1(95)

der(1:10) 1(95) 1(100)

der(1;16;21;1) 1(95) 1(100)

der(18:3) 2(90) 2(95)

der(1:16) 1(90) 1(95)

der(18:17) 1(85) 1(100)

der(19:12) 1(85) 1(100)

der(5) 1(80) 1(100)

i(7p) 1(70) 1(90)

der(14;1;19) 1(95) 0

der(20;15) 1(90) 0

der(15) 1(85) 0

der(1:22) 1(80) 0

der(16:20) 1(45) 0

der(21:7) 1(10) 0

der(4) 0 1(95)

der(16;1;9) 0 1(90)

der(15;3) 0 1(100)

min(10) 0 1(15)

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Table 3. Karyograms of 20 metastatic A13D carcinoma cells

Total number of A13D chromosomes per cell

62 64 61 64 61 64 63 61 62 63 63 62 63 61 62 62 63 61 56 62

Metaphase number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Chromosome no. Chromosome copy number Copy number (% clonal)

1 2 2 2 2 2 2 2 1 2 2 3 2 2 2 1 2 2 2 1 2 2 (80)

2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 (90)

3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 (100)

4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 (100)

5 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 0 1 1 (90)

6 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 3 3 (95)

7 3 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 (95)

8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

9 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (95)

10 1 1 1 2 1 1 1 1 2 1 0 1 1 1 1 1 1 1 2 1 1 (80)

11 1 1 1 2 2 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 1 (80)

12 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 (95)

13 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (100)

14 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 0 1 1 (90)

15 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 (90)

16 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 (100)

17 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 (95)

18 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 2 2 2 2 2 2 (90)

19 2 2 1 2 1 2 2 2 2 2 2 2 2 1 1 2 2 1 2 1 2 (70)

20 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 (100)

21 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 (90)

22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (100)

X 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (100)

Y 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Clonal Markers

der(1;5p) 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 (90)

der(1;11) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (100)

der(14;16p) 1 1 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 (90)

der(7;18) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (100)

i(11p) 1 1 1 0 0 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 (75)

der(8;17) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 (95)

der(9) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 (95)

i(15) 1 0 1 0 1 1 1 0 1 1 1 0 1 1 1 0 1 1 1 1 1 (75)

i(5p) 1 1 1 1 1 1 1 1 1 1 0 1 1 1 2 1 1 1 0 1 1 (85)

der(13;8) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (100)

der(15) 0 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (85)

der(19;12;19) 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 1 0 0 1 (80)

der(1) 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 (90)

der(14;8p) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 (95)

i(12p) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (100)

i(10p) 1 1 1 1 0 1 1 1 0 1 1 1 1 1 0 1 0 1 0 1 1 (75)

i(2q) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 (95)

der(8;10) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (100)

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we analyzed above. The evidence that some multi-chromosomal variants have become sub-clonal in several cells of A13D and A2.1 (Tables 3 and 4) further supports the theory that some new multi-chromosomal rearrangements can be stabilized as new subspecies by selection for autonomy. It is thus likely that the metastases we analyzed here were generated by simultaneous multi-chromosomal rearrangements from parental cancers.

Discussion

In an effort to answer (1) why the karyotypes of metastases are related to those of parental cancers but not to those of other can-cers and (2) why cancers metastasize at rates that often far exceed

average of A13D in 24 of 42 (57%) non-parental chromosomal units and thus in as many chromosomal units as the A13 metas-tases A13D and A13A differ from the parental cancer A13B, namely, in 23 of 49 (47%) and in 26 of 49 (53%) units, respec-tively (Tables 1 and 2).

In addition, we found four apparently new, sub-clonal marker chromosomes in 10%, 15% and 25% of the A13D cells, respec-tively (Table 3), and two apparently new, sub-clonal markers in 10% and 45% of the A2.1 cells, respectively (Table 4).

