FIELD OF THE INVENTION
The present invention relates to protein proto-oncogene isoform B expressed 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 are 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 metros. A 3% per annum rise in breast cancer cases in India has been predicted. By 2015 there will be approximately 2.5 lakh 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, ErbB2 have been earlier identified. In spite of recent advances in the assessment of breast cancer risk, through the identification of crucial susceptibility genes (BRCAl/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 al 2005; Ponzon 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 (Baselga 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.

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 proto-oncogene Isoform B 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 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 a protein 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, preferably proto-oncogene Isoform B and a kit for diagnosing cancer, said kit comprising sample containing protein proto-oncogene Isoform B and buffer components.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

Figure 1: Reference master silver stain image showing 17 spots which were found in
breast cancer patients in Parsi population, Hyderabad, India.

Figure 2: Pooled silver stain gel image from breast cancer Parsi patients [01900172 (age76yrs) - 01900257 (age73yrs)].

Figure 3: Pooled silver stain gel image from breast cancer Parsi patients [01900155 (age59yrs)- 01900209 (age68yrs)].

Figure 4: Pooled silver stain gel image from breast cancer Parsi patients [01900052 (age61yrs) - 01900327 (age60yrs)].

Figure 5: Pooled silver stain gel image from control Parsi patients [01900049 (age76yrs)-01900023(age73yrs)].

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

Figure 7: Mass spectra of Proto-oncogene isoform B.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a protein expressed in cancer.

In another embodiment of the present invention, the protein is proto-oncogene Isofonn B acting as biomarker and the cancer is breast cancer.

The present invention also relates to a gene coding for a protein expressed in cancer.

In another embodiment of the present invention, the gene is a proto-oncogene, the protein is proto-oncogene Isoform B and the cancer is breast cancer.

The present invention also relates to a vector comprising a gene coding for a protein expressed in cancer.

In another embodiment of the present invention, the gene is a proto-oncogene, the protein is proto-oncogene Isoform B and the cancer is breast cancer.

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.

In another embodiment of the present invention, the protein is proto-oncogene Isofonn B and the cancer is breast 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 proto-oncogene Isoform B.

The present invention also relates to a kit for diagnosing cancer, said kit comprising sample containing protein proto-oncogene Isoform B and buffer components.

The present invention is based on identifying proto-oncogene isofonn B new protein expression that correlates with breast cancer (Table 1), Proto-oncogene isofonn B was found to be completely absent in control group. The present invention could be useful to provide a method of diagnosing or determining a predisposition to breast cancer in a subject by determining presence of said protein in a patient-derived biological sample, such as blood plasma.

Table 1: MALDI-TOF-MS identification of newly expressed proteins (P < 0.05) in plasma breast cancer samples from Parsi community, Hyderabad, India

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 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.

Example 2:

Experimental strategy and outcome of experiment:

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 (3-1INL, GE Healthcare) with 80mg protein were used for IEF 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). Spot 11 were cut out from gel and further subjected for tryptic digestion. The mass spectra were obtained using HCCA matrix (Sigma) by MALDI- TOF/TOF (AXIMA TOF2 Shimadzu) (Figure 7). The identity of spot 7 (protein proto- oncogene isoform B) was confirmed by submitting mass spectra to PROTEIN PRSOPECTOR-MS-Fit database (Table 1).

Proto-oncogene isoform B is identified as new protein in breast cancer patients (Figure 1: spot no. 7) in comparison to control normal people.

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). Protein spot no. 7 was identified by mass spectometry technique with the help of tryptic digetsion (Figure 7) and it is confirmed as proto-oncogene isoform B using PROTEIN PROSPECTOR-MS-Fit database (Table 1).

Based on these preliminary results, we are planning to conduct experiment 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 dye (Cy5, Cy3 and Cy2) can be run simultaneously. This helps to compare the differential expression of proteins from one gel only.

Protein proto-oncogene isoform B can be either useful as a target for drug discovery in cancer or as a diagnostic biomarker for early diagnosis of predisposed patients.

The Protein proto-oncogene isoform B 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-El-Leil, MK.; Sadekova, S.; Meloche, S.; and Sauvageau, G (2005) Oncogene, 24, 6890-901.

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

3) Ponzon et al, (1998) 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.

We Claim:
1) A protein expressed in cancer,

2) The protein as claimed in claim 1, wherein the protein is proto-oncogene Isoform B 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 a proto-oncogene, the protein is proto-oncogene Isoform B and the cancer is breast cancer.

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

6) The vector as claimed in claim 5, wherein the gene is a proto-oncogene, the protein is proto-oncogene Isoform B and the cancer is breast cancer.

7) 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.

8) The method as claimed in claim 7, wherein the protein is proto-oncogene Isoform B 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 proto-oncogene Isoform B.

10) A kit for diagnosing cancer, said kit comprising sample containing protein proto- oncogene Isoform B and buffer components.

11) A protein expressed in cancer, a gene coding for the protein, a vector comprising the gene, a method of extracting the protein, a method of diagnosing cancer and a kit as substantially described herein with reference to figures.