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Diagnosis of spindle cell/sarcomatoid melanoma may be challenging due to frequent loss of expression of melanocytic marker(s) and histomorphologic resemblance to various mesenchymal tumours, particularly malignant peripheral nerve sheath tumour (MPNST). Overexpression of PReferentially expressed Antigen in MElanoma (PRAME) supports a diagnosis of melanoma when evaluating challenging melanocytic tumours. PRAME expression in MPNST and other cutaneous sarcomatoid neoplasms, however, has not been well characterised. We aimed to determine the utility of PRAME immunostain in distinguishing spindle cell melanoma from MPNST and other sarcomatoid mimics. PRAME expression was scored by extent (0 to 4+) and intensity (0 to 3) of staining. A strong positive correlation was observed between the extent and intensity scores (r = 0.84). An extent score of 4+, defined by staining in 76-100% of tumour cells, was seen in 56% (23/41) of spindle cell melanomas, 18% (7/38) of MPNSTs, 15% (4/27) of cutaneous sarcomatoid squamous cell carcinomas (SCCs), 33% (5/15) of poorly differentiated cutaneous angiosarcomas, 12% (4/33) of atypical fibroxanthomas (AFXs), 4% (1/25) of pleomorphic dermal sarcomas (PDSs), and none (0/16) of the high-grade cutaneous leiomyosarcomas. A significant difference was found between spindle cell melanoma and all other examined sarcomatoid neoplasms except angiosarcoma. While diffuse (and often strong) PRAME expression is more frequently observed in spindle cell melanoma than MPNST, sarcomatoid SCC, AFX, PDS, and high-grade leiomyosarcoma, its limited sensitivity and specificity caution against its use as a standalone diagnostic marker. PRAME may complement other epigenetic or lineage-specific markers and should only be used as part of an immunohistochemical panel when evaluating these sarcomatoid neoplasms.

Higher doses of olaparib are sufficient to inhibit PAR formation in BRCA1-mutant SUM-149 cells. At higher concentrations of olaparib (1μM), combination treatment of PARP1 inhibition and radiation decreases PAR in a comparable manner to lower doses of olaparib (20nM) in SUM-149 cells.

PDF file - 126K, Supplemental Figure S4. Increase in MEF2c IHC staining in AS and HSA primary tumors.

PDF file - 142K, Supplemental Figure S2. Reduction of phospho-ERK1/2 IHC staining of cutaneous-derived xenograft and cardiac-derived tumorgraft treated with CI-1040.

Angiosarcoma is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. As a model for human angiosarcoma, we studied primary cells and tumorgrafts derived from canine hemangiosarcoma (HSA), which is also an endothelial malignancy with similar presentation and histology. Primary cells isolated from HSA showed constitutive extracellular signal–regulated kinase (ERK) activation. The mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor CI-1040 reduced ERK activation and the viability of primary cells derived from visceral, cutaneous, and cardiac HSA in vitro. HSA-derived primary cells were also sensitive to sorafenib, an inhibitor of B-Raf and multireceptor tyrosine kinases. In vivo, CI-1040 or PD0325901 decreased the growth of cutaneous cell-derived xenografts and cardiac-derived tumorgrafts. Sorafenib decreased tumor size in both in vivo models, although cardiac tumorgrafts were more sensitive. In human angiosarcoma, we noted that 50% of tumors stained positively for phosphorylated ERK1/2 and that the expression of several MEK-responsive transcription factors was upregulated. Our data showed that MEK signaling is essential for the growth of HSA in vitro and in vivo and provided evidence that the same pathways are activated in human angiosarcoma. This indicates that MEK inhibitors may form part of an effective therapeutic strategy for the treatment of canine HSA or human angiosarcoma, and it highlights the use of spontaneous canine cancers as a model of human disease. Mol Cancer Ther; 12(9); 1701–14. ©2013 AACR.

PDF file - 146K, Supplemental Figure S3. Reduction of phospho-ERK1/2 IHC staining of cutaneous-derived xenograft and cardiac-derived tumorgraft treated with sorafenib.

