Enrichment Of Tumor Cells Research Articles

AB34. Circulating tumor cell enrichment and analysis in prostate cancer

The dissemination of circulating tumor cells (CTCs) implicated in the metastatic spread of cancer accounts for the majority of cancer-related deaths. CTCs have been established as a prognostic biomarker and are associated with worse survival outcomes in various cancer types. Microfiltration can be an efficient and antigen-independent method for CTC enrichment. We have developed several generations of microfabricated filtration devices for CTC enrichment. The latest device is a flexible micro spring array (FMSA) device for label-free viable mechanical enrichment of CTCs from whole blood samples obtained from patients with advanced and metastatic carcinoma. CTCs were enriched and analyzed qualitatively and quantitatively based on immunocytochemical determination of phenotype and morphological characteristics. Follow up samples were analyzed during the course of therapy and CTC results were correlated to patient survival and tumor burden. FMSA enriched CTCs obtained from a blood sample may prove to be a prognostic biomarker for prostate cancer.

ClearCell FX: A microfluidic system for label-free circulating tumor cell enrichment.

e22023 Background: Detection and characterization of rare circulating tumor cells (CTCs) are pivotal to early cancer detection and can provide insights into tumor biology leading to better cancer m...

Circulating tumor cells enrichment and characterization in ALK-translocation positive lung cancer.

e19025 Background: Targeted therapy with tyrosine kinase inhibitor (TKI) crizotinib against EML4-ALK (ALK+) non-small cell lung cancer (NSCLC) have impacted cancer therapy as a new paradigm. However, there is still an unmet need to develop affordable and robust technology platforms to assay circulating tumor cells (CTCs), which may form the basis of distant hematogenous metastases. There are relative advantages and pitfalls with various CTC platforms. Methods: We performed pilot studies utilizing two different platforms of CTC detection and enrichment. First, we adopted the CellSearch “positive-selection” platform to profile 11 pts with ALK(+) NSCLC who were treated with crizotinib prospectively. Blood samples were collected (i) pretreatment, (ii) on TKI with CR/PR/SD, and (iii) at disease progression. Second, we evaluated a novel “negative selection” CTCs capture-isolation platform, based on magnetic separation of pan-leukocyte marker CD45+ cells coupled with RBC lysis, to avoid potential bias of predefi...

Size-based enrichment of exfoliated tumor cells in urine increases the sensitivity for DNA-based detection of bladder cancer.

Bladder cancer is diagnosed by cystoscopy, a costly and invasive procedure that is associated with patient discomfort. Analysis of tumor-specific markers in DNA from sediments of voided urine has the potential for non-invasive detection of bladder cancer; however, the sensitivity is limited by low fractions and small numbers of tumor cells exfoliated into the urine from low-grade tumors. The purpose of this study was to improve the sensitivity for non-invasive detection of bladder cancer by size-based capture and enrichment of tumor cells in urine. In a split-sample set-up, urine from a consecutive series of patients with primary or recurrent bladder tumors (N = 189) was processed by microfiltration using a membrane filter with a defined pore-size, and sedimentation by centrifugation, respectively. DNA from the samples was analyzed for seven bladder tumor-associated methylation markers using MethyLight and pyrosequencing assays. The fraction of tumor-derived DNA was higher in the filter samples than in the corresponding sediments for all markers (p<0.000001). Across all tumor stages, the number of cases positive for one or more markers was 87% in filter samples compared to 80% in the corresponding sediments. The largest increase in sensitivity was achieved in low-grade Ta tumors, with 82 out of 98 cases positive in the filter samples (84%) versus 74 out of 98 in the sediments (75%). Our results show that pre-analytic processing of voided urine by size-based filtration can increase the sensitivity for DNA-based detection of bladder cancer.

