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  • Krizman, David  (4)
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  • 1
    Online Resource
    Online Resource
    Abstract A152: Multiplex assay in FFPE tissues to simulateously quantify the human EGF receptor (HER1–4) family proteins: Implications for targeted therapy and resistance to therapy.
    American Association for Cancer Research (AACR) ; 2011
    In:  Molecular Cancer Therapeutics Vol. 10, No. 11_Supplement ( 2011-11-12), p. A152-A152
    Details
    In: Molecular Cancer Therapeutics, American Association for Cancer Research (AACR), Vol. 10, No. 11_Supplement ( 2011-11-12), p. A152-A152
    Abstract: The human EGF receptor family (HER's) consists of two clinically validated drug targets (EGFR and Her2), and two receptors (Her3 and Her4) which are the subject of intensive preclinical and early clinical investigation. Although drugs inhibiting both EGFR and Her2 show significant antitumor activity in the clinic, the acquisition of resistance is a hallmark of these and other targeted therapies. In the case of both targets, one of the emerging resistance mechanisms is the co-expression of other members of the EGFR superfamily. It was recently shown that Her2 co-expression mediates resistance in cetuximab treated head and neck cancer (Sci Transl Med 7(3)99). Similarly, much attention has been paid to Her3 both as a bona fide drug target as well as a resistance mechanism. Finally, Her4, though less well studied appears to be another resistance mechanism. Her3 is usually expressed at much lower levels than EGFR and Her2 (often & lt;10%). Yet, receptor crosstalk and heterodimerization can allow even very low levels of Her3 to exert a large impact on drug response. According to one report (Cancer Epidemiol Biomarkers Prev. 19(4):982), IHC analysis of Her3 may be inadequate to properly quantitate Her3 expression in FFPE tumor tissues. This may be a result of high sequence homology/identity between Her3 and other Her family members. Due to the critical need to accurately quantitate this receptor, we have used a mass spectrometric approach to develop a specific and quantitative SRM assay for Her3 from FFPE tumor tissue. By using trypsin digestion mapping, we identified multiple unique peptide sequences from Her3 which were quantitated by SRM Mass spectrometry. Our assay was preclinically validated using the single most sensitive peptide, quantitating Her3 expression in multiple different cell lines, and human NSCLC primary tumor xenografts. These preclinical studies were then extended by assessing the expression levels of Her3 in two cohorts of clinical tumor tissue which had been treated with Her family antagonists. First, we measured Her3 expression in a set of neoadjuvant gefitinib treated NSCLC tumors. In this cohort, 12/15 tumor showed low but measurable levels of Her3 expression, ranging from 50–100 amol/ug tumor tissue. In a second tissue set, we measured Her3 expression in a cohort of advanced (Stage III-IV) breast cancer tissues which had undergone post resection adjuvant treatment with trastuzumab. These breast cancer samples demonstrated a higher level of expression of Her3, ranging from 50 − 250amol/ug tumor tissue, and 15/18 tumors were Her3 positive. In both studies, the relationship between Her family expression and response to either gefitinib or trastuzumab is currently under study. It is critically important to understand mechanisms of resistance in patients undergoing targeted therapies, and Liquid Tissue-SRM promises to be a platform which can deliver extremely high sensitivity, absolute specificity as well as multiplexing capabilities to assess the four HER family targets in unison. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A152.