Extended methods for immunohistochemistry. Antibodies used in immunohistochemistry experiments are listed with each of the corresponding dilutions, retrieval techniques, and positive controls.

Sustained locoregional control of disease is a significant issue in patients with inflammatory breast cancer (IBC), with local control rates of 80% or less at 5 years. Given the unsatisfactory outcomes for these patients, there is a clear need for intensification of local therapy, including radiation. Inhibition of the DNA repair protein PARP1 has had little efficacy as a single agent in breast cancer outside of studies restricted to patients with BRCA mutations; however, PARP1 inhibition (PARPi) may lead to the radiosensitization of aggressive tumor types. Thus, this study investigates inhibition of PARP1 as a novel and promising radiosensitization strategy in IBC. In multiple existing IBC models (SUM-149, SUM-190, MDA-IBC-3), PARPi (AZD2281-olaparib and ABT-888-veliparib) had limited single-agent efficacy (IC50 > 10 μmol/L) in proliferation assays. Despite limited single-agent efficacy, submicromolar concentrations of AZD2281 in combination with RT led to significant radiosensitization (rER 1.12–1.76). This effect was partially dependent on BRCA1 mutational status. Radiosensitization was due, at least in part, to delayed resolution of double strand DNA breaks as measured by multiple assays. Using a SUM-190 xenograft model in vivo, the combination of PARPi and RT significantly delays tumor doubling and tripling times compared with PARPi or RT alone with limited toxicity. This study demonstrates that PARPi improves the effectiveness of radiotherapy in IBC models and provides the preclinical rationale for the opening phase II randomized trial of RT ± PARPi in women with IBC (SWOG 1706, NCT03598257).

Supplemental table S1. RNA-seq differentially expressed genes modulated by Wnt3a (Table S1A) or Wnt3a+RSPO2 (Table S1B).

Supplementary Table III: Pharmacokinetics Data of AT13387

Supplementary Figure S1; Supplementary Tables S1-S3.

Supplementary Figures S1-S7; Supplementary Tables S1-S6.

Background: Endocrine therapy (ET) fails to induce a response in one half of patients with hormone receptor (HR)–positive metastatic breast cancer (MBC), and almost all will eventually become refractory to ET. Circulating tumor cells (CTC) are associated with worse prognosis in patients with MBC, but enumeration alone is insufficient to predict the absolute odds of benefit from any therapy, including ET. We developed a multiparameter CTC-Endocrine Therapy Index (CTC-ETI), which we hypothesize may predict resistance to ET in patients with HR-positive MBC.Methods: The CTC-ETI combines enumeration and CTC expression of four markers: estrogen receptor (ER), B-cell lymphoma 2 (BCL-2), Human Epidermal Growth Factor Receptor 2 (HER2), and Ki67. The CellSearch System and reagents were used to capture CTC and measure protein expression by immunofluorescent staining on CTC.Results: The feasibility of determining CTC-ETI was initially established in vitro and then in a prospective single-institution pilot study in patients with MBC. CTC-ETI was successfully determined in 44 of 50 (88%) patients. Eighteen (41%), 9 (20%), and 17 (39%) patients had low, intermediate, and high CTC-ETI scores, respectively. Interobserver concordance of CTC-ETI determination was from 94% to 95% (Kappa statistic, 0.90–0.91). Inter- and cell-to-cell intrapatient heterogeneity of expression of each of the CTC markers was observed. CTC biomarker expression was discordant from both primary and metastatic tissues.Conclusions: CTC expression of ER, BCL-2, HER2, and Ki67 can be reproducibly measured with high analytical validity using the CellSearch System. The clinical implications of CTC-ETI, and of the heterogeneity of CTC biomarker expression, are being evaluated in an ongoing prospective trial. Clin Cancer Res; 21(11); 2487–98. ©2014 AACR.See related commentary by Mathew et al., p. 2421

