Christine Kuslich

KRAS mutational status of endoscopic biopsies matches resection specimens

Aims This study was performed to determine systematically whether KRAS mutational analysis in biopsy tissue is a reliable indicator of KRAS status in subsequent corresponding resection specimens. Methods 30 colorectal cancer (CRC) patients with biopsy and corresponding subsequent surgical resection specimens were studied. KRAS mutational analysis was performed on each biopsy sample as well as two separate samples from each resection specimen by PCR and Sanger sequencing. Results Overall, KRAS mutations were identified in 12/30 (40%) of the tumours. There was 100% correlation between biopsy and resection specimens regarding the presence or absence of KRAS mutations. In fact, the same point mutation was identified in both biopsy and corresponding resection specimens in 12/12 (100%) cases. In addition, in two cases, there were two different point mutations detected within the same biopsy specimen. Conclusion This study shows perfect correlation between KRAS mutation status in biopsy and resection specimens from an individual patient, and suggests that biopsy material is adequate for KRAS mutational analysis in CRC patients.

Abstract 444: Subpopulations of circulating microvesicles have different microRNA profiles

The purpose of this study was to determine if circulating microvesicle (cMV) subpopulations as defined by surface protein composition also exhibit distinct microRNA (miR) expression patterns. Circulating microvesicles isolated from a prostate cancer cell line (VCaP) were sorted based on their surface protein composition and were evaluated for differential expression of miRs. Phycoerythrin-labeled antibodies targeting EpCam, CD63, or B7-H3 were used to sort the subpopulations of cMVs by fluorescence-activated cell sorting. The sorted subpopulations of cMVs were subsequently profiled by miR expression. The miR profiles for the EpCam-, CD63-, and B7-H3-positive subpopulations were compared with the profile of the total VCaP cMV population. Differential miR expression patterns were observed across the subpopulations, and all expression patterns were distinct from that observed in the total population. Patterns of both over- and under-expression were seen. Not only can subpopulations of cMVs be distinguished and separated based on surface protein markers, but they can also be differentiated based on their RNA content, in this case miRs. The ability to isolate tissue-specific cMV populations from patient plasma based on surface protein composition and then analyze them based on both surface protein composition and genetic content has considerable diagnostic, prognostic, and theranostic implications. 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 444. doi:10.1158/1538-7445.AM2011-444

Abstract 1568: Plasma-derived circulating microvesicles can both detect and help monitor breast cancer-related pathologies

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Circulating microvesicles (cMV) are membrane-bound structures that play an important role in several biologic processes, including angiogenesis and immune modulation. cMV levels are known to change in response to certain pathologies, and specific subpopulations of cMV can be isolated using antibodies targeted to membrane proteins specific for the secreting cells. We examined whether the levels of specific cMV subpopulations are altered in patients with a disease, specifically breast cancer. In order to characterize individual cancer-associated microvesicles (MVs), advanced breast cancer MVs were compared with normal control MVs. Circulating microvesicles were isolated from breast cancer patients or noncancer control patients, stained with fluorochrome-conjugated antibodies, and analyzed using flow cytometry. Tumor-specific antibodies were paired with process-specific markers, such as DLL4 and VEGFR2 for angiogenic cMV, CTLA4 and FasL for immunosuppressive cMV, and CD80 or CD83 for immunostimulatory cMV, to identify and characterize cMV subpopulations. Using this approach, distinct and informative cMV subpopulations were identified and quantified between breast cancer patients and healthy controls. For example, immunosuppressive MVs were elevated in the cancer patients compared with noncancer controls (68% vs 44% of CD45+ cMV coexpressed CTLA4). Additionally, angiogenic MVs were elevated in cancer plasma, with 44% coexpressing DLL4 and CD31 compared with 2% of cMV from noncancer controls. This study suggests that in addition to aiding diagnosis, monitoring specific subpopulations of cMV may offer important information regarding biologic processes perturbed by breast cancer in an individual patient, without the need for an invasive biopsy or a standard pathology evaluation such as immunohistochemistry, and therefore may also aid in prognosis. 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 1568. doi:10.1158/1538-7445.AM2011-1568

Abstract 4060: Enriching for rrare subpopulations of circulating microvesicles by the depletion of endothelial- and leukocyte-derived microvesicles

