FIELD OF THE INVENTION

The present invention relates to differentially expressed proteins in breast cancer patients.

BACKGROUND AND PRIOR ART OF THE INVENTION

Pointing to the fact that 7.4 million people died of cancer in 2004, the WHO report, which predicts that 83.2 million more people would die of cancer by 2015. Among women, breast cancer was the most common cause of cancer mortality, accounting for 16% of cancer deaths globally. The WHO 2004 report said that breast cancer, along with cervical, colorectal and oral cancers, were the only types for which screening had shown to reduce mortality. In India, breast cancer is the most common form of cancer in women. One in 26 women in India is expected to be diagnosed with breast cancer in their lifetime. WHO predicts that by 2020, India will be on a par with US and Europe in the incidence of breast cancer — one in seven women. At present, an estimated 100,000 women get diagnosed with breast cancer every year in India. Around 30,000 women die of it annually. If a woman has a mother who has suffered from breast cancer, her risk increases about 3-fold. In India, about 50% patients of breast cancer get diagnosed only at stage 3. In the last 40 years, breast cancer cases have gone up by 30-40% across Indian meters. A 3% per annum rise in breast cancer cases in India has been predicted. By 2015 there will be approximately 2.5 laky new cases of breast cancer in India.

Currently screening mammography is an X-ray examination of the breast in a woman who is asymptomatic (has no symptoms of breast cancer). But real challenge is to detect cancer when the cancerous lump is still too small to be felt or to be diagnosed by mammography. Mammography has a variety of shortcomings. In India there is an acute shortage of mammography machines and associated cost factor to conduct mammography.

Early detection of small breast cancers improves a woman's chances of successful treatment and reduces mortality rates by 30%. If cancer is detected in stage 1, chance of survival is 80% while it is 20% when detected in stage 3. Breast cancer is more treatable and curable when detected early. Thus, better diagnostic tools for blood testing for early detection of presence of breast cancer biomarkers has strong clinical significance in terms of enhancing treatment options for treating breast cancer.

Breast cancer associated biomarkers such as BRCA1, BRCA2, Her-2/neu, PTEN, EN2, P53, C-MYC, and ErbB2 have been earlier identified. In spite of recent advances in the assessment of breast cancer risk, through the identification of crucial susceptibility genes (BRCA1/2, PTEN, P53), these account for less than 5% of all breast cancer cases. The advantages and limitations of these approaches have been discussed in the literature (Martin et at 2005; Pontoon et al 1998; Bradbury et al 2002; Ross et al 2003). Most biomarker research has resulted in findings that are inconclusive or insufficient evidence to justify widespread clinical implementation. Among the few biomarker successes two-estrogen receptor (ER) and HER2/neu—have had major impacts on the way in which breast cancer is treated. However, important issues remain unresolved around standards for measuring and reporting these biomarkers. These biomarkers may not be associated with the more commonly occurring sporadic breast cancers (Basel and Norton, 2002).
The discovery of bona fide protein biomarker underlying sporadic breast cancer development remains a major challenge.

Therefore, there is a pressing need to identify a biomarker present in blood during early development of cancer and is having an association with sporadic and familial breast cancers. Biomarker research must aim to substantially improve patient outcomes by accurately identifying those likely to benefit from specific interventions and sparing those who will probably not benefit from these interventions.

A biomarker is defined in this report as; any measurable cellular, subcellular, or humoral factor that demonstrates the presence of malignancy or malignant potential, or predicts tumor behavior, prognosis, or response to treatment.

In the clinical setting, there are many uses for these cancer-related tests; however, not all of the markers are appropriate for every purpose. As they are generally detectable in all healthy individuals, it is not their presence in serum, but their quantity, that makes tumor markers useful. Also, because non-cancerous cells generally produce low levels of tumor-marker molecules, clinical specificity is usually low and due to the low prevalence of cancer, many false-positive results would occur if they were used to screen the mass population. Therefore, panel of plasma tumor markers are recommended for general screening purposes. In cancer diagnosis, these proteins should never be used in vacuity. It is changes in their concentration, over months and years, which are used to assess disease progression and response to treatment, to monitor a patient for recurrence, or to indicate the aggressiveness of a malignancy. While tumor-marker levels that fall within the "normal" reference interval do not necessarily rule out that the cancer is still present, consecutively rising or decreasing levels are a good indicator of suspecting the cancer in early stage. In addition, the markers can aid in the differential diagnosis and serve as an indicator of the primary site for malignancies that have metastasized.