Supplementary Figures S1-S9. S1: MIR22HG expression in lung cancer. S2: MIR22HG expression in lung cancer cell lines. S3: Changing of cell proliferation, colony formation, migration and invasion after MIR22HG knockdown in lung cancers. S4: Western blot showing the changing of AKT, ERK1/2 and STAT3 proteins after MIR22HG siRNA treatment. S5: MIR22HG interacts with YBX1 protein and the correlation of MIR22HG, MET, YBX1 and p21. S6: P21 plays an oncogenic role in promoting tumor cell proliferation, anti-apoptosis and senescence. S7: Genes negatively correlated to MIR22HG are involved in cell cycle/proliferation regulation in primary lung cancer. S8: MIR22HG, CDKN1A (p21), YBX1 and MET mRNA expression in primary tumors and correlation on different lung RNA-seq datasets. S9: Higher expression of p21 mRNA Is related to poor patient survival and p21 protein is co-expressed with Ki-67 in a subset of tumors.

Supplementary figure 1-11

Purpose:Treatment approaches using Hsp90 inhibitors at their maximum tolerated doses (MTDs) have not produced selective tumor toxicity. Inhibition of Hsp90 activity causes degradation of client proteins including those involved in recognizing and repairing DNA lesions. We hypothesized that if DNA repair proteins were degraded by concentrations of an Hsp90 inhibitor below those required to cause nonspecific cytotoxicity, significant tumor-selective radiosensitization might be achieved.Experimental Design:Tandem mass tagged-mass spectrometry was performed to determine the effect of a subcytotoxic concentration of the Hsp90 inhibitor, AT13387 (onalespib), on global protein abundance. The effect of AT13387 on in vitro radiosensitization was assessed using a clonogenic assay. Pharmacokinetics profiling was performed in mice bearing xenografts. Finally, the effect of low-dose AT13387 on the radiosensitization of three tumor models was assessed.Results:A subcytotoxic concentration of AT13387 reduced levels of DNA repair proteins, without affecting the majority of Hsp90 clients. The pharmacokinetics study using one-third of the MTD showed 40-fold higher levels of AT13387 in tumors compared with plasma. This low dose enhanced Hsp70 expression in peripheral blood mononuclear cells (PBMCs), which is a biomarker of Hsp90 inhibition. Low dose monotherapy was ineffective, but when combined with radiotherapy, produced significant tumor growth inhibition.Conclusions:This study shows that a significant therapeutic ratio can be achieved by a low dose of Hsp90 inhibitor in combination with radiotherapy. Hsp90 inhibition, even at a low dose, can be monitored by measuring Hsp70 expression in PBMCs in human studies.

The long noncoding RNA (lncRNA) MIR22HG has previously been identified as a prognostic marker in hepatocellular carcinoma. Here, we performed a comprehensive analysis of lncRNA expression profiles from RNA-Seq data and report that MIR22HG plays a similar role in lung cancer. Analysis of 918 lung cancer and normal lung tissues and lung cancer cell lines revealed that MIR22HG was significantly downregulated in lung cancer; this decreased expression was associated with poor patient survival. MIR22HG bound and stabilized the YBX1 protein. Silencing of MIR22HG triggered both cell survival and cell death signaling through dysregulation of the oncogenes YBX1, MET, and p21. In this MIR22HG network, p21 played an oncogenic role by promoting cell proliferation and antiapoptosis in lung cancers. MIR22HG played a tumor-suppressive role as indicated by inhibition of multiple cell cycle–related genes in human primary lung tumors. These data show that MIR22HG has potential as a new diagnostic and prognostic marker and as a therapeutic target for lung cancer.Significance: The lncRNA MIR22HG functions as a tumor suppressor, with potential use a diagnostic/prognostic marker and therapeutic target in lung cancer. Cancer Res; 78(12); 3207–19. ©2018 AACR.

PDF file, 2676KB, Impact of MK-5108 on phospho-MPM2 staining in ULMS cell lines.

