Mixture Of Tumor Cells Research Articles

Abstract 1881: Drug combination screening of Ipatasertib and Abemaciclib with other targeted agents in complex multicellular tumor spheroids from the NCI-60 and the National Cancer Institute’s Patient-Derived Models Repository

Abstract Drug combinations are frequently used to improve clinical efficacy, to minimize toxicity, and to reduce the development of drug resistance. Here, we investigated the growth inhibitory activities of the pan-Akt inhibitor ipatasertib and the CDK4/6 inhibitor abemaciclib in combination with other targeted agents. Twelve well-characterized patient-derived cancer cell lines from the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov/models/database.htm) and seven established cell lines from the NCI-60 tumor cell line panel (https://dtp.cancer.gov/discovery_development/nci-60/cell_list.htm) were grown as multicellular 3D complex spheroids. The complex spheroids, a mixture of tumor cells (60%), endothelial cells (25%), and mesenchymal stem cells (15%), were grown for 3 days before drug(s) were added. All agents were tested both alone and in combinations at multiple concentrations up to their reported clinical Cmax value and cell viability was assayed using CellTiter-Glo 3D seven days after drug exposure. While abemaciclib had minimal activity as a single agent, ipatasertib was noticeably selective for tumor cells harboring activating PI3K/AKT/mTOR pathway variants. Dual inhibition of the PI3K/AKT/mTOR and RAS/MEK/ERK pathways was one of the most effective combinations. For example, the combination of ipatasertib with the MEK inhibitor selumetinib or the ERK inhibitor ravoxertinib resulted in additive and/or synergistic cytotoxicity in over half the complex spheroid models screened. The V600E variant-specific BRAF inhibitor vemurafenib and the KRAS G12C selective inhibitor sotorasib in combination with ipatasertib showed activity in the one BRAF V600E and two KRAS G12C variant containing complex spheroid models, respectively. Another effective ipatasertib combination was vertical inhibition of the PI3K/AKT/mTOR pathway with the mTORC1/2 kinase inhibitor sapanisertib, which demonstrated additive and/or synergistic responses across multiple complex spheroid models. For abemaciclib, the most successful combination was with the CDK2/7/9 inhibitor BMS-387032, which achieved greater than one log of cytotoxicity in the majority of the complex spheroid models. For the combination of abemaciclib and selumetinib, there was a high correlation between the responses of two patient-derived cell lines grown as complex spheroids and their corresponding patient-derived xenografts (PDX). Additional PDX studies are planned for promising drug combinations from this in vitro screen. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas Steven Dexheimer, Thomas Silvers, Rene Delosh, Julie Laudeman, Russell Reinhart, Chad Ogle, Siddhartha Paul, Nathan P. Coussens, Ralph E. Parchment, Joel Morris, John Wright, Naoko Takebe, Beverly A. Teicher, James H. Doroshow. Drug combination screening of Ipatasertib and Abemaciclib with other targeted agents in complex multicellular tumor spheroids from the NCI-60 and the National Cancer Institute’s Patient-Derived Models Repository [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1881.

Opportunity of Next-Generation Sequencing-Based Short Tandem Repeat System for Tumor Source Identification.

Personal identification using the tumor DNA not only plays an important role in postoperative tissue management but also might be the only accessible source of biological material in forensic identification. Short tandem repeat (STR) is the worldwide accepted forensic marker; however, widespread loss of heterozygosity (L) in tumor tissues challenges the personal identification using the conventional capillary electrophoresis (CE)-based STR typing system (CE-STR). Because the tumors are mixtures of tumor cells and basal cells, we inferred that every germline-originated allele should be detected if the detection method was sensitive enough. Next-generation sequencing (NGS) is known as a highly sensitive application, which might be a promising tool for tumor source identification. In the study, we genotyped and compared the STR results between the platforms, and we found that the concordance was only 91.43%. Higher sensitivity did help identify more germline-originated alleles as expected, and 93.89% of them could be captured by using an NGS-based STR system (NGS-STR). The identity-by-state (IBS) scoring system was applied to generate a new tumor source identification method based on NGS-STR, and the number of loci with 2 identical alleles (A2) proved to be an ideal criterion for the larger area under the receiver operating characteristic (ROC) curve (AUC). Both the sensitivity and specificity were above 98% in the cutoff of A2 to distinguish the paired carcinoma (PC) sample group from the unrelated individual (UI) group, the simulated full sibling (FS) group, and the simulated parent–offspring (PO) group.