Glioblastoma Cell Enrichment Is Critical for Analysis of Phosphorylated Drug Targets and Proteomic–Genomic Correlations

The quality of cancer genomic and proteomic data relies upon the quality of the clinical specimens examined. Here, we show that data derived from non-microdissected glioblastoma multiforme tumor tissue is either masked or not accurate, producing correlations between genomic and proteomic data that lead to false classifications for therapeutic stratification. We analyzed the level of 133 key signaling proteins and phosphoproteins in laser capture microdissected (LCM) primary tumors from a study set of tissues used for the Cancer Genome Atlas (TCGA) profiling efforts, comparing the results to tissue-matched, nontumor cell-enriched lysates from adjacent sections. Among the analytes, 44%, including targets for clinically important inhibitors, such as phosphorylated mTOR, AKT, STAT1, VEGFR2, or BCL2, differed between matched tumor cell-enriched and nonenriched specimens (even in tumor sections with 90% tumor cell content). While total EGFR protein levels were higher in tumors with EGFR mutations, regardless of tumor cell enrichment, EGFR phosphorylation was increased only in LCM-enriched tumor specimens carrying EGFR mutations. Phosphorylated and total PTEN, which is highly expressed in normal brain, was reduced only in LCM-enriched tumor specimens with either PTEN mutation or loss in PTEN copy number, with no differences observed in non-microdissected samples. These results were confirmed in an independent, non-microdissected, publicly available protein data set from the TCGA database. Our findings highlight the necessity for careful upfront cellular enrichment in biospecimens that form the basis for targeted therapy selection and for molecular characterization efforts such as TCGA.

Flexible Micro Spring Array Device for High-Throughput Enrichment of Viable Circulating Tumor Cells

The dissemination of circulating tumor cells (CTCs) that cause metastases in distant organs accounts for the majority of cancer-related deaths. CTCs have been established as a cancer biomarker of known prognostic value. The enrichment of viable CTCs for ex vivo analysis could further improve cancer diagnosis and guide treatment selection. We designed a new flexible micro spring array (FMSA) device for the enrichment of viable CTCs independent of antigen expression. Unlike previous microfiltration devices, flexible structures at the micro scale minimize cell damage to preserve viability, while maximizing throughput to allow rapid enrichment directly from whole blood with no need for sample preprocessing. Device performance with respect to capture efficiency, enrichment against leukocytes, viability, and proliferability was characterized. CTCs and CTC microclusters were enriched from clinical samples obtained from breast, lung, and colorectal cancer patients. The FMSA device enriched tumor cells with 90% capture efficiency, higher than 10(4) enrichment, and better than 80% viability from 7.5-mL whole blood samples in <10 min on a 0.5-cm(2) device. The FMSA detected at least 1 CTC in 16 out of 21 clinical samples (approximately 76%) compared to 4 out of 18 (approximately 22%) detected with the commercial CellSearch® system. There was no incidence of clogging in over 100 tested fresh whole blood samples. The FMSA device provides a versatile platform capable of viable enrichment and analysis of CTCs from clinically relevant volumes of whole blood.

Development Of Methods To Perform Next Generation Sequencing (NGS)-Based Genomics Studies On Multiple Myeloma Clinical Samples

NGS is a powerful tool for interrogating genomic alterations in cancer cells from the scale of single nucleotides up to whole chromosomes. While methods for performing NGS analysis on solid tumors have been widely reported in the literature, fewer methods are available for hematological malignancies, which are often collected and stored in different conditions. Here we report our efforts to overcome the unique challenges of performing a whole exome sequencing (WES) study on hundreds of multiple myeloma (MM) patient samples.As part of Onyx-sponsored clinical trials of the proteasome inhibitor, carfilzomib, bone marrow aspirates were collected from patients and subjected to bead-based CD138 positive selection to enrich tumor cells. The yield of CD138+ cells after enrichment varied greatly from 200,000 to >10,000,000, depending primarily on the disease state and the volume and quality of aspirate received. CD138+ cells were banked in a variety of formats that differed in their suitability for downstream DNA and RNA analysis, including as pellets or re-suspended in reagents such as TRIzol®, RNAlater and Buffer RLT. At the time of abstract submission we have performed extensive analyses of samples stored in TRIzol.TRIzol is renowned for its excellence in preserving RNA integrity while providing a means to simultaneously extract DNA and protein. However, the standard DNA extraction method yields material that is not of sufficient quality for DNA analyses such as WES. We attempted to remedy this problem through three approaches: (1) modifying extraction conditions to improve the quality of the DNA, (2) rescuing the extracted DNA with double-stranding steps prior to library preparation, and (3) applying newer, low input and single-strand library construction methods. Our efforts to modify the extraction conditions were unsuccessful, but by combining DNA rescue and newer library construction methods, we could ultimately generate high quality exomes (as judged by comparison to exomes generated from matched, pelleted samples) from the whole range of clinical samples in our biobank. We believe these methods create new opportunities for the community to interrogate, with the latest NGS approaches, vast numbers of clinical samples banked in the era of gene-expression profiling (GEP). Disclosures:Kwei:Onyx Pharmaceuticals: Employment, Equity Ownership. Degenhardt:Onyx Pharmaceuticals: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership. Tuch: Onyx Pharmaceuticals: Employment, Equity Ownership.

