Patent Application
20200150121
The present invention relates to a method for diagnosing cancer, more particularly a method the for early diagnosis of cancer.
The invention further relates to a kit for carrying out said method.
Neoplastic disease is still one of the leading causes of death in the world today. 90% of cancer deaths are due to the spread of the disease or the development of metastases.
In this regard, the scientific community has focused economic efforts and scientific attention on a group of cells, defined as circulating tumor cells (CTCs) that are available through a non-invasive procedure, i.e. a simple blood draw, and have proved to have significant clinical utility in the prognostic evaluation of the patient and in the early detection of cancer. To date, the diagnosis and assessment of the risk of becoming ill with cancer are compromised by the use of instruments characterized by moderate sensitivity and inadequate accuracy. Similarly, the most advanced radiological techniques do not achieve the spatial resolution necessary to detect a tumor in the early molecular stages of its development. On the other hand, the great variety of types of cancer and the different forms that cancer takes in individual patients are also the main cause of failure of advanced formulations also in the field of nanomedicine (Heidi Ledford, Bankruptcy of nanomedicine firm worries drug developers, Nature 533:304-305, 2016).
Devices and essays have been developed for the detection of cancers (Kaiser J. Liquid biopsy’ for cancer promises early detection. Science. 19; 359-259. (2018)) e per l'analisi e ii monitoraggio della progressione del cancro (Hong X, Sullivan R J, Kalinich M, Kwan T T, Giobbie-Hurder A, Pan S, LiCausi J A, Milner J D, Nieman L T, Wittner B S, et al. Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy. Proc Natl Acad Sci USA. 10:2467-2472. (2018)) and for the identification of relevant personalized pharmacological targets (Brady, S. W., McQuerry, J. A., Qiao, Y., Piccolo, S. R., Shrestha, G., Jenkins, D. F., Layer, R. M., Pedersen, B. S., Miller, R. H., Esch, A., et al. Combating subclonal evolution of resistant cancer phenotypes. Nat. Commun. 8, 1231. (2017)). However, no current approach is competitive with the clinical diagnosis, which is already late compared to the global history of a tumor responsible for little or no control of the neoplastic disease and to its high mortality and health care costs (Carrera P M, Hagop M. K., Blinder V. S. The financial burden and distress of patients with cancer: Understanding and stepping-up action on the financial toxicity of cancer treatment. CA Cancer J Clin. 68, 153-165. (2018).
The object of the present invention is to overcome the problems and limitations of prior art by providing a method for non-invasive cancer diagnosis capable of early detection and diagnosis of cancer with high sensitivity and precision.
These and other objects are achieved with the method for diagnosing cancer and the kit therefor as claimed in the appended claims.
The method for diagnosing cancer according to the invention allows, through a blood draw and the use of a measuring device of the electrochemical type, to detect the defective metabolism of cells as an indicator of abnormal cell division, proliferation and invasion. By measuring the conductivity variation induced by the proportion of titratable free protons (protonation state) contained in cellular catabolites secreted by circulating tumor cells, the method allows to identify cancer patients with a high degree of accuracy and predictive potential.
The method for diagnosing cancer according to the present invention is therefore based on the measurement of the state of molecular protonation of a secretome produced by cells isolated from the peripheral blood (blood tissue) of an individual.
In particular, the method involves a step of producing the secretome, a step of measuring the state of molecular protonation of the secretome, and a step of processing of the data obtained from the measurement to obtain an early detection of cancer.
The secretome production step requires that it is obtained from a cell culture from peripheral blood of the individual, in which, in particular, a non-haematological cellular component present in the peripheral blood is allowed to expand in the cell culture.
During the culture process, at predetermined intervals, a portion of the cell culture medium is replaced by a fresh culture medium and the gradually replaced portions are stored. After a predetermined culture period, the entire culture medium is taken from the culture and the supernatant is obtained therefrom in a known manner. Subsequently, the previously replaced and stored portions of the culture medium are added to this supernatant, resulting in a mixture that constitutes the final total conditioned medium, which is then filtered, thus obtaining the secretome to be analysed.
The use, in this method of diagnosis, of the secretome obtained from a cell culture from peripheral blood of the individual is particularly advantageous because peripheral blood represents, in personalized medicine, the human sample easiest to collect without modification of the clinical conditions of the individual.