Abstract 1028: Combination screening of KRASG12C specific inhibitors with other targeted therapies in patient-derived multicellular tumor spheroids

Abstract KRAS is one of the most frequently mutated oncogenes in cancer and efforts to directly target KRAS have been challenging. However, covalent inhibitors targeting a specific KRASG12C (Gly12 to Cys12) variant have recently been developed and have demonstrated encouraging clinical activity in patients with tumors driven by the mutant protein. Drug combinations are used to improve clinical efficacy, to minimize toxicity, and to reduce the development of resistance. Here, we investigate the growth inhibitory activity of two KRASG12C variant-specific inhibitors, AMG-510 and MRTX-1257, in combination with other targeted therapies. Twenty patient-derived cancer cell lines, which have been well-characterized for their KRAS status and related targets and are available from NCI, were grown as multicellular 3D complex spheroids (https://pdmr.cancer.gov/models/database.htm). The complex spheroids, a mixture of tumor cells, endothelial cells and mesenchymal stem cells, were established for 3 days before drug(s) were added. All agents were tested at concentrations up to their reported clinical Cmax values and cell viability for each individual drug treatment and drug combination was assayed using CellTiter-Glo 3D seven days after drug exposure. As single agents, AMG-510 and MRTX-1257 showed selectivity for the KRASG12C variant containing cancer cell lines. The pancreatic cancer cell line 323965-272-R-J2 carrying a KRASG12C variant exhibited an enhanced sensitivity to the combination of the mTOR inhibitor everolimus and a low concentration of either AMG-510 or MRTX-1257. Additive to greater than additive cytotoxicity for the combination of everolimus and the KRASG12C inhibitors was also observed in the KRASG12C variant melanoma cell line 299254-011-R-J1, while no effect was detected in cell lines with wildtype or other KRAS mutations. Similarly, combining the mTOR kinase inhibitor sapanisertib, which blocks both mTORC1 and mTORC2 activities, with MTRX-1257 also led to a notable reduction in cell viability in all three KRASG12C-containing cell lines. In the KRASG12C variant melanoma cell line 299254-011-R-J1, targeting of the upstream receptor tyrosine kinase FGFR with erdafitinib in combination with the KRASG12C inhibitors produced greater than additive cell killing over the concentration range from 0.1 to 10 µM. Lastly, both KRASG12C inhibitors and abemaciclib, demonstrated additive to greater than additive cytotoxicity in the pancreatic cancer cell line 323965-272-R-J2, but this outcome was not observed with the CDK4/6 inhibitor, palbociclib, or in the other KRASG12C variant cell lines. These preclinical findings provide guidance regarding selection of combination regimens with KRASG12C inhibitors that may result in increased clinical efficacy. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261200800001E. Citation Format: Thomas S. Dexheimer, Thomas Silvers, René Delosh, Julie Laudeman, Russell Reinhart, Chad Ogle, Nathan P. Coussens, John Wright, Beverly A. Teicher, James H. Doroshow. Combination screening of KRASG12C specific inhibitors with other targeted therapies in patient-derived multicellular tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1028.

Recent advances in precision medicine for pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer mortality worldwide. Although advances in systemic chemotherapy for PDAC have improved survival outcomes for patients with the disease, chemoresistance is a major treatment issue for unselected PDAC patient populations. The existence of heterogeneity caused by a mixture of tumor cells and stromal cells produces chemoresistance and limits the targeted therapy of PDAC. Advances in precision medicine for PDACs according to the genetics and molecular biology of this disease may represent the next alternative approach to overcome the heterogeneity of different patients and improve survival outcomes for this poor prognostic disease. The genetic alteration of PDAC is characterized by four genes that are frequently mutated (KRAS, TP53, CDKN2A, and SMAD4). Furthermore, several genetic and molecular profiling studies have revealed that up to 25% of PDACs harbor actionable alterations. In particular, DNA repair dysfunction, including cases with BRCA mutations, is a causal element of sensitivity to platinum‐based anti‐cancer agents and poly‐ADP ribose polymerase (PARP) inhibitors. A deep understanding of the molecular and cellular crosstalk in the tumor microenvironment helps to establish scientifically rational treatment strategies for cancers that show specific molecular profiles. Here, we review recent advances in genetic analysis of PDACs and describe future perspectives in precision medicine according to molecular subtypes or actionable gene mutations for patients with PDAC. We believe the breakthroughs will soon emerge to fight this deadly disease.

A three phase model to investigate the effects of dead material on the growth of avascular tumours

In vivo tumours are highly heterogeneous entities which often comprise intratumoural regions of hypoxia and widespread necrosis. In this paper, we develop a new three phase model of nutrient-limited, avascular tumour growth to investigate how dead material within the tumour may influence the tumour’s growth dynamics. We model the tumour as a mixture of tumour cells, dead cellular material and extracellular fluid. The model equations are derived using mass and momentum balances for each phase along with appropriate constitutive equations. The tumour cells are viewed as a viscous fluid pressure, while the extracellular fluid phase is viewed as inviscid. The physical properties of the dead material are intermediate between those of the tumour cells and extracellular fluid, and are characterised by three key parameters. Through numerical simulation of the model equations, we reproduce spatial structures and dynamics typical of those associated with the growth of avascular tumour spheroids. We also characterise novel, non-monotonic behaviours which are driven by the internal dynamics of the dead material within the tumour. Investigations of the parameter sub-space describing the properties of the dead material reveal that the way in which non-viable tumour cells are modelled may significantly influence the qualitative tumour growth dynamics.

