Microdissected Tumor Tissue Research Articles

Impact of thymidine phosphorylase and CD163 expression on prognosis in stage II colorectal cancer

BackgroundTumor-associated macrophages (TAM) are known to facilitate colorectal cancer (CRC) growth. High macrophage infiltration in thymidine phosphorylase (TYMP) expressing CRC may correspond to poor prognosis. The prognostic impact of the expression CD163, a receptor associated with TAM, and TYMP in stroma, respectively, tumor tissue is not yet established. The aim of this study was to identify the potential associations between TYMP and CD163 expression levels and relapse-free survival (RFS) of patients with stage II CRC, and if microdissection is of importance.MethodsStage II CRC patients, radically resected with relapse (n = 104), were matched to patients with a 5-year relapse-free follow-up (n = 206). Gene expression of TYMP and CD163 was analyzed in snap-frozen tumor tissues and in microdissected formalin-fixed tumor tissues separated into tumor epithelium and stroma.ResultsTYMP expression was high in poorly differentiated tumors, right-sided CRC, and tumors with high microsatellite instability CD163-expressing macrophages near tumor epithelial cells had high expression in poorly differentiated and T4 tumors. High TYMP expression in tumor epithelial cells was in the multivariate analyses associated with shorter relapse-free survival (hazard ratio 1.66; 95% confidence interval: 1.09–2.56; p < 0.05).ConclusionsTYMP expression in tumor epithelial cells was associated with RFS and emphasizes the need for tissue microdissection. Additional studies are needed to establish whether TYMP and CD163 could add clinically relevant information to identify high-risk stage II patients that could benefit from adjuvant chemotherapy.

Development of a microfluidic platform to maintain viability of micro-dissected tumor slices in culture.

One of the issues limiting the development of personalized medicine is the absence of realistic models that reflect the nature and complexity of tumor tissues. We described a new tissue culture approach that combines a microfluidic chip with the microdissected breast cancer tumor. "Tumor-on-a-chip" devices are suitable for precision medicine since the viability of tissue samples is maintained during the culture period by continuously feeding fresh media and eliminating metabolic wastes from the tissue. However, the mass transport of oxygen, which arguably is the most critical nutrient, is rarely assessed. According to our results, transportation of oxygen provides satisfactory in vivo oxygenation within the system. A high level of dissolved oxygen, around 98%-100% for every 24 h, was measurable in the outlet medium. The microfluidic chip system developed within the scope of this study allows living and testing tumor tissues under laboratory conditions. In this study, tumors were generated in CD-1 mice using MDA-MB-231 and SKBR-3 cell lines. Microdissected tumor tissues were cultured both in the newly developed microfluidic chip system and in conventional 24-well culture plates. Two systems were compared for two different types of tumors. The confocal microscopy analyses, lactate dehydrogenase release, and glucose consumption values showed that the tissues in the microfluidic system remained more viable with respect to the conventional well plate culturing method, up to 96 h. The new culturing technique described here may be superior to conventional culturing techniques for developing new treatment strategies, such as testing chemotherapeutics on tumor samples from individual patients.

Microdissected Tissue vs Tissue Slices-A Comparative Study of Tumor Explant Models Cultured On-Chip and Off-Chip.

Simple SummaryCancer drugs have the lowest success rate of approval in drug development programs. In order to address this, predictive preclinical assays that correctly reflect the clinical efficacy of a drug represent an urgent need in both clinical oncology and pharmaceutical research. To address this need, multiple tumor models have been developed, including tumor explant culture platforms, which are the only models that preserve the integrity of the tumor tissue. However, these models have not been fully characterized and multiple variables exist between studies. We investigated the effect of tissue size and culture vessel type on the survival of tumor explants by comparing micro-dissected tumor tissue with corresponding tumor slices. Our results show that the model geometry and culture vessel affect proliferation, apoptosis, and hypoxia in tissue cultures, and must be considered in designing tumor explant culture platforms.Predicting patient responses to anticancer drugs is a major challenge both at the drug development stage and during cancer treatment. Tumor explant culture platforms (TECPs) preserve the native tissue architecture and are well-suited for drug response assays. However, tissue longevity in these models is relatively low. Several methodologies have been developed to address this issue, although no study has compared their efficacy in a controlled fashion. We investigated the effect of two variables in TECPs, specifically, the tissue size and culture vessel on tissue survival using micro-dissected tumor tissue (MDT) and tissue slices which were cultured in microfluidic chips and plastic well plates. Tumor models were produced from ovarian and prostate cancer cell line xenografts and were matched in terms of the specimen, total volume of tissue, and respective volume of medium in each culture system. We examined morphology, viability, and hypoxia in the various tumor models. Our observations suggest that the viability and proliferative capacity of MDTs were not affected during the time course of the experiments. In contrast, tissue slices had reduced proliferation and showed increased cell death and hypoxia under both culture conditions. Tissue slices cultured in microfluidic devices had a lower degree of hypoxia compared to those in 96-well plates. Globally, our results show that tissue slices have lower survival rates compared to MDTs due to inherent diffusion limitations, and that microfluidic devices may decrease hypoxia in tumor models.