Applications and limitations of oncogene mutation testing in clinical cytopathology

In an increased number of settings, cytology represents the only source of sampling and it often substitutes histology as an independent diagnostic modality. Thus, DNA molecular targets to stratify patients for targeted therapy are often evaluated on cytology. In addition, DNA mutational tests may refine indeterminate thyroid and pancreas cytology. This review discusses the applications and limitations of DNA mutational testing on cytology. With respect to histology, most cytological samples have the advantages of a purer population of tumor cells, with low stromal component, a better preserved DNA, and assessing at the same time of sample collection cellular adequacy for DNA testing. However, since in vitro diagnostic tests are licensed only for paraffin-tissue, all mutational assays on cytology are "home brew," requiring a rigorous validation process. This should take into account not only the performance characteristics of the molecular assay but also features inherent to any given cytological samples, such as its source, preparation type, fixation and staining modalities, and the most effective tumor cell enrichment methods. This calls for a change of cytotechnologists and cytopathologists mentality to collect and process the cytological samples not only for microscopy but also to assess clinically relevant molecular markers.

454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples

Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues.

Circulating tumor cell enrichment based on physical properties.

The metastatic dissemination and spread of malignant circulating tumor cells (CTCs) accounts for more than 90% of cancer-related deaths. CTCs detach from a primary tumor, travel through the circulatory system, and then invade and proliferate in distant organs. The detection of CTCs from blood has been established for prognostic monitoring and is predictive of patient outcome. Analysis of CTCs could enable the means for early detection and screening in cancer, as well as provide diagnostic access to tumor tissues in a minimally invasive way. The fundamental challenge with analyzing CTCs is the fact that they occur at extremely low concentrations in blood, on the order of one out of a billion cells. Various technologies have been proposed to isolate CTCs for enrichment. Here we focus on antigen-independent approaches that are not limited by specific capture antibodies. Intrinsic physical properties of CTCs, including cell size, deformability, and electrical properties, are reviewed, and technologies developed to exploit them for enrichment from blood are summarized. Physical enrichment technologies are of particular interest as they have the potential to increase yield and enable the analysis of rare CTC phenotypes that may not be otherwise obtained.

Fibroblast growth factor receptor 1 (FGFR1) and SRY-related HMG-box (SOX2) amplification in squamous cell carcinoma (SCC) of the lung.

11025 Background: SOX2 is a key transcription factor that is amplified in lung SCC. FGFR1 is a receptor tyrosine kinase that promotes cell proliferation, survival and apoptotic resistance through the PLCγ/PKC, RAS/MAPK and PI3K-AKT pathways, respectively. FGFR1 is amplified in 15-25% of lung SCC. Pre-clinical studies of targeted inhibitors showed a growth dependency on FGFR1 amplification both in vitro and in vivo. A European, multicenter clinical trial of second-line treatment with BIBF1120, an FGFR1 inhibitor, will be performed in p with lung SCC with FGFR1 amplification. Methods: We have examined FGFR1 and SOX2 gene copy number (GCN) in 76 lung SCC p by multiplex ligation-dependent probe amplification (MLPA). Genomic DNA (gDNA) was isolated from enriched tumor cells by laser capture microdissection from formalin-fixed paraffin embedded (FFPE) tumor tissue. 50-100 ng of gDNA was analyzed in each of three independent replicates per tumor sample. Two independent probe sets were used for each gene analyzed. For inter-patient GCN comparisons, the results from each patient were normalized against the GCN values derived from FFPE peripheral blood leukocytes. In order to study intra-tumor heterogeneity (TH), we examined FGFR1 and SOX2 GCN in different areas of 4 tumors. In 2 p, TH was examined in serial tumor biopsies and/or resections obtained at different points of disease progression. Results: High FGFR1 amplification was detected in 13/76 (17.10%) and low amplification in 7/76 (9.21%) p. High SOX2 amplification was observed in 38/63 (60.32%) and low amplification in 9/63 (14.28%) p. 46.15% of the FGFR1-amplified tumors were also co-amplified for SOX2. Intra-TH was observed in 2/4 tumors. Survival according to FGFR1 and SOX2 GCN will be presented. In addition, GCN changes in FGFR1, SOX2, PIK3CA, PDGFRA, KDR, EGFR and MET over 10 years of follow-up will be presented for one surgically resected SCC lung p. Conclusions: FGFR1 and SOX2 co-amplification could represent a novel therapeutic target and warrants further research.