RF_Purify: a novel tool for comprehensive analysis of tumor-purity in methylation array data based on random forest regression

BackgroundWith the advent of array-based techniques to measure methylation levels in primary tumor samples, systematic investigations of methylomes have widely been performed on a large number of tumor entities. Most of these approaches are not based on measuring individual cell methylation but rather the bulk tumor sample DNA, which contains a mixture of tumor cells, infiltrating immune cells and other stromal components. This raises questions about the purity of a certain tumor sample, given the varying degrees of stromal infiltration in different entities. Previous methods to infer tumor purity require or are based on the use of matching control samples which are rarely available. Here we present a novel, reference free method to quantify tumor purity, based on two Random Forest classifiers, which were trained on ABSOLUTE as well as ESTIMATE purity values from TCGA tumor samples. We subsequently apply this method to a previously published, large dataset of brain tumors, proving that these models perform well in datasets that have not been characterized with respect to tumor purity .ResultsUsing two gold standard methods to infer purity – the ABSOLUTE score based on whole genome sequencing data and the ESTIMATE score based on gene expression data- we have optimized Random Forest classifiers to predict tumor purity in entities that were contained in the TCGA project. We validated these classifiers using an independent test data set and cross-compared it to other methods which have been applied to the TCGA datasets (such as ESTIMATE and LUMP).Using Illumina methylation array data of brain tumor entities (as published in Capper et al. (Nature 555:469-474,2018)) we applied this model to estimate tumor purity and find that subgroups of brain tumors display substantial differences in tumor purity.ConclusionsRandom forest- based tumor purity prediction is a well suited tool to extrapolate gold standard measures of purity to novel methylation array datasets. In contrast to other available methylation based tumor purity estimation methods, our classifiers do not need a priori knowledge about the tumor entity or matching control tissue to predict tumor purity.

The effect of ALA-PDT on reversing the activation of cancer-associated fibroblasts in cutaneous squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) are important components of the tumor microenvironment, affecting the biological behavior of tumor cells and playing critical roles in tumor growth, invasion, and metastasis. Topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is an established approach for the treatment of non-melanoma skin cancers. It is reported that ALA-PDT treating cutaneous squamous cell carcinoma(cSCC) also induce antitumor immune effect and has an impact on tumor microenvironment. However, the effect of ALA-PDT on CAFs is not well known. In this study, the data showed that the expression of α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP), as well as migratory ability, were elevated in 3T3 fibroblasts co-cultured with tumor cells. Western blot, qRT-PCR and transwell cell migration assay were performed to detect these findings. In vivo, the rate of tumor growth in mice injected with a mixture of tumor cells and 3T3 fibroblasts was higher than that in mice injected with tumor cells only. Furthermore, both in co-cultured 3T3 fibroblasts and CAFs, a reduction in the expression of a-SMA and FAP was observed after ALA-PDT. Same with migratory ability. The findings indicated an inhibitory effect of ALA-PDT on the activation of CAFs in cSCC.

Abstract 3784: Intraoperative changes of low density granulocyte (LDG) in gastrointestinal cancer surgery

Abstract Background: Low density granulocytes (LDG) have been shown to be increased in peripheral blood of autoimmune disease, severe sepsis and cancer. In this study, we examined the frequency and function of the LDG in patients who received abdominal surgery for gastrointestinal malignancy. Method: In 57 patients who received laparotomy due to the gastrointestinal cancer ( 5 esophageal, 25 gastric , 27 colorectal cancer), peripheral blood was collected before and after operative procedure in operation room. After Ficoll-Hypaque centrifugation, intermediate layer which consists mainly of mononuclear cells was collected and immunestained with mAbs to CD66b, CD45 as well as 7AAD and their expressions were examined with flow cytometer. CD66b(+) CD45(+) cells were defined as LDG and the ratios of LDG in total CD45(+) leukocytes were calculated in 7AAD(-) live cell fraction. Next, CD66b(+) LDG and CD66b(-) mononuclear cells were separated with MACS method, and LDG were cultured and examined for extracellular traps (NETs) formation in vitro. Finally, CD66b(-) mononuclear cells were cultured on anti-CD3 mAb coated plate with or without autologous LDG, and proliferation of T lymphocytes was examined with CFSE dilution method. Results: The ratio of the LDG is low (median= 1.14%, 0.62 ~1.66%, n=57) at a beginning of surgery. In contrast, the ratio was markedly increased (median= 8.10%, 5.91~10.3%, n=57, p<0.001) at the end of surgery. The ratio of postoperative LDG showed significant correlation with operation time and positive trend with intraoperative bleeding. The LDG showed relatively week expression of CD11b and CD66b, but highly expressed in IL8 receptor and CD62L, suggesting immature LDG. The short-term culture of the LDG produced massive SYTOX-positive web like stricture which was characteristic for NETs. The mixture of tumor cells on the LDG culture demonstrated that many tumor cells were trapped in the NETs within 5 min incubation and vigorously grew thereafter. Finally, addition of the LDG on mononuclear cell culture strongly suppressed the anti-CD3 stimulated T cell division. Conclusion: LDG with immature phenotype increase in circulating blood of the patients who underwent gastrointestinal cancer surgery, probably due to the surgical stress. The LDG produce NETs which may efficiently trap circulating tumor cells, and can inhibit T lymphocyte mediated-immune reaction. Thus, the LDG provide tumor permissive microenvironment in target organ and have supportive roles on the development of postoperative hematogenous metastasis in gastrointestinal cancer. Citation Format: Yuko Kumagai. Intraoperative changes of low density granulocyte (LDG) in gastrointestinal cancer surgery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3784.

