Chromosomes as Tools of Study of Leukemia Cells

Jérôme Lejeune

Methodological approaches to the study of leukemias the wistar institute press, september 1965


Sommaire

Being a human geneticist, I must apologize for speaking to you about cancer, since geneticists don't know anything about cancer and human geneticists do not know anything about experiments. After what has been said today, I am hesitant about discussing either observations made in humans or theoretical considerations which can be built up from the actual data.

As you probably know, human chromosomes are very interesting little bodies which can be seen very easily with a light microscope. Their significance lies in the fact that if one little chromosome is in excess or if a part of a chromosome is lacking, the person is severely affected. Cytogenetics, therefore, can be used as a tool to investigate the neoplastic process and, more precisely, to try to determine whether there is something peculiar in the neoplastic cells.

We will consider the use of this tool for three general purposes.

The first is a clinical use. There is a strong and very curious relationship between chromosomal abnormalities and leukemia. Individuals who are trisomics for chromosome 21, have about a 20 times greater probability of contracting acute leukemia than normal children. This fact is so well established that it cannot be due to chance. It is possibly related to another particularity of the white cells : the polymorphs of the affected people have an abnormal shape even without any leukemia. Their nuclei have fewer lobulations than normal cells as demonstrated by Turpin and Bernyer (1).

The use of cytogenetics for the clinical study of leukemia was opened by the great discovery made by Dr. Nowell and Dr. Hungerford (2) that in the cells of the bone marrow and blood of people suffering from chronic granulocytic leukemia, there is a tiny chromosome replacing a normal one. This chromosome has been called the Philadelphia chromosome or Ph1 in honor of the town in which it was discovered.

This Ph1 chromosome is only found in blood or in bone marrow of the suffering individual, but never in the cells of their skin or of the rest of the body. Even in the blood, for example, not all the cells carry it; part of the cells have the Ph1 and part of the cells are entirely normal.

It is interesting to follow the evolution of the disease and to compare the number of white cells in the circulating blood with the frequency of the cells carrying the Ph1. If there are more than 20,000 white cells in the blood, a given percentage of the Ph1 positive cells is observed; after treatment, however, if the number of white cells in the blood falls below this level, practically no Ph1 positive cells can be recovered from the blood culture. It seems that they have disappeared. But when the number of white cells increases again, the Ph1 positive cells reappear in the blood. This has been statistically proved in many patients by Tough et al. (3).

Current data indicate that around 247 cases of chronic granulocytic leukemias have been analyzed, and more than 220 show the tiny chromosome in their blood. Thus a very consistent picture emerges.

What becomes of these cells during remission when they disappear from the blood? If correctly searched for, they are still found inside the bone marrow, persisting even when there is a drop in the normal count of white cells, but disappearing from the peripheral blood.

Levin et al. (4) have performed transfusions from individuals carrying these abnormal Ph1 chromosomes to children affected by acute leukemia who were treated with X-ray and anti-metabolites. They could prove that the new cells which were establishing a colony were also carrying this tiny chromosome.

As far as clinical use is concerned, this Ph1 informs us of both the phase of the disease and the actual importance of the clone. An obvious question arises: is there final proof that the cells carrying this abnormality are the malignant cells? I would answer that there is no final proof, but there is a very strong indication that they are.

Sometimes people suffering from chronic granulocytic leukemia exhibit a change in their cells and undergo what is called a blastic transformation. This occurs sooner or later in practically every case of chronic myeloid leukemia. In many instances, this change takes place simultaneously with the appearance of a new imbalance of the chromosomal set. Very often an extra chromosome is found, which looks like a medium sized one. Here we have possibly a tool to determine if the blastic change will occur in the patient. The chromosomal changes seem to be present before the clinical change can be detected by general hematological data.

These additional changes seem to be not entirely at random, and possibly they can be used not only for their clinical interest but also for their experimental interest. For illustration, I'll discuss a different disease, a congenital leukoblastosis.

