An important step in translational research is the validation of molecular findings from in vitro experiments using tissue specimens. However, tissue specimens are complex and contain a multitude of diverse cell populations that interfere with the molecular profiling data of a specific cell type. Laser capture microdissection (LCM) alleviates this issue by providing a valuable tool for the enrichment of a specific cell type within complex tissue samples. However, LCM and molecular analysis from tissue specimens can be complex and challenging due to numerous issues related with the tissue processing and its impact on the integrity of biomolecules in the specimen. The intricate nature of this application highlights the essential role a pathologist plays in translational research by contributing an expertise in histopathology, tissue handling, tissue analysis techniques, and clinical correlation of biological findings. The present review examines key practical aspects in tissue handling, specimen selection, quality control, and sample preparation for LCM and downstream molecular analyses that are a primary objective of the investigative pathologist.
Despite a therapeutic paradigm shift into targeted-driven medicinal approaches, resistance to therapy remains a hallmark of lung cancer, driven by biological and molecular diversity. Using genomic and expression data from advanced non-small cell lung cancer (NSCLC) patients enrolled in the BATTLE-2 clinical trial, we identified RICTOR alterations in a subset of lung adenocarcinomas and found RICTOR expression to carry worse overall survival. RICTOR-altered cohort was significantly enriched in KRAS/MAPK axis mutations, suggesting a co-oncogenic driver role in these molecular settings. Using NSCLC cell lines, we showed that, distinctly in KRAS mutant backgrounds, RICTOR blockade impairs malignant properties and generates a compensatory enhanced activation of the MAPK pathway, exposing a unique therapeutic vulnerability. In vitro and in vivo concomitant pharmacologic inhibition of mTORC1/2 and MEK1/2 resulted in synergistic responses of anti-tumor effects. Our study provides evidence of a distinctive therapeutic opportunity in a subset of NSCLC carrying concomitant RICTOR/KRAS alterations.
Background: Circulating tumor cells (CTC) associated with solid tumors are being studied for their diagnostic and prognostic value. In patients with metastatic tumors, CTC presence in the blood has been putatively associated with short survival. Since blood collection is relatively non-invasive, CTC molecular analysis opens up the possibility of monitoring genotypic changes during cancer treatments. Unfortunately, CTCs are not present in large numbers, often at rates as low as one cell per 106-107 leukocytes. Thus, to perform genotypic biomarker analysis on CTCs, methodologies must be developed to using highly specific and sensitive technologies and an enrichment step to increase analytes to detectable levels. Methods: We developed a methodology for detecting mutations in multiple oncogenes and chemotherapy resistance genes in non-small cell lung cancer (NSCLC) CTC specimens using high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) single nucleotide polymorphism (SNP) analysis (MASSarray; Sequenom, Inc.) to determine cancer-associated genetic mutations in lung cancer specimens. This system allows for up to 10 different somatic mutations to be assayed per well in a 384-well format; requires very little DNA; can be used with whole genome amplified (WGA) DNA; and is sensitive enough to use with small samples such as core needle biopsies (CNB), fine needle aspirates (FNA), and CTCs. We developed a lung cancer assay panel of 13 genes/135 mutations (including AKT1, BRAF, CTNNB1, EGFR, ERBB2, KRAS, MEK1, NRAS, PIK3CA, PIK3R1,PTEN, and STK11) to test for somatic mutations in genes representing multiple pathways known to be involved in lung cancer. All assays can detect a mutation in < 25% of a sample. Results: In the effort to analyze CTCs, we first analyzed 57 NSCLC cell lines with known mutations and confirmed known mutation status. Next, we successfully analyzed DNA from 90 frozen and matched FFPE NSCLC resected tissues. Analysis of unamplified and matched WGA cell line DNA quantity CNB and FNA equivalents gave the same mutational status results. Moving to CTC equivalents, we successfully analyzed cell line DNA and matched WGA DNA equivalents of 100–1000 cells with known EGFR L585R and KRAS G34A mutations and negative control DNA (negative for all assays). Next, WGA methodology for direct CTC cell lysate DNA amplification was developed using CTC cell number equivalents (3 – 200 cells) obtained from a typical clinical blood sample CTC preparation. Then, we directly compared unamplified and matched amplified CTC cell equivalents (50, 100, and 200 cells). Analysis of both unamplified and amplified CTC cell equivalents reported identical mutation status results. We have applied this methodology to spiked blood sample and clinical blood sample CTC fractions. Thus, we demonstrated that we are able to study mutations in multiple genes using small amount of DNA from CTC cell numbers in a high-throughput manner. Conclusion: We developed a robust method for accurately determine cancer-associated genetic mutations in NSCLC CTC cell number equivalent lysates using MALDI-TOF MS SNP analysis which can be applied to better understand the molecular characteristics of lung cancer during treatment and progression. As additional clinical NSCLC CTC samples are collected, we will continue applying this methodology to assess CTC mutation status as potential diagnostic and/or prognostic markers.