    Type of Medium: Online Resource
    ISSN: 1535-7163 , 1538-8514
    URL: Article
    DOI: 10.1158/1535-7163.TARG-11-A152
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2011
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    SSG: 12
    Location Call Number Limitation Availability
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  • 2
    Online Resource
    Online Resource
    Abstract 1514: Comparative quantitation of Warburg Effect proteins in FFPE tumor and stroma tissue from 10 breast carcinoma tumors
    American Association for Cancer Research (AACR) ; 2011
    In:  Cancer Research Vol. 71, No. 8_Supplement ( 2011-04-15), p. 1514-1514
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 71, No. 8_Supplement ( 2011-04-15), p. 1514-1514
    Abstract: The observation that cancer cells can undergo aerobic glycolosis (the Warburg Effect) has been the target of a great deal of research in recent years, specifically directed to development of novel agents which can disrupt this pathway and potentially retard cancer growth. Recent studies, by Lisanti and colleagues, have suggested that the critical site of aerobic glycolysis may not be the tumor epithelial cells, but instead the ‘Warburg Effect’ is occurring in the tumor associated fibroblasts and other stromal cells. In order to better define the importance of ‘tumor’ vs. ‘stroma’ components of the Warburg effect, we have developed a quantitative multiplexed assay which can be performed directly in formalin fixed paraffin embedded (FFPE) patient tissue. This multiplex assay is based on the Liquid Tissue®-SRM technology platform, a combination of tissue microdissection, Liquid Tissue® processing which renders dissected tissue to a completely solubilized tryptic digest, and mass spectrometry-based selected reaction monitoring (SRM). For mass spectrometric analysis, we developed 9 different quantitative assays for each of the Warburg Effect components (LDHA, PKM2, FBA(a), PGKI, LDHA, PMI, EnolaseA, and TPI) as well as Caveolin-1 which was identified as a marker of active aerobic glycolysis in tumor fibroblasts. We then quantitated all of these proteins in multiplex on matched tumor and stromal tissue laser microdissected from 10 different FFPE breast carcinoma tumor samples. This analysis demonstrated broadly divergent levels of glycolytic enzyme expression across the set of tumor samples, and in most cases, much higher enzyme expression in tumor cells as opposed to tumor associated stroma cells. However there were several cases where the stromal level of Warburg protein expression was elevated above matched tumor epithelial cell expression. These results demonstrate that by using the Liquid Tissue® technology, we can separately microdissect tumor and stroma in FFPE tumor sections and characterize the specific tumor/stroma components, be they pharmacodynamic analyses, drug target analysis, or seed vs. soil studies. Second, this technology reinforces the capability of SRM based mass spectrometry studies to function in massively multiplexed fashion, where 20 to 40 or more specific analytes can be quantitated by analysis of 0.5ug of protein lysate from a single FFPE tumor section. Finally, we are in the process of clinically validating this Warburg Effect multiplex assay and will then perform the assay on a larger, completely annotated tumor cohort in order to extend our knowledge of how tumor vs. stroma expression of Warburg effect components impacts tumor progression and therapeutic response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1514. doi:10.1158/1538-7445.AM2011-1514
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2011-1514
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2011
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    Location Call Number Limitation Availability
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  • 3
    Online Resource
    Online Resource
    Abstract 4567: Multiplex assay in FFPE tissues to simultaneously quantify the human EGF receptor (HER1-4) family proteins
    American Association for Cancer Research (AACR) ; 2012
    In:  Cancer Research Vol. 72, No. 8_Supplement ( 2012-04-15), p. 4567-4567
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 72, No. 8_Supplement ( 2012-04-15), p. 4567-4567
    Abstract: The human EGF receptor family (HER's) consists of two clinically validated drug targets (EGFR (HER1) and HER2), and two receptors (HER3 and HER4) which are the subject of intensive preclinical and early clinical investigation. Although drugs inhibiting both EGFR and HER2 show significant antitumor activity in the clinic, the acquisition of resistance is a hallmark of these and other targeted therapies. In the case of both targets, one of the emerging resistance mechanisms is the co-expression of other members of the EGFR superfamily. It was recently shown that HER2 co-expression mediates resistance in EGFR inhibitor (cetuximab) treated head and neck cancer (Sci Transl Med 7(3)99). Similarly, much attention has been paid to HER3 both as a bona fide drug target as well as a resistance mechanism (Oncogene 27, 3944). Finally, HER4, though less well studied may play a role in drug response (Breast Cancer Research 11:R50). We have previously build HER1 and HER2 specific SRM assays. HER3 is usually expressed at much lower levels than EGFR and HER2 (often & lt;10%). Yet, receptor crosstalk and heterodimerization can allow even very low levels of HER3 