Chapter 1    The neoplastic cell

    Higher animals are made up of millions of cells which generally make up the organs. The cells of these organs form the tissues, which can be divided into two classes: connective tissue and parenchyma or functional tissue. Each cell type behaves as an individual species in that each only produces the same kind of cell. We still do not know how, for example, to make a liver cell produce any other kind of cell through karyokinesis. However, it is now thought that there are no genetic differences between cell types; they are only pseudo— species. The non-genetic changes that occur during ontogenesis and which generate these different pseudospecies of cell are called epigenetic changes. According to Harris (196Lf), this pseudospecification of cells is called differentiation.

Another aspect of differentiation is the following:

    In the specialization that appears in each pseudospecies of cell in the adult animal, for example, the cells of the epidermis are not homogeneous, but are made up of basal cells and cells in different stages of keratinization. The reproductive activity of the pseudospecies is usually restricted to the basal cells. The division of these cells produces more basal cells and cells that can no longer divide, but that have the special capacity of producing keratin. Since the cells that produce keratin cannot divide, a new pseudospecies is not generated. The cells that have the capacity of reproduction and, consequently, of maintaining the pseudospecies are usually called trunk cells. Thus, normal differentiation can include the loss of the power of division as in the case of the keratinizing cells of the skin, the neurons of the adult nervous system and the striated muscle cells.

    The mechanisms that control normal differentiation are unknown in many of their aspects. It is clear that the reciprocal action of cells frequently induces the expression of the genes. These reciprocal actions can be mediated at the cell surface. The repression of certain genes from the nucleus and the activation of others can occur, i.e., the transcription of genetic messages can be initiated or suppressed. Alternatively or additionally, the translation of specific messages from the RNA to protein can be initiated or suspended. In any case, the final result is that in the differentiated cell only a small portion of the genome expresses the cell phenotype. That which distinguishes a liver cell from a kidney cell is that only small parts, which are only partially translated, of their common genomes arc expressed.

    Usually, differentiation is quite stable and transmitted to numerous generations of cells. However, the cells that become differentiated have a narrow margin of variability, but can show some phenotypic changes in response to environmental excitation. Bronchial squamous metaplasia is a common example, in which the basal cells are transformed into squamous cells instead of ordinary columnar cells. In general, this is a consequence of cigarette smoking or reflects a vitamin A deficiency. Since it appears to be a reversible modification, it is not considered to be a further differentiation but a modulation which possibly depends on a continuous environmental stimulus more than hereditary cell change.

    In mammals, the differentiation in the various systems ordinarily includes an increase in specialization that is accompanied by a limitation of the potential of the cell for carrying out other functions or for generating other cell types. This increased specialization, however, does not necessarily include a limitation of omnipotentiality. This is present in a highly pronounced form in plant systems. In this case, an extremely specialized cell of an adult plant can regenerate the whole plant, which is to say that the cell has retained its omnipotentiality, just as if it were still a seed cell. Thus, the term differentiation is used slightly ambiguously in mammals to indicate either of these two properties, though they are not necessarily correlated.

    Even though epigenetic organisms can possibly account for a large part and maybe even all of normal differentiation, there is another form of variation in somatic cells. Vie are talking about somatic mutation and it is not known if some functions are dependent on this.

    As will be seen below, neoplasia is a form of abnormal variation of the somatic cells, which is due to somatic mutation and to aberrant and defective differentiation or to both, caused by bio—physico—chemical alterations. The result is a pathological form of hyperplasia.

    The growth in size of a tissue or organ due to an increase in the number of cells is called hyperplasia. Hyperplasia is divided into two different, but frequently overlapping, types: the physiological and the pathological or neoplastic. Physiological hyperplasia is the normal response of a tissue to an entire range of environmental stimuli. Perhaps the most common example is the thickening of the epidermis in response to traumatism, which can be considered a prototypical case.

