
ADVANCES in the study of human chromosomes in the past decade have produced a startling body of evidence that derangements in cellular chromosomal complement are responsible for several important human disease entities, such as Down's disease, Turner's disease, and others. In 1960 Nowell and Hungerford demonstrated a specific chromosomal abnormality characteristic of most cases of chronic granulocytic leukemia.

Over 60 years ago Boveri originally proposed the hypothesis that an imbalance in cellular chromosome content, no matter what physical, chemical, or biological mechanism was responsible for achieving it, might destine such cells to malignant behavior (Boveri, 1929) . In essence this was a proposed explanation of all forms of human cancer. Recently we presented evidence supporting Boveri's hypothesis of a possible specific chromosomal origin of human cancer (Gofman et al., 1967 ). An excess of E16 chromosomes was found in all of 7 human cell lines and in 2 human cancers studied directly.

The purpose of this communication is to provide the data from extensive further tests, in human material, of the relationship of excess E16 chromosome and human cancer.
EXPERIMENTAL

Material
For the study of human chromosomes and cancer the ideal material is fresh cancer tissue directly obtained from surgical specimens. The data for 11 such specimens will be presented. However, for reasons not clear at present, investigators throughout the world, ourselves included, have found that only 10-20% of human cancers lend themselves to such direct study. The difficulties have been of 2 types: (a) an adequate number of mitoses is not present in some of the fresh cancers obtained surgically, and/or (b) the quality of chromosome preparations from some cancers is poor. It is certainly to be hoped that future technological improvements will solve these two problems. At the moment they plague all such investigations. It might appear, superficially, that this problem is obviated through the simple expedient of studying a larger number of cancer cases. This, however, is not a satisfactory solution, for, when 80% of the material does not lend itself to study, the possibility of bias is ever present.
Material
Pending the ultimate technical solution of the problem of handling fresh cancers for chromosome analysis, there exists a supply of very pertinent other material of great relevance for the human cancer problem. This is in the form of the spontaneous human cell lines now available. Cell lines, of human origin, have arisen from explanted material either of non-malignant origin or of malignant origin (American Type Culture Collection, 1967) . Once these lines have become established, they are characterized by essential immortality, in contrast with the limited life-span of normal human cells in similar culture (Hayflick, 1965 ). Further, where tested, such immortal cell lines have been proved to show malignant properties, either by homotransplantation into humans or by heterotransplantation into hamsters (Southam et al., 1957; Handler and Foley, 1956) . Indeed, some investigators feel that cell lines in vitro are the malignancy equivalent to cancer in the living subject (Hayflick, 1965) . Whether this analogy is as thoroughgoing as this is subject to debate, and we shall by no means insist upon it here. It does appear clear, however, that such human cell lines have many properties of malignancy, and, hence, represent suitable material for evaluation of clues concerning human cancer. What is found concerning chromosomal regularities through the study of human cell lines cannot be properly claimed to be a characteristic of human cancers in vivo. But what regularities are found certainly deserve investigation in fresh cancers from humans as being possibly descriptive of a sine qua non of malignant cells. With this proviso in mind, and with no intention of claiming beyond the observations for what they are, the results for the study of chromosomal constitution of 17 human cell lines will be presented here (for every well-documented cell line available to us) (American Type Culture Collection, 1967) .
Methods
The technology for chromosome study by quantitative means is thoroughly described elsewhere (Gofman et al., 1967; Stone, 1967; Stone and Littlepage, 1967; Stone et al., 1969) . In essence, for these studies, human chromosomes are characterized by two parameters of measurement:
Methods
(a) Chromosome length. (b) Centromeric index, defined as the ratio of length of the short arms to the total chromosome length. We may refer to this as " Quantitative Karyotyping " Cla8ssification of chromosomes into the usual well-known " Denver " classes (A1, A2, A3, B, C, D, E16, E17, E18, F, G, X, and Y) is based upon (a) determination of the length and centromeric index parameters for such classes upon chromosomes from normal human material (blood lymphocytes and tissue fibroblasts), and (b) comparison of such parameters for cancer or cell line chromosomes with the limits established, in normals, for the various defined chromosome classes. The study of chromosomes in normal human tissues shows that variability is found to exist for chromosomes of a particular class, e.g. A1 class, both with respect to length and with respect to centromeric index. This variability is present even after a well-known correction designated as the " normalization " correction.
