ES2339524B1 - NEW BIOMARCATOR AS A THERAPEUTIC DIANA IN CANCER DE PULMON. - Google Patents

Abstract

New biomarker as a therapeutic target in lung cancer.

The present invention relates to a prognosis procedure of the evolution of the disease in a subject with lung adenocarcinoma comprising:

The measurement of gene expression of RhoB; or

The measurement of the number of copies of the RhoB gene;

in a sample of said subject with lung adenocarcinoma, characterized in that the detection of overexpression or increase in the number of copies of the RhoB gene in relationship with the expression or number of copies of the same gene in a control sample is indicative of the risk of metastasis, or reappearance or recurrence of neoplastic activity.

The present invention further provides screening procedures for active drugs against lung adenocarcinoma, to determine the efficacy of a pattern of therapeutic treatment in a subject with adenocarcinoma of lung as well as RhoB gene overexpression inhibitors for use as a medicine or for use in the treatment of non-small cell lung cancers (NSCLC).

Description

New biomarker as a therapeutic target in lung cancer.

Field of the Invention

The present invention relates to the area of Cancer diagnosis and therapy. In particular, it refers to the overexpression of the RhoB gene and its clinical impact on cancer of non-small cell lung (NSCLC).

Background of the invention

Lung cancer is the leading cause of cancer-related deaths worldwide. Since it tends to spread, or produce metastases, very early in its evolution, it is a cancer with high lethality and one of the most difficult treatment cancers. Lung cancer can spread to certain organs - particularly the adrenal glands, the liver, the brain, and the bones - the most common organs for metastasis. Despite advances in surgery, chemotherapy and radiotherapy, the survival rate has improved little in the last decade, and long-term survival is very low. It has been established that lung cancer arises as a result of the accumulation of multiple genetic changes in critical genes that control motility, proliferation, differentiation and cell apoptosis. The identification and characterization of these genetic changes that cause the development and progression of lung cancer is of great interest to improve early diagnosis and the development of a novel targeted therapy (Mazieres et al . 2004).

Lung cancers, also known as Bronchogenic carcinomas, are broadly classified into two types: small cell or small cell lung (SCLC) cancers and Non-small or non-small cell lung cancers (NSCLC). This classification is based on the microscopic appearance of the own tumor cells. NSCLCs are the most lung cancers usual, representing approximately 80% of all lung cancers There are three main types of NSCLC that are They call based on the type of cells found in the tumor: adenocarcinomas, squamous cell carcinomas, carcinomas of large cells and mixtures of different types of NSCLC Among lung cancers and particularly NSCLC, the Lung adenocarcinoma is the most frequent, representing a 30-35% of all cases of lung cancer. He Lung adenocarcinoma is defined by the World Organization of the Health (WHO) as "a malignant epithelial tumor with patterns of tubular, acinar, or papillary growth, and / or the production of mucus from tumor cells. "WHO currently recognizes four categories of adenocarcinomas: acinar, papillary, bronchioloalveolar, and solid carcinoma with mucus formation. Most adenocarcinomas appear on the periphery of the lung, and as a result they are frequently asymptomatic in large Part of its evolution. They are often located at the level subpleural and cause pleural retraction and thickening by x-rays

Many studies have examined the potential utility of molecular markers in early diagnosis and targeted therapy. A particularly interesting family of markers are Ras proteins, which regulate important cellular functions that vary from differentiation to cell proliferation. They function as molecular switches, between inactive GDP binding states and active GTP binding states. The ras oncogene plays a fundamental role in the malignant transformation. Ras is mutated resulting in a deficient form of GTPase that leads to its constitutive activation resulting in uncontrolled proliferation in approximately 50% of lung adenocarcinoma. The closely related members of the Ras family, such as small guanosine triphosphatases (GTPases) homologs of Ras (Rho), are also involved in the regulation of a series of cellular processes, such as the organization of the actin cytoskeleton, signaling induced by genotoxic stress, and cell transformation. Recent studies have further confirmed the role of Rho proteins in cancer by demonstrating their involvement in cell transformation, survival, invasion, metastasis, and angiogenesis. In addition, analyzes in human tumors demonstrate the overexpression of several Rho proteins in breast cancers, RhoA in testicular germ cell tumors, and RhoC in pancreatic adenocarcinoma, in melanoma, and in breast cancer (Mazieres et al . 2004) .

While most Rho proteins have proven to have a positive role in proliferation and transformation of malignant tumors, the specific role of RhoB in These processes seem to be more divergent.

On the one hand, Mazieres et al . (2004) showed that RhoB protein was expressed in normal lung and decreased sharply throughout the progression of early stage lung cancer, concluding that loss of RhoB expression occurs very frequently in lung carcinogenesis, reinforcing its potential activity as a tumor suppressor gene.

Sato et al . (2007) analyzed the changes in RhoB NSCLC, demonstrating that RhoB expression is frequently regulated negatively in NSCLCs by multiple mechanisms, and suggesting that RhoB is a tumor suppressor gene in NSCLC.

US7135463 describes the use of RhoB protein and its variants to inhibit growth, migration, invasion, metastasis of cancer cells, cell transformation tumor, and / or to modulate oncogenic signaling, in which the introduction of RhoB directly or indirectly through a nucleic acid sequence encoding RhoB, in a cell transformed tumor or a cancer cell decreases the phosphorylation of Erk and Akt proteins that inhibit the mechanism of PI3-kinase / Akt cell survival and boost the programmed cell death or apoptosis.

Similarly, WO2007011415 describes the use of RhoB protein variants to inhibit growth, metastasis, invasion, migration of cancer cells, the transformation of tumor cells, and / or to modulate the oncogenic signaling. RhoB variant polypeptides are introduced directly or indirectly through a sequence of nucleic acids encoding the RhoB variant polypeptide, in a transformed tumor cell or a cancer cell, in which the RhoB variant polypeptide suppresses tumor growth and boosts apoptosis.

On the other hand, WO2008031910 describes several non-small cell lung cancer cell lines They can experience bone metastases very effectively. In particular, it describes specific cell lines derived from a bronchioalveolar carcinoma cell line (A459) that they share a common gene expression pattern characterized in the overexpression of at least 3, 4, 5, or 6 selected genes between IL11, PITXl, HDAC4, RHOB, ROBO1, and SLC26A2.

Early detection of lung cancer has been a long-term objective pursued by professionals Sanitary and pathologists. Currently, the stage of the disease, based on the TNM system (primary tumor / lymph node / metastasis distant), is the most important individual factor in the Determination of the survival of cancer patients lung. The identification of patients with a higher probability of metastasis, recurrence or recurrence of cancer is a focus Principal in molecular cancer research. As such, and the knowledge of initial molecular changes may have ends predictive, since they frequently reflect the duration of the patient survival or sensitivity to therapy. In this context, there is a need for additional markers that can serve for prognosis in patients with NSCLC, particularly patients with lung adenocarcinoma. In addition, there is a need of validated biomarkers that can be applied directly and Use in clinical oncology.

In an effort to identify and characterize the key target genes involved in the evolution of NSCLC, particularly of lung adenocarcinoma, the inventors have surprisingly found that RhoB plays a critical role in the bone metastasis, by increasing cell motility and boosting the bone colonization RhoB overexpression in adenocarcinoma of lung represents a new and powerful prognostic biomarker potentially useful in the clinical treatment of cancer lung.

Since currently the results of standard treatment of lung adenocarcinoma are generally scarce, there is a need for new therapeutic agents that can prolong or improve the response rate (RR), complete response (CR), partial response (PR), stable disease (SD), time to progression (TTP), progression free survival (PFS), overall survival (GS), or clinical benefit, in patients
affected.

Bisphosphonates are indicated for a wide variety of diseases related to bone metabolism, including osteoporosis, Paget's disease, multiple myeloma, and bone metastases secondary to advanced solid tumors. In patients with tumors, bone metastases can lead to skeletal-related events (SREs), including pathological fractures, compression of the spine, hypercalcemia, bone pain that requires palliative radiotherapy, and orthopedic surgery. These skeletal complications frequently lead to the loss of functional independence and the decrease in the quality of life and, thus, the goal of therapy is to prevent or delay the onset of SREs to preserve independence. Bisphosphonates are the medication of choice for the treatment of osteolytic and osteoblastic lesions associated with bone metastases and have been found to significantly reduce the risk of SREs. (Costa et al . 2007).

In particular, Li et al . (2008) have shown that zoledronic acid, a third-generation nitrogen bisphosphonate, is unable to induce apoptosis, but slows tumor growth and prolongs survival for non-small cell lung cancers.

Turner et al . (2007) discuss the effects of lovastatin, which belongs to the statin group, an HMG-CoA reductase inhibitor on expression, Rho isoform activity, and association with guanine nucleotide dissociation inhibitors, thus providing a molecular mechanism in which statins cause an accumulation of non-pre-piled Rho-GTP.

Hawk et al . (1996) suggested that lovastatin could suppress the formation of lung tumors induced by the chemical carcinogen NNK, possibly in an initial promotional phase, although this suppression did not appear to be related to the presence of mutated K-ras or changes in the expression of K-ras.

WO2004024165 describes a composition Pharmaceutical for the treatment of malignant tumors, in particular multiple myeloma (MM), said composition comprising combination with a bisphosphonate, for example zoledronic acid or a salt or ester, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, for simultaneous use, sequential or separate.

WO2000016809 describes antisense compounds, Compositions and procedures to modulate RhoB expression. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeting nucleic acids that encode RhoB. Procedures for using these compounds for the modulation of RhoB expression and for the treatment of diseases associated with the expression of RhoB.

As part of the exhaustive study on the target Therapeutics of the present invention, ie overexpression of RhoB in NSCLC, such as lung adenocarcinoma, the inventors have surprisingly observed that a combination comprising lovastatin and zoledronic acid significantly improves the overall survival when compared to the administration of Zoledronic acid alone.

In addition, the inventors have also observed that administration of lentivirally transduced lung cells with several rRNAs against RhoB (shRhoB) to immunosuppressed mice it resulted in a decrease in his promising activity, including a sharp reduction in osteolytic lesions and burden of the tumor, compared to the control cells.

As such, the combination comprising lovastatin and zoledronic acid, as well as RNAi agents based on shRhoB, have proven useful in the treatment of NSCLC, such such as lung adenocarcinoma, for example in the slowdown of the progression of bone metastasis.

Summary Description of the Invention

The present invention relates to a prognosis procedure of the evolution of the disease in a subject with lung adenocarcinoma comprising:

The measurement of gene expression of RhoB; or

The measurement of the number of copies of the RhoB gene;

in a sample of said subject with lung adenocarcinoma, characterized in that the detection of overexpression or increase in the number of copies of the RhoB gene in relationship with the expression or number of copies of the same gene in a control sample is indicative of the risk of metastasis, or reappearance or recurrence of neoplastic activity.

In one embodiment, the present invention is refers to a screening procedure for active drugs against lung adenocarcinoma, said procedure comprising:

to)

the RhoB gene expression measurement, or number measurement of copies of the RhoB gene, in a sample with cells from lung adenocarcinoma in the presence of a drug, or measurement of binding affinity between a drug and a polypeptide RhoB;

b)

the comparison of the expression or measured copy numbers of the gene from RhoB, or the binding affinity of step a) to a sample of control;

characterized in that an expression or number of reduced copies of the RhoB gene, or a higher binding affinity in the step a), in relation to the expression or the number of copies of the same gene or the binding affinity of the same polypeptide in a control sample is indicative of the activity of the drug against lung adenocarcinoma.