Abstract LB065: Prostate cancer molecular immunophenotypes in biopsies with high vs average radiation sensitivity as predicted by post-operative radiation therapy outcomes (PORTOS) scores

Abstract Background: The combination of radiation therapy and immunotherapy has emerged as an exciting field of research with the potential for significant clinical benefit. Using pre-treatment prostate biopsies from two clinical trials we explored the hypothesis that a validated gene expression signature of postoperative radiation therapy outcome score (PORTOS) is associated with differences in tumor molecular immunophenotype. In this study, we compared prostate tumors with high vs average PORTOS scores for expression of 32 molecular immunophenoscores that reflect cancer antigen profiles and the cellular composition of tumor infiltrating lymphocytes. Methods: Gene expression profiles were obtained from microdissected tumor tissue from 242 treatment naïve-prostate biopsy cores obtained from 92 patients enrolled in the University of Miami MAST (NCT02242773) and BLaStM (NCT02307058) clinical trials. For each biopsy the PORTOS score (PMID: 27743920) and 32 tumor molecular immunophenoscores (PMID: 28052254) were determined from Affymetrix Human Exon 1.0 ST microarray data using published methods. Results: 14% of the biopsy cores were predicted by the PORTOS signature to be more responsive to radiation therapy. Compared to the cores with average PORTOS scores, the “high PORTOS” biopsies showed a 2 fold or greater level of the tumor immunoactivation markers Act CD4, Act CD8, EC, HLA DPA1 and Tem CD8 and a 2 fold or lower level of the tumor immunosuppression markers MDSC, SC, TIM3 and T reg (Mann-Whitney P value of &amp;lt;0.0001 for all nine comparisons of medians). Discussion: Preliminary studies suggest that prostate cancers with PORTOS scores that predict greater sensitivity to radiation therapy also have immunophenotype profiles consistent with a higher level of overall immunogenicity. Citation Format: Sandra M. Gaston, Sanoj Punnen, Oleksandr Kryvenko, Alan Pollack, Elai Davicioni, Seagle Liu, Radka Stoyanova. Prostate cancer molecular immunophenotypes in biopsies with high vs average radiation sensitivity as predicted by post-operative radiation therapy outcomes (PORTOS) scores [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 LB065.

MiR-139 Modulates Cancer Stem Cell Function of Human Breast Cancer through Targeting CXCR4.

Simple SummaryThe C-X-C motif chemokine receptor 4 (CXCR4) is overexpressed in various cancer stem/progenitor cells via activation of the epithelial-mesenchymal transition (EMT) program to facilitate tumor cell aggressiveness in the premetastatic niche. Through miRNAs microarray and bioinformatics analysis, we confirmed that miR-139 directly interacted with the 3′-untranslated region (3′-UTR) of CXCR4. Overexpression of miR-139 down-modulated CXCR4/p-Akt axis to attenuate invasion and migration of human breast cancer stem cells both in vitro and in vivo. Furthermore, miR-139 expression assessed by quantitative real-time PCR (qRT-PCR) in laser capture microdissected tumor samples significantly correlated with more advanced tumors in patients with breast cancer. Our findings provide support to account for the preferential role of miR-139 in interrupting breast cancer progression, identifying miR-139 as a potential biomarker in prediction of breast cancer invasiveness.Elevated expression of C-X-C motif chemokine receptor 4 (CXCR4) correlates with chemotaxis, invasion, and cancer stem cell (CSC) properties within several solid-tumor malignancies. Recent studies reported that microRNA (miRNA) modulates the stemness of embryonic stem cells. We aimed to investigate the role of miRNA, via CXCR4-modulation, on CSC properties in breast cancer using cell lines and xenotransplantation mouse model and evaluated miR-193 levels in 191 patients with invasive ductal carcinoma. We validated miR-139 directly targets the 3′-untranslated region of CXCR4. Hoechst 33342 fluorescence-activated cell sorting (FACS) and sphere-forming assay were used to identify CSCs. MiR-139 suppressed breast CSCs with mesenchymal traits; led to decreased migration and invasion abilities through down-regulating CXCR4/p-Akt signaling. In lung cancer xenograft model of nude mice transplanted with human miR-139-carrying MDA-MB-231 cells, metastatic lung nodules were suppressed. Clinically, microdissected breast tumor tissues showed miR-139 reduction, compared to adjacent non-tumor tissues, that was significantly associated with worse clinicopathological features, including larger tumor size, advanced tumor stage and lymph node metastasis; moreover, reduced miR-139 level was predominately occurred in late-stage HER2-overexpression tumors. Collectively, our findings highlight miR-139-mediated suppression of CXCR4/p-Akt signaling and thereby affected mesenchymal stem-cell genesis, indicating its potential as a therapeutic target for invasive breast cancer.

Mesonephric and mesonephric-like carcinomas of the female genital tract: molecular characterization including cases with mixed histology and matched metastases.

Mesonephric carcinoma of the cervix is a rare tumor derived from Wolffian remnants. Mesonephric-like carcinomas of the ovary and endometrium, while morphologically similar, do not have obvious Wolffian derivation. Here, we sought to characterize the repertoire of genetic alterations in primary mesonephric and mesonephric-like carcinomas, in the distinct histologic components of mixed cases, as well as in matched primary tumors and metastases. DNA from microdissected tumor and normal tissue from mesonephric carcinomas (cervix, n = 8) and mesonephric-like carcinomas (ovarian n = 15, endometrial n = 13) were subjected to sequencing targeting 468 cancer-related genes. The histologically distinct components of four cases with mixed histology and four primary tumors and their matched metastases were microdissected and analyzed separately. Mesonephric-like carcinomas were underpinned by somatic KRAS mutations (25/28, 89%) akin to mesonephric carcinomas (8/8, 100%), but also harbored genetic alterations more frequently reported in Müllerian tumors. Mesonephric-like carcinomas that lacked KRAS mutations harbored NRAS (n = 2, ovary) or BRAF (n = 1, endometrium) hotspot mutations. PIK3CA mutations were identified in both mesonephric-like (8/28, 28%) and mesonephric carcinomas (2/8, 25%). Only mesonephric-like tumors harbored CTNNB1 hotspot (4/28, 14%) and PTEN (3/13, 23%) mutations. Copy number analysis revealed frequent gains of chromosomes 1q and 10 in both mesonephric (87% 1q; 50% chromosome 10) and mesonephric-like tumors (89% 1q; 43% chromosome 10). Chromosome 12 gains were more frequent in ovarian mesonephric-like carcinomas, and losses of chromosome 9 were more frequent in mesonephric than in mesonephric-like carcinomas (both p = 0.01, Fisher's exact test). The histologically distinct components of four mixed cases were molecularly related and shared similar patterns of genetic alterations. The progression from primary to metastatic lesions involved the acquisition of additional mutations, and/or shifts from subclonal to clonal mutations. Our findings suggest that mesonephric-like carcinomas are derived from a Müllerian substrate with differentiation along Wolffian/mesonephric lines.

