ITMI20110174A1 - PROCEDURE AND EQUIPMENT TO IDENTIFY INDIVIDUALS AT RISK OF PULMONARY AND / OR TO DIAGNOSE A PULMONARY CANCER, AS WELL AS COMPOSITION AND METHOD TO REDUCE OR ELIMINATE THE RISK OF PULMONARY CANCER - Google Patents

Description

DESCRIPTION

â € œProcedure and equipment to identify individuals at risk of lung cancer and / or to diagnose lung cancer, as well as composition and method to reduce or eliminate the risk of lung cancer.â €

FIELD OF THE TROVATO

The present invention relates to methods for identifying and using in prediagnostic and / or diagnostic phases suitable molecular biomarkers identifiable in biological samples, such as for example whole blood, serum, plasma, saliva or bronchial condensate taken from an individual. More in detail, the invention concerns procedures for identifying individuals at risk of cancer, in particular lung cancer. The invention also relates to methods for determining the presence and / or aggression of a tumor, for example lung, in an individual. The invention also relates to diagnostic kits and equipment which can be used to implement one or more steps of the aforementioned methods. Furthermore, the invention relates to pharmaceutical methods and compositions for treating an individual who has been diagnosed with the presence of a tumor, for example a lung tumor. The invention also relates to pharmaceutical methods and compositions for treating an individual who has been identified as having a risk of developing a tumor, for example lung, to reduce and / or eliminate the risk of developing a tumor.

STATE OF THE ART

As is well known, cancers are one of the main causes of death in the world. In particular, lung cancers are the cancers with the highest incidence in the world (they represent about 12% of all new cancer cases) and are the leading cause of cancer death in the world in both men and women. In Europe there are about 400,000 new cases / year (80% men, 20% women). Also in Italy the epidemiology of lung cancer is similar with an incidence of 34,000 cases / year of which about 7,000 cases in women and 27,000 men. Unfortunately, the incidence and mortality are very similar due to the high lethality of lung cancer: global mortality 27,500, including 22,000 men and 5,500 women. These epidemiological data and the scarce curability of the disease underline the importance of identifying methods capable of identifying subjects who may be at risk of developing lung cancer as early as possible. Furthermore, it is of interest to develop methodologies that can assist in the correct diagnosis of tumors, in particular lung tumors, present in an individual subject to examination.

Despite these needs, the tumor markers available today are for diagnostic use, ie they identify patients when the disease has already developed in order to be identifiable with imaging methods (spiral CT). However, these markers are few and non-specific and essentially include biochemical markers such as the evaluation of the CEA protein (Carcino Embryonic Antigen) and of some Cytokeratins such as TP A, TPS and Cyfra 21.1.

A proteomics test (profile of 5 proteins) on serum currently proposed by Vermillion Ine is also known. and used to indicate the probability (score from 1 to 10) that ovarian masses that are malignant in nature. This test is used in women with already a finding of ovarian masses of an unidentified nature

With specific reference to lung cancers, although important improvements have been achieved in the treatment of cancer patients in recent years, there is still a need to develop more effective methods that can lead to an earlier therapeutic intervention in the clinical management of many types of cancer. . Currently, most lung cancers are diagnosed at a late stage when symptoms are clinically evident and, for example with respect to non-small cell lung cancer (NSCLC), only one third of patients with NSCLC have surgically resectable disease. approach that remains the most effective treatment for this type of cancer. Despite recent advances in the treatment of lung cancer after resection and the use of specific treatments for certain molecular targets, the cure rate of non-small cell lung cancer remains low due to relapses in drug-resistant patients or of metastatic diseases.

On the other hand, the efficacy of spiral CT for lung cancer detection in heavy smokers is being evaluated in several ongoing randomized clinical trials in Europe and the United States. Due to its high sensitivity, several critical points remain for its use in current clinical practice, such as the over-diagnosis of indolent nodules, with the consequent high frequency of unnecessary treatments, and the verification of the actual impact on mortality.

In this context, microRNAs (hereinafter miRNAs) have been identified in recent years as a new class of circulating biomarkers which by their nature appear to be very stable and highly tissue specific (Chen X, Celi Res, 2008). MiRNAs are small non-coding RNA molecules (length 19-25 nucleotides), with a regulatory function capable of modulating the expression of multiple target genes involved in various molecular mechanisms, including those involved in transformation processes.

The development of high-throughput technologies has allowed the study of the global expression profiles of miRNAs in cancer (microRNAome) (Cummins JM et al., Proc Nati Acad Sci USA, 2006), revealing that there are hundreds of miRNAs whose expression results deregulated in tumors (Croce CM, Visone R, AJP, 2009; W02009 / 070653, The Ohio State University Research Foundation). In addition to tissue specificity, miRNAs possess high stability, ease of detection and association with known clinical-pathological parameters (Lu J et al., Nature, 2005). Studies were also conducted to determine whether miRNAs were stable, detectable and quantifiable as well as in tissues (whether frozen, formalin- or paraffin-fixed) as well as body fluids. The results of these researches have shown that miRNAs are also present in the bloodstream (whole blood, serum and plasma), where they are found in a stable form protected by endogenous RNase. Circulating miRNAs have been found to be detectable and quantifiable, and studies examining their levels in the biological fluids of cancer patients have reported that some of them have deregulated levels compared to healthy individuals (Heneghan Î — Îœ et al., Ann Surg, 2010; Mitchell PS et al., Proc Nati Acad Sci USA, 2008; Chen X, Celi Res, 2008). Recently some papers have been published that report the profile of circulating miRNAs in the serum and plasma of patients with lung cancer (Hu Z, Clìn Oncol, 2010; Silva J. Eur Respir J, 2010 Shen J, Lab Invest, 2010).

Despite the presence of diagnostic imaging systems and despite studies relating to microRNAs, there remains the need to identify methodologies capable of identifying individuals at risk of developing lung cancer with a certain degree of anticipation and possibly able to predict the development of forms of cancer, particularly lung cancer, which are more aggressive and lethal. There is also a need to improve the degree of reliability of the diagnostic techniques available today.

SUMMARY OF THE FOUND

In this situation, the object of the present invention is to overcome one or more of the limitations of the known methods and products.

Therefore, it is an object of the invention to provide methodologies for the early determination of individuals at risk of developing a tumor, in particular a lung tumor.

Another object of the invention is to provide methods which assist in the diagnosis of tumors, in particular lung tumors, in human subjects.

A further object of the invention is to provide methods which can be easily implemented in the laboratory by analyzing biological samples taken from an individual.

Another object of the invention is to provide methods which allow reliable results to be obtained using blood, serum or plasma samples.

A further object of the invention is to define diagnostic kits and / or equipment that can be used in the methods described above for the identification of human subjects at risk of contracting a tumor and / or for assistance in the diagnosis of tumors present in subjects humans.

It is also an object of the invention to provide pharmaceutical compositions and / or therapies that can be used to treat an individual who has been diagnosed with the presence of a lung tumor.

Finally, it is an object of the invention to provide pharmaceutical compositions and / or therapies to reduce and / or eliminate the risk of developing a lung tumor.

One or more of the specified purposes are substantially achieved by a method and / or by a kit and / or by a composition and / or by an apparatus in accordance with one or more of the appended claims.

Aspects of the invention are described below.

A first aspect concerns a procedure for identifying individuals at risk of lung cancer, said procedure comprising the steps of:

measuring, in at least one biological fluid sample previously taken from a subject, a value of the expression level of a plurality of microRNA molecules;

determining when said measured values of the expression level deviate with respect to a predetermined and respective control criterion.

The microRNA or miRNA molecules are therefore used to predict individuals at risk at a stage where the cancer has not yet manifested.

In a 2nd aspect, said determining phase comprises the sub-phases of calculating a plurality of quotients or real differences determined by carrying out the ratio or respectively the difference between the measured values of the expression levels of a predetermined number of pairs of said microRNA molecules, comparing each of said real quotients or differences with a respective control value, determine the real quotients or differences which deviate from the respective quotient or control difference value.

In a 3rd aspect, in accordance with the 2nd aspect, there is also a phase for determining the number or percentage of real quotients or differences that deviate from the respective control value.

In a 4th aspect according to any of the previous aspects, there is a phase of defining as an individual at risk an individual for whom at least a predetermined number or a predetermined percentage of said real quotients or differences deviates from the respective quotient or difference value control.

In a 5th aspect according to any of the preceding aspects, for each of said calculated quotients a respective control quotient is associated, represented by the ratio of the expression values for the same microRNAs in a control sample relating to a biological fluid of the same type. The control quotient is actually either a known value, or a value determined as the average of values measured over a sufficiently large population of individuals, or a value relative to a fluid sample taken from a healthy individual.

In a 6th aspect according to any of the preceding aspects, the procedure comprises the step of correlating the deviation of a predetermined number or a predetermined percentage of expression levels (i.e. of real quotients or differences) with respect to the corresponding control criteria to the presence or absence of risk that the individual clinically manifests a lung tumor in a fixed time.