In a further embodiment, the present invention relates to a method for determining the effectiveness of a therapeutic treatment regimen in a subject with lung adenocarcinoma, said procedure comprising

to)

the RhoB gene expression measurement, or number measurement of copies of the RhoB gene, in a sample with cells from lung adenocarcinoma of said subject, thereby generating a initial level;

b)

the RhoB gene expression measurement, or number measurement of copies of the RhoB gene, in a second sample with cells from lung adenocarcinoma of the same subject at a time after the administration of the treatment regimen, obtaining from this mode a test level; Y

C)

the comparison of initial and test levels;

characterized in that an expression or number of reduced copies of the RhoB gene at the test level in relation to with the initial level it is indicative that the treatment regimen is effective in the subject.

Inhibitors of the overexpression of the RhoB gene for use as a medicine or for its use in the treatment of NSCLC. Particularly, these inhibitors are selected from:

?

 an antibody or fragment of same that specifically binds to a RhoB protein, or a polypeptide comprising a RhoB binding region;

?

 a mimetic peptide of Rhob polypeptide;

?

 a nucleic acid inhibitor of Rhob gene selected from an antisense oligonucleotide, a ribozyme, and an RNAi agent selected from RNAds and shRNA; or

?

 a combination that comprises lovastatin and zoledronic acid, and / or pharmaceutically salts Acceptable of it.

More particularly, a molecule is provided of siRNA that has at least 70% homology with SEQ ID No. 3, preferably in the form of a retroviral vector, for use in gene therapy of adenocarcinoma of the lung. Too particularly, the combination comprising lovastatin and zoledronic acid, and / or pharmaceutically salts acceptable thereof for use in the treatment of lung adenocarcinoma.

Description of the drawings Figure 1 Isolation of metastatic populations towards the bone

A549 adenocarcinoma cells were inoculated by intracardiac route in immunosuppressed mice. After the radiographic detection of bone metastasis, those were isolated subpopulations, expanded into a crop, and returned to inoculate. This procedure was repeated until the highly metastatic subpopulations M1M1, M1M4, and M1M3. These they returned to inoculate in other groups of mice, as well as the parental population His metastatic ability was assessed by microradiography image analysis of the long bones, and Survival curves free metastasis of Kaplan-Meier

Figure 2 Identification of RhoB in metastatic subpopulations

Through expression microarrays, it was identified RhoB as one of the overexpressed genes in all metastatic subpopulations. Overexpression was validated by RT-PCR and by Western blot analysis.

Figure 3 Preparation of RhoB inhibitor in cancer cells lung

to. Preparation of RhoB inhibition by a lentiviral vector with rRNA specific for RhoB. For the gene Overexpressed a retroviral vector was used.

b. Effect on in vitro proliferation of the RhoB inhibitor and the negative control vector ("scramble"). No changes in proliferation were observed in vitro by MTT between the RhoB inhibitor and the scramble vector.

Figure 4 RhoB inhibition decreases metastasis in vivo

Cells were inoculated with rRNA against RhoB and with a control vector ("Scramble"). After 35 days after inoculation, a significant decrease in area was observed radiographic by image analysis. In addition, it was verified decreased tumor burden by bioluminescence.

Figure 5 Survival rate curves for patients with levels RhoB highs and lows

An immunohistochemistry of biopsies of patients diagnosed with adenocarcinoma with an antibody specific against RhoB.

Figure 6 Pharmacological effect of a combination of lovastatin and zoledronic acid in patients with adenocarcinoma of the lung and cells treated in vitro

A. Vehicle administration guideline, lovastatin (Lov), zoledronic acid (ZA), and a combination of lovastatin and zoledronic acid (Lov + ZA).

B. Overall survival curves at 4 0 days Kaplan-Meier curves showing survival global 40 days after cell inoculation of the different treated groups: Vehicle (control), Lovastatin (Lov) (10 mg / Kg / d), Zoledronic acid (ZA) (3 µg / Kg / d), or a combination (Lov + ZA). * p <0.05, *** p <0.001 vs control; \ dagger p <0.01 vs ZA (Mantel-Cox test). There were no differences between Control and Lovastatin groups.

C. Treatment with A549 TM M1M1 in culture. MTT cells of A54 9 TM M1M1 24 h after the different Pharmacological treatments: vehicle (Control); Lovastatin (Lov) 1  µM; Zoledronic acid (ZA) 10 µM or combination (Lov + ZA) (Lov 1 µM + 10 µM ZA) diluted in culture medium. The seeding density 10,000 cls / ml; 72 h treatment after sowing in 96-well culture plates. ** p <0.001 vs. control; \ dagger p <0.001 vs. Lov or ZA (ANOVA one-way ANOVA followed by Sheffé test). Values, means +/- SEM.

Figure 7

TM M1M1 metastatic cells of A549 adenocarcinoma for 4 days with a vehicle (Control); Lovastatin (Lov) 1 µM and 0.5 µM; zoledronic acid (ZA) 10  µM and 5 µM; and the combinations Lov 1 µM + ZA 10 µM; and 0.5 µM + 5 µM ZA diluted in medium culture. The inoculation lasted 2 days and the density was 2500 cells / well. The photographs taken of the crops that they followed the different treatments mentioned show the effectiveness of the combinations Lov 1 µM + ZA 10 µM and Lov 0.5 µM + 5 µM ZA.

Figure 8

After 24 hours the viability was measured TM M1M1 cell metastatic adenocarcinoma A54 9 in presence of different pharmacological treatments. The treatments were with vehicle (Control), lovastatin (Lov) 1 µM, zoledronic acid (ZA) 10 µM, and the combination Lov 1 µM + 10 µM ZA (Lov + ZA) diluted in culture medium, and applied for 72 h after sowing on culture plates of 96 wells Cell viability was determined by measuring the MTT concentration, a soluble yellow tetrazolium dye in water that transforms into water-soluble violet formazan only in living cell mitochondria. After the solubilization of the resulting formazan crystals with isopropanol, the absorbance of the solution was monitored at 540 nm. The values correlate directly with the number of living cells that remain in the culture when the incubation is complete. Density of inoculation 1000 cells / well; ** p <0.001 vs. control; \ dagger p <0.001 vs. Lov or ZA (one-way ANOVA followed subsequently Tamhane test and contrast). Values, means +/- SEM

Figure 9

TM M1M1 metastatic cells of adenocarcinoma A54 9 for 24 hours with: vehicle (Control); 1 µM Lovastatin (Lov); 10 µM zoledronic acid (ZA); and the combination 1 µM + 10 µM ZA (Lov + ZA) diluted in medium of cultivation The test was performed in a 96-well plate; the cells were seeded (1000 cells / well) and maintained for 72 h after treatment. The activity of caspase-3 by testing with caspasa-Glo 3/7 luminescent. Rehearsals are They performed on 12 replicates for each treatment. ** p <0.001 vs. control, Lov or ZA (one-way ANOVA followed subsequently by Tamhane test and contrast). Values, means +/- SEM. The results show that the combination of lovastatin and zoledronic acid increases caspase activity 3/7.

Figure 10

The human cancer cell line of H727 non-small cell lung derived from carcinoid tumor for 24 h with a vehicle (Control); 1 µM lovastatin (Lov); acid zoledronic (ZA) 10 µM; and the combination Lov 1 µM + ZA 10 µM diluted in culture medium. The incubation lasted 6 days and the density was 5000 cells / well. The photographs taken of the crops that undergo the different previous treatments demonstrate the effectiveness of the combination Lov 1 µM + ZA 10 µM.

The cell viability with MTT of the same human cell line H727 in the presence of them treatments The cells were seeded and treated for 24 h with vehicle (Control); Lovastatin (Lov) 1 µM; Acid zoledronic (ZA) 10 µM or the combination Lov 1 µM + ZA 10 µM (Lov + ZA) diluted in culture medium. Planting density 1000 cells / well; ** p <0.001 vs. control; \ dagger p < 0.001 vs. Lov or ZA (one-way ANOVA followed subsequently by test Tamhane and contrast). Values, means +/- SEM.

Figure 11

The H727 cell line was treated for 24 h with a vehicle (Control); 5 µM lovastatin (Lov); acid zoledronic (ZA) 5 µM; and the combination Lov 5 µM + ZA 5 µM diluted in culture medium. The incubation lasted 6 days and the density was 5000 cells / well. The photographs taken of the crops that undergo the different previous treatments demonstrate the effectiveness of the combination Lov 5 µM + ZA 5 µM.

The cell viability with MTT of the same human cell line of non-small cell lung cancer H727 derived from carcinoid tumor in their presence treatments The cells were seeded and treated for 24 h with vehicle (Control); 5 µM Lovastatin (Lov); Zoledronic acid (ZA) 5 µM or the combination Lov 5 µM + ZA 5 µM (Lov + ZA) diluted in culture medium. 1000 planting density cells / well; ** p <0.001 vs. control; \ dagger p <0.001 vs. Lov or ZA (one-way ANOVA followed subsequently by test Tamhane and contrast). Values, means +/- SEM.

Figure 12

The H727 cell line was treated for 72 h with a vehicle (Control); 5 µM lovastatin (Lov); acid zoledronic (ZA) 5 µM; and the combination Lov 5 µM + ZA 5 µM diluted in culture medium. The incubation lasted 4 days and the density was 5000 cells / well. The photographs taken of the crops that undergo the different previous treatments demonstrate the effectiveness of the combination Lov 5 µM + ZA 5 µM.

The cell viability with MTT of the same H727 cell line in the presence of the same treatments. Cells were seeded and treated for 72 hours with vehicle (Control); 5 µM Lovastatin (Lov); Zoledronic acid (ZA) 5 µM or the combination Lov 5 µM + ZA 5 µM (Lov + ZA) diluted in culture medium. 1000 planting density cells / well; ** p <0.001 vs. control; \ dagger p <0.001 vs. Lov or ZA (one-way ANOVA followed subsequently by test Tamhane and contrast). Values, means +/- SEM.

Figure 13

The human cancer cell line of non-small cell H4 60 derived from a cell carcinoma large for 24 hours with a vehicle (Control); Lovastatin (Lov) 1 µM; 10 µM zoledronic acid (ZA); and the combination Lov 1 µM + 10 µM ZA diluted in culture medium. Incubation It lasted 5 days and the density was 1500 cells / well. The photographs taken of the crops experienced by the different Previous treatments demonstrate the effectiveness of the Lov combination 1 µM + 10 µM ZA.

The cell viability with MTT of the same H460 cell line in the presence of the same treatments. Cells were seeded and treated for 24 hours with vehicle (Control); Lovastatin (Lov) 1 µM; Zoledronic acid (ZA) 10 µM or the combination Lov 1 µM + 10 µM ZA (Lov + ZA) diluted in culture medium. 1000 planting density cells / well; ** p <0.001 vs. control; \ dagger p <0.001 vs. Lov or ZA (one-way ANOVA followed subsequently by test Tamhane and contrast). Values, means +/- SEM.

Figure 14

The H460 cell line was treated for 24 h with a vehicle (Control); 5 µM lovastatin (Lov); acid zoledronic (ZA) 5 µM; and the combination Lov 5 µM + ZA 5 µM diluted in culture medium. The incubation lasted 5 days and the density was 1500 cells / well. The photographs taken of the crops that undergo the different previous treatments demonstrate the effectiveness of the combination Lov 5 µM + ZA 5 µM.

The cell viability with MTT of the same H460 cell line in the presence of the same treatments. Cells were seeded and treated for 24 hours with vehicle (Control); 5 µM Lovastatin (Lov); Zoledronic acid (ZA) 5 µM or the combination Lov 5 µM + ZA 5 µM (Lov + ZA) diluted in culture medium. 1000 planting density cells / well; ** p <0.001 vs. control; \ dagger p <0.001 vs. Lov or ZA (one-way ANOVA followed subsequently by test Tamhane and contrast). Values, means +/- SEM.

