US20140295459A1

US20140295459A1 - Markers for identifying tumor cells, methods and kit thereof

Info

Publication number: US20140295459A1
Application number: US14/304,432
Authority: US
Inventors: Manjiri Bakre
Original assignee: Oncostem Diagnostics (Mauritius) Pvt Ltd
Current assignee: Oncostem Diagnostics (Mauritius) Pvt Ltd
Priority date: 2011-03-27
Filing date: 2014-06-13
Publication date: 2014-10-02
Also published as: JP2014515821A; KR101621036B1; CN103562723A; EP2691776A4; AU2012235767A1; AU2012235767B2; US20120244556A1; US8772455B2; EP2691776A1; WO2012131564A1; SG193630A1; CN103562723B; KR20140015500A

Abstract

The present disclosure relates to a combination of biological markers for identification of prognosis of cancer. The present disclosure further relates to a method of identifying the said markers, a method of predicting prognosis and a method of planning personalized treatment for cancer. The present disclosure further relates to a kit/test comprising the antibodies against/other methods of detecting said markers for the said prediction.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 13/431,919, filed on Mar. 27, 2012, which claims the benefit of the filing date of Indian Application No. 2835/CHE/2010, filed Mar. 27, 2011, the contents of which are incorporated by reference herein.

TECHNICAL FIELD

BACKGROUND

Chemotherapy/Radiotherapy

In the field of oncology, the detection, identification and characterization of cancer cells is an important aspect of diagnosis. Of the many challenges of medicine, none has had a more controversial beginning or has experienced more hard-fought progress than the treatment and cure of cancer. Effective treatment for most patients needed to reach every organ in the body to pin down the metastatic disease. More than 70% of cancer patients undergo chemo/radio therapy.
Despite the path breaking progress in oncology therapy from multiple angles, cancer cure still remains elusive. Advanced solid malignancies remain therapeutic challenges despite maximal therapy, in part, due to the development of resistance to radiation and chemotherapy. Eg Glioblastomas are among the most lethal of cancers with current therapies offering only palliation. Standard-of-care for glioblastoma consists of surgical resection, ionizing external beam irradiation, and chemotherapy. Radiotherapy has been the most effective nonsurgical treatment modality yet recurrence is essentially universal.
However, majority of patients undergoing chemo/radio therapy suffer from un-necessary severe side effects of the treatment. In addition many patients also show resistance to the treatment resulting in treatment failure.
Thus, there exists a need to develop diagnostic tests that can determine a priori the effectiveness of the prescribed chemo/radio therapy. Today, decisions for treatment are made on the basis of clinical parameters such as tumor histology, tumor volume as well as tumor stage and increasingly biological imaging techniques. Radiation/chemotherapy doses and schedules as well as combinations with drugs are prescribed as empirical class solutions under consideration of the tolerance limits of surrounding normal tissues. Since the current standard treatments prescriptions do not take in to account the heterogeneity/individuality of a tumor the therapies fail often and the patient undergoes un-necessary side effects.
Tumors typically have two kinds of cells, CSCs and tumor cells. CSCs constitute only a part of tumors and usually in minority. The bulk of tumor is made up of tumor cells which constantly divide and make the solid big mass called as ‘tumor’.
On the other hand CSCs are quiescent cells which hardly divide and have the ability to self-renew, i.e. divide in such a way that they make a daughter cell which is a perfect copy of them-selves plus make a cells which can go and differentiate into various cells of a particular tumor (Ref: The Biology of Cancer Stem Cells, Neethan A. Lobo et al., Annu. Rev. Cell Dev. Biol. 2007. 23:675-99 and The theoretical basis of cancer-stem-cell-based therapeutics of cancer: can it be put into practice?, Isidro Sanchez-Garcia et al., BioEssays 29:1269-1280)
Chemo/radiotherapies work towards curtailing tumor size by targeting and killing fast dividing cells. Since CSCs hardly divide they do not get killed by these therapies and survive to make a tumor again, which is called as ‘relapse’ of a cancer. (Ref: Chemotherapy and Cancer Stem Cells, Jeremy N. Rich et al. Cell Stem Cell 1, October 2007; Identification of Selective Inhibitors of Cancer Stem Cells by High-Throughput Screening, Piyush B. Gupta et al., Cell 138, 1-15, Aug. 21, 2009; Identification and targeting of cancer stem cells, Tobias Schatton et al., BioEssays 31:1038-1049; TUMOUR STEM CELLS AND DRUG RESISTANCE, Michael Dean et al., Nature Reviews, cancer, Volume 5, April 2005; Cancer stem cells in solid tumors, Patrick C. Hermann et al., Seminars in Cancer Biology (2008)).
From the above references it can be seen that the CSCs have been isolated from fresh tumors based on certain cell surface markers that they have, using FACS (Fluorescent Activated Cell Sorting). These isolated CSCs as few as 100-200 cells were sufficient to initiate a new tumor as against up to 10000-20000 non CSCs/bulk tumor cells. (Ref: Prospective identification of tumorigenic breast cancer cells, Muhammad Al-Haij et al., PNAS, Apr. 1, 2003, vol. 100_no. 7_—3983-3988; Identification of a Cancer Stem Cell in Human Brain Tumors, Sheila K. Singh et al., CANCER RESEARCH 63, 5821-5828, Sep. 15, 2003; Identification of human brain tumour initiating cells, Sheila K. Singh et al., NATURE|VOL|432|18 Nov. 2004). CSCs were identified in mid 90s in blood cancers and first time in solid tumors in 2003 from breast cancers, followed by brain and all other cancers)
Presence of CSCs and lack of drugs directed against them could be one reason cancer cure has been eluding us. Multiple pharmas and biotechs are directing their efforts to invent drugs which will specifically target these CSCs in a tumor to prevent cancer relapse etc. Many of these drugs are in clinical trials.
The resistance to chemo/radio therapy and the treatment failure is due to presence of what are called Cancer Stem Cells (CSC) in the tumor. Tumors are heterogeneous in nature and contain 2 kinds of cells, cancer stem cells and tumor cells which form the bulk of the tumor. While it has been recognized for a long time that not all tumor cells have the potential to initiate a new tumor, or a recurrence after treatment, only recently methodological advances have emerged that eventually allowed identification of CSCs and to investigate their biology. CSCs have been prospectively isolated from a growing number of human cancers, including leukemias and tumors of the breast, brain, colon, head & neck and pancreas. For different tumors it has been shown that transplantation of CSC subpopulations led to higher tumor take rates when compared to unsorted populations from the same tumor.
A CSC is defined as a cell within the tumor that possesses the capacity to self-renew and to generate the heterogeneous lineages of all cancer cells that comprise a tumor. This implies that CSCs are possibly a small subpopulation of rumor cells which are able to expand the CSC pool or differentiate into cancer progenitor cells by symmetric or asymmetric division. However, this point is tumor dependent and in melanomas CSCs contributed to a significantly higher percentage of total tumor cells. The non-stem cells constitute the bulk of all cancer cells within the tumor, but have only limited proliferative potential and are non-tumorigenic.

Properties of CSCs:

CSCs are quiescent cells present in the tumor and thus are functionally different from the rapidly proliferating tumor cells which make up the bulk of the tumor. CSCs by virtue of being quiescent in nature are able to resist the ‘desirable effects’ chemo/radio therapy well as these therapies target the rapidly dividing cells of the tumor. In addition CSCs are endowed with multiple ion channels/transports, higher hypoxia tolerance and diffrential gene expression, etc. which contribute to their chemo/radio resistance phenomenon. An increasing body of data suggests biological differences of CSCs and non-CSCs are crucial to respond to the standard therapies and most pharmas are using these differences to design rational drugs against CSCs. For failure of radiotherapy and chemotherapy treatments, one underlying reason might be a low efficacy of current treatments on eradication of cancer stem cells (CSCs). Growing evidence indicating that CSCs are resistant to cytotoxic/radiation therapies and may thus contribute to treatment failure.
The current technologies that exist in the field similar to the instant disclosure include Oncotype Dx, and Mammaprint. These are similar but they do not detect presence of CSCs. They assess presence of ER/PR and Her-2-neu pathways in patients to assess if a patient needs post-operative chemotherapy. Currently there are no diagnostic tests which detect presence of CSCs in tumors and hence cannot predict relapse time and usefulness of chemo and radiotherapy. In addition, current methods do not offer help in choosing a particular chemotherapy drug/combination.
From a clinical point of view, the direct consequence of this concept is that cancer therapy can cure a patient only if all CSCs are eliminated and that a single surviving CSC can cause a recurrence or metastasis. In addition it also implies that if the tumor is assessed for presence of CSCs before prescribing the chemo/radio therapy there is a fair chance that the oncologist can predict the effectiveness of the treatment, manage the cancer treatment better and reduce the unwanted side effects of the treatment to patients in cases when the therapy is ineffective. Since the discovery of first solid tumor CSCs in 2004 there have been great advances in CSC biology. Predictive tests for content, distribution and sensitivity of CSCs, microenvironmental CSC niches and signatures should be used to allow CSC-based individualized tailoring of therapy within the class solutions.

BRIEF SUMMARY OF THE DISCLOSURE

Accordingly, the present disclosure relates to biological marker selected from a group comprising CD44, CD24, ABCG2, ESA, ABCC4, CD133, Oct-4, Sox-2, APC, β-catenin and P-cadherin or any combination thereof for prognosis of cancer; a kit for prognosis of a subject having cancer or suspected of having cancer, said kit comprising antibody against biological marker selected from a group comprising CD44, CD24, ABCG2, ESA, ABCC4, CDI33, Oct-4, Sox-2, APC, β-catenin and P-cadherin or any combination thereof, optionally along with organic solvent, reagent, secondary antibody, enzyme for performing immunohistochemistry and instruction manual; a method of identifying biological marker on cells in a biological sample being or suspected of being a tumor, said method comprising acts of a) collecting, fixing, sectioning and treating the biological sample with organic solvent, followed by antigen retrieval using predetermined sample dilutions and b) adding primary antibody against biological marker selected from a group comprising CD44, CD24, ABCG2, ESA, ABCC4, CD133, Oct-4, Sox-2, APC, β-catenin and P-cadherin or any combination thereof and adding secondary antibody conjugated with an enzyme and reagents for obtaining a colored reaction or fluorescence for identifying the biological marker; a method of prognosis of a subject having cancer or suspected of having cancer, said method comprising acts of a) collecting biological sample from the subject and identifying expression or absence of receptors selected from a group comprising estrogen receptor, progesterone receptor and Her-2-neu receptor or any combination thereof in cells of the sample to obtain expression identified cells, b) carrying out immunohistochemistry analysis on the cells of step (a) with antibody against biological marker selected from a group comprising CD44, CD24, ABCG2, ESA, ABCC4, CD133, Oct-4, Sox-2, APC, β-catenin and P-cadherin or any combination thereof and c) identifying expression or absence of the receptors and the one or more biological markers in cells of the sample and correlating the identification of the marker with predictive outcome reference table for predicting the prognosis of the subject; and a method of treating cancer, said method comprising acts of a) identifying biological marker selected from a group comprising CD44, CD24, ABCG2, ESA, ABCC4, CD133, Oct-4, Sox-2, APC, β-catenin and P-cadherin or any combination thereof, on tumor cells in a biological sample and predicting prognosis of a subject having cancer or suspected of having cancer and b) based on the prediction, designing a cancer therapy for suppression of the cancer.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to, biological marker selected from a group comprising CD44, CD24, ABCG2. ESA, ABCC4, CD133, Oct-4, Sox-2, APC, β-catenin and P-cadherin or any combination thereof for prognosis of cancer.
In an embodiment of the present disclosure, the cancer is breast cancer.
In another embodiment of the present disclosure, the marker is located on tumor cells of the cancer at locations selected from a group comprising cell membrane, cytoplasm, nucleus, and nuclear membrane or any combination thereof.
The present disclosure, further relates to a kit for prognosis of a subject having cancer or suspected of having cancer, said kit comprising antibody against biological marker selected from a group comprising CD44, CD24, ABCG2, ESA, ABCC4, CD133, Oct-4, Sox-2, APC, β-catenin and P-cadherin or any combination thereof, optionally along with organic solvent, reagent, secondary antibody, enzyme for performing immunohistochemistry and instruction manual.
The present disclosure, further relates to a method of identifying biological marker on cells in a biological sample being or suspected of being a tumor, said method comprising acts of:

- a) collecting, fixing, sectioning and treating the biological sample with organic solvent, followed by antigen retrieval using predetermined sample dilutions and adding primary antibody against biological marker selected from a group comprising CD44, CD24, ABCG2, ESA, ABCC4, CD133, Oct-4, Sox-2, APC, β-catenin and P-cadherin or any combination thereof; and
- b) adding secondary antibody conjugated with an enzyme and reagents for obtaining a colored reaction or fluorescence for identifying the biological marker.

