Abstract:
The present disclosure provides improved methods for identifying breast cancer patients that receive an increased benefit from the addition of a HER 2 -targeted therapy, for example adjuvant trastuzumab, to chemotherapy.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    The present application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/327,460, which was filed on Apr. 23, 2010 and is incorporated herein by reference in its entirety. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0002]    Not Applicable 
       THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT 
       [0003]    Not Applicable 
       INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC 
       [0004]    Not Applicable 
       BACKGROUND OF THE INVENTION 
       [0005]    Currently HER2-targeted therapies such as trastuzumab or lapatinib are only used in the treatment patients diagnosed with HER2 positive breast cancer, which comprise only 15% to 20% of all breast cancer patients. HER2 positivity is defined by either overexpression of HER2 protein, which is determined by immunohistochemical staining (3+ staining score by FDA approved Herceptest assay), or by amplification of the HER2 (ERBB2) gene, which is determined by fluorescence in situ hybridization assay (HER2/CEP17 ratio over 2 using FDA approved PathVysion assay). The current cut-offs for these assays were determined from clinical trials of patients diagnosed with metastatic or advanced breast cancer. 
         [0006]    However, in a trial that tested the worth of addition of trastuzumab to adjuvant chemotherapy in the treatment of stage 2 or 3 breast cancer patients (NSABP trial B-31), even patients diagnosed with breast cancer that was classified as HER2 negative using currently used clinical HER2 assays (IHC and FISH) gained significant benefit from trastuzumab (Paik, et al.,  N. Engl. J. Med.  358:1409-1411, 2008). In this study, degree of HER2 gene amplification or protein expression did not have any correlation with the degree of benefit from trastuzumab, directly challenging the use of currently used HER2 clinical assays (IHC and FISH) for selection of patients for adjuvant trastuzumab or other HER2 targeted therapies. 
         [0007]    Therefore improved predictive tests for HER2-targeted therapies are clearly required. 
       BRIEF SUMMARY OF THE INVENTION 
       [0008]    In order to develop better predictive test for HER2 targeted therapies, whole genome (transcriptome) gene expression profiling was performed on tumor specimens collected from patients enrolled in NSABP trial B-31 using microarrays (Agilent and Affymetrix platforms). As a result of this gene expression profiling effort, it was determined that mRNA expression levels of HER2 (ERBB2) itself is a predictor of the degree of benefit from trastuzumab in NSABP trial B-31. In addition, based on findings from NSABP trial B-31, is was determined that a large number of patients diagnosed with breast cancer that are classified as HER2 negative using current generation HER2 assays (IHC and FISH) are expected to derive benefit from trastuzumab or other HER2 targeted therapies. Therefore, the present disclosure details HER2 assays (based on measurement of HER2 mRNA) that provide a significant improvement over currently used HER2 assays (FISH and IHC) as a predictor of the degree of benefit from HER2 targeted therapies in the treatment of breast cancer in an adjuvant setting (stage 2 or 3 breast cancer). 
         [0009]    Currently, breast cancer samples are assayed for HER2 protein levels or HER2 gene copy number, and based on this analysis the breast cancer samples are classified as “HER2 positive” or “HER2 negative.” Breast cancers that are classified as “HER2 positive” are candidates for treatment with a HER2-targeted therapy, such as trastuzumab, while those that are classified as “HER2 negative” are not candidates for HER2-targeted therapy. However, the inventors have determined that many breast cancers that are currently classified as “HER2 negative” still receive a therapeutic benefit from HER2-targeted therapies, such as trastuzumab. Therefore, the present disclosure provides improved assays that are more accurate in predicting the benefit from addition of a HER2-targeted therapy to chemotherapy. Breast cancer samples that were classified as “HER2 negative” by the assays previously described and used in the clinic are often classified as “HER2 positive” using the presently described HER2 mRNA assays. Therefore, numerous breast cancer patients that would not have been candidates for treatment with a HER2-targeted therapy based on the assays previously described and used in the clinic can be correctly identified as candidates for treatment with HER2-targeted therapies, such as trastuzumab, thus improving breast cancer patient care. 
         [0010]    The present disclosure provides methods of identifying a cancer patient, for example a breast cancer patient, that has an increased benefit from the addition of a HER2-targeted therapy to chemotherapy, comprising assaying a tumor tissue sample from said patient for expression of HER2 mRNA, wherein a normalized HER2 mRNA expression level of about 6.0 or greater is indicative of a cancer patient that has a increased benefit from the addition of a HER2-targeted therapy to chemotherapy. In certain embodiments, normalized HER2 mRNA expression levels of about 6.0 to about 10.5 are indicative of a cancer patient that has an increased benefit from the addition of a HER2-targeted therapy to chemotherapy. In still other embodiments, normalized HER2 mRNA expression levels that are below the levels previously classified as “HER2 positive” are indicative of a cancer patient that has an increased benefit from the addition of a HER2-targeted therapy to chemotherapy. In particular aspects, normalized HER2 mRNA expression levels of about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or about 10.5 or greater are indicative of a cancer patient that has a increased benefit from the addition of a HER2-targeted therapy to chemotherapy. 
         [0011]    In certain aspects of the present disclosure, the HER2-targeted therapy is trastuzumab, while in other aspects of the present disclosure the HER2-targeted therapy is lapatinib. In particular aspects of the present disclosure, the HER2-targeted therapy is combination of trastuzumab and lapatinib. It will be understood to the skilled artisan that other HER2-targeted therapies, either alone or in combination, could be used in conjunction with the teachings of the present disclosure. 
         [0012]    Thus, the present disclosure additionally provides methods of treating breast cancer in a patient in need of such treatment, comprising assaying a breast cancer or tumor tissue sample from said patient for expression of HER2 mRNA, and treating the patient with a HER2-targeted therapy and chemotherapy if the results of the assay indicate a normalized HER2 mRNA expression level of about 6.0 or greater. 
     
