WO2015154715A1

WO2015154715A1 - A method of diagnosis, prognosis or treatment of a cancer

Info

Publication number: WO2015154715A1
Application number: PCT/CN2015/076251
Authority: WO
Inventors: He YU; Yi SHEN; Dionyssios KATSAROS
Original assignee: Anpac Bio-Medical Science (Lishui) Co., Ltd.
Priority date: 2014-04-11
Filing date: 2015-04-10
Publication date: 2015-10-15

Abstract

The present invention provides a method of diagnosis, prognosis or treatment of a cancer, or a predisposition thereto, in a subject, comprising the step of determining a level of RNA linc00472 in a biological sample obtained or obtainable from said subject, which level is indicative of said breast cancer, or said predisposition thereto, in said subject.

Description

A method of diagnosis, prognosis or treatment of a cancer

Cross-Reference of Related Application

This application claims priority to U.S. Application No. US 61/978,788, filed April 11, 2014, the contents of which are incorporated herein by reference in their entireties.

Background of the Invention

Breast cancer is one of the most common malignancies in the world. Each year more than a million women are diagnosed with this devastating disease worldwide. Five-year survival rates of the disease are 84％for patients diagnosed with localized disease, but only 24％for those with non-localized disease. Breast cancer is a highly heterogeneous disease that varies widely from person to person at both cellular and molecular levels. The disease also evolves substantially over time in patients after they receive chemo or hormonal therapies. These features impose significant challenges to the success of patient treatment. A large number of studies have been conducted to search for molecular and genomic features that may confer a better means to differentiate breast cancer with different prognosis or characterize patients with distinct response to specific treatment, but the results are not encouraging as no robust biomarkers have been identified so far for better management of breast cancer patients. Thus, more efforts are needed to improve our understanding of the disease as well as our ability to evaluate patients accurately with regard to their survival outlooks and treatment response.

Long non-coding RNAs (lncRNAs) are a group of newly discovered RNA transcripts which are transcribed from the DNA templates by RNA polymerase II, but devoid of evident open reading frames and codes for protein translation. These RNAs appear to have important biologic functions that have not been fully understood. Emerging evidence suggests that lncRNAs may be a key component of functional genome, regulating many crucial cellular fucntions and activities. LncRNAs exert their actions through a number of mechanisms, which include interacting with chromatins to regulate gene expression, modulating epigenetic regulation, and influencing the activities and locations of other functional molecules such as proteins and other RNA species. Studies have shown disruption of lncRNA actions may result in dysregulation of cell functions, leading to various pathological changes and disease conditions including cancer. XIST is the most studied lncRNA which is found to be involved in the hereditary BRCA-deficient breast cancer even though the exact mechanism remains unknown. Another lncRNA HOTAIR, the first discovered trans-regulatory lncRNA, is frequently dysregulated in several types of malignancies, including breast cancer, hepatocellular carcinoma and colorectal cancer. Recent research suggests that measuring HOTAIR levels in tumor samples may have prognostic values for certain forms of cancer. Our own study on HOTAIR also shows that an intergenic DNA methylation site in the HORAIR gene may have biologic implications in regulation of HOTAIR expression, influencing tumor growth and patient response to treatment. These findings suggest that long non-coding RNAs may play important roles in cancer initiation or tumor progression.

High-throughput microarray technologies have been used lately to improve our understanding of molecular mechanisms in tumorigenesis as well as to strengthen our search for biomarkers for disease characterization and patient management. Using a microarray chip, such as the Affymetrix U133 plus 2.0 array, one can simultaneously analyze the expression of over 47,000 transcripts in a single test of one tumor sample, which significantly increases the efficiency of biomarker search. These high-dimensional gene expression data, however, have not been fully evaluated for their value in prediction of disease prognosis, especially with regard to long non-coding RNAs.

