Individual Tumor Cells Research Articles

Abstract P6-05-28: Nicastrin RNA in situ hybridization (RNAScope®) reveals estrogen receptor negative breast cancer patients at risk of metastatic relapse and could serve as companion diagnostic for anti-nicastrin monoclonal antibody therapy

Abstract Introduction of targeted therapies into oncology requires co development of a drug and its companion diagnostic to achieve clinical efficacy and minimise side effects. Given the aggressive nature of estrogen receptor alpha negative (ERa-ve) breast cancer (BrCa), novel therapeutics are needed. Gamma-secretase enzyme is a relevant target in cancer, and within it, Nicastrin (NCSTN) is amenable to therapeutic intervention using monoclonal antibodies (mAb). NCSTN gene is amplified in a some breast tumours, which correlates with high NCSTN mRNA and adversely impacts overall survival (TCGA, Nature 2012). Immunohistochemistry revealed NCSTN protein overexpression in 47.5% of BrCa (n = 1000), conferring worse prognosis in the ERα-ve cohort. NCSTN genetic depletion in triple negative BrCa cells attenuated tumor growth in vitro and in vivo, invasive capacity, mesenchymal features and cancer stem-cell propagation (Filipovic et al; Lombardo et al). We have developed and characterized anti-NCSTN mAbs in terms of their therapeutic properties, functional effects in vitro and in vivo, and binding epitopes. Here, we have used a novel, bright-field chromogenic in situ hybridization technique (RNAscope), to quantify single-cell NCSTN mRNA levels in ERα-ve BrCa patients (n = 311), and propose this test as a companion diagnostic for targeted anti-NCSTN therapy. Methods:Paraffin BrCa tissue microarrays sections (Nottingham Tenovus ERα-ve cohort; n = 311) were analyzed by RNAScope using a NCSTN-specific probe (GenBank accession number NM_015331.2, and probe region: nt 158-1306). Visualized as brown punctate dots, RNA staining in individual tumor cells was scored semi-quantitatively on a 1-4 scale: 1 = 0-5 dots/cell, 2 = 6-10 dots/cell, 3 = 11-15 dots/cell, 4 = >16 dots/cell. H-score (100-400) capturing the percentage of tumor cells at each staining level was calculated and correlated with clinical and tumour characteristics. Results: NCSTN high H-score >120 correlated with high proliferative index (Ki67) (p = 0.002), high nuclear pleomorphism (p = 0.03) and mitotic score (p = 0.02) and membranous expression of the NCSTN protein (p = 0.05). It further correlated with cytoplsmic BRCA1 positivity (p = 0.025), which is associated with BrCa cell invasion. Importantly, in Kaplan-Meier analyses NCSTN Score>120 conferred a higher probability of metastatic relapse at 10-year follow up (p = 0.019), and retained significance in the Cox regression multivariate analysis, along with tumor grade, stage and size (p = 0.04). Conclusion: RNAScope successfully detected NCSTN mRNA in paraffin embedded BrCa tissues. High NCSTN RNAScope levels may be a novel independent prognostic factor in this patient cohort and one that correlates with established markers of aggressive disease. We will also present data on anti-NCSTN mAb efficacy in cell systems based on NCSTN RNAScope score. We propose that detection of high NCSTN levels by RNAScope in primary BrCa removed at surgery or in subsequent biopsies of disease relapse can be further developed to serve as a clinical companion diagnostic test to assess patient eligibility for treatment using anti-NCSTN therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-28.

Abstract P4-05-12: MultiOmyx™ screening for prognostic indicators of breast cancer