Quantification of intratumoral heterogeneity of HER2 status in breast cancer.

1036 Background: Intratumoral heterogeneity (ITH) occurs as a consequence of epigenetic aberrations in tumor cells with genetic diversity. HER2 ITH can be classified into genetic (a mixture of tumor cells with and without HER2 gene amplification) and epigenetic ITH (a mixture of HER2 gene-amplified tumor cells with and without HER2 protein overexpression). However, the both effects of genetic and epigenetic ITH on HER2-targeted therapy have not been clearly demonstrated. In order to implement ITH as a referenced factor for treatment selection, the ITH quantification is necessary. Gene-protein assay (GPA), in which immunohistochemistry and dual in situ hybridization are simultaneously performed on a single slide, allows bright-field analyses of both gene and protein status. We aimed to quantify HER2 ITH by the combination of gene and protein status and clarify its clinical significance. Methods: Fifty three patients with HER2-positive breast cancer, who underwent neoadjuvant trastuzumab with chemotherapy, were examined. Five representative microscopic images were captured from a GPA slide of a pre-therapeutic biopsy material. All evaluable tumor cells in the images were scored according to the HER2 status determined by the combination of gene copy number and protein expression (Table). We investigated the relationship between the HER2 scores and pathological complete response (pCR) to the neoadjuvant treatment by the logistic analysis. Results: The average of HER2 scores, indicating the degree of the HER2 status, varied from 2.21 to 5.98. It was significantly related to pCR (Estimate: 1.21, Std. error: 0.46, RR: 3.34, P=0.009, 95%CI: 1.35-8.25). The standard deviation of HER2 scores, indicating the degree of the HER2 ITH, varied from 0.13 to 1.37. It was significantly related to pCR (Estimate: -2.09, Std. error: 0.83, RR: 0.12, P=0.012, 95%CI: 0.02-0.63). Conclusions: HER2 ITH, quantified by GPA, is a predictive factor for the therapeutic effect to trastuzumab-based treatment in HER2-positive breast cancer. [Table: see text]

Abstract 3298: Distinct microbiome populations within breast cancer microenvironments

Abstract INTRODUCTION: The tumor microenvironment consists of a complicated interplay between a heterogeneous mixture of tumor cells and the surrounding tissue and there is a burgeoning interest in the role of the tissue microbiota within this microenvironment. Our group has investigated the breast tumor microbiome using the unaligned (non-human) reads from 796 RNASequencing data obtained from The Cancer Genome Atlas (TCGA) repository of Breast Cancer. METHODS: Unmapped sequences for 720 TCGA tumor samples and 76 paired normal samples were analyzed with Kraken v0.10.5 using the RefSeq 16S and standard genomes. To minimize false discoveries Kraken results were filtered to species that have bacterial genomic and 16S read support. Cluster analysis of the T-distributed Stochastic Neighbor Embedding (tsne) was performed and consensus voting of 26 maximum scoring metrics identified 3 distinct clusters. Differential abundance analysis was performed with the edgeR package. Covariate association analysis was performed for tumor clinical characteristics, as well as sample heterogeneity characteristics. RESULTS: There were three microbiome clusters we identified using TCGA breast samples. The average silhouette width of the third cluster was deemed statistically insignificant (0.071), suggesting further stratification of this cluster is necessary. The remaining two TCGA clusters (467) shared an average silhouette width of 0.39, using Bray's dissimilarity index. Differential abundance analysis of the two clusters after Bonferroni correction identified 450 significant species. The biomes were represented by 63 triple negative, 93 HER2+, 258 Luminal, and 50 tumor adjacent normals. Concordant cluster assignment was observed in 93.7 +/- 7.3% of the normal-tumor pairings, averaged across clinical subtypes. We observed clear delineation of biome populations at the order taxonomic level, which was not associated with the clinical subtype or the tumor/tumor adjacent pairings. We found enrichment of Burkholderiales in the first biome and enrichment of Bacillales and Lactobacillales within the second biome. The second biome appears to be deplete of representatives from the Asian population (p value 0.005) and survival associations were observed to be significantly reduced for HER2 patients associated with Biome 1 (p value 0.025). Geneset enrichment analysis of the expression signatures indicated drug metabolism and chemical carcinogenesis pathways to be associated with biome 2. CONCLUSIONS: In our study, we observed two significantly different microbiomes among TCGA breast cancer samples that were not associated with the clinical subtypes. Current efforts include validating these biomes, using 16S sequencing on matching fresh frozen paraffin embedded samples. Additionally, we are evaluating the influence of the biomes in independent adjuvant and neo-adjuvant studies with next generation sequencing data. Citation Format: Kevin J. Thompson, Jason Sinnwell, Xiaojia Tang, James N. Ingle, Matthew P. Goetz, Krishna R. Kalari. Distinct microbiome populations within breast cancer microenvironments. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3298.