A child who was trisomic for 21 exhibited a leukoblastosis at the age of two years. A clone containing 54 chromosomes was found in the blood and in the bone marrow (5). In the blood and bone marrow different types of cells were detected. The main type was trisomic 21, with 47 chromosomes. After classifying the cells by their number of chromosomes, it was found that a little acrocentric was first in excess. Then two of them were observed and, progressively, we saw the appearance of a large acrocentric, then two. With still greater numbers of chromosomes, a medium-sized, then two appeared; finally, a small metacentric was visible, then two.

The experimental interest of such observations is that what at first glance appeared to be a random change in the cells of the individual seems to make sense and to follow some evolutive pattern if enough data are available.

This type of evolution was observed in 1963 and quite similar patterns have been observed repeatedly in other case's of acute congenital leukemia in 21-trisomics. It seems that this pattern, if not typical, has some propensity to repeat itself in different individuals.

Cytogenetics has experimental interest be-cause the hypothesis that a change in the karyotype has something to do with the properties of the cell can be tested. For example, in a case that will soon be published, we were able to follow the evolution during one and a half years. At the beginning it was a chronic myeloid leukemia with the Ph1 chromosome. Following treatment, the number of white cells fell but, after a remission, rose again. Finally, there was a transformation with a predominance of promyelocytes. Around this time, an extra 18 chromosome was observed. The treatment with Myleran was effective for a while; however, after the appearance of other extra chromosomes, neither Myleran nor 5.278 R.P. was active any more, and the child eventually died.

This observation is not unique, and it seems there could be a very strong correlation between the action of drugs and the karyotype of cells. For example, 6-mercaptopurine was effective on cells having the Ph1 and three extra chromosomes in a case of Court-Brown and Tough (6), but not on cells containing only the Ph1. The latter cells instead were affected by Myleran and prednisolone.

For the moment this quasi-experimen-tal approach to see if the alteration of the karyotype of the cells really induces a change in their behavior is very crude and relies upon pure observation. But if we remember that it is well known that Myleran is efficient during the chronic stage and not during the blastic trarus formation stage, then the whole story corroborates what has been found by the hematologists. Indeed, it is difficult to go much further in the experimental use of cytogenetics in human leukemias, and we have to see now the heuris-tic value of this research - i.e., what the real interest of looking at chromosomes is and where it can lead us.

To evaluate the real value of cytogenetic studies, the first question is whether there is always a chromosomal change once the neoplastic process has occurred. Unfortunately, there is no answer to that question. Generally, and in most of the cases, chromosomal abnormalities are found which are characteristic of each particular cancer. But, in the case of negative results, it is very difficult to tell if really neoplastic cells indeed had a normal karyotype. We know for certain that in our cultures there is a very severe selection against the abnormal cells. After a while it can be said that the cancer is " cured " in vitro, since only normal cells are growing well in the ordinary medium. Thus, the few negative results which are known are of a dubious significance.

The second question is: if chromosomal aberrations are really present in neoplasia, what can be said about their role? We can foresee that few rules should be respected if, really, there were a strong correlation between the chromosomal changes and the neoplastic process.

The first one is the likelihood that there would be what we call un variant commun (a common variant). If the chromosomal change is really related to the change of cell behavior, then the same change in behavior should be found in relation to the same change in chromosomes. Indeed, the case of the Ph1, which is found unfailingly only in chronic granulocytic leukemia, is an extremely good example of what could theoretically be expected - i.e., a variant commun.

The second thing we must discover is whether or not these abnormal karyotypes are related to the normal karyotype by particular pathways, as we have seen in one case; moreover, we have to determine if this clonal evolution can be regarded as a general phenomenon. In all the cases in which enough mitoses have been studied, the cells have had the same chromosome number but they have not had the same formula. Nevertheless, if a general view of the differences is adopted, it can often be surmised that the cells are probably derived from each other by accumulating abnormalities step by step. Therefore, a reconstruction of the past history of the clone can be attempted. If enough data are available, as in the case previously mentioned, a stepwise evolution of the chromosomal changes can be detected, after going back to the normal karotype.