OBJECTS OF THE INVENTION

The main object of the present invention is to obtain a protein expressed in cancer.

Another object of the present invention is to obtain a protein expressed in cancer,
wherein the protein is differentially expressed acting as biomarker and the cancer is
breast cancer.

Yet another object of the present invention is to obtain a gene coding for a protein
differentially expressed in cancer.

Still another object of the present invention is to obtain a vector comprising a gene
coding for a protein expressed in cancer.

Still another object of the present invention is to obtain a method of extracting a protein expressed in cancer.

Still another object of the present invention is to obtain a method of diagnosing cancer.

Still another object of the present invention is to obtain a kit for diagnosing cancer.

STATEMENT OF THE INVENTION

Accordingly, the present invention relates to differential proteins expressed in cancer; a gene coding for a protein expressed in cancer; a vector comprising a gene coding for a protein expressed in cancer; a method of extracting a protein expressed in cancer, said method comprising steps of: (a) extracting plasma protein from cancer patient; and (b) depleting IgG and albumin abundant proteins followed by desalting to obtain the protein expressed in cancer; a method of diagnosing cancer, said method comprising step of identifying the expression of protein in cancer patient and a kit for diagnosing cancer, said kit comprising sample containing the said protein and buffer components.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

Figure 1: Location of differentially expressed ten proteins in plasma breast cancer
samples from Parsi community, India.

Figure 2: Pooled silver stained gel image from breast cancer Parsi patients [01900172
(age 76 yrs)-01900257 (age 73 yrs)]

Figure 3: Pooled silver stained image from breast cancer Parsi patients [01900155 (age
59yrs)-01900209 (age 68yrs)].

Figure 4: Pooled silver stained image from breast cancer Parsi patients [01900052 (age
61 yrs)-01900327 (age 60yrs)]

Figure 5: Pooled silver stained image from control Parsi patients [01900049 (age
76yrs)-01900023 (age 73yrs)]

Figure 6: Pooled silver stained image from control Parsi patients [01900036 (age
59yrs)-01900020 (age 58yrs))

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification of differentially expressed proteins (up and down regulation) that correlates with breast cancer (Table 1). These proteins could be useful to provide a method of early diagnosis in breast cancer in a subject by detemining presence of said protein/proteins in a patient-derived biological sample, such as blood plasma.

Table 1 MS identification of differentially expressed proteins by peptide mass fingerprinting (P<0.05) in plasma breast cancer samples from Parsi community, India

The present invention also relates to a method of extracting a protein expressed in cancer, said method comprising steps of:

a) extracting plasma protein from cancer patient; and

b) depleting IgG and albumin abundant proteins followed by desalting to obtain the protein expressed in cancer.

The present invention also relates to a method of diagnosing cancer, said method comprising step of identifying the expression of protein in cancer patient, preferably using differentially expressed proteins.

The present invention also relates to a kit for diagnosing cancer, said kit comprising sample containing the said protein and buffer components.

The invention is further elaborated with the help of following examples. However, these examples should not be construed to limit the scope of the present invention.

Example 1:

Experimental design in brief:

The twelve samples (female; 6 cases vs 6 control) were used in this experiment were collected from Hyderabad, India, 2007. Cases and control were selected and pooled based on age matching criteria viz: 50-60yrs, 60-70yrs and >70yrs.

Experimental strategy and outcome of experiment:

Proteomic profiling of blood plasma samples from affected Pars! patients from breast cancer as revealed by 2-D analysis. Plasma protein extract from breast cancer patients and normal patients were initially depleted for IgG and Albumin abundant proteins using
Enchant Multi-Protein Affinity Separation Kit (PALL laboratories) and desalted further by using 2-D clean up kit (GE Healthcare). 18cm IPG strip (3-11NL, GE Healthcare) with 80g protein were used for lEF and standard 12.5% SDS-PAGE for the second dimension. Five Gels were stained with MS compatible silver stain (Figure 2 to Figure 6) and gel images were scanned and analyzed by Image Master 2-D platinum v 6 (GE Healthcare). Five spots (spots no. 7, 11, 13, 14 and 16) were found to be newly expressed protein spots in breast cancer patients (Figure 1).