    It is known that any agent that eliminates cells, kills them, or both, in superficial epidermal strata causes an increase in the reproductive activity of the basal stratum. In this stratum the increased production of cells recomposes the superficial strata, returning them to their normal states. This restitution and excess are called compensating hyperplasia. When the stimulus which initially started the phenomenon is not iterative, the mitotic activity in the basal stratum declines and finally the hyperplasia disappears. Therefore, physiological hyperplasia is conditioned by the continuous application of an exterior stimulus.

    It is evident that in a tissue in equilibrium with respect to its mass, the production of new cells should be exactly equal to the cell mortality rate. In a tissue like the epidermis, this means that on the average, half of the cell progeny that are produced by the basal stratum have to follow the path of cell differentiation, which leads to the formation of keratin, and, finally, to the death of the cells. As a result, hyperplasia has a self—limiting effect only when this proportion is reached.

    The fact that the elimination of the superficial strata activates mitosis in the basal strata led to the hypothesis that the former inhibits the latter. How it is well known that the keratinizing strata produce a mitosis inhibitor, which has been partially purified and is called chalone. It does not appear to be specific to animal species, but different kinds of chalone occur in different tissues.

    Chalones are inhibitory agents that have short-range effects, i.e., near their points of origin, which distinguishes them from those hormones that have long-range effects. There are also regular effects at the level of cell contact collectively called contact inhibition of mitosis. It is clear, then that there are many mechanisms that interact and control the growth and division of cells.

    These mechanisms are the bio—physico—chemical components of the organism that are found in perfect equilibrium. With this we mean to say that the pH, the osmotic pressure, the oncotic state, surface tension, electrical conductivity, etc. which are the physical elements 0-f the organism, will not vary outside of normal limits, as is so with the oxygen, C02, K, Na, Ca, Mg, etc., protein, lipids and carbohydrates in the organism.

    Physiological hyperplasia differs from neoplasia in that the latter implies a change in the intrinsic process of cell heredity. This cell alteration has the result of a. race of cells less subject to the mechanisms of normal tissue regulation. For example, in the skin this can mean that the neoplastic cells can produce less chalone, that they will be less sensitive to the inhibitory effect of it, or both. In this class of cells, cell differentiation is defective, since little less than 50% of the daughter cells of the basal stratum evolve towards keratinization. Of course, a decrease in the proportion of differentiated cells is accompanied by a lower level of chalone, though this does not always occur. Some neoplastic hyperplasias show a rate of mitosis lower than normal, but the tissue keeps growing because of the imbalanced relation between renovation and differentiation of the trunk cells.

    How, to begin our description of neoplasia, we can define it as the form of hyperplasia caused, at least partially, by an intrinsic hereditary abnormality of the affected cells, which can be modified by a bio—physico—chemical disequilibrium affecting the physical elements and factors or the chemical ones. Neoplasms can he transplanted from one animal to another by inoculation with living neoplastic cells. This can be done infinitely, as long as the immune reaction for the neoplastic cells can be suppressed in some way. With the help of chromosomic or antigenic markers the cells of the new neoplasms, which result in the inoculated animals, are ordinarily the progeny of the transplanted cells and not of the receptor cells. In the human, metastases show similar characteristics: the neoplastic cells are transported in the blood or in the lymph to places far from their original site of introduction and at the new sites produce neoplasms of the type of the progenitor cells.

    Cancer is a discompensation or disequilibrium, bio— physico—chemical in nature, affecting the whole organism, which is inherited and constitutes the bio—physico—chemical "terrain." When an organism has this terrain, it does not mean that the disease is propagated either by the lymphatic or by the circulatory system to sites far from its origin. The cells feed on this bio—physico—chemical terrain, besides which their intracellular constitution is also altered by it making the cycle vicious, though the disequilibrium is of the whole organism. There is no metastasis but the same disease; it is just that there is greater chemical affinity in the other affected site, and for this reason the disease is manifested there, as well.