In an embodiment of the present disclosure, the cancer is breast cancer.
In another embodiment of the present disclosure, the organic solvent is selected from a group comprising alcohol and xylene or any combination thereof.
In yet another embodiment of the present disclosure, the collecting, fixing, sectioning and treating is carried out under predetermined conditions by conventional immunohistochemistry technique.
The present disclosure further relates to a method of prognosis of a subject having cancer or suspected of having cancer, said method comprising acts of:

- a) collecting biological sample from the subject and identifying expression or absence of receptors selected from a group comprising estrogen receptor, progesterone receptor and Her-2-neu receptor or any combination thereof in cells of the sample to obtain expression identified cells;
- b) carrying out immunohistochemistry analysis on the cells of step (a) with antibody against biological marker selected from a group comprising CD44, CD24, ABCG2, ESA, ABCC4, CD133, Oct-4, Sox-2, APC, 1-catenin and P-cadherin or any combination thereof; and
- c) identifying expression or absence of the receptors and the one or more biological markers in cells of the sample and correlating the identification of the marker with predictive outcome reference table for predicting the prognosis of the subject.

In an embodiment of the present disclosure, the immunohistochemistry analysis is carried out by conventional method and wherein the identification of markers is carried out by visualizing a colored reaction or fluorescence obtained at completion of the method due to staining of the cells from the sample.
In another embodiment of the present disclosure, the correlating is based on parameters selected from a group comprising percentage of staining, intensity of staining and location of staining or any combination thereof; and wherein the location of the staining is selected from a group comprising cell membrane, cytoplasm, nucleus, and nuclear membrane or any combination thereof.
In yet another embodiment of the present disclosure, the correlating comprises multiplying the percentage of staining with the intensity of staining to arrive at a predictive score in order to predict the prognosis as being good or bad depending on the location of expression of the biological marker.
In still another embodiment of the present disclosure, the predictive score is selected from a group comprising low score ranging from about 1 to about 80, moderate score ranging from about 81 to about 150 and high score ranging from about 150 to about 300.
In still another embodiment of the present disclosure, the predictive outcome reference table is individually or a combination of tables selected from a group comprising 1, 1A, 2, 2A, 3, 3A, 4, 4A, 5, 6, 6A, 7, 7A, 8, 9 and 10 or any combination of tables thereof.
The present disclosure further relates to a method of treating cancer, said method comprising acts of:

- a) identifying biological marker selected from a group comprising CD44, CD24, ABCG2, ESA, ABCC4, CD133, Oct-4, Sox-2, APC, β-catenin and P-cadherin or any combination thereof, on tumor cells in a biological sample and predicting prognosis of a subject having cancer or suspected of having cancer; and
- b) based on the prediction, designing a cancer therapy for suppression of the cancer.

The present disclosure is a diagnostic test that assesses the tumor sample using certain CSC specific markers using immunohistochemistry and reverse transcription polymerase reaction (RT-PCR) as a technique to find out the presence of CSCs/drug resistant cells in the given tumor which are indicative of the response of the patient to standard therapies. Examples of markers which includes but is not limited to by the following markers: CD44, CD133, CD24, Oct 4, Sox2, and ion transporters/channels present on CSCs such as the ABC family of transporters namely ABCG2 and ABCC4. ESA, APC, P-cadherin, B-catenin (phospho, total and unphospho).
The present disclosure has utility in the field of oncology for the early detection of tumors. The diagnosis/prognosis of a possible cancer will help oncologist in planning the chemo and prescribing alternate targeted treatment. The patient will be further spared from unwanted side effects of the expensive treatment.
The present disclosure also relates to markers used to identify the CSCs and a combination of these markers and methodologies to detect such Cancer Stem Cells (CSCs).
A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the disclosure.
The following examples represent various markers which may be used either individually or in combination with each other for prognosis of breast cancer. The examples provided herein illustrate the kinds of combinations which are possible for analysing and arriving at prognosis of a subject having or suspected of having cancer. The tables provided herein are combined for the sake of representation and clarity as to how to arrive at an interpretation by perceiving either a combination of markers or the markers individually. Any table from any combination illustrated herein may be used either single or in combination with any other marker provided herein for an interpretation which may not have been explicitly illustrated by way of the examples herein. All such possible combinations and interpretations of such combinations fall within the scope of the instant disclosure. A person skilled in the art would therefore be able to envisage the prognosis of a subject by way of examining the sample, arriving at results, and comparing the results with the interpretation of the markers provided herein, for accurate prognosis and strategize the course of further treatment based on such prognosis.

EXAMPLES

In the present disclosure, IHC (immunohistochemistry) is performed with two antibodies against CD44 and CD24 to understand if any CSC signature in these patients can be seen. The signature being looked for is CD44⁺/CD24^−low
However, these two markers are known to be present on CSCs based on FACS analysis, which only detects surface/membrane associated expression. FACS can only detect membrane expression, so even though CD24 expression is seen in cytoplasm in many cases, it is considered as negative or it is marked as cytoplasmic. However, IHC can detect presence of CD24 in both membrane and cytoplasm and in many cases expression can be seen in the cytoplasm in addition to membrane or at times in cytoplasm only. Therefore, in cases where the expression is entirely cytoplasmic as disclosed in some of the tables of the instant disclosure, the results in them can be considered as CD24− (CD24 negative) if detected using FACS, unlike using IHC in the instant disclosure. Because of detection based on IHC in the instant disclosure, membrane as well as cytoplasmic, and nuclear membrane expression is detected.
In an embodiment of the present disclosure, it is to be noted that cancer/tumour node status/stage N0 also at times reflects bad outcome. However, as common practice, usually increase in node stage Eg: N2 or N1 reflect bad outcome, whereas NO is considered to have no metastasis to the adjoining nodes and thus resulting in good outcome/having less severe form of cancer/tumor. Nonetheless, table 10 (patient history) disclosed in the instant disclosure clearly depicts that there are bad outcomes with lower node stage tumors and vice versa as well. Hence, the node status cannot be completely relied upon and thus a deeper analysis of the sample is required since good or bad prognosis cannot be solely carried out based on such priorly known techniques, as node status identification. Therefore, the instant disclosure clearly reflects that need and importance of identification of markers present on cancer cells which may be combined with previously known techniques such as the tumor node status. Thus, as is apparent from the instant disclosure, a combination of the % of cells stained, intensity of staining and location of the respective markers is to be considered for arriving at the correct prognosis of a patient sample.
In another embodiment of the present disclosure, it is also to be noted that the markers reflected below are to be identified, not only in the cancer stem cells/cancer initiating cells/tumor initiating cells of the patient sample, but instead also to encompass all the tumor cells of the sample.

Example 1

The technique used for the identification of markers in a patient sample in the present disclosure involves Immunohistochemistry (IHC) or RT-PCR.
The process of identifying the Cancer Stem Cells from the tumor is carried out using the technique as below:
In an embodiment of the present disclosure, the organic solvents, reagents, secondary antibody and enzymes mentioned in the detailed protocol below are only for the purposes of illustration and should not be construed to be limiting in nature. The instant disclosure envisages and encompasses all possible combinations and alternatives of such solvents, reagents, secondary antibody and enzymes known and commonly used by a person skilled in the art.
To further provide clarity on the process employed in the present disclosure, the detailed protocol of the immunohistochemistry is provided as below:

IHC Protocol

A] Coating Slides and Cutting a Section of FFPE Block:

- 1. Wash new glass slides with tap water.
- 2. Dip the slides with 1% acid alcohol solution (1 ml HCl 99 ml of 70% ethanol) for 5 mins.
- 3. Wash the slides with distilled water once to make sure the acid alcohol is washed off.
- 4. Dry the slides.
- 5. Dip the slides in 10% Poly-L-lysine solution (PLL) in water for 15 minutes at room temperature (RT).
- 6. Remove the slides and dry them at RT.
- 7. Take a 3-5 micron section of the FFPE tumor block on these PLL coated slides using Leica Microteome.
- 8. Incubate the section at 60 C for 3 hrs or over night.

B] De-Paraffinization Protocol:

- 1. Dip the slides with tumor section in Xylene-I for 10 mins at RT.
- 2. Dip the slides in Xylene-II for 10 mins at RT.
- 3. Dip the slides in Xylene-III for 10 mins at RT.
- 4. Dip the slides in 100% alcohol for 5 mins at RT.
- 5. Dip the slides in 100% alcohol for 5 mins at RT.
- 6. Dip the slides in 70% alcohol for 5 mins at RT.
- 7. Dip the slides in 70% alcohol for 5 mins at RT.
- 8. Dip the slides in D/W for 5 mins at RT.
- 9. Dip the slides in 3% H2O2 in methanol at RT for 20-30 mins.

C]Antigen Retrieval and Immunostaining:

This step will be different for each antibody/marker. Following the protocol for antibody A and B. The buffer for antigen retrieval for both antibodies is as follows: 1 mM EDTA+10 mM Tris-Cl buffer at pH 7.4

- 1. For antibody A: The antigen is best retrieved by using pressure cooker conditions in Tris-EDTA buffer 3 whistles (depending on your pressure cooker, the number whistles can be adjusted. Please avoid over cooking).

For antibody B: The antigen is best retrieved by using microwave conditions as follows:

- 800 Watts for 6 mins
- 800 watts for 6 mins
- 200 watts for 15 mins

However, one can also try pressure cooker method as above.

- 2. After the antigen retrieval, cool the slides to RT in the buffer itself for 30 mins.
- 3. Wash the slides in distilled water for 2 mins.
- 4. Wash the slides in TBST (Tris-Buffered Saline with 0.1% Tween 20) for 5 mins.
- 5. Wash the slides in TBST for 5 mins.
- 6. Block the slides with 3% BSA solution/if you are using a kit for the secondary antibody then use the blocking given by the kit as appropriate.
- 7. Add primary antibody diluted in buffer (see below) for 60 mins at RT.
- 8. Wash the slides in TBST for 5 mins at RT.
- 9. Repeat step #8 twice more.
- 10. Add secondary antibody i.e. HRP conjugated anti-mouse antibody at the appropriate dilution as per kit. Incubate at RT for 30-60 mins.
- 11. Wash in TBST for 5 mins at RT.
- 12. Repeat step #11 twice more.
- 13. Add the substrate DAB etc. as per the recommendation by the kit.
- 14. Wash off the excess substrate as per kit.
- 15. Counter stain with haematoxylin.
- 16. Wash off excess stain by dipping in 100% alcohol for 5 mins.
- 17. Repeat step #16.
- 18. Dry the slides.
- 19. Wash in xylene once for 5 mins at RT.
- 20. Mount in DPX/appropriate mounting medium.
- 21. Score/grade the slides as per the sheet.

Recipes for Solutions:

- 1. Acid alcohol: 1 ml of cone HCl+99 ml of 70% alcohol.
- 2. Antigen retrieval buffer: 10mMEDTA pH 8.0+10 mM Tris-Cl pH 7.4.
- 3. Primary Antibody dilution buffer: 10 mM Tris-Cl pH7.4+0.9% NaCl+0.5% BSA.
- 4. Blocking buffer: 3-5% BSA in Tris-Cl pH 7.4 OR use blocking solution given by the kit OR as per your protocol.
- 5. TBST: 10 mM Tris-CL pH 7.4+0.9% NaCl+0.1% Tween-20.