Summary of the Invention

Non-coding RNAs (ncRNAs) are a group of RNA transcripts which do not encode proteins, but have important biological functions. Dysregulation of these RNAs affect the functions of normal cells, leading to abnormal cellular activities and adverse health consequences. Small ncRNAs such as microRNAs were initially found to play pivotal roles in regulation of post-transcriptional levels of messenger RNAs. Disruption of this regulation has been linked to a number of human diseases including cancer. Recently, a new class of ncRNAs has been discovered, and their functions are found to be equally important in control of cell functions and activities. This group of ncRNAs is called long ncRNAs (lncRNAs) which have nucleotide sequnces much larger than microRNAs. LncRNAs regulate essential cellular activities such as cell growth, proliferation, differentiation, migration and apoptosis. The potential role of lncRNAs in cancer has recently been suggested by a number of studies, but the findings are still inconclusive, especially with regard to their clinical applications. To further examine the clinical implications of lncRNAs in cancer prognosis and patient treatment, we analyzed several public databases to search for lncRNA expression in association with clinical and pathological features of breast cancer. Our search results in identification of an intergenic lncRNA (lincRNA) , the expression of which was associated with tumor grade, hormone receptor status and lymph node mestastasis. These results are highly consistent across several publicly available databases. Based on the preliminary finding, we designed a real-time RT-PCR, and used the qPCR to analyze the expression of this lincRNA in 348 fresh-frozen tumor samples from patients diagnosed with primary breast cancer. Our analysis shows that the lincRNA was significantly different by disease stage, tumor grade, tumor size and hormone receptor status. High expression was associated with well differentiated tumors (low grade) and less aggressive diseases (positive ER or PR status, early stage disease, smaller tumors and good molecular subtypes) . Furthermore, our survival analysis indicates that patients with high expression had better disease-free and overall survivals compared to those with low expression, and the association with disease-free survival was independent of patients’ clinical and pathological characteristics. Finally, patients who received adjuvant chemo-or hormonal therapy also responded better to the treatment if the lincRNA expression was high. Based on these observations, we propose that measuring tissue levels of this lincRNA in breast (or ovarian) cancer patients can help to predict disease prognosis and patient response to adjuvant treatment, which improves the efficiency of clinical management of breast cancer patients. This lincRNA may also serve as a potential target for cancer.

In one aspect, the present invention provides a method of diagnosis, prognosis or treatment of a cancer, or a predisposition thereto, in a subject, said method including the step of determining a level of RNA linc00472 a in a biological sample obtained or obtainable from said subject, which level is indicative of said breast cancer, or said predisposition thereto, in said mammal.

Preferably, said level of said RNA linc00472 in said biological sample is at least partly decreased compared to a corresponding level of the RNA linc00472 in a biological sample obtained or obtainable from a subject that does not have said breast cancer, or said predisposition thereto.

Preferably, said level of said RNA linc00472 in said biological sample is associated with tumor grade, tumor size, disease stage, hormone receptor status, lymph node mestastasis, disease outcomes and subject’s response to adjuvant treatment.

Preferably, said level of said RNA linc00472 in said biological sample comprising well differentiated tumors is at least partly increased compared to a corresponding level of the RNA linc00472 in a biological sample comprising non differentiated tumors.

Preferably, said level of said RNA linc00472 in said biological sample comprising low tumor grade is at least partly increased compared to a corresponding level of the RNA linc00472 in a biological sample comprising high tumor grade.

Preferably, said level of said RNA linc00472 in said biological sample comprising smaller size of tumor is at least partly increased compared to a corresponding level of the RNA linc00472 in a biological sample comprising bigger size of tumor.

Preferably, said level of said RNA linc00472 in said biological sample at earlier disease stage is at least partly increased compared to a corresponding level of the RNA linc00472 in a biological sample at later disease stage.

Preferably, said level of said RNA linc00472 in said biological sample comprising positive hormone receptor is at least partly increased compared to a corresponding level of the RNA linc00472 in a biological sample comprising negative hormone receptor.

Preferably, said level of said RNA linc00472 in said biological sample comprising positive nodal status is at least partly decreased compared to a corresponding level of the RNA linc00472 in a biological sample comprising negative nodal status.

Preferably, said level of said RNA linc00472 in said biological sample comprising good molecular subtype is at least partly increased compared to a corresponding level of the RNA linc00472 in a biological sample comprising worse molecular subtype.

Preferably, said subject with higher level of said RNA linc00472 having a better disease-free and overall survivals compared to those with lower level.

Preferably, said subject with higher level of said RNA linc00472 having a better response to adjuvant treatment compared to those with lower level.

Preferably, said adjuvant treatment is chemo or hormonal therapy.

Preferably, said subject is human.

Preferably, said cancer is breast cancer and/or ovarian cancer.

In another aspect, the present invention provides a kit for cancer diagnosis, prognosis or treatment, said kit comprising one or more probes, primers, antibodies and/or other reagents for detecting: a RNA linc00472 nucleic acid, or a fragment thereof. Preferably, said cancer is breast cancer and/or ovarian cancer.

Preferably, said kit is suitable for use with the method mentioned above.

