Ribograma v17

RIBOGRAMA A project with potential In the case of colorectal cancer, it is possible to do relevant tests without an invasive procedure, because the feces contain colonocytes that can be separated by washing with a suitable solvent, so that we have laboratory access to colonocytes.

A review of medical literature has shown that it could be useful to analyze the colonocytes to determine the concentration of free ribosomes, both at one time (snapshot) and over an interval (readings once each six months). We call the curve of the numerical values of free ribosomes over time the “ribograma� for the purpose of this article.

We think that the ribograma contains useful information about the degree of malignization, and that it could be useful to determine when to take clinical measures to reduce the chance of cancer, for example the prescription of aspirin, non-steroid anti-inflammatory drugs, statins, or metformin. We see also potential for the ribograma to be used as an indicator for procedures such as colonoscopy or recommendation about lifestyle.

At this time, this thought process is considered sufficient so as to commence a project in order to obtain the experimental evidence that will determine the usefulness of the ribograma in clinical decision making.

Funding desired It is estimated that 5 million euros, spent over a period of 5 years, will be sufficient to obtain sufficient experimental results in order to determine whether the ribograma could be helpful in saving lives and improving quality of life. If so, it could help save a very large number of lives, as the number of persons for example in Europe who are presently dying from colorectal cancer is approximately 200,000 each year. Also it could improve the quality of life of a much larger group of people if this noninvasive method can make it possible to reduce presently applied invasive tests.

The 5 million euros would be used to finance 4 to 5 persons working on the project over 5 years. This cost can be achieved if the project is carried out in Portugal, Spain or UK, and is directed by the author of this project. However, because Portugal is an advanced country with lower labor costs than neighbor countries in the European Union and because the author of the project is Portuguese and lives manly in Portugal. All communications and publications about this project will be in English.

The money will be spent on arranging massive participation from persons aged over 50, preparation of ribogramas, determination of normal population bands of the ribograma, analysis of cases who are outside the normal bands or have a trend taking them outside the normal band. It is also intended to review the helpfulness of clinical measures adopted if the ribograma shows a trend towards malignization. It is also intended to review final end

1

effects, in order to determine whether the use of this method has the effect of saving lives and effects on costs and benefits.

Intellectual property rights A collection device has been designed, which is fitted to the toilet seat, and enables samples to be taken without the participant handling feces. Furthermore the collection device carries out automatically the washing of the feces and the obtention of a solution of colonocytes which can be delivered to the laboratory for analysis.

Intellectual property rights belong to the author of the project. This refers to the collection devices to be used. Also a canon could be charged to laboratories that are carrying out this test, this would be a small charge per test, and the income so resulting could be shared between the author of the project and the agency that funds the project.

Thinking process At the present time, the basis for this project is a thinking process, based on analysis of over 1,000 published articles and other knowledge of the author about oncogenic processes.

There have not yet been any publications or experimental results, only a doctoral thesis by the author.

Usefulness If, as expected by the author of the project, a research study shows that the ribograma improves clinical results, this could be an enormous improvement in the screening and management of colorectal cancer, which could potentially save many lives and improve quality of life for many more.

o.o..o.o.o‌o.o.o.o.o.o.o.o.o.o.o.o.o.o.o.o.o.o.o.o.o..o.o.o.o.o.o.o.o.o.o.o.o.o.o.o.o.o.o..o.o.o .o..o.o.o.o..o..o.o..o.o.o.o..o.o.o..o.o..o

RIBOGRAMA (*): NEW CONCEPT TO SURVEY CANCER CONCEPT AND APPLICATIONS IN the COLON AND RECTUM ONCOLOGIC CLINIC

A. Ferreira-AlemĂŁo, MD Ph.D. UNIVERSIDAD COMPLUTENSE DE MADRID Facultad de Medicina Madrid ribograma@gmail.com www.ribograma.com + 351 93 671 54 88

ABSTRACT The purpose of this research, which has been presented as a Doctoral Thesis, with the above title, is the following: -a draft method of diagnosis, screening and monitoring the colon and rectum cancer founded on technical and current concepts of molecular biology. This allows a "pre-carcinoma in situ" diagnostic since it is based on the phenotype of mucosa cells composed of very high quantities of free ribosomes in the cytoplasm, 2

which can be quantified by means of flow cytometry, after isolation, and labeled with fluorochromes. The repeated records of those quantities of free ribosomes establish a graphic curve that represents a malignant trend, which above a certain level of concentration, allows to say that the cells in a tissue (of the colon and rectum, e.g..) can be considered to be in the process of developing carcinoma in the colon-rectum mucus, before any macroscopic viewing (endoscopy). This means that we are facing a new method, on a clear biomolecular level, as shown in this Thesis, which allows a good improvement on the macroscopy (endoscopy).This method exceeds the scope of other non-invasive methods of the colon-rectum (test of occult blood in feces and test of DNA mutations in the same feces, the last of which, has been clinically practiced for many years in the USA and is, at the moment, only at an initial phase in Europe). Thanks to this method, it is possible to have a screening program (through a noninvasive method with great acceptance by the population), in which all citizens who present a greatly increasing curve of RIBOGRAMA must be (mandatorily) checked with a diagnostic/therapeutic endoscopy and, at the same time, remain under observation and therapy. With a proper food survey and diet with some antiproliferative drugs (non-steroidal anti-inflammatory, acetylsalicylic acid, metformine and statins), with proven efficiency in the prevention and reduction of the formation of polyps or premalignant trend, representing savings in the therapeutic inversion of cancer.

INTRODUCTION RIBOGRAMA is a new concept in modern biomolecular clinical oncology based on the transition from the subjective and qualitative understanding of the processes in the tumor genesis of cells to a digital and numericalsuitable way for computer analysis. One place to start, as well, is the published data of the 60's and 70's, and research related to the accumulation of free ribosomes under the chemical induction of tumor growth, through cell initiating, with 7,12-dimethylbenz(a)anthracene and promotion caused by 12-O-tetradecanoyl-phorbol-13-acetate in the interfollicular area of the back skin of mice. However, their description was considered to be important literary and scientific results, at that time, but did not allow a quantitative description of the process. According to the trend noted in the cells, characteristics of cancer were observed with heavily increasing number of free ribosomes. In the studies carried out by this author, the process of studying cell carcinogenesis is then placed in a new level with the digital definition of pathological processes, taking place with free ribosomes. It becomes an objective visualization that is read out by creating a graphic curve, with numerical values corresponding to the RIBOGRAMA which gives concrete values of the trend to the cancer. Analyzing the construction of the curve, using statistically derived banded numbers of free ribosomes one finds an abnormal and marked increase in their number, and the transition to the dangerous increase in the cancer process which, at this stage, is a diagnostic tool available in the arsenal of modern clinical oncology prior to the cancer biomarkers and to the pathomorphologic changes in the tissues under study. To calculate the number of free ribosomes the flow cytometry method is used, after their isolation and labeling with fluorochromes. Giving mathematical direction, it allows the statistical computer analysis to process the received material, the formation of different groups of persons according to the degree of risk, taking into account various factors such as the levels of height in the graphic curves, representing the quantities of free ribosomes (RIBOGRAMA representation).

THE NOVELTY OF THE WORK RIBOGRAMA is a non-invasive laboratory method that studies the dynamics of the current cancer process, through its screening and monitoring at the cellular level, i.e. even before the appearance of early histological and clinical manifestations in the body. The method is based on the author’s study of the literature in the field of electron microscopy which utilizes the latest molecular biology, biomedical engineering and informatics. The study is based on the calculation and statistical analysis of the dynamics of the change in the number (density) of free ribosomes in the cytoplasm of eukaryotic cells, the so-called phenotype of the analyzed organs and systems and the construction of individual RIBOGRAMA graphic curve. It is shown that in the cell a very big increase in the biogenesis of free ribosomes (and the simultaneous increase of RNA) indicates the presence of cancer.

3

MATERIALS AND METHODS The method is based on a retrospective analysis of 206 basic and well-documented scientific observation studies in the field of electron microscopy of cells with a malignant phenotype by using the database of research published literature in PubMed. It is based on detailed changes in malignant tumor cells and tissues. A method of quantification of free ribosomes was developed (those responsible for the internal cell economy), with help of the principle of flow cytometry after isolation and labeling them with fluorochrome staining. With a sequence of records obtained with the numerical values representing the quantity of free ribosomes, over a time period, it is possible to do a mathematical graphic analysis of the dynamics of free ribosomes biogenesis and then to interpret the process of carcinogenesis, by reading the RIBOGRAMA graphic curve. In this way trend values are obtained and their changes, giving evidence of the existence and nature of a malignant process.