Endothelial- and leukocyte-derived circulating microvesicles (cMV) represent a majority of the cMV present in the blood. The purpose of this study was to determine if depletion of these more common cMV populations is possible, allowing for the enrichment and analysis of the remaining subpopulations of cMV. Circulating microvesicles were isolated from the blood plasma of breast cancer patients. Magnetic beads conjugated to antibodies specific for either CD31 or CD45 were used to deplete the isolated endothelial- and leukocyte-derived cMV. The remaining cMV population was characterized with a multiplexed immunoassay against a panel of 20 different antigens. In addition to the expected depletion of the biomarker used to remove the targeted cMV in the remaining cMV population, a concomitant depletion of associated biomarkers was observed. In the case of the cMV population in which CD31 had been used to remove endothelial-derived cMV, for instance, there was a significant concomitant depletion of DLL4, a highly associated endothelial marker. This was not the case with general epithelial microvesicle markers, such as CD9, which were still very much present in the cMV population remaining after depletion. These findings suggest a potential method for a more complete characterization of biomarker profiles associated with cMV derived from disease-associated cells and provide the foundation for a novel cMV-based strategy for disease detection through a noninvasive blood test. 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 4060. doi:10.1158/1538-7445.AM2011-4060

Abstract 4156: Detection of lung cancer from plasma using the biosignature of circulating microvesicles

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Circulating microvesicles (cMV) are cell-derived structures that are abundantly present in the blood. Tumor cells produce large quantities of cMV, and their abundance has been shown to correlate with tumor invasiveness and resistance to therapy. This study attempts to understand the origin, composition, and potential clinical utility of cMV, analyzing their protein composition in patients with non-small cell lung cancer (NSCLC) and identifying a cMV-based biosignature that can be used to predict the presence of tumors from a blood sample. cMV were isolated from plasma samples that were obtained from an initial cohort of 65 patients with NSCLC and from 46 control samples. A discovery panel of 63 specific biomarkers was used to develop an assay with high specificity and sensitivity based on 4 cMV surface proteins. Using a novel cMV-based multiplexed analysis platform we optimized a threshold level for 4 significant cMV subpopulations to effectively distinguish lung cancer patients from controls. The multiplex assay included 1 general cMV marker (CD81) and 3 lung cancer-associated biomarkers: the lung epithelial C-type lectin members SPD and SPA, plus the sialoprotein osteopontin. The decision tree analysis, based on specific cut offs, showed a sensitivity of 81% and a specificity of 90%. These results provide initial evidence that the identification of biosignatures in distinct subpopulations of cMV may offer a powerful blood-based approach for the detection and monitoring of specific disease states, such as NSCLC. 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 4156. doi:10.1158/1538-7445.AM2011-4156

Abstract 4954: Comparison of miR expression patterns in plasma, serum, and cell line cMV

Circulating microvesicles (cMV) isolated from either blood plasma or serum can yield genetic material sufficient for informative microRNA (miR) expression analysis. The ability to purify RNA from cMV isolated from several sources was compared, and the miR expression patterns were determined. cMV were isolated from the blood plasma and serum of men with and without prostate cancer, as well as from healthy and prostate tumor tissue and prostate cancer cell lines. Total RNA was extracted from the plasma and cell line cMV using the Qiagen miReasy kit and from the serum cMV using the ExoMir extraction method. The miR expression was determined for each source using Agilent V3 miRNA microarrays. Each sample (300 ng) was hybridized to the microarrays, and the acquired data were analyzed with the GeneSpring software package. Expression patterns for miR from cMV isolated from the plasma and serum of men with and without prostate cancer were evaluated for significant differences. Hierarchical clustering on both samples and genes demonstrated a distinct expression pattern for miR from plasma cMV compared with miR from cell line and tumor tissue cMV. These findings demonstrate that cMV isolated from the peripheral blood offer a unique and potentially clinically relevant source of miR for blood-based analysis. 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 4954. doi:10.1158/1538-7445.AM2011-4954

Abstract 817: KRAS and BRAF mutation spectrum in 1,035 FFPE tumor samples submitted for clinical testing

genes are associated with poor prognosis and non‐ response to anti‐EGFR therapies in patients with advanced cancers. Clinical guidelines have recommended that KRAS mutational status be determined in all patients with metastatic colorectal cancer that are being considered for anti‐EGFR therapies In this study a pathologist reviewed an Mutation pectrum o Samples Tested for KRAS KRAS and BRAF in -EGFR Antibody Resistance KRAS Mutations in Different Tumor Types

Abstract 3018: Identifying and characterizing subpopulation of exosomes to provide the foundation for a novel exosome-based cancer diagnostic platform