In a 7th aspect, according to the previous aspect, the set time is between one and three years, more optionally between 12 and 28 months. In other words, the method in accordance with the invention is able to significantly anticipate the determination of the risk of contracting a tumor compared to traditional techniques (such as spiral CT) which must wait for the onset of the disease at the level of lacerations or nodules of several mm. .

In an 8th aspect according to any of the preceding aspects, the procedure comprises the step of correlating the deviation of a predetermined number or a predetermined percentage of expression levels with respect to the corresponding control criteria to the presence or absence of risk that the individual you clinically develop aggressive lung cancer within a set time.

In a 9th aspect, according to the previous aspect, the set time is between one and three years, more optionally between 12 and 28 months. In other words, the method in accordance with the invention is able to significantly anticipate the determination of the risk of contracting an aggressive tumor with respect to traditional techniques (such as spiral CT) which must wait for the onset of the disease at the level of lacerations or nodules of different mm.

In a 10th aspect according to any of the preceding aspects, calculating said plurality of real quotients or differences comprises using the expression values of a predetermined number or a predetermined percentage of the miRNAs referred to in Table I and / or referred to in Table II.

In an 11th aspect according to any of the 6th, 7th or 10th aspects, calculating said plurality of real quotients or differences comprises using the expression values of a predetermined number or a predetermined percentage of the miRNAs belonging to the following list

hsa-mirR-140-5p

hsa-mirR-660

hsa-mirR-320

hsa-mirR-28-3p

hsa-mirR-45 1

hsa-mirR-30c

hsa-mirR-92a

hsa-mirR-197

hsa-mirR-221

hsa-mirR-19b

hsa-mirR-15b

hsa-mirR-30b

hsa-mirR-17

hsa-mirR-45 1

hsa-mirR-106a

In a 12th aspect according to any of the 8th, 9th or 10th aspects, calculating said plurality of real quotients or differences comprises using the expression values of a predetermined number or a predetermined percentage of the miRNAs belonging to the following list

hsa-mirR-221

hsa-mirR-45 1

hsa-mirR-197

hsa-mirR-486-5p

hsa-mirR-28-3p

hsa-mirR-221

hsa-mirR-660

hsa-mirR-106a

hsa-mirR-140-5p

hsa-mirR-16

In a 13th aspect according to any one of the preceding aspects calculating said plurality of real quotients comprises determining a predetermined number or a predetermined percentage of quotients between the values of the expression levels, such quotients being selected in the group comprising quotients between values of expression levels of pairs of microRNAs referred to in Table III, optionally in which at least 20% are determined, more optionally at least 50% and even more optionally all the quotients referred to in Table III.

In a 14th aspect according to the previous aspect, determining a predetermined number or a predetermined percentage of quotients involves calculating at least 20% of the real quotients referred to in Table III and in which it includes a phase of defining as an individual at risk of lung cancer, optionally in a period between one and three years from the collection of the biological fluid sample, an individual for which at least 30%, optionally at least 50%, of the calculated real quotients deviate from the respective control quotient value.

In a 15th aspect according to any of the 13th or 14th aspects in which the quotients are chosen from those in the following list

Qi = hsa-mirR-106a / hsa-mirR-451

Q2 = hsa-mirR-140-5p / hsa-mirR-320

Q3 = hsa-mirR- 140-5p / hsa-mirR-45 1

Q4 = hsa-mirR- 140-5p / hsa-mirR-660

Q5 = hsa-mirR- 140-5p / hsa-mirR- 92a

Q6 = hsa-mirR- 15b / hsa-mirR-92a

Q7 = hsa-mirR- 17 / hsa-mirR-451

Q8 = hsa-mirR- 197 / hsa-mirR-451

Q9 = hsa-mirR- 19b / hsa-mirR-660

Qio = hsa-mirR-221 / hsa-mirR-660

Qn = hsa-mirR-28-3p / hsa-mirR-660

Qi2 = hsa-mirR-30b / hsa-mirR-92a

Q13 = hsa-mirR-30c / hsa-mirR-451

Q14 = hsa-mirR-30c / hsa-mirR-660

In a 16th aspect according to any of the preceding aspects calculating said plurality of real quotients comprises determining a predetermined number or a predetermined percentage of quotients between the values of the expression levels, said quotients being selected in the group comprising quotients between values of expression levels of pairs of microRNAs referred to in Table IV, optionally in which at least 30% are determined, more optionally at least 50% and even more optionally all the quotients referred to in Table IV.

In a 17th aspect according to the previous aspect, determining a predetermined number or a predetermined percentage of quotients involves calculating at least 30% of the real quotients referred to in Table IV and in which the procedure includes a phase of defining as an individual at risk of aggressive lung cancer, optionally in a period between one and three years from the collection of the biological fluid sample, an individual for which at least 50% optionally at least 75% of said real calculated quotients deviate from the respective control quotient value .

In an 18th aspect according to any of the 16th or 17th aspects in which the quotients are chosen from those in the following list

Qi = hsa-mirR-106a / hsa-mirR-660

Q2 = hsa-mirR-140-5p / hsa-mirR-486-5p

Q3 = hsa-mirR-16 / hsa-mirR-197

Q4 = hsa-mirR-197 / hsa-mirR-486-5p

Qs = hsa-mirR-197 / hsa-mirR-660

Q6 = hsa-mirR-221 / hsa-mirR-45 1

Q7 = hsa-mirR-221 / hsa-mirR-660

Qs = hsa-mirR-28-3p / hsa-mirR-451

Q9 = hsa-mirR-28-3p / hsa-mirR-486-5p

Q10 = hsa-mirR-28-3p / hsa-mirR-660

In a 19th aspect according to any one of the preceding aspects, the steps of said process are carried out in vitro.

In a 20th aspect according to any of the foregoing aspects said biological fluid is one selected from the group comprising: whole blood, a fraction of blood, plasma, serum.

In a 21st aspect according to any of the foregoing aspects said lung cancer is one selected from the group comprising: small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma (ADC), bronchial carcinoma alveolar (BAC), squamous cell lung cancer (SCC), large cell carcinoma (LC).

In a 22 ° aspect according to any of the preceding aspects, said biological fluid sample comes from a smoker who, at the time of sampling, does not show lung cancer if subjected to diagnostic methods by image, in particular does not show nodules larger than 5mm when subjected to spiral CT.

A 23rd aspect concerns a medical kit for the determination of biomolecular markers present in a sample of human biological fluid, said kit comprising a platform having a plurality of sites each of which is intended to receive a respective discrete quantity of said fluid sample biological, each of said sites comprising a reagent capable of binding with at least one respective of the microRNAs of Table I and / or Table II.

In a 24th aspect according to the preceding aspect said reagent includes at least one selected from the group comprising:

- a polynucleotide comprising a nucleotide sequence of at least one of the microRNAs listed in Table I and / or Table II, - a polynucleotide comprising a nucleotide sequence complementary to that of at least one of the microRNAs listed in Table I and / or Table II ,

- molecular probe configured to recognize a sequence of at least one of the microRNAs listed in Table I and / or Table II.

A 25th aspect concerns a medical device comprising:

- a unit defining a seat to receive one or more of the kits referred to in aspect 23 ° or 24 °,

- means to determine the value of the expression level of the microRNAs referred to in Table I and / or Table II,

- means for calculating the values of the real quotients between values of expression levels of pairs of microRNAs referred to in Table III and / or referred to in Table IV.

In a 26th aspect according to the previous aspect the means for determining the value of the level of expression include one of the techniques selected in the group:

Quantitative Real-time PCR, Microfluidic cards (microfluidic chips),

Microarrays (microchips),

RT (reverse transcription) - quantitative or semiquantitative PCR, Northern blot,

Solution Hybridization,

Sequencing.

A 28th aspect involves an in vitro procedure to identify individuals at risk of cancer and / or to determine the presence and / or aggression of a tumor in an individual, said procedure including the steps of:

measuring, in at least one biological fluid sample previously taken from a subject, a value of the expression level of a plurality of microRNA molecules;

calculating a plurality of real quotients determined by making the ratio between the measured values of the expression levels of a predetermined number of pairs of said microRNA molecules;

comparing each of said real quotients with a respective control value.

In a 29th aspect in accordance with the 28th aspect the procedure comprises determining the number or percentage of real quotients that deviate from the respective control value, defining as an individual in which a form of tumor is present, an individual for the which at least a predetermined number or a predetermined percentage of said real quotients deviate from the respective control value.

In a 30 ° aspect in agreement with the 29 ° for each of said calculated quotients a respective control value is associated, represented by the ratio of the expression values for the same microRNAs in a control sample relating to a biological fluid of the same type.

In a 31st aspect according to the 30th or 29th aspect, calculating said plurality of real quotients includes using the expression values of a predetermined number of miRNAs:

referred to in Table I and / or Table II and / or Table V and / or Table VI.

In a 32nd aspect in accordance with any one of the 29th to 31st aspects calculating said plurality of real quotients comprises determining a predetermined number or a predetermined percentage of quotients among the values of the expression levels, such quotients being selected in the group including the quotients referred to in Table III and / or Table IV and / or Table VII and / or Table VIII.