    From what has been said, it is clear that neoplasia is a disturbance that is characterized by the abnormal behavior of the cells and by abnormal reciprocal actions caused by the factors described in the paragraph above. Neoplastic cells do not behave in the highly integrated manner characteristic of normal cell conglomerates in higher mammals. Thus, neoplasia might be considered an incomplete cellular reversion to a more primitive, ancestral cell type, in which some of the regulating mechanisms normally active in the metazoarian cell are either missing or defective. This is seen from a cellular point of view.

    Before discussing in more detail the features of neoplastic cells, we should consider, even if briefly, the pseudo—neoplasias which resemble closely the true neoplasias.

    To this end, it is not sufficient that the definition of neoplastic hyperplasia indicate a hereditary cell change, but it should also specify that this change is found in the cells that directly constitute the neoplasia. Otherwise, disturbances closely related to but not usually thought of as true neoplasias would not be excluded. A disturbance that resembles neoplasia is pernicious anemia. This disease is characterized by the massive hyperplasia of immature erythrocytes in the bone marrow. The anemia observed in peripheral blood is the result of the immaturity of these cells. The ratio of trunk cells to maturing (non-reproductive) cells has been changed. However, independently of the fact that the disease is due to an alteration in cell heredity, the lesion is not found in the cells of the hematopoietic system as they are in hyperplasia, but in the gastric cells that normally carry the factor that is necessary for the absorption of vitamin B12. This vitamin, in turn, is necessary for the maturation and differentiation of the red cells. Therefore, pernicious anemia is very similar, in most of its features, to a true neoplasia. Not only does this chemical alteration occur in this disturbance, but other chemical elements. which undergo changes that are not in the same proportion as in cancer, nor are they the same ones that are altered in cancer; the same occurs with all diseases; the chemical and physical compounds are what change to different extents, intra and extracellularly in different diseases. Pernicious anemia is also a sickness that can be corrected by injecting a factor for cell maturity.

    Chronic physiological hyperplasia can resemble neoplasia due to its histological (physico—chemical) characteristics and the distinction can be very difficult to make, even for the experienced pathologist. The two kinds of hyperplasia, physiological and neoplastic, are frequently encountered at the same time, to a variable degree, in one and the same lesion. The cells with a hereditary alteration often respond, to a certain degree, to environmental stimuli, as well.

    It is often thought that the growth of a tumor is simply exponential. However there is proof that this is not exactly true. In the great majority of experimental tumors it can be seen that the growth rate of the tumor diminishes with time. To describe this, different investigators have proposed different complicated mathematical functions. These curves indicate that even the most malignant tumors and those that grow fastest reach plateaus with little subsequent growth if the animals that have them live long enough, though unfortunately the animal’s death usually occurs when the tumor is still growing exponentially. More benign turners often make it possible to reach the growth plateau while the animal is still alive and, unless the neoplasia shows subsequent progress, the lesion can remain virtually the same size. From our point of view what happens is that on the one hand: a) while the organ— ism is alive, the cancerous cell, intra or extracellularly will be physico—chemically imbalanced with relation to the medium. b) death comes because of an excess of intracellular toxic substances because they can no longer be neutralized or eliminated. Thus, physically what is seen is that the cells keep growing, due to the mechanisms described, but this growth will not cease while the organism is alive; when it dies the cells stop reproducing, but the growth of the cells is proportional to the degree of intracellular intoxication.

    Immunity can alter the rate of tumoral growth, and this will be discussed below. Immunity is no more than a chemical mechanism which produces chemical reactions in the organ— ism. However, in relation to the growth curves, it might be relevant to point out here that in experimental tumors two different trunk cell populations have been identified. One of them does not reproduce, but if the immune response is suppressed, part of this non—reproductive subpopulation begins to reproduce (Decosse and Gelfant, 1968). It seems that the immune reaction was able to block (reversibly) the karyokinesis in a small, though variable, percentage of the tumoral cells. It is possible that a mechanism such as this contributes to complicate tumoral growth curves.

    Neoplasia can affect any tissue or organ whose cells can divide. This alteration can be slight, in which case the neoplastic cells vary little from the normal ones, or it can be so serious that the differentiated cell be absolutely unrecognizable when compared to the distorted neoplastic cells. These different degrees of aberration in cells are usually divided into two or three categories.

