Correlation of molecular markers with clinical outcome in gbm patients radiation treated with or without gefitinib

Abstract

Interestingly, for prognosis, the significant biomarkers for Gefitinib-treated GBM patients (RTOG 0211) appeared to differ compared to historical, RT and non-Gefitinib-treated GBM patients. In Gefitinib-treated patients, those with higher levels of nuclear pAKT driven by PTEN loss, higher levels of nuclear pMAPK, and lower levels of nuclear pmTOR had significantly worse clinical outcomes. In contrast, in non-Gefitinib-treated patients, patients with PTEN-deficiency, and higher levels of EGFRvIII, total EGFR, IGFR1, NFkB and lower levels of nuclear Survivin appeared to have adverse clinical outcomes, highlighting the treatment-dependency of these biomarkers.

1 - 27 . (canceled) 
     
     
         28 . A method of determining a prognosis of a patient harboring a glioblastoma multiforme (GBM) tumor comprising:
 assessing a subcellular concentration of one or more protein biomarkers selected from a group consisting of EGFRvIII, EGFR, IGF-1R and NFkB in a tissue specimen comprising GBM cells obtained from a patient harboring a GBM tumor by comparing the concentration of the one or more protein biomarkers to a concentration for the same protein biomarker in a control tissue specimen or a plurality of tissue specimens obtained from a plurality of patients harboring GBM;   classifying a patient having a high level of one or more of said protein biomarkers as having a poor prognosis if the patient is treated with radiation therapy alone, and   withholding from the patient classified as having a poor prognosis the administration of radiation therapy alone.   
     
     
         29 . The method of  claim 28 , further comprising:
 assessing a concentration of PTEN in the tissue specimen comprising GBM cells obtained from the patient harboring a GBM tumor,   affirming a classification of a patient having a low level of PTEN as having a poor prognosis if the patient is treated with radiation therapy alone.   
     
     
         30 . A method of determining a prognosis of a patient harboring a glioblastoma multiforme (GBM) tumor comprising:
 assessing a subcellular concentration of two or more protein biomarkers selected from a group consisting of EGFRvIII, EGFR, IGF-1R and NFkB in a tissue specimen comprising GBM cells obtained from a patient harboring a GBM tumor;   comparing the concentration of the two or more protein biomarkers to (a) a concentration for the same two or more protein biomarkers in a control tissue specimen, or (b) a concentration for the same two or more protein biomarkers in a plurality of tissue specimens obtained from a plurality of patients harboring a GBM tumor;   classifying the patient having a high level of the two or more protein biomarkers as having a poor prognosis if the patient is treated with radiation therapy alone, and   withholding from the patient classified as having a poor prognosis the administration of radiation therapy alone.   
     
     
         31 . The method of  claim 30 , further comprising:
 assessing a relative concentration of PTEN in the tissue specimen comprising GBM cells obtained from the patient harboring a GBM tumor,   affirming a classification of a patient having a low level of PTEN as having a poor prognosis if the patient is treated with radiation therapy alone.   
     
     
         32 . The method of  claim 30  in which the concentration of the two or more protein biomarkers of the patient are compared with the concentration of the same two or more protein biomarkers in a plurality of tissue specimens obtained from a plurality of patients harboring a GBM tumor. 
     
     
         33 . A method of determining a prognosis of a patient harboring a glioblastoma multiforme (GBM) tumor comprising:
 assessing a subcellular concentration of one or more protein biomarkers selected from a group consisting of nuclear pAKT, nuclear pmTOR, nuclear pMAPK and Src in a tissue specimen comprising GBM cells obtained from a patient harboring a GBM tumor by comparing the concentration of the one or more protein biomarkers to a concentration for the same protein biomarker in a control tissue specimen or a plurality of tissue specimens obtained from a plurality of patients harboring GBM;   classifying a patient having a high level of nuclear pAKT, nuclear pMAPK, low level of nuclear pmTOR and/or intermediate or high level of Src as having a poor prognosis if the patient is treated with radiation therapy combined with gefitinib, and   withholding from the patient classified as having a poor prognosis the administration of radiation therapy in combination with gefitinib.   
     