In a 33rd aspect in accordance with the 32nd determining a predetermined number or a predetermined percentage of quotients involves calculating at least 20% of the real quotients referred to in Table VII and in which it includes a phase of defining as an individual in which it is a lung tumor is an individual for which at least 30% of the predetermined number of real quotients as per Table VII that have been calculated deviates from the respective control value.

In a 34th aspect in accordance with the 33rd or 32nd, determining a predetermined number or a predetermined percentage of quotients involves calculating at least 20% of the real quotients referred to in Table VIII and in which it includes a phase of defining as an individual in which an aggressive lung tumor is present an individual for which 50%, optionally at least 60%, of the real quotients that have been calculated deviate from the respective control value.

In a 35th aspect in accordance with the 34th or 33rd or 32nd, determining a predetermined number or a predetermined percentage of quotients involves calculating at least 20% of the real quotients referred to in Table III and in which it includes a phase to define as an individual at risk of lung cancer, optionally in a period between one and three years from the collection of the biological fluid sample, an individual for whom at least 30%, optionally at least 50%, of the real calculated ratios deviate from to the respective control quotient value.

In a 36th aspect in accordance with the 35th or 34th or 33rd or 32nd, determining a predetermined number or a predetermined percentage of quotients involves calculating at least 30% of the real quotients referred to in Table IV and in which includes a phase of defining as an individual at risk of aggressive lung cancer, optionally in a period between one and three years from the collection of the biological fluid sample, an individual for whom at least 50%, optionally at least 75% of said calculated real quotients deviate from the respective control quotient value.

In a 37th aspect in accordance with any of the preceding 28th to 36th aspects said biological fluid is one selected from the group comprising: whole blood, a fraction of blood, plasma, serum, saliva or bronchial condensate.

In a 38th aspect in accordance with any of the previous 28th to 37th aspects wherein said tumor is a lung tumor selected in the group comprising:

small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma (ADC), bronchoalveolar carcinoma (BAC), squamous cell lung cancer (SCC), large cell carcinoma (LC).

In a 39 ° aspect in accordance with any of the previous aspects from the 28 ° to the 38 ° said biological fluid sample comes from an individual smoker who, at the time of sampling, shows lung cancer if subjected to diagnostic methods such as image, in particular shows lesions larger than 5mm if subjected to spiral CT.

In a 40th aspect there is a medical kit for the determination of biomolecular markers present in a sample of human biological fluid, said kit comprising:

a platform, for example a support for receiving fluid samples, having a plurality of sites each of which is intended to receive a respective discrete quantity of said biological fluid sample;

each of said sites comprising a reagent capable of binding with at least one respective of the microRNAs of Table I and / or Table II and / or Table V and / or Table VI.

In a 41st aspect in accordance with the previous aspect said reagent includes at least one selected from the group comprising:

- a polynucleotide comprising a nucleotide sequence of at least one of the microRNAs of Table I and / or Table II and / or Table V and / or Table VI;

- a polynucleotide comprising a nucleotide sequence complementary to that of at least one of the microRNAs of Table I and / or Table II and / or Table V and / or Table VI; - a molecular probe configured to recognize a sequence of at least one of the microRNAs referred to in Table I and / or Table II and / or Table V and / or Table VI.

In a 42 ° aspect there is a medical device comprising:

- a unit defining a seat to receive one or more of the kits referred to in the previous claim,

- means for determining the value of the expression level of the microRNAs referred to in Table I and / or Table II and / or Table V and / or Table VI;

- means for calculating the values of the real quotients between values of expression levels of microRNA pairs, said quotients being selected from those of Table III and / or of Table IV and / or of Table VII and / or of Table VIII.

In a 43rd aspect according to the previous aspect the means for determining the value of the expression level include one selected in the group:

Quantitative Real-time PCR, Microfluidic cards (microfluidic chips),

Microarrays (microchips),

RT (reverse transcription) - quantitative or semiquantitative PCR, Northern blot,

Solution Hybridization, Sequencing,

In a 44th aspect, there is a method for treating an individual who has been diagnosed with lung cancer or who has been identified as having a risk of developing lung cancer, respectively for the treatment of said lung cancer or for reducing and / or eliminate the risk of developing lung cancer, this method including the following steps:

- measure an expression level of at least one miRNA listed in Table I or Table II or Table V or Table VI present in a sample of biological fluid previously taken from that individual,

- determining the miRNAs having measured values of the expression level which deviate from a predetermined and respective control criterion;

- altering the expression level of miRNAs whose expression levels deviate from the respective control criterion.

In a 45 ° aspect according to the previous aspect, said phase of alteration of the level of expression of miRNAs includes administering to the individual an effective quantity of at least one of the miRNAs listed in Table I or in Table II or in Table V or in Table VI if the measured expression level of such miRNA (s) is below a respective control expression level.

In a 46 ° aspect according to the aspect 44 ° or 45 ° said phase of alteration of the level of miRNA expression includes administering to the individual an effective quantity of at least one composition to inhibit the expression of at least one of the listed miRNAs in Table I or Table II or Table V or Table VI if the measured expression level of one or more of such miRNAs is above the control expression level.

In a 47th aspect by the 45th aspect the method comprises restoring the expression level values to the control expression level for the under-expressed miRNAs with respect to the respective control expression level.

In a 48th aspect according to any one of the 44th to 47th aspects the method comprises administering a therapeutically effective amount of a composition comprising at least one of the miRNAs listed in Table I or Table II or Table V or Table VI chemically synthesized (mimetic miRNA) or recombinant.

In a 49th aspect according to any one of the 44th to 48th aspects the method comprises reducing the expression level values to the control expression level for the overexpressed miRNAs with respect to the respective control expression level.

In a 50th aspect according to any one of the 44th to 49th aspects the method comprises administering a therapeutically effective amount of a composition comprising at least one inhibitor of a microRNA of Table I or Table II or Table V or Table YOU.

In a 51st aspect according to the above aspect said inhibitor comprises double stranded RNA.

In a 52nd aspect according to the previous aspect the method comprises short interfering RNAs (siRNAs), antisense nucleic acids (anti-miRNA oligonucleotides (AMOs), enzymatic RNA molecules (ribozymes).

In a 53rd aspect according to any one of the aspects from the 50th to the 52nd this inhibitor is addressed to a specific microRNA product and interferes with the expression, for example by inhibition of translation or induction of degradation, of a target gene of the microRNA.

In a 54th aspect according to any of the foregoing aspects, the step of determining miRNAs having measured expression level values that deviate from the respective control criterion comprises:

- calculate a plurality of real quotients determined by making the ratio between the measured values of the expression levels of a predetermined number of pairs of said microRNA molecules, these quotients being selected from the group comprising the quotients referred to in Table III and / or Table IV and / or Table VII and / or Table VIII,

- determine the real quotients that deviate from the respective control value, - identify the miRNAs involved in the real quotients that deviate from the respective control value.

A 55th aspect concerns a pharmaceutical composition to treat an individual who has been diagnosed with lung cancer or who has been identified as having a risk of developing lung cancer, respectively for the treatment of said lung cancer or to reduce and / or eliminate the risk of developing lung cancer,

this composition including:

- at least one of the miRNAs listed in Table I or Table II or Table V or Table VI, and / or

- at least one inhibitor of the expression of at least one of the miRNAs listed in Table I or Table II or Table V or Table VI.

In a 56th aspect according to the preceding aspect, the composition comprises a therapeutically effective quantity of at least one of said miRNAs listed in Table I or in Table II or in Table V or in Table VI.

In a 57th aspect according to any one of the aspects from 55th to 56th said quantity is capable, for the miRNAs under-expressed with respect to the respective control expression level, to restore the expression level values to the respective control expression level .

In a 58th aspect according to any one of the 55th to 57th aspects said therapeutically effective quantity comprises chemically synthesized or recombinant miRNAs of Table I or Table II or Table V or Table VI.

In a 59th aspect according to any one of the 55th to 57th aspects, the composition comprises a therapeutically effective amount of said inhibitor of the expression of at least one of the miRNAs listed in Table I or in Table II or in Table V or in Table VI, said quantity being capable, for miRNAs overexpressed with respect to the respective control expression level, to reduce the expression level values to this respective control expression level.

In a 60th aspect according to the preceding aspect, said inhibitor comprises double-stranded RNA, optionally short interfering RNAs (siRNA), and / or antisense nucleic acids, and / or enzymatic RNA molecules (ribozymes).

In a 61 ° aspect according to the 59 ° or 60 ° aspect said inhibitor is addressed to a specific microRNA product and interferes with the expression (by inhibition of translation or induction of degradation) of a target gene of the microRNA.

In a 62 ° a pharmaceutical composition is provided according to any of the aspects from 55 ° to 61 ° for the preparation of a medicament usable in one of the therapeutic methods referred to in any of the aspects from 44 ° to 54 °.

In a 63rd aspect according to the previous aspect, the therapeutic method is a method of treating an individual who has been diagnosed with the presence of lung cancer.