Ewing sarcomas are characterized by the presence of EWS/ETS fusion genes in the absence of other recurrent genetic alterations and mechanisms of tumor heterogeneity that contribute to disease progression remain unclear. Mutations in the Wnt/β-catenin pathway are rare in Ewing sarcoma but the Wnt pathway modulator LGR5 is often highly expressed, suggesting a potential role for the axis in tumor pathogenesis. We evaluated β-catenin and LGR5 expression in Ewing sarcoma cell lines and tumors and noted marked intra- and inter-tumor heterogeneity. Tumors with evidence of active Wnt/β-catenin signaling were associated with increased incidence of tumor relapse and worse overall survival. Paradoxically, RNA sequencing revealed a marked antagonism of EWS/ETS transcriptional activity in Wnt/β-catenin–activated tumor cells. Consistent with this, Wnt/β-catenin–activated cells displayed a phenotype that was reminiscent of Ewing sarcoma cells with partial EWS/ETS loss of function. Specifically, activation of Wnt/β-catenin induced alterations to the actin cytoskeleton, acquisition of a migratory phenotype, and upregulation of EWS/ETS–repressed genes. Notably, activation of Wnt/β-catenin signaling led to marked induction of tenascin C (TNC), an established promoter of cancer metastasis, and an EWS/ETS–repressed target gene. Loss of TNC function in Ewing sarcoma cells profoundly inhibited their migratory and metastatic potential. Our studies reveal that heterogeneous activation of Wnt/β-catenin signaling in subpopulations of tumor cells contributes to phenotypic heterogeneity and disease progression in Ewing sarcoma. Significantly, this is mediated, at least in part, by inhibition of EWS/ETS fusion protein function that results in derepression of metastasis-associated gene programs. Cancer Res; 76(17); 5040–53. ©2016 AACR.

Addressing drug resistance is a core challenge in cancer research, but the degree of heterogeneity in resistance mechanisms in cancer is unclear. In this study, we conducted next-generation sequencing (NGS) of circulating tumor cells (CTC) from patients with advanced cancer to assess mechanisms of resistance to targeted therapy and reveal opportunities for precision medicine. Comparison of the genomic landscapes of CTCs and tissue metastases is complicated by challenges in comprehensive CTC genomic profiling and paired tissue acquisition, particularly in patients who progress after targeted therapy. Thus, we assessed by NGS somatic mutations and copy number alterations (CNA) in archived CTCs isolated from patients with metastatic breast cancer who were enrolled in concurrent clinical trials that collected and analyzed CTCs and metastatic tissues. In 76 individual and pooled informative CTCs from 12 patients, we observed 85% concordance in at least one or more prioritized somatic mutations and CNA between paired CTCs and tissue metastases. Potentially actionable genomic alterations were identified in tissue but not CTCs, and vice versa. CTC profiling identified diverse intra- and interpatient molecular mechanisms of endocrine therapy resistance, including loss of heterozygosity in individual CTCs. For example, in one patient, we observed CTCs that were either wild type for ESR1 (n = 5/32), harbored the known activating ESR1 p.Y537S mutation (n = 26/32), or harbored a novel ESR1 p.A569S (n = 1/32). ESR1 p.A569S was modestly activating in vitro, consistent with its presence as a minority circulating subclone. Our results demonstrate the feasibility and potential clinical utility of comprehensive profiling of archived fixed CTCs. Tissue and CTC genomic assessment are complementary, and precise combination therapies will likely be required for effective targeting in advanced breast cancer patients.Significance: These findings demonstrate the complementary nature of genomic profiling from paired tissue metastasis and circulating tumor cells from patients with metastatic breast cancer. Cancer Res; 78(4); 1110–22. ©2017 AACR.

PDF file, 1041KB, Impact of combining MK-5108 with chemotherapeutic reagents on cell proliferation in ULMS cell lines.

