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Introduction Metaplastic carcinoma of the breast (MCB) is a heterogeneous group of malignant tumors comprising less than 1% of all breast carcinomas encompassing tumors with purely epithelial, as well as epithelial and mesenchymal components, which may be entirely metaplastic or admixed with carcinoma. There is no agreed upon optimal treatment regime for these tumors, which overall have a worse prognosis than similar stage invasive ductal carcinoma (IDC). Research into MCB has been limited by the rarity of this tumor; however as the majority of these tumors do not express ER, PR or Her2 (triple negative) and response to traditional chemotherapy is limited, there is a need for further research to identify predictive biomarkers and new targets for treatment. We report our findings of multigene mutation profiling in a series of 21 MCB. Methods DNA was extracted from tumors of twenty one patients with MCB, genotyped using the Oncocarta v1.0 kit a sensitive, high throughput mass spectrometry based assay which detects a panel of 238 specific mutations in 19 oncogenes including KRAS, BRAF, PIK3CA, MET, AKT1 & ERBB2 and processed on the Sequenom compact MassARRAY platform. In the case with heterologous differentiation, separate cores were taken from the spindle cell, osteo- and chrondrosarcomatous areas. Results Histological review revealed three pure spindle cell cases with the remainder showing mixed patterns of spindle, squamous and IDC, no special type. A single case showed heterologous osteosarcomatous and chondrosarcomatous differentiation. Ten mutations were detected in these 21 MCB. PIK3CA gene mutations (six H1047L and one E545K mutation) were seen in seven patients; and KRAS gene mutations (two G12V and one G12A) were observed in three patients. No mutations were detected in the case with heterologous elements in the separate areas of spindle, osteo- and chondrosarcomatous differentiation. Conclusions PIK3CA mutations were seen in 33% and KRAS mutations were seen in 14% of MCB cases in this series. Given the general lack of effective chemotherapies in MCB, these findings suggest that the PI3K pathway may be a promising therapeutic target worthy of further investigation. We plan to also undertake detailed mutational analyses using next generation sequencing to identify further potential therapeutic targets in these rare and difficult to treat malignancies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-08-03.

Investigation of the expression pattern of DNA-repair protein excision repair cross-complementation group 1 (ERCC1) has been reported to allow selection of patients with non-small cell lung cancer who are likely to benefit from cisplatin-based therapy. Recent evidence suggests that ERCC1 expression may also find prognostic use in patients with nasopharyngeal carcinoma (NPC). We evaluated ERCC1 expression and genotype from NPC tissues and peripheral blood mononuclear cells of patients with NPC. ERCC1 expression was detected in 61/77 cases (79.2%) with varying intensities, where high ERCC1 expression significantly associated with worse relapse-free survival (RFS) (HR 2.34, 1.06-5.16, p=0.036). In addition, the presence of ERCC1 C118T genotype was significantly associated with favorable RFS and overall survival (OS) in a subgroup of patients with undetectable post-treatment plasma EBV DNA. These findings support a prognostic role for ERCC1 examination in NPC. However, the invasive nature of obtaining biopsy samples for tumor marker studies is a major hindrance of this approach. To facilitate the investigation of ERCC1 expression in a noninvasive manner, we have developed a negative selection immunomagnetic method for isolating circulating tumor cells from patient blood. Cell line spike-in experiments reveal a mean recovery rate of 66% for NPC cells with greater than 99% removal of non-targeted blood cells. Specificity of CTC identification was confirmed by detection of the expression of the Epstein-Barr virus encoded small RNA (EBER) in the CTCs. This method also allowed the simultaneous analysis of the expression of multiple protein markers including CD45, cytokeratin and ERCC1. It is expected that the development of noninvasive methodologies for tumor marker studies will facilitate their clinical application for improved patient care and monitoring in the future. Note: This abstract was not presented at the meeting. Citation Format: Edwin P. Hui, Brigette BY Ma, KC Allen Chan, Charles ML Chan, SC Cesar Wong, Ka Fai To, Herbert HF Loong, Frankie KF Mo, Roger KC Ngan, Anthony TC Chan. Examination of ERCC1 status in circulating tumor cells as a prognostic tool for patients with nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3776. doi:10.1158/1538-7445.AM2017-3776