In a 64 ° aspect according to the 62 ° aspect the therapeutic method is a method of treating an individual who has been identified as having a risk of developing lung cancer to reduce and / or eliminate the risk of developing lung cancer.

In accordance with a 65 ° aspect according to any of the preceding aspects, it is envisaged that, as a variant of the invention and alternatively to the real quotients (in the procedure, in the medical kit and in the equipment), real differences are determined by making the difference between the values measured the expression levels of a predetermined number of pairs of said microRNA molecules. In this case, each of the differences is compared with a respective control value to then determine the differences that deviate from the respective control value. All the aspects previously described are applied equally by substituting real differences between the same miRNA pairs in the attached tables to the real quotients.

DETAILED DESCRIPTION

Some specific and therefore non-limiting aspects of the invention are described below. In particular, the steps of the research conducted by the inventors are described, the results obtained and various exemplary aspects of the invention are reported.

Definitions

The following terminology when used in the description, claims and drawings of the present patent application assumes the meaning given below.

Individuals: human subjects.

MicroRNA or miRNA: small non-coding RNA molecules (length 19-25 nucleotides). In particular, it refers to miRNAs present in biological samples of human tissue, for example whole blood, plasma, serum, saliva or bronchial condensate.

MiRNA quotients or â € œmiRNA ratiosâ €: real quotients determined by making the ratio between the measured values of the expression levels of predetermined pairs of microRNA molecules.

Aggressive tumor: tumor, such as lung cancer, with a poor prognosis that is capable of leading to death in 90% of patients within 5 years of diagnosis of the disease.

Individual at risk of cancer (aggressive or not depending on the cases studied): individual who has a risk of more than T80% of developing a tumor within the reference period (1-3 years) following the taking of the biological sample. for example a lung tumor, detectable with techniques such as spiral CT.

Research

The inventors investigated the miRNA expression profile in plasma of individuals enrolled in spiral CT screening protocols. This investigation was conducted in order to verify the capacity of miRNAs as a new class of molecular biomarkers for:

- predicting the risk of developing cancer, especially lung cancer, e

- the diagnosis of cancer, in particular lung cancer, and therefore as a prognostic aid to discriminate patients with indolent or aggressive lung lesions.

Plasma samples collected from smokers older than 50 years of age were used between one and two years before the spiral CT scan of the presence of lung cancer in the same individuals. Plasma samples taken at the onset of the disease (detected by spiral CT scan) were also used. Plasma samples were obtained from patients who developed lung cancer with different characteristics in terms of clinical aggression (indolent nodules or advanced and metastatic tumors), as well as from individuals who remained disease-free throughout the screening.

In a first phase of the research, they identified which microRNAs were present in the plasma using Applied Biosystems' TaqMan <®> model â € œmicrofluidic cardsâ €. Of the 378 microRNAs analyzed, 100 were found to be stably present in the plasma of healthy smoking individuals used as a control group. Therefore, being able to use a large number of initial data, there is a general agreement on the possibility of normalizing the expression levels of the individual microRNAs on the average of the expression levels of the 100 microRNAs for each individual (Mestdagh P et al. Gemme Biol, 2009). The data obtained with this type of normalization were compared with those obtained by normalizing on potential housekeeping microRNAs (for example mir-16, mammU6, RNU44 or RNU48).

The inventors then decided to use no longer the values of the expression levels of the individual microRNAs, but the relationships between pairs of them. The threshold cycle value (Ct) obtained from qReal-Time PCR with SDS 2.2.2 <®> (Applied Biosystems) software was transformed into the corresponding expression value (2). Then the quotient between the value of the measured expression level of each pair of possible microRNAs was calculated, obtaining 4950 total ratios: the 4950 total ratios are given by the formula 100 * 99/2 as the ratio between two miRNAs and its reciprocal contain the same information. Finally, the variation of these ratios (called â € œmiRNA ratiosâ €) in the plasma of the different classes of patients was analyzed to identify plasma biomarkers.

The results showed that the microRNAs most present in the ratios that discriminate between the classes of patients are the same as those resulting from the analyzes carried out by normalizing the average value of the expression levels of the 100 microRNAs for each individual, thus validating the method based on quotients or â € œmiRNA ratiosâ € for the quantification of the involved microRNAs.

In more detail, in order to identify biomarkers in plasma capable of predicting the appearance of lung cancer, the inventors studied the expression profile of circulating microRNAs in samples collected up to two years before the diagnosis of the disease and at the time of surgery in patients of two independent clinical trials, as mentioned above, for the early diagnosis of lung cancer in high-risk individuals (age> 50 years and smokers) by spiral CT scan. In the first training set consisting of 40 plasma samples from 19 patients and 27 plasma samples from healthy control individuals grouped into 5 different pools, miRNA expression levels were analyzed with TaqMan MicroRNA Assays (Applied Biosystems) in order to identify significantly different â € œmiRNA ratiosâ € (p <0.05) between plasma samples collected pre-disease, at the time of surgery and from healthy individuals.

The specificity and sensitivity of the microRNA signatures or â € œsignaturesâ € thus obtained were compared with the validation set composed, as described above, from 32 plasma samples from 22 patients and 54 plasma samples from healthy control individuals, grouped in 10 different pools.

For the generation of the signatures or â € œsignaturesâ € used to predict the aggressiveness of the disease, the inventors grouped the two cohorts (training set and validation set) in order to obtain a sufficient number for statistical analyzes. The cases with poor prognosis were first compared to the respective controls and the signatures or â € œsignaturesâ € thus obtained were then tested to assess their effective ability to discriminate patients with poor and good prognosis.

Tests and tests performed - materials and equipment used

As already mentioned, the microRNA signatures or â € œsignatureâ € identified in the different analyzes were validated on two independent sets made up of high-risk individuals (smokers over the age of 50) enrolled in two different clinical trials for early detection lung cancer with low-dose spiral CT scan: a first set or training set consisting of 40 plasma samples from 19 patients and 27 plasma samples from healthy control individuals grouped in 5 different pools and a second set or validation set ( i.e. in a second set of individuals) consisting of 32 plasma samples from 22 patients and 54 plasma samples from healthy control individuals, grouped into 10 different pools. Figure 1 summarizes the pathological clinical characteristics of the training set and validation set selected for the analysis of miRNA expression levels in plasma samples.

To determine the microRNA profile in plasma samples, total RNA was extracted from 200Î1⁄41 plasma using the mirVanaâ „¢ PARISâ„ ¢ Kit (Ambion), eluting in 50Î1⁄41 elution buffer.

The expression levels were determined by q-Real Time PCR starting from 3Î1⁄41 of eluate using first the Megaplexâ „¢ Pools Protocol on â € œmicrofluidic cardâ €, type A (Applied Biosystems), then the Multiplexâ„ ¢ Pools Protocol (Applied Biosystems).

All data were extrapolated with the Sequence Detection System software (SDS 2.2.2 <®> Applied Biosystems) by setting the threshold manually to 0.2 and the baseline between 3 and 18 cycles (out of a total of 40).

In addition to standard molecular biology equipment, Real-time quantitative PCR 7900-HT (Applied Biosystems) and GeneAmp <®> 9700 Sequence Detection System (Applied Biosystems) were used.

Results

The inventors have identified miRNAs, in particular those referred to in Table I attached herein, as molecular biomarkers for assessing the risk of lung cancer within 1-3 years of biological fluid sampling. The inventors have also identified that the quotients between the miRNA expression values are ideal molecular biomarkers to be investigated for the assessment of the risk of contracting a lung tumor within 1-3 years of taking a sample of biological fluid such as whole blood, serum, plasma. , saliva or bronchial condensate.

Using the miRNA expression levels listed in Table I, the inventors specifically identified the quotients between measured values of the expression levels relative to the microRNA pairs listed in Table III. These quotients can be used to assess the risk of contracting lung cancer within 1-3 years of taking a sample of biological fluid, giving extremely reliable prediction results.

More in detail, by calculating a sufficient number of real quotients selected from those of Table III, for example at least 20% of them, and optionally at least 50%, it is possible to study their trend with respect to control quotients. An individual is defined at high risk of lung cancer, which can manifest itself in a detectable way by spiral CT scan in a period between one and three years from the collection of the biological fluid sample, an individual for which at least 30% of the real calculated quotients deviates from the respective control quotient value.

The inventors have also identified miRNAs, particularly those listed in Table II attached herein, as biomarkers for assessing the risk of contracting aggressive lung cancer within 1-3 years of biological fluid sampling. Furthermore, also in this case, specifically the quotients between the miRNA expression values have been identified as ideal molecular biomarkers to be investigated for the assessment of the risk of contracting an aggressive lung tumor within 1-3 years of taking a sample of biological fluid such as blood. whole, serum, plasma, saliva or bronchial condensate. In addition, using the miRNA expression levels in Table II, the inventors identified the quotients between measured values of expression levels relative to pairs of microRNAs in Table IV for assessing the risk of contracting an aggressive lung cancer within 1-3 years from sampling of biological fluid. More in detail, by calculating a sufficient number of real quotients selected from the quotients referred to in Table IV, for example at least 30%, and optionally at least 50%, it is possible to study the trend of these quotients with respect to quotients of check. An individual is defined as at risk of contracting an aggressive lung cancer in a period between one and three years from the collection of the biological fluid sample, if in that individual at least 50%, optionally at least 75%, of said real calculated ratios deviates from the respective control quotient value.