Figure 15

The H460 cell line was treated for 72 h with a vehicle (Control); 5 µM lovastatin (Lov); acid zoledronic (ZA) 5 µM; and the combination Lov 5 µM + ZA 5 µM diluted in culture medium. The incubation lasted 3 days and the density was 1500 cells / well. The photographs taken of the crops that undergo the different previous treatments demonstrate the effectiveness of the combination Lov 5 µM + ZA 5 µM.

The cell viability with MTT of the same H460 cell line in the presence of the same treatments. Cells were seeded and treated for 72 hours with vehicle (Control); 5 µM Lovastatin (Lov); Zoledronic acid (ZA) 5 µM or the combination Lov 5 µM + ZA 5 µM (Lov + ZA) diluted in culture medium. 1000 planting density cells / well; ** p <0.001 vs. control; \ dagger p <0.001 vs. Lov or ZA (one-way ANOVA followed subsequently by test Tamhane and contrast). Values, means +/- SEM.

Description of the invention

The present invention relates to a prognosis procedure of the evolution of the disease in a subject with lung adenocarcinoma comprising:

to)

the RhoB gene expression measurement; or

b)

the measurement of the number of copies of the RhoB gene;

in a sample of said subject with lung adenocarcinoma, characterized in that the detection of overexpression or increase in the number of copies of the RhoB gene in relation to the expression or number of copies of the same gene in a control sample is indicative of risk for metastasis, or reappearance or recurrence of neoplastic activity.

       \ global \ parskip0.900000 \ baselineskip
    

The term "prognosis of disease progression" refers to providing information regarding the impact of the presence of lung adenocarcinoma, for example, as determined by the methods of the present invention, in the future health of a subject, or in other words, the prognosis of the evolution of the disease provides a prediction of how the disease will progress in a subject and whether there is a possibility of recovery. As such, the methods of the present invention, as will be described below, can be used to determine the progression of cancer; for example, from lung carcinoma in situ to metastasis, in particular to bone, or recurrence or recurrence of neoplastic activity.

In an embodiment of the present invention, the prognosis of the evolution of the disease includes the determination of a clinical outcome selected from the group that consists of response speed (RR), complete response (CR), partial response (PR), stable disease (SD), time to progression (TTP), overall survival (OS), and clinical benefit, comprising complete response (CR), partial response (PR), and stable disease (SD).

The term "response speed (RR)" is refers to the percentage of patients whose cancer decreases or It disappears after treatment.

The term "complete response (CR)" is refers to the disappearance of all signs of cancer in treatment response This does not always mean that cancer is I have healed. It is also called complete remission.

The term "partial response (PR)" is refers to a decrease in the size of a tumor, or in the extent of cancer in the body, in response to treatment. I also know called partial remission.

The term "stable disease (SD)" is refers to a cancer that does not grow or decrease in extent or gravity.

The term "time to progression (TTP) "is a synonym for" progression-free survival (PFS) "and refers to a measurement of time after it has diagnosed or treated a disease, until the disease It starts to get worse. Here the measurement for each patient is equal to time elapsed since the beginning of treatment in a patient in a test - as defined in the test protocol - up to the detection of a tumor progression - as defined in the test protocol - or the occurrence of any fatality, which be first. If patient observation was stopped (for example, at the end of the study) after a period and no event, then this observation time is called "censored."

The term "global survival (OS)" is a synonym for "time to death (TTD)" or "rate of survival "and refers to the percentage of subjects in a study that have survived for a defined period of time. This percentage is usually indicated along with the period of time from the date of diagnosis or treatment. Habitually, overall survival is measured from the day of surgery until the death related to the tumor. Here, the measurement for each patient is equal to the time elapsed since the beginning of the treatment of a patient in a test - as defined in the protocol - until the appearance of any fatality. If it stopped patient observation, for example, at the end of the study, after a period t and the patient survived this time, then this observation time t is called "censored."

The term "subject" refers to a organism affected by lung cancer, particularly cancer of non-small cell lung, more particularly adenocarcinoma of lung. This subject may be a mammal, preferably a human, rat, mouse, or a non-human primate. Usually the subject He is a patient.

The term "metastasis" refers to the movement or expansion of cancer cells of an organ or tissue to another. Cancer cells normally expand through of the bloodstream or lymphatic system. The term "metastasis" also refers to the formation of foci of secondary tumors in progressive growth in discontinuous points to the primary lesion. The metastatic process is a mechanism with multiple stages in which a metastatic cancer cell escapes of the primary tumor, enters the circulation, invades a point of distant tissue and develops a macroscopic tumor at the site Diana.

The term "neoplastic activity" is refers to an unregulated or abnormally regulated cell proliferation regulated and the process of angiogenesis or the formation of new vasculature that supports a neoplasm in the microenvironment Endothelial around the neoplasm. The term "neoplasm" is refers to tumor cells, cells that have a proliferation unregulated or abnormally regulated as a result of the instability or genetic mutation, and an endothelium in which endothelial cells have an unregulated proliferation or abnormally regulated as a result of a pathogenic state.

The term "sample", as used in the present invention, it refers to a composition that obtains or derives from a subject of interest that contain an entity cellular and / or other molecular entity to be characterized and / or be identified based on, for example, physical characteristics, biochemical, chemical and / or physiological. Any suitable sample in which the overexpression of the RhoB gene or the number of copies thereof is included in the scope of the present invention

In one embodiment, the sample is a material biological obtained from a subject, and can be any sample Organic tissue sample, cell sample, body fluid, body fluid precipitate, or an isolated wash sample from a subject who has lung cancer or at the risk of cancer of lung (for example, based on family history or history staff, such as chronic smoker). A sample can be obtained from a subject in any of the ways used in devices clinicians to obtain a sample comprising the cell or acid required nucleic, ie DNA or RNA. For example, the samples are they can get from recent surgical samples, frozen or paraffin samples or biopsies of an organ or tissue which comprise the appropriate nucleic acid or cell to be tested. The suitable samples used in procedures for detection or Cancer prognosis can be obtained from a sample of breast, ducts or lobes of the breast tissues, sample of gastrointestinal tract (for example, a biopsy of a polyp), a liver sample, a sample of pancreatic tissue, a sample of bone marrow, a skin sample, a lymph node sample, a kidney sample, a lung sample, a muscle sample, a bone sample, a brain sample, or the like. The sample it can be derived from a biological fluid, for example, blood, marrow bone, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymphatic fluid, serum, plasma, pancreatic juice, urine, kilo, stool, ejaculate, sputum, aspirated nipples, fluid from the ducts, saliva, samples obtained with duct swabs organic, colon wash samples, and brush samples. The tissues and body fluids can be collected using any of the procedures known in the art. If appropriate, the Sample can be obtained by using a procedure of aspiration.

Among the usual samples for detection or prognosis of lung adenocarcinoma are included, without limitation, cells or tissue (for example, from a biopsy or autopsy), solid lung tumors, sputum, cough, wash bronchoalveolar, bronchial brushes, buccal mucosa, blood peripheral, whole blood, red blood cell concentrates, platelet concentrates, leukocyte concentrates, proteins red blood cells, blood plasma, platelet rich plasma, a plasma concentrate, a precipitate of any fraction of the plasma, a supernatant of any fraction of the plasma, fractions of blood plasma proteins, proteins or other components purified or partially purified blood, serum, tissue or fine needle biopsy samples and pleural fluid, etc. isolated of a subject with lung cancer, or any other tumor, or any other extract thereof, obtained from a test subject, Healthy volunteer or experimental animal.

In some embodiments, it may be desirable. separate lung cancer cells from cells that are not from lung cancer in a sample. In some embodiments, it may be desirable not to separate lung cancer cells from cells They are not from lung cancer in a sample. In some embodiments, a sample may be substantially free of Tumor cells, for example, contain less than 1, 5, 10, 20, 30, 40, or 50% of tumor cells compared to non-cells tumor in the sample. In some embodiments, the cells do not Tumors can be epithelial cells or lymphocytes. In the case of evaluate the prognosis of a subject with lung cancer, is it is preferable to use a sample that contains the cancer cell of lung. The sample can be purified cells from a tissue. In addition, biological samples can be obtained from a subject or subject at different time points, including before, during and / or after a treatment.

A sample may also include, without limitation, products produced in cell cultures by cells normal or transformed, for example, through DNA technology recombinant or monoclonal antibodies. A sample can also be a cell or a cell line created in conditions Experiments that are not isolated directly from a subject. A Sample can also be cell free, derived or synthesized artificially A sample can be from a cell or tissue known to be cancerous, suspected of being cancerous, or to be Believes that it is not cancerous (for example, normal or control).

The term "expression" of the RhoB gene is refers to the process of converting coded genetic information in the RhoB gene in RNA, for example, mRNA, rRNA, tRNA, or mRNA, a through gene transcription, that is, through action enzymatic RNA polymerase, and for genes that encode proteins, in the protein through the translation of mature mRNA. Gene expression can be regulated in many stages of the process. "Positive regulation" or "activation" refers to the regulation that increases the production of expression products gene, that is, RNA or protein, while "regulation negative "or" repression "refers to the regulation that Decrease production Molecules, for example, factors of transcription, which are involved in positive regulation or the negative regulation are often called "activators" and "repressors", respectively.

RhoB gene expression can be measured at protein level, mRNA level, or cDNA level.

Protein level measurement

RhoB derived protein expression can be measured using any of the immunoassays available by the art. Examples of immunoassays useful in determining the level of protein expression include, but are not limited to, immunoprecipitation, radioimmunoassay, Western blotting, immunofluorescent assay, enzymatic immunoassay, chemiluminescent assay, immunohistochemical assay, and immunoabsorbent assay. enzyme bound (ELISA). In addition, the above immunoassays can be used in combination, such as immunoprecipitation followed by Western blotting. The above procedures are described in Principies and Practice of Immunoassay , Price and Newman, eds., Stochton Press, last ed. Such assays may be direct, indirect, competitive, or non-competitive immunoassays as described by the technique (Oelbrick, N. (1984) J. Clin. Chem. Clin. Biochem ., 22: 895-904). The protein to be analyzed by said procedures can be obtained directly from the sample. Alternatively, the protein can be obtained as a crude lysate or can be further purified by methods known to those skilled in the art, including immunoaffinity chromatography using antibodies to the RhoB protein (Sambrook, J. et al (2001) in "Molecular Cloning: A Laboratory Manual ", Cold Spring Harbor Press, Plainview, NY). Alternatively, the levels
RhoB protein can be detected by immunohistochemistry of fixed or frozen tumor sections.

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For the detection of RhoB protein by immunoassay, polyclonal or monoclonal anti-RhoB antibodies can be used. If polyclonal antibodies are desired, serum containing polyclonal antibodies to the RhoB protein can be used or the polyclonal antibodies can be purified from antigens present in the serum by immunoaffinity chromatography. Alternatively, monoclonal antibodies directed against RhoB can be easily produced by one skilled in the art. The methods of producing monoclonal or polyclonal antibodies are known to one skilled in the art (Goding, JW (1996) Monoclonal antibodies: Principies and Practice, Plodermic Press, Inc., NY, NY, pages 56-97; Hurn, BAL et al . (1980) Meth. Enzymol. , 70: 104-141).

Suitable immunogens that can be used to produce polyclonal or monoclonal antibodies include Cellular used prepared from cells transfected with a recombinant RhoB protein, a partially or substantially purified recombinant or native RhoB protein, or peptides derived from the amino acid sequence of RhoB. Vectors and their use in the production of recombinant proteins are known to those skilled in the art (Sambrook, J. et al . (2001) in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview, NY).

Antibodies or antigen binding fragments can also be produced by genetic engineering. The technology for the expression of both heavy chain and light chain genes in E. coli is the subject of PCT patent applications; Publication number WO 901443, WO 901443, and WO 9014424 and in Huse et al . (1989) Science , 246: 1275-1281.

Alternatively, the antibodies anti-RhoB can be induced by administration of anti-idiotype antibodies as immunogens. From practical way, an anti-RhoB antibody is used purified to induce an anti-idiotype antibody in an animal Guest.