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Exosomes are endosome-derived vesicles between 40-100 nm in diameter that are secreted by most cell types and can be distinguished from other types of microvesicles released from the cell by a characteristic protein composition. Furthermore, it is known that exosomes transport mRNAs, microRNAs (miRs) and proteins, all of which can be used to identify the cell from which they are derived and can be exploited for noninvasive molecular profiling. Differential expression of exosomal miRs between cancer and normal patient samples has been described previously. In this study we identified various exosome subpopulations by their particular surface protein topography. Subsequently, we characterized plasma-derived exosomal RNA content of each subpopulation for their specific association with a cancer phenotype. We have exploited the protein topography and RNA content of exosomes found in plasma from patients with cancer, benign prostatic hyperplasia (BPH), and unaffected individuals in order to characterize and identify the exosome subpopulations that are indicative of a given disease state. We intend to use the various biosignatures of these exosome subpopulations to develop a diagnostic platform to aid in the screening and diagnosis of various cancers. Using flow cytometry (FACS) and cell sorting techniques, we separated plasma-derived exosomes into protein-specific subpopulations by using membrane-specific protein biomarkers (e.g. EpCam). We consistently found that exosomes from prostate cancer (PCa) patients had the highest percentage of exosomes labeled with EpCam, PSMA and CD-9 compared to exosomes from normal, BPH and colorectal cancer (CRC) patients. Additionally, for each subpopulation of exosomes separated by FACS, we used quantitative expression profiling of miRs to identify expression signatures specific to cancer patients. We found that the RNA content of various subpopulations of exosomes, defined by their membrane protein biosignature, was unique. In an exosome subpopulation where proteins CD-9 and CD-81 are on the surface, miR 141 is significantly overexpressed in exosomes from PCa patient plasma compared to exosomes derived from normal plasma. Interestingly, miR 9 was significantly overexpressed in exosomes from BPH plasma in EpCam and PSMA exosomes but not in exosomes of the same subpopulation isolated from normal and PCa plasma, supporting the idea that exosome biosignatures can be used to distinguish between BPH and PCa. Additionally, miR 491 was overexpressed in EpCam expressing exosomes derived from colon cancer plasma compared to normal and PCa. These findings highlight the potential of exosome biosignature profiles for use as a diagnostic marker of disease and provide the foundation for a novel exosome-based diagnostic platform that can be used through a non-invasive blood test. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3018.

Abstract 3246: Important differences between exosome biosignatures from prostate cancer patient plasma samples and prostate cancer cell lines

Exosomes are endosome-derived vesicles secreted by many cell types, including tumor cells. The vesicles are formed intracellularly by invagination followed by fusion with the multivesicular body (MVB). The MVB ultimately merges with the plasma membrane, leading to exocytosis of the exosomes, which consequently have a similar membrane protein composition as their cell of origin; this provides the exosomes with unique biosignatures. Furthermore, these circulating exosomes participate in cellular communication by transporting mRNAs, microRNAs and proteins to target cells where they can elicit biological responses. This study compared the biosignatures identified from prostate cancer (PCa) cell line-derived exosomes to those found in exosomes isolated from the plasma of PCa patient samples. To develop an exosome-specific protein and RNA signature for PCa, we studied RNA and surface membrane protein profiles by flow cytometry (FACS) of exosomes derived from four prostate cancer cell lines, VCaP, 22Rv1, LNCaP and DU145. The exosome biosignatures identified from the cell lines were then measured in plasma exosomes isolated from PCa patients. We found that there was variability between the four cell line exosome-specific mRNA expression levels and exosome surface protein content. The mRNA exosome biosignatures identified in the four cell lines were not found in the exosomes from plasma samples of patients with PCa. Using a combination of antibodies for B7H3, PSMA and CD63 by FACS, we identified a protein signature from all prostate cancer cell line exosome populations that defined a specific subpopulation of exosomes containing all 3 proteins on their surface. Interestingly, this same exosome subpopulation was not found in exosomes derived from patients with PCa. Additionally, exosome-specific mRNA expression of two mRNA transcripts often found to be overexpressed in PCa, STEAP1 and SPINK1, were consistently identified in exosomes derived from prostate cancer cell lines VCaP and 22Rv1, but were only found in the plasma derived exosomes from one out of eight patients with PCa. Our results show that prostate cancer cell lines are not appropriate models for the development of exosome-specific disease profiles in prostate cancer; this will be investigated in additional cell lines. We find that patient samples are essential to identify disease-specific exosome biosignatures. These findings support the development of a versatile diagnostic platform based on exosome-specific biosignatures found in the plasma of patient samples for prostate and other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3246.