The use of miRNA quotients therefore allows us to reliably predict the development of lung cancer, in particular of the more aggressive form, in high-risk individuals (over 50 years and heavy smokers) up to two years before the disease is at a visible stage with the best imaging techniques available today (spiral CT scan). It should also be noted that the method using the calculation of quotients or â € œmiRNA ratiosâ € described above can be implemented with a simple blood sample and therefore totally non-invasive, allowing analyzes to be carried out quickly and economically.

In addition to the above, the inventors have identified that miRNAs, and in particular those listed in Table V, play a role as biomolecular markers for determining the current presence of a lung tumor in an individual, ie for diagnostic purposes. The inventors also found that in this case the quotients between values of expression levels of miRNA pairs are valid biomarkers with diagnostic and prognostic function. In detail, the miRNAs of Table V were used to determine the quotients of Table VII which represent quotients between measured values of expression levels relative to pairs of microRNAs and which are used to determine the current presence (diagnosis) of lung cancer in an individual. More in detail, by determining at least 20% of the real quotients referred to in Table VII, it is then possible to define that an individual has a lung tumor if at least 30% of the real quotients (referred to in Table VII) that have actually been calculated deviate from the respective control value.

Finally, the inventors have identified that miRNAs, and in particular those listed in Table VI, can be used as biomolecular markers for determining the current presence of an aggressive lung tumor in an individual (prognosis). The inventors have also found that in particular the quotients between values of expression levels of miRNA pairs are valid biomarkers with diagnostic and prognostic function even in the case of aggressive tumor. In detail, the miRNAs in Table VI were used to determine the quotients referred to in Table VIII where quotients are listed between the measured values of the expression levels relating to pairs of microRNAs referred to in Table VI used for the determination of the current presence of a aggressive lung cancer in an individual. In detail, by detecting at least 20% of the real quotients referred to in Table VIII, an individual in which an aggressive lung tumor is present can be defined as an individual for which at least 60% of the real quotients that have been calculated deviate from the respective value of check.

Therefore, the use of the methodology described can also help to solve the problem of over-diagnosis and over-treatment of patients not at risk.

More specifically, in a context of surveillance of the disease by spiral CT, the use of a test based on this method could allow to select only a subgroup of patients at high risk of developing the disease to be kept under closer control subsequently. Furthermore, the ability of the test to predict patients who will develop a more aggressive disease, frequently not diagnosed by CT scan, allows these individuals to be directed directly to targeted drug programs (including smoking cessation), and / or to the use of diagnostic tests. more specific based on metabolic-biological characteristics such as PET with different tracers or body MR, or to a different local treatment such as stereotaxic radiotherapy, or other treatments.

Finally, the use of â € œmiRNA ratiosâ € is an easily applicable method with potential for current clinical use and which avoids the use of more extensive and complex analyzes.

Examples

1. MiRNA-based signature identification capable of identifying individuals at risk of developing lung cancer (Figure 4A)

Plasma samples collected 1-2 years earlier from patients who were later diagnosed with tumors by spiral CT were analyzed and compared to control pools, which consisted of healthy individuals.

In the training set a â € œsignatureâ € of 14 miRNA quotients or â € œmiRNA ratiosâ € composed of 14 microRNAs able to correctly discriminate 18 out of 20 pre-disease samples of individuals who will subsequently develop the disease (sensitivity of the 90%), while only one control pool was found to be positive for this â € œsignatureâ € (specificity of 80%). In the validation set the sensitivity was found to be 80% while the specificity was 90% (AUC-ROC = 0.85, p <0.001).

The miRNA quotients of this first example are listed below and are also reported in Figure 4A.

Qi = hsa-mirR-106a / hsa-mirR-451

Q2 = hsa-mirR-140-5p / hsa-mirR-320

Q3 = hsa-mirR-140-5p / hsa-mirR-451

Q4 = hsa-mirR- 140-5p / hsa-mlrR-660

Q5 = hsa-mirR-140-5p / hsa-mirR-92a

Q6 = hsa-mirR-15b / hsa-mirR-92a

Q7 = hsa-mirR-17 / hsa-mirR-451

Qs = hsa-mirR-197 / hsa-mirR-451

Q9 = hsa-mirR- 19b / hsa-mirR-660

Qio = hsa-mirR-221 / hsa-mirR-660

Qn = hsa-mirR-28-3p / hsa-mirR-660

Qi2 = hsa-mirR-30b / hsa-mirR-92a

Qi3 <=> hsa-mirR-30c / hsa-mirR-451

Q i4 = hsa-mirR-3 Oc / hsa-mirR-660

The predictive ability of this `` signature '' was validated in samples collected up to 28 months before the diagnosis of the disease with spiral CT and the most frequently deregulated microRNAs were: mir-660, mir 140-5p, mir 451, mir- 28-3p, mir-30c and mir-92.

2. Signature identification or â € œsignatureâ € based on miRNAs capable of having diagnostic value (Figure 4B)

Plasma samples collected at the time of surgery or at the time of detection of the disease by spiral CT were compared with the control pools. In the training set, a panel of 16 â € œmiRNA ratiosâ €, composed of 13 microRNAs, correctly classifies 16 out of 19 patients with a sensitivity of 84% and a specificity of 80%. In the validation set the sensitivity is 75% and the specificity is 100% (AUC-ROC = 0.88, p <0.0001).

A lower sensitivity in the validation set may be correlated with the presence of a greater number of small indolent nodules, which would include two patients whose samples are mis-classified by both the risk `` signature '' in the pre-samples. disease, which from that of diagnosis in samples in the presence of disease.

The miRNA quotients of this second example are listed below and are also reported in Figure 4B.

Qi <=> hsa-mirR-106a / hsa-mirR-140-3p

Q2 = hsa-mirR-106a / hsa-mirR-30c

Q3 = hsa-mirR-106a / hsa-mirR-486-5p

Q4 = hsa-mirR-140-3p / hsa-mirR-17

Q5 = hsa-mirR- 140-5p / hsa-mirR-660

Q6 = hsa-mirR- 15b / hsa-mirR-660

Q7 = hsa-mirR- 15b / hsa-mirR-92a

Q8 = hsa-mirR-17 / hsa-mirR-30c

<3⁄4 = hsa-mirR-17 / hsa-mirR-451

Qio = hsa-mirR-17 / hsa-mirR-486-5p

Qn = hsa-mirR- 19b / hsa-mirR-451

Qi2 = hsa-mirR- 19b / hsa-mirR-660

QB = hsa-mirR- 19b / hsa-mirR-92a

QH = hsa-mirR-21 / hsa-mirR-92a

Q15 = hsa-mirR-28-3p / hsa-mirR-660

Qi6 <=> hsa-mirR-28-3p / hsa-mirR-92a

This â € œsignatureâ € of diagnosis was then used to verify the presence of disease in plasma samples collected prior to detection of the disease by spiral CT scan. In the training set 11 out of 20 (55%) of the cases are classified as having disease and, very interestingly, of these 11, 10 are either cases with a bad prognosis or belonging to patients in whom the tumor has been identified in the years later than screening, ie where more aggressive tumors with a worse prognosis have been identified. In the validation set very similar results were obtained, since in 10 out of 15 (66.6%) pre-disease samples presented the â € œsignatureâ € of the presence of disease. There are only 4 â € œmiRNA ratiosâ € in common between the â € œsignatureâ € of risk and diagnosis and the microRNAs involved are also partially different: mir-17, mir-660, mir92a, mir-106a, mir-19b are the microRNAs most deregulated at the time of lung cancer diagnosis.

3. Identification of a miRNA-based signature or â € œsignatureâ € for risk of developing aggressive lung cancer

The microRNA profiles of the pre-disease samples with poor prognosis were analyzed and 10 â € œmiRNA ratiosâ € were identified capable of recognizing 5 out of 5 patients in the first set, 4 out of 5 in the validation set and with a specificity of 100 in both. %. Note that mir-221, mir-660, mir-486-5p, mir-28-3p, mir-197, mir-106a, mir-451, mir-140-5p, and mir-16 are the deregulated microRNAs.

The miRNA quotients of this third example are listed below and are also reported in Figure 4C.