P114: Intratumor heterogeneity of driver mutations in lung adenocarcinoma

Background Lung cancers are characterized by the genetic alterations that often affect a common group of oncogenic signaling pathways. Identification of biologically significant genetic alterations in lung cancer that lead to activation of oncogenes and inactivation of tumor suppressor genes has the potential to provide further therapeutic opportunities (Cooper, 2013). In this context, it is important that molecular aberrations may act as negative predictors of sensitivity to treatment. The discovery of driver oncogenes, such as activating mutations in the epidermal growth factor receptor gene (EGFR), has made personalized medicine for lung cancer a reality. Despite the high initial results, NSCLC patients acquire resistance to tyrosine kinase inhibitors in the worldwide. There are several findings suggesting that the effect of tyrosine kinase inhibitors could be limited to patients harboring KRAS mutation. To date, a limited information regarding link of morphological portrait of adenocarcinomas with bearing of simultaneously concurrent mutations of KRAS and EGFR genes. The detection of concurrent gene mutation is usually carried out in a mixture of tumor cells. We plan to analyze EGFR and KRAS mutations in all clonal components (or morphological structures) of NSCLC tumors isolated by laser microdissection. So, we suggest that NSCLC clonal components might bear two concurrent EGFR and KRAS mutations simultaneously or different clonal components might bear either EGFR or KRAS mutations. The data obtained can predict resistance to EGFR-TKIs and affect therapeutic decision making. Materials and methods The tumor samples were obtained from archived formalin-fixed paraffin-embedded tissue blocks. Paraffin was removed by xylene extraction, and the sample was subsequently lysed by proteinase K. The region containing the highest percentage of tumor cells (at least 50%) was dissected. Microscopes Axio Scope A1 and Axio Star plus (Carl Zeiss, Germany) was used to perform histological analysis. If necessary, tumour cells were carefully selected and removed from the samples by laser microdissection using a P.A.L.M. microlaser instrument (PALM AdhesiveCaps, P.A.L.M., Bernried, Germany). EGFR (exon 19 deletion and 21 L858R) and KRAS (Gly12Cys, Gly12Ser, Gly12Arg, Gly12Val, Gly12Asp, Gly12Ala, Gly13Asp) gene amplification was based on RT-PCR on CFX96 Real-Time System (Bio-Rad). Results A total of 115 patients with stage (IIIB–IV) of NSCLC with EGFR-TKI- sensitive mutations were eligible for the analysis. A rare coexistence of KRAS and EGFR mutations were observed in 3 patients (2.6%). The first sample was described as nonmucinous solid adenocarcinoma and has deletion of 19 exon of EGFR gene and G12C mutation of KRAS gene. The second sample was described as mucinous lepidic adenocarcinoma and has deletion of 19 exon of EGFR gene and G12D mutation of KRAS gene. The third sample was described as mucinous adenocarcinoma with solid and acinar monoclonal components and also has deletion of 19 exon of EGFR gene and G12C mutation of KRAS gene. Of all the samples, only one had two different monoclonal components. Unfortunately, this sample was not available due to biopsy and few numbers of cells for microdissection. Fiala et al. (2013) reported that KRAS mutations were not only as a negative prognostic factor and also as a biomarker predicting resistance to EGFR-TKIs, especially G12C. Patient with nonmucinous solid adenocarcinoma with deletion of 19 exon and G12C (EGFR and KRAS genes, respectively) is unlikely to benefit from receiving gefitinib or erlotinib. Conclusion The data obtained show that further studies are required to improve personalized therapy for NSCLC patients. The frequency and type of KRAS and EGFR mutations were first assessed in NSCLC patients from West Siberia. This work was supported by a Grant from the OPTEK Company (No 122/2014/51/Nvs ).

Abstract POSTER-TECH-1119: Phylogenetic portraits of high grade serous ovarian cancer

Abstract High Grade Serous Ovarian Cancer (HGSOvCa) is characterized by genomic instability resulting from deficient DNA repair processes primarily due to TP53 mutation and BRCA abnormalities. Genomic instability has been shown to result in significant intratumoral heterogeneity, with temporally and spatially separated tumours exhibiting unique mutational profiles. A key unanswered question is whether genomic instability produces functionally relevant driver mutations, or whether the tumours complement of driver mutations predates the emergence of instability. We sought to reconstruct the evolutionary histories of the tumours of 8 HGSOvCa patients using spatial and temporal profiling of somatic mutations. For each patient, we collected multiple tumour biopsies resected from primary and metastatic sites at the time of surgery. We then used whole genome shotgun sequencing to identify somatic point mutation, rearrangement and copy number changes present at each site. We used novel computational techniques to unmix the signal produced by sequencing of a mixture of tumour cells, and infer the genomic architecture of clonal populations in each tumour sample. Additionally, we used phylogenetic inference techniques to reconstruct the ancestral relationships between the genomically distinct clones identified in each patient. We classified mutations as occurring ancestrally (before clonal divergence) or occurring on a descendent lineage. We identified multiple well known driver mutations occurring in descendent lineages of the tumour phylogeny, including mutations in CDKN2A, BCL6 and ERBB2. Aberrant site specific expression patterns were concomitant with genomic changes. Furthermore, we identified instances of convergent evolution, including independent deletion of wild type RAD51B in a patient with a germline RAD51B mutation. Reconstruction of clonal phylogenies enabled inference of metastatic spread. In one patient, a minor subclone of a right ovary tumour acquired an ERBB2 amplification, then subsequently populated all other metastatic sites sampled by the experiment. Finally, from our analyses we concluded that in many HGSOvCa, genomic instability and associated copy number changes result in the deletion of a significant number of point mutations, with implications for phylogenetic reconstruction using traditional techniques. Citation Format: Andrew McPherson, Andrew Roth, David Huntsman, Jessica McAlpine, Sohrab Shah. Phylogenetic portraits of high grade serous ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1119.