Abstract Over the last three decades, the incidence of early-stage breast cancer has doubled while the incidence of late stage breast cancer has slightly decreased; however, the standard treatment for both has not changed significantly in the last 10 years. Of those diagnosed with early-stage breast cancer, 30% will develop recurrent disease, though there is currently no diagnostic available to identify these cases. Conversely, identifying late stage patients with a low- risk for recurrence could spare them from post-surgical chemotherapy. We have developed a technology that allows in situ staining and analysis of up to 60 proteins on a single tissue slide. In this exploratory study we evaluated 25 known biomarkers on a cohort of more than 800 breast cancer cases assembled onto three tissue arrays. The cohort spans clinical grades and breast cancer subtypes, and is thus useful in novel associations of biomarker expression across a variety of clinical features. Twenty-five markers were chosen based on literature reports for marker association with disease recurrence and represent a range of biological features including signaling pathways and cell phenotypes. The slides were sequentially stained with antibodies that were directly conjugated to Cy3 and Cy5 dyes, imaged, evaluated for quality and subject inclusion, and subsequently analyzed for statistical correlations of expression values to clinical features. The images were then processed using a single cell analysis algorithm which allows for quantification of individual tumor cells and signal intensities for the markers to be extracted from sub-cellular domains (nucleus, cytoplasm, and membrane of each cell) allowing unique patterns of protein expression to be determined. Univariate Cox proportional hazards analysis was first applied to determine those features with strongest association with ‘death due to disease’. Of the 25 markers analyzed, several markers (e.g. API3, AMPH2, beta-catenin, CD44, CEACAM5, CK15, CK19, cMET, Her2, Ki67, TRIM29) were found to have strong association with poor outcome (FDR < 0.05). After adjusting for clinical variables (stage, number of positive nodes, grade), a number of markers (e.g. CD44, CEACAM5, cMET, TRIM29) still show a strong association with poor clinical outcome (FDR < 0.05, Cox proportional hazards model). CEACAM5 is part of the MammostratTM panel of markers used for determining prognosis in ER positive breast cancers after Tamoxifen® treatment. CD44, a cancer stem cell marker, and TRIM 29, a transcriptional regulatory factor involved in carcinogenesis, have been associated with basal-like breast cancers which have the worst prognosis of all the breast cancer subtypes. High levels of cMET have been associated with poor prognosis for all breast cancer subtypes and cMET is being explored as a therapeutic target. The MultiomyxTM methodology enabled us to verify a subset of biomarkers that are clinically relevant to breast cancer outcome. Using this technology could lead to identification of novel biosignatures that stratify patients and enable precision medicine that results in better treatment decisions and prognosis for patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-12.

Abstract P2-11-03: A tumor microenvironment of metastasis (TMEM) biomarker in early breast cancer provides prognostic information that is complementary to IHC4: A prospective validation study

Abstract Background: Multiparameter gene expression assays (eg, Oncotype DX®) provide prognostic and predictive information in estrogen receptor (ER)+ Her2- negative disease, but are driven largely by proliferation and ER signaling captured by immunohistochemistry (IHC) assays such as IHC4 (Cuzick et al. J Clin Oncol. 2011 10;29:4273-8). However, these assays do not reflect the capacity of individual tumor cells to metastasize, nor interaction with their microenvironment. Using a bench-to-bedside approach, we have identified in animal models and human breast cancer by IHC methods a microanatomic landmark (TMEM) precisely where Mena-overexpressing invasive tumor cells intravasate and hematogenously metastasize by directly interacting with endothelial cells and macrophages (Robinson et al. Clin Cancer Res 2009;15:2433-41). In the present study, we performed a prospective validation study to establish analytic and clinical validity of TMEM in comparison with IHC4 in accordance with recommended guidelines (Simon et al. JNCI 2009;101:1446-52). Methods: A case-control study was performed nested within a cohort of 3760 invasive ductal breast carcinomas, in which cases (subsequent distant recurrence) and controls (no recurrence) were selected using incidence density sampling matched on age at and calendar year of primary diagnosis, resulting in 481 subjects (259 case-control pairs). TMEM and IHC4 were performed as previously reported, and stained slides were read independently by 5 pathologists blinded to outcome. Odds ratios (OR)(95% CI) for the association with TMEM were estimated using logistic regression, with adjustment for clinical variables, including age, tumor size, grade, number of positive lymph nodes, ER, PR, and Her2 expression, lymphovascular invasion (LVI), and use of chemotherapy or hormonal therapy. The ORs for TMEM was compared with those for IHC4. Results: in a multivariate model adjusted for other covariates, TMEM was associated with increased risk of distant metastasis in 62% of subjects with ER+/Her2- tumors (OR [95%CI]high vs. low tertile = 2.70 [1.39-5.26], Ptrend = 0.004), whereas IHC4 had a borderline positive association (OR10 unit increase = 1.06 [1.00-1.13]); the association for TMEM persisted after adjustment for IHC4 (OR [95%CI]high vs. low tertile = 2.68 [1.31-5.49], Ptrend = 0.004). TMEM scores ranged up to a nearly 200-fold from lowest to highest, and did not correlate with tumor size, number of positive lymph nodes, nor IHC4 score (Spearman correlation 0.12 for IHC4). The AUC for a TMEM composite score (0.77), derived from a multivariate logistic regression model with TMEM and clinical variables, was significantly higher than those for TMEM alone (0.65) and for IHC4 (0.62). Neither TMEM nor IHC4 was independently associated with metastatic risk in the triple negative (20%) or HER2+ (18%) tumors. Conclusion: TMEM score, a mechanism-based assay, is positively associated with risk of distant metastasis in ER+/Her2- breast cancer and provides prognostic information that is complementary to IHC4 and other clinicopathologic risk factors. Additional validation studies designed to establish clinical validity are currently being planned. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-03.