Alternatively to immunoassays, antibodies can be used in situ to detect RhoB protein in the sample. The antibodies can be used in direct or indirect immunofluorescence. Alternatively, the RhoB protein can be detected in situ by the use of radiolabeled anti-RhoB antibody or by the use of an unlabeled anti-RhoB antibody followed by a second radiolabeled antibody reactive to the anti-RhoB antibody.

Antibodies can also be used to immunoprecipitate RhoB protein from a mixture of proteins The use of immunoprecipitation as a sensitive technique and specific to detect and quantify target antigens in mixtures of Protein is well known to those skilled in the art (see, Molecular Cloning, A Laboratory Manual, 2d Edition, (2001) Cold Spring Harbor Press).

Antibodies can also be fixed to solid supports for use in the isolation of Rhob protein by immunoaffinity chromatography. The techniques for immunoaffinity chromatography are well known in the art (Harlow, E. and Lañe, D. (1888) "Antibodies: A Laboratory Manual ", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and include techniques for attaching antibodies to solid supports, of way they maintain their immunoselective activity; the techniques used may be those in which the antibodies are adsorb to the support, as well as those in which the antibodies They are covalently attached to the support.

An antibody suitable for the measurement of Rho B gene expression at the protein level is Rho B (119) sc-180 commercially available from Santa Cruz Biotechnology, Inc. Rho B (119) is a polyclonal antibody of affinity purified rabbit developed against a peptide that maps in an internal Rho B region of human origin.

The antibodies described above and the Binding fragments thereof can be supplied in a kit of useful diagnosis for the detection of alterations in expression Rhob protein.

RNA level measurement

RhoB gene expression can be controlled at the level of messenger RNA (mRNA) according to known procedures in the technique The procedures that use hybridization of RhoB transcript nucleic acid probes, including the microarray technology, gel visualization, and transfer by Northern, they can be used to measure the presence and / or RhoB mRNA level. MRNA can also be evaluated using amplification techniques, such as RT-PCR or Real-time quantitative RT-PCR.

The hybridization analysis, which is based on the specificity of nucleotide interactions, uses oligonucleotides or cDNA to selectively identify or capture DNA or mRNA from the specific sequence composition. The amount of mRNA or cDNA hybridized to a capture sequence known can be determined quantitatively or qualitatively, thus providing information about the representation Relative RhoB protein. You can also perform the hybridization to arrays or chips of both DNA, cDNA, oligonucleotides or RNA, where these can be produced according to any procedure suitable known by technique. For example, the procedures of Production of large oligonucleotide arrays are described in US5,134,854, and US5,445,934 using directed synthesis techniques by light Using a computer controlled system, it converts a heterogeneous array of monomers, through simultaneous coupling in a number of reaction sites, in a heterogeneous array of polymers.

Alternatively, microarrays are generated by deposition of presynthesized oligonucleotides on a solid substrate, for example as described in WO95 / 35505.

Sequence-based procedures are an alternative. These procedures began with the use of expressed sequence markers (ESTs), and now include procedures based on short markers, such as analysis in series of gene expression (SAGE) and MPSS (Massively Parallel Signature Sequencing). As such, mRNA expression levels in A test sample can be determined by generating a library of ESTs from said test sample. The enumeration of the relative representation of ESTs in the library can be used to approximate the relative representation of a gene transcript in the starting sample. The results of the EST analysis of a test sample can then be compared with the EST analysis of a reference sample comprising control cells to determine expression levels relative of a polynucleotide encoding RhoB.

SAGE (Velculescu et al ., Science (1995) 270: 484) involves the isolation of unique short sequence markers from a specific location in each transcript. Sequence markers are concatenated, cloned and sequenced. The frequency of particular transcripts in the starting test sample is reflected by the number of times the associated sequence marker is found in the sequence population.

MPSS, described by Brenner et al ., Nature Biotechnology 18: 630-634 (2000), is a sequencing strategy that combines non-gel-based sequencing with in vitro cloning of millions of templates in separate 5 µm microspheres. diameter. First, a DNA template microsphere library is constructed by in vitro cloning. This is followed by the assembly of a flat array of the microspheres containing templates in a high density flow cells (usually greater than 3 x 10 6 microspheres / cm 2). The free ends of the cloned templates in each microsphere are analyzed simultaneously, using a fluorescence-based sequencing procedure that does not require separation of DNA fragments. This technique allows the efficient identification and isolation of many hundreds of thousands of individual sequences, the generation of a "library" of small RNAs. The abundance or frequency of occurrence of each different sequence of a "library" of small RNAs is indicative of the amount in the original sample from which the RNA was obtained. In addition, by comparing the sequences, which are usually 17-20 nucleotides in length, with a genomic DNA database, it is possible to determine the points in the DNA that act as the origin for small RNAs. Comparisons with genome annotations, cDNA databases, and other data can often be used to identify the largest RNA precursors of small RNAs. More significantly, MPSS provides the ability to direct the biology of small RNA to a broad genome scale.

CDNA level measurement

RhoB gene expression can also be control at the level of cDNA. RhoB gene expression levels are determined using the polymerase chain reaction of reverse transcriptase (RT-PCR). The RT-PCR is a well known technique in the sector that is based on the fact that the enzyme reverse transcriptase transcribes so reverse the mRNA to form cDNA, which can then be amplify in a standard PCR reaction. The protocols and kits for carry out the RT-PCR are well known by the subject matter experts and are commercially available.

RT-PCR can be carried out in a non-quantitative way. Said point RT-PCR final measures changes in expression levels using three Different procedures: relative, competitive and comparative. These traditional procedures are well known in the art, for example, visualization of the amplicon in an electrophoresis in gel.

In an alternative embodiment, the RT-PCR is carried out in real time and in a manner quantitative Real-time PCR is a technique used to monitor the progress of a real-time PCR reaction. The same time, a relatively small amount of PCR product (DNA, cDNA or RNA). Real-time PCR is based on the detection of fluorescence produced by a molecule reporter that increases as the reaction progresses. This occurs due to the accumulation of the PCR product with each cycle amplification These fluorescent reporter molecules include fluorochromes that bind to double stranded DNA or probes of specific sequence

Quantitative real-time RT-PCT or real-time PCR has been described extensively in the literature (see, Gibson et al for an initial example of the technique) and a number of techniques are possible to control the PCR product. These techniques include the use of hydrolytic probes (Taqman®), hairpin probes (Molecular Beacons), pairs of probes for the FRET technique (LightCycler), primers incorporating a hairpin probe (Scorpion), system Plexor ™, hairpin primers (Amplifluor), primers incorporating complementary sequences of DNAzymes that divide an reporter substrate included in the reaction mixture (DzyNA), or fluorescent dyes (SYBR Green etc.). All these probes, primers, or fluorochromes are commercially available and well characterized.

In one embodiment, the present invention provides primers with SEQ ID NO 4-6 for Perform real-time RT-PCR or PCR. The primers with SEQ ID NO 4 and 5 are particularly suitable for use in the expression profile of the RhoB gene using RT-PCR

one

As mentioned herein invention, real-time quantitative RT-PCR or Real-time PCR produces a fluorescence reading that can be Control continuously. The real time techniques are advantageous because they keep the reaction in a "single tube". This means that there is no need for further analysis. to get results, which leads to more results quickly. In addition, keeping the reaction in a "unique" medium tube "reduces the risk of cross contamination and allows a quantitative output from the procedures herein invention. This can be particularly important in the clinical arrangement of the present invention.

The recognition of multiple alterations of DNA can increase the effective identification of cancer. Probes allow multiple types of DNA to be measured in the same sample (Multiplex PCR), since fluorescent dyes with different emission spectra can be attached to different probes. PCR Multiplex allows internal controls to be co-amplified. These hybridization probes allow an impossible level of discrimination to obtain with SYBR Green, since they will only hybridize to true targets in a PCR and not to primer-dimers or others non-specific products

Variants of the basic PCR technique such as nested PCR, and the like are also included within the scope of the present invention. Examples include isothermal amplification techniques such as NASBA, 3SR, TMA, and triamplification, all known in the industry and commercially available. Other suitable amplification procedures include ligase chain reaction (CSF) (Barringer et al , 1990), Methylation Specific Multiple Ligand-dependent Probe Amplification assay (MS-MLPA) (Nygren et al ., Nucleic Acids Res 2005; 33 (14) e128; www.mlpa.com), selective amplification of target polynucleotide sequences (US6,410,276), polymerase chain reaction with priming with consensus sequences (US4,437,975), chain reaction of polymerase with arbitrary priming (WO90 / 06995), pyrosequencing, and amplification by shear displacement (WO2004 / 067726).

Gene expression in a test sample It can also be analyzed using the methodology of "differential display" (DD). This family of techniques is based in the random amplification of cDNA fragments generated by restriction digestion, where the cDNA fragments are used as unique identifiers of genes, coupled with the information about the length of the fragment or the location of the fragment in the expressed gene. The relative representation of a gene expressed in a sample can then be estimated based on the relative representation of the fragment associated with the gene that is in the group of all possible fragments. The procedures and Compositions for carrying out DD are known in the art, see, for example, US5,776,683; and US5,807,680.

The sequences of genomic DNA, protein, mRNA, and cDNA of the RhoB gene, or a region thereof, are provided. then. In the same way, the primers and probes described in the present invention they are provided as SEQ ID NO. 4-6.

The nucleotide sequence of genomic DNA from Human RhoB (NC_000002.10) (5 '\ rightarrow3', 2367 bp) is provides in the following sequence SEQ ID No. 1:

2

3

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The amino acid sequence of RhoB protein Human (NP_004031.1) (196 aa) is provided in the following sequence SEQ ID No. 2:

4

The nucleotide sequence of RhoB mRNA Human (NM_004040.2) (5 '\ rightarrow3', 2384 bp) is provided in The following sequence SEQ ID No. 7:

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5

6

The nucleotide sequence of the RhoB cDNA Human (CCDS1699.1) (5 '\ rightarrow3', 591 bp) is provided in the following sequence SEQ ID No. 8:

7

The sequences of genomic DNA, protein, mRNA, CDNA, primers, and probes presented in the present invention they also comprise those sequences that have at least one 70% homology with respect to the sequences in SEQ ID NO. 1-8, preferably at least 80%, and more preferably at least 90%, 95%, 96%, 97%, 98%, 99% of homology Alternatively, the forms generated by splicing alternatively, they are also within the scope of this invention.

The term "homology" refers to the level of similarity between the nucleic acid or amino acid sequences in terms of percentage of positional identity of nucleotides or amino acids, respectively, that is, the similarity or identity of the sequences. The percentage of homology between two sequences can be calculated according to the FASTA or BLAST algorithms (Altschul et al . "Basic local alignment search tool" J. Mol. Biol. 1990, 215, 403-410, http: //www.ncbi. nlm.nih.gov/blast; Lipman et al . "Rapid and sensitive protein similarity searches". Science 1985, 227, 1435-1441. http://www.ebi.ac.uk), which are incorporated into the present invention by reference

Any other suitable technique to measure the Rhob gene expression at the level of protein, mRNA, or cDNA as well It is within the scope of the present invention.

In an alternative embodiment of the present invention, the prognosis procedure of the evolution of the disease in a subject with lung adenocarcinoma comprises the measurement of the number of copies of the RhoB gene.

Measurement of RhoB gene copy number

One methodology for determining the copy number of the RhoB gene in a sample is in situ hybridization, for example, fluorescence in situ hybridization (FISH) (see, Angerer, 1987 Meth. Enzymol 152: 649).

Generally, in situ hybridization comprises the following main stages: (1) fixation of the tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility to the target DNA, and to reduce non-specific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove fragments of unbound nucleic acids in hybridization, and (5) detection of hybridized nucleic acid fragments. The probes used in such applications are usually labeled, for example, with radioisotopes or fluorescent reporters. Preferred probes are sufficiently long, for example, from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, to allow specific hybridization with the target acid or nucleic acids under stringent conditions.