Methods
The need for a normalization correction in chromosome studies arises from the fact that during metaphase (the phase of cell mitosis in which chromosomes are measured), chromosomes undergo progressive shortening, presumably due to, coiling. As a result, the individual cells studied will vary from each other in the extent to which such metaphase " contraction" has occurred. Variability, even after a perfect normalization correction, is due to several factors; (a) errors of measurement, (b) biological variation, and (c) variability due to preparation of slides for study. Even with such sources of variability, it is possible to set up limiting values of length and of centromeric index so as to allow satisfactory " karyotyping " of chromosomes into the well-defined classes. A small proportion of " misclassifications " occur, and even these are correctable through inter-class correction factors, established for normals. Shown in Fig. 1(a) is a " cutting line " diagram which shows the boundary limits, both for length and for centromeric index, which define the various chromosome classes. Established to be consistent with "' Denver " karyotype classification in our first 1082 normals, it has continued to provide correct karyotypes in the subsequent 752 cases. We consider this cutting line diagram now quite satisfactory, and all cell lines and cancer data are calculated utilizing these cutting lines. Our studies indicate that it is difficult, if not impossible, to segregate E17 from E18 chromosomes. Accordingly, we classify them together as E(17-18) chromosomes. While the Denver System separates these into E17 and E18 chromosomes, inspection of published karyotypes makes the arbitrary separation, rather than measured differences, quite apparent. Also shown in Fig. 1(b) (Miles, personal communication) . All these suggestions are possible explanations. The fact remains, however, that label them whatever one wishes, chromosomes of constant dimensional characteristics and normalized to account for differential contraction consistently appear as a possible " specific common chromosomal pathway for the origin of human malignancy". Until there is some technological breakthrough which provides less debatable karyotypes than a study of past literature shows is produced by the much-vaunted " eye of the experienced cytogeneticist ", we shall for the sake of simplicity continue calling these objects " E-16", with full awareness that the genetic content of all such objects is currently unknown.
Numbers of cells studied
It is distressing that a large fraction of the cytogenetic literature is based upon studies of one or a few cells per case. If one considers the biological and technical variability inherent in the material, it is certain that numerous literature assignments into classes are in grave error. Furthermore, it is commonplace to find published karyotypes in which chromosomes with obviously similar measurements are found in differing assigned classes. It must be remembered that science is barely into the second decade of such work, and semi-quantitative approaches are common in the early explorations of a new and exciting field.
Numbers of cells studied
For our purposes, however, quantitation is the essence of the problem of evaluating Boveri's hypothesis. We must, therefore, study a sufficient number of cells, taking inevitable variability into account, such that the standard error of each chromosome frequency is sufficiently low that small differences can be perceived in comparing cell lines with normals or cancers with normals. We wish to be in such a position that if a cancer cell averages 05 chromosome or more per cell higher or lower than a normal cell, we can measure such a difference and be certain to a probability of less than 1 in 100 that the measured difference is real. human metaphases has demonstrated that certain limits of centromeric index and of len are never (or almost never) exceeded by chromosomes encountered in normal cells. Th( fore, chromosomes falling outside such limits are defined as marker chromosomes. In 1 manner marker classes 1, 3, 4, 5, and 15 are shown to have domains in the cutting line diagi based upon length and centromeric index. The remaining marker classes; 2, 6, 7, 8, 9, 11, 12, 13, and 14 have additional defining features shown in Fig. 1(b) . The various mar chromosomes are presumed to arise by several mechanisms, including deletions of a port of a chromosome, translocations of a part of one chromosome to another chromosome, intei breaks and rejoiniing within a single chromosome, and various combinations of these oc( rences. It is virtually impossible, other thari by sheer speculation, to know what nor chromosome genetic material is incorporated into any particular marker chromosc class.
Numbers of cells studied
Actually we study a sufficient number of cells so that even much smaller ences can, in general, be reliably determined. For cell lines or norma supply is essentially unlimited so that only labor precludes studying ver) numbers of cells. In the case of fresh cancers, the amount of tissue ai quality of the preparations may preclude obtaining an ideal number of c( study. In these instances, it is not possible to obtain additional materia particular case.
Numbers of cells studied
A word is indicated concerning the reporting of results on the chrom Bone marrow from an untreated case of chronic granulocytic leukemia One case of chronic granulocytic leukemia was studied. The chromosome preparations are from bone marrow, taken before any therapy was started. This case definitely shows the classical Philadelphia chromosome.