Nanowire substrate-based laser scanning cytometry for quantitation of circulating tumor cells.

We report on the development of a nanowire substrate-enabled laser scanning imaging cytometry for rare cell analysis in order to achieve quantitative, automated, and functional evaluation of circulating tumor cells. Immuno-functionalized nanowire arrays have been demonstrated as a superior material to capture rare cells from heterogeneous cell populations. The laser scanning cytometry method enables large-area, automated quantitation of captured cells and rapid evaluation of functional cellular parameters (e.g., size, shape, and signaling protein) at the single-cell level. This integrated platform was first tested for capture and quantitation of human lung carcinoma cells from a mixture of tumor cells and leukocytes. We further applied it to the analysis of rare tumor cells spiked in fresh human whole blood (several cells per mL) that emulate metastatic cancer patient blood and demonstrated the potential of this technology for analyzing circulating tumor cells in the clinical settings. Using a high-content image analysis algorithm, cellular morphometric parameters and fluorescence intensities can be rapidly quantitated in an automated, unbiased, and standardized manner. Together, this approach enables informative characterization of captured cells in situ and potentially allows for subclassification of circulating tumor cells, a key step toward the identification of true metastasis-initiating cells. Thus, this nanoenabled platform holds great potential for studying the biology of rare tumor cells and for differential diagnosis of cancer progression and metastasis.

In Vivo Evolution of Tumor-Derived Endothelial Cells

The growth of a malignant tumor beyond a certain, limited size requires that it first develop an independent blood supply. In addition to providing metabolic support, this neovasculature also allows tumor cells to access the systemic circulation, thus facilitating metastatic dissemination. The neovasculature may originate either from normal blood vessels in close physical proximity to the tumor and/or from the recruitment of bone marrow-derived endothelial cell (EC) precursors. Recent studies have shown that human tumor vasculature ECs may also arise directly from tumor cells themselves and that the two populations have highly similar or identical karyotypes. We now show that, during the course of serial in vivo passage, these tumor-derived ECs (TDECs) progressively acquire more pronounced EC-like properties. These include higher-level expression of EC-specific genes and proteins, a greater capacity for EC-like behavior in vitro, and a markedly enhanced propensity to incorporate into the tumor vasculature. In addition, both vessel density and size are significantly increased in neoplasms derived from mixtures of tumor cells and serially passaged TDECs. A comparison of early- and late-passage TDECs using whole-genome single nucleotide polymorphism profiling showed the latter cells to have apparently evolved by a process of clonal expansion of a population with a distinct pattern of interstitial chromosomal gains and losses affecting a relatively small number of genes. The majority of these have established roles in vascular development, tumor suppression or epithelial-mesenchymal transition. These studies provide direct evidence that TDECs have a strong evolutionary capacity as a result of their inherent genomic instability. Consequently such cells might be capable of escaping anti-angiogenic cancer therapies by generating resistant populations.

Hemostatic gelatin sponge is a superior matrix to matrigel for establishment of LNCaP human prostate cancer in nude mice

Matrigels, solubilized basement membrane preparations, are often used to support tumor development in animal models. However, tumors formed by a mixture of tumor cells and Matrigel may vary significantly. The purpose of this study was to compare tumor development and growth of LNCaP human prostate cancer cells mixed with Matrigel or in gelatin sponges. LNCaP cells were mixed with Matrigel or absorbed into VETSPON, a gelatin sponge, and inoculated into the subcutis of nude mice. Tumor incidence and growth rate were determined. Gene expression and cell growth and survival in tumor lesions were evaluated by immunohistochemistry (IHC), immunoblotting, and RT-PCR. All mice (12/12) inoculated with LNCaP cells in VETSPON produced tumors, compared to 70% (19/27) of mice injected with the cells with Matrigel. Tumor volume also varied less with VETSPON implants. No significant differences were observed in gene expression, cell growth, apoptosis, and microvessel density in tumors established from the two types of implants. However, in samples collected on days 1 and 4, more cells in Matrigel implants than those in VETSPON implants were stained positive for cleaved-caspase 3 and -PARP1. Expression of VEGF-A, HIF-1α, and Bcl-2 was elevated in the early VETSPON implants. These data indicate that VETSPON promotes tumor cell survival at the early stage of implantation and suggest that the gelatin sponge is superior to Matrigel in supporting development and progression of human prostate cancer in nude mice. This model should be useful for preclinical studies in nude mice using LNCaP cells.