Abstract 5858: Multiplatform analyses identify upregulated hematopoietic cell kinase activity in poorly prognostic high-grade serous ovarian cancer and its role in regulating tumor aggressiveness

Abstract High-grade serous ovarian cancer (HGSOC) is the most predominant and lethal subtype of ovarian cancer. While most patients initially respond to standard-of-care treatment, the majority will eventually relapse. Despite improved treatment strategies, the prognosis has not improved significantly and the overall 5-year survival rate remains ~31%. To understand the underlying mechanisms that govern the distinctions between tumors with good and poor prognoses, we implemented a proteogenomic approach to analyze gene expression, proteomic and phosphoproteomic profiles of HGSOC tumors available in the TCGA and the CPTAC studies. Our analyses identified upregulated hematopoietic cell kinase (HCK) signaling along with innate immunity related and ERBB activity in poor prognostic tumors. These results were confirmed using independent datasets to demonstrate HCK up-regulation in tumor samples compared to borderline tumors and/or normal samples. Further analyses of micro-dissected HGSOC tumor tissue determined that HCK was specifically upregulated in tumor epithelial cells. Importantly, differential analysis of CPTAC phospho-proteomic data identified significant enrichment of phosphorylated HCK in the primary tumors of patients with tumor recurrence within 1 year compared to tumor-free patients. Interestingly, in-silico analyses suggest that HCK may play a dichotomous tumorigenic role as HCK was strongly associated with pro-survival signaling pathways as well as enrichment of pro-tumorigenic immune signaling. Consistent with these results, in vitro studies in NIH-OVCAR3 and CAOV3 cell lines with high endogenous HCK expression demonstrated that HCK silencing decreased cell proliferation, invasion, and colony formation. Conversely, HCK overexpression significantly induced proliferation and clonogenic capacity in OVSAHO cells (low HCK). These results suggest that HCK is essential for HGSOC proliferation and survival. Reverse phase protein array (RPPA) analysis was next used to identify potential mechanisms by which HCK modulates HGSOC tumorigenesis. Our analyses identified a number of candidate proteins that are significantly downregulated following shRNA-mediated silencing of HCK including PD-L1, CD44, and NOTCH3. Further in silico analyses demonstrated significant enrichment of PD-L1 protein expression in HGSOC tumors with high HCK activity suggesting a potential role for HCK in regulating the tumor microenvironment. These latter analyses also identified a significant correlation between HCK and CD44 mRNA and protein expression in human tumors. Importantly, experimental studies in CAOV3 cells confirmed that shRNA silencing of HCK inhibited PD-L1, CD44 and NOTCH3 protein expression; these results were confirmed by lentiviral overexpression of HCK which resulted in increased CD44 and NOTCH3 expression in OVSAHO cells. Given that CD44 and NOTCH3 signaling have been shown to mediate tumor cell proliferation and survival, our data, which indicate that CD44 and NOTCH3 expression are regulated by HCK activity, suggest that these genes may contribute to HCK mediated HGSOC tumorigenesis. Our data collectively implicate HCK as a novel driver of HGSOC development and progression and may represent a novel therapeutic opportunity in this subset of patients. Citation Format: Christen A. Khella, Michael L. Gatza. Multiplatform analyses identify upregulated hematopoietic cell kinase activity in poorly prognostic high-grade serous ovarian cancer and its role in regulating tumor aggressiveness [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5858.

Abstract 2332: Tumor infiltrating lymphocytes, immunoSeq, and CMS classification in the molecular epidemiology of colorectal cancer study