Numbers of cells studied
Cell line from Burkitt's lymphoma A chromosome preparation was made from a suspension culture (Jijoye) of a cell line derived from a case of Burkitt's lymphoma. This apparently malignant disease is of special interest because of the high likelihood of its being of viral origin.
The Experimental Results-Human Cell Lines
The Boveri concept hypothesizes a chromosomal imbalance that may be the sine qua non of malignancy. This could be an excess number of a specific class of chromosomes per cell, a deficiency in number of such chromosomes per cell, or an imbalance in the ratio of one class of chromosomes to some other class. If such a sine qua non exists for all malignancy, the same chromosomal imbalance should exist for all cell lines and all cancers. From our previously reported studies of 7 human cell lines plus 2 freshly obtained human cancers, E16 chromosomes were consistently elevated, expressed as average number of E16 chromosomes per cell.
The Experimental Results-Human Cell Lines
Even after correcting for any increase in total number of chromosomes per cell, both in cell lines and in fresh cancers, the E16 chromosome level was still significantly elevated in all cell lines and fresh cancers. Lastly, imbalances, expressed as the ratio of one class of chromosomes to another class, were consistently found only for ratios involving E16 chromosomes. the satisfactory character of the cutting lines of Fig. 1(a) .
The Experimental Results-Human Cell Lines
As was anticipated over 60 years ago by Boveri, numerous highly sigi elevations and depressions in mean number of specific chromosome clas found for the various cell lines studied.* But isolated elevations or depi are of no real interest in evaluation of the Boveri hypothesis. If a pa chromosome imbalance is to be a sine qua non of malignant behavior, ti balance must be present in all the cell lines and cancers studied. TI question we must ask, for the human cell lines, is " Are there any chror elevations (or depressions) in mean number per cell that are found in all lines? " The answer is that only for the E16 chromosome class can cot behavior be found for all the cell lines presented here. No other chror class is significantly elevated or significantly depressed in all the cell lines mean chromosome number per cell is elevated in every cell line studied. * Detailed analyses for every chromosome class is available to the interested reader upoi to the authors. The elevation in mean number of E 16 chromosomes per cell is highly significant in every case (for the t values in Table IV , all P values are <0 0001). Further, the elevation in E16 level is marked, averaging 3-10-fold for the entire group.
The Experimental Results-Human Cell Lines
The only moderate elevation is in RPMI-2650, where a 360% elevation in E16 level is noted.
The Experimental Results-Human Cell Lines
Chromosome levels corrected for total number of chromosomes per cell Inspection of Table IV shows that almost every cell line shows a mean total number of chromosomes greater than the 46 chromosomes characteristic of normal cells. The only exceptional cell line is RPMI-2650 which does show 46 total chromosomes, but with an abnormal distribution of chromosomes including E16 elevation. The question can be raised whether the observed E 16 elevations in the other cell lines is simply a reflection of the increase in total number of chromosomes per cell. This is definitely not the case. First, if such were the explanation, it is odd that no chromosome class other than E 16 shows consistent elevation in all the cell lines. Second, by direct test it is readily demonstrable that the E16 elevation is significantly and appreciably greater than that expected from the elevation in total chromosome number per cell.
The Experimental Results-Human Cell Lines
We shall henceforth refer to the number of chromosomes expected after correcting for total number of chromosomes per cell as the " corrected expectation ".
The Experimental Results-Human Cell Lines
Such analysis has been carried through for every chromosome class in every cell line studied. Again, only for E 16 chromosomes is consistent behavior observed-namely, an elevation of E 16 mean chromosome number above the "corrected expectation ". The mean E16 level is 2-03 times the "corrected E16 expectation ".
The Experimental Results-Human Cell Lines
Gartler (1968) has raised the question of whether several of the cell lines American Type Culture Collection might not be independent, but were pi derived by culture contamination with HeLa cells. His evidence is basec studies of isozymes of glucose-6-phosphate dehydrogenase and phosphc mutase. For argument sake, let us assume this may be true. If true indeed of great interest that there are such gross chromosomal comp.
