Patent Application
20140235466
This invention pertains generally to the fields of molecular biology and medical diagnosis. More particularly this invention provides novel biomarkers for the detection of pancreatic cancer comprising at least one messenger RNA and/or up-regulated peptide in pancreatic cancer. The present invention provides biomarkers capable of discriminating between non-neoplastic pancreatic tissues and ductal adenocarcinoma tissues and can be correlated with a probable diagnosis of pancreatic cancer as well as assessing the efficacy of ongoing therapies for this disease.
Pancreatic ductal adenocarcinoma (PDAC), also known as pancreatic cancer, has been identified as the fourth leading cause of cancer related death in the United States. This disease is more common in the elderly and only 20% of patients have localized tumors and potentially curable. Importantly, the survival rate in patients at 5 years is less than 5% [1], making the PDAC one of the deadliest solid tumors. The dreadful prognosis of PDAC is directly linked to the lack of methods for early diagnosis and effective antineoplasic treatments. The patient survival rate substantially improves if the tumor is diagnosed in early stages of the disease when they are amenable to surgical intervention. Recently it was demonstrated that the progression of PDAC, from the generation of the tumor cell of origin to the first appearance of metastatic clone, occurs in an average of 15 years, allowing a hypothetical time window for early diagnosis of the disease [2]. In this context it is important to identify molecular biomarkers to detect the tumor at an early stage and whose implementation can be transferable to routine clinical use. The antigen CA 19-9 is the only biomarker that has clinical utility, and it is used to monitor treatment response and detection of disease recurrence after treatment, but has little use as a method for early diagnosis of pancreatic cancer and is not specific to this cancer. Therefore, there is an urgent need to identify new strategies for early diagnosis of PDAC.
The methodology of suppression subtractive hybridization coupled with microarray allows the identification of specific genes of tumor cells whose expression is relatively low [3]. The genes identified by this methodology may have a role in the carcinogenesis process, and therefore have the potential to be used as therapeutic targets or tools for specific diagnosis of pancreatic cancer.
The present invention provides methods for diagnosing the presence or absence of cancer in patients by detecting expression levels of one or more genes in tissues where expression of these genes is indicative of the presence of disease especially pancreatic cancer.
The present invention provides for the identification of genes whose gene products are specifically expressed in pancreatic ductal adenocarcinoma tissues and not in normal tissues or tissue adjacent to the tumor.
In one embodiment, the present invention provides for the measurement of parameters, alone or in combination, which may be correlated with a probable diagnosis of cancer.
In another embodiment, the present invention provides new therapeutic methods for the treatment of pancreatic cancer; particularly, the reduction of the expression or interfering with the biological function of some of the described genes, the monitoring of the progression of the disease and the a method to determine the efficacy of drugs used in treatment.
In yet another embodiment, the present invention provides a method to evaluate the efficacy of treatment of pancreatic cancer, comprising the steps of administering a pharmaceutical compound or specific surgical procedure, followed by comparing the gene expression profile in blood samples containing the patient's tumor cells with the profile obtained in healthy cells or tissue.
The expression of specific sets of genes in a cell is subject to spatial and temporal changes that allow the coordination of cellular processes and the maintenance of normal cell function. The pathological changes associated with the carcinogenic process and tumor progression are associated with alterations in the expression of genes that can influence cell behavior. These pathological changes can also be used as indicators or markers of the disease, thus allowing the potential diagnosis, monitoring and treatment of disease.
The present invention describes the changes in gene expression in pancreatic cancer compared to non-neoplastic tissue of the pancreas. Surgically removed tissues of malignant tumors of the pancreas were studied using a microarray platform coupled with the methodology of suppression subtractive hybridization to identify specific gene expression in tumor tissues. Gene expression profiling results are described in
The present invention provides methods to detect genes expressed specifically in tumor tissues compared to non-tumor tissue.
In order to identify differentially expressed genes in pancreatic cancer, we performed a paired analysis of 6 tumor tissues and their neoplastic tissues. We identified 150 genes up-regulated in tumor tissues by microarray analysis coupled with the strategy of suppression subtractive hybridization.
At the protein level, by immunohistochemical staining, PLEKHM1 expression is up-regulated in PDAC tissues compared to normal tissues and non-neoplastic pancreatic (
To evaluate the specific expression of PLEKHM1 in pancreatic cancer, immunohistochemical stains were used on other organs using a TMA of normal tissues, where we observed that PLEKHM1 is not expressed in a normal pancreas, however, is expressed by prostate cell types (prostate), thymus (thymus), amygdala (tonsil), cervix (uterus cervix) and skin (skin), the latter being the organ where there is a greater expression of PLEKHM1 (
On the other hand, another study findings was the identification of WNT9A expressed in pancreatic cancer specifically. WNT9A belongs to the family of WNT proteins and has been linked to the process of chondrogenesis and joint integrity in murine models [4], as well as morphogenesis and cell proliferation in liver of avian models [5]. In zebrafish has been linked to the development of the palate and lower jaw [6]. The WNT9A role in cancer has not been studied in depth.