Abstract Background: Tumor infiltrating lymphocytes (TILs) are prognostic and predictive biomarkers in colorectal cancer and are associated with improved prognosis and response to immunotherapy. While TILs are routinely assessed by pathologists, a standardized technique (immunoSEQ, Adaptive Biotechnologies) that leverages targeted next-generation sequencing can also be used to quantify and characterize the T-cell receptor (TCR) repertoire of individual colorectal cancers. In a large, population-based study of incident colorectal cancer, the host immune responses were measured by an expert pathologist and ImmunoSEQ to understand the relationships between TILs, TCRs/cell and specific subgroups of colorectal cancer. Methods: Incident cases of adenocarcinoma of the colon or rectum from the Molecular Epidemiology of Colorectal Cancer (MECC) study included 1,000 cancers that were uniformly evaluated for TILs and other histopathologic features by one pathologist. FFPE-derived DNA from microdissected tumor tissue was extracted and sequenced using ImmunoSEQ analysis for the same 1,000 individuals. A resulting quantitative metric from this assay includes TCRs/cell, a measure of rearranged T cell quantity relative to all nucleated cells in a tumor sample. Gene expression in snap-frozen tissue available from 342/1,000 MECC colorectal cancers was measured with Affymetrix Human Genome U133 Arrays (U133A and U133 Plus2.0) as previously described. CMS classification was performed using the R package 3.5.1, CMS classifier, randomForest 4.6-14. Multivariate analysis assessed CMS by age, gender, TILs/HPF, TCRs/cell, MSI status, BRAF and KRAS mutational status. Results: TILs/HPF and TCRs/cell were significantly correlated among all 1000 cases (r=0.5, p&amp;lt;0.001). Among the 342 cases with available expression profiles, CMS1 constituted 12.0% of all CRC, with CMS2 (41.8%), CMS3 (8.5%), and CMS4 (13.7%) and unclassified (24%) representing the remaining distribution. There were statistically significant differences in the molecular and histopathologic features of colorectal cancers by CMS subgroups. MSI-H tumors were most frequently observed within CMS1 cancers (56.6% of CMS1 were MSI-H), with lower representation among CMS2 (1.5%), CMS3 (10%), CMS4 (3.5%), and unclassified CRC (9.5%) (p&amp;lt;0.0001). In addition, BRAF positive tumors were more frequently observed within the CMS1 group (12.2%, p =0.0065) and KRAS positive tumors within the CMS3 group (31%, p&amp;lt;0.0001). Consistent with prior reports, TILs/HPF were significantly higher in the CMS1 group (mean=7.7, p&amp;lt;0.0001). Similar statistically significant trends were observed across classes for TCRs/cell (mean=0.16, p=0.04). Conclusions: Subtypes of CRC have distinct histopathologic and molecular features that can be distinguished by expression profiles and immunoSEQ. Citation Format: Marilena Melas, Charalampos Lazaris, Stephanie L. Schmit, Asaf Maoz, Rebeca Sanz Pamplona, Chenxu Qu, Joel K. Greenson, Rork Kuick, Flavio Lejbkowicz, Hedy S. Rennert, Christopher P. Walker, Chase M. Bowen, Diane M. Da Silva, W. Martin Kast, Gregory E. Idos, Kevin J. McDonnell, Victor Moreno, Gad Rennert, Stephen B. Gruber. Tumor infiltrating lymphocytes, immunoSeq, and CMS classification in the molecular epidemiology of colorectal cancer study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2332.

Cancer-on-a-chip for Drug Screening.

The oncology pharmaceutical research spent a shocking amount of money on target validation and drug optimization in preclinical models because many oncology drugs fail during clinical trial phase III. One of the most important reasons for oncology drug failures in clinical trials may due to the poor predictive tool of existing preclinical models. Therefore, in cancer research and personalized medicine field, it is critical to improve the effectiveness of preclinical predictions of the drug response of patients to therapies and to reduce costly failures in clinical trials. Three dimensional (3D) tumor models combine micro-manufacturing technologies mimic critical physiologic parameters present in vivo, including complex multicellular architecture with multicellular arrangement and extracellular matrix deposition, packed 3D structures with cell-cell interactions, such as tight junctions, barriers to mass transport of drugs, nutrients and other factors, which are similar to in vivo tumor tissues. These systems provide a solution to mimic the physiological environment for improving predictive accuracy in oncology drug discovery. This review gives an overview of the innovations, development and limitations of different types of tumor-like construction techniques such as self-assemble spheroid formation, spheroids formation by micro-manufacturing technologies, micro-dissected tumor tissues and tumor organoid. Combination of 3D tumor-like construction and microfluidic techniques to achieve tumor on a chip for in vitro tumor environment modeling and drug screening were all included. Eventually, developmental directions and technical challenges in the research field are also discussed. We believe tumor on chip models have provided better sufficient clinical predictive power and will bridge the gap between proof-of-concept studies and a wider implementation within the oncology drug development for pathophysiological applications.

Micro-dissected tumor tissues on chip: an ex vivo method for drug testing and personalized therapy.

In cancer research and personalized medicine, new tissue culture models are needed to better predict the response of patients to therapies. With a concern for the small volume of tissue typically obtained through a biopsy, we describe a method to reproducibly section live tumor tissue to submillimeter sizes. These micro-dissected tissues (MDTs) share with spheroids the advantages of being easily manipulated on-chip and kept alive for periods extending over one week, while being biologically relevant for numerous assays. At dimensions below ~420 μm in diameter, as suggested by a simple metabolite transport model and confirmed experimentally, continuous perfusion is not required to keep samples alive, considerably simplifying the technical challenges. For the long-term culture of MDTs, we describe a simple microfluidic platform that can reliably trap samples in a low shear stress environment. We report the analysis of MDT viability for eight different types of tissues (four mouse xenografts derived from human cancer cell lines, three from ovarian and prostate cancer patients, and one from a patient with benign prostatic hyperplasia) analyzed by both confocal microscopy and flow cytometry over an 8-day incubation period. Finally, we provide a proof of principle for chemosensitivity testing of human tissue from a cancer patient performed using the described MDT chip method. This technology has the potential to improve treatment success rates by identifying potential responders earlier during the course of treatment and providing opportunities for direct drug testing on patient tissues in early drug development stages.