In sum, we deduce from these data that the stochastic appear-ances of new multi-chromosomal rearrangements in the cancers studied here is proof-of-principle that such multi-chromosomal rearrangements could also generate the kind of metastases that

Table 3. Karyograms of 20 metastatic A13D carcinoma cells

Clonal Markers (continued)

der(12;22;19) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (100)

der(1;5q) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 (95)

der(15;3) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (100)

i(12q) 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 (90)

der(10q) 1 1 1 0 0 1 0 1 1 1 1 0 1 0 0 0 1 0 0 0 1 (50)

Sub-Clonal Markers

der(10;15) 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 (15)

der(20;19) 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 (10)

der(10;19) 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 1 1 0 1 1 (25)

Non-Clonal Markers

dmin(22) 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 (5)

der(7p) 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 “

der(14;5) 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 “

der(5;10) 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 “

der(9;2) 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 “

der(10;21) 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 “

der(1;15) 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 “

der(?;2) 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 “

der(5) 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 “

der(17p) 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 “

der(10;19) b 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 “

der(10;19) c 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 “

der(20;12) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 “

der(2;10p) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 “

der(18;19) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 “

der(1;2) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 “

der(8;9) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 “

der(19;2) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 “

der(10;14) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 “

der(9;12) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 “

der(6;11) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 “

der(3;8;14) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 “

der(19) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 “

der(10) 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 “

der(7) 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 “

(continued)

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Table 4. Karyotypes of 20 cells of the liver metastasis A2.1 of a pancreatic cancer

Total number of A2.1 chromosomes per cell

63 63 64 65 61 64 65 66 64 66 61 63 61 72 60 62 62 61 66 66

Metaphase number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Chromosome no. Chromosome copy number Copy number (% clonal)

1 1 1 1 1 1 1 1 1 0 2 1 1 1 2 1 1 1 1 1 1 1 (85)

2 3 3 3 3 3 3 3 3 2 3 3 3 3 3 3 3 3 3 3 3 3 (95)

3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 (100)

4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 (100)

5 3 3 3 3 2 2 3 2 3 3 3 2 3 3 3 3 3 3 3 3 3 (80)

6 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 (100)

7 2 2 1 2 2 2 2 2 2 2 3 3 2 3 2 2 2 2 3 3 2 (70)

8 3 3 3 3 2 3 3 3 4 3 3 3 2 3 3 3 2 3 3 3 3 (80)

9 2 2 2 2 2 2 3 2 2 2 2 2 2 2 1 2 2 2 3 2 2 (85)

10 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 (100)

11 3 3 3 3 3 3 3 3 3 3 3 4 3 3 3 3 3 3 3 3 3 (95)

12 2 2 2 2 2 2 2 2 2 1 3 3 2 3 2 2 2 2 2 2 2 (80)

13 3 3 4 4 3 4 3 4 4 4 3 3 3 4 2 4 4 3 4 4 4 (55)

14 2 2 2 2 2 2 3 2 1 1 1 2 1 3 2 1 1 2 2 2 2 (60)

15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (100)

16 2 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 (90)

17 2 2 2 2 2 2 2 2 2 3 2 2 2 3 2 2 1 2 2 2 2 (85)

18 2 2 2 2 2 2 2 2 3 2 2 2 1 2 2 2 2 2 2 2 2 (90)

19 1 1 1 1 1 1 1 1 1 1 2 1 2 1 1 1 1 1 1 1 1 (90)

20 3 3 3 3 2 3 3 3 3 3 2 3 3 4 3 3 3 3 3 3 3 (85)

21 2 1 2 2 1 2 2 2 2 2 2 1 2 2 2 1 2 3 1 1 2 (65)

22 2 2 2 2 2 2 2 1 2 2 2 1 2 3 2 1 2 2 2 2 2 (80)

X 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 (100)

Y 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Clonal Markers

der(6;15) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (100)

der(1:10) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 (95)

der(1;16;21;1) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 (95)

der(18:3) 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 1 2 2 2 (90)

der(1:16) 1 1 0 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 (90)

der(18:17) 1 1 1 1 1 1 1 1 1 0 1 1 1 0 1 1 1 0 1 1 1 (85)

der(19:12) 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 0 1 1 1 1 (85)

der(5) 1 1 1 1 1 1 1 2 2 1 0 1 1 1 1 1 0 1 1 1 1 (80)

i(7p) 1 1 2 1 1 1 1 1 2 1 0 1 1 1 0 1 1 1 0 0 1 (70)

der(14;1;19) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 (95)

der(20;15) 1 1 1 1 1 1 1 1 2 1 1 1 1 2 1 1 1 1 1 1 1 (90)

der(15) 1 1 1 1 1 1 0 1 1 1 1 1 1 0 1 1 1 2 1 1 1 (85)

der(1:22) 1 2 1 1 1 1 1 1 0 1 1 1 1 2 1 2 1 1 1 1 1 (80)