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         [0013]      FIG. 1 . A plot of the log hazard ratio of trastuzumab in B-31 patients in relation to expression levels of HER2 mRNA. 
           [0014]      FIG. 2 . A plot of the mRNA levels of samples classified as HER2 negative and HER2 positive from the B-31 and B-28 studies. 
           [0015]      FIG. 3 . A plot showing the correlation between HER2 mRNA expression levels measured by the Nanostring method (nCounter assay) and the QuantigenePlex method. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0016]    Based on findings from NSABP trial B-31, a large number of patients diagnosed with breast cancer that are classified as HER2 negative using current generation HER2 assays (IHC and FISH) derived benefit from trastuzumab, a HER2-targeted therapy. Therefore, the present disclosure details HER2 assays (based on measurement of HER2 mRNA) that provide a significant improvement over currently used HER2 assays (FISH and IHC) as a predictor of the degree of benefit from HER2-targeted therapies in the treatment of breast cancer in an adjuvant setting (stage 2 or 3 breast cancer). In order to develop better predictive test for HER2 targeted therapies, whole genome (transcriptome) gene expression profiling was performed on tumor specimens collected from patients enrolled in NSABP trial B-31 using microarrays (Agilent and Affymetrix platforms). As a result of this gene expression profiling effort, it was determined that mRNA expression levels of HER2 (ERBB2) were a more accurate predictor of the degree of benefit from trastuzumab. 
         [0017]    Although specific techniques for the quantitation of HER2 mRNA levels are discussed in the Example below, it will be understood by the skilled artisan that any technique currently used for quantitation of mRNA levels can be used in the practice of the present invention. 
         [0018]    Therapeutic formulations are provided as pharmaceutical preparations for local administration to patients or subjects. The term “patient” or “subject” as used herein refers to human or animal subjects (animals being particularly useful as models for clinical efficacy of a particular composition). Selection of a suitable pharmaceutical preparation depends upon the method of administration chosen, and may be made according to protocols well-known to medicinal chemists. 
         [0019]    The term “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the platinum-based therapeutic agents, its use in the therapeutic compositions is contemplated. Supplementary active ingredients or therapeutic agents can also be used with the platinum-based therapeutic agents. 
         [0020]    As used herein, “pharmaceutically-acceptable salts” refer to derivatives of the disclosed compounds wherein one or more components of the disclosed compounds are modified by making acid or base salts thereof. Examples of pharmaceutically-acceptable salts include, but are not limited to: mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Thus, the term “acid addition salt” refers to the corresponding salt derivative of a component that has been prepared by the addition of an acid. The pharmaceutically-acceptable salts include the conventional salts or the quaternary ammonium salts of the component formed, for example, from inorganic or organic acids. For example, such conventional salts include, but are not limited to: those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. Certain acidic or basic compounds may exist as zwitterions. All forms of the active agents, including free acid, free base, and zwitterions, are contemplated to be within the scope of the present disclosure. 
         [0021]    A protein or antibody can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein), and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. 
         [0022]    In addition, the disclosed compositions or components thereof can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, dextran, and the like. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application. 
         [0023]    The dosage unit forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be suitably fluid. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. 
         [0024]    Sterile injectable solutions are prepared by incorporating the disclosed compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the dosage unit plus any additional desired ingredient from a previously sterile-filtered solution thereof. 
         [0025]    In certain aspects the present disclosure encompasses methods of treating or managing cancer, which comprise administering to a patient in need of such treatment or management a therapeutically effective amount of a disclosed composition or dosage unit thereof. In certain embodiments, such a compound or dosage unit is referred to as an active agent. Use of the disclosed compositions in the manufacture of a medicament for treating or preventing a disease or disorder is also contemplated. The present disclosure also encompasses compositions comprising a biologically or therapeutically effective amount of one or more of the disclosed compounds for use in the preparation of a medicament for use in treatment of cancer. 