Further, the specific sample dilutions, organic solvents and experimental conditions employed for antigen retrieval are provided as below, only for the purposes of illustration. A person skilled in the art would be able to comprehend the various parameters and conditions that are employed and thus all the various alternatives and substitutes known to the person having skill in the art for arriving at a protocol for such analysis are also under the purview of the instant disclosure:

- Antibody A—Pressure cooker (PC), Tris-EDTA (TE) pH 7.4 buffer, 2 whistles at setting 2 and 1 whistle at setting 1 (Steamed for 20 mins at about Temp. 100° C.+10 mins at 121° C. at pressure about 10 psi); 1:1400 dilution.
- Antibody B—Microwave, 50 mM Citrate buffer pH 6.0; Microwave/Cit 6.0/7 min at ‘high’ setting-700 W; 5 min at High-750 W; 15 min at ‘defrost’ setting-200 W; Dilution 1:50.
- Antibody C—2N HCl, RT; 1:300 dilution.
- Antibody E—Water bath at 90 C for 30 mins in 50 mM Citrate buffer pH 6.0; Dilution 1:600.
- Antibody F—Pressure Cooker, Citrate buffer pH-6: 1 whistle at 1 setting on the heater and boil 20 mins at 1 setting (Steamed for: 40 mins at about 100° C. pressure+10 mins at about 10 psi at 121° C.); Dilution 1:200.
- Antibody G—2N HCl, RT 15 minutes, 1:50 dilution.
- Antibody H—PC/Cit 6.0/2 whistles at 2 setting & 1 whistle at 1 setting; Dilution 1:1000.
- Antibody I—Pressure cooker (2 whistles at 2 minutes, 1 whistle at 1 setting), Cit 6.0; Dilution 1:1000.
- Antibody J—Pressure Cooker, Cit 6.0—Pressure cooker (2 whistles at 2 minutes. 1 whistle at 1 setting); Antibody dilutions of 1:50.
- Antibody K—Water bath at 90 C for 30 minutes in Citrate, pH 6.0; 1:100 dilutions.
- Antibody L—Pressure cooker (2 whistles at 2 minutes, 1 whistle at 1 minute), TE buffer at 7.4 pH, prediluted antibody from company.
- Antibody O—Pressure cooker in Citrate buffer pH 6.0 at 2 whistles at 2 setting and 1 whistle at 1 setting.

Example 2

The antibodies being used in the instant disclosure are selected from one or more of the following:

- CD44: (Ref: CD44 Std./HCAM Ab-4 (Clone 156-3C11))
- CD24: (Ref: (CD24 (GPI-linked surface mucin) Ab-2 (Clone SN3b)
- ABCG2: (Ref: NB-11093511 from Novus Biologicals)
- ESA: (Ref: Novocastra Epithelial Specific Antigen)
- ABCC4: (Ref: ABCC4 monoclonal antibody (M03), clone 1B2)
- CD133: (Ref: PAB-12663 from Abnova)
- Oct-4: (Ref: OCT4 Antibody (NB110-90606))
- Sox-2: (E-18600 from Spring Biosciences)
- APC: (Ref: Adenomateous Polyposis Coli gene product)
- P-cadherin: (Ref: Anti-P cadherin antibody [56C1], prediluted (ab75442))
- B-catenin: (Ref: Anti-Active-β-Catenin (anti-ABC), clone 8E7) JBC-1870057.

In all the tables below from tables 1-9, the intensity of cells stained, have the values ranging from 1 to 3. Here, intensity 1=very light brown colour of slide stained, intensity 2=medium brown colour of slide stained and intensity 3=dark brown colour of slide stained. Intermediate colour observation is referred as 1-2 or 2-3 etc.
Further, the tables provided herein selected from a group comprising table 1, 1A, 2, 2A, 3, 3A, 4, 4A, 5, 6, 6A, 7, 7A, 8, 9 and 10, either individually or any combination of tables thereof may be used by a person skilled in the art to interpret and arrive at relevant results for prediction of prognosis for a subject having or suspected of having cancer. These tables individually or in combination may also be referred to as predictive outcome reference table in the instant disclosure.

TABLE 1

		Antibody A	Antibody B
	Serial No	CD44	CD24

	of Patients	% of cells	intensity	location	% of cells	intensity	location

ER+/PR+	1	20	1-2	M	60	1.5	M
GOOD					80	1.5	C
OUTCOME	2	20	2-3	M	10	1.5	M
					40	1.5	C

3

Negative

55

1.5

C

	4	35	2	M	65-70	1.5	C
	5	5	3	M	85	3	M
					50	2	C
	6	40	2-3	M	80	2-3	C
					20	1-2	M
	7	40	3	M	35-40	3	C
					25-30	2	M
	8	10	1.5	C	75	2	C
					35-40	1.5	M
	9	20	3	M	25	1.5	M
					15	EACH	C
ER+/PR+	10	20	1.5	M	95	1.5	C
BAD		60	2	C
OUTCOME	11	95	3	M	<10	1	M
					90	1	C
	12	70	3	M	50	2-3	M
					70	1-2	C
	13	90	2.5	M	95	2.5	C
	14	75	1.5	M	95	1.5	C
					80	1.5	M
	15	60	2.5	M	70	1.5	C
	16	80-85	3	M	10	1.5	C
					20	2	M
	17	65-70	3	M	55	1.5	C
	18	90	3	M	50	1	M
					50	1	C
ER−/PR−	19	100	2-3	M	70	1	M
GOOD					70	1	C
OUTCOME	20	90	3	M	50	1-2	M
					70	3	C
	21	80	3	M	60	1-2	M
					70	1	C
	22	40	2.5	M	95	1.5	C
					60	1.5	M
	23	95	3	M	60	2-3	M
					90	2-3	C
	24	95	3	M	50	1.5	C
					20	1.5	M

25

90

3

M

Negative

	26	95	3	M	10	1.5	M
	27	80	3	M	45	1.5	C
					35	1.5	M
ER−/PR−	28	<5	1	M	60-70	1-2	M
BAD					40	1-2	C
OUTCOME	29	75	3	M	20	1-2	C
		20	EACH	C
	30	5	1-2	M	50	2-3	M
					90	2-3	C
	31	30	2.5	M	40	2-3	M
		50	2	C	60	2-3	C
	32	70	1.5	M	95	2.5	C
		40	1.5	C	55	1.5	M
	33	60	2.5	M	95	2	C
		40	1.5	C	50	2	M
	34	45	2.5	M	90	1.5	C
		30	1.5	C	45	1.5	M

35

30

2.5

M

Negative

36	35	3	M	50	2	C
	15	1-2	C	30	1.5	M

TABLE 1A

ER/PR	Expression level and location	Potential

Status	Outcome	Marker A	Marker B	use

P/P	Good	Low	Moderately high	Less
		expression	when expression	aggressive
		in M	in C alone	follow-up
		65	91.5
			When expressed
			in MC, C is
			higher than M
			M-65
			C-108
	Bad	High	Moderate	More
		expression	when expression	aggressive
		in M	in C alone	treatment
		213	142
			When expressed
			in MC, C is
			higher than M
			M-57
			C-82
N/N	Good	High	When expressed	Less
		expression	in MC, C is	aggressive
		in M	higher than M	treatment
		238	M-82
			C-119
	Bad	Low	When expressed	Clues for
		when expression	in MC, C is much	better
		in M alone	higher than M	follow-tip
		20	M-91
		When expressed	C-148
		in MC, M is much
		higher than C
		M-131
		C-58.5

Expression of A/CD44 should be scored at the invasive edge and not at DCIS edge
M: cell membrane
C: cytoplasm
N: nucleus
NM: Nuclear membrane
Ranges: Low: 1-80; Moderate: 81-150; High 150-onwards

Formula Applied for Scoring:

Total of Column A (% of cells)/number of patient samples=score A
Total of Column B (intensity)/number of patient samples=score B
Score A multiplied by Score B=Final score for that Marker in that category.
The above scoring and formula has been used keeping in mind the entities only in a specific field (location of the marker) and not across all the patient samples of that category. For Example: in ER−/PR− status of patients with bad outcome for Marker A, the scoring for predicting the prognosis is carried out in the following manner:
For arriving at an interpretation of expression of Marker A in Membrane and Cytoplasm together, only 6 out of the 9 samples are considered, as the remaining 3, constitutes samples having expression only in the Membrane.
Similarly, for arriving at an interpretation of expression of Marker A in Membrane alone, only 3 out of the 9 samples are considered, as the remaining 6, constitutes samples having expression both in Cytoplasm and Membrane, and not in Membrane alone, which is the required criteria for the prediction in this category.
Similar strategy and conversion is employed for interpreting results of all the samples, across all the markers and ER/PR status.

Note: ER+/PR+ Good Outcome:

It is critical to assess the presence or absence of marker A at invasive edge. It is possible that the entire tumor with DCIS focus has HIGH expression of A but the invasive edge has low or no expression and therefore it is the actual expression at the invasive edge that is very important for the interpretation of results for Marker A.

Table 1 and 1A Interpretation:

Patient sample is segregated based on ER+/PR+ status as one group and ER−/PR− status as another group. On selection and segregation of such expression, the test sample is checked for the presence of either individual markers or a combination of markers (for example as captured here: CD44 in combination with CD24 is taken into account) mentioned in Table 1.
The result for good and bad prognosis/outcome should ideally show the marker status after taking into account the expression of staining intensity in conjunction with location of the marker and % of staining as disclosed in Table 1.
A pattern of good/bad outcome as depicted in Table 1A can be arrived at, by correlating the 3 entities namely % of staining, staining intensity and location of the marker in the sample after carrying out the process steps disclosed as aforementioned under Example 1. It is to be noted that it is critical to assess the presence or absence of marker A at invasive edge. It is possible that the entire tumor with DCIS focus has HIGH expression of A but the invasive edge has low or no expression and therefore, this aspect is very important for the interpretation of results for Marker A.
If the results obtained after conducting the aforementioned process steps on the test sample coincide with the marker expression results for good outcome (as disclosed in Table 1), the treatment module followed for the patients having good outcome as per Table 10 (patient history) can be referred to and treatment strategy for such patients will therefore require lesser follow up or less aggressive treatment strategy. However, in case of sample results coinciding with marker expression results for bad outcome (as disclosed in Table 1), then such patients will require more aggressive follow ups along with strategic treatment module to be followed OR an alternate treatment approach needs to be employed or conceived which can essentially comprise developing and administering antibodies specific to these combination of markers (here CD44 and CD24) to curtail its expression
Increasingly the world over it has been realised that there is a subset of patients in ER+/PR+ group who tend to have bad outcome. Our above mentioned results can segregate ER+/PR+ women with potential good and bad outcome based on expression of CD44 and CD24. However, a person skilled in the art would be able to comprehend that based on the above mentioned results, one way to treat these patients is to prescribe anti-CD44 antibodies since CD44 is highly expressed in the cell-membrane in these patients with a hope of long term disease free survival. On the other hand, in ER−/PR− patients with potential bad outcome, there is low over all expression of CD44, and so antibodies to CD44 will not help much. Nevertheless, it is important to identify them and treat them appropriately with more targeted drugs, frequent and thorough follow-ups etc. to ensure long term disease free survival. Similar strategy can be employed for treating cancer patients with varied marker expressions, some of which are illustrated in the tables below.
It is also to be noted that Good outcome from across ER/PR status cannot be combined. As aforementioned, typically +/+ patients are considered good prognosis cases and hence are need not be treated aggressively. Typically, follow ups for +/+ patients can be carried out every 6-12 months which can be too long for the +/+ bad outcome group. Thus, with the aforementioned interpretation of outcome, it can be inferred that there can be more frequent follow-ups to reduce cancer recurrences between two follow-ups, called interval recurrences, especially for the +/+ bad outcome cases/patients. On the other hand −/− cancers are aggressive cancers and hence patients with good outcome need not be aggressively treated. Whereas, −/− patients with bad outcomes, can have more detailed, effective and innovative follow-ups to catch the metastasis as early as possible and start the treatment.