Another alternative methodology to determine the DNA copy number is comparative genomic hybridization (CGH). In comparative genomic hybridization procedures, mark a "test" group of nucleic acids with a first marker, while a second test group (for example, of a normal cell or tissue) is marked with a second marker. The Hybridization ratio of nucleic acids is determined by the ratio of the binding of the first and second markers to Each probe of a group. Differences in proportion are detected of the signals of the two markers, for example, due to the gene amplification in the test group, and the proportion provides a measure of the number of copies of the RhoB gene, corresponding to the specific probe used. Can be generated a cytogenetic representation of the variation in the number of copies of DNA using CGH, which provides relationships of fluorescence along the length of DNA chromosomes differentially labeled test and genomic DNA from reference.

Hybridization protocols suitable for use with the methods of the present invention are described, for example, in Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl Acad. Sci. USA 85: 9138-9142; 430402EPAE PO Pub. No. 430: 402; Methods in Molecular Biology, Vol. 33: In Situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, NJ (1994).

Amplification-based assays can also be used to measure the copy number of the RhoB gene. In such tests, the corresponding RhoB nucleic acid sequence acts as a template in an amplification reaction (for example, Polymerase Chain Reaction or PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison with appropriate controls provides a measure of the copy number of the RhoB gene, corresponding to the specific probe used, according to the principle described above. Real-time quantitative PCR procedures using Taqman probes are known in the art. Detailed protocols for real-time quantitative PCR are provided, for example, for RNA in: Gibson et al ., 1996, A novel method for real time quantitative RT-PCR. Genome Res. 10: 995-1001; and for DNA in: Heid et al ., 1996, Real time quantitative PCR. Genome Res. 10: 986-994.

Southern blotting can also be used to measure the copy number of the RhoB gene. With this methodology, the DNA is analyzed in agarose or acrylamide gels to fractionate the DNA according to the size followed by the transfer of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. Then, the immobilized DNA is hybridized with a labeled probe to detect DNA samples complementary to the probe used. The DNA can be divided with restriction enzymes before electrophoresis. After electrophoresis, the DNA can be partially stripped and denatured before or during the transfer to the solid support. Southern blots are a common tool for molecular biologists (J. Sambrook et al ., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, p. 9.31-9.58 [1989]).

A powerful procedure for determining the number of copies of DNA uses microarray-based platforms. Microarray technology can be used because it offers high resolution. For example, traditional CGH generally has a mapping resolution limited to 20 Mb; whereas in CGH based on arrays, the fluorescence ratios of the differentially labeled test DNAs and genomic reference DNA provide a "locus per locus" measurement of the variation in the number of copies of DNA, thus achieving greater mapping resolution Details of a microarray procedure can be found in the literature. See, for example, 6232068USB US 6,232,068; Pollack et al ., Nat Genet, 1999, 23 (1): 41-6.

Any other suitable technique to measure the RhoB gene copy number is also included in the Scope of the present invention.

It has been observed that overexpression or increase in the number of copies of the RhoB gene in relation to the expression or the number of copies of the same gene in a control sample are Useful markers for the prognosis of lung adenocarcinoma. In particular, said overexpression or increase in the number of copies of the RhoB gene is indicative of the risk of metastasis, or recurrence or recidivism of neoplastic activity.

The "overexpression" of a RhoB gene or a "higher" level or "high" level of a polynucleotide or RhoB protein refers to a level of polynucleotide or protein RhoB which, compared to the level of RhoB control, is of highest detectable form. Similarly, an "increase" in the number of copies of the RhoB gene refers to a level of number of RhoB copies that, compared to a control level of RhoB, is detectably higher.

Overexpression or increase in the number of copies of the RhoB gene are relative terms that mean that find a detectable difference (beyond the contribution of the noise in the system used to measure it) in the amount of expression or number of copies of the RhoB gene relative to a certain line base. In the present invention, the baseline is gene expression measure or the number of copies of a control sample, whose term It is defined below.

The comparison can be carried out by statistical analysis on the numerical measurements of the expression or number of copies of the RhoB gene; or, it can be done through of the visual examination of the experimental results by qualified researchers.

For example, expression intensities gene of a diseased tissue can be compared with intensities of expression generated from normal tissue of the same type (for example, sample of diseased lung tissue vs. lung tissue sample normal). A relationship of these intensities of expression indicates the times that gene expression changes between test samples and of control.

In an embodiment of the present invention, the RhoB gene overexpression is determined when it exists so minus a difference of 1.5 times in its expression in relation to the expression of the same gene in a control sample. In other embodiment of the present invention, an increase in the number of RhoB gene copies are determined when there is at least one 1.5 times difference in its number of copies in relation to the number of copies of the same gene in a control sample.

Student's t-test is an example of a test robust statistic that can be used to find differences significant between two groups. The lower the value of p, the evidence that the gene shows a difference is more conclusive between the different groups A value of p less than 0.05 by the t test shows that the gene is significantly different. A more conclusive evidence is a value of p less than 0.05 after taking into account the correction of Sidak. For a large number of samples in each group, a value of p less than 0.05 after the randomization / permutation test is the evidence most conclusive of a significant difference.

A "control sample" refers to a sample of biological material representative of healthy subjects without Cancer. The expression level of the RhoB gene or the number of copies of the RhoB gene in a control sample is desirably habitual of the general population of normal subjects without cancer of The same species. This sample can be collected from a subject with the objective of being used in the procedures described in the present invention or, it can be any biological material representative of normal subjects without cancer obtained for others reasons, but nevertheless, suitable for use in the Procedures of the present invention. Can also be obtained a control sample from normal tissue of the subject that You have cancer or are suspected of having cancer. A control sample it can also refer to a certain level of gene expression of RhoB or the number of copies of the RhoB gene representative of the population without cancer, which have been previously established based on the measurements of normal subjects without cancer. As such, a sample of control does not need to be a material, but can be data or information related to a control sample. This data or Information is usually stored in electronic form or paper. Alternatively, a biological control sample may refer to a sample that is obtained from a different individual or it can be a normalized value based on baseline values found in a population. In addition, a control sample can be defined by age, sex, ethnicity or other demographic parameters specific. In some situations, control is implicit in the concrete measurement For example, it is considered a procedure of detection that can only be detected by RhoB or the number of copies of the gene of RhoB when a higher level than usual is present in a normal subject without cancer, for example, a trial immunohistochemistry, to assess the level of RhoB or the number of copies of the RhoB gene compared to the level of control or the number of copies of the RhoB gene, since the level of control or Number of copies is natural and known in the essay.

The present invention further relates to the use of a RhoB gene, or a region thereof, as a marker of prognosis of metastasis, or recurrence or recurrence of Neoplastic activity in a subject with lung adenocarcinoma.

The present invention further provides a procedure for screening active drugs against lung adenocarcinoma, said procedure comprising:

to)

the RhoB gene expression measurement, or number measurement of copies of the RhoB gene, in a sample with cells from lung adenocarcinoma in the presence of a drug, or measurement of binding affinity between a drug and a polypeptide RhoB;

b)

the comparison of the expression or measured copy numbers of the gene from RhoB, or the binding affinity of step a) to a sample of control;

characterized in that a reduced expression or copy number of the RhoB gene, or a greater enzymatic activity in step a), in relation to the expression or the number of copies of the same gene or the binding affinity of the same polypeptide in a sample of control is indicative of the activity of the drug against adenocarcinoma of
lung.

As such, the present invention provides procedures to identify compounds or agents that can be use to treat disorders characterized by, or associated with an expression or reduced copy number of the RhoB gene. These procedures usually include the screening stage of a candidate, test compound or agent to identify compounds which are an agonist or antagonist of a RhoB polypeptide, and specifically for the ability to interact with (for example, bind to) a RhoB polypeptide, to modulate the interaction of a RhoB polypeptide and a target molecule, and / or to modulate expression of RhoB nucleic acid and / or RhoB polypeptide activity. The candidates, test compounds or agents that have one or more of These capabilities can be used as drugs to treat disorders characterized by an expression or number of copies reduced RhoB gene.

An expression or reduced number of copies of the RhoB gene marker in a lung adenocarcinoma also it can serve to predict the response of that cancer to therapies specific. This molecular marker can be used to Select the most appropriate therapy for the subject.

As such, the present invention provides also a procedure to determine the effectiveness of a pattern of therapeutic treatment in a subject with lung adenocarcinoma, comprising said procedure

to)

the RhoB gene expression measurement, or number measurement of copies of the RhoB gene, in a sample with cells from lung adenocarcinoma of said subject, thereby generating a initial level;

b)

the RhoB gene expression measurement, or number measurement of copies of the RhoB gene, in a second sample with cells from lung adenocarcinoma of the same subject at a time after the administration of the treatment regimen, obtaining from this mode a test level; Y

C)

the comparison of initial and test levels;

characterized in that an expression or number of reduced copies of the RhoB gene at the test level in relation to with the initial level it is indicative that the treatment regimen is effective in the subject.

The present invention further relates to the use of a nucleotide molecule or antibody that interact with DNA, RNA, RhoB protein, or a fragment thereof, for the prognosis of disease progression in a subject with lung adenocarcinoma, for active drug screening against lung adenocarcinoma, or to determine the efficacy of a therapeutic treatment regimen in a subject with adenocarcinoma of lung Said nucleotide molecule is a nucleotide that has at least 70% homology with respect to SEQ ID NO. 4-6, preferably at least about one 80, 85, 90, 95, 96, 97, 98, 99, or 100% homology with respect to SEQ ID NO. 4-6. The antibody molecule that interacts with DNA, RNA, Rhob protein, or a fragment of themselves, it can be monoclonal, polyclonal, chimeric, humanized or bispecific, or is conjugated to a second antibody, or a fragment thereof. Particularly, the antibody molecule is the polyclonal antibody Rho B (119), or an antibody molecule which preferably has at least 80% and more preferably at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of homology with the polyclonal antibody Rho B (119).

A "fragment" is a part of a Natural, native or endogenous mature Rhob sequence in length complete that has one or more amino acid residues removed. He residue or amino acid residues removed may be in any part of the polypeptide, including at the end N-terminal or C-terminal end, or internally. Said fragment will have at least one property Biological in common with RhoB. RhoB fragments will have usually a consecutive sequence of at least 10, 15, 20, 25, 30, 40, 50 or 60 amino acid residues that are identical to RhoB sequences isolated from a mammal, including SEC RhoB ID NO: 2.

As used in the present invention, the term "antibody" is interchangeable with "immunoglobulin" and refers to immunoglobulins of the form general found in vertebrate species, including mammals, such like humans, primates, rodents, rabbits and many other species in those that have been identified said immunoglobulins. "Antibody" means molecules that correspond to complete immunoglobulins, as well as fragments thereof, such like Fab, F (ab ') 2, and Fv, which are able to join the epitopic determinant

The term "monoclonal antibody" is refers to a population of antibody molecules that contain just a kind of an antigen binding site capable of immunoreact with a particular epitope of an antigen, while that the term "polyclonal antibody" refers to a population of antibody molecules containing multiple species of antigen binding sites capable of interacting with a particular antigen Thus, a composition with monoclonal antibodies usually show a single affinity of binding for a particular antigen with which it immunoreacts.

The procedures to produce and screen Specific antibodies using hybridoma technology are routine and known in the art. In a non-limiting example, it can immunize mice with a polypeptide of the present invention or a cell that expresses said peptide. Once a immune response, for example, specific antibodies are detected for the mouse serum antigen, the mouse spleen is collected and Splenocytes are isolated. Then the splenocytes are fused by known techniques to any myeloma cell adequate. The hybridomas are selected and cloned by limited dilution Next, the hybridoma clones are assay by methods known in the art for cells that secrete antibodies capable of binding to a polypeptide of the present invention Ascites fluids, which contain in general high levels of antibodies can be generated by immunization of mice with positive hybridoma clones.