         [0026]    As used herein, and unless otherwise indicated, the terms “treat,” “treating,” and “treatment” contemplate an action that occurs while a patient is suffering from cancer, which reduces the severity of one or more symptoms or effects of cancer, or a related disease or disorder. As used herein, and unless otherwise indicated, the terms “manage,” “managing,” and “management” encompass preventing, delaying, or reducing the severity of a recurrence of cancer in a patient who has already suffered from the cancer. The terms encompass modulating the threshold, development, and/or duration of the cancer, or changing the way that a patient responds to the cancer. 
         [0027]    As used herein, and unless otherwise specified, a “therapeutically effective amount” of a compound is an amount sufficient to provide any therapeutic benefit in the treatment or management of cancer, or to delay or minimize one or more symptoms associated with cancer. A therapeutically effective amount of a compound means an amount of the compound, alone or in combination with one or more other therapy and/or therapeutic agent, which provides any therapeutic benefit in the treatment or management of cancer, or related diseases or disorders. The term “therapeutically effective amount” can encompass an amount that cures cancer, improves or reduces cancer, reduces or avoids symptoms or causes of cancer, improves overall therapy, or enhances the therapeutic efficacy of another therapeutic agent. 
         [0028]    Toxicity and therapeutic efficacy of the described compounds and compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are preferred. Compounds that exhibit toxic side effects may be used in certain embodiments, however, care should usually be taken to design delivery systems that target such compounds preferentially to the site of affected tissue, in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. 
         [0029]    Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. In certain aspects of the present disclosure, the dosages of such compounds Ile within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the disclosed methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels may be measured, for example, by high performance liquid chromatography. 
         [0030]    When therapeutic treatment is contemplated, the appropriate dosage may also be determined using animal studies to determine the maximal tolerable dose, or MTD, of a bioactive agent per kilogram weight of the test subject. In general, at least one animal species tested is mammalian. Those skilled in the art regularly extrapolate doses for efficacy and avoiding toxicity to other species, including human. Before human studies of efficacy are undertaken, Phase I clinical studies help establish safe doses. Additionally, the bioactive agent may be complexed with a variety of well established compounds or structures that, for instance, enhance the stability of the bioactive agent, or otherwise enhance its pharmacological properties (e.g., increase in vivo half-life, reduce toxicity, etc.). 
         [0031]    In certain embodiments of the present disclosure, the effective dose of the composition or dosage unit can be in the range of about 10 mg/kg to about 0.01 mg/kg, about 10 mg/kg to about 0.025 mg/kg, about 10 mg/kg to about 0.05 mg/kg, about 10 mg/kg to about 0.1 mg/kg, about 10 mg/kg to about 0.25 mg/kg, about 10 mg/kg to about 0.5 mg/kg, about 10 mg/kg to about 1 mg/kg, about 10 mg/kg to about 2.5 mg/kg, about 10 mg/kg to about 5 mg/kg, about 5 mg/kg to about 0.01 mg/kg, about 2.5 mg/kg to about 0.01 mg/kg, about 1 mg/kg to about 0.01 mg/kg, about 0.5 mg/kg to about 0.01 mg/kg, about 0.25 mg/kg to about 0.01 mg/kg, about 0.1 mg/kg to about 0.01 mg/kg, about 0.05 mg/kg to about 0.01 mg/kg, about 0.025 mg/kg to about 0.01 mg/kg, about 5 mg/kg to about 0.025 mg/kg, about 2.5 mg/kg to about 0.05 mg/kg, about 1 mg/kg to about 0.1 mg/kg, about 0.5 mg/kg to about 0.25 mg/kg, or about 3 mg/kg to about 0.1 mg/kg, or so. Thus, in particular embodiments, the effective dose of the composition or dosage unit is about 0.01 mg/kg, about 0.025 mg/kg, about 0.05 mg/kg, about 0.075 mg/kg, about 0.1 mg/kg, about 0.25 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 5 mg/kg, about 7.5 mg/kg, or about 10 mg/kg, or so. 
         [0032]    The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. 
       Example 1 
       [0033]    In The National Surgical Adjuvant Breast and Bowel Project (“NSABP”) clinical trial B31 cohort, the HER2 assays currently used in routine clinical practice to select patients for HER2 targeted therapies (namely IHC and FISH assays) failed to predict the degree of benefit from trastuzumab, and surprisingly, as shown in Table 1, even patients diagnosed with HER2 negative tumors gained the same degree of benefit as those with HER2 positive breast cancer defined by current HER2 assays (IHC and FISH) (Paik, et al.,  N Engl. J. Med.  358:1409-1411, 2008). This data underscores the need to develop a new predictive test that can be used to predict the degree of benefit from HER2 targeted therapies in adjuvant setting. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
             