Association of mesenchymal phenotype in circulating tumor cells with poor prognosis in metastatic colorectal cancer.

e14567 Background: Although the prognostic relevance of circulating tumor cells (CTCs) is well established in metastatic colorectal cancer (mCRC), their clinical use is currently constrained by the phenotypic definition of such cells that is largely limited to epithelial markers. Our objective in this study is to characterize the prognostic significance of a larger group of CTCs including those with characteristics of epithelial-mesenchymal transition (EMT) or cancer stem cells (CSCs) in a cohort of pre-treated mCRC patients (pts). Methods: Peripheral blood was collected in AdnaCollect preservation tubes from pts for immunomagnetic tumor cell enrichment using the AdnaTest ColonCancer Select kit and multiplex qRT-PCR analyses of tumor-associated epithelial (CEA, EPCAM, EGFR), cancer stem cell (ALDH1) and mesenchymal (TWIST1, AKT2, PIK3CA) transcripts. Results: In the initial phase, 41 previously treated mCRC pts (median prior therapies 3) were enrolled over a 4 month period and were followed prospectively. In this cohort, there was a low rate of CEA, EPCAM, and EGFR expression in the CTCs (12%, 2%, and 0%, respectively), consistent with loss of epithelial markers. Conversely, ALDH1, an established CSC marker was expressed in 30% of pts, with only one pt’s CTCs expressing both ALDH1 and CEA expression. ALDH1 expression was associated with very poor prognosis (HR 24.6, p=0.037) and 57% 60-day mortality, compared to 0% 60-day mortality for pts with no detectable ALDH1 expression. The mesenchymal markers of TWIST1, AKT2, and PIK3CA were strongly correlated with ALDH1 expression (Spearman r=0.55, 0.59, 0.63, respectively, p &lt; 0.01 for each). Conclusions: The majority of CTCs detected in refractory mCRC pts have lost epithelial markers. Acquisition of markers associated with EMT or CSCs appear to be associated with poor prognosis, and may provide important insights into better prognostication and also biology of refractory colorectal cancer. Further enrolment into this study is ongoing and updated results will be presented.

Filtration parameters influencing circulating tumor cell enrichment from whole blood.

Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·104∶102∶1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ∼10 mbar for a 1 cm2 track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC.

Abstract 4141: Tumor cell enrichment is critical for assessing cell signaling pathways in glioblastoma multiforme.

Abstract Introduction &amp; Methods: Glioblastoma tumor cells are generally assumed to be mixed with few distinct non-tumorigenic cell types and few cells of the microenvironment in general, owing to their location in the brain and the high tumor cell density compared to normal brain parenchyma. However, even small cell populations can have a significant impact on the abundance of phosphorylated proteins. We have analyzed the level and/or phosphorylation of 133 proteins in laser capture microdissected glioblastoma primary tumors (n=39) and compared the results to non-tumor cell enriched, whole tissue lysates. Results: A large portion (44 percent) of endpoints were significantly different between matched tumor cell enriched and non-enriched samples - even when limiting the investigation to tumor samples with at least 90 percent tumor content. EGFR protein levels, which is highly expressed in tumor cells compared to normal brain parenchyma, increased in enriched and non-enriched samples following EGFR mutation, while EGFR phosphorylation was only significantly increased in tumor cell enriched samples. In contrast, PTEN, which has a higher expression in normal brain compared to tumor cells, was only found to be reduced in tumor cell enriched samples from patients with either PTEN mutation or loss in PTEN copy number, with no difference seen in non-enriched samples. Patient stratification by either phosphorylated EGFR, Akt, mTOR, Stat1, VEGFR2 or Bcl-2 based on non-tumor cell enriched samples was dramatically different from tumor cell enriched samples, showing significant misclassification for up to 40 percent of patients even in high tumor content samples. Conclusions: This study demonstrates the critical necessity for selected tumor cell population procurement when investigating protein levels and phosphorylation in glioblastoma. Only tumor cell enriched samples correctly reflected expected tumor biology in the face of EGFR and PTEN mutations. Moreover, tumor cell enrichment had a dramatic effect on the stratification of clinical patient samples that would form the basis for targeted therapy selection. Citation Format: Claudius Mueller, Ana C. deCarvalho, Tom Mikkelsen, Valerie Calvert, Virginia Espina, Lance A. Liotta, Emanuel F. Petricoin III. Tumor cell enrichment is critical for assessing cell signaling pathways in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4141. doi:10.1158/1538-7445.AM2013-4141