TABLE 2

		Antibody A	Antibody B	Antibody F
	Serial No	CD44	CD24	ABCC4

	of Patients	% of cells	intensity	location	% of cells	intensity	location	% of cells	intensity	location

ER+/PR+	1	20	1-2	M	60	1.5	M	20	1-2	M
GOOD					80	1.5	C
OUTCOME	2	20	2-3	M	10	1.5	M	30	1.5	M
					40	1.5	C

3

Negative

55

1.5

C

Negative

	4	35	2	M	65-70	1.5	C	60	1.5	C
								35	EACH	M
	5	5	3	M	85	3	M	50	2	M
					50	2	C
	6	40	2-3	M	80	2-3	C	20	2	M
					20	1-2	M	20	2	C
	7	40	3	M	35-40	3	C	20	2	M
					25-30	2	M	30	2	C
	8	10	1.5	C	75	2	C	50	1-2	C
					35-40	1.5	M	each	1-2	M
	9	20	3	M	25	1.5	M	15	1.5	M
					15	EACH	C	15
ER+/PR+	10	20	1.5	M	95	1.5	C	50	1.5	C
BAD		60	2	C

OUTCOME

11

95

3

M

<10

1

M

Negative

				90	1	C
12	70	3	M	50	3-3	M	70	2	M
				70	1-2	C
13	90	2.5	M	95	2.5	C	90	3	C
							50	2	M
14	75	1.5	M	95	1.5	C	80	1.5	M
				80	1.5	M

	15	60	2.5	M	70	1.5	C	Negative
	16	80-85	3	M	10	1.5	C	Negative

					20	2	M
	17	65-70	3	M	55	1.5	C	40	1.5	C

18

90

3

M

50

1

M

Negative

					50	1	C
ER−/PR−	19	100	2-3	M	70	1	M	20	1.5	M
GOOD					70	1	C
OUTCOME	20	90	3	M	50	1-2	M	40-50	1.5	M
					70	3	C

21

80

3

M

60

1-2

M

Negative

70

1

C

22

40

2.5

M

95

1.5

C

Negative

				60	1.5	M
23	95	3	M	60	2-3	M	70	2	M
				90	2-3	C

24

95

3

M

50

1.5

C

Negative

20

1.5

M

25

90

3

M

Negative

	26	95	3	M	10	1.5	M	25	1.5	M
	27	80	3	M	45	1.5	C	85	3	M
					35	1.5	M
ER−/PR−	28	<5	1	M	60-70	1-2	M	30	1.5	M
BAD					40	1-2	C
OUTCOME	29	75	3	M	20	1-2	C	60	2	M
		20	EACH	C				20	2	C

30

5

1-2

M

50

2-3

M

Negative

				90	2-3	C
31	30	2.5	M	40	2-3	M	20	1.5	M
	50	2	C	60	2-3	C
32	70	1.5	M	95	2.5	C	60	1.5	M
	40	1.5	C	55	1.5	M
33	60	2.5	M	95	2	C	80	2.5	M
	40	1.5	C	50	2	M
34	45	2.5	M	90	1.5	C	60	1.5	C
	30	1.5	C	45	1.5	M	35	each	M

35

30

2.5

M

Negative

50

1.5

C

36	35	3	M	50	2	C	75	1.5	C
	15	1-2	C	30	1.5	M	55	EACH	M

TABLE 2A

ER/PR	Expression level and location	Potential

Status	Outcome	Marker A	Marker B	Marker F	use

P/P	Good	Low	Moderately high	Low	Less
		expression	when expression	when expression	aggressive
		in M	in C alone	in M alone	follow-up
		65	91.5	49
			When expressed	When expressed
			in MC, C is	in MC, C is
			higher than M	higher than M
			M-65	M-56
			C-108	C-72
	Bad	High	Moderate	Low	More
		expression	when expression	when expression	aggressive
		in M	in C alone	in C alone	treatment
		213	142	67.5
			When expressed	Moderate
			in MC, C is	when expression
			higher than M	in M alone
			M-57	135
			C-82	When expressed
				in MC, C is much
				higher than M
				M-100
				C-270
				When compared
				to ER + GOOD
N/N	Good	High	When expressed	Moderate	Less
		expression	in MC, C is	expression	aggressive
		in M	higher than M	in M	treatment
		238	M-82	98
			C-119	OR No Expression
				at all
	Bad	Low	When expressed	Moderate	Clues for
		when expression	in MC, C is much	when expression	better
		in M alone	higher than M	in M alone	follow-up
		20	M-91	86
		When expressed	C-148	When expressed
		in MC, M is much		in MC, C is
		higher than C		higher than M
		M-131		M-85
		C-58.5		C-88

Expression of A/CD44 should be scored at the invasive edge and not at DCIS edge
M: cell membrane
C: cytoplasm
N: nucleus
NM: Nuclear membrane

Table 2 and 2A Interpretation:

Patient sample is segregated based on ER+/PR+ status as one group and ER−/PR− status as another group. On selection and segregation of such expression, the test sample is checked for the presence of either individual markers or a combination of markers (for example as captured here: CD44 and CD24 in combination with ABCC4 is taken into account) mentioned in Table 2.
The result for good and bad prognosis/outcome should ideally show the marker status after taking into account the expression of staining intensity in conjunction with location of the marker and % of staining as disclosed in Table 2.
A pattern of good/bad outcome as depicted in Table 2A can be arrived at, by correlating the 3 entities namely % of staining, staining intensity and location of the marker in the sample after carrying out the process steps disclosed as aforementioned under Example 1. It is to be noted that it is critical to assess the presence or absence of marker A at invasive edge. It is possible that the entire tumor with DCIS focus has HIGH expression of A but the invasive edge has low or no expression and therefore, this aspect is very important for the interpretation of results for Marker A.
If the results obtained after conducting the aforementioned process steps on the test sample coincide with the marker expression results for good outcome (as disclosed in Table 2), the treatment module followed for the patients having good outcome as per Table 10 (patient history) can be referred to and treatment strategy for such patients will therefore require lesser follow up or less aggressive treatment strategy. However, in case of sample results coinciding with marker expression results for bad outcome (as disclosed in Table 2), then such patients will require more aggressive follow ups along with strategic treatment module to be followed OR an alternate treatment approach needs to be employed or conceived which can essentially comprise developing and administering antibodies specific to these combination of markers (here CD44 and CD24 in combination with ABCC4) to curtail its expression.
Increasingly the world over it has been realised that there is a subset of patients in ER+/PR+ group who tend to have bad outcome. Our above mentioned results can segregate ER+/PR+ women with potential good and bad outcome based on expression of CD44 and CD24. However, a person skilled in the art would be able to comprehend that based on the above mentioned results, one way to treat these patients is to prescribe anti-CD44 antibodies since CD44 is highly expressed in the cell-membrane in these patients with a hope of long term disease free survival. On the other hand, in ER−/PR− patients with potential bad outcome, there is low over all expression of CD44, and so antibodies to CD44 will not help much. Nevertheless, it is important to identify them and treat them appropriately with more targeted drugs, frequent and thorough follow-ups etc. to ensure long term disease free survival. Similar strategy can be employed for treating cancer patients with varied marker expressions, some of which are illustrated in the tables below.
It is also to be noted that Good outcome from across ER/PR status cannot be combined. As aforementioned, typically +/+ patients are considered good prognosis cases and hence are need not be treated aggressively. Follow ups for +/+ patients can be carried out every 6-12 months which can be too long for the +/+ bad outcome group. Thus, with the aforementioned interpretation of outcome, it can be inferred that there can be more frequent follow-ups to reduce cancer recurrences between two follow-ups, called interval recurrences, especially for the +/+ bad outcome cases/patients. On the other hand −/− cancers are aggressive cancers and hence patients with good outcome need not be aggressively treated. Whereas, −/− patients with bad outcomes, can have more detailed, effective and innovative Follow-ups to catch the metastasis as early as possible and start the treatment.
ABCC4 is a membrane transporter. Cells use it to get the chemotherapy drugs pumped out, hence when the expression of this marker is high the cells tend to be more resistant to CT (chemotherapy). Also, it is been noted that the cytoplasmic expression of this transporter also helps the cells to pump out the drugs which should be kept in mind while treating the patients. Patients with high M or M+C expression of ABCC4 will be more resistant to drugs and hence perhaps are going to have bad prognosis/outcome and therefore should be treated accordingly.

TABLE 3

		Antibody I	Antibody J
	Serial No	Oct-4	Sox-2

	of Patients	% of cells	Intensity	location	% of cells	intensity	location

ER+/PR+

1

90

3

N

20

3

N

GOOD	2	Negative	Negative
OUTCOME	3	Negative	Negative

4	Negative	15	2.5	N
5	Negative	40	3	N
6	Negative	40	3	N

7

60

3

N

Negative

	8	Negative	Negative
	9	Negative	Negative

ER+/PR+	10	70	2-3	N	Negative
BAD	11	90	3	N	Negative
OUTCOME	12	70	2-3	N	Negative
	13	65	1-2	C	Negative
	14	30	1-2	N	Negative
	15	75	1-2	N	Negative
	16	30-35	3	N	Negative
	17	25-30	1-2	N	Negative
	18	80	3	N	Negative

ER−/PR−

19

65

1-2

N

25

3

N

GOOD

20

Negative

OUTCOME	21	Negative	10	3	N
	22	Negative	10	3	N

	23	Negative	Negative
	24	Negative	Negative

25

75

1-2

N

Negative

26

Negative

80

3

N

27

15

1-2

N

Negative

ER−/PR−	28	80	2.5	N	45	3	N
BAD	29	40	1-2	N	25	3	N

OUTCOME

30

Negative

50

3

N

31

Negative

32	25	1-2	N	Negative
33	20	1-2	N	Negative
34	25	2	N	Negative

35

20

3

N

10

3

N

	36	55	1-2	N	Negative

TABLE 3A

ER/PR	Expression level and location	Potential

Status	Outcome	Marker I	Marker J	use

P/P	Good	No expression	Moderate	Less
		at all	expression	aggressive
			in N	follow-up
			84
			Or NO expression
			at all
	Bad	Moderate	No expression	More
		expression	at all	aggressive
		in N		treatment
		118
N/N	Good	Low	Moderate	Less
		expression	expression	aggressive
		in N	in N	treatment
		78	93
		Or NO expression	Or NO expression
		at all	at all
	Bad	Low	Moderate	Clues for
		expression	expression	better
		in N	in N	follow-up
		76	98
			Or NO expression
			at all

M: cell membrane
C: cytoplasm
N: nucleus
NM: Nuclear membrane

Table 3 and 3A Interpretation:

Patient sample is segregated based on ER+/PR+ status as one group and ER−/PR− status as another group. On selection and segregation of such expression, the test sample is checked for the presence of either individual markers or a combination of markers (for example as captured here: Oct-4 in combination with Sox-2 is taken into account) mentioned in Table 3.
The result for good and bad prognosis/outcome should ideally show the marker status after taking into account the expression of staining intensity in conjunction with location of the marker and % of staining as disclosed in Table 3.
A pattern of good/bad outcome as depicted in Table 3A can be arrived at, by correlating the 3 entities namely % of staining, staining intensity and location of the marker in the sample after carrying out the process steps disclosed as aforementioned under Example 1.
If the results obtained after conducting the aforementioned process steps on the test sample coincide with the marker expression results for good outcome (as disclosed in Table 3), the treatment module followed for the patients having good outcome as per Table 10 (patient history) can be referred to and treatment strategy for such patients will therefore require lesser follow up or less aggressive treatment strategy. However, in case of sample results coinciding with marker expression results for bad outcome (as disclosed in Table 3), then such patients will require more aggressive follow ups along with strategic treatment module to be followed OR an alternate treatment approach needs to be employed or conceived which can essentially comprise developing and administering antibodies specific to these combination of markers (here Oct-4 and Sox-2) to curtail its expression.
It is also to be noted that Good outcome from across ER/PR status cannot be combined. As aforementioned, typically +/+ patients are considered good prognosis cases and hence are need not be treated aggressively. Follow ups for +/+ patients can be carried out every 6-12 months which can be too long for the +/+ bad outcome group. Thus, with the aforementioned interpretation of outcome, it can be inferred that there can be more frequent follow-ups to reduce cancer recurrences between two follow-ups, called interval recurrences, especially for the +/+ bad outcome cases/patients. On the other hand −/− cancers are aggressive cancers and hence patients with good outcome need not be aggressively treated. Whereas, −/− patients with bad outcomes, can have more detailed, effective and innovative Follow-ups to catch the metastasis as early as possible and start the treatment.