RESULTS The descriptions of the morphologic aspects of cells in each tissue, under study, were critically analyzed (in the qualitative aspect). In this way, six different groups of expressions were created from this search, relative to the quality content of free ribosomes in cancer cells (in the 1st group - numerous free ribosomes - 36 observations; in the 2nd group- rich in free ribosomes - 5 observations; in the 3rd group - abundant free ribosomes-36 observations; in the 4th group - many free ribosomes -19 observations; in the 5th group - increased number of free ribosomes 28 observations; in the 6th group - free ribosomes - 70 observations). In this search there were some other terms that have been used, which were not so frequent in the chosen groups among the 206 cases collected and they have not been highlighted. Of the available scientific evidence, under the morphologic aspect (Electron Microscopy), the six groups of expressions from the search do not allow to have an accurate mathematical analysis to identify the risk of evidence - based trends in cancer process - which, in the end, has not allowed the adoption of the correct tactics of treatment in the clinic. The proposed present quantitative method follows the construction and analysis of recording quantities of free ribosomes, from a certain tissue, of a patient under study, which allows analyzing and predicting the dynamics of directions and trends of a cell group, after making a RIBOGRAMA graphic curve. With the construction of a patient RIBOGRAMA graphic curve it becomes possible to quantify the enlarged quantity of free ribosomes - in the time evolution aspect – and to have the time of each numerical change and, subsequently, the possible transformation and transition to the phase of clinical manifestations (before the change of blood markers and histological changes), distinguished from the normalization process or apoptosis, we are monitoring the behavior of a future cancer cell.

DISCUSSION A PERSPECTIVE ON THE BIOLOGY OF MALIGNANT CELLS Several lines of evidence, some well established and recent, support emphatically the idea that RNA content is high in cancer cells and genetic events that lead to cancer are often linked, directly or indirectly, to the ribosome biogenesis (20,21).On the other hand, the inverse problem is observed in the cases of "idiopathic ineffective anemia� (IIE - Ineffective erythropoiesis idiopathic), where there are insufficient rates of cell proliferation, and patients have erythroblasts containing only 70% of normal levels rRNA (27). Then, in a situation of excessive cell proliferation (aggressive cancers) rRNA levels are amplified, whereas in a situation of insufficient cell proliferation (certain anemias marrow hypoplasia) rRNA levels are depressed. There have been developing oncobiologic investigations which have shown that the expression of the alterations of rDNA (ribosomal deoxyribonucleic acid) and ribosomal protein genes are associated with the development of tumors and cytological studies show that the nucleolus of cells cancer are increased because they have an increased transcriptional activity, representing predictive aspects of cell proliferation rate and prognosis of patients (28,29).These researches have shown that cancer cells with high expression of these ribosomal proteins have a higher content of ribosomes. The genetic alteration associated with cancer development very often involves changes in signaling pathways that leads to 4

the rDNA effect (28, 29). The scenario described above reiterates what is known by cancer biologists: ribosome biogenesis and tumor genesis are closely linked.

ACCUMULATION OF RIBOSOMES IN THE PROCESS OF ONCOGENESIS In the nineteen sixties and seventies studies were published on free ribosomes and ribosomes bound to membranes (42,43) and its accumulation during the induction of growth in many organs and tissues (33-36). In the sequence of these investigations, studies have been published in the attempt to quantify the accumulation of ribosomes in interfollicular areas of the dorsal skin of mice during chemically induced neoplastic growth in two phases, initiation by 7,12-dimethylbenz (a)anthracene and promotion caused by 12-O-tetradecanoyl-phorbol-13acetate (52).The epidermis is an epithelial surface and like other epithelia which are in direct contact with the external environment, such as the linings of the respiratory and gastrointestinal tracts, show a high incidence of cancer. Thus, the epidermis has served as a useful model to establish a comprehensive development of the role of ribosome accumulation during neoplastic growth (47).

POTENTIAL BIOMEDICAL APPLICATIONS RESULTING FROM THE QUANTIFICATION OF RIBOSOMES The preparation of ribosomes, depending on the tissue under study, can be done through some adaptive modifications, published in previous studies (37-41). The preparation of ribosomes can be used for many purposes, for example, to study the growth and development of tissues induced by hormone action (50), the study of gene expression including synthesis of ribosomal proteins which are translational control (44-45) studies of the assembling of the ribosome(51), studies of differential expression of ribosomal proteins of the mucosa of the colon and rectum, normal and neoplastic (46), among many other potentials, and most likely, in nonmalignant diseases. The identification of changes in the structure of the cytoplasm, such as the sharp increase in the number of ribosomes (quantitative method) accumulated in the cytoplasm of a community of cells of a given tissue, may be one of the phenotypic expressions in malignancy of the fine structure of the transformed cells, which may be essential to characterize the evolution of cell behavior. In this sense, for example, a profile study of the graphical changes in the number of ribosomes per cell (or per unit volume) in a defined time period, within an average of exfoliated cells of colorectal mucosa of a patient, which will form a standard curve/ average number of ribosomes per unit volume in the RIBOGRAMA in relation to the cells lining the colon and rectum, isolated and separated from the feces. Like many fields of life sciences, biology of cancer is an exponentially growing field and highly complex, including work that ranges from molecular biology to epidemiology and oncogenes environment.

Survival rates for various cancers, once they become clinically observable, have shown a modest improvement in recent decades. There is a strong motivation to integrate different fields of knowledge in cancer biology and introduce a new conceptual and theoretical framework that can improve the understanding the dynamics of tumor researches so as to develop better therapeutic measures. It will be important that mechanistically predictive models are produced based on tumor dynamics that can abstract the significance of the relatively advanced biological details for oncogenesis and tumor progression. While it is true that cancer is a multifaceted disease with a variety of close "triggers" in different tissues and different patients, there is also a strong possibility that cancers share a central feature originating from a common cellular machinery from which the cells depend for their proliferation (19).The most visible aspect of aggressive malignancies is increased cell proliferation, which has at its core a marked increase in protein synthesis. In the process of normal mitogenic response, there is a transient and cyclical increase in the rate of general protein synthesis. The overall increase in protein synthesis is a necessary phenomenon, observed and controlled before cell division, leading to duplication of content and the increase in size before the normal mitosis. Thus, the average size of the cells is maintained during physiological proliferative response. One of the key mechanisms in the loss of control of the protein synthesis in transformed cells is the inability to decrease the number of ribosomes that are correlated with cell proliferation in fresh culture media without added growth factors to serum (1). Many researchers have observed, in tissue cultures, differences in growth properties between normal cells and their malignant counterparts, one of which points to the failure of these by showing a cyclic variation of cellular and biochemical parameters through the cell cycle or 5

growth cycle (3-6). Previously, other researchers have given attention to changes that occur in the rate of protein synthesis and function of machines that are "translational" within the cell, relative to the cell cycle, once such changes are necessary for transitions and growth and multiplication in normal conditions.

THE NUCLEOLUS In this context, it makes sense to talk about the fact that many pathologists during the routine examination and evaluation of the biopsy material often pay attention to the nucleoli (the sites where rRNA is synthesized and ribosomes are "assembled"), as, in malignant neoplasms, nucleoli are altered in some way in relation to the number and size. Thus, the associations of altered nucleoli in neoplastic disturbances have been referred to research of more than fifty years ago (2). The nucleolus is a functional, well-defined structural unit in the interphase cell in which ribosomal genes are located and where ribosomal RNA synthesis (7) occurs. Among other functions, the nucleolus, which is a key organelle, coordinates the synthesis and assembly of ribosomal units. The production of ribosomes is an important metabolic activity and thus the role of the nucleolus is closely linked to cell growth. Recent data suggests that the nucleolus also plays an important role in cell cycle regulation, senescence and stress responses (30). Ribosome biogenesis involves the synthesis of rRNA, maturation and assembling of RNA and ribosomal proteins in ribosomal subunits, small and large. This process is regulated through the cell cycle, primarily at the level of rRNA synthesis (31). RDNA transcription reaches its peak during the S phase and G2, and is interrupted when the cell enters mitosis and is reactivated when cells exit mitosis (32).In the last ten years significant knowledge has been acquired about the significance of changes in the nucleolus in tumor pathology, through studies of changes in distribution of a group of nucleolar proteins, proteins AgNOR (Argyrophilic Nucleolar region Organiser) in malignant tissues. These proteins are required for ribosome biogenesis and are selectively stained by silver methods (8). The two most important proteins nucleolin and protein B23 are involved in rRNA synthesis and processing. (9).On studies of tumor biopsy fragments for in situ observation of cytohistological and isolated proteins, it is verified that the amount of AgNOR proteins increases progressively when resting cells enter the mitotic cycle from G1 to the end of S phase (10 - 12). Also, there is evidence that the amount of AgNOR proteins are directly related to the rapidity of cell proliferation in cell lines establishing human cancer (13-15).The NOR (Nucleolar Organiser region) are sites of rRNA transcription, modification post-transcipcional of RNA transcripts and its "assembly" in functional ribosomes. The numbers of NORs expressed in a tissue are related to the rate of cell proliferation, with differentiation and neoplastic transformation. This has been used to demonstrate the potential malignancy and to assess prognosis and malignant aggression (16-18).Quiescent hematopoietic cells such as the not activated T and B lymphocytes, and pluripotent stem cells, are small with a lower content of protein and RNA than their counterparts in the state of proliferation. During the transition from G0 to G1 there is a mandatory increase in cell mass and in the number of ribosomes (48) with an increase in the rate of protein synthesis, due in part to increased initiation factor eIF-4E (49).In skin tumors in mice induced by the application of tumor promoters, the RNA: DNA relation and RNA content (percentage of dry mass contributed by RNA) were 2 to 3 times higher than in normal tissues (22). In several types of leukemia, the cell RNA content was strongly correlated with accelerated cell growth kinetics and the prognosis of the patient (23).In a study of gynecologic cancers in which the neoplastic tissues were compared with their normal counterparts, the content of DNA and RNA content in the neoplastic tissues were increased 1.6 and 2.4 times, respectively (24). Similarly, cellular RNA content has increased by a factor of 1.4 in neuroblastoma cells myc-transfected cells relative to normal (25). In another study of breast cancer, the test of cell DNA content was normal, but the cellular RNA content was well correlated with tumor grade, histological type, with the hormonal status and patient survival (26).