Abstract 53: FGFR1 and SOX2 amplification in squamous cell carcinoma (SCC) of the lung.

Abstract SRY-related HMG-box (SOX2) gene is a key transcription factor that coordinates development, differentiation and self-renewal of normal non-alveolar epithelium of the airway. SOX2 amplification has been reported in lung squamous cell carcinoma (SCC). Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that, upon activation, promotes cell proliferation, survival and apoptotic resistance through PLCγ/PKC, RAS/MAPK and PI3K-AKT pathways, respectively. FGFR1 is also amplified in 15-25% of lung SCC and pre-clinical data with targeted inhibitors showed a growth dependency on FGFR1 amplification either in vitro and in vivo. A clinical trial with BIBF1120 (which also inhibits FGFR1), will be developed in the Netherlands and Spain in second line SCC of the lung with FGFR1 amplified by FISH. Genetic heterogeneity in solid tumors is a major research area and therefore its assessment in SCC of the lung is of great relevance. We have examined FGFR1 and SOX2 gene copy number (GCN) in 76 lung SCC by multiplex ligation-dependent probe amplification (MLPA). Analysis of mutations in DDR2, PIK3CA, BRAF, EGFR, KRAS, and TP53 is ongoing. Genomic DNA (gDNA) was isolated from enriched tumor cells by laser captured microdisection from formalin-fixed paraffin embedded (FFPE) tumor tissue sections. 50-100 ng of gDNA was analyzed in each of the three independent replicates per tumor sample. Two independent probe sets were used for each gene analyzed. For inter-patient GCN value comparisons, the tumor results from each patient was normalized against the GCN values derived from FFPE peripheral blood leukocytes control. We also used MLPA technique to study intra-tumor heterogeneity (TH) by analyzing FGFR1 and SOX2 in different tumor areas. In particular, in 4 cases FGFR1 and SOX2 were co-amplified. Also, TH was examined in serial tumor biopsies and/or resections obtained at different points of disease progression in two patients. Of the 76 patients evaluable for FGFR1 and SOX2 GCN, FGFR1 high amplification was detected in 13/76 (17.10%) and low amplification in 7/76 (9.21%). SOX2 presented high amplification in 38/63 (60.32%) and low amplification in 9/63 (14.28%). Interestingly, 46.15% of the FGFR1-amplified tumors were also co-amplified for SOX2. An important degree of TH was found in 2 of the 4 tumors analyzed. GCN changes in FGFR1, SOX2, PIK3CA, PDGFRa, KDR, EGFR and MET over 10 years follow-up will be presented for one surgically resected SCC lung cancer patient. Frequencies of FGFR1 and SOX2 gene amplifications detected are in agreement with previously published data. Survival according to FGFR1 and SOX2 status will also be presented. FGFR1 and SOX2 co-amplification, together with the TH, warrants further research and could represent a novel therapeutic target. Citation Format: Pedro Méndez, José L. Ramirez, Amaya Gascó, Teresa Morán, Enric Carcereny, Miquel Taron, José Luís Mate, Irene Sansano, María Pérez, Mónica Botia, Montserrat Tierno, Harry J.M Groen, Rafael Rosell. FGFR1 and SOX2 amplification in squamous cell carcinoma (SCC) of the lung. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 53. doi:10.1158/1538-7445.AM2013-53