Moreover, thanks to the fact that the culture process used involves the expansion of the non-haematological cellular component present in the peripheral blood, the secretome obtained from this culture process provides information not only on the metabolic and biochemical state of blood cells but also and above all of non-haematological cells carrying valuable information derived from tissues other than haematic tissue.
The step of measuring the state of molecular protonation of the secretome involves the use of a measuring device of the electrochemical type comprising at least one measurement point, or preferably several measurement points, each comprising a conductive polymer channel between a respective first and second electrode. Said channel acts as a conducting channel of a respective transistor and is connected to a gate electrode of the transistor through the secretome. At least one measurement of channel current I(t) is then performed at each measurement point. If several channel current measurements are performed at one measurement point, these are performed at different potentials Vds applied between the first and second electrode of the measurement point. A modulation m and a time constant τ are obtained from each measurement of channel current I(t). The modulation m is given by a final intensity Ids of channel current normalized with respect to the initial intensity Ids0, and the time constant τ is given by the time it takes for the channel current I(t) to reach a predetermined percentage of the final intensity Ids. The above values of modulation m and time constant τ are indicative of the protonation state of the secretome.
The step of measuring the state of molecular protonation of the secretome also provides to make Marangoni convection flows develop in the secretome, before performing said at least one measurement of channel current I(t), so that, when the above measurement of channel current is performed, different molecular species (mainly proteins and nucleic acids) contained in the secretome are distributed in different positions within the secretome according to their dimensions and diffusion coefficients D. These Marangoni flows develop thanks to the fact that the measuring device on which the secretome is positioned has a superhydrophobic surface, so that the secretome positioned on the measuring device assumes an almost spherical drop-like shape, which wets all the measurement points of the measuring device, connecting each measurement point to the gate electrode.
In addition, the step of measuring the state of molecular protonation of the secretome preferably provides to place the secretome drop symmetrically with respect to the plurality of measurement points of the measuring device.
The step of measuring the state of molecular protonation of the secretome further provides to select, among the several pairs of modulation m and time constant τ obtained from the measurements of channel current I(t) performed at different measurement points, a pair of values of modulation m and time constant τ, said pair being representative of the analyzed secretome. Such selection is effected, for example, by means of statistical analyses of known type.
The step of processing the data obtained from the measurements described above involves classifying a secretome, based on the value of modulation m and time constant τ associated with that secretome, distinguishing whether a secretome comes from a healthy individual, an individual having cancer or an individual at risk of getting cancer.
In case there are pairs of values of modulation m and time constant τ associated to respective secretomes of different individuals, the processing step provides to apply clustering algorithms to these pairs of values of modulation m and time constant τ and to group, by applying said algorithms, these secretomes, classifying them in secretomes from healthy individuals, individuals having cancer or individuals at risk of getting cancer.
These and other features and advantages of this invention will be more evident from the following description of preferred embodiments given by way of non-limiting example with reference to the annexed drawings, in which elements designated by same or similar reference numeral indicate elements that have same or similar functionality and construction, and in which:
A method for the early diagnosis of cancer in an individual based on the measurement of the state of molecular protonation of a secretome produced by cells isolated from the peripheral blood (haematic tissue) of the individual is described below.
The method provides a step of producing the secretome, a step of measuring the state of molecular protonation of the secretome and a step of processing the data obtained from the measurement to obtain an early diagnosis of cancer.
The production of the secretome from peripheral blood requires the application of a protocol of isolation, culture and brief in vitro expansion of the non-haematological cellular component, present in low concentrations, in the peripheral blood, described in Italian patent application No. 10201724609.
In accordance with this protocol, the cell culture is preferably maintained for fourteen consecutive days, with partial replacement of the culture medium. In particular, every 48 hours, preferably, the cell culture is checked and 10% of the total volume of the culture medium is taken and replaced with fresh culture medium. The volumes taken are stored in special cuvettes and preferably stored at 4° C. At the end of the fourteen days, the total volume of the culture medium is taken, for example by means of a centrifuge at 1870 rpm operated for about fifteen minutes. After centrifugation, the pellet (i.e. the material remaining on the bottom) is separated from the supernatant (i.e. the material remaining in suspension). The volumes of the culture medium previously taken at 48-hour intervals are added to the supernatant taken after centrifugation, resulting in a mixture that constitutes the final total conditioned medium, which is then filtered, stored and preserved preferably at a temperature of −80° C., thus obtaining the secretome to be analyzed.