If we assume that this clonal evolution has biological meaning, we could expect that it would follow some basic regulations. Indeed, these regulations are still unknown, but some of them can possibly be foreseen. The reasoning is based on the notion that if the genetic material is increased, the enzymatic activity which is under the control of this material should also be increased in the abnormal cells.

Let us say, for example, that for a particular enzyme, controlled by a particular gene, a normal diploid individual has two activity units of this enzyme in each cell. If this chromosome is present in triplicate, the cell would have three activity units, etc. Surely the relation is more complicated than this simple arithmetic, but at least there should be some relationship of this kind. (The possibility of a non-linear relationship between gene-dose and enzyme activity does not change the logic of the argument.)

A ? B ? C

A ? B ? D

Let us suppose a given metabolite A is transformed in B, B being transformed in C, or in D. Let us suppose also that enzymes controlling each step are made by different genes located on different chromosomes.

Then, if the transformation of B to C is accelerated (the chromosome carrying the particular enzyme is in excess), and if the supply of A is not increased, a relative deprivation in D should result. If D has to be produced in a certain amount so that the cell can survive, it follows that before C is increased, the production of A itself should have been increased.

Obviously this over-simplified example does not pretend to represent any particular situation.

The only interest in the argument is that if extra chromosomes do something on the enzvmatic activity of the cells, then absolutely random changes should not occur. In other words, some cornbinaisons interdites should never occur. The systematic analysis of karyotypic changes could then show, for example, that chromosome No. 1 is never found in excess before chromosome No. 18 has been put in excess. Statistically speaking, a typical bias in the order of acquisition (or loss) of genetic material could be observed and would be an indirect, but interesting, way of testing the clonal evolution hypothesis. Similarly, a statistical approach could also eventually reveal that chromosomal evolution is following some mechanical rules, such as the preferential duplication of the supernumerary chromosomes (7).

The whole speculation just presented could have some implications if it were found to represent at least a part of the reality. The most obvious one would be the use of a new tool in analyzing the genie con-tent of the chromosomes. Excess or lack of given chromosomes, if related to changes in given enzymatic activities, would suggest on what part of the genome the genes con-trolling these enzymes are located.

If the hypothesis of combinaison interdites had some significance, these maps, even if very crude, could be of great interest. Simple karyotypic analysis would then reveal the "sensitive biochemical pathways" that the cells have overcome, and the artificial changes of the equilibrium (by anti-metabolites or, on the contrary, excess of some metabolite) could reverse the selective advantage acquired by the abnormal cells.

In other words, karyotypic analysis could help to choose what drug should be the most efficient in any given case.

These speculations, based on some observation data, have been developed to show that cytogenetics is potentially a very efficient tool in research.

Having myself raised so many basic questions, I will not try to escape the fundamental one: what is the real meaning of these chromosomal changes ? All the carcinogenic agents from the virus to chemicals, from radiation to aging, have in common the production of chromosomal damage. Hence, the basic question cannot be eluded: are the chromosomal changes the sequence or the cause of cancer?

No formal answer is available, but the cytogeneticist observing these tremendous changes of genetic patrimony of malignant cells is led to conclude that the chromosomal aberrations are probably neither the cause nor the consequence of cancer - they are the neoplastic process by itself.

Haut

Discussion

Haut

DR. NOWELL

I would like to make a comment which may have application to some of the earlier discussions here today.

In the viral tumors of animals, those produced by RNA viruses show much less in the way of chromosome abnormalities than do those due to DNA viruses. This may conceivably suggest that whereas in the DNA virus tumors there is direct interaction between the virus and the genetic material of the cell, in the RNA virus tumors, such as the murine leukemias, more indirect mechanisms may be operative.