FEATURES OF MALIGNANCY versus QUANTIFICATION OF FREE RIBOSOMES One verifies that the descriptions of Electron Microscopy of cells with phenotypic characteristics of malignancy vary by greater or lesser degree of differentiation of its texture, or morphology, but these descriptions are subjective, depending on the observer who is doing the research or pathology exam. In terms of molecular biology of cancer, malignant transformation of normal cells is the progressive acquisition of a number of specific 6

genetic changes acting by disobeying strongly antitumor mechanisms in all normal cells, which include: a) regulation of signal transduction; b) - cell differentiation; c) - apoptosis; d) - DNA repair; e) - cell cycle progression; f) - angiogenesis; g) - cell adhesion. Regarding the descriptions of EM, these mechanisms of malignant transformation are judged only by subjective criteria, in which the characteristics cannot be described in mathematically parametrized form, as happens with the descriptions listed in TABLE 1.

Similar to the properties of the malignant transformed cells growth in cell culture or in vivo, when biology cell techniques were observed, molecular biology does not allow a rigorous quantitative assessment, because it depends on subjective criteria, without numerical decision rules as shown in the properties which are listed in TABLE - 1, (properties of transformed malignant cells growing in cell culture and / or in vivo). With free ribosomes it is possible to quantify them by counting, using the technique of flow cytometry.

TABLE-1 Properties of transformed malignant cells growing in a culture of cells and/or in vivo 1. Cytological abnormalities similar to cancer cells in vivo, including increased cytoplasmic basophilia; number and size of the grown nuclei; nucleus-cytoplasmic ratio increased; formation of clusters and cords of cells; 2. Altered growth characteristics: a.“Immortality “is transformed cells in culture. The transformed malignant cells become “immortal" in that they can be transferred in culture indefinitely. b. dependent Inhibition decrease in cell density or loss “contact inhibition “. Transformed cells often grow to more than their normal counterparts until high density, and they can pile up in culture rather than stop growing when they touch each other. c. Decreased serum needs. The transformed cells require decreased serum levels of growth factors in culture to replicate comparably to the non-transformed cells d. Loss of dependency to anchor and acquiring the ability to grow in soft agar. Transformed cells may lose their need to grow attached to surfaces and can grow as free colonies in a semisolid medium e. Loss of cell cycle control. Transformed cells do not stop in G1 or at G1 / S boundary of the cell cycle when subjected to metabolic growth constraints. f. Resistance to apoptosis (programmed cell death) 3. Changes in the structure and function of the cell membrane - including augmented agglutination by the plant lecithins, alteration in the composition of cell surface glycoproteins, proteoglycans, glycolipids, and mucins; appearance of tumor-associated antigens; and increased uptake of amino acids, hexoses and núcleosidos.

4. Loss of cell-cell and cell-extracellular matrix that promotes cell differentiation interactions.

5. Loss of response to anti-differentiation-inducing agents and altered cellular receptors for these agents.

6. Disruption of signal transduction mechanisms, including constitutive growth receptors, cascades of phosphorylation and dephosphorylation mechanisms instead of a controlled function.

7. Increased expression of oncogenic proteins due to translocation, amplification and chromosomal mutation 8. Loss of protein products of tumor suppressor genes due to deletion or mutation.

9. Genomic reading errors which provokes overproduction of growth-promoting substances, eg, IGF-2.

10. Increased production or deregulation of growth factors, e.g., TGF- alpha, tumor angiogenesis factor, PDGF, hematopoietic growth factors (e.g., CSFs, interleucins) 11. Genetic instability, leading to progressive loss of regulated cell proliferation, increased invasion and increased metastatic potential. The “switched“genes may be involved in this effect 12. Alteration of enzyme pictures. The transformed cells have increased levels of enzymes involved in the synthesis of nucleic acids and produce higher levels of lytic enzymes, e.g., proteases, collagenases and glycosidase levels 13. Production of oncodevelopment gene products. Many malignant transformed cells growing in culture or in vivo produced increased amounts of oncofetal antigens (e.g. carcinoembryonic antigen), placental hormones (e.g., chorionic gonadotrophin ) or fetal-placental isoenzyme type (e.g., placental alkaline phosphatase).

14. Capacity to produce experimental animal tumors. This is the sine qua non that defines malignant transformation in vitro. If the cells thought to be transformed do not produce tumors into appropriate host animal, then they cannot be defined as "malignant." However, the failure to grow in an animal model does not exclude the fact that they can be tumorigenic in an animal of a different kind.

15. Capacity to prevent antitumor immune response of the host.

7

_____________________________________________________________________________ Given these characteristics, with the evolution of the variable time, it is not possible to obtain a numerical record, which might constitute a set of values, a graph or trend expression that translates mathematical and reproducibly dynamic state trend growth and proliferation of a set of cells belonging to a given tissue, as in the case of the mucosa of the colon and rectum, which is the object of the scope of this study.

THE RIBOGRAMA GRAPHIC CURVE (THE ESSENCE OF THE METHOD) In the framework of figure n.1 there are two axes designed to represent the two Cartesian coordinates (horizontal axis and vertical axis) with which one is building a graphical curve. This curve is characterized by two variables for the free ribosomes of cytological community of cells under study (the epithelial cells of colorectal mucosa or colonocyte). These variables are:"quantity� (number) average of free ribosomes in the cytoplasm of each cell and "time" over which the behavior of certain variations of the average "quantity� (number) of free ribosomes is observed. There will be the need to determine, experimentally, graphic profiles of numerical values (average quantity/number of free ribosomes) that can be set as normal, from which one can draw a curve-middle-register.

Thus, it is possible to make comparative studies of large population groups, which will be possible to see multiple potential trends of malignant disease (by increases in the curve) in a community of cells in a tissue under investigation. In studies correlating electron microscopy and biochemical data (usually quantitative), the morphological (electron microscopy) is more limited and dependent of descriptions based on subjective criteria.

This criterion (electron microscopy approach) is subjective because it does not allow a statistical measurement of data, with mathematical rigor, preventing a parametric correlation with morphological data for biochemical data.

The outline of the graph is a record of the quantitative assessment of free ribosomes in the static and dynamic sense, as information collected from sub cellular fractions, which can be correlated with other parameters of quantitative cell biological measurement (e.g., some tumor markers, e.g. CEA.).From the knowledge of the range of values " medium quantity (number) of free ribosomes" considered within the normal range (range A) one may consider that other intervals in the outline of the figure are represented by B - level increased, C - level alarm, D clinical level, A - low level and 0 absent. In the horizontal axis are inserted multiple multiples of a time period, for example, multiples of three months, (1, 2, 3... 10) starting from the beginning of the monitoring records of the values of "quantity (number) average free ribosomes of cells under study. Descriptions of measurements of the cells with phenotypic characteristics of malignancy vary by greater or lesser degree of differentiation of their texture or morphology, but these descriptions are subjective, depending on the observer who does the research.

The same happens when free ribosomes suffer a progressive variation in the amount, moving from the bottom of a crypt to the apex of intestinal villi. Free ribosomes can be counted, or their quantity can be measurable and comparable over time. The free ribosomes can be quantified by counting them, using flow cytometry techniques.

Thus, the procedure for screening and early diagnosis of colorectal cancer, which the author advocates in this document, is based on monitoring the quantitative profile graph of the change in the number of ribosomes per cell (or per unit volume) in a defined period of time, within an average of the exfoliated cells of colorectal mucus, isolated and separated from the feces of a patient, whose records will form a sequential curve pattern/average number of free ribosomes per unit volume, after an adequate isolation of free ribosomes, using certain standard techniques. Harvest of feces: A significant number of cells remain intact and viable in the feces and they can be isolated. It has been shown that these cells are representatives of the entire colon and can be very useful in clinical research in the process of the disease. Exfoliated colon cells (colonocytes exfoliated): The colonic cells exfoliated in feces can be collected in a transport medium at room temperature in a isolated collector suitable for such a role, thereby obtaining cells for detection and making it possible to study the number (quantity) of free ribosomes in a predefined pattern mucosa epithelial cell. The application of molecular biology techniques to exfoliated cells of the colon and rectum profoundly increases the sensitivity of the detection of colorectal cancer or its tendency to malignant transformation, enabling its use in diagnosis and screening.