Background and aims Resistance to platinum-based chemotherapy remains a major obstacle in the treatment against human ovarian cancer. In this study, binary combinations of platinum compounds: cisplatin, carboplatin and oxaliplatin with phytochemicals: artemisinin, thymoquinone, curcumin, lupeol and oleanolic acid have been applied to human ovarian cancer cell lines: A2780, A2780cisR, and A2780ZD0473R using three different sequences of administration (0/0h, 0/4h and 4/0h) where 0/0h means both the drugs added at the same time, 0/4h means platinum added first followed phytochemical 4 h later and 4/0h means the converse. Methods Cytotoxicity was determined based on MTT reduction MTT assay. Median effect analysis was done to characterize combined drug action and combination Index (CI) was used as a measure of synergism or antagonism whereas cellular accumulation of platinum, platinum to DNA binding levels and proteomic studies were done to provide insight into drug resistance and synergism from combined drug action. Results The combinations of platinum and the phytochemicals produced sequence- and concentration-dependent synergism in the ovarian tumour models. The degree of synergism was greater in the resistant cell lines than the parent cell line. It was found that combination with phytochemicals also increased intracellular accumulation of platinum and the level of platinum binding with DNA. Proteomic study identified a number of proteins believed to be associated with cisplatin resistance in ovarian cancer cells A2780 and A2780cisR. The expression of the proteins were either down- or up-regulated in A2780cisR cell line as compared to A2780 cell line but fully or partially restored after treatment with synergistic combinations. Conclusion The results of the study indicate that appropriately sequenced combinations of platinum drugs and phytochemicals can provide a means of overcoming drug resistance in ovarian cancer. Citation Format: Safiah I. Althurwi, Jun Qing Yu,, Philip Beale, Charles Chan, Fazlul Huq. Sequenced combinations of platinum drugs and phytochemicals towards overcoming drug resistance in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2534. doi:10.1158/1538-7445.AM2015-2534

Osteosarcoma (OS) is an aggressive pediatric bone malignancy that affects 400 children per year in the United States. It has one of the lowest 5 yr. survival rates of any pediatric cancer. In order to gain a better understanding of the pathogenesis of OS, we have established a panel of patient derived xenografts (PDX). Through a three institution collaboration, we have obtained 52 OS patient samples, 38 pre-chemotherapy biopsies and 16 post induction chemotherapy resections. These samples were implanted into immunocompromised mice either subrenal or intradermal and allowed to expand. We were able to generate 20 PDX samples (38% take rate) and have characterized 7 of them. This includes one pre and post-chemo pair from the same patient. The PDX samples had similar histology to the original patient biopsy including formation of osteoid, which is a hallmark of OS. Tumor propagating cells (TPCs) are a rare subset of cells with in the tumor that are thought to contain the properties necessary to propagate and give rise to all cells found in the original tumor. TPCs are also thought to be resistant to chemotherapy and thus, it is important to identify and study these cells to gain a better understand the biology that underlies the TPCs ability to propagate the tumor. To identify the TPCs in OS, we selected a panel of cell surface markers that have been associated with TPCs in other cancer types and assessed for their expression across multiple OS PDX samples. CD49f, CD146 and CD90 were expressed heterogeneously in most PDX samples. There was also a high degree of overlap in the expression of these three markers in a subset of OS cells. To determine if the heterogenenous staining observed has a biological significance, we sorted CD49f positive and negative cells and implanted them in serial dilutions into immunocompromized mice. Although no difference in tumor instance was observed, CD49f + tumor cells formed significantly larger tumors than those arising from CD49f- tumor cells. Thus, CD49f expression may mark a more proliferative subset of cells within the bulk tumor. Further studies are currently ongoing to define the functional relevance of CD146 and CD90 expression and their role in tumor propagation and tumor morphology using OS PDX samples. To further characterize these OS PDX samples, we have employed RNA sequencing technology. To date we have obtained RNAseq on 4 pre-chemotherapy and 3 post-chemotherapy patient samples. Analysis of this dataset is ongoing and may provide insight into mechanisms of chemoresistance. Citation Format: Leanne C. Sayles, Justin Vincent-Tompkins, Charles Chan, Irving Weissman, E. Alejandro Sweet-Cordero. Using primary osteosarcoma samples to study tumor heterogeneity. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A78.

Translocations involving members of the Ets family of transcription factors are common in prostate cancer and sarcomas, yet their effects on signal rewiring in cancer are unclear. Translocations between EWS and the Ets transcription factor Fli1 are found in Ewing's sarcoma, an aggressive tumor found in children and young adults. To elucidate the mechanism of transformation by EWS-Fli1, we have developed a novel approach that leverages the advantages of both mouse and human model systems. A knock-in mouse in which a Fli1-cDNA was introduced downstream of the endogenous EWS allele was generated. To our knowledge, this is the first described genetically engineered model in which EWS-Fli1 (EF) can be expressed conditionally from the native EWS promotor. Expression of EF in MEFs and MSCs from this model demonstrates that MSCs tolerate EF expression better than MEFs, consistent with prior data suggesting that MSCs are the likely cell of origin of Ewing's sarcoma. Ongoing analysis of well-defined MSC subsets may provide additional information regarding the cell of origin. In parallel, we have analyzed the consequences of expression of EF in human MSCs derived from pediatric patients. Deep-sequencing of these human pediatric MSCs (hpMSCs) has identified many novel coding and non-coding transcripts that are specifically upregulated in hpMSCs as a consequence of expression of EF. We have used functional analysis using hMSC and ES cell lines to validate the role of these genes in EWS pathogenesis. Some, but not all of these novel transcripts are also upregulated in mouse MSCs and MEFs that express EF. In summary, a cross-species approach that combines mouse genetics with functional genomics has identified novel targets for ES specific therapeutic development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1427. doi:1538-7445.AM2012-1427