The isolation treatment prior to cell cultivation ensures the presence, in the culture, of a non-haematological component, if present, together with the haematological component proper to the haematic tissue, as shown in
The composition of the culture in the extracellular space is characterized by different molecular products (or species) released by the cultured cells. The molecular species present in the secretome are mainly proteins and nucleic acids. With reference to
During the cultivation period, the seeded cells divide and increase in number with a duplication time that depends on their replication capacity. By increasing the number of cells over time, the amount of molecules they secrete in the culture medium increases proportionally. If one cell population grows more than another one, the former conditions the culture medium more than the latter and the secretome will have a molecular composition that reflects the cell functions and metabolism of the prevailing cell population.
These considerations underline that, as observed by the Applicant, in a cell culture derived from an individual with cancer, the resulting presence of transformed cells in the culture will tend to characterize the secretome more than the normal cell component because the latter has a lower proliferative capacity than the tumor.
In this regard, the comparison, conducted by the Applicant, between the secretome and the interstitial fluid taken directly from the tumor tissue showed a strong correlation (with a Pearson correlation factor r of 0.9) in terms of types and amounts of proteins present in the two samples taken from the same individual.
The above data support that the composition of the secretome of the cell culture obtained from peripheral blood taken from individuals with neoplastic disease is comparable to that of the interstitial fluid of the primary tumor mass.
Finally, in order to assess whether the characteristics related to ionic conductivity related to the protonation state are different when comparing the secretome obtained from the cell culture derived from the peripheral blood of a healthy individual compared to that of an individual with cancer, the Applicant has studied the isoelectric point of the proteins contained therein, i.e. the pH value at which these proteins have no net electrical charge. This analysis has shown that the isoelectric point of the secretome of cell cultures obtained from the peripheral blood of ill individuals is different from the same derived from healthy individuals.
On the basis of these premises, the secretome obtained according to the above-illustrated procedure is analyzed by measuring the protonation state thereof. This analysis is carried out by means of a measuring device 10 capable of highlighting the different ionic conductivity of the ionized molecules contained in the secretome and of providing, depending on the concentration and intermolecular complexation capacity of these molecules, information suitable for early detection of cancer.
The measuring device 10 for measuring the protonation state of the secretome, shown in
With particular reference to
The entire measuring device 10 is covered with a thin film of conductive polymer 26, preferably PEDOT:PSS, possibly with solvents suitable for increasing conductivity (e.g. ethylene glycol 10% by weight and DBSA (dodecyl benzene sulfonic acid) 0.1% by weight), which creates a conducting channel 28 between the nano-electrodes 14a-b of a measuring pillar 15-19. The device 10 is finally covered with a thin hydrophobic polymer film 27, for example a film made of C4F8 (Teflon).
The geometric arrangement of the pillars 11 and the hydrophobic film 27 make the device 10 superhydrophobic, so that a biological solution placed on the device has contact angles close to 180°, assuming an almost spherical drop-like shape 50.
Thanks to the superhydrophobic properties of the device 10, within the drop 50 of secretome sample solution, depending on its curvature, convective flows, so-called Marangoni flows, are developed that transport the molecular species contained within the drop in a differential way, modifying the distribution thereof. In particular, the molecular species contained within the drop move according to their diffusion coefficient D and their dimensions, as described by Einstein's relation D=KBT/6πηa (where KB is Boltzmann's constant, T is the temperature, η is the viscosity of the solution and a is the radius of the molecule in solution), this movement distributing the molecular species over the surface of the measuring device 10 at different points.
In addition, the variable pitch of the grating of pillars 11, with higher density of pillars in the central part of the device 10, allows self-centering of the drop 50 with respect to the substrate (
The distributed arrangement of the measurement points 15-19 allows measurements of the ion current of the secretome sample solution to be made with a spatial as well as a temporal resolution. To carry out these ion current measurements, a multiple organic electrochemical transistor geometry with a common gate has been applied, in which the channel 28 made of PEDOT:PSS between the nano-electrodes 14a-b of each measuring pillar 15-19 is the conducting channel of each transistor, and all the conducting channels 28 are put in contact, through the drop 50 of secretome sample solution, with a common gate, for example a conductive filament made of platinum (not shown), placed on top of the drop 50.
The device 10 therefore exploits the combination of its superhydrophobic properties, combined with the characteristics of the drop 50 of secretome sample solution, and a matrix of organic electrochemical sensors 15-19 based on a transistor architecture.