In this connection, the chromosome findings in the human leukemias may be somewhat paradoxical. Acute childhood leukemias would seem to be currently the best candidate for a human RNA-virus tumor and yet, as Dr. Lejeune has indicated, the childhood leukemias show the most extensive chromosome changes of any of the human leukemias.

Haut

DR. BARSKI

In our laboratory we are following malignant cell transformation in vitro, and at the same time we are controlling the karyotype of the transformed cells.

I must say, and this can be disappointing from your point of view, our observations are not conclusive about relation between identifiable chromosomal alterations and malignant cell transformation. We studied in particular cultures of lung tissue from different rodents. During the in vitro adaptation process, we always see chromosomal transformation well before appearance of malignancy. In mice for example we see drastic changes in the chromosome members, then appearance of new, abnormal chromosomes and no malignancy. This was observed repeatedly in at least eight different and independent explantation series; only later on, in these already chromosomally transformed cell populations, do we see appearance of malignant clones.

When we are checking systematically the karyotype of virus induced tumors, we see that some cells appear as having normal karvotype; some show quantitative and more rarely qualitative variation. On the basis of rather careful idogrammic analysis of these changes we assume that they are random. This study was accomplished in particular on adenovirus-induced tumors in hamsters.

Haut

DR. LEJEUNE

I would say that random chromosomal changes appearing before malignancy are the best proof that one can give for the hypothesis that chromosomes have something to do with the transformation process. This system is free of the fantastic selection pressure which is inside the body. Then cultured cells are allowed to develop combinaisons interdites which could have no success as malignant cells. The chromosomal changes can play around until they get, just by chance, the right combination, the only one that they would have found in the individual to give a malignant cell. That goes remarkably well with what has to be expected. If selection is suppressed, these changes proceed by a random trial and error process. But if the transformation occurs inside the body, then the permissible path-way is extremely narrow.

To the second point that in the transformation by virus there are cells which look normal and cells which appear to have random chromosomal changes, I would add that before we did special research for this clonal evolution, we did not suspect that a mixture of cells with 48 to 54 chromosomes were at all interrelated. Apparent disorder is obvious, but there is logic underneath, even if it is not easy to find.

I would guess that after malignancy of the virus-transformed cells is established, there is a better consistency of the karyotype shift, and I think this is what you do find.


Haut

Literature cited

1. Turpin, R., et C. Bernyer 1947. De l'influence de l'hérédité sur la formule d'Arneth - (cas particulier du mongolisme). Revue d'Hematol., 2: 189.

2. Nowell, P. C., and D. A. Hungerford 1960 Chromosome studies on normal and leukemic human leukocytes. J. Nat. Cancer Inst, 25: 85-109.

3. Tough, I. M., W. M. Court-Brown, A. G. Baikie, K. E. Buckton, D. G. Harnden, P. A. Jacobs and J. A. Williams. 1962 Chronic myeloid leukaemia - cytogenetic studies before and after splenic irradiation. Lancet, ii: 115-120.

4. Levin, R. H., J. Wang, J. H. Tjio, P. P. Carbone, E. Frei III and E. J. Freireich 1963. Persistent mitosis of transfused homologous leucocytes in children receiving antileukemic therapy. Science, 142: 1305-1311.

5. Lejeune, J., R. Berger, M. Haines, J. Lafourcade, J. Vialatte, P. Satge et R. Turpin 1963 Constitution d'un clone à 54 chromosomes au cours d'une leucoblastose chez un enfant mongolienne. C. R. Acad. Sci, 256: 1195-1197.

6. Court-Brown, W. M., and I. M. Tough 1963 Cytogenetic studies in chronic myeloid leukemia. Adv. Cancer Res., 7: 351-361.

7. Lejeune, J. Chapter VIII, in Le Chromo-somes Humains; R. Turpin and J. Lejeune, Gauthier Villars Edit., Paris, 1965.