Q 1 = hsa-mirR-106a / hsa-mirR-660

Q2 = hsa-mirR- 140-5p / hsa-mirR-486-5p

Q3 = hsa-mirR- 16 / hsa-mirR- 197

Q4 = hsa-mirR- 197 / hsa-mirR-486-5p

Q5 = hsa-mirR- 197 / hsa-mirR-660

Q6 = hsa-mirR-221 / hsa-mirR-451

Q7 = hsa-mirR-221 / hsa-mirR-660

Qs = hsa-mirR-28-3p / hsa-mirR-451

Q9 = hsa-mirR-28-3p / hsa-mirR-486-5p

Qio = hsa-mirR-28-3p / hsa-mirR-660

This â € œsignatureâ € was then tested on the pre-disease samples of the patients with good prognosis in the training set and in the validation set. This â € œsignatureâ € classifies, respectively, in the two sets, 33.3% and 45% of the champions; Figure 2 represents a Kaplan-Meier survival curve of patients with or without the `` signature '' of risk of aggressive disease: the curve with aggressive `` signature '' is represented in a continuous line and identified by RAD + (risk of aggressive disease ) while the curve without â € œsignatureâ € of risk of aggressive disease is represented in a discontinuous line and identified by RAD- (risk of aggressive disease -) in plasma samples collected 1-2 years before the detection of the disease by CT scan spiral.

Interestingly, most of the samples identified belong to individuals who developed cancer between the third and fifth year of screening, regardless of the grade of the cancer. This supports the previous observation on tumor and normal lung tissue samples, where a different microRNA profile was present in the tumor and normal tissue of the same patients, respectively. It should be noted that among the patients with cancer diagnosed in the second year of screening (all stage I and Ib cancers), only one case with stage Ib cancer had the â € œsignatureâ € of aggressive risk.

4. Identification of a miRNA-based signature or â € œsignatureâ € for prognosis of patients identified by spiral CT scan (Figure 4P)

The samples of patients with poor prognosis collected at the time of diagnosis of the disease were analyzed, finding a signature of 10 â € œmiRNA ratiosâ €, all containing mir-486-5p, which identified 7 out of 8 patients with poor prognosis in the training set, 2 out of 3 in the validation set and no control pool in both datasets.

The miRNA quotients of this fourth example are listed below and are also reported in Figure 4D.

Qi = hsa-mirR-106a / hsa-mirR-486-5p

Q2 = hsa-mirR-126 / hsa-mirR-486-5p

Q3 = hsa-mirR-142-3p / hsa-mirR-486-5p

<(> 3⁄4 = hsa-mirR-148a / hsa-mirR-486-5p

Q5 = hsa-mirR-15b / hsa-mirR-486-5p

Q6 = hsa-mirR-17 / hsa-mirR-486-5p

Q7 = hsa-mirR- 197 / hsa-mirR-486-5p

Qs = hsa-mirR-21 / hsa-mirR-486-5p

Q9 = hsa-mirR-221 / hsa-mirR-486-5p

Qio = hsa-mirR-28-3p / hsa-mirR-486-5p

Furthermore, only 2 out of 11 and 2 out of 13 patients with a good prognosis, respectively of the first and second set, are positive for this â € œsignatureâ €; Figure 3 shows a Kaplan-Meier survival curve of patients with or without the â € œsignatureâ € of presence of aggressive disease (respectively identified with continuous line from PAD +, acronym which stands for presence of aggressive disease, and with discontinuous line from PAD -, abbreviation for the presence of aggressive disease -) in plasma samples collected at the time of detection of the disease by spiral CT scan.

Furthermore, this â € œsignatureâ € was used to classify the pre-disease samples in both datasets. Half of the patients with a bad prognosis also have this â € œsignatureâ € of aggression, while for those with a good prognosis of the 6 positives for this â € œsignatureâ € 5 are of tumors identified after the third year of screening.

It is noted that mir-486-5p, compared with mir-21, mir-126, mir-15b, mir-148a, mir-142-3p, mir-17, mir-197, mir-221, mir-28-3p and mir-106a, always appears to be under-expressed in the plasma of patients with a poor prognosis. From the results reported above, the inventors deduced that the microRNAs present in the plasma are useful for detecting the presence of lung cancer even 1-2 years before detection with spiral CT and also to predict the development of the most aggressive types of lung cancer. indicating the possibility of selecting high-risk individuals on the basis of circulating microRNA profiles.

Kits and equipment for the determination of biomolecular markers present in a sample of human biological fluid that can be used for risk determination.

The inventors have also devised medical kits useful for effectively and simply implementing the methods described above for determining the risk of contracting a tumor, for example using a blood sample taken from an individual.

In its general form, the kit comprises a platform having a plurality of sites, each of which is intended to receive a respective discrete quantity of said biological fluid sample (for example whole blood, serum, plasma, saliva or bronchial condensate). Each site includes a reagent capable of binding with at least one respective of the microRNAs listed in Table I and / or Table II, so as to allow detectability with the equipment described below. From the structural point of view, the platform can be in support of the micro-fluidic card type with the miRNAs of interest and with channels for the distribution in the respective sites of a predetermined number of biological fluid samples.

For example, this includes at least one selected in the group comprising:

- a polynucleotide comprising a nucleotide sequence of at least one of the microRNAs listed in Table I and / or Table II, - a polynucleotide comprising a nucleotide sequence complementary to that of at least one of the microRNAs listed in Table I and / or Table II ,

- molecular probe configured to recognize a sequence of at least one of the microRNAs listed in Table I and / or Table II.

The described medical kit can then be used with a medical equipment comprising a unit defining a seat for receiving one or more kits and means for determining the value of the expression level of the microRNAs referred to in Table I and / or Table II. The means of determining the value of the level of expression include one of the techniques selected in the group:

Quantitative Real-time PCR Microfluidic cards (microfluidic chips)

Microarrays (microchips)

RT (reverse transcription) - Quantitative or semiquantitative Northern blot PCR

Solution Hybridization Sequencing

The apparatus may also present means for calculating the values of the real quotients between the values of expression levels of microRNA pairs referred to in Table III and / or referred to in Table IV; such means can comprise a program and a processing unit in which the program contains instructions which, when executed by the computer, allow the calculation of the quotients. Alternatively, an analog circuit capable of carrying out the same calculations can be provided.

Kits and equipment for the determination of biomolecular markers present in a sample of human biological fluid that can be used for tumor diagnosis.

The inventors have also devised medical kits useful for effectively and simply implementing the methods described above for tumor diagnosis, for example using a blood sample taken from an individual.

In its general form, the kit comprises a platform having a plurality of sites, each of which is intended to receive a respective discrete quantity of said biological fluid sample (for example whole blood, serum, plasma, saliva or bronchial condensate). From the structural point of view, the platform can be in support of the micro-fluidic card type with the miRNAs of interest and with channels for the distribution in the respective sites of a predetermined number of biological fluid samples. Each site comprises a reagent capable of binding with at least one respective of the microRNAs of Table V and / or Table VI.

For example, the reagent includes at least one selected from the group comprising:

- a polynucleotide comprising a nucleotide sequence of at least one of the microRNAs of Table V and / or Table VI; - a polynucleotide comprising a nucleotide sequence complementary to that of at least one of the microRNAs of Table V and / or Table VI;

- a molecular probe configured to recognize a sequence of at least one of the microRNAs referred to in Table V and / or Table VI.

The medical kit described can then be used with a medical equipment comprising a unit defining a seat for receiving one or more of the kits referred to in the previous claim, and means for determining the value of the expression level of the microRNAs referred to in Table V and / or Table VI. The means of determining the expression level value include one selected in the group:

Quantitative Real-time PCR, Microfluidic cards (microfluidic chips),

Microarrays (microchips),

RT (reverse transcription) - Quantitative or semiquantitative PCR, Northern blot,

Solution Hybridization, Sequencing.

The apparatus also provides means for calculating the values of the real quotients between values of expression levels of pairs of microRNAs, said quotients being selected from those of Table VII and / or of Table VIII. Such means can comprise a program and a processing unit in which the program contains instructions which, when executed by the computer, allow the calculation of the quotients. Alternatively, an analog circuit capable of carrying out the same calculations can be provided.

Compositions and therapies

The inventors then developed a method to treat an individual who has been diagnosed with lung cancer or who has been identified as having a risk of developing lung cancer, respectively to treat said lung cancer or to reduce and / or eliminate. the risk of developing lung cancer.

The method comprises the following steps of measuring an expression level of at least one miRNA listed in Table I or Table II or Table V or Table VI present in a sample of biological fluid previously taken from that individual, and then determining the miRNAs having measured values of the expression level that deviate from a predetermined and respective control criterion. The evaluation of the deviation with respect to a control criterion can use the methodologies of the miRNA quotients described above for the various cases.

Once the over-expressed or under-expressed miRNAs have been determined, the method envisages altering the expression level of the miRNAs whose expression levels deviate from the respective control criterion.

For example, to alter the expression level of miRNAs, it is possible to administer to the individual a pharmaceutical composition having an effective quantity of at least one of the miRNAs listed in Table I or in Table II or in Table V or in Table VI if the level of measured expression of such miRNA (s) is below a respective control expression level.

Alternatively or in addition to the above, it is also possible to administer to the individual a pharmaceutical composition having an effective amount of at least one composition to inhibit the expression of at least one of the miRNAs listed in Table I or Table II or Table V or Table VI if and for those miRNAs whose measured expression level is above the control expression level.