     
         34 . A method of determining a prognosis of a patient comprising:
 assessing the concentration of two or more protein biomarkers selected from a group consisting of pAKT, pmTOR, and pMAPK in a tissue specimen obtained from a patient harboring a GBM tumor;   comparing the concentration of the two or more protein biomarkers to (a) a concentration for the same one or more protein biomarkers in a control tissue specimen or (b) a concentration for the same one or more protein biomarkers in a plurality of tissue specimens obtained from a plurality of patients harboring a GBM tumor;   wherein (A) high levels of nuclear pAKT, nuclear pMAPK, and/or a low level of nuclear pmTOR indicates a poor prognosis for the patient when treated with radiation therapy combined with gefitinib, or (B) low levels of nuclear pAKT, nuclear pMAPK, and/or a high level of nuclear pmTOR indicates a better prognosis for the patient when treated with radiation therapy combined with gefitinib; and   withholding or administering treatment with radiation therapy in combination with gefitinib depending on the prognosis.   
     
     
         35 . A method of determining a prognosis of a patient harboring a glioblastoma multiforme (GBM) tumor comprising:
 a) incubating a tissue sample comprising GBM cells obtained from a patient harboring a GUM tumor with a first stain that specifically labels a first marker defining a first subcellular compartment, a second stain that specifically labels a second marker defining a second subcellular compartment and a third stain that specifically labels a marker different from the first and second markers;   b) obtaining a high resolution image of each of the first, the second and the third stain in the tissue sample;   c) assigning a pixel of the image to the first subcellular compartment based on the first stain intensity, the second subcellular compartment based on the second stain intensity, or to neither a first nor second compartment;   d) measuring the intensity of the third stain in each of the pixels assigned to either the first or the second subcellular compartments or both;   e) determining a staining score indicative of the concentration of the biomarker in the first or the second subcellular compartments or both by dividing a subset of pixels assigned to the third stain by a number of pixels assigned to both the first and the second stains; and   f) predicting the prognosis of the patient based on the staining score for the biomarker in the first or the second compartments, or both;   wherein (1) the biomarker is selected from the group consisting of nuclear pAKT, cytoplasmic PTEN, nuclear PTEN, EGFRvIII, EGFR, IGF-1 R, nuclear pmTOR, nuclear pMAPK, survivin and NFkB;   (2) the patient having (a) a high level of nuclear pAKT or nuclear pMAPK or (b) a low level of cytoplasmic or nuclear PTEN is classified as having a poor prognosis when treated with radiation therapy combined with gefitinib; or   (3) classifying the patient with a high level of nuclear pmTOR as having good prognosis when treated with radiation therapy combined with gefitinib; and   withholding or administering the treatment depending on the prognosis.   
     
     
         36 . The method of  claim 35 , wherein the patient having an intermediate level of EGFR, a high level of NFkB and an intermediate or high level of SRC is classified as having a poor prognosis when treated with radiation therapy combined with gefitinib 
     
     
         37 . The method of  claim 35 , wherein the first subcellular compartment is cytoplasm. 
     
     
         38 . The method of  claim 37 , wherein the first stain comprises a stain for GFAP. 
     
     
         39 . The method of  claim 35 , wherein the second subcellular compartment is nucleus. 
     
     
         40 . The method of  claim 39 , wherein the second stain comprises a stain for DAPI. 
     
     
         41 . A kit comprising: a) a first stain specific for pAKT, PTEN, EGFRvIII, EGFR, IGF-1R, pmTOR, pMAPK, survivin, Src and/or NFkB and b) a second stain specific for a first subcellular compartment of a cell. 
     
     
         42 . The kit of  claim 41  in which the second stain comprises a stain for GFAP. 
     
     
         43 . The kit of  claim 41 , further comprising instructions for using the kit. 
     
     
         44 . A method of identifying a patient harboring a glioblastoma multiforme (GBM) tumor who is suitable for treatment with a gefitinib in combination with radiation therapy, comprising:
 assessing concentrations of two or more biomarkers selected from the group consisting of pAKT, pmTOR, pMAPK, PTEN, EGFR, Src and NFkB in a subcellular compartment of a tissue specimen comprising glioblastoma multiforme cells obtained from a patient harboring a GBM tumor,   comparing the concentrations of the two or more biomarkers to the concentrations of the same two or more biomarkers in a plurality of tissue specimens obtained from a plurality of patients harboring a GBM tumor;   wherein (A) a high level of nuclear pmTOR or PTEN indicates that the patient is likely to benefit from treatment with gefitinib in combination with radiation therapy; or (B) a low level of nuclear pAKT, nuclear pMAPK, EGFR, Src and NFkB indicates that the patient is likely to benefit from treatment with gefitinib with radiation therapy; and   administering to the patient indicated as likely to benefit from treatment with gefitinib in combination with radiation therapy.