TABLE 4

		Antibody K	Antibody 0
	Serial No	APC	B-Catenin

	of Patients	% of cells	Intensity	Location	% of cells	Intensity	Location

ER+/PR+	1	60	2	M	35-40	1-2	N
GOOD		80	3	C	45	EACH	M
OUTCOME		30	3	N
	2	70	2	C	15	1-2	M
					10	2	N

3

85

3

C

Negative

	50	2	M
4	85	2	C	20	1.5	M
	70	2-3	NM

5

60

2

M

—

90

2.5

C

6

10

1-2

M

Negative

	90	3	C
7	80	3	C	10	2	N
	30	2-3	M

8

90

3

C

Negative

		50	3	N
	9	75	2	M	35	1.5	M
		30	1-2	C
ER+/PR+	10	85	3	C	50	1.5	C
BAD		50	2-3	NM
OUTCOME	11	65	2	M	80	2	M
		85	2.5	C
		65	2.5	NM
	12	60	2	M	50	2	C
		80	2	C	25-30	1-2	M

13

90

3

C

—

	14	70	3	C	20	1.5	M
		60	2-3	M

15

90

2-3

C

—

50

2

M

16

70

1-2

C

—

		60	2	N
	17	50	1-2	C	40	2	M
	18	80	2.5	C	65	3	M
		50	2.5	NM
ER−/PR−	19	30	2	M	75	1-2	M
GOOD		60	1-2	C
OUTCOME	20	70	2-3	M	60	1-2	M
		90	2-3	C
	21	50	2	M	20	1-2	M
		70	1.5	C
	22	60	3	C	50	1.5	C
		60	2	M
	23	90	3	C	75	1-2	M
		80	2.5	M

24

85

3

C

—

75

2-3

M

25

85

3

C

—

30

2

M

26

80

1-2

C

Negative

	27	70	2	C	60	3	M
		70	2	M
ER−/PR−	28	80	2-3	M	50	1-2	M
BAD		55	1.5	C
OUTCOME	29	40	1.5	C	60	2	M
		40	2	M
	30	90	2-3	C	60	1-2	M
		40	2	M
		65	2.5	NM
	31	95	3	C	60	1-2	M
		60	2-3	M
	32	80	3	C	75	2	M
		70	2-3	M
		50	2-3	NM
	33	90	3	C	80	1.5	C
		85	2	M	40	EACH	M

34

95

3

C

—

		60	2	M
	35	30	1-2	C

	36	90	2	C	Negative

TABLE 4A

ER/PR	Expression level and location	Potential

Status	Outcome	Marker K	Marker O	use

P/P	Good	When expressed	Low	Less
		in MC, C is much	when expression	aggressive
		higher than M	in M alone	follow-up
		M-86	41
		C-225	When expressed
		Expression also	in N and M, M
		seen in N in	and N are at
		various levels	most equally
			expressed
			M-45
			N-43
	Bad	Moderate	Moderate	More
		when expression	expression	aggressive
		in C alone	in M	treatment
		140	102
		When expressed	Expression
		in MC, C is	in C is
		higher than M	also seen
		M-114
		C-200
		When expressed
		in C-NM, C is
		higher than NM
		NM-138
		C-246
N/N	Good	When expressed	Moderate	Less
		in MC, C is	expression	aggressive
		higher than M	in M alone	treatment
		M-116	104
		C-152
	Bad	When expressed	Moderate	Clues for
		in MC, C is	expression	better
		higher than M	in M alone	follow-up
		M-130	104
		C-150
		Moderate
		when expression
		in C alone
		108
		When expressed
		in C, M and NM, C
		is highest, followed
		by NM and M
		M-110
		NM-145
		C-238

M: cell membrane
C: cytoplasm
N: nucleus
NM: Nuclear membrane

Table 4 and 4A Interpretation:

Patient sample is segregated based on ER+/PR+ status as one group and ER−/PR− status as another group. On selection and segregation of such expression, the test sample is checked for the presence of either individual markers or a combination of markers (for example as captured here: APC in combination with B-Catenin is taken into account) mentioned in Table 4.
The result for good and bad prognosis/outcome should ideally show the marker status after taking into account the expression of staining intensity in conjunction with location of the marker and % of staining as disclosed in Table 4.
A pattern of good/bad outcome as depicted in Table 4A can be arrived at, by correlating the 3 entities namely % of staining, staining intensity and location of the marker in the sample after carrying out the process steps disclosed as aforementioned under Example 1.
If the results obtained after conducting the aforementioned process steps on the test sample coincide with the marker expression results for good outcome (as disclosed in Table 4), the treatment module followed for the patients having good outcome as per Table 10 (patient history) can be referred to and treatment strategy for such patients will therefore require lesser follow up or less aggressive treatment strategy. However, in case of sample results coinciding with marker expression results for bad outcome (as disclosed in Table 4), then such patients will require more aggressive follow ups along with strategic treatment module to be followed OR an alternate treatment approach needs to be employed or conceived which can essentially comprise developing and administering antibodies specific to these combination of markers (here APC and B-Catenin) to curtail its expression.
It is also to be noted that Good outcome from across ER/PR status cannot be combined. As aforementioned, typically +/+ patients are considered good prognosis cases and hence are need not be treated aggressively. Follow ups for +/+ patients can be carried out every 6-12 months which can be too long for the +/+ bad outcome group. Thus, with the aforementioned interpretation of outcome, it can be inferred that there can be more frequent follow-ups to reduce cancer recurrences between two follow-ups, called interval recurrences, especially for the +/+ bad outcome cases/patients. On the other hand −/− cancers are aggressive cancers and hence patients with good outcome need not be aggressively treated. Whereas, −/− patients with bad outcomes, can have more detailed, effective and innovative Follow-ups to catch the metastasis as early as possible and start the treatment.

	TABLE 5

		Antibody G
	Serial No of	CD133

	Patients	% of cells	Intensity	Location

ER+/PR+	1	60	2.5	N
GOOD		30	1.5	M
OUTCOME	2	50	1.5	C
	3	90	1.5 EACH	C
		20		M
	4	30-35	2	M
		50	1.5	C
	5	70	2	M
		50	2	C
	6	50	2	M
		60	2	C
	7	40	2	M
		70	2-3	C
	8	45	1-2	C
	9	65	2	M
ER+/PR+	10	80	1.5	C
BAD	11	60	1-2	C
OUTCOME	12	25	2	M
		80	2	C
	13	80	2	C
	14	90	2	M
		30	1.5	C
	15	70	1.5 EACH	M
		30		C
	16	15	1.5 EACH	M
		35		C
	17	30	1.5	M

	18	Negative
ER−/PR−	19	NEGATIVE

GOOD	20	70	2	M
OUTCOME		60	2	C
ER−/PR−	21	25	1-2	M
	22	65	1.5 each	C
		65		M
	23	55	2	M
		75	2	C
	24	60	2	M
		25	1-2	C
	25	60	1.5	C
		40	1.5	M
	26	20	1.5	M
	27	55	1.5 EACH	C
		45		M
ER−/PR−	28	60	1-2	M
BAD		30	1-2	C
OUTCOME	29	20	2	M
ER−/PR−		20	2	C
	30	50	1.5 Each	M
		50		C
	31	55	1.5 Each	C
		10		M
	32	70	2	M
		45	1.5	C
	33	80	2 each	C
		75		M
	34	65	2	C
	35	40	2	C
		20	3	N
	36	50	2	C

	TABLE 6

		Antibody L
	Serial No of	P-Cadherin

	Patients	% of cells	Intensity	Location

ER+/PR+

1

20

2-3

M

GOOD	2	Negative
OUTCOME	3	Negative

4

20

2

M

	5	Negative
	6	Negative

	7	50	3	M
	8	10	3	M
	9	40	3	M
ER+/PR+	10	20	3	M
BAD	11	80	3	M
OUTCOME	12	20	3	M

	13	Negative
	14	Negative

	15	90	3	M
	16	30	2.5	M
	17	45	2.5	M
	18	90-100	3	M
ER−/PR−	19	50	3	M
GOOD	20	55	3	M
OUTCOME	21	50	3	M
ER−/PR−	22	80	3	M
	23	70	2	M
	24	70	3	M
	25	65-70	3	M

26

Negative

	27	80	3	M
ER−/PR−	28	80	3	M
BAD	29	15-20	3	M
OUTCOME	30	15	3	M

ER−/PR−

31

Negative

32	20	3	M
33	75	3	M
34	<5	3	M
35	45	2.5	M

	36	Negative

TABLE 6A

		Expression level
ER/PR		and location
Status	Outcome	Marker L	Potential use

P/P	Good	Moderate	Less
		expression	aggressive
		in M	follow-up
		84
	Bad	High	More
		expression	aggressive
		in M	treatment
		162
N/N	Good	High	Less
		expression	aggressive
		in M	treatment
		195
	Bad	Moderate	Clues for
		expression	better
		in M	follow-up
		111

M: cell membrane
C: cytoplasm
N: nucleus
NM: Nuclear membrane

Table 6 and 6A Interpretation:

Patient sample is segregated based on ER+/PR+ status as one group and ER−/PR− status as another group. On selection and segregation of such expression, the test sample is checked for the presence of either individual markers or a combination of markers (for example as captured here: P-cadherin alone is taken into account) mentioned in Table 6.
The result for good and bad prognosis/outcome should ideally show the marker status after taking into account the expression of staining intensity in conjunction with location of the marker and % of staining as disclosed in Table 6.
A pattern of good/bad outcome as depicted in Table 6A can be arrived at, by correlating the 3 entities namely % of staining, staining intensity and location of the marker in the sample after carrying out the process steps disclosed as aforementioned under Example 1.
If the results obtained after conducting the aforementioned process steps on the test sample coincide with the marker expression results for good outcome (as disclosed in Table 6), the treatment module followed for the patients having good outcome as per Table 10 (patient history) can be referred to and treatment strategy for such patients will therefore require lesser follow up or less aggressive treatment strategy. However, in case of sample results coinciding with marker expression results for bad outcome (as disclosed in Table 6), then such patients will require more aggressive follow ups along with strategic treatment module to be followed OR an alternate treatment approach needs to be employed or conceived which can essentially comprise developing and administering antibodies specific to these combination of markers (here P-cadherin alone) to curtail its expression.
Increasingly the world over it has been realised that there is a subset of patients in ER+/PR+ group who tend to have bad outcome. Our above mentioned results can segregate ER+/PR+ women with potential good and bad outcome based on expression of P-cadherin. However, a person skilled in the art would be able to comprehend that based on the above mentioned results, one way to treat these patients is to prescribe anti-P-cadherin antibodies since P-cadherin is highly expressed in the cell-membrane in these patients with a hope of long term disease free survival. On the other hand, in ER−/PR− patients with potential bad outcome, there is lower over all expression of P-cadherin, and so antibodies to P-cadherin will not help much. Nevertheless, it is important to identify them and treat them appropriately with more targeted drugs, frequent and thorough follow-ups etc. to ensure long term disease free survival. Similar strategy can be employed for treating cancer patients with varied marker expressions, some of which are illustrated in the tables below.
It is also to be noted that Good outcome from across ER/PR status cannot be combined. As aforementioned, typically +/+ patients are considered good prognosis cases and hence are need not be treated aggressively. Follow ups for +/+ patients can be carried out every 6-12 months which can be too long for the +/+ bad outcome group. Thus, with the aforementioned interpretation of outcome, it can be inferred that there can be more frequent follow-ups to reduce cancer recurrences between two follow-ups, called interval recurrences, especially for the +/+ bad outcome cases/patients. On the other hand −/− cancers are aggressive cancers and hence patients with good outcome need not be aggressively treated. Whereas, −/− patients with bad outcomes, can have more detailed, effective and innovative Follow-ups to catch the metastasis as early as possible and start the treatment.