In this way it is possible to restore the expression level values to the control expression level for the under-expressed miRNAs compared to the respective control expression level and / or to reduce the expression level for the over-expressed miRNAs.

In order to restore the level of under-expressed mRNAs, a therapeutically effective amount of a composition comprising at least one of the miRNAs listed in Table I or Table II or Table V or Table VI can be administered chemically synthesized (mimetic miRNA) or recombinant.

In order to reduce the expression level values to the control expression level for the overexpressed miRNAs relative to the respective control expression level, a therapeutically effective amount of a composition comprising at least one inhibitor of a microRNA from Table can be administered. I or Table II or Table V or Table VI. This inhibitor includes for example one or more of the following: double-stranded RNA, optionally short interfering RNAs (siRNA), antisense nucleic acids (antimiRNA oligonucleotides (AMOs), enzymatic RNA molecules (ribozymes). a specific microRNA product and interferes with the expression (by inhibition of translation or induction of degradation) of a target gene of the microRNA.

The administration of the above compositions (synthetic microRNAs or mimetic miRNAs and microRNA inhibitors) can for example take place by means of viral systems or nanoparticles containing microRNA or microRNA inhibitor conjugated covalently with lipids or encapsulated in liposomes.

The routes of administration can be the following, possibly also combined with each other:

a) intranasal instillation

b) inhalation (aerosol)

c) systemic (injection or infusion)

d) direct inoculation into the tumor (where present and visible)

e) intrapleural administration

f) endopleural administration

At the dosage level it is possible to proceed with continuous and prolonged dosage over time. Since miRNA molecules are â € œnaturallyâ € present in the organism, no relevant toxicities are expected.

Table I: microRNAs used to assess the risk of developing lung cancer (within 1-3 years of biological fluid sampling)

hsa-miR-45 1

hsa-miR-320

hsa-miR-660

hsa-miR-92a

hsa-miR-106a

hsa-miR-140-5p

hsa-miR-15b

hsa-miR-17

hsa-miR-197

hsa-miR-19b

hsa-miR-221

hsa-miR-28-3p

hsa-miR-30b

hsa-miR-30c

hsa-miR-145

hsa-miR-16

hsa-miR-133a

Table II: microRNAs used to assess the risk of developing aggressive lung cancer (within 1-3 years of biological fluid sampling)

hsa-miR-106a

hsa-miR-660

hsa-miR-140-5p

hsa-miR-486-5p

hsa-miR-16

hsa-miR-197

hsa-miR-22 1

hsa-miR-45 1

hsa-miR-28-3p

Table III: quotients between measured values levels of expression pairs of microRNAs used to assess the risk of contracting lung cancer (within 1-3 years of biological fluid sampling)

Qi = hsa-miR-106a / hsa-miR-660

Q2 = hsa-miR-106a / hsa-miR-451

Q3 = hsa-miR-106a / hsa-miR-320

Q4 = hsa-miR-106a / hsa-miR-92a

Qs = hsa-miR- 140-5p / hsa-miR-320

Qe = hsa-miR- 140-5p / hsa-miR-451

Q7 = hsa-miR- 140-5p / hsa-miR-660

Qg = hsa-miR- 140-5p / hsa-miR-92a

Q9 = hsa-miR-30b / hsa-miR-451

Qio = hsa-miR-30b / hsa-miR-660

Qn = hsa-miR-30b / hsa-miR-92a

Qi2 = hsa-miR-30b / hsa-miR-320

Qi3 = hsa-miR-28-3p / hsa-miR-320

IQ4 = hsa-miR-28-3p / hsa-miR-451

Qi5 = hsa-miR-28-3p / hsa-miR-660

Qi6 = hsa-miR-28-3p / hsa-miR-92a

Qn = hsa-miR- 15b / hsa-miR-451

Qig = hsa-miR- 15b / hsa-miR-660

Qi9 = hsa-miR- 15b / hsa-miR-320

(3⁄4ο = hsa-miR- 15b / hsa-miR-92a

Q2I = hsa-miR- 17 / hsa-miR-451

Q22 = hsa-miR- 17 / hsa-miR-660

Q23 = hsa-miR- 17 / hsa-miR-320

Q24 = hsa-miR- 17 / hsa-miR-92a

Q25 = hsa-miR- 197 / hsa-miR-451

Q26 = hsa-miR- 197 / hsa-miR-92a

Q27 = hsa-miR- 197 / hsa-miR-660

Q2g = hsa-miR- 197 / hsa-miR-320

Q29 = hsa-miR- 19b / hsa-miR-660

Q3O = hsa-miR- 19b / hsa-miR-320

Q3i = hsa-miR-19b / hsa-miR-451

Q32 = hsa-miR-19b / hsa-miR-92a

Q33 = hsa-miR-221 / hsa-miR-660

Q34 = hsa-miR-221 / hsa-miR-451

Q35 = hsa-miR-221 / hsa-miR-320

Q36 = hsa-miR-221 / hsa-miR-92a

Q37 = hsa-miR-145 / hsa-miR-660

Q38 = hsa-miR-145 / hsa-miR-45 1

Q39 = hsa-miR-145 / hsa-miR-320

Q4O <=> hsa-miR-145 / hsa-miR-92a

Q4i = hsa-miR-30c / hsa-miR-451

Q42 = hsa-miR-30c / hsa-miR-660

(3⁄43 = hsa-miR-30c / hsa-miR-320

Q44 = hsa-miR-30c / hsa-miR-92a

Q45 = hsa-miR-133a / hsa-miR-660

Q46 = hsa-miR-133a / hsa-miR-451

Q47 = hsa-miR-133a / hsa-miR-320

Q4g = hsa-miR-133a / hsa-miR-92a

Q49 = hsa-miR-16 / hsa-miR-660

Q5O <=> hsa-miR- 16 / hsa-miR-451

Q5I = hsa-miR-16 / hsa-miR-320

Q52 = hsa-miR-16 / hsa-miR-92a

Table IV: quotients between measured values levels of expression pairs of microRNAs used to assess the risk of developing aggressive lung cancer (within 1-3 years of biological fluid sampling)

Qi = hsa-miR-106a / hsa-miR-660

Q2 = hsa-miR-106a / hsa-miR-451

Q3 = hsa-miR- 106a / hsa-miR-486-5p

Q4 = hsa-miR-106a / hsa-miR-16

Q5 = hsa-miR- 140-5p / hsa-miR-486-5p

Q6 = hsa-miR- 140-5p / hsa-miR-660

Q7 = hsa-miR- 140-5p / hsa-miR-451

Q8 = hsa-miR-140-5p / hsa-miR-16 (3⁄4 = hsa-miR-197 / hsa-miR-451 Qio = hsa-miR-197 / hsa-miR-486-5p Qi 1 = hsa -miR-197 / hsa-miR-660 Qi2 = hsa-miR-197 / hsa-miR-16 Qn = hsa-miR-221 / hsa-miR-45 1 Qu = hsa-miR-221 / hsa-miR-660 Qi5 = hsa-miR-221 / hsa-miR-486-5p Qi6 = hsa-miR-221 / hsa-miR-16 Qn = hsa-miR-28-3p / hsa-miR-45 1 Qi8 = hsa-miR- 28-3p / hsa-miR-486-5p Qi9 <=> hsa-miR-28-3p / hsa-miR-660 Q2O = hsa-miR-28-3p / hsa-miR-16 Table V: microRNAs used for determination presence of current lung cancer in an individual

hsa-miR-106a

hsa-miR-140-3p

hsa-miR-486-5p

hsa-miR-17

hsa-miR-660

hsa-miR-15b

hsa-miR-92a

hsa-miR-451

hsa-miR-19b

hsa-miR-28-3p

hsa-miR-133a

hsa-miR-16

hsa-miR-101

Table VI: MicroRNAs Used to Determine Present Presence of Aggressive Lung Cancer in an Individual

hsa-miR-106a

hsa-miR-142-3p

hsa-miR-148a

hsa-miR-15b

hsa-miR-17

hsa-miR-197

hsa-miR-21

hsa-miR-221

hsa-miR-28-3p

hsa-miR-660

hsa-miR-19b

hsa-miR-486-5p

hsa-miR-126

Table VII: quotients between measured values of microRNA pairs expression levels used to determine the current presence of lung cancer in an individual