Use of touch imprint cytology as a simple method to enrich tumor cells for molecular analysis

A relative excess of nonneoplastic cells in frozen carcinoma samples is often a cause of false-negative results in molecular assays. Given the greater cohesiveness of epithelial tumor cells compared with nonneoplastic epithelium and mesenchymal stroma, the authors hypothesized that tumor procurement by touch imprinting would provide a simple, cost-effective method of obtaining enriched neoplastic cells compared with frozen whole-tumor samples. Eleven adenocarcinomas with known KRAS gene mutations were tested. Two sets of 8 touch imprint (TP) slides and 1 frozen whole-tumor sample (FS), both with a corresponding hematoxylin and eosin-stained slide, were obtained from each tumor. DNA from unstained TP and FS samples was tested for KRAS exon 2 mutations by Sanger sequencing. The percentage of carcinoma cells was determined by light microscopy of hematoxylin and eosin-stained slides. The fold increase in the mutant-enriched DNA in TP versus FS samples was determined by calculating the height ratio between the mutant and wild-type peaks on the sequencing electropherogram. Using light microscopy, TP demonstrated a 1.1-fold to 3.5-fold (mean, 1.8-fold) enrichment in neoplastic cells compared with the FS. The mutant-to-wild-type peak height ratio was 1.4-fold to 7.1-fold (mean, 3.1-fold) higher in TP compared with the corresponding FS samples. The average amount of extracted DNA ranged from 145 ng to 7.9 μg per TP slide. The procurement of carcinoma samples by TP is rapid, simple, and inexpensive; consistently provides a tumor-enriched sample; is an excellent source of high-quality tumor DNA; and could compensate for the relatively low sensitivity of direct sequencing. Cancer (Cancer Cytopathol) 2013;121:354-360. © 2013 American Cancer Society.

Imaging cytometry for counting circulating tumor cells: comparative analysis of the CellSearch vs ImageStream systems

Circulating tumor cell (CTC) enumeration is important clinically for identifying prognostic and predictive factors in patients with solid cancers. The CellSearch device (Veridex) is an immunomagnetic CTC selection and enumeration system used in clinical practice. The ImageStream (Amnis) combines the strengths of flow cytometry and fluorescent microscopy in a single platform and has potential application for CTC counting. The performance in CTC enumeration was compared between the ImageStream and CellSearch systems. Various numbers of PANC-1 tumor cells were spiked into 7.5 mL of peripheral blood from a healthy donor. Before cell analysis by the ImageStream, tumor cell enrichment was performed by immunomagnetic selection with anti-EpPCAM. Anti-CD45 and anti-CK markers were used to discriminate between tumor cells and leukocytes. The ratios of tumor cells recovered from each dilution were calculated for both methods. The Wilcoxon rank test was applied to compare the results of the two methods and the reference value. The results of the two tested methods differed significantly from the reference value, but did not differ between them. Nevertheless, lower level of trueness and precision was observed in ImageStream when fewer numbers of CTCs were analyzed. Our results suggest that ImageStream platform for CTC enumeration has a potential value for the early diagnosis of disseminated disease, but needs an improvement of precision for the enumeration of low number of CTC.

Abstract C89: ESRP1-CD44v-xCT axis as a potential therapeutic target for the metastasis