MTR-18PREDICTIVE BIOMARKERS OF BEVACIZUMAB RESPONSE IN RECURRENT GLIOBLASTOMA PATIENTS

BACKGROUND: Bevacizumab (BEV) plus chemotherapy has shown activity in recurrent glioblastoma (GBM). However, the prognosis varies and only one third of patients have a durable clinical response to BEV combination therapy. Recent findings from a randomized phase-3 study (AVAglio) indicate that patients with the proneural GBM subtype have a survival benefit when treated with BEV in combination with standard treatment. However, no validated biomarkers able to predict BEV response have been identified and the biology reflecting a clinical BEV response is poorly understood. The primary objective of this study was to evaluate the predictive and prognostic value of GBM subtypes in recurrent GBM patients treated with BEV therapy. The secondary objective was to identify biomarkers able to predict response to BEV therapy in recurrent GBM patients. METHODS: A total of 90 recurrent GBM patients treated with BEV combination treatment according to previously published protocols were included. Inclusion criteria: BEV plus irinotecan treatment in the period between May 2005-2011; available GBM tissue (according to WHO); response evaluable (RANO). RNA was extracted from laser microdissected tumor tissue and analyzed by the NanoString platform covering 800 genes. Raw data was assigned to molecular subtypes for each of the samples using the PAMR classifier model, previously trained on the AVAglio dataset. By performing a t-test, comparing gene profiles of patients responding versus progressing on BEV novel candidate biomarkers were identified. Candidate biomarkers were analyzed by logistic regression and Cox regression modelling response and survival endpoints, respectively. Biomarkers associated with response were added to a prognostic model consisting of three independent prognostic factors: Corticosteroid use, neurocognitive deficit and multifocal disease. RESULTS: Molecular subtypes were not associated with response or survival. However, two independent predictive biomarkers (gene1 down-regulated and gene2 up-regulated in responders, respectively) of BEV response and survival were identified. Results will be presented.

Abstract S6-06: The genomic landscape of male breast cancers

Abstract Background: Male breast cancer (MaBC) accounts for &amp;lt;1% of all breast cancers and its genomic landscape has yet to be characterized. The majority of MaBCs are ER-positive invasive ductal carcinomas of no special type and, unlike female breast cancers (FBC), rarely display HER2 gene amplification or a triple-negative phenotype. Given the relative rarity of MaBCs, treatment decisions for MaBC patients are often extrapolated from trials carried out with FBC patients. Here we employed targeted capture massively parallel sequencing to define the repertoire of somatic mutations and gene copy number alterations (CNAs) in MaBCs. Methods: Subtyping of the 64 MaBCs included in this study was performed by means of immunohistochemistry using the definitions described in the latest St. Gallen’s consensus report. DNA extracted from microdissected tumor and adjacent normal tissue were subjected to massively parallel targeting sequencing of all exons of 273 genes most frequently mutated in FBCs or directly related to DNA repair. Somatic mutations were defined using a combination of MuTect, SomaticSniper, MutationSeq and Haplotype Caller. Selected mutations were validated with Sequenom MassARRAY. CNAs were identified using Varscan2 and GISTIC2.0. Pathway and network analysis of mutations/CNAs was performed using Ingenuity Pathway Analysis and HOTNET. The genomic landscape of MaBCs was compared with that of FBCs of the same subtype analyzed as part of The Cancer Genome Atlas project. Results: All MaBCs were ER-positive and HER2-negative. Using the St. Gallen’s criteria, 37.5% and 62.5% were classified as luminal A-like or luminal B-like, respectively. The genes most frequently mutated in MaBCs were PIK3CA, GATA3, FLG and PLEC, with PIK3CA being the only significantly mutated gene as defined by MutSigCV (q=0.003). CNA analysis revealed recurrent gains of 1q and 8q and loss of 16q. GISTIC2.0 identified significantly recurrent high-level amplifications in 1q25.3, 8p11 (FGFR1, ZNF703), 8q24.3 (DEPTOR), 17q23 (PPM1D) and 15q26 (IGF1R) and deletions in 11q22 (ATM) and 21q22.12 (RUNX1). HOTNET analysis of the genes mutated and/or targeted by gene amplifications in MaBCs revealed significantly altered subnetworks involving genes related to DNA repair, PI3K and FGF signaling pathways. The most frequently mutated genes in luminal A-like MaBCs were PIK3CA and MLL3, while those of luminal B-like MaBCs were PIK3CA and GATA3. MLL3 and GATA3 mutations were only found in luminal A-like MaBCs (p=0.039) and luminal B-like MaBCs (p=0.048), respectively. Although the mutational landscapes of MaBCs and luminal FBCs were qualitatively similar, PIK3CA and TP53 were less frequently mutated in MaBCs (p&amp;lt;0.001), whereas genes found to be recurrently mutated in FBCs, such as MAP2K4 and NCOR1, were not mutated in MaBCs. Conclusions: MaBCs are preferentially of luminal subtype and are characterized by recurrent mutations in PIK3CA, GATA3, FLG and PLEC. Genetic alterations in MaBCs often target DNA repair and FGF signaling pathways. The known drivers of luminal FBCs appear to be less frequently altered in MaBCs. Given these important differences between MaBCs and FBCs, caution should be exercised in the extrapolation of biologic and clinical implications from studies in FBCs to the management of MaBCs. Citation Format: Salvatore Piscuoglio, Melissa Murray, Charlotte KY Ng, Elena Guerini Rocco, Luciano G Martelotto, Francois-Clement Bidard, Carey A Eberle, Nicola Fusco, Rita A Sakr, Leticia De Mattos-Arruda, Raymond Lim, Timour Baslan, James Hicks, Tari A King, Edi Brogi, Larry Norton, Britta Weigelt, Clifford A Hudis, Jorge S Reis-Filho. The genomic landscape of male breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S6-06.

Expanded KRAS and NRAS assays increase detection of mutations that predict for resistance to therapy in colorectal cancer patients.