The rest of other twelve spots were differential expressed spots (Figurel). All 17 spots were removed and further subjected for tryptic digestion. The mass spectra were obtained using HCCA matrix by MALDI-TOF/TOF (AXIMA T0F2, Shimadzu). The identities of proteins were confirmed by submitting mass spectra to PROTEIN PRSOPECTOR-MS-Fit database (Table 1).

3 age-wise pooled gel images from 6 breast cancer samples (Figures 2, 3, and 4) and 2 age-wise pooled control images (Figures 5 and 6) are enclosed. Spot no. 7 newly appeared spot in breast cancer patients (Figure 1).

Based on these preliminary results, an experiment are to be conducted with more than 50 breast cancer samples with the help of differential in-gel electrophoresis (DIGE) technique. Advantage of using DIGE technique is to minimize technical variation. With the help of DIGE technique, 3 protein samples tagged with 3 different fluorescent dyes (Cy5, Cy3 and Cy2) can be run simultaneously. This helps to compare the differential expression of proteins from one gel only.

Protein identified here can either be useful as a target for drug discovery in cancer or as a diagnostic biomarker for early diagnosis of predisposed patients. The protein identified can be isolated and the level of expression can be further altered. This can be accomplished by amplification of the gene coding for the protein using an appropriate vector system.

REFERENCES:

1) Martin, NL.; Saba-EI-Leil. MK.; Sadekova, S.; Meloche, S.; and Sauvageau, G (2005) Oncogene, 24, 6890-6901.

2) Baselga, J. and Norton, L. (2002). CancerCell, 1, 319-322.

3) Ponzon et aL, 0998) Eur. J.Cancer 34 (7):966-967

4) Bradbury et al (2002) Lancet Oncol. 3:2

5) Ross et al (2003) Expert. Rev. Mol. Diagn. 3(5):573-585.

Claim:

1) A protein differentially expressed in cancer.

2) Tile protein as claimed in claim 1, wherein the protein is a differentially expressed protein, preferably Ig gamma-1 chain C region protein, Endothelia converting enzyme-like 1, Immunoglobulin kappa light chain VLJ region, Unnamed protein product, Zinc finger protein 425, Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit), is form CRA_b, Immunoglobulin kappa light chain IGKC, Zinc finger protein 473, B-lymphocyte activation-related protein, Nuclear transcription factor, X-box binding-like 1, Isoform CRA_b and other similar proteins, acting as biomarker and the cancer is breast cancer.

3) A gene coding for a protein expressed in cancer.

4) The gene as claimed in claim 3, wherein the gene is an oncogene, the protein is differentially expressed protein in cancer and the cancer is breast cancer.

5) A vector comprising a gene coding for a protein differentially expressed in cancer.

6) The vector as claimed in claim 5, wherein the gene is an oncogene, the protein is differentially expressed protein in cancer and the cancer is breast cancer.

7) A method of extracting a protein expressed differentially in cancer, said method comprising steps of:

a) extracting plasma protein from cancer patient; and

b) depleting IgG and albumin abundant proteins followed by desalting to obtain the protein expressed differentially in cancer.

8) The method as claimed in claim 7, wherein the protein is a differentially expressed protein in cancer and the cancer is breast cancer.

9) A method of diagnosing cancer, said method comprising step of identifying the expression of protein in cancer patient, preferably Ig gamma-1 chain C region protein, Endothelin converting enzyme-like 1, Immunoglobulin kappa light chain VLJ region, Unnamed protein product. Zinc finger protein 425, Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit), isoform CRA_b, Immunoglobulin kappa light chain IGKC, Zinc finger protein 473, B-lymphocyte activation-related protein, Nuclear transcription factor, X-box binding-like 1, isoform CRA_b and other similar proteins.

10) A kit for diagnosing cancer, said kit comprising sample containing a protein differentially expressed in cancer and buffer components.