Fixed potentials are sequentially applied to the pairs of electric tracks 20a-b, 21a-b, 22a-b, 23a-b, 24a-b of each transistor or measurement point 15-19, so that a current (Ids) is passed in the channel 28 of each transistor. This current is measured separately for each of the channels 28, i.e. for each of the measurement points 15-19. By applying a positive potential to the gate electrode (Vgs=1 V) the ions in the drop 50 of secretome sample solution are forced to enter the channel 28 made of PEDOT:PSS of the transistors. Since it is known that the conductivity of PEDOT:PSS decreases in the presence of positive ions, a decrease in current proportional to the concentration of ions in the vicinity of the channels 28 is obtained in the channels 28. In particular, the application of the positive potential (Vgs) to the gate electrode, determines that the positive ions (M+) of the drop 50 penetrate into the channel 28 made of PEDOT:PSS using its de-doping as per the following reaction:
PEDOT+:PSS−+M++e−=>PEDOT0+M+:PSS−
The de-doping process causes a decrease in the value of the module of the channel current |Ids| due to a decrease in the holes available for conduction. The positive ion M+ allows the reduction of the oxidized state of the PEDOT+ to PEDOT0. This process is reversible and when the Vgs potential is switched off the ions return to diffuse in the drop 50 and bring the polymer back to the previous doping state.
The penetration, or injection, of the ions into the conducting channel 28 occurs when the gate potential Vgs is switched on, generating a signal of reduction of the channel current I(t) with exponential trend (
With reference to
By measuring the values of modulation m and time constant τ, information on the molecular species in solution is obtained. In particular, m is inversely proportional to mass and directly proportional to the charge of a molecular ion whereas r is directly proportional to mass and inversely proportional to the charge of a molecular ion.
By crossing the above qualitative data (association of a trajectory in the m-r plane with a particular molecular species) and quantitative data (values of m and r) obtained for each measurement point 15-19 of the measuring device 10, a detection is obtained that allows comparison of the ionic strength at the different measurement points 15-19 of the measuring device 10.
The method for measuring the protonation state (PS) of a secretome sample solution and the subsequent processing for obtaining a diagnosis is described in more detail below.
A change in current I(t) over time measured by the measuring device 10 is associated with the transport of ionized molecular species within the solution and depends in particular on five parameters (p1-p5), namely the diffusion coefficient D (p1), the charge z of the molecular species within the solution (p2), the Marangoni convection flows carrying the species within the solution drop 50 (p3), the position and density of the measurement points 15-19 on the substrate 12 of the device 10 (p4) and the intensity of the electromotive force (EMF), i.e. the potential Vds applied between the first 14a and the second 14b electrode of the measurement points 15-19 for the measurement of the current I(t)(p5).
The parameters p1 and p2 are physical characteristics of the molecular species under examination, which are to be determined and which encode the protonation state of specific molecular species of the secretome. The parameters p3, p4, p5 are manufacturing parameters and can be suitably modulated by choosing the size of the drop 50 and the hydrophobicity of the surface 27 of the device 10, the number and type of measurement points 15-19 in the device 10, the intensity of the electromotive force. By modulating the parameters p3, p4, p5 it is possible to optimize the response of the device 10 and maximize the sensitivity of the function I(t).
The steps of the protonation state (PS) measurement method are as follows:
After a careful analysis, the parameters p3, p4, p5 are chosen that guarantee the best response of the device 10.
Measurements are made on different sample solutions in a small range of parameters around p3, p4, p5 and each measurement is coded in a current-time curve I(t), as described above.
The curves I(t) are analyzed and the two variables modulation m and time constant τ are extracted from each curve, which are characteristic of a specific state of protonation.
Using a known statistical analysis (ANOVA) for each sample solution, a single pair of m-τ values (representative pair) out of all the measured pairs is obtained.
The representative pairs of each sample solution analyzed are reported in an m-τ plane. Different sample solutions, which express different protonation states (Ps), will be characterized by a different representative m-r pair and will therefore be positioned in different areas of the m-r plane (
Clustering algorithms based on Euclidean metrics are applied to the representative pairs shown in the m-r diagram. These algorithms group the sample solutions into distinct groups on the basis of their similarity. The sample solutions are thus classified into solutions from healthy individuals, individuals having cancer or individuals at risk of getting cancer on the basis of the group to which they belong (
| Number | Date | Country | Kind |
|---|---|---|---|
| 102018000010263 | Nov 2018 | IT | national |