    The neoplasias that are not very differentiated, so that they are not very different in form and behavior from the original tissue, in general are called benign, independently of the fact that they can sometimes be fatal. These neoplasias are, in general, very slow—growing and the individual cells can be morphologically indistinguishable from the normal corresponding cells in extreme cases.

    On the other hand, neoplasias with a marked cell atypia and observable deficiency in differentiation (anaplasia) are called malignant, though it is possible to cure them. A malignant neoplasm is a cancer. Those cancers of endodermic origin are called carcinomas, while those of mesodermic origin, with some exceptions, are called sarcomas. The cells in a malignant neoplasm are generally aneuploid and show a whole range of chromosomal anomalies. Frequently, abundant pleomorphism (variation in form from cell to cell) can be found. The cells are generally bigger than normal, their nuclei are large and multiple nuclei are common; the size of nucleus/size of cytoplasm ratio is large. Often, the rough endoplasmic reticulum is deficient, showing an increase in the number of free ribosomes. Mitoses are frequent in histological sections and they are often abnormal. A characteristic feature of the malignant cell is the tendency that it has to lose, to a variable degree, its normal adhesion to neighboring cells. The proximity of the cells is diminished and the ionic communication between them is reduced. The cell has an elevated propensity for emigrating considerable distances from its original location. This tendency can be shown by the presence of neoplastic cells in lymph vessels, blood vessels, and the pleural and peritoneal cavities. In this fashion they can propagate and originate new, distant secondary sites of neoplastic growth. This process, called metastasis, is what frequently causes the failure of attempts at surgical extirpation. With surgical excision of the neoplasm the bio—physico—chemical terrain is not modified and remains cancer—prone. Given the clinical importance of the real or potential ability to metastasize, it is considered the distinctive feature of malignancy.

    Between these two extremes one finds a group of neoplasias which have the cytological and morphologic characteristics of the malignant type, without, however, tending to invade other cells. Lesions of this nature are called in situ carcinomas. We have seen many patients that 1) when the diagnosis was correct, a certain period after surgical intervention either the lesion reappears in the same place or in another part of the body. This shows that the term "in situ" is very relative because cancer is a bio—physico—chemical disequilibrium of the whole organism.

    In situ carcinomas illustrate spectacularly that there is no correspondence between the cytological characteristics of malignancy and the effective propensity towards metastasis. Not even an experienced pathologist can evaluate adequately potential malignancy by studying the cytological evidence, and, in particular, the histological evidence, for there is no unique criterion for arriving at this verdict.

    For the evaluation of malignant neoplasias it has been useful to assign them to histological categories. To this end, Broders introduced a scale where neoplasias are classified from 1 to 4 according to the percentage of undifferentiated cells: the most differentiated type of lesion is classified as 1 and the totally anaplasic one as 4. These degrees of differentiation have been shown to have statistical significance in the prognosis of different kinds of malignancy. Exfoliated, fixed and pigmented epithelial cells are useful for the diagnosis, especially in the case of the cervix. With the Pap test these cells are evaluated and placed on a scale from 0 to V according to the cell or group of cells that is most malignant. Class V corresponds to a certain carcinoma and class 0 is totally normal (Papanicolau, 1958).

    In spite of the fact that sometimes the pathologist is asked to classify with the greatest urgency whether a given enoplasia is benign or malignant on the basis of cytological or histological criteria, it should be emphasized that these classes are arbitrary and there is no clear line that separates them biologically. The propensity to metastasize, just as many other biological manifestations, is a function of probability and not an absolute fact.