The basic concept of the tumor stem cell model

Both theoretical and experimental evidence support the concept of a tumor stem cell (TSC) model - a rare population of self-renewing stem cells among the heterogeneous mixture of tumor cells essential for solid tumor growth and progression. However, this growing evidence is not by itself convincing without an adequate molecular mechanism explaining how these TSCs maintain their stemness, since there is always a possibility that the so-called solid tumor TSCs may be a highly tumorigenic fraction of the heterogeneous mixture of tumor cells. The lack of knowledge of how TSCs maintain their state of stemness is therefore a major limitation that requires attention in order to improve our understanding on the stem cell model of tumor growth. An emerging and provocative concept in tumor biology is that a rare population of tumor stem cells exists among the heterogeneous population of cells within tumors. The TSC model suggests that proliferative potential and growth patterns of many human tumors may depend upon a small proportion of tumor stem cells that lead to repopulation following cytotoxic therapy. In this review, we will briefly discuss the basic concept of the TSC model and the emerging findings of the existence of the TSC fraction, even in established tumor cell lines.

Abstract 2182: Differential transfection on a cell chip for high throughput analysis of paracrine gene effects in angiogenesis and tumor invasion

Abstract Introduction: Tumor invasion and angiogenesis are key processes in tumor progression. Previously, transcriptome analyses identified numerous genes which are associated with tumor progression. The role of these genes in tumor invasion and angiogenesis is mostly unknown. Here we describe for the first time a cell chip-based high throughput analysis of paracrine gene functions in tumor invasion and angiogenesis. Method: Expression plasmids of tumor progression-associated genes, gelatin and transfection reagent were spotted onto slides using a microarray robot. Subsequently mixtures of tumor cells and target cells (e.g. endothelial cells for angiogenesis, fibroblasts for tumor invasion) were seeded onto the slides. The conditions were adjusted that exclusively the tumor cells but not the target cells got transfected. Restriction of paracrine activities on the different transfection spots was achieved by an overlay with appropriate polymer matrices to decrease spot to spot diffusion. Immunocytochemical detection of the proliferation marker Ki67 and matrix metalloproteinases in the target cells indicated paracrine activities of transfected genes on angiogenesis (increased endothelial cell proliferation) and invasion (increased expression of matrix metalloproteinases in fibroblasts). Results: Using epithelial tumor cells and fibroblasts the differential transfection on the cell chip could be successfully established. Model experiments with ectopic expression of IFN-gamma in the tumor cells and the detection of IFN-gamma-induced genes in fibroblasts showed that paracrine gene effects could be sensitively detected. An overlay with polymeric matrices restricted locally the diffusion of IFN-gamma on the respective transfection spots. With this method 150 transfections could be carried out on a single chip in order to investigate paracrine gene functions. Conclusion: Differential transfection on a cell chip provides a novel and efficient high throughput technology to determine paracrine gene functions in angiogenesis and tumor invasion. 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 2182.

Equivalency of RT-PCR and immunohistochemistry: fact or factoid

Management of breast cancer relies heavily on determination of predictive and prognostic markers. Analysis of estrogen and progesterone receptors was originally performed using ligand-binding assays (LBA); this has been replaced by immunohistochemistry (IHC) in the last couple of decades. In recent days, RT-PCR assays for ER and PR have been proposed as a superior alternative to IHC. Several studies have attempted to analyze the concordance between these different types of assays. In this issue, investigators from the Hellenic Oncology Group examines the expression and predictive significance of ER, PR and microtubule-associated protein tau (MAP-tau) by quantitative RT-PCR (qRT-PCR) in a series of 274/279 breast cancer patients enrolled in clinical trial HE 10/97 [1]. In these analyses, MAP-tau strongly correlated with ER and PR mRNA status (Spearmann r = 0.52 and 0.64, P \ 0.001). However, they find only a modest correlation between RT-PCR and IHC results for ER (kappa = 0.41) and a fair correlation for PR (kappa = 0.33). Significantly, no correlation was observed between increasing ER and PR mRNA and increasing benefit from endocrine therapy in 203 ER and/or PR IHC-positive patients receiving adjuvant hormone therapy (Wald P = 0.54 for ER, 0.51 for PR). As has been acknowledged by the authors, the findings in this study are at variance with prior reports [2–4] with regards to concordance between IHC and qRT-PCR. These differences seen in the qRT-PCR results may be ascribed to a variety of pre-analytical and analytical parameters, which are briefly discussed below. Analysis for both these assays was performed on formalin fixed paraffin-embedded (FFPE) tissues obtained from multiple institutions; it can be safely presumed that these were associated with a variety of fixation and processing protocols. The time duration of warm ischemia, length of exposure to formalin, and exact temperatures used in tissue processing and embedding are thought to be important for intactness of both RNA and protein. In addition, the age of the paraffin block has been shown to directly correlate with amount of fragmentation of RNA [5]. Akin to the choice of antigen retrieval methods available for IHC, multiple kits are available for extraction of RNA from FFPE materials. Abramowitz et al. [6] have recently performed a comparative analysis of different commercially available kits and demonstrated significant differences in quantities of RNA obtained and reproducibility of results. In their study, variation in the digestion/ incubation time (3 h versus overnight) resulted in significant differences in reproducibility even when using the same kit. Tumor tissue is a composite mixture of tumor cells, tumor microenvironment and non-malignant elements, which include normal breast epithelium and in situ carcinoma. As morphologic assessment is a critical part of IHC methods, expression is only analyzed in tumor cells. The pathologist has the capacity to exclude expression in normal elements and ductal carcinoma in situ, which therefore do not contribute to the final results. Assays such as LBA and qRT-PCR that require tissue digestion, do not permit morphologic distinction of tumor cells from nonmalignant This is a commentary to doi:10.1007/s10549-008-0144-9.