Abstract P4-06-12: Simultaneous phenotypic and genotypic heterogeneity at the cellular level approached by HER2 gene-protein assay (GPA) in the invasive ductal carcinoma of the breast: A new approach for cancer pathobiology

Abstract Introduction: Trastuzumab therapy has been given to the HER2 positive breast cancer (BC) patients (IHC 3+ and/or amplified HER2 gene). Heterogeneity of HER2 expression has been a problem when the recurrence is considered. Recently, the combined HER2 IHC and dual color ISH (DISH) method has been introduced as a gene-protein assay (GPA) (Roche Diagnostics Inc.). This method enables us to analyze heterogeneous expression of HER2 protein and HER2 gene at the individual cell level on the same tissue sections. This study is aimed to elucidate the relevance of GPA in studying the gene-protein(GP) heterogeneity of HER2 status at the individual cells of BCs and further to pursue the cellular GP heterogeneity as a possible cause of stage IV post-therapeutic recurrence. Materials and Methods: Formalin-fixed, paraffin-embedded sections from 17 HER2 IHC 3+ cases (5 immediate post-operative cases and 12 recurrence cases) and 11 HER2 IHC 2+ cases (9 immediate post-operative cases and 2 recurrence cases) were analyzed with HER2 GPA using BenchMark ULTRA (Roche Diagnostics K.K.). All slides were scored for HER2 protein expression (0 to 3+) and the ratio of HER2/CEN17 of 100 cells from the following areas: one hot spot and two non-hot spots. HER2 protein and gene status of individual cells were grouped from A to F according to the following definition: Group A: HER2 IHC positive (3+), DISH positive (HER2/CEN17 ratio ≥2.0) Group B: HER2 IHC positive (3+), DISH negative (<2.0) Group C: HER2 IHC equivocal (2+), DISH positive (≥2.0) Group D: HER2 IHC equivocal (2+), DISH negative (<2.0) Group E: HER2 IHC negative (0 or 1+), DISH positive (≥2.0) Group F: HER2 IHC negative (0 or 1+), DISH negative (<2.0) Results: HER2 GPA technology clearly demonstrated the HER2 heterogeneity at the individual cell level of invasive BCs. The GP heterogeneity was apparent in the HER2 3+ and 2+ immediate post-operative cases, particularly more obvious with HER2 IHC 2+ cases. For IHC 3+ cases (table 1), groups A and C are prominent with the HER2 GP heterogeneity compared with the other groups. For IHC 2+ cases (table 2), the HER2 GP heterogeneity was more obvious with prominent groups D and F than others. Table 1: Distribution of groups of HER2 status by GPA Cases with HER2 IHC 3+CasesABCDEFPost-operative79.22.88.407.02.6Recurrent50.83.017.06.17.68.9Post-operative and recurrent cases showing hetelogeneity as individual cell level For the recurrent cases, the GP heterogeneity was similar but more pronounced comparing to immediate post-operative tumors in IHC 3+ cases, and GP heterogeneity in recurrent cases was more pronounced in IHC 2+ cases. Conclusions: HER2 GPA system is an appropriate and essential technology to study the GP heterogeneity at the individual tumor cell level of BCs. We hypothesize that the GPA system may possibly predict the recurrence of post-therapeutic stage IV tumors by the presence of more pronounced HER2 GP heterogeneity. Table 2: Distribution of groups of HER2 status by GPA Cases with HER2 IHC 2+CasesABCDEFPost-operative01.714.944.78.630.2Recurrent007.00.548.044.5 Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-06-12.

B Cell Receptor with Variant Surface Isotypes Transduce Functional Signals by Elevating Phospho-ERK1/2 Levels in Hairy Cell Leukemia with Mutant BRAF