Tumor biomarker evaluation, such as EGFR or KRAS mutation status, is a main factor in non-small cell lung carcinoma (NSCLC) treatment decisions. For most NSCLC patients, tumor-specific biomarkers have not been identified for effective therapy. This has been due in part to difficulties obtaining the DNA quantity and quality from archival FFPE clinical specimens necessary for high-throughput (HT) mutation analysis. Activating mutations and copy number variation (CNV) analysis of key lung cancer-related oncogenes in NSCLC FFPE tissues will allow for identification of subsets of patients with oncogenotypes of which treatment can be personalized. Thus, development of improved DNA recovery methods from FFPE tissues is imperative. The goal of this study was to improve DNA recovery from archival specimens for analyses by HT genotyping technologies. DNA was extracted/isolated from 2 normal human epithelial bronchial/4 NSCLC cell lines and 389 resected FFPE NSCLC tissues using improved SPRI-TE/DNA cleanup and compared to standard method (SM). Detailed tissue characteristics [whole tissue (WT), manual microdissected (MMD), artificial core needle biopsy (aCNB)], thickness (10 or 5 μm), tumor area (cm2), % tumor cells, and % fibrotic/necrotic tissue was recorded. DNA recovery quantity, quality and amplification were assessed. HT genotyping methodologies for analysis of FFPE DNA included 1) aCGH/qPCR for CNV, 2) DNA amplification for sequencing, and 3) Sanger, pyrosequencing, and MALDI-TOF MS SNP analysis for KRAS mutation status. Data were analyzed by unpaired t-test with Welch's correction, one-way ANOVA, and linear regression model for multivariable analyses. Statistical significance was defined as p≤0.05. DNA recovery using improved SPRI-TE/DNA cleanup was 5X higher than SM. DNA recovery was significantly higher (p≤0.05) from: a) 2 x 10 μm sections WT vs. MMD or aCNBs, b) tissue area >1 cm2, and c) decreasing % fibrosis. aCGH was successfully performed using DNA from FFPE NSCLC primary tumors (PT) and brain metastases (BM), identified multiple chromosomal regions with significant CNV. BM demonstrated significantly higher frequency of CN gains than PT in 3 chromosomal regions: 8q24.3 (100% vs. 40%), 19q13.33 (90% vs. 20%), and 20q13.12 (60% vs. 0%). DNA amplification (WGA vs. matched non-WGA) was evaluated for sequencing and resulted in correct mutation calls (p≤0.05). KRAS mutation analysis by 3 methods resulted in 100% identical mutation calls. Using our improved SPRI-TE/DNA cleanup, recovery of quantities and qualities of DNA from FFPE NSCLC tissues for precise HT genotyping analysis was achieved. Best predictor of optimal DNA quantity recovery is tumor area >1 cm2. FFPE NSCLC tissue DNA is suitable for use in HT genotyping assays and discovery of oncogene-specific genotypes which could be applied to personalized medicine. This work was supported by The V Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3195. doi:1538-7445.AM2012-3195
Background: Rictor (RPTOR independent companion of MTOR, complex 2) is a highly conserved protein and is a critical component for proper assembly and functionality of the mTORC2 complex. The goal of our current study is to characterize the functional consequences of genomic alterations of RICTOR in advanced refractory NSCLC. Our preliminary data suggest that Rictor alterations have the potential to not only signal canonically through AKT, but also provide cancer cells with alternate, more advantageous oncogenic signaling via non-canonical mechanisms. Methods: We correlated genomic data (DNA hybrid capture based next generation sequencing (NGS), Foundation Medicine, Inc.), gene expression profiling, and clinical outcome in the context of the ongoing BATTLE-2 clinical trial of targeted therapies in chemo-refractory NSCLC (198 cases). We further (1) surveyed early stage NSCLC (230 cases) in The Cancer Genome Atlas (TCGA) database and performed two-way hierarchical clustering comparing gene expression profiling in amplified vs. diploid cases; (2) utilized a single-nucleotide polymorphism array to select RICTOR amplified and diploid NSCLC cell lines; (3) assessed Rictor protein and RNA expression by Western blot and qRT-PCR, respectively; and (4) performed RICTOR knockdown using siRNA followed by migration, invasion, and clonogenic assays. Results: In the BATTLE-2 cases, we identified 15% of RICTOR alterations (9% amplifications, 6% mutations, mutually exclusive) preferentially associated with resistance to all therapies (AKTi+MEKi, erlotinib+AKTi, sorafenib, or erlotinib). In the TCGA we found: (1) 10% of RICTOR amplifications and 3% mutations; (2) significant correlation between amplification and elevated RICTOR gene expression; and (3) a putative functional gene expression signature associated with RICTOR amplification. In diploid cell lines we found concordance between AKT phosphorylation and activation of other downstream mTORC2 targets (i.e. SGK1 and PKCα), but in RICTOR amplified cell lines we witnessed a discordant activation of these pathways, and thus were able to define unique signaling class systems in our cell lines harboring RICTOR alterations. Furthermore, following RICTOR knockdown in our amplified cell lines, a reduction in clonogenic, migratory, and invasive capacity was seen, suggesting that RICTOR amplification may provide a survival advantage in select cancer cells by tipping the signaling balance toward a non-canonical oncogenic pathway (AKT-independent). Conclusion: Rictor alterations may define a new molecular NSCLC subtype with distinct biology that expose unique avenues for therapeutic intervention. Ongoing studies are underway to explore specific therapeutic strategies, non-canonical signaling and Rictor mutations. Supported by: NHI-NCI CA155196 & 2P50CA070907-16A1 Citation Format: Dennis Ruder, Vassiliki Papadimitrakopoulou, Kazuhiko Shien, Neda Kalhor, J. Jack Lee, Waun K. Hong, Ximing Tang, Luc Girard, John D. Minna, Lixia Diao, Jing Wang, Nana E. Hanson, James Sun, Vincent Miller, Garrett Frampton, Roy S. Herbst, Ignacio I. Wistuba, Julie G. Izzo. Rictor alterations elicit non-canonical signaling mechanisms contributing to tumorigenicity and therapeutic resistance in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3576. doi:10.1158/1538-7445.AM2015-3576
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