Abstract Metastasis of tumor cells is a multi-step phenomenon including dissociation from primary site, intravasation, long-term circulation in the blood, extravasation, and colonization in the pre-metastatic niche in the distant organs. It is quite important that cancer cells are always exposed to redox stress throughout this muti-step metastatic phenomenon. This is why metastasis occurs only when cancer cells are refractory to the high level of reactive oxygen species (ROS) in the tumor microenvironment. CD44, one of the cancer stem cell (CSC) markers, makes diversity in isofroms due to the alternative splicing via epithelial splicing regulatory protein1 (ESRP1) or transcription factor grainyhead-like 2 (Grhl2). In the clinical perspective, a high expression level of ESRP1 or Grhl-2 in the tumor cells is generally associated with a poor prognosis in breast cancer patients. In addition, epigenetic modifications including DNA and histone methylation at metastasis-related genes have been also considered as a cause of cancer metastasis. This is why the depletion of CD44variant isoform (CD44v) is not necessarily accompanied by epithelial-mesenchymal transition (EMT), given that EMT has widely been recognized to expand the CSC population. Besides, CD44 is not only a surface marker of CSCs but also a functional molecule; for instance, the interaction between CD44v6 and c-Met, the receptor of hepatocyte growth factor (HGF), activates the Ras-dependent survival signal of tumor cells. We have recently identified that CD44 variant 8-10 (CD44v9) stabilizes the localization of xCT glutamate-cystine transporter at the cellular membrane, thereby increasing the intracellular level of reduced glutathione (GSH). In fact, enrichment of CD44v9-positive tumor cells is frequently recognized in the metastatic foci and recalcitrant cases. The expression level of CD44v9 is positively correlated with the Akt/PI3K/mTOR signal activity of metastatic breast tumor cells. Akt/PI3K/mTOR signal is related to cholesterol synthesis and to adaptation to the hypo-nutrition, or hypoxia. In the case of metastasis of breast cancer cells to the lungs, tumor-entrained neutrophils (TEN) accumulate in the lung to inhibit lung metastasis through the increased redox stress in the NADPH-dependent manner. Thus, the presence of xCT transporter promotes the GSH synthesis and the protection from tumor stroma-derived ROS, which plays a fundamental role in the adaptive resistance to conventional cancer therapy for the metastasis. We have also revealed that ESRP1-dependent alternative splicing of CD44 mRNA increases both the intracellular GSH level and the metastatic potential of the breast cancer cells. We have demonstrated that depletion of ESRP1 or Grhl2 in metastatic breast cancer cells adversely affects the metastatic potential, since they fail to express CD44v9. Notably, even the tumor cells which are lack of CD44v9 are able to form the metastasis to lungs, but the ability of survival and proliferation in the microenvironment rich in redox stress are considerably lower. Akt/PI3K/mTOR activity is significantly suppressed compared with the aggressive metastatic foci mainly composed of CD44v9-positive tumor cells. Therapies targeted to alternative splicing of CD44 as well as those that target CD44v-mediated stabilization of xCT might be expected to imapir the adaptive resistance to ROS in the metastatic stem-like cancer cells and thereby rendering them susceptible to currently available cancer treatments. Considering the high dependency to lipid synthesis In the survival and proliferation of metastatic breast cancer cells, HMG-CoA reductase inhibitors contribute to the synergistic inhibition of the metastatic exacerbation. Citation Format: Go J. Yoshida, Kenji Tsuchihashi, Toshifumi Yae, Osamu Nagano, Hideyuki Saya. ESRP1-CD44v-xCT axis as a potential therapeutic target for the metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C89.

Comparison of Direct Sequencing, PNA Clamping-Real Time Polymerase Chain Reaction, and Pyrosequencing Methods for the Detection ofEGFRMutations in Non-small Cell Lung Carcinoma and the Correlation with Clinical Responses to EGFR Tyrosine Kinase Inhibitor Treatment

BackgroundThe aims of this study were to evaluate the abilities of direct sequencing (DS), peptide nucleic acid (PNA) clamping, and pyrosequencing methods to detect epidermal growth factor receptor (EGFR) mutations in formalin-fixed paraffin-embedded (FFPE) non-small cell lung carcinoma (NSCLC) samples and to correlate EGFR mutational status as determined by each method with the clinical response to EGFR tyrosine kinase inhibitors (TKIs).MethodsSixty-one NSCLC patients treated with EGFR TKIs were identified to investigate somatic mutations in the EGFR gene (exons 18-21).ResultsMutations in the EGFR gene were detected in 38 of the 61 patients (62%) by DS, 35 (57%) by PNA clamping and 37 (61%) by pyrosequencing. A total of 44 mutations (72%) were found by at least one of the three methods, and the concordances among the results were relatively high (82-85%; kappa coefficient, 0.713 to 0.736). There were 15 discordant cases (25%) among the three different methods.ConclusionsAll three EGFR mutation tests had good concordance rates (over 82%) for FFPE samples. These results suggest that if the DNA quality and enrichment of tumor cells are assured, then DS, PNA clamping, and pyrosequencing are appropriate methods for the detection of EGFR mutations.

Nanotechnology of Circulating Tumor Cell Enrichment and Detection

Nanotechnology of Circulating Tumor Cell Enrichment and Detection