Kidney cancer is the sixth leading cause of cancer death in the USA, and the primary tumor is frequently accompanied by metastasis to lungs, liver, brain and bones. Importantly a large number of kidney cancer cases are characterized by loss of von Hippel-Lindau (VHL) gene, which leads to stabilization of hypoxia-inducible factors (HIFs), which contribute to tumor progression and metastasis by multiple mechanisms. CUB-domain-containing protein 1 (CDCP1) was shown to be expressed on the cell surface of metastatic cell lines and to increase the number of nodules formed by lung adenocarcinoma cells and melanoma in lungs in tail vein injection experiments, enhance peritoneal dissemination of scirrhous adenocarcinoma, and to induce metastasis in the chicken embryo metastatic model. In the present study we investigated the role of CDCP1 protein in clear cell renal cell carcinoma (CC-RCC) and found it to be upregulated in this disease entity by a mechanism of VHL loss through HIFs. Interestingly, we found CDCP1 protein expressed not only on the membrane of kidney cancer cell lines, but also to be secreted into the media as a full length isoform. Importantly, in the knockdown experiments we found CDCP1 to promote CC-RCC cell migration in vitro, the process known to be the key during metastasis. Accordingly CDCP1 participates in the signal transduction pathway leading to PKCδ phosphorylation in CC-RCC, stimulating migration. The role of PKCδ in CDCP1-dependent migration was verified by the PKCδ knockdown experiments, which phenocopy the effect of CDCP1 knockdown; as well as migration rescue experiments, where the impairment of migration caused by CDCP1 downregulation was rescued by the overexpression of constitutively active mutant of PKCδ. Furthermore, we showed a correlation of CDCP1 cell surface expression in primary tumor with poor patient outcome. Finally, we found that suramin, a drug, which showed the clinical benefit for CC-RCC patients at premetastatic stages, causes the downregulation of CDCP1 at the protein level. Thus, the further investigation of the role of CDCP1 protein in kidney cancer metastasis is important to validate CDCP1 as a therapeutic target. 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 LB-357. doi:10.1158/1538-7445.AM2011-LB-357

Objectives: Recently, we discovered isolated cytokeratin 20 (CK20) positive cells in pN0 lymph nodes (LN) from patients with colorectal cancer (CRC) and molecular dissection of this micrometastasis pathway is essential for us to understand which metastasis-related mRNA genes are involved. Using tumor metastasis quantitative polymerase chain reaction (QPCR) arrays, vascular endothelial growth factor (VEGF) mRNA has the highest median fold change in CK20 positive pN0 LN when compared to CK20 negative pN0 LN from patients with CRC (AACR Infection and Cancer abstract number B47). In this study, we validate the QPCR array results by measuring VEGF mRNA concentration in an independent set of pN0 patients with CRC and examine the prognostic significance of VEGF mRNA in the same cohort of patients. Materials and Methods: Twenty-nine paraffin-embedded pN0 LN specimens with CK20 positive cells and the same number of specimens without CK20 positive cells from patients with CRC detected by immunostaining were retrospectively retrieved from the Department of Pathology, Queen Elizabeth Hospital, Hong Kong Special Administrative Region. Total RNA was extracted from each sample for the quantification of VEGF mRNA using primers and a Taqman minor groove binder probe which labeled with a carboxyfluorescein reporter dye at the 5′-end and a nonfluorescent quencher at the 3′-end (Applied Biosystems, Foster City, USA). All 58 pN0 patients with CRC were follow-up for 48 months from their respective diagnosis for recurrent or metastatic CRC and a recurrent/metastatic curve was plotted using the median copy number of VEGF mRNA as the cut-off point. Results: The median concentration of VEGF mRNA in CK20 positive pN0 LN (35710 copy numbers, range: 5189-51240 copy numbers) was significantly higher than those in CK20 negative pN0 LN (1654 copy numbers, range: 210-4450 copy numbers) (p < 0.0001, Mann Whitney test). Using the median VEGF mRNA copy number (35710) of 29 CK20 positive pN0 patients with CRC as the cut-off point to plot the recurrent/metastatic curves, the time to recurrence or metastasis was significantly shorter for 14 patients with VEGF mRNA concentration > 35710 copy numbers (median recurrence/metastasis = 29.5 months) than for 15 patients with VEGF mRNA concentration ≤ 35710 copy numbers (p < 0.05, log-rank test). Conclusions: VEGF mRNA concentration is significantly correlated to the time of recurrence/metastasis and it may be able to predict the risk of recurrence/metastasis in pN0 patients with CRC. 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 853.