WNT9A expression was analyzed in adenocarcinoma pancreatic ductal tissues by immunohistochemistry. WNT9A expresses in non-neoplastic pancreatic ductal cells with a granular staining pattern of supranuclear location, suggestive of location in the endoplasmic reticulum, however, this expression is low or absent in most small ducts (
Adenocarcinoma ductal pancreatic tissues and non-neoplastic adjacent tissues to the tumoral lesion were collected rapidly after surgical resection and frozen at −80° C. until use. Tissue microarrays (TMA) for pancreatic cancer containing tissues of 30 patients, including 2 cores of tumoral tissue for each core of non-neoplastic tissue from the same patient (Array AccuMax A307). The analysis of expression in normal tissues was performed using a TMA Pantomic Normal Tissues MN0661, built with 33 normal tissues in duplicate (Pantomics Inc.).
Total RNA was extracted from PDAC tissues using Trizol reagent (Invitrogen Corp., Carlsbad, Calif., USA) followed by purification using RNeasy mini kit columns (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Human Universal Reference RNA and Human Pancreas Total RNA were purchased from Clontech (Palo Alto, Calif., USA). RNA integrity was assessed by denaturing agar and ultraviolet spectrophotometer. Total RNA from PDAC and non-neoplastic tissues were used as tester and commercial Human Pancreas Total RNA served as driver. First strand cDNA was synthesized from 1 ug of total RNA using Super SMART PCR cDNA Synthesis Kit (Clontech, Palo Alto, Calif., USA) and SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, Calif., USA) following the supplier's protocol. SSH procedures were essentially the same described in the manufacturer's instructions except for the use of a modified nested PCR Primer 5 2R 5′ CTAATACGACTCACTATAGGGCTCGAGCGGCC-3′ in the secondary PCR, which includes a T7 promoter site to carry out In Vitro transcription of the subtractive amplicon. After secondary PCR, subtractive cDNA was purified using E.Z.N.A Cycle Pure Kit (Omega Bio-Tek, Norcross, Ga., USA). For in vitro transcription, 300 ng of the purified cDNA subtractive cDNA was used as template. The newly aRNA was synthesized and labeled with aminoallyl-UTP and AlexaFluor 647 (Invitrogen, Carlsbad, Calif., USA) using the SuperScript Indirect RNA Amplification System (Invitrogen, Carlsbad, Calif., USA). aRNA for the Direct strategy was amplified and labeled directly from 1 ug of total RNA using the same system described above. Labeled aRNA from Direct and SSH-strategy were employed for hybridization on 48.5K Exonic Evidence Based Oligonucleotide (HEEBO) arrays, Purchased from Microarray Inc. (Nashville, Tenn., USA). Prior to hybridization, slides were pre-blocked with 5×SSC, 0.1% BSA and 0.1% SDS. Fluorescent-labeled probe were mixed with 1× hybridization solution (5×SSC, 50% formamide; 0.1% SDS, and 0.01% salmon sperm DNA) and heated at 95° C. for 2 min. Samples were hybridized on microarray slides for 16 hrs at 42° C. Slides were scanned using a ScanArray Gx (Perkin Elmer, Waltham, Mass., USA).
Microarray signal intensity was evaluated by SpotReader Software (Niles Scientific, Portola Valley, Calif., USA). Normalization was performed in R statistical environment using Limma package (www.r-proyect.org). Raw data from individual arrays were processed using standard and normexp background correction [7] and printtiploess normalization [8]. Global scale normalization function using median absolute deviation was used for normalization between arrays [9]. Heatmaps were constructed using MeV software [10].
The following antibodies were used in this study for the validation of the corresponding gene candidate: PLEKHM1 (Human Atlas Protein HPA021558, dilution 1:75); WNT9A (Human Atlas Protein HPA011223; 1:25). Histological sections of 4 um thick sections were mounted on Super Frost slides, incubated at 65° C. for 60 min, dewaxed, blocked with 1% hydrogen peroxide for 10 min and rehydrated by successive incubations in graded alcohols. Antigen retrieval was performed by incubating the slides in a buffer of Tris/EDTA pH 9.0 (10 mM Tris, 1 mM EDTA) and heated in a microwave at 750 W for 10 min. Subsequently, the samples were incubated in serum 1% in TBS buffer to block nonspecific staining. The presence of the antigen was evaluated by incubating the samples for 45 min with primary antibody followed by detection with secondary antibody conjugated to peroxidase complex system EnVision poly-HRP (DAKO) for 45 min. The samples were then incubated with DAB+chromogen (Dako) for 15 minutes for color development and finally contrasted with hematoxylin.
The analysis of immunohistochemical staining (IHC) was performed using the automated ACIS III (DAKO) to digitize and quantify IHC staining. The ACIS III software can recognize separately brown pixels (positive staining) and blue pixels (hematoxylin counterstain). For analysis of the staining TMAs generate a score depending on the intensity of staining and the percentage of cells showing immunoreactivity within the core.