Sub-Clonal Markers

der(16:20) 0 0 1 0 1 1 1 1 0 1 0 0 0 0 0 1 0 0 1 1 1 (45)

der(21:7) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 (10)

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By extension, the speciation theory further predicts that metastases may share specific gene mutations with parental can-cers, particularly since metastases are only about 30 cell genera-tions away from parental cancers by the time they are diagnosed and analyzed. This prediction has recently been confirmed by targeted sequencing of several oncogenes including Kras70 and by whole-genome sequencing in several labs, including that of two of us (C.I.-D. and J.B.), which sequenced the pancreatic cancers studied here in references 27–29 (see Introduction and Materials and Methods). For example, Tie et al. found that the “Mutation status was highly concordant between primary cancer and metas-tasis from the same individual.”70

By contrast, the common “metastasis-specific, per se,”29 muta-tions or metastasis-specific chromosome alterations, predicted by conventional mutational theories62 have not been found here nor in recent comparisons of the sequences and chromosome copy numbers of metastases.27,29,31-33

Evidence for saltational origins of metastases from cancers. In view of (1) the stochastic origins of metastases (Introduction, “Kinetics of the origins of metastases from cancers”) and (2) the difficulties of reconciling the many non-clonal karyotypic inter-mediates predicted by sequential mutational theories with the intrinsic instability of non-clonal and thus non-autonomous karyotypes, we have proposed here a saltational mechanism of origin for metastases (Results, “How are the new multi-chromo-somal rearrangements of metastases generated in cancer cells?”). According to this proposal, stochastic, simultaneous multi-chromosomal rearrangements of cancer karyotypes generate new autonomous subspecies, such as metastases.

This proposal is based on the multi-chromosomal differences between metastases and parental cancers described here and on the intrinsic flexibility of the cancer karyotypes described here and in other studies previously (Introduction). In addition, our proposal borrows from classical evolutionary theories.71-73

Testing this view, we found that non-clonal (and thus unsta-ble) karyotypic rearrangements with as many new chromosomal units as in authentic metastases arise spontaneously in the cancer cells studied here (Tables 3 and 4). Thus stochastic multi-chro-mosomal rearrangements can be seen as proof-of-principle for the saltational theory of metastasis.

those of conventional mutations, we have tested here a new the-ory of metastasis. This theory postulates that metastases are sub-species of cancers. Based on experimental tests conducted to test this theory, these two questions can now be answered as follows.

Metastases karyotypically related to parental cancers but not to others. The speciation theory of metastasis predicts that metastases are subspecies of cancers and thus phylogenetically related to those of parental cancers. At the same time, this the-ory predicts that the karyotypes of metastases from independent cancers are not related to each other. In short, the theory holds that metastases are individual species with individual karyotypes and phenotypes, which are related to those of parental cancers but not to those of independent cancers.

This phylogenetic metastasis theory thus explains the many karyotypic relationships between metastases and parental cancers described by us here and those listed in the Introduction.1-18

The theory further explains that the species-specific indi-viduality of each metastasis is the probable reason why so many searches for metastasis-specific commonalities, predicted by mutational theories,62 have been unsuccessful.63 The following examples illustrate this point: (1) An extensive comparison of the transcriptomes of 65 breast cancers found that, “These pat-terns provided a distinctive molecular portrait of each tumor.” But, “Gene expression patterns in two tumor samples from the same individual were almost always more similar to each other than either was to any other sample.”64 A similar comparative study also ended up with numerous metastasis-specific tran-scripts.65 (2) Another study of breast cancers observed, “primary tumors and M1 cells (metastases) harbored different and char-acteristic chromosomal imbalances.”66 (3) A recent comparison of the chromosome copy numbers of primary medulloblastomas and metastases showed “in each case we observed clonal genetic events in the metastatic tumor(s) that were not present in the matched primary tumor. We also observed genetic events in the primary tumor that were absent from the matched metastases.”67 (4) Many comparative biochemical and therapeutic assays have observed complex individual phenotypes setting apart metastases from each other and from parental cancers.63,68,69 (5) Accordingly, a recent review points out “the distinct programs governing met-astatic colonization may number in the dozens.”23