In another aspect, the present invention provides a method of designing, engineering, screening for, or otherwise producing a cancer therapeutic agent, said method including the step of identifying a candidate agent which at least partly modulates the expression and/or activity of a RNA linc00472 nucleic acid.

Effective of the Invention

Breast cancer remains to be a devastating disease worldwide. Every year nearly 40,000 breast cancer patients die from the disease in the US alone, accounting for about 17％of women who are diagnosed with the disease each year in the US. Recent research has substantially improved our understanding of the disease. We now know that breast cancer is a highly heterogeneous disease, and moreover it evolves over time when interacting with therapeutic agents. Molecular subtype of breast cancer is a relatively new classification used to predict disease outcome and patient response to adjuvant treatment. This classification provides valuable insights into the biologic behaviors of breast cancer, especially concerning the reaction of tumor cells to targeted therapies. However, to certain patients, accurate prediction of disease prognosis and timely prescription of effective treatment remain to be a challenge. We still need new biomarkers that can provide additional information on the disease with regard to better prediction of disease outcome and treatment response. The burden of breast cancer can be further lessened if we are able to continuously lower the disease mortality through improving patient survival. We believe the current invention will help to achieve this goal because our findings are based on a new class of molecules which seem to have profound biologic influences on cell activities and functions. Non-coding RNAs, especially long non-coding RNAs (lncRNAs) , are discovered quite recently to have an important impact on genome functions. Cell transformation and cancer development are believed to be caused by a collection of genomic abnormalities which occur physically and functionally over time. Any biologic molecules which have extensive influence on genome function are suspected to play important roles in the process of cancinogenesis or tumor growth.

In addition to the significant biologic implication of lncRNAs, our investigation also shows another unique feature which makes the discovery distinct. It is that our reserach finding is based on very consistent observations that are derived from multiple datasets involving diverse patient populations. To investigate the effect of lncRNAs on cancer, we studied several microarray databases which are publicly available for information on high-dimensional gene expression data. Our research results in identification of a unique intergenic lncRNA (linc00472) that is shown consistently to be associated with clinical and pathological features of breast cancer. Furthermore, to confirm our findings, we designed a specific PCR assay and analyzed the expression of this lincRNA in over 300 breast tumor samples. The results of our own study are in complete agreement with our analysis of microarray database, showing that high expression of linc00472 was associated with less aggressive tumors and patients with early stage disease. In addition to that, our data further suggest that this lincRNA was associated with disease recurrence and survival outcomes. Patients with high expression had lower risks of disease recurrence and death. The association with relapse remained significant after other known prognostic and predictive factors were adjusted in the analysis. Finally, patients with high expression of linc00472 had better responses to adjuvant chemotherapy or hormonal therapy. Based on these observations, we believe that linc00472 can serve as a valuable prognostic and predictive biomarker for management of breast cancer patients.

As used herein, the term “linc00472” (SEQ ID NO: 1) refers to a "RefSeq RNA" which can be found in the Reference Sequence (RefSeq) database, a collection of publicly available nucleotide sequences and their protein products built by the National Center for Biotechnology Information (NCBI) . The RefSeq database provides an annotated, non-redundant record for each natural biological molecule (i.e. DNA, RNA or protein) included in the database. Thus, a sequence of a RefSeq RNA is well-known and can be found in the RefSeq database at http: //www. ncbi. nlm. nih. gov RefSeq/, Accession numbers: GI: 223468561. Therefore, the sequence of “linc00472” is readily available from publicly available sources.

Brief Descriptions of the Figures

Figure 1 shows LINC00472 genomic location and analysis by a real-time RT-PCR Figure 2 shows overall survival of 302 breast cancer patients by linc00472 expression (p＝0.003)

Figure 3 shows disease-free survival of 302 breast cancer patients by linc00472 expression (p<0.001)

Figure 4 shows association of linc00472 expression with breast cancer survival

Figure 5 shows association of linc00472 expression with breast cancer survival

Figure 6 shows association of linc00472 expression with breast cancer survival

Detailed Description of the Invention

Taking this opportunity, we screened the high-dimensional gene expression data deposited in the Gene Expression Omnibus (GEO) , and selected 9 different datasets for bioinformatic analysis. After comparing the expression of tens of thousands of genes using an online software GEO2R, we discovered an uncharacterized lncRNA which is an intergenic lncRNA (lincRNA) named linc00472 (Figure 1) . The expression of linc00472 was distinct between tumors and normal tissues, between patients who were dead and alive, as well as between tumors with different tumor grades, hormone receptor status, and lymph node involvement (Part 1 -Association of linc00472 expression with clinical and pathological features in Detailed Description of the Invention) . The expression was also associated with patient survival, i.e. higher expression and better survival outcomes (Part 2 -Association of linc00472 expresson with breast cancer survival in Detailed Description of the Invention) . The findings are relatively consistent across the selected datasets (Part 3 -Meta-analysis forest plot in Detailed Description of the Invention) . In our meta-analysis, molecular subtypes of breast cancer were also associated with linc00472 expression, higher expression in better subtypes. The consistency across studies and disease features underscores the potential significance of this lincRNA in breast tumor biology as well as possible implication in clinical management of breast cancer patients.