Purpose: This study was performed to discover prognostic genomic markers associated with postoperative outcome of stage I to III non–small cell lung cancer (NSCLC) that are reproducible between geographically distant and demographically distinct patient populations.Experimental Design: American patients (n = 27) were stratified on the basis of recurrence and microarray profiling of their tumors was performed to derive a training set of 44 genes. A larger Korean patient validation cohort (n = 138) was also stratified by recurrence and screened for these genes. Four reproducible genes were identified and used to construct genomic and clinicogenomic Cox models for both cohorts.Results: Four genomic markers, DBN1 (drebrin 1), CACNB3 (calcium channel beta 3), FLAD1 (PP591; flavin adenine dinucleotide synthetase), and CCND2 (cyclin D2), exhibited highly significant differential expression in recurrent tumors in the training set (P < 0.001). In the validation set, DBN1, FLAD1 (PP591), and CACNB3 were significant by Cox univariate analysis (P ≤ 0.035), whereas only DBN1 was significant by multivariate analysis. Genomic and clinicogenomic models for recurrence-free survival (RFS) were equally effective for risk stratification of stage I to II or I to III patients (all models P < 0.0001). For stage I to II or I to III patients, 5-year RFS of the low- and high-risk patients was approximately 70% versus 30% for both models. The genomic model for overall survival of stage I to III patients was improved by addition of pT and pN stage (P < 0.0013 vs. 0.010).Conclusion: A 4-gene prognostic model incorporating the multivariate marker DBN1 exhibits potential clinical utility for risk stratification of stage I to III NSCLC patients. Clin Cancer Res; 17(9); 2934–46. ©2011 AACR.

Table S1. Antibodies used for CTC and tissue studies. Table S2. Stage 2 stopping rules for the cumulative number of successfully assayed patients. Table S3. A. CTC-Enumeration Points based on CTC Enumeration B. CTC-Bio-Pointsa Footnotes:aOnly aliquots with CTC {greater than or equal to}5/7.5 ml WB evaluated bLow or negative ER expression is weighed more than BCL-2, HER2, and Ki67 due to its fundamental role in endocrine responsiveness. C. Calculation of CTC-ETI Scores. D. CTC-ETI Categories. Footnote: Abbreviations: CTC= circulating tumor cells; ER = estrogen receptor; ET= Endocrine Therapy; MBC = metastatic breast cancer; ET= endocrine therapy. Table S4. CTC-ETI determination in cultured human breast cancer cell lines spiked into normal human blood and evaluated by the CellSearch® system. Table S5. Examples of low, intermediate, and high CTC-ETI from patient samples. A. LOW CTC-ETI Patient # 5 (Primary Tissue: ER 90% positive, HER2 negative by FISH; Metastatic tissue: ER positive, HER2 negative by FISH)* B. INTERMEDIATE CTC-ETI Patient #4 (Primary Tissue: ER {greater than or equal to}95% positive, HER2 0 by IHC; Metastatic tissue: insufficient tissue to evaluate ER and HER2)* C. HIGH CTC-ETI Patient # 3 (Primary Tissue: ER 60% positive, HER2 1+/2+ by IHC; Metastatic tissue: ER 75% positive; HER2 1+ by IHC)* Footnote: *ER and HER2 as per initial clinical laboratory using the laboratory's criteria for positivity or negativity. Table S6.< CTC-ETI for each patient. Footnote: aAF =Analytical Failure (CTCbTF=Technical Failure (Machine failure in at least one of 4 aliquots). Table S7. Concordance between biomarkers in primary and metastatic tissues and CTC. aKappa score for concordance between CTC and tissue biomarker for respective biomarker.

Purpose: LRP1 (low-density lipoprotein receptor–related protein 1) is a broadly expressed receptor that binds multiple extracellular ligands and participates in protein clearance. It is expressed in numerous cancers, but its role in lung cancer has not been characterized. Here, we investigate the relationship between LRP1 and lung cancer.Experimental Design:LRP1 mRNA levels were determined in lung tumors from several large, multicenter studies. LRP1 protein localization was determined by immunohistochemical analysis of lung tumor microarrays. Normal fibroblasts, fibroblasts treated with the LRP1 inhibitor RAP (receptor-associated protein), and Lrp1 null fibroblasts were cocultured with 3 independent lung cancer cell lines to investigate the role of LRP1 on tumor cell proliferation.Results:LRP1 mRNA levels are significantly decreased in lung tumors relative to nontumorous lung tissue. Lower expression of LRP1 in lung adenocarcinomas correlates with less favorable clinical outcome in a cohort of 439 patients. Immunohistochemical analysis shows that LRP1 is primarily expressed in stromal cells in 94/111 lung cancers, with very little protein found in cancer cells. A growth-suppressive function of mouse embryonic fibroblast (MEF) cells was observed in 3 lung cancer cell lines tested (H460, H2347, and HCC4006 cells); growth suppression was blocked by the LRP1 inhibitor RAP. Lrp1 deletion in fibroblasts reduced the ability of MEF cells to suppress tumor cell mitosis. In a validation set of adenocarcinomas, we confirmed a significant, positive correlation between both LRP1 mRNA and protein levels and favorable clinical outcomes.Conclusions:LRP1 expression is associated with improved lung cancer outcomes. Mechanistically, stromal LRP1 may non–cell autonomously suppress lung tumor cell proliferation. Clin Cancer Res; 17(8); 2426–33. ©2011 AACR.