             
               
                   
                   
               
               
                   
                 Treatment (events/total events) 
                   
               
             
          
           
               
                   
                 Central 
                   
                 Chemo plus 
                 RR 
                   
                 Interaction 
               
               
                 End Point 
                 HER2 Assay 
                 Chemo 
                 Trastuzumab 
                 (95% CI) 
                 p-value 
                 p-value 
               
               
                   
               
             
          
           
               
                 DFS 
                 Positive 
                 163/875 
                 85/804 
                 0.47 
                 &lt;0.001 
                 0.47 
               
               
                   
                   
                   
                   
                 (0.37-0.62) 
               
               
                   
                 Negative 
                 20/92 
                 7/82 
                 0.34 
                 0.014 
               
               
                   
                   
                   
                   
                 (0.14-0.80) 
               
               
                 Overall 
                 Positive 
                  55/875 
                 38/804 
                 0.66 
                 0.047 
                 0.08 
               
               
                 Survival 
                   
                   
                   
                 (0.43-0.99) 
               
               
                   
                 Negative 
                 10/92 
                 1/82 
                 0.08 
                 0.17 
               
               
                   
                   
                   
                   
                 (0.01-0.64) 
               
               
                   
               
             
          
         
       
     
         [0034]    In Table 1, the end points were disease-free survival (“DFS”) or overall survival. The central HER2 assay results were defined as negative if they were negative by both fluorescence in situ hybridization (PathVysion™, Vysis) and immunohistochemical analysis (Herceptest™, Dako), and were defined as positive if either test was positive. Chemotherapy denotes 4 cycles of doxorubicin plus cyclophosphamide followed by 4 cycles of paclitaxel. The 95% confidence intervals (“CI”) and p-values were adjusted according to the number of positive nodes and estrogen-receptor status from the univariate Cox proportional-hazards model for each subgroup in the NSABP B-31 trial. 
         [0035]    In order to develop a predictive test for the degree of benefit from trastuzumab or other HER2-targeted therapies, whole genome (trasnscriptome) gene expression profiling was performed on formalin fixed paraffin embedded tumor blocks collected from NSABP trial B-31, which tested the value of adding trastuzumab to standard adjuvant chemotherapy in the treatment of stage 2 or stage 3 breast cancer. The B-31 trial was largely enriched for HER2 positive breast cancer (90%), but also included HER2 negative breast cancer (10%). 
         [0036]    The available tumor blocks from NSABP B-31 were divided into two randomly selected cohorts of discovery and validation sets. Microarray gene expression profiling was performed using both Agilent and Affymetrix arrays, and formal statistical tests (in Cox proportional hazard models controlling for clinical variables such as estrogen receptor status, tumor size, age, and number of metastatic axillary lymph nodes) were performed to test the interaction between gene expression and trastuzumab benefit. Since HER2 is a known target for trastuzumab, the initial a priori hypothesis was that HER2 (ERBB2) mRNA expression level is a linear predictor of the degree of benefit from trastuzumab, and improves upon the current generation of IHC- or FISH-based HER2 assays as a predictor of the degree of benefit from trastuzumab. 
         [0037]    There are two independent oligonucleotide probes that hybridize to HER2 (ERBB2) mRNA in the Agilent microarray and three probes in the Affymetrix microarray. All five probes showed statistically significant interaction with trastuzumab as shown in Table 2, with interaction p-values ranging from 0.0075 to 00036. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Microarray Platform 
                 Probe 
                 Interaction p-value 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Agilent 
                 a_24_p284420 
                 0.00092 
               