A functional BCR is essential for survival of normal B-cells, in response to cognate antigen or tonic, antigen-independent stimuli. Cross-linking of BCR by antigen triggers key phosphorylation events in the early signalosome. Mammalian wild type BRAF, a member of the RAF cytosolic kinase family (A-RAF, B-RAF, C-RAF) is critical to BCR function, mediating RAS-RAF-MEK-ERK or mitogen-activated protein kinase (MAPK) pathway signal transduction, via phosphorylation of ERK1/2. The MAPK pathway is a central conduit for survival and proliferation. Importantly, use of inducible deletion models have shown that wild type BRAF is the predominant kinase that transduces BCR signals to activate ERK1/2, with C-RAF playing an accessory role and A-RAF displaying a x20-30 fold lower basal activity in ERK1/2 stimulation. Tonic BCR survival signals in normal B-cells are also transduced via phosphorylation of ERK1/2, but do not require antigen stimuli. Whether the BCR plays a comparable role in sustaining survival in malignant B-cells is less well characterized and remains a central question in understanding the origins and progression of mature B-cell neoplasms.Hairy cell leukaemia (HCL) is a rare B cell leukaemia with characteristic hair-like cytoplasmic projections on tumor cells, displaying distinctive activation markers. A striking feature of monoclonal HCL tumors is expression of multiple variant immunoglobulin heavy chain (sIgH) isotypes as components of the BCR on individual tumor cells, defining the major subset of disease (mult-HCL). Most mult-HCL tumors exhibit IGV somatic hypermutation with a low level of on-going mutations and AID expressed, implicating initial contact with antigen via the BCR. The functional relevance of the BCR to transformation and survival however has as yet not been mapped in HCL. Seminal exome sequencing data in typical HCL identified mutant BRAF V(600)E as almost universal in this tumor. Consistent with mutant BRAFV(600)E, levels of phosphorylated ERK (p-ERK) are raised in hairy cells. As there is an essential requirement for wild type BRAF in transducing BCR signals, the question arises how mutant BRAF may affect BCR function in HCL, given requirements for dimerization of wild type BRAF for ERK1/2 activation, and whether ERK1/2 activation can be enhanced by functional BCR signals for downstream effector signals in HCL.To examine this, we evaluated BCR signalling in mult-HCL (n=10), in cases uniformly displaying mutated BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD+ mult-HCL, IgD mediated persistent Ca2+ flux, which was also evident via >1 sIgH isotype, linked to increased ERK1/2 activation and BCR endocytosis (4/4 cases). In sIgD-vemult-HCL however (6/6 cases), BCR-mediated signals activating ERK1/2 for downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal a clear discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing mult-HCL (3/3 cases), only a single sIgL was fully functional.We next examined effects of anti-BCR stimuli on mult-HCL tumor cell survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both sets (4/4 cases) to establish a consistent outcome in these replicate cases. IgD stimuli, in marked contrast retained tumor viability with no evidence for pre-apoptotic effects (2 cases; 1 mult-HCL and 1 sIgD only-HCL).Despite mutant BRAF, BCR signals amplify ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL lacking IgD, a role for antigen in stimulating the BCR for tumor persistence appears unlikely. It remains feasible that mutant BRAF in these cases retains tonic signals through activated ERK1/2 for survival, but this remains speculative at present. Surface IgD emerges with potential to transduce BCR signals for tumor survival and persistence in-vivo, but given its enigmatic role even in normal B-cells, its relevance to HCL progression is unclear.These observations raise the possibility that mutant BRAF may be a mechanism to bypass any antigen-dependent BCR signalling constraints in mult-HCL. Disclosures:No relevant conflicts of interest to declare.

POBRAD Trial: Prospective trial evaluating outcomes of immediate implant breast reconstruction using an acellular dermal matrix

undertaken after administration of both the magnetic and standard tracers. We have defined the trial with 80% power, a 5% one-sided significance and a proportion discordance of 0.052. Results: 169 SLNB procedures were undertaken on 160 patients. The identification rate was 95% (152/160) with the combined technique and 94% (151/160) with the magnetic technique (95% upper confidence limit 4.4%; 0.62% difference; 6.88% discordance). Of 23% (36/160) of patients with lymph node involvement, 16% (26/160) had at least 1 macrometastasis and 6% (10/160), a micrometastasis only. Another 2% (3/160) had individual tumour cells. Of 402 lymph nodes removed, 295 (73%) were true sentinel nodes. Lymph node retrieval rate was 2.5 nodes per patient overall, 1.8 nodes per patient with the standard technique and 2.0 nodes per patient with the magnetic technique. Conclusion: The magnetic technique is a feasible technique for SLNB, non-inferior to the combined technique.

Gut neuroendocrine tumor blood qPCR fingerprint assay: characteristics and reproducibility

We have developed a PCR-based tool that measures a 51-gene panel for identification of gastroenteropancreatic (GEP) neuroendocrine neoplasms (NENs) in peripheral blood. This manuscript assesses the robustness (performance metrics) of this tool with a specific focus on the effects of individual parameters including collection, storage, acid suppressive medication [proton pump inhibitor (PPI)], age, sex, race and food on accuracy. Performance metrics were evaluated using a gold standard (mRNA derived from three individual human neuroendocrine tumor cell lines) and clinical samples using qPCR. One hundred percent of the 51 transcripts were amplified in the gold standard (NEN cell line-derived mRNA) (CQ<35, average efficiency 1.94). The inter- and intra-assay variations were 1%-2%. In clinical samples, 50 of 51 targets (98%) were amplified. The inter- and intra-assay reproducibility ranged between 0.4% and 1.2%. The coefficient of variation (CV) was 5.3%. Expression of the reference gene, ALG9, was robust [low variation, low M-value, high (99.5%) PCR efficiency] and unaffected by sample processing. Test meals, long-term PPI use (>1 year), age, sex and ethnicity had no effect on the signature. Expression of two genes, ALP2 and CD59 correlated strongly with RNA integrity (R=0.72, p<0.001) and could be used to assess storage and processing. The 51 marker gene signature was robust and reproducible, exhibiting acceptable inter- and intra-assay metrics (<5%). Feeding, PPI intake, age, sex and ethnicity do not affect the signature. Expression levels of APLP2 and CD59 are effective surrogate markers of proper sample collection and processing.