519 Background: The selection of targeted therapies is guided by the analysis of somatic mutations. The identification of RAS activating mutations can be used to examine tyrosine kinase inhibitor therapeutic eligibility and prognosis. Beyond known mutations in RAS exon 2 (codons 12 and 13), the identification of additional mutations in RAS exons 3 and 4 (codons 61, 117 and 146) also predict for resistance to EGFR therapy in colorectal cancer (CRC). Meta-analysis supports screening for these additional mutations in any screening strategy prior to administration of EGFR mAb therapy in metastatic CRC patients (Sorich, MJ, et al., Ann Oncol, Aug 12, 2014). Methods: We expanded our existing allele-specific KRAS and sequencing-based NRAS assays to include codons 61, 117, and 146 and analytically validated these assays to CAP/CLIA standards. DNA, from microdissected colon tumor tissue that was wild-type for RAS exon 2, was tested for exon 3 and 4 mutations and included over 15 additional mutations. Results: Forty-two (9.1%) samples were identified as bearing either an exon 3 or 4 KRAS mutation amongst 461 colon cancer specimens. Exon 4 codon 146 mutations were more prevalent than three of the commonly screened exon 2 mutations: G12A, G12R, and G12S. Five (1.8%) of samples were identified as carrying exon 3 or 4 NRAS mutations amongst 272 colon cancer specimens. This included a single codon 146 mutation in exon 4. As is seen with exon 2, RAS mutations at exons 3 and 4 were mutually exclusive of activating BRAF mutations with ~10% of patients harboring V600E. The collection of additional data studying KRAS and NRAS mutation status is currently ongoing. Conclusions: The KRAS expanded coverage contributed an additional 5.5% to overall burden of specimens bearing mutations. The NRAS expanded coverage contributed an additional 1.8% to the mutational burden. These analyses in clinical cohorts support the observations made in a trial population (Douillard JV, et al. NEJM 369:1023-34, 2013). The expanded RAS coverage identifies additional patients unlikely to respond to EGFR-targeted therapies that would otherwise have been assessed as “no mutation detected” in using assays restricted to RAS exon 2.

Abstract A211: Multiethnic screening for irinotecan sensitivity in FFPE tissue from colorectal cancer patients.

Abstract The selection of chemotherapies is guided by the analysis of molecular targets in solid tumors for well-known somatic mutations identified in microdissected FFPE tissue. Combining a molecular targeting panel with select pharmacogenomic targets provides additional clinical information useful in customized patient management. We have developed a combined pharmacogenomic/molecular target approach using FFPE tissue from colon cancer patients. Our test, based on FDA guidance for Irinotecan administration to metastatic colorectal cancer (CRC) patients, is a recommended prescreening assay for UGT1A1 [TA] promoter variations to mimimize neutropenia. Meta-analysis of Irinotecan treated CRC patients has demonstrated an association between the [TA]7 allele (UGT1A1*28) and elevated levels of neutropenia in both homozygous and heterozygous carriers suggesting lowered dosing regimens (Liu, et al Pharmacogenomics J, 2013). Additional alleles, while uncommon in Caucasians, contribute to decreased levels of UGT1A1 enzymatic activity, and are restricted to genetic subgroups. Collectively these alleles influence the pharmacodynamic t1/2 of Irinotecan but are often not widely screened. Thus, a screening palate encompassing multiple ethnic-specific variants provides improved predictive information in a cosmopolitan population of cancer patients. As these polymorphisms are germline changes, UGT1A1 genotypes can be derived from either traditional whole blood assessment or more conveniently from FFPE surgical tissue at the time of diagnosis. We have screened paired blood/tumor samples from CRC patients to examine potential loss of heterozygosity in tumor specimens at this locus. In parallel, we screened microdissected tumor and adjacent normal tissue from FFPE samples to verify the potential of using tumor specimens as a routine alternative to blood. The use of FFPE tissue from CRC patients as part of a molecular work-up can provide genotypes for multiple UGT1A1 alleles of reduced activity (including *28), and may be better at predicting neutropenia in distinct Caucasian, Asian or other genetically distinct patient populations. The development of FFPE-based tests for pharmacogenomic tests such as UGT1A1 premised on genetic ancestry provides actionable information that can be used to maximize patient responses. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A211. Citation Format: Jack Hsiang, Rita El-Khoueiry, Heinz-Josef Lenz, Stephanie H. Astrow, Garrett P. Larson. Multiethnic screening for irinotecan sensitivity in FFPE tissue from colorectal cancer patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A211.

Distinct molecular features of colorectal cancer in Ghana

Objectives: While colorectal cancer (CRC) is common, its incidence significantly varies around the globe. The incidence of CRC in West Africa is relatively low, but it has a distinctive clinical pattern and its molecular characteristics have not been studied. This study is one of the first attempts to analyze molecular, genetic, and pathological characteristics of colorectal cancer in Ghana. Methods: DNA was extracted from microdissected tumor and adjacent normal tissue of 90 paraffin blocks of CRC cases (1997–2007) collected at the University of Ghana. Microsatellite instability (MSI) was determined using fragment analysis of ten microsatellite markers. We analyzed expression of mismatch repair (MMR) proteins by immunohistochemistry and sequenced exons 2 and 3 of KRAS and exon 15 of BRAF. Results: MSI analysis showed 41% (29/70) MSI-High, 20% (14/70) MSI-Low, and 39% (27/70) microsatellite-stable (MSS) tumors. Sequencing of KRAS exons 2 and 3 identified activating mutations in 32% (24/75) of tumors, and sequencing of BRAF exon 15, the location of the common activating mutation (V600), did not show mutations at codons 599 and 600 in 88 tumors. Conclusions: Our study found a high frequency of MSI-High colorectal tumors (41%) in Ghana. While the frequency of KRAS mutations is comparable with other populations, absence of BRAF mutations is intriguing and would require further analysis of the molecular epidemiology of CRC in West Africa.

Application of Selected Reaction Monitoring for Multiplex Quantification of Clinically Validated Biomarkers in Formalin-Fixed, Paraffin-Embedded Tumor Tissue

One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R(2) > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies.

Abstract 1939: Identification of microRNAs related to chemosensitivity in ovarian cancer using Next Generation Sequencing.