    Cancer is a general bio—physico—chemical disequilibrium of the whole organism that is inherited and which constitutes the terrain in which neoplasias may arise. Once the organism has this prepared terrain, it does not mean that the disease is propagated to distant sites. There is no metastasis itself, because it is the same disease except that there is greater chemical affinity at this other site (tissue, organ, etc.) and it is for this reason that the disease manifests itself again, though before metastasis can happen, the neoplastic cells should already have invaded the normal tissue that surrounds them. In the beginning, at least, healthy—looking tissues inhibit the growth and the emigration of small neoplastic cell groups. With the passage of time, the characteristics of the cells change in such a way that this inhibitive effect and the neoplasia can grow, spread or both. Though the nature of this inhibition is unknown, it is known that cell— to—cell contact phenomena exist as well as substances that have inhibitory effects over short distances.

    In a characteristic way, the neoplastic cells are less adhesive to others than normal cells. This fact is accompanied by a lower calcium content in the plasmatic membrane. Some malignant cells produce hyaluronidase, which can foster the process of invasion. The cells also acquire a more negative surface charge and this can also contribute to lessening aggregation.

    The great majority of neoplastic cells which get into the blood die without forming a new malignant focus (Zeidman, 1965). For metastasis to occur, the neoplastic cells should adhere to the vascular endothelium. This adherence is determined by factors such as the size of the cell or group of cells, the diameter of the capillary and the "glutinosity" of the capillary wall. This glutinosity is conditioned in part by factors related to blood coagulation.

    Metastases are not distributed randomly in the organism, for all of the types and sites of malignancy have patterns and characteristic routes that the metastasis most probably will take. These routes are determined by the physical and chemical compounds that are present in the tissue or organ, the degree of surface tension and intracellular pH, as well as the concentration of the different chemical elements that make up this tissue or organ. The different patterns are conditioned partially by purely mechanical circumstances such as the location of the primary neoplasia and the magnitude of the capillary layer in the different organs. Many cancerous tumors have the propensity to metastasize in the lungs because the capillary layer of the lungs is the first filter through which the neoplastic cells pass after having entered into circulation (Southam et al., 1967).

    Beside these mechanical factors, other patterns are explicable only taking into consideration the ‘terrain’ that is most receptive to a specific neoplasia (Southam et al.,1967). Given that the physico—chemical environment is different from one organ to another, it would be strange if this were not the case.

    It has been reported that many neoplastic tumors propagate through the lymphatic system, but the role of the lymphatic ganglia has been discussed very much (Cribe, 1968). The great frequency of metastasis in such ganglia casts a doubt on the idea that they are defensive barriers. But it can be argued that the tumor lodges in a lymphatic ganglion and only replaces it when the hypothetical defensive potential of the ganglion has been used up. For now it should be pointed out that the presence of a tumor in a regional lymphatic ganglion is, in general, a sign of a grave prognosis indicating the expectance of a shorter life for the patient. There are two reasons for this: metastases in the ganglia indicate an aggressive or malignant neoplasia, and metastasis represents a neoplasia that has already begun to disseminate itself and that can propagate extensively, which reflects, basically, an increase in the intracellular concentration of toxic chemical substances modifying even more electrical conductivity and the intracellular pH.

    With respect to alternations in the cell chromosomes, given that the neoplastic eel]. is affected by a hereditary defect, the study of the kariotypes of the different tumors could reveal a distinctive lesion that was the direct result of the basic defect or its cause. With only one exception this does not appear to be so.

    It is true that all of the malignant and many of the benign neoplasias show abnormalities in the kariotype. These abnormalities encompass a wide range of phenomena which includes deletions, translocations and more complicated arrangements in aneuploid as well as euploid cells. No particular alteration, except one, seems to be related to a specific type of neoplasia. The great majority of neoplasms are aneuploid and frequently more than one modal number of chromosomes can appear in one lesion, though, generally, one mode statistically predominates. In spite of this, sometimes neoplasms, some of which are malignant, have chromosomal complements that are completely normal, quantitatively and qualitatively, as far as can be detected with the normal techniques (Nowell, 1965).