Table 4. Karyotypes of 20 cells of the liver metastasis A2.1 of a pancreatic cancer

Non-Clonal Markers

der(5q) 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 (5)

der(6;22) 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 “

der(?) 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 “

der(15:14) 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 “

der(12:16) 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 “

der(10:1:16) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 “

der(12:19:8) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 “

der(1:2) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 “

der(8) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 “

der(6:17) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 “

der(7p) 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 “

(continued)

1164 Cell Cycle Volume 11 Issue 6

Materials and Methods

Pancreatic cancer cells and metastases. The pancreatic cancers and corresponding metastases studied here have been recently isolated and described by Jones et al.30 and Yachida et al.29 Specifically, we have analyzed here the concurrent pancreatic can-cers A13B and A13A and a lung metastasis A13D. The primary A13B was bigger than a grapefruit by the time the much smaller A13A cancer was isolated as a peripheral subclone or metastasis. A13D was sequenced and termed Pa04C by Jones et al.30 and Yachida et al.29 In addition, we have analyzed two liver metasta-ses of an unknown parental pancreatic cancer, A2.1 and A2.2. A2.1 has been sequenced and termed Pa01 by Jones et al.30 and Yachida et al.29

Karyotyping cancer cells by hybridization of metaphase chromosomes with chromosome-specific color-coded DNA probes. One to two days before karyotyping, cells were seeded at about 50% confluence in 3 mL RPMI 1640 medium containing 5% fetal calf serum in a 5-cm culture dish. After reaching ~75% confluence, Colcemid (KaryoMax Colcemid solution; Gibco Invitrogen) was added to 50 ng per mL medium. The culture was then incubated at 37°C for 4–8 h. Subsequently, cells were dis-sociated with trypsin, incubated in 0.075 molar KCl at 37°C for 16 min, fixed with freshly mixed glacial acetic acid-methanol (1:3, vol. per vol.) and pipetted with plastic pipette tips onto microscope slides following published procedures.50,51 Slides with suitable metaphase chromosomes were then hybridized with chromosome-specific, color-coded DNA probes as described by the manufac-turer (MetaSystems) and by us previously in references 50 and 51.

Conclusions

Based on the inherent flexibility of cancer karyotypes and on the close karyotypic relationships between metastases and parental pancreatic cancers, we have advanced here the theory that metas-tases are autonomous subspecies of cancers. Testing this theory, we found that metastases are karyotypically related to parental cancers but unrelated to independent cancers. Metastases dif-fered from the parental cancers studied in 30 to 50% of their clonal chromosomal units. Since non-clonal karyotypic vari-ants of autonomous cancers, which sequential mutation theo-ries postulate as intermediates between cancers and clonal subspecies, are unstable, we propose here that simultaneous multi-chromosomal alterations of cancers generate metastases. Testing this view, we found in the pancreatic cancers studied here spontaneous, non-clonal rearrangements with as many new chromosomal units as in authentic metastases. We conclude that metastases are autonomous subspecies of cancers with clonal, species-specific karyotypes and phenotypes, which are generated by simultaneous multi-chromosomal rearrangements, much like conventional species. The species-specific individualities of metastases explain why so many searches for commonalities have been unsuccessful.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Consistent with a valid theory, the saltational multi-chromo-somal theory of the origin of metastases would directly explain the peculiar kinetics of metastasis, namely, that they are unex-pectedly “rapid”43 or arise at “high rates”44 and at “early and late stages of genetic divergence,”40 that they unpredictably change via “nonlinear progressions”8,15 and follow a “nongenetic mecha-nism”38 and “abrupt”36 or “punctuated clonal expansions”24 and generate “unpredictable” phenotypes36,37 as originally described by Foulds and then in other studies listed in the Introduction.