Molecular Targeting and Treatment of Composite EGFR and EGFRvIII-Positive Gliomas Using Boronated Monoclonal Antibodies

The purpose of the present study was to evaluate the anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), cetuximab, (IMC-C225) and the anti-EGFRvIII mAb, L8A4, used in combination as delivery agents for boron neutron capture therapy (BNCT) of a rat glioma composed of a mixture of cells expressing either wild-type (F98(EGFR)) or mutant receptors(F98(npEGFRvIII)). A heavily boronated polyamidoamine dendrimer (BD) was linked by heterobifunctional reagents to produce the boronated mAbs, BD-C225 and BD-L8A4. For in vivo biodistribution and therapy studies, a mixture of tumor cells were implanted intracerebrally into Fischer rats. Biodistribution studies were carried out by administering (125)I-labeled bioconjugates via convection-enhanced delivery (CED), and for therapy studies, nonradiolabeled bioconjugates were used for BNCT. This was carried out 14 days after tumor implantation and 24 h after CED at the Massachusetts Institute of Technology nuclear reactor. Following CED of a mixture of (125)I-BD-C225 and (125)I-BD-L8A4 to rats bearing composite tumors, 61.4% of the injected dose per gram (ID/g) was localized in the tumor compared with 30.8% ID/g for (125)I-BD-L8A4 and 34.7% ID/g for (125)I-BD-C225 alone. The corresponding calculated tumor boron values were 24.4 mug/g for rats that received both mAbs, and 12.3 and 13.8 mug/g, respectively, for BD-L8A4 or BD-C225 alone. The mean survival time of animals bearing composite tumors, which received both mAbs, was 55 days (P < 0.0001) compared with 36 days for BD-L8A4 and 38 days for BD-C225 alone, which were not significantly different from irradiated controls. Both EGFRvIII and wild-type EGFR tumor cell populations must be targeted using a combination of BD-cetuximab and BD-L8A4. Although in vitro C225 recognized both receptors, in vivo it was incapable of delivering the requisite amount of (10)B for BNCT of EGFRvIII-expressing gliomas.

Stromal matrix metalloproteinase-9 regulates the vascular architecture in neuroblastoma by promoting pericyte recruitment.

Advanced stages of neuroblastoma show increased expression of matrix metalloproteinases MMP-2 and MMP-9, that have been implicated in many steps of tumor progression, suggesting that they play a contributory role. Using pharmacological and genetic approaches, we have examined the role of these MMPs in progression of SK-N-BE (2).10 human neuroblastoma tumors orthotopically xenotransplanted into immunodeficient mice. Mice treated with Prinomastat, a synthetic inhibitor of MMPs, showed an inhibition of tumor cell proliferation in implanted tumors and a prolonged survival (50 versus 39 days in control group, P < 0.035). Treatment with Prinomastat did not affect formation of liver metastases (P = 0.52) but inhibited intravascular colonization by the tumor cells in the lung by 73.8% (P = 0.03) and angiogenesis in both primary tumors and experimental liver metastases. The primary tumors from Prinomastat-treated mice showed a 39.3% reduction in endothelial area detected by PECAM/CD31 staining in tumor sections (P < 0.001), primarily due to the presence of smaller vessels (P = 0.004). MMP-2 is expressed by neuroblastoma tumor cells and stromal cells, whereas MMP-9 is exclusively expressed by stromal cells, particularly vascular cells. To examine the contribution of MMP-9 to tumor angiogenesis, we generated RAG1/MMP-9 double-deficient mice. We observed a significant inhibition of angiogenesis in the immunodeficient RAG1/MMP-9 double-deficient mice orthotopically implanted with tumor cells (P = 0.043) or implanted s.c. with a mixture of tumor cells and Matrigel (P < 0.001). Using an FITC-labeled lectin, we demonstrated an inhibition in the architecture of the tumor vasculature in MMP-9-deficient mice, resulting in fewer and smaller blood vessels. These changes were associated with a 48% decrease in pericytes present along microvessels. Taken together, the data demonstrate that in neuroblastoma, stromally derived MMP-9 contributes to angiogenesis by promoting blood vessel morphogenesis and pericyte recruitment.