Purpose: Circulating tumor cells (CTC) are prognostic in metastatic breast cancer (MBC). We tested whether EpCAM-based capture system (CellSearch) is effective in patients with triple-negative (TN) MBC, and whether CTC apoptosis and clustering enhances the prognostic role of CTC.Experimental Design: CTC enumeration and apoptosis were determined using the CXC CellSearch kit at baseline and days 15 and 29 in blood drawn from TN MBC patients who participated in a prospective randomized phase II trial of nanoparticle albumin-bound paclitaxel (nab-paclitaxel) with or without tigatuzumab. Association between levels of CTC and patient outcomes was assessed using logistic regression, Kaplan–Meier curves, and Cox proportional hazards modeling.Results: Nineteen of 52 (36.5%), 14 of 52 (26.9%), and 13 of 49 (26.5%) patients who were evaluable had elevated CTC (≥5 CTC/7.5 mL whole blood) at baseline and at days 15 and 29, respectively. Patients with elevated versus not elevated CTC at each time point had worse progression-free survival (PFS; P = 0.005, 0.0003, 0.0002, respectively). The odds of clinical benefit response for those who had elevated versus low CTC at baseline and days 15 and 29 were 0.25 (95% CI: 0.08–0.84; P = 0.024), 0.19 (95% CI: 0.05–0.17; P = 0.014), and 0.06 (95% CI: 0.01–0.33; P = 0.001), respectively. There was no apparent prognostic effect comparing CTC apoptosis versus non-apoptosis. Presence of CTC cluster at day 15 and day 29 was associated with shorter PFS.Conclusions: CTC were detected using CellSearch assay in approximately one-third of TN MBC patients. Elevated CTC at baseline and days 15 and 29 were prognostic, and reductions in CTC levels reflected response. Clin Cancer Res; 21(12); 2771–9. ©2015 AACR.See related article by Forero-Torres et al., p. 2722

Supplementary Figure 1-5. Supplementary Figure 1. Example of cluster image using the CellSearch® system. Supplementary Figure 2. REMARK diagram of the 19 patients who had elevated CTC (Group B/C) at baseline that were used to study "CTC-response". Supplementary Figure 3. PFS for TIG-treated patients versus the control group according to baseline CTC levels: CTC

Detailed methods and reagents lists for lentiviral transduction, AQUA, in situ hybridization and microscopy.

Supplementary Methods about RNAs extraction and quantitative real-time RT-PCR, proteins extraction, immunoblot analysis and tissue array, cell cycle analysis by flow cytometry, and MIR22HG lentiviral expression construct transfection.