    Nowadays, the only exception that is known is the one discovered by Nowell and Hungerford in which a specific chromosomal alteration characterizes a particular type of neoplasia. In patients with chronic granulocytic leukemia it was found that the neoplastic cells characteristically showed the small abnormal chromosome Philadelphia (ph) , which in appearance derives from the G group by the loss of approximately half of its longest branch. This abnormality is restricted to leukemias persisting during an entire sickness, independently of the fact that there can be additional. changes in the kariotype during the terminal phase. This alteration has not been observed in individuals with other types of leukemia nor in persons without leukemia.

    It can be said, to summarize, that almost all neoplasias show anomalies of one kind or other in their chromosomes, though the usual lack of specific anomalies indicates that those that do exist can represent the consequences of mitotic events that occur during neoplastic growth. Therefore, they probably have little or no causal importance, but are very important for the progress of the neoplasia. The alterations in the equilibrium of the genes, caused by variable states of aneuploidia probably have profound effects on the behavior of cells (Hitosumachi et al, 1971).

    It seems improbable that a malignant neoplasia should develop from normal tissues without any intermediate benign steps. The number of these steps is still not definitely known and might be variable. The time frame for the alteration of the patterns of differentiation and growth from normality to malignancy is very variable. On some occasions a benign neoplasia can appear that never progresses to malignancy during the life of the host patient. On others, transitional changes can appear in the parent cells that cannot be detected by the techniques now in use, until the cells are plainly malignant. Only with the Oncodiagnosticator can a propensity for cancer be detected. Perhaps it can be inferred that benign stages though aberrant, precede the presence of malignancy in many if not all cases.

    The change of the biological properties of the cells that constitute a neoplasia is called neoplastic progress. Of course, almost all of the neoplasias grow and spread, but none of these changes is an integral part of progress: only strictly cellular changes are part of the term (Foulds, 1954).

    In the same way that the neoplasia begins as a consequence of hereditary alterations of the affected cells, all of the morphological, biochemical and conduction properties that distinguish malignant cells are probably the result of progressive hereditary modifications (Klein and Klein, 1957; Law, 1952; Patterson et al, 1969). There exists a proclivity of these properties to act so as to associate genetic characteristics randomly, but most probably the different properties are not found, together in identical proportions in any malignant neoplasia. Neoplasias not only vary to a great extent among themselves, but even within one neoplastic tumor the variation from one region to another can be very great. This is a function of surface tension, osmotic gradient, pH, etc. and of the chemical composition. As a consequence, when a neoplasia is judged histologically, the evaluation should be in the most anaplastic field of the microscope that can be found, because this kind of field determines the potential malignancy of the tumor but never the real malignancy. In this form the chemical elements that are the direct and indirect causes of the malignancy cannot be measured qualitatively or quantitatively.

    The variability that can be observed from one region to another in the same neoplasm can be a clonal phenomenon, that is each different region can represent the descendence of one single divergent cell that results in a relatively strange change, analogous, if not identical, to somatic mutation. It is probable that the progress of a neoplasia can be caused by the appearance, at random, of clones of divergent cells and by their subsequent amplification through natural selection (Klein and Klein, 1957). For us, the increased bio—physico— chemical disequilibrium is what augments the degree of intracellular intoxication.

    If the neoplasia progresses due to ‘the selection of more and more divergent cells that appear at random, would it be possible to account for the regression of progress by a similar mechanism? It would be logical to accept that a set of neoplastic cells that can yield even more malignant variants could, eventually, generate variants that are more normal. These variants would not be observable unless they were not selectively disadvantaged in the environment. At the moment, this is no more than wishful thinking, though some neoplasias, for example the neuroblastoma of childhood, seem to be able to become more normal or more differentiated. This would constitute a form of regression of the neoplasia’s progress. The way to do this is through Donatian Therapy in which the bio— physico—chemical terrain that favors progress is altered.

    That the hereditary modification producing a neoplastic cell has a biochemical basis is a fact that virtually does not need any demonstration. The fundamental aspects of this biochemical basis have not yet been discovered, though some hypotheses have been formulated. For us it is a fact that finds proof in the cures we have achieved through the use of Donatian Therapy.

part 2