TABLE 7

		Antibody A	Antibody B	Antibody I	Antibody J
	Serial No	CD44	CD24	Oct-4	Sox-2

of Patients

% of cells

intensity

location

% of cells

intensity

location

% of cells

intensity

location

% of cells

intensity

location

ER+/PR+	1	20	1-2	M	60	1.5	M	90	3	N	20	3	N
GOOD					80	1.5	C

OUTCOME

2

20

2-3

M

10

1.5

M

Negative

40

1.5

C

3	Negative			55	1.5	C	Negative	<5%	2-3	N
4	35	2	M	65-70	1.5	C	Negative	15	2.5	N
5	5	3	M	85	3	M	Negative	40	3	N

50

2

C

6

40

2-3

M

80

2-3

C

Negative

40

3

N

20

1-2

M

7

40

3

M

35-40

3

C

60

3

N

Negative

25-30

2

M

8

10

1.5

C

75

2

C

Negative

35-40

1.5

M

9

20

3

M

25

1.5

M

Negative

15

EACH

C

ER+/PR+

10

20

1.5

M

95

1.5

C

70

2-3

N

Negative

BAD

60

2

C

OUTCOME

11

95

3

M

<10

1

M

90

3

N

Negative

				90	1	C
12	70	3	M	50	2-3	M	70	2-3	N	5	3	N
				70	1-2	C

	13	90	2.5	M	95	2.5	C	65	1-2	C	Negative
	14	75	1.5	M	95	1.5	C	30	1-2	N	Negative

80

1.5

M

	15	60	2.5	M	70	1.5	C	75	1-2	N	Negative
	16	80-85	3	M	10	1.5	C	30-35	3	N	Negaitive

20

2

M

	17	65-70	3	M	55	1.5	C	25-30	1-2	N	Negative
	18	90	3	M	50	1	M	80	3	N	Negative

					50	1	C
ER−/PR−	19	100	2-3	M	70	1	M	65	1-2	N	25	3	N
GOOD					70	1	C

OUTCOME

20

90

3

M

50

1-2

M

Negative

70

3

C

21

80

3

M

60

1-2

M

Negative

10

3

N

70

1

C

22

40

2.5

M

95

1.5

C

Negative

10

3

N

60

1.5

M

23

95

3

M

60

2-3

M

Negative

90

2-3

C

24

95

3

M

50

1.5

C

Negative

<5

2-3

N

20

1.5

M

25

90

3

M

Negative

75

1-2

N

Negative

26

95

3

M

10

1.5

M

Negative

80

3

N

27

80

3

M

45

1.5

C

15

1-2

N

Negative

					35	1.5	M
ER−/PR−	28	<5	1	M	60-70	1-2	M	80	2.5	N	45	3	N
BAD					40	1-2	C
OUTCOME	29	75	3	M	20	1-2	C	40	1-2	N	25	3	N
		20	EACH	C

30

5

1-2

M

50

2-3

M

Negative

50

3

N

90

2-3

C

31

30

2.5

M

40

2-3

M

Negative

	50	2	C	60	2-3	C
32	70	1.5	M	95	2.5	C	25	1-2	N	Negative	<5%
	40	1.5	C	55	1.5	M

33

60

2.5

M

95

2

C

20

1-2

N

Negative

40

1.5

C

50

2

M

34

45

2.5

M

90

1.5

C

25

2

N

Negative

30

1.5

C

45

1.5

M

35

30

2.5

M

Negative

20

3

N

10

3

N

36

35

3

M

50

2

C

55

1-2

N

Negative

		15	1-2	C	30	1.5	M

TABLE 7A

ER/PR	Expression level and location	Potential

Status	Outcome	Marker A	Marker B	Marker I	Marker J	use

P/P	Good	Low	Moderately high	No	Moderate	Less
		expression	when expression	expression	expression in	aggressive
		in M 65	in C alone	at all	N 84	follow-up
			91.5		Or NO
			When expressed		expression
			in MC, C is		at all
			highter than M
			M-65 C-108
	Bad	High	Moderate when	Moderate	No	More
		expression	expression in	expression	expression	aggressive
		in M 213	C alone 142	in N 118	at all	treatment
			When expressed
			in MC, C is
			higher than M
			M-57 C-82
N/N	Good	High	When expressed	Low	Moderate	Less
		expression	in MC, C is	expression in	expression in	aggressive
		in M 238	higher than M	N 78	N 93	treatment
			M-82 C-119	Or NO	Or NO
				expression	expression
				at all	at all
	Bad	Low	When expressed	Low	Moderate	Clues for
		when	in MC, C is	expression	expression in	better
		expression	much higher	in N 76	N 98	follow-
		in M alone 20	than M-91 C-148		Or NO	up
		When expressed			expression
		in MC, M is			at all
		much higher
		than C
		M-131 C-58.5

Expression of A/CD44 should be scored at the invasive edge and not at DCIS edge
M: cell membrane
C: cytoplasm
N: nucleus
NM: Nuclear membrane

Table 7 and 7A Interpretation:

Patient sample is segregated based on ER+/PR+ status as one group and ER−/PR− status as another group. On selection and segregation of such expression, the test sample is checked for the presence of either individual markers or a combination of markers (for example as captured here: CD44 in combination with CD24, Oct-4 and Sox-2 is taken into account) mentioned in Table 7.
The result for good and bad prognosis/outcome should ideally show the marker status after taking into account the expression of staining intensity in conjunction with location of the marker and % of staining as disclosed in Table 7.
A pattern of good/bad outcome as depicted in Table 7A can be arrived at, by correlating the 3 entities namely % of staining, staining intensity and location of the marker in the sample after carrying out the process steps disclosed as aforementioned under Example 1. It is to be noted that it is critical to assess the presence or absence of marker A at invasive edge. It is possible that the entire tumor with DCIS focus has HIGH expression of A but the invasive edge has low or no expression and therefore, this aspect is very important for the interpretation of results for Marker A.
If the results obtained after conducting the aforementioned process steps on the test sample coincide with the marker expression results for good outcome (as disclosed in Table 7), the treatment module followed for the patients having good outcome as per Table 10 (patient history) can be referred to and treatment strategy for such patients will therefore require lesser follow up or less aggressive treatment strategy. However, in case of sample results coinciding with marker expression results for bad outcome (as disclosed in Table 7), then such patients will require more aggressive follow ups along with strategic treatment module to be followed OR an alternate treatment approach needs to be employed or conceived which can essentially comprise developing and administering antibodies specific to these combination of markers (here CD44 in combination with CD24, Oct-4 and Sox-2) to curtail its expression
Increasingly the world over it has been realised that there is a subset of patients in ER+/PR+ group who tend to have bad outcome. Our above mentioned results can segregate ER+/PR+ women with potential good and bad outcome based on expression of CD44 and CD24. However, a person skilled in the art would be able to comprehend that based on the above mentioned results, one way to treat these patients is to prescribe anti-CD44 antibodies since CD44 is highly expressed in the cell-membrane in these patients with a hope of long term disease free survival. On the other hand, in ER−/PR− patients with potential bad outcome, there is low over all expression of CD44, and so antibodies to CD44 will not help much. Nevertheless, it is important to identify them and treat them appropriately with more targeted drugs, frequent and thorough follow-ups etc. to ensure long term disease free survival. Similar strategy can be employed for treating cancer patients with varied marker expressions, some of which are illustrated in the tables below.
It is also to be noted that Good outcome from across ER/PR status cannot be combined. As aforementioned, typically +/+ patients are considered good prognosis cases and hence are need not be treated aggressively. Follow ups for +/+ patients can be carried out every 6-12 months which can be too long for the +/+ bad outcome group. Thus, with the aforementioned interpretation of outcome, it can be inferred that there can be more frequent follow-ups to reduce cancer recurrences between two follow-ups, called interval recurrences, especially for the +/+ bad outcome cases/patients. On the other hand −/− cancers are aggressive cancers and hence patients with good outcome need not be aggressively treated. Whereas, −/− patients with bad outcomes, can have more detailed, effective and innovative Follow-ups to catch the metastasis as early as possible and start the treatment.

	TABLE 8

		Antibody C
	Serial No of	ABCG2

	Patients	% of cells	Intensity	Location

ER+/PR+	1	95	3	N
GOOD		30	1.5	M
OUTCOME	2	75	3	N
	3	95	1.5	C
		35	1.5	M
	4	90	2.5	C
		35	1.5	M
	5	50	2	M
		80	2	C
	6	<5	2	M
		80	2-3	C
	7	80	3	N
		60	3	C
	8	90	3	N
		70	2	C
		10	1-2	M
	9	45	3	N
		35	2	M
ER+/PR+	10	95	1.5 EACH	C
BAD		45		N
OUTCOME	11	80	2	C
		90	3	N
	12	20	1.5	M
		50	1.5	C
		40	2.5	N
	13	90	2.5	C
	14	90	1.5	C
		70	1.5	M
	15	95	1.5 EACH	N
		95		C
		20		M
	16	30	3	N
	17	60	2	C
		80	3	N
	18	50	1.2	C
		80	3	N
ER−/PR−	19	80	3	Nu
GOOD	20	40	2	M
OUTCOME		80	2-3	C
ER−/PR−		75	3	Nu
	21	65	2.5	N
		40	1.5	C
		30	2	M
	22	75	1.5	N
		90	1.5	C
		65	1.5	M
	23	30	2	M
		65	3	C
	24	85	1.5	C
		50	1.5	M
	25	75	2.5	N
		80	1.5	C
		20	1.5	M
	26	45	2	N
	27	95	2	C
		20	2	N
		20	2	M
ER−/PR−	28	50	1.5 each	N
BAD		65		C
OUTCOME		20		M
ER−/PR−	29	40	2-3	C
		20	3	Nu
	30	70	2	C
	31	70	2.5	C
		10	2	M
	32	30	2 EACH	N
		60		M
		40		C
	33	95	1.5	C
		10	1	M
	34	75	1.5	C
	35	80	2	C
		30	3	N
	36	85	2	C
		40	2	N

	TABLE 9

		Antibody E
	Serial No of	ESA

	Patients	% of cells	Intensity	Location

ER+/PR+	1	90	3	M
GOOD	2	90	3	M
OUTCOME	3	95	3	M
	4	95	3	M
	5	90	3	M
	6	95	3	M
	7	95	3	M

8

—

	9	45	3	M
ER+/PR+	10	95	3	M
BAD	11	95	3	M
OUTCOME	12	75	3	M
	13	95	3	M
	14	95	3	M
	15	95	3	M
	16	90	3	M
	17	55	3	M
	18	100	3	M
ER−/PR−	19	85-90	3	M
GOOD	20	95	3	M
OUTCOME	21	90	3	M
ER−/PR−	22	95	3	M
	23	90	3	M
	24	95	3	M
	25	95	3	M
	26	85	3	M
	27	35	3	M
ER−/PR−	28	100	3	M
BAD	29	50	3	M
OUTCOME	30	85	3	M
ER−/PR−	31	90	3	M
	32	95	3	M
	33	95	3	M
	34	95	3	M
		70	1.5	C
	35	75	3	M
	36	90	3	M