8

To complement the description being made and in order to help better understand the features of this method, accompanying this specification, as an integral part thereof is a set of drawings and it is explained in an illustrative mode to represent the RIBOGRAMA graphic curve:

ll Figure n.1 - the figure shows an example of a scheme in which the axes represent the two cartesian coordinates related with the two variables: the quantity of free ribosomes (per cell or per unit of volume) and the time in which there is a variation of them. One constructs a curve to represent the change in the quantity of free ribosomes per cell (or per unit volume) in a defined time period, within an average of the exfoliated cells of colorectal mucosa of a patient.

→ Curve that starts at the normal level, when reaching the increased level it indicates the advisability of a prophylactic study on the reasons or causes that determine its appearance.

The cells at this level (increased) still have no security features of malignancy, but their increased number of ribosomes continues growing. The cell is transformed into a malignant phenotype (alarm level). At this point it is essential to make a detailed study to survey and identify possible mutations responsible for the uncontrolled growth of ribosome biogenesis in the process of carcinogenesis; → Segment of the curve derived from the anterior (segment a-b), which corresponds to the phenotypic alterations and to visible light microscopy in which cells present already with phenotypic features of malignancy; → Segment of the curve, continuing the anterior segment, which represents the level of signs and symptoms of the CRC disease (clinical level); → This segment of the curve corresponds to a change in direction of the curve a-b that results from preventive action and treatment (medications and special diet); → corresponds to a curve in which cells grow (multiply or proliferate) in a selfmonitoring mechanisms, with increased proliferation, as in the case of the seminiferous tubules, or the endometrium. For this increased level of ribosome biogenesis the control is within normal physiological mechanisms (non-malignant); → Normal curve; → This work is intended to study the item colorectal cancer (CRC), in which ribograma curve is high, so it is not the time to talk about the meaning of it at low level. Still it will be 9

useful to study conditions of degeneration and involution of cells and tissues, either the intestinal mucosa, or other extra-intestinal tissue.

RIBOGRAMA AS A BIOMARKER The RIBOGRAMA METHOD is a real biological biomarker that monitorises the behavior/phenotype of a community of cells under study/treatment (e. g. breast, colorectal tract, upper digestive tube, lung, prostate, gynecologic tract, etc), before metastatic cancer explosion. If this biomarker is adopted, we can analyze someone's tumor tissue before treatment and see how they respond to medication and follow the rules of personalized treatments. The RIBOGRAMA METHOD is a strong tool of the chemistry, manufacturing and control of the new (and/or orphan) drugs, as well the old medicines, because it is based on the mechanisms of the universal biological macromolecules in the cytoplasm, like the free ribosomes. The RIBOGRAMA METHOD is an in vitro predictive biopharmaceutical tool for drug therapeutics choice, especially in sensivity to cancer cell lines and to stem cell cultures (in view to the sensivity of cell upstream machinery to the free ribosomes), since one can read the height of the RIBOGRAMA curve as it changes in response to a certain action of a substance. The RIBOGRAMA METHOD helps to make a quick decision in the choice of the best antiproliferative drug in cancer therapy and the understanding of the behavior of human induced pluripotent stem cells to the drug action. The goal of RIBOGRAMA is an innovative biomolecular method for very early diagnosis (not invasive) of cancer, which is based on a simple new biomolecular approach, similar for all types of cancer, particularly hematological cancers and cancers affecting the lung, breast, gastrointestinal tract, pancreas, gynecology and urogenital tracts.

It will enhance the life of patients and improve the management of cancer disease, allowing the monitoring and the efficiency of any cancer treatment and other non-cancer diseases. According with our present understanding of the multiple known basic mechanisms of the uncontrolled cell growth and metastasis, there is a growing big number of multiple pathways, from the malignant tumor growth to angiogenesis and metastasis that complicates the design of a transversal method of diagnosis of cancer of the eukaryote cells. There is a very wide source of information on cell signaling pathways (up - and downstream in the cell) which makes the communications in the cellular biomolecular processes complex and difficult to read in its global context, such as in signal transduction resources, information on Akt signaling, apoptosis, cancer pathways, caspase cascades and pathways, the cell cycle, cell death pathway, cell stress, cyclic nucleotides, cytoskeleton, extracellular matrix signaling, G proteins, gene regulation, growth factors, hormones, immune cell signaling, ion channels, lipids, multi-drug resistance, neurobiology, neurotransmission, nitric oxide, phosphorylation, receptor signaling and receptor signal pathways, secondary messengers and signaling molecules. The number, the cross-talk and complexity of the signaling paths in the function of the cellular biomolecular network naturally became an obstacle to design a transversal method to diagnose a hyperproliferative state of a community of cells. With the RIBOGRAMA METHOD it is possible to design a curve with the quantities of free ribosomes in the cytoplasm of a community of cells under study, allowing in advance the monitoring of its biomolecular response, in a global and integrated fashion, allowing to know if we are in a state of hyperproliferation or in a diminishing of the state of proliferation. Defects in cell-signaling pathways allow cancer cells to alter their normal programmes of proliferation, transcription, growth, migration, differentiation and death. Tumour formation and progression occur through a range of defects that develop both within and outside the cancer cell. Many different anticancer agents have therefore been developed to target proteins that act in many of these pathways. These very important aspects about the cell behavior can`t give a monitoring quantitative information like the RIBOGRAMA. RIBOGRAMA versus TUMOR MARKERS IN THE DIAGNOSIS AND MONITORING ACCURACY IN CLINICAL PRACTICE In the figure above it can be regarded as the biomolecular path located between the initial position of the DNA (where the genes, which are more than sixty thousand, but of whom are only known slightly more than three hundred with known functions, some were little studied) and the final position, which are the proteins. Genes work 10

in "concert" with each other, with the result that the uncertain variable functions which might be attributed to the genes are usually limited to certain safe functionality As it remains to determine most of the functions of the genes it is unreliable to make a diagnosis based on some known genes, either for malignant diseases, or for non-malignant diseases, since it is much more what you do not know than what is already known. Having regard to the variable in time, to monitor any parameters of a cellular community, considering the upstream position of ribosomes, on the basis of biomolecular cellular machinery, from the outset there are countless variables that exist in the nature of the genome (some known and others unknown) which do not allow to be graphical / mathematical, rather than what is achieved with the quantification of free ribosomes (introducing the concept of RIBOGRAMA). On the other hand, in a position downstream of the ribosomes, the proteins that may be considered markers of a certain line of cancerous cells are the gene products of a cell line; not allowing a very early diagnosis, which does not happen with the slope of the curve of the RIBOGRAMA that allows you to monitor your ascent curve. The RIBOGRAMA is, for those reasons, a strong, safe and convenient diagnostic tool, for screening, monitoring and prognosis in vitro, in clinical practices and bioanalytical studies. Tumor markers are, usually, proteins, that are usually produced by the cancer cells. Some tumor markers are specific for one type of cancer, while others are seen in several cancer types. Many of the well-known markers are seen in non-cancerous conditions as well as cancer. Tumor Markers are biochemical substances elaborated by tumor cells either due to the cause or effect of malignant process. These markers can be normal endogenous products that are produced at a greater rate in cancer cells or the products of newly switched on genes that remained quiescent in the normal cells. Consequently, they cannot be used to diagnose cancer. There are only a handful of well-established tumor markers that are being routinely used by physicians.

Many other potential markers are still being researched. Some marker tests cause great excitement when they are first discovered but, upon further investigation they prove to be no more useful than the markers already in use. The goal is to be able to screen for and diagnose cancer early, when it is the most treatable and before it has had a chance to grow and spread. So far, no tumor marker has gained acceptance in the medical practice as a general screen, including the Prostate Specific Antigen (PSA). The markers are either not specific enough (too many false positives, leading to expensive and unnecessary follow-up testing) or they are not elevated early enough in the disease process. In 1968 the World Health Organization recommended ten principles to be followed when countries consider developing national screening programs. The essence of these is that the disease should be important, well understood to be able to be recognized and tested for at an early stage.