The Experimental Results-Human Cell Lines
differences, both in total number and distribution into the various classe, noted in Table IV but with all cell lines showing marked E16 chromosome It can be argued, therefore, that whether the cell lines originated indepen( or whether some may have derived from HeLa cells, they can evolve to di chromosome composition, but that if they are to become " immortal " cel capable of producing cancer on heterotransplantation, they must possess ti chromosome excess. Thus, we do not believe Gartler's concern over origin cell lines would alter the importance of the demonstrated E16 excess in I cell lines. And, of course, Gartler's evidence is totally irrelevant for the st confirmatory evidence derived from the E16 excess in the 11 fresh cancers, HeLa contamination is out of the question.
Experimental resUlts-freshly obtained human cancers
All the data presented to this point confirm our previous hypothesis t E16 chromosome level elevation, absolute or relative to other chromosome characterizes human cell lines-cell lines regarded by many as malignan vitro. How well does this hypothesis hold up in freshly obtained human cz This latter represents the ultimately desirable test material, for with such m the argument cannot be raised that long-continued cell culture accounts i chromosome findings, rather than a relationship of chromosome constitutio malignancy per se. Eleven fresh cancers have now been studied, in evei with chromosome preparations made between 2-20 hours after excision malignant tissue or withdrawal of the malignant effusion. The nature material was described in Table II . The E16 data for all the 11 fresh canc presented in Table V . Ten of the 11 fresh cancers show appreciable and highly significant elevations in absolute E16 chromosome level. One case, EA-225, cannot be proved to show an absolute elevation in E16 level. This one cancer is extremely unusual in that it has only 37*13 total chromosomes per cell. Such cancers with total chromosome numbers this low have been reported before, but they are extremely rare. As will be noted below, when the E 16 level is considered relative to the total number of chromosomes, the elevation in E16 level compared with the " corrected expectation " for 37 total chromosomes is highly significant.
Experimental resUlts-freshly obtained human cancers
Analysis made of every other chromosome class demonstrate, for the 11 fresh cancers, as for the 17 cell lines, no chromosome class other than E16 shows consistent elevation-nor does any other class show consistency in the form of a depressed chromosome level per cell. These analyses are available upon request.
Experimental resUlts-freshly obtained human cancers
E16 chromosome levels corrected for total number of chromosomes per cell
Experimental resUlts-freshly obtained human cancers
The " corrected expectation " for any class of chromosomes is the number of chromosomes of that class expected after correcting for total number of chromosomes per cell. Ten of the 11 fresh cancers show significant E16 chromosome elevation above the "corrected expectation." Case EA-225, with the very low total of 37 chromosomes per cell meets this criterion. Case EB-216, with a demonstrable E16 level elevation on an absolute basis fails to meet the more rigorous criterion of elevation above " corrected expectation ". E-16 levels in S V-40 virus altered human cells Shein and Enders (1962) first demonstrated that the virus of Simian origin, SV-40, can regularly accomplish the alteration of normal human cells to immortal malignant-behaving cell lines. This same SV-40 virus has been proved definitively to produce cancer in two species, including newborn hamsters and Mastomys.
Experimental resUlts-freshly obtained human cancers
In the " materials " section above one human cell line was described which was produced in vitro by the action of SV-40 viruses upon normal human cells in culture. The chromosome analysis for this cell line, together with comparisons with chromosome levels in normal controls is presented in Table VI . It is clear from these data that the E16 chromosome level is markedly elevated in the cell line produced by SV-40 alteration of normal cells, approximately 2-39 times the normal cell E16 content. Since the total number of chromosomes in the SV-40 altered cells is 81-73 chromosomes per cell, the " corrected expectation " for E16 chromosomes is [(81.73/46- 00) x 2.05] = 3-64 per cell. But the observed E16 chromosome content is 4 90 chromosomes per cell. The difference, 1-26 chromosomes per cell, is still large, and the probability of this not being real, with a t value = 6-71 is P < 0*0001. Thus, not only is the E16 chromosome level absolutely elevated, but also it is elevated markedly even after correcting for total number of chromosomes per cell.
Experimental resUlts-freshly obtained human cancers
Further inspection of the data of Table VI shows that more marker classes of chromosomes are represented at significant occurrence frequencies than for any other human cell line or human cancer presented earlier in this communication.