To confirm our initial finding, we designed a real-time RT-PCR (Figure 1) and analyzed the expression of linc00472 in 348 fresh frozen tumor samples from women diagnosed with primary breast cancer. The results of our qPCR analysis show that the lincRNA expression was inversely correlated with tumor size, grade and disease stage. Higher expression was associated with smaller tumors, lower tumor grades and earlier stage diseases (Clinical Study Table 1 in Detailed Description of the Invention) . Our analysis also demonstrates a positive correlation between hormone receptor status and linc00472 expression； patients with tumors of positive hormone receptors had higher expression of this lincRNA compared to those with negative hormone receptors (Clinical Study Table 1) . Furthermore, we found the expression associated with disease outcomes. Patients with high expression of linc00472 had better disease-free and overall survival compared to those with low expression (Figure 2, 3 and Clinical Study Table 2 in Detailed Description of the Invention) . More specifically, patients whose tumor expression of linc00472 in the top 25 percentile had more than 60％reduction in risks of relapse and death comparing to those whose tumor expression at the bottom 25 percentile. The risk reduction in relapse wasalso observed when disease stage, tumor grade, receptor status and other clinical features of the patients were adjusted in analysis. Finally, we noticed that the expression of linc00472 was associated with patient's response to adjuvant treatment. Patients with high expression had better responses to adjuvant chemo-or hormonal therapy than those with low expression (Clinical Study Table 3 in Detailed Description of the Invention) . Among the patients who had information on molecular subtypes (n＝204) , linc00472 expression was high in luminal A, luminal B and normal breast-like tumors, but low in triple negative or Her2/neu positive tumors (data not shown) , which is consistent with the findings of other clinicopathological features. Based on these observations, we propose that measuring the expression of linc00472 in breast tumors can guide the assessment of disease prognosis and prediction of patient response to adjuvant treatment (either hormonal therapy or chemotherpary) , and the assessment is independent from the existing clinical, pathological and molecular prognostic and predictive indicators.

Part 1: Association of linc00472 expression with clinical and pathological features

Platform: Affymetrix Human Genome U133 plus 2.0 Array and U133A Array

Method: Using the online software GEO2R, we compared two or more groups of samples in order to identify genes that are differentially expressed across experimental conditions. Results are presented as a table of genes ordered by significance. Genes with the smallest P-values will be the most reliable.

Results:

2. Dataset #1 (13 normal tissues and 20 tumor tissues)

LINC00472 expression in tumor versus normal samples:

2. Dataset #2 (20 normal tissues and 10 tumor tissues)

LINC00472 expression in tumor versus normal samples:

3. Dataset #3 (90 breast cancer samples)

Linc00472 expression in Grade 3 versus Grade 1-2 tumors:

4. Dataset #4 (107 breast cancer samples)

LINC00472 expression in Grade 3 versus Grade 1-2 tumors:

LINC00472 expression in lymph node metastasis positive versus negative:

5. Dataset #5 (111 breast cancer samples)

LINC00472 expression in Grade 3 versus Grade 1-2 tumors:

6. Dataset #6 (327 breast cancer samples)

LINC00472 expression in dead versus alive patients:

LINC00472 expression in lymph node metastasis positive versus negative:

7. Dataset #7 (116 breast cancer samples)

LINC00472 expression in lymph node metastasis positive versus negative:

8. Dataset #8 (54 breast cancer samples)

LINC00472 expression in lymph node metastasis positive versus negative:

9. Dataset #9 (310 breast cancer samples)

LINC00472 expression in Grade 3 versus Grade 1-2 tumors:

Part 2 -Association of linc00472 expression with breast cancer survival

Results:

1. Dataset #1 (GSE20685: 327 breast cancer samples)

Platform: U133 plus 2.0

Figure 4 shows association of linc00472 expression with breast cancer survival and the analysis are:

2. Dataset #2 (GSE25055: 310 breast cancer samples)

Platform: Affymetrix Human Genome U133A Array

Figure 5 shows association of linc00472 expression with breast cancer survival and the analysis are:

3. Dataset #3 (61 breast cancer samples)

Platform: Affymetrix Human Gene 1.0 ST Array

Figure 6 shows association of linc00472 expression with breast cancer survival, and the analysis are:

Part 3 -Meta-analysis forest plot

Part 4:

Clinical Study Table 1 -Association of linc00472 expression with clinical and pathological features of breast cancer

Clinical Study Table 2 -Association of linc00472 expression with disease-free and overall survival

Clinical Study Table 3 -Linc00472 expression and patient response to adjuvant treatment

Part 5: LINC00472 expression in ovarian cancer:

Our work also suggested that long non-coding RNA LINC00472 expression may have prognostic value in the clinical management of breast cancer. As an extension of our research, we examined LINC00472 expression in ovarian cancer and evaluated their associations with clinical and pathologic features and patient survival. The findings are in agreement with that in breast cancer, indicating that high LINC00472 expression was associated with less aggressive ovarian tumors (Table 4) and more favorable disease outcome (Table 5) .

Table 4. Association of LINC00472 with clinic pathological factors in our study

Table 5. Association of LINC00472 with ovarian cancer survival in our study

*adjusted for age, disease stage, and Tumor grade.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. All publications referenced herein are incorporated by reference in their entireties.

Claims

A method of diagnosis, prognosis or treatment of a cancer, or a predisposition thereto, in a subject, said method including the step of determining a level of RNA linc00472 a in a biological sample obtained or obtainable from said subject, which level is indicative of said breast cancer, or said predisposition thereto, in said subject.
The method of Claim 1 , wherein said level of said RNA linc00472 in said biological sample is at least partly decreased compared to a corresponding level of the RNA linc00472 in a biological sample obtained or obtainable from a subject that does not have said breast cancer, or said predisposition thereto.
The method of Claim 1, wherein said level of said RNA linc00472 in said biological sample is associated with tumor grade, tumor size, disease stage, hormone receptor status, lymph node mestastasis, disease outcomes and subject’s response to adjuvant treatment.
The method of Claim 3, wherein said level of said RNA linc00472 in said biological sample comprising well differentiated tumors is at least partly increased compared to a corresponding level of the RNA linc00472 in a biological sample comprising non differentiated tumors.
The method of Claim 3, wherein said level of said RNA linc00472 in said biological sample comprising low tumor grade is at least partly increased compared to a corresponding level of the RNA linc00472 in a biological sample comprising high tumor grade.
The method of Claim 3, wherein said level of said RNA linc00472 in said biological sample comprising smaller size of tumor is at least partly increased compared to a corresponding level of the RNA linc00472 in a biological sample comprising bigger size of tumor.
The method of Claim 3, wherein said level of said RNA linc00472 in said biological sample at earlier disease stage is at least partly increased compared to a corresponding level of the RNA linc00472 in a biological sample at later disease stage.
The method of Claim 3, wherein said level of said RNA linc00472 in said biological sample comprising positive hormone receptor is at least partly increased compared to a corresponding level of the RNA linc00472 in a biological sample comprising negative hormone receptor.
The method of Claim 3, wherein said level of said RNA linc00472 in said biological sample comprising positive nodal status is at least partly decreased compared to a corresponding level of the RNA linc00472 in a biological sample comprising negative nodal status.
The method of Claim 3, wherein said level of said RNA linc00472 in said biological sample comprising good molecular subtype is at least partly increased compared to a corresponding level of the RNA linc00472 in a biological sample comprising worse molecular subtype.
The method of Claim 3, wherein said subject with higher level of said RNA linc00472 having a better disease-free and overall survivals compared to those with lower level.
The method of Claim 3, wherein said subject with higher level of said RNA linc00472 having a better response to adjuvant treatment compared to those with lower level.
The method of Claim 12, wherein said adjuvant treatment is chemo or hormonal therapy.
The method of Claim 1, wherein said subject is human.
The method of Claim 1, wherein said cancer is breast cancer and/or ovarian cancer.
A kit for cancer diagnosis, prognosis or treatment, said kit comprising one or more probes, primers, antibodies and/or other reagents for detecting: a RNA linc00472 nucleic acid, or a fragment thereof.
The kit of Claim 16, wherein said kit is suitable for use with the method of any one of Claims 1-15.
A method of designing, engineering, screening for, or otherwise producing a cancer therapeutic agent, said method including the step of identifying a candidate agent which at least partly modulates the expression and/or activity of a RNA linc00472 nucleic acid.