Qi = hsa-miR-106a / hsa-miR-140-3p

Q2 = hsa-miR- 106a / hsa-miR- 10 1

<3⁄4 = hsa-miR- 106a / hsa-miR-486-5p

Q4 = hsa-miR- 106a / hsa-miR- 16

Q5 = hsa-miR-106a / hsa-miR-660

(3⁄4 = hsa-miR-106a / hsa-miR-92a

Q7 = hsa-miR- 106a / hsa-miR-45 1

Qs = hsa-miR- 133a / hsa-miR-451

Q9 = hsa-miR- 133a / hsa-miR-92a

Qio = hsa-miR- 133a / hsa-miR- 140-3p

Qn = hsa-miR-133a / hsa-miR-101

Q12 = hsa-miR-133a / hsa-miR-486-5p

Qn = hsa-miR-133a / hsa-miR-660

QH = hsa-miR- 133a / hsa-miR- 16

Qi5 = hsa-miR- 15b / hsa-miR-660

Qi6 = hsa-miR-15b / hsa-miR-92a

Qiv = hsa-miR- 15b / hsa-miR-451

Qis = hsa-miR-15b / hsa-miR-140-3p

Qi9 ~ hsa-miR- 15b / hsa-miR-101

Q2O = hsa-miR- 15b / hsa-miR-486-5p

Q2I = hsa-miR- 15 b / hsa-miR- 16

Q22 <=> hsa-miR- 17 / hsa-miR- 101

(3⁄43 = hsa-miR- 17 / hsa-miR-451

Q24 = hsa-miR- 17 / hsa-miR-486-5p

Q25 = hsa-miR- 17 / hsa-miR-660

Q26 = hsa-miR- 17 / hsa-miR-92a

Q27 = hsa-miR- 17 / hsa-miR- 16

Q28 = hsa-miR- 17 / hsa-miR- 140-3 p

Q29 = hsa-miR- 19b / hsa-miR-45 1

Q30 = hsa-miR- 19b / hsa-miR-660

(3⁄4ι = hsa-miR-19b / hsa-miR-92a

Q32 = hsa-miR-19b / hsa-miR-486-5p

Q33 = hsa-miR-19b / hsa-miR-101

Q34 = hsa-miR-19b / hsa-miR-16

(3⁄45 = hsa-miR-19b / hsa-miR-140-3p

Q36 = hsa-miR-28-3p / hsa-miR-660

(3⁄47 = hsa-miR-28-3p / hsa-miR-92a

Q3S = hsa-miR-28-3p / hsa-miR-451

Q39 = hsa-miR-28-3p / hsa-miR-140-3p

Q4o <=> hsa-miR-28-3p / hsa-miR-486-5p

Q4I = hsa-miR-28-3p / hsa-miR-101

Q42 = hsa-miR-28-3p / hsa-miR-16

Table VIII: quotients between measured values levels of expression pairs of microRNAs used to determine the presence of current aggressive lung cancer in an individual

Qi = hsa-miR-106a / hsa-miR-486-5p

Q2 = hsa-miR-106a / hsa-miR-19b

(3⁄4 = hsa-miR-106a / hsa-miR-660

Q4 = hsa-miR-142-3p / hsa-miR-486-5p

Q5 = hsa-miR-142-3p / hsa-miR-660

Qe = hsa-miR-142-3p / hsa-miR-19b

Q7 = hsa-miR-148a / hsa-miR-486-5p

Qg = hsa-miR-148a / hsa-miR-660

Q9 = hsa-miR- 148a / hsa-miR- 19b

Qio = hsa-miR- 15b / hsa-miR-486-5p

Qn = hsa-miR- 15b / hsa-miR- 19b

Qi2 = hsa-miR- 15b / hsa-miR-660

Qi3 = hsa-miR- 17 / hsa-miR-486-5p

IQ4 = hsa-miR- 17 / hsa-miR-660

Qi5 = hsa-miR- 17 / hsa-miR- 19b

Qi6 = hsa-miR- 197 / hsa-miR-486-5p

Qn = hsa-miR- 197 / hsa-miR-660

Qi8 = hsa-miR-197 / hsa-miR-19b Qi9 = hsa-miR-21 / hsa-miR-486-5p Q2O = hsa-miR-21 / hsa-miR-660 Q2I = hsa-miR-21 / hsa -miR-19b Q22 = hsa-miR-221 / hsa-miR-486-5p Q23 = hsa-miR-221 / hsa-miR-660 Q24 = hsa-miR-221 / hsa-miR-19b Q25 = hsa-miR -28-3p / hsa-miR-486-5p Q26 = hsa-miR-28-3p / hsa-miR-660 Q27 = hsa-miR-28-3p / hsa-miR-19b Q2g = hsa-miR-126 / hsa-miR-486-5p Q29 = hsa-miR-126 / hsa-miR-660 Q30 = hsa-miR-126 / hsa-miR-19b

Claims (13)

CLAIMS 1. In vitro procedure to identify individuals at risk of cancer and / or to determine the presence and / or aggression of a tumor in an individual, said procedure comprising the steps of: measuring, in at least one biological fluid sample previously taken from a subject, a value of the expression level of a plurality of microRNA molecules; calculating a plurality of real quotients determined by making the ratio between the measured values of the expression levels of a predetermined number of pairs of said microRNA molecules; comparing each of said real quotients with a respective control value. A method according to the preceding claim comprising: determining the number or percentage of real quotients which deviate from the respective control value, define as an individual in which a form of tumor is present, an individual for which at least a predetermined number or a predetermined percentage of said real quotients deviate from the respective control value. 3. Process according to claim 2, wherein for each of said calculated quotients a respective control value is associated, represented by the ratio of the expression values for the same microRNAs in a control sample relating to a biological fluid of the same type. Process according to any one of claims 2 or 3, wherein calculating said plurality of real quotients comprises using the expression values of a predetermined number of miRNAs: referred to in Table I and / or Table II and / or Table V and / or Table VI. A method according to any one of claims 2 to 4 wherein calculating said plurality of real quotients comprises determining a predetermined number or a predetermined percentage of quotients between the values of the expression levels, said quotients being selected in the group comprising the quotients of which to Schedule III and / or Schedule IV and / or Schedule VII and / or Schedule VIII. 6. Process according to the previous claim in which determining a predetermined number or a predetermined percentage of quotients involves calculating at least 20% of the real quotients referred to in Table VII and in which it comprises a step of defining as an individual in which a lung cancer an individual for which at least 30% of the predetermined number of real quotients referred to in Table VII that have been calculated deviates from the respective control value. 7. Process according to claim 5 or claim 6 wherein determining a predetermined number or a predetermined percentage of quotients involves calculating at least 20% of the real quotients referred to in Table VIII and in which it comprises a step of defining as an individual in which An aggressive lung tumor is an individual for which 50%, optionally at least 60%, of the real quotients that have been calculated deviate from the respective control value. 8. Process according to any one of claims 5 or 6 or 7 in which determining a predetermined number or a predetermined percentage of quotients involves calculating at least 20% of the real quotients referred to in Table III and in which it comprises a step of defining as an individual at risk of lung cancer, optionally in a period between one and three years from the collection of the biological fluid sample, an individual for whom at least 30%, optionally at least 50%, of the real calculated ratios deviate from the respective value of control quotient. 9. Process according to any one of claims 5 or 6 or 7 or 8 in which determining a predetermined number or a predetermined percentage of quotients provides for calculating at least 30% of the real quotients referred to in Table IV and in which it comprises a step of defining as an individual at risk of aggressive lung cancer, optionally in a period between one and three years from the collection of the biological fluid sample, an individual for which at least 50%, optionally at least 75% of said real calculated ratios deviate from the respective control quotient value. 10. Process according to claim any one of the preceding claims wherein said biological fluid is one selected from the group comprising: whole blood, a fraction of blood, plasma, serum, saliva or bronchial condensate; and / or in which said tumor is a lung tumor selected from the group comprising: small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma (ADC), bronchoalveolar carcinoma (BAC), squamous cell lung cancer (SCC), large cell carcinoma (LC). 11. Process according to any one of the preceding claims, in which said biological fluid sample is from a smoker who, at the time of sampling, highlights lung cancer if subjected to diagnostic imaging methods, in particular shows lesions of dimensions greater than 5mm if subjected to spiral CT. 12. Medical kit for the determination of biomolecular markers present in a sample of human biological fluid, said kit comprising: a platform having a plurality of sites each of which is intended to receive a respective discrete quantity of said biological fluid sample; each of said sites comprising a reagent capable of binding with at least one respective of the microRNAs of Table I and / or Table II and / or Table V and / or Table VI; optionally wherein said reagent includes at least one selected from the group comprising: - a polynucleotide comprising a nucleotide sequence of at least one of the microRNAs of Table I and / or Table II and / or Table V and / or Table VI; - a polynucleotide comprising a nucleotide sequence complementary to that of at least one of the microRNAs of Table I and / or Table II and / or Table V and / or Table VI; - a molecular probe configured to recognize a sequence of at least one of the microRNAs referred to in Table I and / or Table II and / or Table V and / or Table VI. 13. Medical equipment comprising: - a unit defining a seat to receive one or more of the kits referred to in the previous claim, - means for determining the value of the expression level of the microRNAs referred to in Table I and / or Table II and / or Table V and / or Table VI; - means for calculating the values of the real quotients between values of expression levels of microRNA pairs, said quotients being selected from those of Table III and / or of Table IV and / or of Table VII and / or of Table VIII, optionally where the means of determining the expression level value includes one selected in the group: Quantitative Real-time PCR, Microfluidic cards (microfluidic chips), Microarrays (microchips), RT (reverse transcription) - quantitative or semiquantitative PCR, Northern blot, Solution Hybridization, Sequencing.