Abstract Ovarian cancer is the most common cause of gynecologic malignancy related mortality in women. The combination of tumor reductive surgery and chemotherapy is the main therapeutic strategy for ovarian cancer. However, especially in the most of advanced cases, complete site reduction is impossible to achieve. In those cases, the sensitivity to chemotherapy strongly affects their prognoses. MicroRNAs (miRNAs), which are approximately 22-nucleotide non-coding RNAs, regulate protein expression by translational inhibition and degradation of their target mRNAs. The expression of miRNAs is dysregulated in various types of cancers, suggesting that miRNAs are involved in carcinogenesis and cancer progression. The purpose of present work was to identify miRNAs which are related to chemo-sensitivity in ovarian cancer. Using Next Generation Sequencing (NGS), miRNA profiling was performed in 38 advanced high-grade serous ovarian cancers (HGSOCs). RNAs including miRNAs were procured from the epithelial component of the laser microdissected tumor tissue removed from patients undergoing primary surgery without neo-adjuvant chemotherapy. A total of 1935 miRNAs and miRNA variants were detected by NGS and normalized expression data of each miRNAs and miRNA variants were used for further analyses. Comparing the copy numbers of each miRNAs between chemo-refractory group (patients with disease progression in the period of first line chemotherapy, n=7) and chemo-sensitive group (patients with disease progression in more than 6 months after completion of first line chemotherapy, n=13) by Mann-Whitney U test, 77 miRNAs and miRNA variants were found to be dysregulated with significant differences (p&amp;lt;0.05). The 10 most significant miRs identified by NGS were selected for further validation studies by real-time reverse transcription PCR (RT-PCR) analysis on the same sample set. To determine whether miRNAs could be used as a prognostic marker HGSOC, expression levels of those 10 miRNAs were quantified by RT-PCR using 95 HGSOC samples including the 38 samples used in the discovery set. The results showed that mir-625-3p expression levels were significantly reduced in the chemo-refractory group compared with the chemo-sensitive group as indicated by the NGS data (n=20, p=0.029), and by the RT-PCR data (n=63, p=0.004). Furthermore, Kaplan-Meier survival analysis with log-rank test indicated that low expression of mir-625-3p (less than mean expression) was significantly associated with shorter progression-free survival (n=95, p=0.007) and shorter over-all survival (n=95, p&amp;lt;0.001) as compared with the high expression group (more than mean expression). Our results strongly suggest that mir-625-3p might be a prognostic marker that can be used to predict chemo-sensitivity in patients with HGSOC. Further characterization of mir-625-3p as a therapeutic target for HGSOC is in progress. Citation Format: Tetsushi Tsuruga, Chi Lam Au Yeung, Cecilia S. Leung, Tsz-Lun Yeung, Kwong K. Wong, Rosemarie Schmandt, Ngai Na Co, Karen H. Lu, Samuel Mok. Identification of microRNAs related to chemosensitivity in ovarian cancer using Next Generation Sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1939. doi:10.1158/1538-7445.AM2013-1939

978 PTEN DELETION AND MUTATION IN CLINICALLY INSIGNIFICANT AND SIGNIFICANT PROSTATE CANCER: AN ANALYSIS BY MATE-PAIR NEXT GENERATION AND SANGER SEQUENCING

You have accessJournal of UrologyProstate Cancer: Basic Research (V)1 Apr 2013978 PTEN DELETION AND MUTATION IN CLINICALLY INSIGNIFICANT AND SIGNIFICANT PROSTATE CANCER: AN ANALYSIS BY MATE-PAIR NEXT GENERATION AND SANGER SEQUENCING R. Jeffrey Karnes, john cheville, george vasmatzis, and william sukov R. Jeffrey KarnesR. Jeffrey Karnes Rochester, MN More articles by this author , john chevillejohn cheville Rochester, MN More articles by this author , george vasmatzisgeorge vasmatzis Rochester, MN More articles by this author , and william sukovwilliam sukov Rochester, MN More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.560AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Deletion of PTEN is associated with worse prognosis in prostate cancer. However, little is known about the impact of PTEN mutations, and there is limited data regarding the frequency and clinical impact of deletion and mutation in clinically insignificant tumors compared to significant tumors. METHODS We identified 40 prostatectomy cases with adenocarcinoma consisting of 9 clinically insignificant tumors (defined as GS6 and tumor volume <0.5 cm3), 7 cases of large volume (>1.0 cm3) GS6, 23 cases of GS7, and 1 case of GS8) and performed massively parallel mate-pair next generation sequencing (NGS) on whole genome-amplified DNA extracted from laser capture microdissected tumor tissue. Sequence analysis of exonic regions was performed using standard Sanger methodology. Follow-up information was available for 31 patients. Tumor-free survival from date of prostatectomy was assessed using the Kaplan-Meier method and the log-rank test. RESULTS PTEN deletion detected by mate-pair NGS occurred in 10 cases and three of these cases showed additional deleterious mutations (2 nonsense and 1 frameshift) in the remaining PTEN allele by Sanger sequencing. Although not mutually exclusive, alterations in PTEN occurred more frequently in the setting of ERG rearrangements (70%). PTEN abnormalities were absent in clinically insignificant GS6, and large volume GS6 but were common in GS7 or higher tumors (10 of 23 cases; 43%)(p=0.032). Radiographic or biochemical recurrence occurred in 5 patients with PTEN abnormalities (HR=4.65, p=0.036), including metastatic recurrences in 2 of the 3 patients with confirmed bi-allelic loss of PTEN. The overall 5-year tumor-free survival rate among patients with Gleason 7-8 tumors was 57% with PTEN abnormalities and 79% in the remaining cases (p=0.039). CONCLUSIONS PTEN abnormalities were absent in clinically insignificant and large volume GS6 tumors, but occurred in a significant number of GS7 cases, Although our series is small, patients that had PTEN deletion and mutation has a significantly worse outcome.. These results suggest that PTEN abnormalities identify a subset of prostatic adenocarciomas at higher risk for progression, for which molecular and cytogenetic testing for PTEN abnormalities may provide significant prognostic information. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e402 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information R. Jeffrey Karnes Rochester, MN More articles by this author john cheville Rochester, MN More articles by this author george vasmatzis Rochester, MN More articles by this author william sukov Rochester, MN More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract B92: Molecular characterization of colorectal cancer in Ghana