Polyclonal antibodies are produced usually by immunization of a suitable mammal, such Like a mouse, rabbit or goat. Mammals are often preferred larger, since the amount of serum that can be collected is higher. An antigen is injected into the mammal. This induces the B lymphocytes produce specific IgG immunoglobulins for antigen. This polyclonal IgG is purified from mammalian serum. In instead, monoclonal antibodies derive from a single line mobile.

The term "chimeric antibody" is refers to an antibody in which the constant region comes from a antibody of a species (usually human) and the region variable comes from an antibody of another species (usually rodent). The procedures for producing chimeric antibodies are known in the art.

The term "humanized antibody" as used in the present invention refers to antibody molecules in which amino acids have been substituted in the regions that are not antigen-binding in order to be as close as possible to a human antibody, while still maintaining the original binding capacity. Depending on the context, this expression may also include "primate" antibodies, in which a first antibody obtained from a
Non-primate organism has been modified to closely resemble a primate immunoglobulin.

A "bispecific" or bifunctional antibody is an artificial hybrid antibody that has two different heavy / light chain partners and two different binding sites. Bispecific antibodies can be produced by a series of procedures including hybridoma fusion or Fab 'fragment binding. See, for example, Songsivilai & Lachmann, Clin Exp. Immunol. 79: 315-321 (1990); Kostelny et al ., J. Immunol. 148, 1547-1553 (1992).

A further embodiment of the present invention provides a kit for the forecast of the evolution of the disease in a subject with adenocarcinoma of the lung that comprises reagents to measure expression or number of copies of the RhoB gene. Said kit may additionally comprise reagents suitable for microarray analysis. In particular, The kit according to the present invention comprises Rho B (119): sc-180 Santa Cruz Biotechnology, Inc. Rho B (119) is an affinity purified rabbit polyclonal antibody developed against a peptide that maps to an internal region of RhoB of human origin. Alternatively, the kit according to the present invention comprises primers with SEQ ID No. 4-6 to perform the RT-PCR or quantitative PCR on time real.

The present invention further provides a isolated RhoB gene amplicon, where the amplicon comprises more than a copy of a polynucleotide selected from:

to)

a polynucleotide encoding the polypeptide set forth in SEQ ID NO. 2;

b)

a polynucleotide set forth in SEQ ID NO. one;

C)

a polynucleotide that has at least 70% homology to the polynucleotide of a) or b); Y

d)

a polynucleotide that is overexpressed in adenocarcinoma cells of lung that has at least 70% homology with the polynucleotide of a) or b).

The present invention further relates to RhoB gene overexpression inhibitors for use as medicine. Said use as a medicine or treatment method includes administration to subjects with NSCLC or subjects likely to develop NSCLC including adenocarcinoma of lung, squamous cell carcinoma, or cell carcinoma large of an effective amount of an overexpression inhibitor of the RhoB gene to combat conditions associated with NSCLC, in particular to avoid the development after bone metastasis, or the reappearance or recurrence of neoplastic activity.

The present invention also relates to the use of RhoB gene overexpression inhibitors or any subgroup thereof in the manufacture of a medicine for the NSCLC treatment including lung adenocarcinoma, carcinoma squamous cell, or large cell carcinoma, in particular to prevent further development in bone metastases, or the reappearance or recurrence of neoplastic activity. Said of another way, the present invention provides inhibitors of overexpression of the RhoB gene for use in the treatment of NSCLC including lung adenocarcinoma, cell carcinoma squamous, or large cell carcinoma, in particular to avoid the development after bone metastasis, or the reappearance or recidivism of neoplastic activity.

The term "treat", as used in The present invention relates to inverting, alleviating or inhibiting the progress of the disorder or condition to which the term applies, or one or more symptoms of such disorders or condition. The term "treatment" refers to the act of treating, which has been defined above.

The term "therapeutically amount effective "or" effective amount "as used in the present invention, mean the amount of compound or component or active pharmaceutical agent that obtains the biological response or medical in a tissue, system, animal or human that is being sought, in the light of the present invention, by a researcher, veterinarian, doctor or other clinical professional, which includes the improvement of symptoms of the disease being treated

Since the present invention includes combinations comprising two or more agents, the "amount therapeutically effective "is that amount of agents taken together, so that the combined effect provides the desired biological or medical response.

The term "inhibitor" is intended to include antagonists and, refers to a molecule that direct or indirectly reduces RhoB gene overexpression, or the Biological activity of a RhoB polypeptide or protein. The RhoB gene overexpression inhibitors may include antibodies, nucleic acids, or other molecules.

In one embodiment, the inhibitors of RhoB gene overexpression can be selected from:

to)

a antibody or fragment thereof that specifically binds to a RhoB protein, or a polypeptide comprising a region of binding to RhoB;

b)

a Rhob polypeptide mimetic peptide;

C)

a RhoB gene inhibitor nucleic acid selected from a antisense oligonucleotide, a ribozyme, and an RNAi agent selected from ARNds and ARNsh; or

d)

a combination comprising lovastatin and zoledronic acid, and / or salts pharmaceutically acceptable thereof.

The antibody molecule that binds specifically a RhoB protein can be monoclonal, polyclonal, chimeric, humanized or bispecific, as described above in the present invention.

The term "antibody fragment" is more specifically refers to these fragments and / or polypeptides of immunoglobulin type that do not comprise an immunoglobulin complete.

A "polypeptide comprising a region of specific binding to RhoB "means a polypeptide that incorporates one or more binding regions that specifically bind to the RhoB protein of SEQ ID NO. 2. A classic type of junction region antigen specific is a determining region of Complementarity (CDR) found in an immunoglobulin. The CDRs are short amino acid sequences that specifically bind to antigen in question and provide the basis for the selectivity of binding of the polypeptide in which it resides. CDRs are identified usually from immunoglobulins, but they can be generated by other means.

Antigen specific binding regions they also include relatively short amino acid sequences that bind to an antigen, even if these sequences do not derive from CDRs. WO2004 / 044011, incorporated in the present invention by reference, provides an example of how said binding regions Antigen-specific (non-CDRs) can be identified and develop. There are other procedures known to experts in the matter. Once developed, the binding region specifies RhoB can be used in a wide variety of structures and known and futuristic frameworks, including any isotype of immunoglobulin or fragment thereof, and other polypeptides of structure or framework that are not immunoglobulins to provide the specificity of antigen binding. All these polypeptides are considered in the present invention as "polypeptides that they comprise a region of specific binding to RhoB ".

A "mimetic peptide" is a non-agent synthetically derived peptide created on the basis of knowledge of the critical residues of a subject's polypeptide which can mimic the normal function of the polypeptide. Mimetics peptide can break the binding of a polypeptide to its receptor or to other proteins and thus interfere with normal function of a polypeptide. For example, a RhoB mimetic would interfere with normal RhoB function.

"Nucleic acid sequence", such as used in the present invention, refers to a oligonucleotide, nucleotide or polynucleotide, and fragments or parts thereof, whose polymeric components may be DNA, RNA, modified nucleotides, nucleotide mimetics, or combinations thereof; and can be of genomic or synthetic origin, and can be single or double chain, and represent the chain of meaning or antisense

The term "antisense" as it is used in the present invention, refers to sequences of nucleotides that are complementary to a DNA or RNA sequence specific, usually a target mRNA. They bind mRNA through the Watson-Crick base pairing, and inhibit its target message through degradation by ribonuclease (RNasa) H or through interference with the translation machinery. Antisense oligonucleotides can be optionally stabilized. with phosphorothionates, methylphosphonates, nanoparticles of polyalkylcyanoacrylate, and 2 'modifications of the sugars. He term "antisense chain" is used in reference to a nucleic acid chain that is complementary to the "chain of meaning. "Antisense molecules can be produced by any procedure, including synthesis by binding of gene of interest in reverse orientation to a viral promoter that It allows the synthesis of a complementary chain. Designation "negative" is sometimes used in reference to the string antisense, and "positive" is sometimes used in reference to the chain of meaning.

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As used in the present invention, the term "ribozyme" means an RNA molecule that has a enzymatic activity that is capable of dividing or splicing others RNA molecules separated in a nucleotide base sequence in a specific way. In reference to an RNA molecule catalytic or enzymatic is meant an RNA molecule that has complementarity in a substrate binding region with the mRNA of RhoB, and also has enzymatic activity that is active for split and / or splice that mRNA, altering it in this way.

The term "RNAi agent" as used in the present invention, refers to compounds and compositions that can act through a mechanism of RNA interference (see, for a general reference, He and Hannon, (2004) Nat. Genet. 5: 522-532). Habitually RNAi agents such as short interference RNA are used (SiRNA), double stranded RNA (RNAs), short hairpin RNA (ARNsh), also sometimes referred to as hairpin RNA Small, others are in development. When they are introduced in a cell or are synthesized in a cell, RNAi agents are incorporated in a macromolecular complex that uses RNAi agent chains to mark and divide strands of RNA that contain the sequence complementary (or substantially complementary).

As contemplated in the present invention, antisense oligonucleotides, triple helix DNA are designed, RNA aptamers, RNAi agents such as siRNA, siRNA, and siRNA, Ribozymes and single stranded RNA to inhibit RhoB expression, so that the nucleotide sequence chosen from the inhibitory molecule to cause synthesis inhibition endogenous RhoB protein.

For example, based on this description, you can use the knowledge of the nucleotide sequence of RhoB to design an siRNA molecule that inhibits the expression of RhoB without too much experimentation. Similarly, ribozymes can be synthesized to recognize nucleotide sequences specific to a protein of interest and divide it (Cech. J. Amer. Med Assn. 260: 3030 (1988)). The techniques for the design of said molecules for use in targeted expression inhibition genetics are well known to those skilled in the art.

In an embodiment of the present invention, the RhoB gene overexpression inhibitor is an mRNA molecule which has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO. 3, as is provided below.

8

Antisense oligonucleotides, ribozymes, and RNAi agents can be prepared by any method known in the art for the synthesis of nucleic acid molecules. Techniques for chemical synthesis, such as chemical synthesis of solid phase phosphoramidite, are included. Alternatively, antisense oligonucleotides, ribozymes, and RNAi agents can be generated by in vitro transcription of DNA sequences encoding the relevant molecule. Such DNA sequences can be incorporated into a wide variety of vectors with suitable RNA polymerase promoters.

The antisense oligonucleotides, ribozymes, and RNAi agents can be released to cells by application direct, such as transfection or formulated in liposomes. The Conventional transfection techniques include a variety of recognized techniques in the sector to introduce nucleic acid exogenous (eg, DNA or RNA) in a host cell, including coprecipitation with calcium phosphate or calcium chloride, transfection mediated by DEAE-dextran, lipofection, (micro) injection, or electroporation.

Liposomes can be prepared using conventional techniques including sonication, dialysis with chelates, homogenization, solvent infusion coupled with extrusion, freeze-thaw extrusion, microemulsification, and others. The reagents used for cross-link a liposome or other agent that contains lipids with antisense oligonucleotides, ribozymes, and RNAi agents comprise a phospholipid derivative to anchor at one end of the cross-linking in the lipid layer and a reactive group in the other end to provide a point of attachment to the target biomolecule. Polymerized liposomes or liposomes can also be used. polymer coated. Such polymers can stabilize the liposome, reduce your body clearance, and / or reduce your immunogenicity The liposome can be loaded with a residue functional such as a diagnostic or therapeutic agent during or After its formation. The agent can be contained in the aqueous nucleus of the liposome or can be incorporated or bound to its surrounding membrane

Alternatively, the release of antisense oligonucleotides, ribozymes, and RNAi agents is performed through a gene therapy strategy in which vectors, including plasmids, cosmids, bacteriophages, or viral vectors, particularly retroviral, lentiviral, or adenoviral vectors, are they release a cell or subject; or cells that direct the expression of the molecules, for example, cells in which a vector that directs the expression of the molecule has been introduced, are administered to the subject. The vectors that direct the in vivo synthesis of antisense oligonucleotides, ri-
Bozimas, and RNAi agents can be introduced into cell lines, cells or tissues constitutively or inducibly.