Medical support and treatment must be available and be more beneficial if given at an early stage. The health benefits must be greater than any harm done by the screening process which itself must be cost effective. Some people are at a higher risk for particular cancers because they have inherited a genetic mutation. While not considered as tumor markers, there are tests that look for these mutations in order to estimate the risk of developing a particular type of cancer. BRCA1 and BRCA2 are examples of gene mutations related to an inherited risk of breast cancer and ovarian cancer. Screening for early diagnosis has also led to lower mortality for diseases such as breast cancer and cervical cancer. Many malignancies, however, are still diagnosed after the metastatic process has already started, indicating a poor prognosis. Tumor markers are usually proteins associated with a malignancy and might be clinically usable in patients with cancer. A tumor marker can be detected in a solid tumor, in circulating tumor cells in peripheral blood, in lymph nodes, in bone marrow, or in other body fluids (ascites, urine, and stool). A tumor marker may be used to define a particular disease entity, in which case it may be used for diagnosis. Tumor markers comprise a wide spectrum of biomacromolecules synthesized in excess concentration by a wide variety of neoplastic cells. The markers could be endogenous products of highly active metabolic malignant cells or the products of newly switched on genes, which remained unexpressed in early life or newly acquired antigens at cellular and sub-cellular levels. The appearance of tumor marker and their concentration are related to the genesis and growth of malignant tumors in patients. An ideal tumor marker should be highly sensitive, specific, and reliable with a high prognostic value, organ specificity and it should correlate with tumor stages. However, none of the tumor markers reported to date has all these characteristics. In 11

spite of these limitations, many tumor markers have shown excellent clinical relevance in monitoring efficiency of the different modes of therapies during the entire course of illness in cancer patients. Additionally, determination of markers also helps in early detection of cancer recurrence and in the prognostic. A tumor marker produced by the tumor and, when present in significant amounts, indicates the presence of a cancer. They may be present as intracellular substances in tissues or may be released into the circulation and appear in serum. Continuing search for suitable tumor markers in serum, tissue and body fluids during neoplastic process is of clinical value in the management of patients with various malignancies. The spectrums of biochemical tumor markers reported to date are very wide.

PROCEDURES FOR PREPARATION AND COUNTING FREE RIBOSOMES TECHNIQUE AND STEPS TO REALIZATION OF THE METHOD For the counting of free ribosomes in the unit volume under study the following techniques and/or phases will be employed: 01 - HARVEST FECES - COLLECTOR to harvest the stool sample, a design has been made for a feces collector, which is fitted into the opening rate of the toilet, which contains an easy closure device in opaque plastic-type material. Thus, the patient produces a unique ejection of feces, at home, directly to the appropriate container (adaptable to the closure rate and easy-zipper type), which is securely closed, without any manipulation of stools to be sent to a lab that runs the techniques of the method.02 - COLLECTION OF EXFOLIATED COLONOCYTES. The stool is formed by a mixture of undigested alimentary waste, microflora, endogenous secretions and exfoliated cellular components of the intestinal tract. When neoplasm is developed, the mucosal cells and tissue fragments are scaly and released in the feces of the intestinal lumen, and it’s then constituted as the sample for the test. There are techniques of isolation and collection of viable exfoliated human colonocytes intact from the stools, which are extensively studied and standardized so that, with appropriate adaptations, they can be used for the preparation and production of free ribosomes. 03 - RUPTURE OF TISSUES AND CELLS. The process of Subcellular fractionation has two stages: - Rupture of cells or tissues to obtain a cell lysate (or tissue) in which the desired fraction is in proper condition for purification. - Separation of the desired fraction of the remaining components of the cell or tissue using any criteria such as a density difference (gradient centrifugation or differential centrifugation), the presence of an antigen, etc.. - Techniques of tissue and cell breaking. The first step in the purification of most proteins and subcellular structures is the disruption of tissues or cells to obtain a cell lysate in which the protein or multiprotein complex subcellular structure is in conditions where isolation is permitted. This is accomplished using procedures commonly known as soft mechanical homogenization. These methods produce the rupture of cell membranes, gently, thereby releasing the cellular contents. The most common homogenization procedures are: - Sonication. It involves the application of ultrasound to a cell suspension. The intense agitation destroys the cell membranes. Depending on frequency, intensity and energy applied it can also destroy even the subcellular structures containing solubilized protein complexes. Cold is usually applied to prevent overheating of the samples that could cause denaturation of proteins. - Use of detergents for solubilisation of cell membranes. Cells are passed through small diameter holes that cause the breakdown of cell membranes.04 – HOMOGENIZATION. To prepare the tissues for cell fractionation, we must first suspend in an appropriate medium (a buffer solution, isotonic saline or sugar). To maintain the integrity of the organelles and enzymes during the fractionation procedure, all solutions and glassware should be kept cold. After slicing the leaves, the tissue is prepared for the homogenization, a process that breaks the link cell and releases the cell contents without damaging the suspension. The cell suspension is called homogenized constituents. Homogenizers - it happens with the breaking of the cells with the help of a rotary piston that fits perfectly with the thick walls of a special glass tube, the homogenizer. There are devices in which homogenization is calibrated for separation between the plunger and the glass wall to produce homogenized with a particle size. 05 - SUBCELLULAR FRACTIONATION Subcellular fractionation, which can encompass a set of methods and techniques, aims to obtain pure fractions or enriched in a specific cellular component, be it an organelle (mitochondria, nuclei, peroxisomes, ribosomes ...), a fraction of membranes (total membrane, plasma basolateral domain, apical domain ,...), or multiprotein complexes (actin cytoskeleton, 12

microtubules, nuclear pores, etc. ..). The subcellular fractionation is a technique that involves the separation of different cell components by differential centrifugation. To do this, the cells are crushed and centrifuged several times. Each centrifuge separates a distinct organelle from the cell (nucleus 1). Observe the speed at which sediments may know each organelle sedimentation coefficient expressed in units of S (Svedberg) (e.g. 70s ribosomes). The cell fractionation is used to investigate the biochemistry and physiology of organelles outside the complex environment of the intact cell. 06 – CENTRIFUGATION The centrifuges are tools to bring to the samples strong forces inducing sedimentation of the particles which have a density greater than that of their surrounding medium. In general they differ depending on the margins of acceleration applied to the samples submitted: centrifugal (from a few g to approx 3000xg), SUPER (or high-speed centrifuges range up to 20,000xg) and ultracentrifuges (up to 60,0000xg). The centrifuges are often controlled in a temperature chamber to prevent overheating of the samples due to friction. In ultracentrifuges, extreme speeds (over 100,000 rpm), are necessary to make a strong vacuum in the centrifuge chamber to prevent heating of the rotor and sample. In a centrifuge the determinant element 5 is the rotor spinning device and in which tubes they are placed. The parameters to be present in any centrifuge, which determine the conditions, are: A - Volume of solution in centrifuge, which will determine the type of tubes and rotors to use. B - Chemical nature of the solution, which will determine the nature of the tube to use. C - Differential in density between the particle sedimentation and density of the medium in which it is located. In general, the height difference before (shorter and less force of acceleration) sediment.

When the difference is very small it can be spun by hundreds of thousands of g for hours. Due to differences in size and density, each cell component is subject to a centrifugal force. The force generated by the centrifuge is expressed as relative centrifugal force (RCF) in units of g. The RCF is a function of centrifuge speed in revolutions per minute (rpm) and the particle distance from the axis of rotation. 07 – SEDIMENTATION RATE It is possible to exploit the difference in speed to sediment the particles for a spin in an environment where there is a density gradient, being lower in the top and largest at the bottom. After a while the different particle populations are at different depths 5 tube. Making a small hole in the bottom of the can, different fractions can be collected containing the different populations separated. This is the foundation of the preparative ultra-centrifugation, which determines the settling velocity of a particle (measured in units Svedberg, S). After uniformly centrifuging the sample, it will have separated into two fractions: supernatant (‘supernatant’) homogeneous fraction that has not settled and the pellet (‘pellet’) which has been affixed in the bottom of the tube. 08 - DIFFERENTIAL CENTRIFUGATION. Differential centrifugation: In differential centrifugation, the homogenate is centrifuged repeatedly at high speed, gradually sedimenting small particles. The first is a 600g centrifugation for 10 minutes, which settles the nuclear fraction. This pellet or ball contains the nucleus, intact cells and tissue. The next separation is the post-nuclear supernatant or liquid particles suspended in the uniform pellet nuclear, which is transferred to another centrifuge tube and centrifuged at 10,000 g for 30 minutes in a refrigerated centrifuge. The material regimented called mitochondrial fraction contains mitochondria and micro bodies. Differential centrifugation is based on the existence of different particles in suspension that differ in density of the medium. If centrifuged in mild conditions (recently, little acceleration force) sediment particles larger and / or more dense.