Supplementary Methods

Supplementary Figures. Figure S1. Clinical trial study design. Figure S2. Examples of biomarker staining of cultured human breast cancer cell lines after spike and capture from normal human blood using the CellSearch® system. A semi-quantitative scale was established based on relative expression of each biomarker: negative (0), weak (1+), intermediate (2+), and high (3+). Footnote: *hormone depleted condition. Figure S3. Demonstration of appropriate antibody staining of cultured human breast cancer cells with known positive and negative biomarker expression after spike and capture from normal human blood using the CellSearch® System. A: ER staining for MCF-7 and Sk-Br-3. B: BCL- 2 staining for MCF-7 and Sk-Br-3. C: HER2 staining for Sk-Br-3 and MCF-7. D: Ki-67 staining for MCF-7. Figure S4. Composite "CTC-Bio-Score" for different cell lines. A: Hormone depleted MCF-7 cells (Luminal-type). B: MDA-MB-231 (Basal-like type). C: Sk-Br-3 (HER2 positive). Figure S5. Heterogeneity of ER expression within the cultured MCF-7 human breast cancer cell line. Cells were cultured in hormone depleted media for 48 hours, harvested, and spiked into 7.5 ml of normal human blood. The blood was then processed using CellSearch® and stained for DAPI, CK, CD45, and ER. Examples of strong (2+, 3+), weak (1+), and no staining (0) are displayed. Apparent staining in the bottom panel is due to high gain analysis by the automated fluorescent microscopy, as indicated by very high background. Figure S6. Coefficient of variation of CTC-enumeration by mean CTC count. Figure S7. Heterogeneity between primary tissue and CTC for patient #4. The CTC galleries displayed were selected from over 1,000 images to illustrate CTC-ER heterogeneity.

Supplementary Figure S1. Clinical timelines. Clinical timelines and treatment from first diagnosis until enrollment into MI-CTC-ONCOSEQ for the 11 metastatic breast cancer patients; Supplementary Figure S2. Sanger sequencing of WGA CTC DNA and Western blot confirming the presence of A569S mutation; Supplementary Figure S3. MCF-7 (ESR1 A569S) are not estrogen independent over 5 days in hormone-free conditions; Supplementary Figure S4. The tamoxifen metabolites 4-hydroxytamoxifen and endoxifen have no increased agonistic effect in MCF-7 cells over-expressing ER-A569S; Supplementary Figure S5. Inhibition of estradiol-stimulated MCF-7 parental or A569S- overexpressing cells. Supplementary Table S1. Oncomine Comprehensive Panel (OCP) target genes by presence of the MseI restriction site; Table S2. NGS of BT-474 cells spiked into blood and purified identifies expectedTP53 mutation; Supplementary Table S3. Results of CTC purification by DEPArray; Supplementary Table S4: Mean CTC Next Generation Sequencing parameters; Supplementary Table S5. NGS-identified prioritized mutations in individual and pooled CTC samples; Supplementary Table S6. 2 x 2 contingency tables of concordance for alterations in CTC vs tissue; Supplementary Table S7. Patient #2 analysis of primary, clinical metastatic tissue, research biopsy, pt-DNA by ddPCR; Supplementary Table S8. Primer/probe sequences used in Sanger sequencing and ddPCR.

Supplementary Figures S1-S4; Supplementary Tables S1-S4.

Supplementary Data from Molecular Classification and Prognostication of Adrenocortical Tumors by Transcriptome Profiling

Purpose: Uterine leiomyosarcoma (ULMS) is a poorly understood cancer with few effective treatments. This study explores the molecular events involved in ULMS with the goal of developing novel therapeutic strategies.Experimental Design: Genome-wide transcriptional profiling, Western blotting, and real-time PCR were used to compare specimens of myometrium, leiomyoma, and leiomyosarcoma. Aurora A kinase was targeted in cell lines derived from metastatic ULMS using siRNA or MK-5108, a highly specific small-molecule inhibitor. An orthotopic model was used to evaluate the ability of MK-5108 to inhibit ULMS growth in vivo.Results: We found that 26 of 50 gene products most overexpressed in ULMS regulate mitotic centrosome and spindle functions. These include UBE2C, Aurora A and B kinase, TPX2, and Polo-like kinase 1 (PLK1). Targeting Aurora A inhibited proliferation and induced apoptosis in LEIO285, LEIO505, and SK-LMS1, regardless of whether siRNA or MK-5108 was used. In vitro, MK-5108 did not consistently synergize with gemcitabine or docetaxel. Gavage of an orthotopic ULMS model with MK-5108 at 30 or 60 mg/kg decreased the number and size of tumor implants compared with sham-fed controls. Oral MK-5108 also decreased the rate of proliferation, increased intratumoral apoptosis, and increased expression of phospho-histone H3 in ULMS xenografts.Conclusions: Our results show that dysregulated centrosome function and spindle assembly are a robust feature of ULMS that can be targeted to slow its growth both in vitro and in vivo. These observations identify novel directions that can be potentially used to improve clinical outcomes for this disease. Clin Cancer Res; 18(12); 3352–65. ©2012 AACR.