Mechanisms of Metastatic Tumor Dormancy.

Tumor metastasis can occur years after an apparent cure due to a phenomenon known as metastatic tumor dormancy; in which tumor masses or individual tumor cells are growth restricted for extended periods of time. This period of dormancy is induced and maintained by several mechanisms, including: (1) Tumor microenvironment factors such as cytokine expression, immunosurveillance and angiogenesis; (2) Metastasis suppressor gene activity; and (3) Cancer therapeutics. Disseminated tumor cells (DTC) are the key cells that result in dormant tumors. However, many challenges exist towards isolating DTCs for mechanistic studies. The main DTC that may represent the dormant cell is the cancer stem cells (CSC) as they have a slow proliferation rate. In addition to limited knowledge regarding induction of tumor dormancy, there are large gaps in knowledge regarding how tumors escape from dormancy. Emerging research into cancer stem cells, immunotherapy, and metastasis suppressor genes, may lead to new approaches for targeted anti-metastatic therapy to prevent dormancy escape. Overall, an enhanced understanding of tumor dormancy is critical for better targeting and treatment of patients to prevent cancer recurrence.

Does Aquaporin-1 expression have clinical significance in serous epithelial ovarian cancer?

To assess the relationship between Aquaporin-1 (AQP1) expression and clinicopathological variables in serous epithelial ovarian cancer (EOC). Serous EOC cases treated in our institution between January 2007 and December 2009 were included in the study. A semi-quantitative immunohistochemical method was used to determine AQP1 expression levels, intratumoral microvessel density (IMD) and AQP1/IMD ratios. The relationship between these parameters and clinicopathological variables were assessed. P values less than 0.05 was considered statistically significant. A total of 55 cases of serous EOC were included in the study. AQP1 was strongly expressed in the membranes of microvessels and small vessels within all tumor tissues. In a few cases, AQP1 expression was also observed in the membrane of interstitial cells and in individual tumor cells. A positive correlation was detected between preoperative CA125 levels and the expression of AQP1 (R: 0.277, p<0.05). AQP1 expression was similar between FIGO stage I-II and FIGO stage III-IV cases (p > 0.05). A significant relationship did not exist between AQP1 expression and FIGO stage, lymph node metastasis or ascites volume (p>0.05). In this study, AQP1 expression did not have a significant association with important clinicopathological variables in serous EOC. Future studies are needed to determine AQP1 expression in other histological types of EOC.

Determination of EGFR mutations in single cells microdissected from enriched lung tumor cells in peripheral blood

A minimally invasive and repeatable approach for real-time epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized therapy of late stage lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single circulating tumor cells (CTCs). Using a model CTC system of spiked tumor cells in whole blood, we evaluated EGFR mutation determination in single tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked tumor cells enriched from 7.5ml whole blood. Whole-genome amplification provided sufficient DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55% (11/20) for exon 19 deletion, 45% (9/20) for T790M, and 85% (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80% of the amplicons. EGFR mutation determination from single captured tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the tumor's EGFR status, multiple individual tumor cells should be assayed.

Utility of Intraoperative Frozen Section Examination of Sentinel Lymph Nodes in Ductal Carcinoma In Situ of the Breast

BackgroundIntraoperative frozen section (IFS) examination of sentinel lymph nodes (SLN) is useful in selecting patients with invasive breast cancer for immediate axillary lymph node dissection. However, whether IFS evaluation of the SLNs in ductal carcinoma in situ (DCIS) of the breast has any value has not been previously assessed. MethodsClinicopathologic data from patients with DCIS who underwent resection with SLN biopsy (2004-2010) were collected to assess the sensitivity, specificity, and accuracy of IFS, and its impact on axillary management. ResultsA total of 267 patients with DCIS underwent resection with SLN biopsy and IFS evaluation. Preoperative pathology was DCIS (n = 231), DCIS with microinvasion (n = 24), and DCIS with other lesions (n = 12). Fifty-two (19.5%) patients had invasive breast cancer on final pathology. SLN metastases were identified in 13 (4.8%) patients; however, only 4 (1.5%) were IFS positive. IFS examination was negative in 263 (98.5%) patients. Among patients with SLN metastases, the most common pattern of metastases was either micrometastasis (n = 6) or immunohistochemistry-positive individual tumor cells (n = 4), whereas 3 patients had a macrometastasis. IFS examination was falsely negative in 9 of these 13 patients for a false-negative rate of 69.3%, and a sensitivity and specificity of 31% and 100% respectively. Nine of the 13 patients underwent axillary lymph node dissection and only 1 patient had further axillary metastasis. ConclusionsSLN metastases in DCIS is rare and most commonly involves SLN micrometastasis or immunohistochemistry-positive individual tumor cells. SLN IFS evaluation in DCIS has a low yield and sensitivity, and can be safely omitted to reduce operative duration and cost.