When the supernatant of the first centrifugation is centrifuged again in terms of more time and acceleration force, it settles back denser particles present and so on. Increasing severity in the centrifuge may be applied and one gets a collection of sediments that are successively particle fractions of different size and / or density. 09 EQUILIBRIUM SEDIMENTATION - This is usually performed using the ultracentrifuge. This is the separation of particles according to their buoyant density. The sample above is available or mixed with a density gradient steeper than the previous case, which contains a very high concentration of sucrose or cesium chloride. On centrifugation each subcellular component will shift up or down until it reaches a position where its density equals that of their environment (position of neutral buoyancy) and it did not move anymore. As a consequence, it produces a series of discrete bands, the closest one in the bottom of the tube containing the larger particles with buoyant density. This method is also called equilibrium density gradient. Different substances are used to achieve the density gradients, such as sucrose, coffee, and cesium chloride. The gradients can be self-generated such as those based on cesium chloride formed in the centrifugation process itself, or preformed such as sucrose or 13

percolator. In the latter case the preparation is done before centrifugation through a gradient-forming device that consists of two cells connected by the base and with agitation in solutions that fit with the two extremes of concentration. As it is pulled from one another replacing the fluid removed, the concentration changes linearly. An alternative is discontinuous gradient centrifugation, which creates a stepwise gradient inside a tube that successively stacks volumes and homogeneous solutions of different densities. In the interfaces of the different layers floating particles (less dense) will accumulate in the upper layer and on the bottom (denser) the particles that have sunk. 10 - PREPARATION OF FREE RIBOSOMES AND LABELING WITH FLUOROCHROMES. It should be noted that the translation of genetic information into polypeptides by the ribosome is a universal and fundamental cellular process. The structural and biochemical evidence of ribosome dynamics strongly supports its functional importance during protein synthesis and is described as conventional molecular biology techniques that allow you to separate cellular ribosomes (free and bound to membranes) from other subcellular organelles There are several studies that describes various ways of labeling ribosomes. The preparation of ribosomes, depending on the tissue under study, can be done through some adaptive modifications, published in previous studies. 11 - QUANTIFICATION OF FREE RIBOSOMES WITH THE USE OF FLOW CYTOMETER. IN SUMMARY The advantages of the RIBOGRAMA test, and other tests is that it is a non invasive test, and obviously twelve factors by themselves allow a good participation rate of patients and determines a preference for health professionals, because there are no false positives or false negatives. A significant number of cells in the mucosa of the colon and rectum remains intact and viable in the feces and can be isolated. It has been shown that these cells are only representatives of the entire colon and can be very useful in clinical research in the process of the disease Exfoliated colonocytes (cells lining the colon and rectum) in the stool of patients under study can be collected in a transport medium (feces collector designed and patented for this method), at room temperature and isolated, for the study of the number of cells (quantity) of free ribosomes which are determined mathematically in a standard medium, from a half predefined pattern resulting from the epithelial cells of normal mucosa (obtained in a study of more than two thousand patients without pathology). The application of molecular biology techniques of exfoliated cells of the colon and rectum profoundly increases the sensitivity of the detection of colorectal cancer or its tendency to malignant transformation, enabling its use in diagnosis and in tracking. With this RIBOGRAMA method it is possible to lower significantly, for the first time in the last fifty years, the incidence, mortality and morbidity of the disease CRC, because this new method allows you to go and meet the tendency of malignancy (or already in the state of malignancy) before any endoscopy, which will be negative in the eyes of the endoscopist. Relatively, the test of fecal occult blood is a crucial aspect: - The test of fecal occult blood, in the case of a positive test result, the blood is detected in the blood vessels of the submucosa, which means that there is an invasion of the vasculature beneath the malignant mucosa , and the cancer has invaded the circulation, which means the dissemination of malignant cells in both the blood and lymphatic circulation, where they will miss the potential existence of micrometastasis, which signifies and represents the uncertainty in the forecast, because of the difficulty of establishing through imaging methods whether or not there are micrometastasis in circulation. Relative to testing DNA mutations in the stool there is a crucial aspect: The test for identifying DNA mutations in stool defines only the genetic alteration of the major genes known to be responsible for malignant potential DNA colonocytes. It is known in molecular genetics that the genetic alterations of genes responsible for pre-defined as malignancy of colon rectal mucosa does not necessarily induce malignant transformation of cells where they occur as there is simultaneously a process of gene repair, a phenomenon in opposition to the emergence of mutations. - In the stool DNA particles can come from other parts of the digestive tract, such as small intestinal mucosa of the gastric and duodenal mucosa, the mucosa of the bile ducts, the mucosa of the excretory ducts of the pancreas, the oesophageal mucosa and respiratory mucosa, once here the mucus and sputum are virtually swallowed. So the existence of numerous false positives is not surprisingly. In the scientific literature on the sensitivity of this test there are variations which may lie between values of fifty percent to eighty-five percent of sensitivity. From the discussion on the existing non-invasive tests in the market (fecal 14

occult blood test and identification of DNA mutations in the stool) it is concluded that the RIBOGRAMA test is far superior to the traditional non-evasive tests in the current market, highlighting its total sensitivity (100%).

Additionally the RIBOGRAMA test is a quantitative test allowing a dynamic comparison over time; this is not possible with the other two tests. – The RIBOGRAMA curve has a numerical basis (may be reproducible in equivalent circumstances), which corresponds to a non-subjective language. Analysis of trends and variations found in this curve will be a sure indicator of the degree of development and propensity for malignant transformation. The phenotype corresponds to a quantitative basis that can provide quantitative information on levels of risk and significance, prior to the “open", malignization, information on the risk profile and the heredofamiliar profile food dietary and lifestyle habits. Theoretically, in comparison to the existing non-invasive tests in use (occult blood in feces and DNA mutation analysis of coloncytes) the ribosome test has major advantages over the existing tests: I. Knowing that it will be studied as an isolated coloncyte with its own specific technique, the specificity of the results is 100% in relation to the counting of free ribosomes; II. The existence of amount levels of free ribosomes, based on criteria of risk and significance, allowing time for preventive and therapeutic attitudes towards a tendency of malignancy, while in the tests of discovery of DNA mutations in the stool, the answer is only positive / negative, not allowing a quantification by levels or degrees of significant risk .

III. The test of DNA mutations in stool has false positives and false negatives, which vary to a significant extent, according to the authors; IV. The positive test for fecal occult blood has a high percentage of false positives and false negatives which is not negligible; V. When a tumor is bleeding, this means that the mucosal barrier has already been destroyed by erosion / invasion of the submucosa, with its blood and lymph vessels, ensuring no certainty as to the metastizacion and lymph node, or no tumor Tis is a / 0; VI. Mutations in the DNA testing in the stool can be of other organs (respiratory tract, gastrointestinal tract, etc...), allowing no guarantee of eventual location of a tumor; VII. Furthermore, mutations in these tests (DNA mutations in the stool) may be non-mutant phenotypes, which contribute to the many false positives; VIII - The counting test of free ribosomes, and the corresponding significantly increased levels of the number of free ribosomes in colonocytes isolated from feces represents the existence of a real uncontrolled increased synthesis of proteins, and cannot, therefore, be false positives, because a tumor depends crucially on the excessive production of cytoplasmic proteins in the growth process, and that in the cell can only be done through the free ribosomes.

OTHER BENEFITS In addition for the diagnostic method to be specific for the disease of CRC, with no false positives or false negatives, it will be used to modify personal and family behavior based on persuasive and convincing information in case of a significant elevation of the curve by providing guidance from the RIBOGRAMA method which allows to know if there is or not the need for conducting diagnostic and therapeutic attitudes for the groups at risk (RIBOGRAMA elevated curves). There will be no more blind endoscopies, i.e. diagnostic endoscopy will only be undertaken in specific cases of elevation of the RIBOGRAMA curve, with corresponding and significant savings in patient discomfort and the elimination of missed working days and costs associated with diagnostic tests and acidents during colonoscopies. This means, of course, reliable information on risks and opportunities for possible malignant lesions of the colon, rectal mucosa, and well in advance of any invasion of the submucosa is the most important point for malignant dissemination, because at this moment blood and lymph vessels of the submucosa may already be invaded by micrometastasis in the process of malignancy. In addition to the advantages described above, these foundations for monitoring of pathogenicity produce a reduction in uncertainties about the possibility of diagnosis in cases where otherwise one would be surprised with the disease who did not do the test, this means of influence will also be helpful in the remaining relatives of a patient with significantly elevated RIBOGRAMA curve. In the concrete conditions of patients with significant elevation of the curve of the RIBOGRAMA curve and if the colonoscopy does not show any suspected lesion of possible malignancy, this means that the patient is in the process of developing a potential increased proliferation of some point and 15