The saltational origins of subspecies of cancer can also solve the alleged “progression puzzle,”23,74 namely, the origins of pheno-types that do not confer selective advantages to the parental can-cer such as metastasis and drug-resistance. These puzzles derive from the theory that multiple sequential mutations generate such multigenic phenotypes. In contrast, the saltational theory pre-dicts that multiple unselected, and thus unpredictable, pheno-types hitchhike with the chromosomal rearrangements that are the basis for subspeciations.57,75

The multi-chromosomal origin of metastases from cancers—a model for the origin of cancers from normal cells? Since cancers have individual, clonal karyotypes that often differ from those of the normal cells of origin in many new chromosomal units,17,76 it seems likely that they also arise by a multi-chromosomal saltational mechanism, rather than by the sequential evolution of multiple, unstable karyotypic intermediates. The logic of this argument is in fact the same as that advanced above against sequential evolu-tion of multiple non-clonal karyotypic precursors of metastasis. The only difference would be that carcinogenesis starting from highly stable normal karyotypes would be much less likely, and thus slower, than metastasis from flexible cancer karyotypes.

Indeed, Peyton Rous was perhaps the first to make a case for a saltational origin of cancers in 1959, at least indirectly.77 In this paper Rous specifically objected to “sequential mutations” as the cause of cancer, because (1) the origins of cancers are “abrupt and discontinuous episodic phenomena,” and because (2) there are no intermediates; “most fatal human cancers are far removed from the normal in character and almost no growths fill the gap in between, much less a gradual series of them, as one might expect were they the outcome of random somatic mutations.” Rous fur-ther argued against mutations, because none have been found that allow cells to “proliferate independently of the organism,” which defines cancer according to Rous and Julian Huxley.78 The lack of functional proof for somatic mutations that can generate autonomous growth remains an unmet obligation of the muta-tion theory to this very day.17,79-81 Nevertheless, the proponents of the mutation theory have singled out this very paper as the Achilles heel of Rous’ scientific career.82-84

Final answers to Rous’ challenge would, however, depend on an experimental system that allows fast evolutions of new cancer karyotypes. Since the end-results of the evolutions of new cancers from normal cells and of the evolutions of new metastases from cancers are the same, namely, the generation of new autonomous karyotypes, we propose that the evolution of metastases from cancers can be seen as a time-lapse model of the evolution of cancers from normal cells and possibly even of new species from normal precursors.71-73,85,86

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Acknowledgements

We gratefully acknowledge Bert Vogelstein (Johns Hopkins Medical Institutions, Baltimore) for encouragement and for offer-ing cultures of the pancreatic cancers and metastases studied here. We thank Kavon Karrobi [University of California at Berkeley (UC Berkeley)] and Rainer Sachs (Dept. Mathematics and Physics, UC Berkeley) for critical reviews of the manuscript. Further we thank Alfred Boecking (Universitätsklinikum Düsseldorf, Duesseldorf, Gemany), Alice Fabarius (University of Heidelberg at Mannheim, Germany), Richard Harland (Dept. Molecular and Cell Biology, UC Berkeley), Ruediger Hehlmann (University of Heidelberg at Mannheim, Germany), Josh Nicholson (Virginia Polytechnic Institute and State University, Blacksburg, VA), Jerry Pollack (University of Washington, Seattle), David Rasnick (Berkeley,

former visiting scholar at UC Berkeley) and Richard Strathmann (University of Washington, Seattle) for valuable information and Josh Nicholson in particular for many timely alerts and critical discussions. Bong-Gyoon Han (Lawrence Berkeley Lab) is grate-fully acknowledged for the preparation of Figure 1. Our research would not have been possible without the generous support from the philanthropists Dr. Christian Fiala (Vienna, Austria), Rajeev and Christine Joshi (London, UK), Robert Leppo (San Francisco), Peter Rozsa of the Taubert Memorial Foundation (Los Angeles), Howard Urnovitz (Chronix Biomedical, San Jose, CA), a Foundation that prefers to remain anonymous, an anony-mous sponsor from Connecticut, other private sources and the Forschungsfonds der Fakultaet fuer Klinische Medizin, University of Heidelberg at Mannheim, Germany.

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