               
                   
                 Agilent 
                 a_23_p89249 
                 0.00063 
               
               
                   
                 Affymetrix 
                 234354_x_at 
                 0.0013 
               
               
                   
                 Affymetrix 
                 216836_s_at 
                 0.00036 
               
               
                   
                 Affymetrix 
                 210930_s_at 
                 0.0075 
               
               
                   
                   
               
             
          
         
       
     
         [0038]    Based on these findings, a new HER2 mRNA assay was developed using nanostring platform (Geiss, et al.,  Nat. Biotechnol.  26:317-325, 2008). The test is based on a commercially available technical platform from Nanostring but with custom designed probe sets including a specific set of reference genes (ACTB, RPLP0, H2ASY, SNRP70) to normalize the expression value of HER2 mRNA. This proprietary set of reference genes were selected from data mining of microarray data from NSABP trial B-27. 
         [0039]    All available tumor blocks from the B-31 trial were examined, and formal statistical tests for interaction between HER2 mRNA and trastuzumab were performed. Nanostring-based HER2 mRNA was strongly predictive of the degree of benefit from trastuzumab in B-31. To illustrate this, log hazard of trastuzumab in B-31 patients is plotted in relation to expression levels of HER2 mRNA ( FIG. 1 ).  FIG. 1  shows a linear prediction of the degree of benefit from trastuzumab added to chemotherapy by the level of expression of HER2 mRNA in the treatment of breast cancer. Values above zero on the Y-axis means no benefit, and negative values on the Y-axis mean benefit from trastuzumab. HER2 mRNA levels in  FIG. 1  are based on nanostring assays, but other methods of measurement showed similar plots. 
         [0040]    With increasing amounts of HER2 mRNA expression in the tumor tissue, there is an increasing degree of benefit from trastuzumab added to chemotherapy in B-31. The cut-off of trastuzumab benefit can be determined from  FIG. 1  with confidence intervals. The cut off based on B-31 data is 8.5 normalized HER2 mRNA expression level with a confidence interval of 6 to 10.5. 
         [0041]    When this cut-off was applied to all breast cancer (B-31 study and B-28 study, which also compares 4 cycles of arimycin (doxorubicin) plus cyclophosphamide versus 4 cycles of AC followed by four cycles of TAXOL® (paclitaxel)), it became evident that a significant proportion of HER2 negative patients would benefit from trastuzumab ( FIG. 2 ).  FIG. 2  shows the identification of breast cancer patients who may benefit from trastuzumab in adjuvant setting (stage 2 or stage 3) based on HER2 mRNA measurement. The cut off derived from the nanostring HER2 mRNA assay is applied to a scattergram of tumors that are classified as either HER2 positive or HER2 negative by current clinical HER2 assays (IHC or FISH). The dotted line is the cut-off. It is clear that most breast cancers express HER2 mRNA at levels above the dotted line, suggesting that a significant proportion of patients with breast cancer are expected to benefit from trastuzumab. 
         [0042]    Since HER2 mRNA expression levels linearly correlate with the degree of benefit from trastuzumab, this assay can be utilized to estimate the degree of benefit from trastuzumab before starting the treatment, and this information will help clinicians and patients decide whether to use HER2-targeted therapies, as well as considering other therapies. While the data in this Example is based on HER2 mRNA expression levels measured using either Agilent or Affymetrix arrays, or nanostring platform, the results are applicable broadly to any measure of HER2 mRNA, since a close correlation was demonstrated between HER2 mRNA measured by nanostring and other methods such as Quantigene Plex assay that were performed in a subset of B-31 samples ( FIG. 3 ).  FIG. 3  shows the correlation between HER2 mRNA expression levels measured by Nanostring method (nCounter assay) and QuantigenePlex method. 
         [0043]    All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.