mucosa which is still not visible microscopically (endoscopy). So once this is well established, the cause-effect of certain foods has statistically caused a significant increase in the proliferation of tissues, such as the colorectal mucosa, and patients with RIBOGRAMA curves of a significant elevation should undergo a food survey conducted by themselves in such cases also within the study of what will be concrete answers on the deviations from normality for some types of food and its cultural influence in the way of cooking, where it is possible to route these patients for a query of dietary advice. Thus, the method helps to educate people about the impact that certain foods have when repeated. The scientific system enables high utilization of postoperative monitoring in cases of colorectal surgery for CRC. Today it is unequivocally proven that aspirin and nonsteroidal antiinflammatory drugs have a strong antiproliferative effect on colorectal mucosa. Thus, in a patient with a significant elevation of the RIBOGRAMA curve one can act through two simple steps: a)- Diet without content of foods that may cause hyperproliferation of the colorectal mucosa, oriented by the information and conclusions of the survey of food habits; b)- Reduce high RIBOGRAMA through drugs already used in other therapeutic domains which have been approved by the appropriate government agencies ADVANTAGES OF THE RIBOGRAMA METHOD OVER OTHER NON-INVASIVE METHODS OF CRC The method of RIBOGRAMA (count of free ribosome of exfoliated colonocyts of colon rectal mucosa that mixes with the stool inside the intestine) is a stool test, based on techniques of cell and molecular biology, it has a strong and important advantage over other stool tests for diagnosis and tracking (Test of fecal occult blood test and identification of DNA mutations in the stool). Because of these characteristics, the RIBOGRAMA test is easily accessible to patients, because of its simplicity and practicality as they will show. In the RIBOGRAMA test there are several advantages: a) – no invasive act; b) - no need on the part of patients, any manipulation of their own feces in their harvest, c) - no annoying odor of feces after the collection of these as it has an industrial design in this regard, d) - no need for bowel preparation, e) - no need for cleansing enemas, f) - not necessary the use of cathartics, g) - not necessary the displacement of the patient to medical consultation or medical examination center, h) - the patient does not have to alter their lifestyle, i) - the patient does not have to alter their routine daily activities or work, j) - the patient has no need to alter the pace and type of feeding, k) - the patient does not have to interrupt or modify any medication, l) - there are no false negative results or false positives, because in the test specification, merely counting the colonocytes’ free ribosomes, which are the cells that are maligned in the cases of CRC or process in proceedings of malignancy. RIBOGRAMA curve has a numerical basis (that may be reproducible in circumstances equivalent), which corresponds to a non-subjective language. Analysis of trends and changes found in that curve will be a reliable indicator of the degree of development and propensity for malignancy; The RIBOGRAMA phenotype corresponds to a quantitative basis that can provide quantitative information on levels of risk and significance, prior to the "open" malignancy and an information on the profile risk should include the heredofamiliar profile, food and dietary habits and the lifestyle. Compared to existing noninvasive tests in use (occult blood in the stool and analysis of DNA mutations on the colonocytes) the testing of ribosome count has, theoretically, some features that are major advantages over existing tests: I. Knowing that it will be studied as isolated coloncytes its own specific technique, the specificity of the results is 100% relative to the counting of free ribosome; II. The existence of levels of amounts of free ribosome, based on criteria of risk and significance, allows time for preventive and therapeutic attitudes towards a tendency of malignancy, while in the tests of discovery of DNA mutations in the stool, the answer is only positive / negative not allowing a quantification by levels of significant degree of risk ; III. The test of DNA mutations in the stool has false positives and false negatives, which vary to a significant extent, according to the authors; IV. The positive test for fecal occult blood has a high percentage of false positives and false negatives are not negligible; V. When a tumor is bleeding, this means that the mucosal barrier has already been destroyed by erosion / invasion of the submucosa, with their blood and lymph vessels, ensuring no certainty as to the metastizacion and lymph node, or no tumor Tis is a / 0; VI. Mutations in the DNA testing in the stool can be of other organs (respiratory tract, gastrointestinal tract, etc...), allowing no guarantee of eventual location of a tumor; VII. Furthermore, mutations in 16

these tests (DNA mutations in the stool) may be non-mutant phenotypes, which contributes to the many false positives; VIII. The counting test of free ribosome, the corresponding levels significantly increased the number of free ribosome in colonocytes isolated from feces, representing the existence of a real uncontrolled increased synthesis of proteins, it cannot, therefore, be false positives, because a tumor depends crucially on the excessive production of proteins in the growth process, and that the cell can only be done by free ribosome. The relative diagnostic value, of the non-invasive methods available today, for example - the test for occult blood in the feces (for colorectal cancer) and/or DNA mutations (or any other organ and system) are very time-consuming, with false-negative and false-positive results, and the need of special conditions, equipment and personnel, etc. colonoscopy (which has a certain percentage of risk) in the study of colon and rectum cancer - or other organs makes the RIBOGRAMA a method much more preferable, because it is painless, highly informative and rigorous in clinical oncology and laboratory research, and also allows us to conduct massive research in thousands of patients, making all things being equal, practical and very economic to the patients and their countries. Tumor markers are proteins, which, usually, are produced by the cancer cells, it means that when we start using tumor markers it is already established a state of cancer in the cells under study. Furthermore, the method allows inducing and seeing the change of the RIBOGRAMA curve, under external stimuli to the cell, allowing in advance knowing the way how a community of cancer cells responds to the chemotherapeutic agents. The dynamic and mathematic accuracy control of the method allows interpreting the success of a treatment with a special diet and proper selection of drugs by quantitative change in the RIBOGRAMA graphic curve. We can analyze and monitor the subsequent construction of guide lines and its analysis in the dynamics of a treatment. It allows the discharge of patients in various groups of degrees of cancer risk, their follow-up and regroups during their treatment. It is possible to monitor large number of observations and groups of patients and make effective analysis of trends, both in conservative and in surgical treatments. The RIBOGRAMA METHOD is a real biological biomarker to monitorise the behavior/phenotype of a community of cells under study/treatment (e. g. breast, colorectal tract, upper digestive tube, lung, prostate, gynecologic tract, etc), before metastatic cancer explosion. If this biomarker is adopted, we can analyze someone's tumor tissue before treatment and see how they respond to medication and follow the rules of personalized treatments.

CONCLUSIONS 1. A Significant great sequential increase in the accumulation of free ribosome in the cytoplasm of cells of any tissue is one of the phenotypic characteristics of malignant transformation of cells under study; 2. The RIBOGRAMA method reveals the limits of normal concentration of free ribosome, to determine their concentration per unit volume. A great increase in their quantities is a sign, at the cellular level, of malignant transformation, long before clinical manifestations - or the appearance of blood specific tumor markers, or histological transformation of tissues and DNA mutations; 3. A Phenotypic expression in the cytoplasm, in a significant great increase in the number of free ribosome(by levels of concentration) is a sign of tumor growth and may be considered as a prognostic and a dynamic factor in cancer patients; 4. These RIBOGRAMA data confirm that there is a correlation between different numerical content of free ribosomes in the cells and their dependence on the degree of tissue differentiation (morphological and physicochemical studies); 5. A Quantitative analysis of free ribosomes, their dynamic performance in the form of a graphic curve RIBOGRAMA- allows analyzing and predicting the trend of malignization in each case, the choice of the most appropriate treatment and getting the correct orientation and reasoned in the diet and drug therapy in each case.

References 1. Stanners C.P., Adams M.E., Harkins J.L. and Pollard J.W. (1979). J. Cell Physiol.. 100, 127 – 138.

2. Haumeder E.(1933), L.Krebsforsch, 40 , 105 – 116 3. Sakiyama, H., S. K. Gross and P. W. Robbins (1972). Glycolipid synthesis in normal and virus-transformed 17

4. Bose, S. K. and B. J. Zlotnick (1973). Growth - and density – dependent inhibition of deoxyglucose transport in Balb 3T3 cells and its absence in cells transformed by murine sarcoma virus. Proc. Nat. Acad. Sci. (U.S.A)., 70, 2374 – 2778.

5. Hirschberg, C. B, B. A. Wolfund and P.R. Robbins (1975). Synthesis of glycolipids and phospholipids in hamster cells: Dependence on cell density and the cell cycle. J. Cell. Physiol., 85, 31 – 40.

6. Chou, I. N., O´Donnell, P. H. Black and R.O Robbin (1977), Cell density-dependent secretion of plasminogen activator by 3T3 cells. J. Cell. Physiol., 91, 31 – 32.

7. Hadjiolov A.A.(1995) The nucleolus and ribosome biogenesis. Cell Biology Monographs. Wien, New York, Springer, 1 – 267.

8. Howell W.M. (1982) Selective staining of nucleolus organizer regions (NORs). The Cell Nucleus. Edited by Busch H, Rothblum L.New York, Academic Press, 89 – 112.

9. Olson M.O.J. (1990). The role of proteins in nucleolar structure and function. The Eukaryotic Nucleolus: Molecular Biochemistry and Macromolecular Assemblies. Edited by Strauss PR, Wilson SH. Caldwell, New York, Telford Press Inc. , 519 – 559.

10. Pession A., Farabegoli F., Treré D., Novello F., Montanaro L., Sperti S., Rambelli F., Derenzini M. (1991) The Ag- NOR proteins and transcription and duplication of ribosomal genes in mammalian cell nuclei. Chromosoma, 100, 242 – 250.