Enrichment of putative prostate cancer stem cells after androgen deprivation: Upregulation of pluripotency transactivators concurs with resistance to androgen deprivation in LNCaP cell lines

Prostate cancer stem cells (PCSC) offer theoretical explanations to many clinical and biological behaviors of the disease in human. In contrast to approaches of using side populations and cell-surface markers to isolate and characterize the putative PCSC, we hypothesize that androgen deprivation leads to functional enrichment of putative PCSC. Human prostate cancer lines LNCaP, LAPC4 and LAPC9 were depleted of androgen in cell cultures and in castrated SCID mice. The resultant androgen deprivation-resistant or castration-resistant populations, in particular in LNCaP and its derivative cell lines, displayed increased expression of pluripotency transactivators and significantly higher tumorigenicity. Individual tumor cell clones were isolated from castration-resistant bulk cultures of LNCaP (CR-LNCaP) and tested for tumorigenicity in male SCID mice under limiting dilution conditions. As few as 200 cells were able to form spheres in vitro, and generate tumors with similar growth kinetics as 10(6) LNCaP or 10(4) CR-LNCaP cells in vivo. These putative PCSC were CD44(+) /CD24(-) and lack the expression of prostate lineage proteins. When transplanted into the prostate of an intact male SCID mouse, these putative PCSC seemed to show limited differentiation into Ck5(+) , Ck8(+) , Ck5(+) /Ck8(+) , and AR(+) cells. On the other hand, stable transduction of LNCaP with retrovirus encoding Sox2 led to androgen-deprivation resistant growth and down-regulation of major prostate lineage gene products in vitro. Concurrence of overexpression of pluripotency transactivators and resistance to androgen deprivation supported the role of putative PCSC in the emergence of prostate cancer resistant to androgen deprivation.

Lymph node metastases in endocervical adenocarcinoma: Correlation with Silva pattern of invasion.

e16518 Background: Endocervical adenocarcinoma (EA) is the 2nd most common uterine cervical malignancy (after squamous cell carcinoma). Early stage tumors are defined by depth of invasion (DOI); although used to guide nodal dissection, DOI not an entirely reliable criteria, and most patients have negative nodes. Thus, pathologic criteria that better predict risk of nodal metastases are needed. A prior study of 352 cases from 12 US and international institutions, with complete pathological and clinical follow-up averaging 52 months, found classifying EA by a newly proposed ‘Silva Pattern of Invasion’ (SPI) is the best predictor of nodal metastases. SPI are defined as A: no destructive stromal invasion or lymph-vascular invasion (LVI); B: small foci (&lt;4 mm) of fragmented, angulated glands, solid aggregates or individual tumor cells associated with desmoplastia or LVI; and C: diffuse destructive invasion. Pattern A, 25% of cases, had no nodal metastases or recurrences. Approximately 25% were B, with 5% nodal metastases, 2% recurrence, and no tumor deaths. Pattern C cases: nodal metastases 23%, recurrences 21%, and death from tumor 9%. SPI appeared simple to diagnose, but a formal assessment of reproducibility is needed. Methods: Reproducibility for diagnosing SPI, and for the standard measurement of DOI is compared. Four Gynecological Pathologists, who have read the initial manuscript, but not involved in the original study nor from the participating institutions, and blinded to clinical information examine 1or 2 hematoxylin and eosin-stained glass slides from 90 cases from the original series. Results: 30 cases each of Pattern A, B, and C are reviewed. Kappa statistics will be estimated. With 90 samples, we will have &gt;80% power to detect a true kappa of 0.7 in a test of the null hypothesis that kappa = 0.5 vs the alternative that kappa≠0.5 with equal frequencies in each SPI category. Conclusions: A good reproducibility outcome would indicate that the SPI could be applied in general practice. Accurate use of SPI requires examination of the entire lesion unless pattern B or C is found in the biopsy specimen. Use of the SPI system to guide nodal dissection will require validation in a large series.

Abstract 1464: In vitro culturing of CTCs/DTCs in personalized cancer management.