Supplemental table S2. qRT-PCR primer sequences.

PDF file, 673KB, Impact of MK-5108 on the IC50 of gemcitabine or docetaxel in ULMS cell lines.

Purpose: The chemopreventive effects of selenium have been extensively examined, but its role in cancer development or as a chemotherapeutic agent has only recently been explored. Because selenium-binding protein 1 (SELENBP1, SBP1, hSP56) has been shown to bind selenium covalently and selenium deficiency has been associated with esophageal adenocarcinoma (EAC), we examined its role in EAC development and its potential effect on chemosensitivity in the presence of selenium.Experimental Design: SELENBP1 expression level and copy number variation were determined by oligonucleotide microarrays, real-time reverse transcription-PCR, tissue microarrays, immunoblotting, and single-nucleotide polymorphism arrays. Bisulfite sequencing and sequence analysis of reverse transcription-PCR–amplified products explored epigenetic and posttranscriptional regulation of SELENBP1 expression, respectively. WST-1 cell proliferation assays, senescence-associated β-galactosidase staining, immunoblotting, and flow cytometry were done to evaluate the biological significance of SELENBP1 overexpression in selenium-supplemented EAC cells.Results: SELENBP1 expression decreased significantly in Barrett's esophagus to adenocarcinoma progression. Both epigenetic and posttranscriptional mechanisms seemed to modulate SELENBP1 expression. Stable overexpression of SELENBP1 in methylseleninic acid–supplemented Flo-1 cells resulted in enhanced apoptosis, increased cellular senescence, and enhanced cisplatin cytotoxicity. Although inorganic sodium selenite similarly enhanced cisplatin cytotoxicity, these two forms of selenium elicited different cellular responses.Conclusions: SELENBP1 expression may be an important predictor of response to chemoprevention or chemosensitization with certain forms of selenium in esophageal tissues. Clin Cancer Res; 16(7); 2009–21. ©2010 AACR.

Supplementary Tables S1-S3. S1: MIR22HG level and clinical variables from validation set of 101 LUADs measured by qRT-PCR. S2: genomic alterations of lung cancer cell lines used in this study. S3: Primer and siRNA sequences, as well as antibodies used in this study.

Purpose: Our understanding of adrenocortical carcinoma (ACC) has improved considerably, yet many unanswered questions remain. For instance, can molecular subtypes of ACC be identified? If so, what is their underlying pathogenetic basis and do they possess clinical significance?Experimental Design: We did a whole genome gene expression study of a large cohort of adrenocortical tissues annotated with clinicopathologic data. Using Affymetrix Human Genome U133 Plus 2.0 oligonucleotide arrays, transcriptional profiles were generated for 10 normal adrenal cortices (NC), 22 adrenocortical adenomas (ACA), and 33 ACCs.Results: The overall classification of adrenocortical tumors was recapitulated using principal component analysis of the entire data set. The NC and ACA cohorts showed little intragroup variation, whereas the ACC cohort revealed much greater variation in gene expression. A robust list of 2,875 differentially expressed genes in ACC compared with both NC and ACA was generated and used in functional enrichment analysis to find pathways and attributes of biological significance. Cluster analysis of the ACCs revealed two subtypes that reflected tumor proliferation, as measured by mitotic counts and cell cycle genes. Kaplan-Meier analysis of these ACC clusters showed a significant difference in survival (P < 0.020). Multivariate Cox modeling using stage, mitotic rate, and gene expression data as measured by the first principal component for ACC samples showed that gene expression data contains significant independent prognostic information (P < 0.017).Conclusions: This study lays the foundation for the molecular classification and prognostication of adrenocortical tumors and also provides a rich source of potential diagnostic and prognostic markers.

Supplementary Data from Decreased Selenium-Binding Protein 1 in Esophageal Adenocarcinoma Results from Posttranscriptional and Epigenetic Regulation and Affects Chemosensitivity