Abstract Colorectal cancer is the fourth most common cancer in men and the third most common cancer in women worldwide with significant variation between different parts of the globe. Although colorectal cancer incidence is relatively low in West Africa, the most recent publications from Nigeria and Ghana note significant increase of colorectal cancer incidence in these countries since 1970. These studies also show that colorectal cancer in West Africa has distinctive pattern with young age of onset and predominantly left-sided tumors, yet the spectrum of molecular changes in colorectal tumors has not been characterized in West African populations. The goal of this study was to investigate molecular characteristics of colorectal cancer in Ghana. We retrieved 90 formalin-fixed paraffin embedded (FFPE) colorectal cancer blocks from the University of Ghana Medical School (Accra) diagnosed between 1997 and 2007. Ten 5 μm recuts and H&amp;E slide were prepared for each block and reviewed by the pathologist to determine areas of at least 70% tumor cellularity and areas of adjacent normal tissue. DNA was extracted from the microdissected tumor and normal tissue. Only 71 samples had both tumor and normal tissue within the available specimen. Ten microsatellite marker loci (D10S197, BAT26, beta-catenin, D18S58, BAT40, D2S123, D17S250, BAT25, TGF-b-RIIF, and D5S346F) were used for microsatellite instability (MSI) testing using fluorescently labeled primers and fragment analysis. Tumors with at least one mononucleotide marker and a minimum of three available markers were considered for analysis. The tumors with instability in at least 30% of markers were called MSI-High, less than 30% classified as MSI-Low, and tumors with all stable markers were called microsatellite stable (MSS). Sequencing BRAF and KRAS was repeated twice from both directions and analyzed by two independent readers using Sequencher from GeneCodes (Ann Arbor, MI) and Mutation Surveyor from SoftGenetics (State College, PA) to avoid misinterpretation. Out of 71 colorectal cancer pairs of normal and tumor tissue, MSI testing was informative for 70 pairs. An exceptionally large proportion of cancers were MSI-High (29/70, 41.4%), with 14/70 (20%) MSI-Low tumors, and 27/70 (38.6%) MSS tumors. Sequencing of exons 1 and 2 of KRAS in 75 tumors detected 24 (32%) activating mutations (G12D, G12V, G12C, G12S, G13D, Q61R, and Q61K). Surprisingly, the sequencing of exon 15 of BRAF, the location of frequent activating mutation (V600E), did not show any mutations at codons 599 and 600 in 88 tumor samples. Our study showed a very high frequency of MSI-high colorectal tumors (41.4%), consistent with the higher rates of MSI-High cancers observed in some studies of colorectal cancer in African American patients. The frequency of KRAS mutations is comparable to one found in African Americans, but absence of BRAF mutations is intriguing and requires further analysis of the epidemiology and biology of colorectal cancer in West Africa. Citation Format: Leon Raskin, Jonathan C.B. Dakubo, Nicole Palaski, Joel Greenson, Stephen Gruber. Molecular characterization of colorectal cancer in Ghana. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B92.

Proteomics signature of early stages in pancreatic cancer

s / Pancreatology 12 (2012) 502–597 514 Dept. of Medicine, Div. of Gastroenterology, Endocrinology and Metabolism, Philipps-University Marburg, Marburg, Germany 2 Institute for General Pathology, University Hospital, Tuebingen, Germany Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive pancreatic cancer with an overall 5-year survival rate of <5%. Despite significant advances in treatment of the disease during the past decade, the median survival rate (w6 months) has hardly improved, warranting the need to identify novel targets for therapeutic approaches. Our previous data from microarray analyses of microdissected pancreatic tumor tissues as well as parallelized cell-based assays indicated a previously unknown role for Plac8 inpancreatic cancer initiation and progression. Plac8 isa rather smallproteinwhosephysiological functions remain largelyunclear. Aim:Main aim of this project is to characterize gene Plac8 with respect to its role in PDAC. The study also intends to dissect out the pathway(s) involved in the functional role. Methods: qPCR, tissue microarrays, RNAi, cell proliferation and viability assays, FACS analysis, Western blot, shRNA inducible clones. Results: qRT-PCR as well as tissue microarray data confirmed that Plac8 has a strong ectopic expression in pancreatic cancer tissues and further demonstrated that this high Plac8 expression is also retained in the majority of pancreatic tumor cell lines in vitro, with negligible expression in non-transformed cell lines. Functional effects of Plac8 were investigated after transient knockdown in a variety of different transformed and non-transformed cell lines. Proliferation and viability were strongly impaired by down-regulation of Plac8. Western blot and flow cytometry did not show any involvement of classical apoptosis pathway (Caspase-3 and PARP cleavage). Flow cytometry analyses and time course experiments with cell cycle inhibitors demonstrated strong attenuation of cell cycle progression after Plac8 knockdown, although intriguingly classical checkpoint mechanisms (p21, p53, Chk1, Cdc25A) were not activated, thereby suggesting involvement of hitherto unknown mechanisms. Generation of stable inducible clones as well as “multiple” transgenic mice (pertinent to human PDAC condition) for further in vivo experiments is in progress. Conclusion: Our experimental data shows that ectopic expression of Plac8 is indispensible for proliferation, viability and cell cycle progression in pancreatic cancer cells. A clear insight into pathophysiological role(s) of Plac8 in the onset and progression of pancreatic cancer is expected to emerge with the generation of transgenic mice.