Abstract Subsequent detection, quantification and molecular characterization of CTCs has an enormous potential in numerous of oncology areas. But detection and isolation of CTCs is technically quite challenging. The whole dissemination process encompasses also individual tumor cells which are found in relatively big quantities in peritoneal washings (PW) and pleural effusions (PE), so called disseminated tumor cells (DTCs). Thanks in vitro cultures of separated CTCs/DTCs we were able to introduce additional chemosensitivity testing for the purpose of individualized cancer treatment. We present an isolation and cultivation of CTC/DTC of non-small cell lung cancer and gastrointestinal cancers (esophageal cancer, colorectal cancer, gastric cancer, pancreatic cancer) describing cultivation methodology on patients case studies. The cancer cells were separated from body fluids (PW, PE, blood) using the differences in size of cancer cells if compared to other nucleated cells detected in body cavities. After reaching the confluence the chemosensitivity of cell cultures has been tested by xCELLigence system (RTCA, Roche) for different concentrations of single cis-platinum, gemcitabine and fluorouracyl (incubation time 24-72 hours) or their combinations. The dynamic real-time cell growth monitoring enables parallel or sequential combinations of administered drugs with a direct outcome for patients care. Conclusions: We assume that developing cultivation strategies for CTCs/DTCs could bring us closer to the definition of the CTC/DTC-function within the dissemination process with a perspective to use this knowledge in personalized cancer treatment. Citation Format: Vladimir Bobek. In vitro culturing of CTCs/DTCs in personalized cancer management. [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 1464. doi:10.1158/1538-7445.AM2013-1464

Abstract SY05-01: Genetic and non-genetic mechanisms contribute to long-term clonal growth dynamics and therapy resistance.

Abstract SY05-01: Genetic and non-genetic mechanisms contribute to long-term clonal growth dynamics and therapy resistance.

Abstract 3512: Dielectrophoretic capture and genetic analysis of individual disseminated solid tumor cells.

Abstract Background: The purpose of this study was to develop a novel approach for isolation and genetic analysis of individual circulating and disseminated tumor cells (CTC, DTC) from patient samples. This work is motivated by our desire to develop non-invasive molecular diagnostics and understand single tumor cell heterogeneity. Methods: Here we used the DEPArray microelectronics and microfluidics platform for the manipulation and capture of individual tumor cells. A suspension of live or fixed cells, prelabeled with fluorochrome-conjugated antibodies, was loaded into the DEPArray cartridge, and application of non-uniform electrical fields (dielectrophoresis) was employed to trap individual cells into movable micro-cages. Cell images were acquired via the incorporated fluorescence microscope and CCD camera, and cells were selected based on morphology and expression of tumor antigens. To determine sensitivity and specificity of the capture process, cell lines were spiked into normal donor white blood cells (WBC) over a 5-log range. Pre-enrichment by FACSAria was employed when the starting tumor cell concentration was below 1 in 1,000 WBC. The whole genome amplification (WGA) product of single cells was then subjected to targeted sequencing. To apply this approach to patient samples, we obtained blood and bone marrow samples from patients with refractory high-risk neuroblastoma, a cancer with a high rate and quantity of distant metastatic disease. Results: Single tumor cells were recovered from all spiking experiments, including spiked samples with a tumor cell concentration as low as 1 in 106 WBC. Recovery of EpCAM-negative single cells was accomplished with the neuroblastoma cell lines SY5Y, NB1643, and Kelly labeled with an antibody for the disialoganglioside GD2, the breast cancer cell line MCF-7 with a HER2 antibody, and for the lung cancer cell line H292 with EGFR labeling. Recovery of EpCAM-positive cells was achieved with the colon carcinoma cell line SW480. Captured single cells were WGA'd and specificity was verified using Sanger sequencing. For the 2 neuroblastoma spiking experiments for which sequencing has been completed, the known ALK mutation (F1174L) was detected in 100% of cells. The known KRAS mutation (G12V) was detected in 100% of single SW480 cells sequenced to date. No mutations were detected in isolated WBC. We next analyzed the post-Ficoll fraction of 12 patient bone marrow and 1 patient blood sample, with a median tumor cell concentration of 0.119% (range 0.003 - 80.0%). A minimum of 10 single tumor cells were captured per patient. Genome-wide analysis of DNA and RNA copy number and mutation status from these samples is ongoing. Conclusion: These data demonstrate the development of a novel workflow for the reliable capture and genetic analysis of rare CTC. Efforts are ongoing to join this approach with a more comprehensive genome-wide analysis of single cells from neuroblastoma and other cancers. Citation Format: Erica L. Carpenter, JulieAnn Rader, Kateryna Krytska, Jacob Ruden, Eric Rappaport, Yael Mosse, Francesca Fontana, Manuela Banzi, Gianni Medoro, Nicolo Manaresi, Peter O'Dwyer, John Maris. Dielectrophoretic capture and genetic analysis of individual disseminated solid tumor cells. [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 3512. doi:10.1158/1538-7445.AM2013-3512

