We hypothesize that circulating micrometastases (MM) in peripheral blood (PB) and bone marrow (BM) can be detected and characterized for ErbB receptor expression by molecular and antibody-based assays, thus enabling serial assessments of anti-ErbB treatment effects, including overall antitumor efficacy and mechanistic effects. Indeed, MM may function as a """"""""super-surrogate tissue"""""""" in that these cancer cells may be more predictive of treatment effects than is the primary tumor in many clinical situations. For example, in the adjuvant therapy of cancer, in which any residual cancer is occult by definition, it is impossible to assess disease status or response to treatment using conventional methods. Detection and characterization of MM, the putative targets of adjuvant therapy, can provide a direct indication of treatment effects. Similarly, in advanced cancer, current methods to characterize molecular targets typically rely on previously obtained primary tumor tissue rather than on the metastatic cells themselves. Because post-treatment sampling can be easily performed, MM-based assays can be used to probe target-specific events associated with anti-ErbB therapies, such as ErbB receptor down modulation, expression of variant isoforms (truncated EGFR and ErbB2), internalization, and drug delivery. We further hypothesize that methods to isolate MM for representational amplification of DNA and RNA can be developed for molecular profiling of MM, thereby helping to elucidate the process of metastatic progression as well as alterations associated with treatment response or resistance. Our highly interdisciplinary and collaborative team will detect circulating MM using a multimodality battery of assays based upon immunocytochemistry (ICC), flow cytometry (FC), and reverse transcription-polymerase chain reaction (RT -PCR). These MM detection systems have previously been developed and validated for specificity and sensitivity, and are undergoing comparative evaluation for prognostic impact in ongoing clinical studies. In this project, we will adapt this MM-based assay platform to provide novel assessments of ErbB receptor expression. By applying three independent approaches for MM detection and ErbB characterization in both PB and BM, we will perform a comprehensive evaluation of MM-based ErbB analysis to determine the most promising method(s) to assess ErbB-targeted treatment effects. We will apply these novel techniques in a series of clinical studies, including cross-validations in untreated patients and in the assessment of various therapeutic strategies. We will evaluate our micrometastasis-based methods in conjunction with standard chemotherapy, anti-ErbB2 monoclonal antibody (trastuzumab), anti-ErbB2 immunoliposomes containing doxorubicin, and other ErbB-targeted agents.
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