TABLE 10

Patient history

	Serial No
	of
	Patients	Age	Stage	Grade	ER/PR/Her2	MRM/CT/RT	Patient status

ER+/PR+	1	54, Post	IDC-2,	T2N2M0,	P/P/P	11/03: MRM; 12/03-04/04: CT-FAC x6;	No mets
Good			small foci	pT2NaM2		04/04: RT; 10/04 on Tamx, c/o lower	and Alive:
Out-			DCIS,			back ache
come			L Br
	2	26	IDC-2, L	T2N0M0	P/P/N	Quadrectomy in Nov 04, BCT/RT/CT-	Alive, most
			Br	(Stage 2a)		FAC, on TAM 20γ HS; 2007-Normal,	likely Ca
						3 yrs of TAM: Dec 2009-Normal, TAM	free
						to stop after 5 yrs of usage; 2011-All
						normal
	3	44,	IDC-3	T2N1M0.	20% P/20% P	H/o DM, Hypothyroid, HT, Lap.	No mets
		Premeno.	lymphatic	pT2N3M0		Cholecystectomy; 01/04 Trucut Biopsy,	and alive:
			emboli &	(Stage-3a)		Bone scan-N, MRM; 02-06/04 CT-
			perineural			FEC x6; 04/04 RT; 07/09 on extended
			invasion;			Adj Hormonal therapy; 06/10
			R Br			Anastrozole for 6 yrs L-Mammo:
						some mass present, no intervention adv.;
						08/11: R-Mammo-N, Inj Zolodronic acid
	4	48	ICD-2; L	pT2N0M0	P/P/P	MRM in 08/2005: Adj CT-FEC x6 in 08-12/2005;	Alive
			Br			12/2005 Start Tamx 20 mg/d × 5 yrs;
						12/2005-01/2006 RT 45 Gy/25#
	5	55, Post	IDC-3,	T2N2M0	P/P	12/02: Exi. Biopsy L Br, MRM, Bone	No mets
			L Br			scan-Normal; 01-06/03: High risk CT-	for 6 yrs
						AC x6; 03-05/03: RT; 06/03 Started	and alive
						Tamx; 03/09: Breathless, no wt gain,
						R Br, L MRM - Normal, adv. PET-CT
						scan
	6	54, Post	IDC-3,	T2N0M0	P/P	lump in R br for 2 weeks. MRM 05/03;	no mets
			R Br	pT2N0		CT-AC x4 06-08/03; on Tamoxifen for	and alive
						7 yrs total & anastrazole for 5 yrs: 10/06
						all Normal; 09/08 all normal, 07/09
						Fatty changes in liver, 02/11 L br
						Normal; 02/11 BMD-osteoporosis L & R
						br Normal, on Zoledromide fro 01/10
						til 12/11
	7	52/54,	IDC,	multifocal	P/P	R Br Lumpectomy in ′99, again in 2001	No mets
		Post	L Br	T1N1M0		whih was found Normal. Post surgery	and alive, R
						CT/RT: Was put on TAM but stopped	Br in ′91, ′00
						and started Tab Anastrazole; Mother	(found to be
						had Cancer: Cholecystectomy on 05/04/08	Normal),
							Mother had
							cancer
	8	62	IDC-3	T2N0	P/P	K/c/o Ca Br; L-MRM 03/2005; Adj CT-	Alive and
						Taxol x4; Pt on Shelcal: Stazonex, inj	doing well
						Zolastra: anastrazole ect tablets and	and no mets
						regular F/up since 2005; Last F/up in
						07/2011 and next
						due in 07/2012.
	9	40	IDC-III;	T2N1MX	N/P30%/P	Lump in R breast for 6 months; R-MRM on	All
			R Br			08/2005; Adj CT-TACx6 09-12/2005. Adj RT	Normal,
						50.4 Gy/28#s 01-02/2006; 2006-2009 on Tamx;
						Switched to Aromasin in 02/2009;
						Last F/up 04/2011 all Normal.
ER+/PR+	10	41,	ILC.	T2N0M0	P/P	04/03 c/o Lump R Br(6mths), H/o	Mets and
Bad		Premeno.	R Br			Hypothyroid, Trucut Biopsy, 04/2003 MRM,	alive; Local
Out-						Bone scan-N. 04-03/2003 CT-AC x6; RT;	recurrence
come						09/2007 local recurrence R ch. wall &	within 4 yrs,
						Wide exi of R chest wall lesion. CT-FECx6	defaulted
						11/07-02/2008; 03/08 No hormonal	on CT
						Rx past 4 yrs, Adv Anastozole; Not
						been taking Anastozole; F/up 05 2010 R&L
						br N & on Anastrazole now; 05/2011 Br N, U/S
						N continue Anastrozole; All Normal;
						09/11 Cardio checkup-N, pain in L neck,
						No node identified clinically, Sonography,
						TSH, FT4- All N, Adv. see Endocrinologist
						for Thyroxene replacement.
	11	54, Post	IDC-3,	T2N0M0	P/P	H/O Hypothyroid, vomitting at least 1/day	Diag./MRM
			R Br			for 15 days (03/01), c/o Thyrotoxicosis in	06/04, Bone &
						Gastritis; MRM (R Br) 06/04, Post op. Green	Brain Mets,
						color urine, No fam history; CT-FACx6	expired in
						(07-10/04); C/o Seizures, Brain CT, given	4 yrs 09/08
						anticonvulsants 06/08, RT 06/08. completed
						Palli WBRT 30Gy/10# 2 weeks; 2x CT-
						Gem+Cis (11 & 30/07/08); Br Ca (Stage 4) +
						Intracranial Mets, on Hormonal therapy,
						refused treatment; completes Palli RT & CT-
						Gem + Cis 29/08/08; altered sensorium,
						fever, vomit, bluish color of arm, 2units
						of FFP + 2 red blood transfusion,
						discharged for Palli care at home 18/09/08
	12	43, Post	ILC,	T2N2M0	P/P	04/04: Lump in Br; 05/04: MRM: 05-09/04:	Expired in 3.5
			R Br			CT-FAC x6 09-11/04: RT 50.4GY/#; 09/04:	yrs. Bone,
						Started Tamx; 02/05: came back w pain, on	lung, live
						Novoldex; 10/05 Bone scan-muliple mets	mets within
						adv RT; 06/06: Bone, liver, lung, mets;	1-2 yrs
						03/07; CT-Gem+Cis x6; 06/07: RT 30Gy/
						10#; 09/07: Intracranial CT-Methotrexate;
						09/07: Expired
	13	58, Post	ILC;	—	P/80% P	02/02 c/o sever back pain, Op.	Mets and dead;
			L Br			Laminectomy, surg. D9 vertebra, found	Came w bone
						Br mass; Trucut Biopsy for Br, Diag.	mets, possible
						ILC w mulitple bone mets, recd RT for	met recurrence
						spial lesions, pan-CK + ive; 02/02 CT-	within 4
						Dexona; 06/06 On Letrozole, c/o pain in	years?
						R shoulder, lower back ache, reed, Palli
						RT 20Gy/5#/1 wk
ER+/PR+	14	66	IDC-3;	T2N1M0	80% P/N/100% P	H/o Hystrectomy (′87), Ht, Hypothyroid;	Mets and dead;
Bad			R Br	(L Br)		02/03: Lump, MRM+BCS, RT; 04-09/03	Progressive
Out-						On Herceptin & Xeloda, locally	disease despite
come						recurrent T nodules ++ all over	CT, RT,
						reconstructed Br: 06/03 Nodules regressed,	Hormonal
						RT; 10/03 L Br-N, skin healthy 3weekly	therapy, local
						does of Herceptin; 12/03 R Br noticed,	recurrence in
						FNAC +ve; 01/04 Local recurrencem recd	<1 yr
						FAC, Tamx, Letrozole, started on Palli
						Gemcitabine; 02/04 C/o Met AdenoCa
						advanced disease, recd. Palli CT x6, planned
						CT-5FU+Levoflex/weekly; 04/04 Palli
						MRM (R) + Exi. L side nodules, Toilet
						Mast (R); 09/04 only on supportive care,
						c/o severe pain in neck & chest wall, un-
						conciousness, giddiness, duee to locally
						recurrent disease, adv. Brain CT
	15	61	IDC-3,	pT3N2aMx	P/5% P/P	R Br TCB in 09/03; R MRM 09/03; CT-	Recurred in 3
			R Br			FECx3 09/03-11/03; RT 50.4 Gy 11/03-	yrs to L br and
						01/04: CT-FECx3 11/03-01/04; Normal	to bone in 5
						F/U 05/04; Ricurred in L Br 2007; L-	yrs:
						MRM 2007; aggressive disease as −/−/+;	Likely expired
						Bone mets in 03/09: Now metastic
						progressive disease; Likely expired as per
						Dr Patil since metastic dis in 2009
	16	48	IDC3;	T2N2aM0	P/P/N	08/2000 L MRM wax, Clearance; Adj	Alive with
			L Br			CT-ACx3 IN 08-10/2000; Defaulted	recurrent local
						on CT; Locally advanced dis in 2003;	disease
						04/2006 Recurrence of IDC of Br in soft
						tissues of Chest wall; RT 50.4 + 10 GY/
						30#s; Defaulted on CT; 12/2009 Ch wall
						recurrence; 12/2009 CT-FECx6 in till
						04/2010; On Lterozole 2.5 mg × 3 mths;
						Regular F/up; Last in 10/2011
ER+/PR+	17	58	ILC-2A;	pT2N0Mx	P/P/N	Lumpectomy on 05/2005; Neo adj CT-	Metastic
Bad			R Br			FACx6 06-09/2005 Interstitial brachy	disease/Dead
Out-						of R Br ??/; 02/2006 F/up N
come	18	55	IDC-3;	T2N1	P/P(20%)/N	MRM on 02/2005	Mets and
			L Br				dead
ER−/PR−	19	49, Post	IDC-3, L	T2N0M0	N/N	Lumpectomy on 03/03, have	Alive, 9 yrs
Good			Br, high			lumpectomy sample, MRM 04/03	since MRM
Out-			mitotic
come			case, no
			embolic or
			invasion
			but
			necrosis
			present
	20	62, Post	IDC-3, L	T4N0Mx	N/N	Mammo, Trucut (01/01/04), Lump, MRM	8 yr since
			Br			(L Br) 6 & 7/01/04; CT-FACx6 (17/01-	MRM
						10/05/04), RT 50.4 Gy/28# (02/06-08/07/04);	(L Br),
						08/06- In remission; 08/07-c/o UTI, vomiting,	currently
						fever, adv CT scan, Mammo; 10/07- Inj	c/o pain in
						Avastin; 04/08- R-Br Normal; 07/09 - lower	R Br
						back pain, bone scan-no Met;01/12- 8 yrs
						since MRM, now pain in R Br, adv Mammo.
	21	49,	IDC, R Br	T2N2M0	N/N	08/03: Lump R Br(1.5 mths), MRM;	Alive & no
		Premeno.				09-12/03 CT-FEC x6; 10/03 RT 50.4 GY/	mets
						26# 02/06: All Normal; 11/06: Accident,
						Rt should bruise
ER−/PR−	22	61,	IDC-3 w	pT2N1Mx	N/N	10/08 F/u L Br, R MRM - N	No mets
Good			DCIS foci:				and alive
Out-			R Br
come	23	51,	IDC-3, R	T2N1M0,	N/N/N	91. ′00: FNAC for Lump L, R Br-	Alive with
		Premeno.	Br	pT3N2M0		Neg(repectively); H/o tubectomy; 08/03:	Mets;
						c/o Lump in Br(2 yrs), pain only during	1st FNAC
						menstural cycle, MRM (R); 08/03-01/04	done in ′91,
						CT-FEC x6; 12/03 RT; 04/04 Recurrent Br	then ′00,
						Ca Nodule- exi biopsy; 06/08 5 yrs since	MRM in 03,
						MRM, All Normal; 06/09: Bone scan-	local
						Normal; 06/11: Multiple neck nodules-	recurrence in
						Met Ca; 07/11; Local RT, start Capecitabine,	1 yr, mets in
						09/11; CT, 1 week break then start	8 yrs
						Capecitabine; 12/11: 5 cycles of
						Capecitabine; 01/12; ---?
	24	43	IDC-3 +	T2N1M0	N/N	L-MRM 02/2003; Adj CT-ACx4 03-05/2003;	Alive with
			DCIS,			Adj RT 50.4 Gy/28 # + Boost RT 14 Gy/7#	mets
			L Br			in 07/2003; CT-Pacli x4 08-09.2003:	CHECK
						Liver mets 08/2008; Alive with mets
	25	47	IDC-3	pT2N0M0	N/N/P	06/2001 L Br surgery & B/L
						oophorectomy; Adj CT-ECx4 07-09/2001;	Alive with
						Adj RT 50.4 + 10 Gy/33#s 10/2001;	local mets
						On Tamx 2002-2004 & all normal F/up;
						Local recurrence 06/2005; L MRM 08/2005;
						Adj CT-Docx6 09-12/2005; Adj RT 50.4 Gy/
						28# 01-02/2006; 11/2008 F/up Normal;
						05/2011 F/up normal.
	26	57	IDC;	T2N0M0;	N/N/N	R-MRM 02/2006; Adj CT-ACx4 IN 03-	Doing well
			Medullary	Stg 2A		05/2006; Adj CT-Paclix12 05-08/2006;
			Ca: R Br			Adj RT to R ch wall 50.4 Gy/28# 08-10/2006;
						On regular F/up in 2007+2009, all normal,
						minor c/o swelling in arms w pains; 05/2010
						local recurrence to L breast;
ER−/PR−	27	35	IDC-III;	T2N1M0;	N/N/P	Had L. BC Surgery 01/2006; Adj CT-TACx6	Doing well
Good			L Br	Stg 2B		in 02-05/2006; CT Pacli + Herceptin x12
Out-						in 05-08/2006;
come
ER−/PR−	28	68,	IDC-3,	T3N1M0	N/N	06/03: Lump in R Br(3 wks), MRM, Bone	Mets and dead;
Bad		Post	R Br			scan-possible skeletal mets; 06/03-01/04:	Bone mets
Out-						CT-AC x6; 06/04 No c/o Mets, Facial (R)	within 1 year
come						Palsy, severe weakness, no other Neuro
						complaints; 10/04 severe pain L shoulder,
						Bone scan-multiple skeletal mets, adv. Palli
						CT-Taxane and RT
	29	50	R Br	T4bN1M1	—	06/2000: Diag Br Ca w pulmonary met in	Brain mets in
						Hyd, 01-04/03 Palli CT-FAC; 04/03: R Br-	3 yrs; dead
						mass reduced in size; 11/03 c/o vomit,
						headache, disorientation- Brain mets, given
						Palli RT
	30	42,	IDC-3,	T2N1M0	N/N	MRM 08/03, adj CT-FEC x1/FEC x6;	Barin mets in
		Premano.	R Br			RT taken; 06/05 H/0 headache; Recurred in	1.8 yrs yrs of
						22 months in brain, crainiotomy done 06/05	MRM; Dead
	31	55,	IDC-2,	T4N2M0	N/N/P	Neo CT-AC x1/RT + Tamoxifen x4/	Mets and dead
		Post	R Br			MRM-3c/Her2 + & Xeloda x1; Met: L Br,	L Br, Bones
						Bones in 10 months/CT- Herpceptin +	in 10
						Vinorelbrine	months
	32	38,	ILC +		N/N/	02/03 FNAC, Lump, MRM; 02-08/03 CT-AC	Mets and dead;
		Premeno.	IDC −			x6; 03/03 RT 50.4 GY/28#; 02/04 - All	Bone (3 yrs)
			3 + DCIS;			Normal; 07/05 vomiting, headache, L facial	and
			L Br			pain, adv Dexona, Hy6drocort; 10/05 feels	subsequently
						week, mild pallor, Bone marrow asp & Bx-	brain mets
						met Ca adv CT-Docetaxel/week; 06/06 FNAC	(4 yrs)
						R Br-IDC, tiny brain mets, bone marrow-met	Mets and dead;
						Ca, started on Xeloda; 07/06 severe	bone (3 yrs)
						headache (1 mth). FNAC R Br(swelling) +	and
						ve, Mets in contralateral Br, bone marrow,	subsequently
						brain. Carcinomatous meningitis, Adv	brain mets
						contuinue CT, Palli RT spinal, cranial, femur:	(4 yrs)
						12/06 Bone scan-some lesions regressed,
						some increased.
ER−/PR−	33	45,	IDC-3 w	T3N1Mx,	N/N	c/o Br lump-6 mths, H/o Hashinoto's	Mets and dead;
Bad		Premeno.	DCIS	pT3N2aM0		Thyroiditis, Thyromegaly (2 yrs); 07/03:	Expired within
Out-			foci,	(Stage-3a)		MRM; 07-12/03: CT-FEC x6; 09/03: RT	2 yrs with
come			L Br			50.4 Gy/28# given after 3rd CT; 11/03:	Brain, liver
						cold, anemia w Mycoplasma infection, given	mets
						washed RBC; 09/03/05: c/o Rt Hemiparesis
						(1 week), loss of appetite & weakness. Bone,
						brain, chest scan show mets. Adv Palli RT;
						11/03/05: Expired, COD: Mulitiple brain sec.,
						Pri Br Ca w Liver mets
	34	62	IDC-3,	T3N1Mx;	N/N/P	Br lump since 2003; 06/06 lung mets;	Had multiple
			R Br	Stg 4		Neoadj CT-TACx4 in 10/06; T increased	regimes of
						in size, CT changed to Gem + Cisplatin ×	CT. Mets
						03/07; on Xeloda since 07/07: T increased	and dead
						in size; Start Capcitabine/Vincristine/
						Gem+ irinotecan by 07/08-> Palliative
						R MRM 07/08: Lung mets; cough &
						itching in 05/09 advised Xeloda + Tykerb;
						10/09 endoxan, celecoxib; 07/10 disease
						progression to LN mets + cough
	35	60	IDC-3b,	T4N2M1:	TBD	06/2004 came w lump in R br + bone & brain	Multiple CTs;
				IV		mets; Neo-adj CT-Doce + epirubiein x3	Metastatic
						06-08/2004; Progressive disease thus changed	adeno-
						to salvage CT Gemcitabine + Vinorelbine	carcinoma,
						08/2004; T mass increased, CT changed to	dead
						palliative CT-TACx4 01-03/2005: Had HT
						also, Patient passed away on 17.8.2005
	36	44	IDC-3;	TxN1M1	N/N/P2+	B/L Lump excision in 08/2005: R-MRM on	Mets
			R Br			11/2005: Adj CT-FECx6 11/05-02/06: RT	and
						50.4/28# 03-05/2006; 12/2006 brain mets &	dead
						crainiotectomy done; 01/2007 RT to brain
						45 Gy/25#s; 02/2007 c/o cough B/L lung mets;
						Not willing for injectible CT hence oral CT
						advised