A "vector" molecule is a molecule of nucleic acid into which heterologous nucleic acid can be inserted which can then be introduced into an appropriate host cell. The vectors preferably have one or more origins of replication, and one or more sites where DNA can be inserted recombinant Vectors often have adequate means whereby cells with vectors can be selected of those without vectors, for example, that encode genes from drug resistance

A "host cell", as used In the present invention, it refers to a prokaryotic cell or eukaryotic containing heterologous DNA that has been introduced into the cell by any means, for example, electroporation, precipitation with calcium phosphate, microinjection, transformation, viral infection, and the like.

"Heterologous" as used in the present invention means "of different natural origin" or It represents an unnatural state. For example, if a host cell with a DNA or gene derived from another organism, particularly from another species, that gene is heterologous with respect to the host cell and also with respect to the descendants of the host cell that carry that gene. Similarly, heterologous refers to a nucleotide sequence derived from and inserted into the same original natural cell type, but that is present in a unnatural state, for example a number of different copies, or under the control of different regulatory elements.

Additional gene therapy protocols may involve isolating a population of cells, for example, stem cells or cells of a subject's immune system, optionally expanding the cells in tissue culture, and administering a gene therapy vector to the cells in vitro. Next, the cells can be returned to the subject. Optionally, cells expressing the desired antisense oligonucleotide, ribozyme, or RNAi agent may be selected in vitro before being introduced into the subject. Alternatively, a population of cells can be used, which can be cells of a cell line or of an individual that is not the subject. The isolation procedures of stem cells, immune system cells, etc., of a subject are well known in the art. Such procedures are used, for example, for bone marrow transplantation, peripheral blood stem cell transplantation, etc., in individuals undergoing chemotherapy.

In a further embodiment of the present invention, the RhoB gene overexpression inhibitor is a combination comprising lovastatin and zoledronic acid, and / or salts pharmaceutically acceptable thereof.

The term "and / or" refers to an "or" not exclusive, that is, "A and / or B" includes "A and B", so like "A or B". As such, the expression "lovastatin and acid zoledronic, and / or pharmaceutically acceptable salts of same ", refers to the following combinations:

to)

lovastatin and acid zoledronic;

b)

a pharmaceutically acceptable salt of lovastatin and acid zoledronic;

C)

lovastatin and a salt pharmaceutically acceptable zoledronic acid; Y

d)

a pharmaceutically acceptable salt of lovastatin and a salt pharmaceutically acceptable zoledronic acid.

Lovastatin is a commercial product that Can be provided by TCI Europe NV, Sigma companies Aldrich, among others. The CAS registry number of lovastatin is 75330-75-5, and its structural formula is:

9

Zoledronic acid, which is also made reference as zoledronate, it is a commercial product that can be provided by Interchim Ambinter companies, ACIC Europe Limited, among others. The CAS registration number of the acid zoledronic is 118072-93-8, and its structural formula is:

10

The term "pharmaceutically acceptable" means that a compound or combination of compounds must be compatible with the other ingredients of a formulation, and not be Referrals for the patient. For therapeutic use, salts of lovastatin or zoledronic acid are those in which the Counterion is pharmaceutically acceptable.

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Acid addition salts pharmaceutically acceptable as mentioned above they intend to understand forms of therapeutically non-toxic acid addition salts active that lovastatin and zoledronic acid are each able to form. Acid addition salts pharmaceutically acceptable can be obtained properly by treatment of the basic form of lovastatin and zoledronic acid with said appropriate acid. Appropriate acids comprise, by for example, inorganic acids such as hydro acids, for example acid hydrochloric or bromhydric, sulfuric, nitric, phosphoric and acids Similar; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and acids Similar. Conversely, such salt forms can be converted by treating with an appropriate base in the base form free.

In an embodiment of the present invention, the combination comprising lovastatin and zoledronic acid, and / or salts pharmaceutically acceptable thereof can be administered in simultaneously, separately or sequentially.

As such, the present invention relates to in addition to a combination of separate pharmaceutical formulations in kit shape For example, a kit can comprise two formulations separate pharmaceuticals comprising: 1) lovastatin or salts pharmaceutically acceptable thereof; and 2) zoledronic acid or pharmaceutically acceptable salts thereof. The kit can also comprise a container for separate formulations, such as a compartmentalized bottle or a paper container of compartmentalized aluminum Additional examples of containers They include syringes, boxes, bags and the like. Usually a kit includes instructions for component administration separated. The kit form is particularly advantageous when separate components are preferably administered in forms of different dosage (for example, oral and parenteral), it administered at different dosage intervals, or when you want the adjustment of the individual components of the combination by clinical professional.

In general, it is contemplated that it can be used an effective daily amount of zoledronic acid of approximately 0.5 to about 2000 mg. An effective daily amount of Lovastatin can range from about 5 mg to approximately 400 mg

The exact dose and frequency of administration of overexpression inhibitors of the gene of RhoB depend on the specific compound or combination used, the specific condition in treatment, the severity of the condition in treatment, age, weight, sex, extent of the disorder and the general physical condition of the specific patient, as well as another medication that the individual may be taking, as is well known by experts in the field. In addition, it is clear that such effective daily amount may decrease or increase depending on the response of the treated subject and / or depending on the evaluation of the physician prescribing the compounds of the present invention. The variations of the effective daily amount mentioned above They therefore serve only as guides.

The following non-limiting examples help illustrate the principles of the invention.

Examples

Animals and cell cultures . Nude nude mice of four weeks of life were acquired from Harlan (Barcelona, Spain) and kept in specific pathogen-free conditions. The A549 adenocarcinoma cell line was obtained from Dr. Gazdar. All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum.

RNA isolation, cDNA synthesis and hybridization with microarray

RNA was extracted using TRIzol Reagent (Life Technologies) of three 10 cm culture plates per cell line and It was purified using the RNeasy kit (Qiagen, Hilden, Germany). The Purity and quality was evaluated using RNA 6000 nanochips (Agilent Technologies, Germany). Next, the DNA was used to synthesize complementary probes (cDNA) using the kit cDNA synthesis of a cycle (Affimetrix). Subsequently synthesized cRNA and was labeled with cDNA as a template using the kit IVT marking (Affimetrix), and was purified by GeneChip Sample Cleanup Module (Affimetrix). The labeled cRNAs were fragmented and hybridized to high density oligonucleotide microarrays of GeneChip of Affimetrix Human Genome U133 2.0 according to protocols described in Gene Expression Analysis Technical Manual (http://www.affimetrix.com). 3 microarrays were hybridized with 3 independent biological replicas per 3-line cell line different (called M1M1, M1M3, and M1M4). For each line cell, a group of three mRNA extractions was used independent to hybridize each oligonucleotide microarray of High density human GeneChip. As controls 6 were used parental tumor cell lines: 3 transfected with the Luciferase vector and 3 untransfected. In total, a hybridized set of 15 samples: 6 controls and 9 metastatic lines.

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Quantitative real-time PCR

Total RNA of 70-80% was isolated of confluent cultures using Trizol RNA (2 µg) and DNase I, it was treated and transcribed in reverse using transcriptase Reverse Superscript II and oligo primers (dT). A Assay-on-Demand? (Applied Biosystems) using a Gene sequence detection system Amp 7300 (Applied Biosystems) for RHOB according to recommendations manufacturer. The average threshold cycle value (Ct) of the triplicate samples was used to calculate expression gene. PCR products were normalized to levels of β-actin. The experiment was repeated three times. with identical results.

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Analysis of microarray data: normalization, calculation of the signal, significant differential expression and grouping of sample / gen profiles

The analysis of microarray data was performed using the strategy and procedures described in the literature (Castellano et al . 2007). In summary, the RMA algorithm was used for base correction, intra- and intermicroarray normalization and expression signal calculation (2-4). Once the absolute expression signal was calculated for each gene (that is, the value of the signal for each set of probes) in each microarray, a procedure (called SAM) (Tusher et al . 2001) was applied to calculate the differential expression significant and find the sets of probes for genes that characterize highly metastatic samples. The procedure uses permutations to provide a robust statistical inference of the most significant genes and provides p-values adjusted to multiple tests using FDR (Benjamini et al . 1995). The main test was performed by contrasting 4 control samples against 9 highly metastatic samples. However, with 3 different metastatic lines (M1M1, M1M3, MM1M4) performed in triplicate, multiple SAM analyzes were performed to find the genes that showed a significant differential expression that were also stable in different SAM tests. A cut of FDR <0.20 was used for all differential expression calculations. All these procedures were applied using the statistical tools "R" and Bioconductor. After identifying the set of probes for differentially expressed genes, the corresponding array of expression values for all microarray hybridizations was analyzed using the "hclust" clustering algorithm implemented in "R". This algorithm performs a hierarchical clustering analysis with complete binding to look for similarities between the sets of probes based on their expression values in the different microarray of chips analyzed. The algorithm classifies probe sets into correlated groups that have similar expression profiles or expression signatures. This global strategy allowed the selection of significant key genes with similar expression profiles.

Metastasis trials : ic was performed as described previously (González et al . 2007).

Bioluminescence imaging and analysis . Mice were anesthetized and 1.5 mg of D-Luciferin was injected intraperitoneally in 100 µl of PBS. The image was completed at 2 min exactly for each group of mice with an Xenogen IVIS system coupled with a Living Image acquisition and analysis software (Xenogen Inc.). The photon flow was calculated for each mouse by using a circular region of interest for each hind leg. Base values (from mouse injected with luciferin without tumor cells) were subtracted from each
measurement.

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Proliferation test

Cell proliferation was evaluated by a MTT test according to the manufacturer's recommendations (Roche Diagnostics GmbH, Mannheim, Germany). The experiments are repeated three times and the data were represented as the average of wells per sextuplicate ± SEM.

Microcomputerized radiographic and tomographic analysis (\ muCT)

A low X-ray was done anesthesia, supine cube position on a film radiographic sensitive (MIN-R, Eastman Kodak). The mice were exposed to X radiation at 20 kV for 20 s using a Faxitron instrument (model MX-20; Faxitron, Buffalo Grove, IL, USA). The area of osteolytic metastasis was evaluated with a computerized image analysis system, AnalySIS® (Soft imaging system GmbH, Münster, Germany). Be they captured images scanned on a high x-ray film resolution with double magnification at 1,200 ppi using an Epson scanner Expression 1680 Pro (Long Beach, CA, USA). The quantification of metastatic area was performed twice after calibration by Two independent observers. For the curves of Kaplan-Meier metastasis was valued as time elapsed until the first appearance of signs of cachexia and dificulty to walk.

All femorotibial joints were analyzed using a µCT system (micro CAT ™ II, Siemens Preclinical Solutions, Knoxville, TN, USA) at 75.0 kVp and 250.0 uA. The scans were performed at a resolution of 10 µm. 2D CT images were reconstructed using a standard retro-convolution projection procedure with a Shepp-Logan filter. The images were stored in 3D groups with a voxel size of 19 µm x 19 µm x
23 µm.