High-resolution, serial intravital microscopic imaging of nanoparticle delivery and targeting in a small animal tumor model

Nanoparticles are under active investigation for the detection and treatment of cancer. Yet our understanding of nanoparticle delivery to tumors is limited by our ability to observe the uptake process on its own scale in living subjects. We chose to study single-walled carbon nanotubes (SWNTs) because they exhibit among the highest levels of tumor uptake across the wide variety of available nanoparticles. We target them using RGD (arginine-glycine-aspartic acid) peptide which directs them to integrins overexpressed on tumor vasculature and on the surface of some tumor cells (e.g., U87MG as used here). We employ intravital microscopy (IVM) to quantitatively examine the spatiotemporal framework of targeted SWNT uptake in a murine tumor model. IVM provided a dynamic microscale window into nanoparticle circulation, binding to tumor blood vessels, extravasation, binding to tumor cells, and tumor retention. RGD-SWNTs bound to tumor vasculature significantly more than controls (P<0.0001). RGD-SWNTs extravasated similarly compared to control RAD-SWNTs, but post-extravasation we observed as RGD-SWNTs eventually bound to individual tumor cells significantly more than RAD-SWNTs (p<0.0001) over time. RGD-SWNTs and RAD-SWNTs displayed similar signal in tumor for a week, but over time their curves significantly diverged (p<0.001) showing increasing RGD-SWNTs relative to untargeted SWNTs. We uncovered the complex spatiotemporal interplay between these competing uptake mechanisms. Specific uptake was delimited to early (1-6 hours) and late (1-4 weeks) time-points, while non-specific uptake dominated from 6 hours to 1 week. Our analysis revealed critical, quantitative insights into the dynamic, multifaceted mechanisms implicated in ligand-targeted SWNT accumulation in tumor using real-time observation.

New experimental model of gastric carcinogenesis in rats with the use of formaldehyde and hydrogen peroxide mixture

This study was designed to develop an animal model of gastric cancer by using a mixture of formaldehyde and hydrogen peroxide. Thirty‐five outbred rats were used. In experimental group, 1.0 ml of the formaldehyde (0.4%) and hydrogen peroxide (0.4%) mixture (1:1) were administered to 30 rats by intragastric gavage on the 1st and 3rd day every 2 weeks for 6 months. Five non‐treated rats served as a control. The mortality rate among treated rats was 13.3%. We found that 6 months after the onset of treatment the 4 of 5 rats had gastric tumor, while after 7.5 months all survived 21 rats showed malignant tumors of the stomach. The tumors were primarily localized within the gastric mucosa, though the invasion of individual tumor cells and their clusters in the muscularis propria and serosa as well as in the lymphatic and blood vessels were also seen. In most cases, the infiltrated gastric mucosa areas (2 to 3) had rigid gastric folds and were 0.7×1.0cm to 1.5×3.0cm in size. We observed signet‐ring cell carcinomas in 80.9% of rats, well‐differentiated tubular adenocarcinoma in 66.6% of rats and moderately differentiated tumors in 23.8% of rats. Metastasis in regional lymph nodes were noted in 3 rats (14.3 %), while 6 animals (28.6%) revealed peritoneal dissemination of the small intestine. In addition, 19.0% of rats showed tumors of the lungs. Therefore, these data demonstrate that our animal model can be used to study gastric carcinogenesis.

Synchrotron X-ray fluorescence studies of a bromine-labelled cyclic RGD peptide interacting with individual tumor cells.

The first example of synchrotron X-ray fluorescence imaging of cultured mammalian cells in cyclic peptide research is reported. The study reports the first quantitative analysis of the incorporation of a bromine-labelled cyclic RGD peptide and its effects on the biodistribution of endogenous elements (for example, K and Cl) within individual tumor cells.

Kidney: Metanephric adenoma

Grossly, the tumors are solid, gray to tan in color, and homogenous on cut surface. Most tumors are well-demarcated but lack a capsule. Microscopically, the tumor is chiefly composed of tightly packed primitive tubules and acina, with frequent papillary or glomeruloid structures. The individual tumor cells have scant cytoplasm, round-to-oval nuclei with smooth chromatin, inconspicuous nucleoli. Stromal hyalinization, dystrophic calcification and psammoma bodies are occasionally seen. Mitotic figures are rare or absent. The tumor cells are positive for WT1, PAX2 and CD57, but negative for CK7, CD56 and AMACR.