Claims

We claim:

1. A method of prognosis of a subject having breast cancer or suspected of having breast cancer, said method comprising acts of:

a) collecting biological sample from the subject and identifying expression or absence of receptors selected from a group comprising estrogen receptor, progesterone receptor and Human Epidermal Growth Factor Receptor receptor or any combination thereof in cells of the sample to obtain expression of identified cells;

b) carrying out immunohistochemistry analysis on the cells of step (a) with antibody against a combination of: biological markers consisting of CD44 and P-cadherin; along with at least one biological marker selected from a group consisting of ABCC4, β-catenin, ABCG2, CD133 and Oct-4 or any combination thereof;

c) identifying expression or absence of the receptors and the biological markers in cells of the sample and correlating the identification of the markers with a corresponding outcome based on combination of predictive outcome reference table nos. 11, 12, 13, 14, 15 and 16 for predicting the prognosis of the subject:

TABLE 11 MARKER ER+/PR+ ER−/PR− RESULTS CD44 YES — GOOD OUTCOME LOW EXPRESSION OF CD44 IN MEMBRANE YES — BAD OUTCOME HIGH EXPRESSION OF CD44 IN MEMBRANE OR MERE CYTOPLASMIC/NUCLEAR EXPRESSION CD44 — YES GOOD OUTCOME HIGH EXPRESSION OF CD44 IN MEMBRANE — YES BAD OUTCOME LOW EXPRESSION OF CD44 IN MEMBRANE OR MERE CYTOPLASMIC/NUCLEAR EXPRESSION

TABLE 12 MARKER ER+/PR+ ER−/PR− RESULTS ABCG2 — YES GOOD OUTCOME HIGH EXPRESSION OF ABCG2 IN NUCLEUS — YES BAD OUTCOME LOW EXPRESSION OF ABCG2 IN NUCLEUS

TABLE 13 MARKER ER+/PR+ ER−/PR− RESULTS ABCC4 YES — GOOD OUTCOME LOW EXPRESSION OF ABCC4 IN MEMBRANE YES — BAD OUTCOME HIGH EXPRESSION OF ABCC4 IN MEMBRANE

TABLE 14 MARKER ER+/PR+ ER−/PR− RESULTS CD133 — YES GOOD OUTCOME (HER2 −ve) LOW EXPRESSION OF CD133 IN MEMBRANE — YES BAD OUTCOME (HER2 −ve) HIGH EXPRESSION OF CD133 IN MEMBRANE CD133 — YES GOOD OUTCOME (HER2 +ve) HIGH EXPRESSION OF CD133 IN MEMBRANE — YES BAD OUTCOME (HER2 +ve) LOW EXPRESSION OF CD133 IN MEMBRANE

TABLE 15 MARKER ER+/PR+ ER−/PR− RESULTS P-CADHERIN YES — GOOD OUTCOME LOW EXPRESSION OF P- CADHERIN IN MEMBRANE YES — BAD OUTCOME HIGH EXPRESSION OF P- CADHERIN IN MEMBRANE P-CADHERIN — YES GOOD OUTCOME HIGH EXPRESSION OF P- CADHERIN IN MEMBRANE — YES BAD OUTCOME LOW EXPRESSION OF P- CADHERIN IN MEMBRANE

TABLE 16 MARKER ER+/PR+ ER−/PR− RESULTS β-CATENIN YES — GOOD OUTCOME LOW EXPRESSION OF β- CATENIN IN MEMBRANE YES — BAD OUTCOME HIGH EXPRESSION OF β- CATENIN IN MEMBRANE

Wherein, the low expression represents percentage intensity of staining of the cells between 0 to 40%, and the high expression represents percentage intensity of staining of the cells between more than 40% and upto 100%.

2. The method as claimed in claim 1, wherein the immunohistochemistry analysis is carried out by conventional method and wherein the identification of markers is carried out by visualizing a colored reaction or fluorescence obtained at completion of the method due to staining of the cells from the sample.

3. The method as claimed in claim 1, wherein the correlating is based on parameters selected from a group comprising percentage of staining, intensity of staining and location of staining or any combination thereof; and wherein the location of the staining is selected from a group comprising cell membrane, cytoplasm, nucleus, and nuclear membrane or any combination thereof.

4. The method as claimed in claim 1, wherein the correlating comprises multiplying the percentage of staining with the intensity of staining to arrive at a predictive score, or wherein the correlating is carried out based on percentage of cells stained in order to predict the prognosis as being good or bad depending on the location of expression of the biological markers.

5. The combination as claimed in claim 1, wherein the combination of biological markers consisting of CD44, ABCC4, P-cadherin and β-catenin prognose breast cancer in a subject having cells expressing estrogen receptor or progesterone receptor or both.

6. The combination as claimed in claim 1, wherein the combination of biological markers consisting of CD44, ABCG2, CD133 and P-cadherin optionally along with Oct-4 prognose breast cancer in a subject having cells not expressing estrogen receptor and progesterone receptor.

7. The combination as claimed in claim 1, wherein the combination of biological markers consisting of CD44, CD133 and P-cadherin prognose breast cancer in a subject having cells expressing Human Epidermal Growth Factor Receptor 2, and not expressing estrogen receptor and progesterone receptor.

8. A method of treating breast cancer, said method comprising acts of:

a) identifying a combination of biological markers on tumor cells in a biological sample and predicting prognosis of a subject having breast cancer or suspected of having breast cancer; and

b) based on the prediction, designing a cancer therapy for suppression of the cancer;

wherein the combination comprises biological markers consisting of CD44 and P-cadherin; along with at least one biological marker selected from a group consisting of ABCC4, β-catenin, ABCG2, CD133 and Oct-4 or any combination thereof.

9. A kit for prognosis of a subject having breast cancer or suspected of having cancer, said kit comprising antibody against combination of: biological markers consisting of CD44 and P-cadherin; along with at least one biological marker selected from a group consisting of ABCC4, β-catenin, ABCG2, CD133 and Oct-4 or any combination thereof, optionally along with organic solvent, reagent, secondary antibody, enzyme for performing immunohistochemistry and instruction manual.

10. The kit as claimed in claim 9, wherein the organic solvent is selected from a group comprising alcohol and xylene or a combination thereof.

11. The kit as claimed in claim 9, wherein the combination of biological markers consisting of CD44, ABCC4, P-cadherin and β-catenin prognose breast cancer in the subject having cells expressing estrogen receptor or progesterone receptor or both.

12. The kit as claimed in claim 9, wherein the combination of biological markers consisting of CD44. ABCG2, CD133 and P-cadherin optionally along with Oct-4 prognose breast cancer in the subject having cells not expressing estrogen receptor and progesterone receptor.

13. The kit as claimed in claim 9, wherein the combination of biological markers consisting of CD44, CD133 and P-cadherin prognose breast cancer in the subject having cells expressing Human Epidermal Growth Factor Receptor 2, and not expressing estrogen receptor and progesterone receptor.