11. Derenzini M., Sirri V., Pession A., Treré D., Roussel P., Ochs R.L., Hernandez- Verdun D. (1995) Quantitative changes of the two major AgNOR proteins, nucleolin and protein B23, are related to stimulation of rADN transcription. Exp Cell Res, 219, 276 – 282.

12. Sirri V., Roussel P., Gendron M.C, Hernandez-Verdun D. (1997) Amount of the two major Ag-NOR proteins, nucleolin, and protein B23 is cell-cycle dependent. Cytometry, 28, 147 – 156.

13. Treré D., Pession A., Derenzini M. (1989). The silver-stained proteins of interphasic nucleolar organizer regions as a parameter of cell duplication rate. Exp Cell Res 184, 131 – 137.

14. Derenzini M., Pession A., Treré D. (1990) The quantity of nucleolar silver stained proteins is related to proliferating activity in cancer cells. Lab Invest, 63, 137 – 140.

15. Öfner D., Hittmair A., Marth C.R., Ofner C., Totsch M., Daxenbichler G., Mikuz G., Margreiter R., Schmid K.W. (1992) Relationship between quantity of silver-stained nucloelar organizer regions associated proteins (AgNORs) and population doubling time in ten breast cancer cell lines. Pathol Res Pract, 188, 742 – 746.

16. Ochs R.L. and Smetana K. (1989) Fibrillar center distribution in nucleoli of PHA-stimulated human lymphocytes. Exp Cell Res, 184, 552 – 557.

17. Arrighi F.E., Lau Y.F. and Spallone A. (1980) Nucleolar activity in differentiated cells after stimulation.

Cytogenet Cell Genet, 26, 244 – 250.

18. Sirri V., Hernandez-Verdun D., Roussel P. (2000) Cyclin-dependent kinases govern formation and maintenance of the nucleolus. J Cell Biol, 156, 969 – 981.

19. Hanahan D., Weinberg R.A. (2000). The hallmarks of cancer. Cell, 100, 57 – 70.

20. Darzynkiewicz Z. (1998) Cellular ARN content, a feature correlated with cell kinetics and tumor prognosis.

Leukemia, 2, 777 – 787.

21. Ruggero D, Pandolfi P.P. (2003). Does the ribosome translate cancer? Nature Reviews Cancer, 3, 179 – 192.

22. De Young L.M., Argyris T.S., Gordon G.B.(1977) Epidermal ribosome accumulation during two-stage skin tumorigenesis. Cancer Research, 37, 388 – 393.

23. Chu T.Y., Hwang K.S., Yu M.H., Lee H.S., Lai H.C., Liu J.Y. (2002) A research-based tumor tissue bank of gynecologic oncology: Characteristics of nucleic acids extracted from normal and tumor tissues from different sites. Internacional Journal of Gynecological Cancer, 12, 171 – 176.

24. Boon K. et al. (2001) N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis. EMBO Journal, 20, 1383 – 1393.

25. Linaggar A.K., Kemp B.L., Sneige N., Hurr K.G., Steck K., Tu Z.N., Pritsche H.A., Singletary S.E., Balch C.M. (1996) Bivariate ARN and ADN content analysis in breast carcinoma: Biological significance of ARN content.

Clinical Cancer Research, 2, 419 – 426.

26. Lourenco G., Embury S., Schrier S.L., Kedes L.H. (1978). Decreased ribosomal ARN content and in vitro ARN synthesis in purified bone marrow erythoblasts of patiens with idiopathic ineffective erythropoiesis and DiGuglielmo disease. America Journal of Hematology, 5, 169 – 182.

27. Derenzini M., Sirri V., Treré D. (1994). Nucleolar organizer regions in tumor cells. Cancer Journal, 7, 71 – 77.

28. Pomeroy S.L. et al. (2002). Prediction of central nervous system embryonal tumor outcome based on gene expression. Nature, 415, 436 – 442.

18

29. Derenzini M., Treré D., Pession A., Montanaro L., Sirri V., Ochs R.L. (1998) Nucleolar function and size in cancer cells. American Journal of Pathology, 152, 1291 – 1297.

30. Andersen J.S., Lam Y.W., Leung A.K., Ong S.E., Lyon C.E., Lamond A.I., Mann M. (2005) Nocleolar proteome dynamics. Nature, Jan 6; 433(7021), 77 – 83.

31. Hannan K.M., Hannan R.D. and Rothblum L.I. (1998). Transcription by ARN polymerase I. Front Biosci, 3, d376 – d398.

32. Grummt I. (1999). Regulation of mammalian ribosomal gene transcription by ARN polymerase I. Prog Nucleic Acid Res Mol Biol, 62, 109 – 154.

33. Johnson L.F., Abelson H.T., Green H. and Penman S. (1974) Changes in ARN relation to growth of the fibroblast. I. Amounts of mARN, rARN, and tARN in resting and growing cells. Cell, 1, 95 – 100.

34. Loeb J.N. and Yeung L.L. (1975) Synthesis and degradation of ribosomal ARN in regenerating liver. J Exptl Med, 142, 575 – 587.

35. Malt R.A. and LeMaitre D.A. (1968) Turnover of ARN in the renoprival kidney. Am J Physiol, 214, 1041 – 1047.

36. Tata J.H. (1967. The formation and distribution of ribosomes during hormone-induced growth and development. Biochem J, 104, 1 – 16.

37. Blobel G. and Potter V.R.(1967) Studies on free and membrane-bound ribosomes in rat liver. I. Distribution as related to total cellular ARN. J Mol Biol, 26, 279 – 292.

38. Mueller S.N. and Argyris T.S. (1975) Free and membrane-bound ribosomes in normal and methylcholantrenetreated mouse epidermis. Lab Invest, 32, 209 – 216.

39. Argyris T.S. and Nevar C. (1975). Further studies on the isolation of ribosomes from normal and methylcholantrene-treated mouse epidermis. J Invest Dermatol, 65, 334 – 336.

40. Sharma D.S. R., Reid I., Verney E. And Sidransky H. (1972) Studies on the nature of attachment of ribosomes to membranes in liver. I. Influence of Ethionine, Sparsomycin, CCl and Puromycin on membranebound polyribosomal disaggregation and on detachment of membrane-bound ribosomes from membranes. Lab Invest, 27, 39 – 47.

41. Sjostrand F.S. (1967) In: Electron Microscopy of cells and tissues, 444 – 445, New York: Academic Press, Inc. 42. Blobel G. and Potter V.R. (1966) Nuclei from rat liver: isolation method that combines purity with high yield.

Science, 154, 1662.

43. Blobel G. and Potter V.R. (1967) Studies on free and membrane-bound ribosomes in rat liver. J Mol Biol, 26, 279.

44. Amaldi F. and Pierandrei-Amaldi P. (1996) Top genes: A translationally controlled class of genes including those coding for ribosomal proteins. In “Cytoplasmic Fate of Eukaryotic mARN” (P. Jeanteur, ed.), Prog Mol Subcell Biol, 18, 1 – 17. Springer-Verlag, Heidelberg.

45. Meyuhas O., Avril D. and Sharma S.(1996) Translational control of ribosomal protein mARN in eukaryotes. In “Translational control”, (J.W.B.Hershey, M.B. Mathews and N. Sonenberg, eds.), 363 – 388. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 46. Kasai H., Nadano D., Hidaka E., Higuchi K., Kawakubo M., Sato T.A. and Nakayama J. (2003) Differential expression of ribosomal proteins in human normal neoplastic colorectum. Journal of Histochemistry and Cytochemistry, 51, 567 – 574.

47. Raick A.N. (1974). Cell diferentiation and tumor-promotor action in skin carcinogenesis. Cancer Research, 34, 2915 – 2925.

48. Zetterberg, A. (1997) Cell growth and cell cycle progression in mammalian cells. In: Apoptosis and Cell Cycle Control in Cancer (ed. N.S.B. Thomas). BIOS Scientific Publishers Ltd., Oxford, pp. 17-36.

49. Mao, X., Green, J.M., Safer, B., Lindsten, T., Frederickson, R.M., Miyamoto, S., Sonenberg, N. and Thompson, C.B. (1992) Regulation of translation initiation factor gene expression during human T cell activation.

J. Biol. Chem. 267: 20444-20450.

50. Mueller, Stephen N. and Argyris, Thomas S.(1975), Free and Membrane-Bound ribosomes in normal and Methycholanthrene-Treated mouse epidermis; Copyright by the Internacional Academy of Pathology.

51. Greco, Anna and Madjar, Jean-Jacques, preparation of Ribosomes and Ribosomal proteins from cultured cells; A laboratory handbook, Second edition, vol.2.

52. De Young, Lawrence M., Argyris, Thomas S. and Gordon, Gerald B., (1977), Epidermal Ribosome Accumulation during Two-Stage Skin Tumorigenesis, Cancer Research 37, 388-393.

19

* (Patents and copyrights are N ยบ 200701216 (2) MCT Esp. 02/18/2010 - SPAIN)

20