to exert a large impact on drug response. According to one report (Cancer Epidemiol Biomarkers Prev. 19(4):982), IHC analysis of HER3 may be inadequate to properly quantitate HER3 expression in FFPE tumor tissues. This antibody inadequacy may be a result of high sequence homology/identity between HER3 and other HER family members. Due to the critical need to accurately quantitate this receptor, we have used a mass spectrometric approach to develop a specific and quantitative SRM assay for HER3 from FFPE tumor tissue. These preclinical studies are being extended by assessing the expression levels of the complete HER family in two cohorts of clinical tumor tissue which had been treated with HER family antagonists. First, we measured HER3 expression in a set of neoadjuvant gefitinib treated NSCLC tumors. In this cohort, 12/15 tumor showed low but measurable levels of HER3 expression, ranging from 50-100 amol/ug tumor tissue. In a second tissue set, we measured HER3 expression in a cohort of advanced (Stage III-IV) breast cancer tissues which had undergone post resection adjuvant treatment with trastuzumab. These breast cancer samples demonstrated a higher level of expression of HER3, ranging from 50 - 250amol/ug tumor tissue, and 15/18 tumors were HER3 positive. In both studies, the relationship between HER family expression and response to either gefitinib or trastuzumab is currently under study. Analysis of HER4 in these cohorts is ongoing. It is critically important to understand mechanisms of resistance in patients undergoing targeted therapies, and Liquid Tissue-SRM promises to be a platform which can deliver extremely high sensitivity, absolute specificity as well as multiplexing capabilities to assess the four HER family targets in unison. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Asso ciation for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4567. doi:1538-7445.AM2012-4567
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2012-4567
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2012
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
    Location Call Number Limitation Availability
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  • 4
    Online Resource
    Online Resource
    Abstract 5129: Mapping the activity of oncogenic signaling networks with phospho-specific Liquid Tissue® mass spectrometry
    American Association for Cancer Research (AACR) ; 2011
    In:  Cancer Research Vol. 71, No. 8_Supplement ( 2011-04-15), p. 5129-5129
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 71, No. 8_Supplement ( 2011-04-15), p. 5129-5129
    Abstract: Many of the current targeted therapies in oncology target the activity of either receptor tyrosine kinases (Her2, EGFR, IGF1R, cMet, FGFR, PGDFR) or cytoplasmic tyrosine kinases (cSrc, AurA, PI3K, MEK, Raf, Akt). The current slate of clinically useful diagnostic tests measure target expression either directly by immunohistochemistry or indirectly by extrapolation from the level of gene or mRNA expression/amplification. These assays are limited by lack of quantitation, and no assay can directly assess the activation state of the signaling pathway components. The lack of information regarding target activation and downstream signal transduction can be overcome using the Liquid Tissue®-SRM technology platform. This approach enables relative and absolute quantification of multiple proteins and their phosphorylation status directly in formalin fixed tissue. In order to fill this diagnostic ‘gap’ we have used this platform to develop a quantitative multiplexed phospho-target assay format which measures the specific phosphorylation state of many clinically relevant oncogenic kinases (EGFR, cMet, Her3, Erk, cSrc, Mek, Akt, p70S6K) directly in FFPE tumor tissue. This phosphopeptide multiplex assay was initially preclinically validated on the A431 tumor cell line which harbors an amplification of the EGFR gene. These cells were stimulated with a dose range of EGF (50-200ng/ml) or in a time course study (EGF 50ng/ml for 5-30min). Confluent, EGF stimulated cells were then formalin fixed, subjected to Liquid Tissue® processing, and then phospho-enriched using TiO2 magnetic beads. The resulting enriched phosphopeptides were then analyzed by mass spectrometry. This method demonstrated the feasibility, and reproducibility of this method for quantitating EGFR pY1197, EGFRpT693, AKT pS473, p-p70S6K pS447, ERK pT202/pY204. We extended this study by performing phosphoenrichment and mass spectrometric analysis of human tumor xenografts, primary human tumor NSCLC explants and clinical trial tissue from EGFR inhibitor treated NSCLC and colorectal cancer patients. In each case we were able to enrich and measure the phosphorylation of a large set of important oncogenic kinases. Our intention is to develop this diagnostic tool to provide a multiplex assay format in formalin fixed tissue that can be applied from preclinical to clinical studies that will impact both targeted drug development and patient stratification needs in this era of personalized healthcare. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5129. doi:10.1158/1538-7445.AM2011-5129
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2011-5129
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2011
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
    Location Call Number Limitation Availability
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