Experimental resUlts-freshly obtained human cancers
Since marker chromosomes are presumed to arise by injuries to normal chromosome classes, the inference is that the SV-40 virus has produced extensive chromosomal injury during the process of alteration of normal cells to a malignant cell line. That such is the case is evident from careful study of many metaphases not included in Table VI . Chromosome pulverization, chromosome distortion recognition, and other anomalous chromosome distortions are noted rei many metaphases unsuitable for analysis. The true chromosomal alt in toto is even more extensive, therefore, than presented in Table VI . As; of the extensive representation of markers, it would be particularly hazar assign much meaning to ratios of one chromosome class to another in these virus-altered cells.
Experimental resUlts-freshly obtained human cancers
Possible Exceptions (?) to the E16 Hypothesis Chronic granulocytic leukemia Nowell and Hungerford's (1960) discovery of the Philadelphia chromos chronic granulocytic leukemia was the first specific chromosome abnormz a malignant disease. This chromosome is considered to represent a G chron from which approximately 40%0 of the DNA content has been deleted. The no indication reported of an E16 chromosome excess. We have now I opportunity to examine a case of chronic granulocytic leukemia by quani chromosome analysis. No excess of E-16 chromosomes was demonstrable
Experimental resUlts-freshly obtained human cancers
The deletion of some G chromosomal material means that the G + Y lower than normal in chronic granulocytic leukemia. Therefore, even normal absolute E16 level, the ratio E16/G + Y may well be higher, in effec normal. It is to be noted that this ratio, E16/G + Y, is high in every hurr line and every one of the 11 fresh cancers studied. It is, therefore, possib chronic granulocytic leukemia may not represent an exception to the E16 hypothesis. Final decision must await determination of whether the E16/G + Y imbalance is a necessary or sufficient condition for malignancy.
Experimental resUlts-freshly obtained human cancers
Burkitt's lymphoma Burkitt's lymphoma is a special case among malignancies in two major respects. First, virus or virus-like particles are commonly demonstrable in involved tissues. Second, cell lines drived from Burkitt's lymphoma grow only in suspension culture, in contrast to those described above, which all grow as monolayers, but which may also grow in suspension culture. One such cell line (JIJOYE) has been subjected to quantitative chromosome analysis. The E16 level was within normal limits both in the Burkitt's lymphoma cells with 46 and those with 47 chromosomes. Every cell, however, shows a specific marker chromosome (Marker 12, Fig. 1(b) ), the genetic content of which is unknown. If E16 genetic material is present in this marker chromosome, Burkitt's lymphoma would be consistent with all the cell lines and cancers. If not, this will represent a distinct exception to the E16 hypothesis.
Experimental resUlts-freshly obtained human cancers
Corroborative evidence from the literature In a problem of the magnitude of evaluation of the E16 hypothesis it is clear that no one laboratory is ultimately going to be able to provide the breadth of observations required. Unfortunately, in the semi-quantitative state of chromosome studies broadly, there are very few reports of chromosomes in malignancy where quantitative characterization of chromosomes is provided. In general the reports are of the " cut-out " karyotype variety for one or a few cells per case.
Experimental resUlts-freshly obtained human cancers
In the absence of quantitative parameters it is commonly found that chromosome assignments are equivocal. Regardless of their reported interpretation, we feel there are several suggestive and some straightforward corroborative examples presented in recent literature (Messinetti et al., 1968; Ayraud and Kermarec, 1968; McAllister et al., 1969; Crossen et al., 1969; Rigby, 1968, Ponten and Saksela, 1967) .
Experimental resUlts-freshly obtained human cancers
Our sincere thanks to the many people who contributed in a variety of indispensable ways to this research effort. Dolores Piluso, Margaret Soderberg and Erma Kovich provided much help in the laborious process of tracing metaphases. Frank Fickel designed, executed, and maintained our microprojection hardware with technical and photographic assistance from David Dixon and Richard Krumhansl of the Technical Photography Group at LRL, Livermore. Stuart Stone provided the sophisticated character recognition programs required for computer measurements and calculations, with the help of A. Landon Bruce. James L. Littlepage provided electronic scanning of the tracing photographs. Charlie Pierce gave us excellent service as a reference librarian. And special thanks to Lillian Rachlin, M.D., and her staff of the Department of Surgery, U.S.
Experimental resUlts-freshly obtained human cancers
V'eterans' Hospital, Livermore, California, for supplying us with large numbers of fresh cancer specimens. This work was supported by the UT.S. Atomic Energy Commission. 

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