Knock-down constructions

Lentiviral constructions were obtained for Human RhoB rRNA from Sigma (St. Louis, MO, USA). To get viral particles, packaging cells were transfected with 8 µg of DNA for each type of fusion by the procedure with clasicote phosphate according to the manufacturer's recommendations. Be used the vector pLKO "scramble" as a control (which contains a shRHOB sequence in which the shifts have been randomly changed nucleotide bases). Two days after transfection, the supernatants were centrifuged for 10 minutes at 600 g and were filtered through a 0.45 cellulose acetate filter pore \ mum. For transduction infection, they were seeded A549 to 1x105 cells and incubated overnight with viral supernatants in the presence of 4 µg / ml of polybrene (Sigma). Forty-eight hours after infection, they were incubated cell populations in medium containing the antibiotic appropriate for two additional weeks. The groups resistant to antibiotic expanded and frozen in each passage mobile.

Isolation of colonies derived from a single cell (SCC) of long bones

Metastatic marrow cells were isolated Bone of the lower extremities. Mice were sacrificed according to the approved protocols of the Local Animal Committee (University of Navarra, protocol 003-04). Be they removed the long bones, and washed all the tissues soft. The marrow cells were released by "flushing", introducing 5-10 ml of medium α-MEM containing 2 x penicillin / streptomycin with a 27 gauge needle in the epiphysis distal through the bone marrow compartment. The cell clusters were disaggregated by passing the medium that contained the cells through the 27 gauge needle syringe G. Cells were seeded in expanded 10 or 15 cm plates for 5 days in a medium containing 0.4 mg / ml of G-418. This procedure was performed separately. for each femur and tibia of 7 mice per group. SCCs were counted under the light of a microscope after staining with violet crystal.

Histological analysis

The hind legs were removed, removed carefully soft tissues of the femur, were fixed for 24 h in 10% neutral buffered formalin, dehydrated and descaled in Osteosoft® solution. After full dehydration with alcohol, bones were included in paraffin and 5 µm sections were stained with H&E.

RhoB expression and prediction value in patients with adenocarcinoma Patients

For this analysis, 35 were selected individuals who separated into two selected groups based on Progressive Disease (PD) group compared to a group Without Disease (DF) after surgery. The PD group included patients with a history of a primary lung cancer completely resected who relapsed (recurrence or metastasis) after a 6 month period without illness after surgery. The DF patients were the rest of individuals with lung cancer Primary completely resected without signs of disease. The patients who performed any treatment, whether radiotherapy or chemotherapy, after surgery they were excluded from the analysis. The study protocol was approved by the Local Ethical Committee. For Each patient obtained consent and questionnaires for writing (background and follow-up). Inclusion criteria were: histology (NSCLC: adenocarcinoma), and absence of cancer in the 5 years prior to the surgical intervention of cancer lung, except melanoma or skin cancer. The diagnosis Histological was determined according to the WHO classification. Staging Pathological tumors were performed according to the international system for lung cancer. The follow-up period continued until on October 31, 2007. The recidivism time was calculated. from surgery to the date of recidivism.

Immunohistochemistry (IHC)

Tissue sections fixed to formalin and embedded in paraffin for procedures immunohistochemicals Paraffin was extracted from tissues and sections were hydrated through a graduated series of ethanol. The endogenous peroxidase activity was stopped with hydrogen peroxidase 3% for 10 minutes. Microwave Antigen Extraction it was carried out with EDTA buffer (mM, pH 8) for 2 x 15 min. The non-specific binding sites were blocked with normal serum of 5% goat in TBS-Tween (wash buffer, Dako) for 30 minutes Sections were incubated with antibody rabbit polyclonal anti-RhoB (sc-180, Santa Cruz Biotechnology, Inc) throughout the night at 4 ° C. The dilution of work was: 1:75. After rinse with TBS, the sections were incubated with the complex Envision polyclonal (Dako). The peroxidase activity was demonstrated by diaminobenzidine. Finally, the sections were washed in water, were slightly contrasted with hematoxylin, dehydrated and they were mounted on DPX. The tissues that expressed different levels of RhoB were included in each immunohistochemical band to unify the possible intensity mismatch.

Immunostaining Evaluation

Two observers independently and to blinds evaluated the extent and intensity of staining in the samples. For RhoB, the extension was scored as the percentage of positive cells (0-100%) and the intensity of Staining was evaluated compared to an external positive control known (tidal 1+, light; 2+, moderate and 3+, intense). The Discordant independent readings were resolved by simultaneous review by both observers.

Statistical Analysis The log-rank test was used to calculate the statistical significance (p-value) of the differences observed between the Kaplan-Meier curves. To study the differences in proliferation rates, tumor growth, differences in the metastatic area were analyzed using the Kruskall-Wallis test with the Dunn multiple comparison test. Values were expressed as the mean ± SEM and the statistical significance was defined as p <0.05 (*), p <0.01 (**), p <0.001 (***). Complementary methods provide additional information.

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References

Mazieres et al . ( 2004 ) Loss of RhoB Expression in Human Lung Cancer Progression. Clinical Cancer Research 10: 2742-2750.

Sato et al . ( 2007 ). RhoB is frequently downregulated in non-small-cell lung cancer and resides in the 2p24 homozygous deletion region of a lung cancer cell line. Int J Cancer . 120 (3): 543-51.

Li et al . ( 2008 ). Lung Cancer 2008 Feb; 59 (2): 180-91.

Turner et al . ( 2008 ). Effects of lovastatin on Rho isoform expression, activity, and association with guanine nucleotide dissociation inhibitors. Biochemical Pharmacology, 75, 405-413.

Hawk et al . ( 1996 ). Inhibition of lung tumor cell growth in vitro and mouse lung tumor formation by lovastatin. Cancer Letters , 109 (1,2), 217-222.

Castellano E, De Las Rivas J, Guerrero C, Santos E. Transcriptional networks of knockout cell Unes identify functional specificities of H-Ras and N-Ras: significant involvement of N-Ras in biotic and defense responses. Oncogene 2007 ; 26: 917-33.

Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 2001 ; 98: 5116-21.

Benjamini Y, Hochberg Y. Controlling the false discovery rate: A practical and powerful approach to multiple testing. J RoyStat Soc 1995 ; (Ser B) 57: 289-300.

González I, Vicent S, of Alava E, Lecanda F. EWS / FLI-1 oncoprotein subtypes impose different requirements for transformation and metastatic activity in a murine model. J Mol Med 2007 ; 85: 1015-29.

Yin JJ, Selander K, Chirgwin JM, et al . TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development. J Clin Invest 1999 ; 103: 197-206.

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\hskip-.1em\dddseqskip

cacttctgtc ccaatgtgcc catcatc

\hfill

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cacttctgtc ccaatgtgcc catcatc

 \ hfill 

27

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<210> 7

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<211> 2384

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<212> DNA

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<213> Homo sapiens

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<400> 7

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30

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<210> 8

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<211> 591

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<212> DNA

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<213> Homo sapiens

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<400> 8

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32

Claims (17)

1. Evolution forecast procedure of the disease in a subject with lung adenocarcinoma that understands:

to)

the RhoB gene expression measurement; or

b)

the measurement of the number of copies of the RhoB gene;

in a sample of said subject with lung adenocarcinoma, characterized in that the detection of overexpression or the increase in the number of copies of the RhoB gene in relation to the expression or the number of copies of the same gene in a control sample is indicative of the risk of metastasis, or recurrence or recurrence of neoplastic activity.

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2. Method according to claim 1, in which the prognosis of the evolution of the disease includes the determination of a clinical outcome selected from the group that consists of response speed (RR), complete response (CR), partial response (PR), stable disease (SD), time to progression (TTP), overall survival (OS), and clinical benefit, comprising complete response (CR), partial response (PR), and stable disease (SD). 3. Procedure according to any of the claims 1-2, wherein the measurement of the RhoB gene expression is determined at the level of protein, mRNA or CDNA 4. Use of a RhoB gene, or a region of the same, as a marker of the prognosis of metastasis, or recurrence or recurrence of neoplastic activity in a subject with adenocarcinoma of lung 5. Procedure for drug screening active against lung adenocarcinoma, said said process:

to)

the RhoB gene expression measurement, or number measurement of copies of the RhoB gene, in a sample with cells from lung adenocarcinoma in the presence of a drug, or measurement of the binding affinity between a drug and a polypeptide RhoB;

b)

the comparison of the expression or measured copy numbers of the gene from RhoB, or the binding affinity of step a) to a sample of control;

characterized in that a reduced expression or copy number of the RhoB gene, or a higher binding affinity in step a), in relation to the expression or the number of copies of the same gene or the binding affinity of the same polypeptide in a sample Control is indicative of the activity of the drug against lung adenocarcinoma.

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6. Procedure to determine the effectiveness of a therapeutic treatment regimen in a subject with adenocarcinoma of lung, said procedure comprising:

to)

the RhoB gene expression measurement, or number measurement of copies of the RhoB gene, in a sample with cells from lung adenocarcinoma of said subject, thereby generating a initial level;

b)

the RhoB gene expression measurement, or number measurement of copies of the RhoB gene, in a second sample with cells from lung adenocarcinoma of the same subject at a time after the administration of the treatment regimen, obtaining from this mode a test level; Y

C)

the comparison of initial and test levels;

characterized in that a reduced expression or number of copies of the RhoB gene at the test level in relation to the initial level is indicative that the treatment pattern is effective in the subject.

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7. Use of a nucleotide molecule or antibody that interacts with DNA, RNA, RhoB protein, or a fragment thereof, for the forecast of the evolution of the disease in a subject with lung adenocarcinoma, for the active drug screening against lung adenocarcinoma, or to determine the efficacy of a therapeutic treatment regimen in a subject with lung adenocarcinoma. 8. Use according to claim 7, wherein the nucleotide molecule is a nucleotide that has at least one 70% homology with SEQ ID NO. 4-6. 9. Use according to claim 7, wherein the antibody molecule is an antibody that has at least one 70% homology with RhoB (119). 10. Kit for the forecast of the evolution of the disease in a subject with lung adenocarcinoma comprising a nucleotide that has at least 70% homology to the SEC ID NO. 4-6, or an antibody molecule that has at least 70% homology with RhoB (119). 11. Amplicon of the isolated RhoB gene, in which the amplicon comprises more than one copy of a polynucleotide selected from:

to)

a polynucleotide encoding the polypeptide set forth in SEQ ID NO. 2;

b)

a polynucleotide set forth in SEQ ID NO. one;

C)

a polynucleotide that has at least 70% homology to the polynucleotide of a) or b); Y

d)

a polynucleotide that is overexpressed in adenocarcinoma cells of lung that has at least 70% homology with the polynucleotide of a) or b).

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12. Use of overexpression inhibitors of RhoB gene, for the manufacture of a medicament for non-small cell lung cancer (NSCLC) treatment, where inhibitors are selected from:

to)

a antibody or fragment thereof that specifically binds to a RhoB protein, or a polypeptide comprising a region of binding to RhoB;

b)

a RhoB polypeptide mimetic peptide;

C)

a RhoB gene inhibitor nucleic acid selected from a antisense oligonucleotide, a ribozyme, and an RNAi agent selected from ARNds, siRNA and siRNA; or

d)

a combination comprising lovastatin and zoledronic acid, and / or salts pharmaceutically acceptable thereof.

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13. Use of overexpression inhibitors of RhoB gene according to claim 12, wherein the inhibitor is a mRNA molecule that has at least 70% homology to the SEQ ID No. 3. 14. Use of the overexpression inhibitor of RhoB gene according to claim 13, wherein the shRNA is in form of an oligonucleotide or vector. 15. Use of the overexpression inhibitor of RhoB gene according to claim 14, wherein the vector is a plasmid, cosmid, bacteriophage, or a virus. 16. Use of inhibitors according to claim 12, in which the inhibitor is a combination comprising lovastatin and zoledronic acid, and / or pharmaceutically salts acceptable thereof, and said combination is for the simultaneous, separate or sequential administration. 17. Use of inhibitors according to claim 16, where the NSCLC is selected from lung adenocarcinoma, squamous cell carcinoma and large cell carcinoma.