Patient-matched Tumor Research Articles

Automated Genotyping of Biobank Samples by Multiplex Amplification of Insertion/Deletion Polymorphisms

The genomic revolution in oncology will entail mutational analyses of vast numbers of patient-matched tumor and normal tissue samples. This has meant an increased risk of patient sample mix up due to manual handling. Therefore, scalable genotyping and sample identification procedures are essential to pathology biobanks. We have developed an efficient alternative to traditional genotyping methods suited for automated analysis. By targeting 53 prevalent deletions and insertions found in human populations with fluorescent multiplex ligation dependent genome amplification, followed by separation in a capillary sequencer, a peak spectrum is obtained that can be automatically analyzed. 24 tumor-normal patient samples were successfully matched using this method. The potential use of the developed assay for forensic applications is discussed.

Tumor-Infiltrating FoxP3+ Tregs and CD8+ T Cells Affect the Prognosis of Hepatocellular Carcinoma Patients

Purpose: Tumor-infiltrating lymphocytes are considered to represent a host immune response against tumor. This study was carried out to analyze the effect of both FoxP3+ regulatory T cells (Tregs) and CD8+ T lymphocytes in prognostic value of hepatocellular carcinoma (HCC) patients. Methods: Expressions of FoxP3, CD4, CD8 and CD34 in patient-matched tumors and peritumoral tissues were assessed by immunohistochemistry for 54 HCC patients. The prognostic effect of groups with high and low numbers was evaluated by the Kaplan-Meier and Cox model analysis using median values as a cutoff. Results: Compared with the corresponding peritumoral tissue, the density of intratumoral Tregs was significantly higher, while the density of intratumoral CD8+ T cells was lower (p < 0.001 and p = 0.013, respectively). In addition, tumor-infiltrating Tregs were positively correlated with microvessel density in tumors (r = 0.334, p = 0.020). The high intratumoral Tregs density group showed a significantly lower survival rate (overall survival, p = 0.018; disease-free survival, p = 0.029). Multivariate Cox analysis revealed that intratumoral Tregs density was an independent prognostic factor for HCC. Conclusions: Tumor-infiltrating Tregs may promote HCC progression by fostering angiogenesis and decreasing CD8+ T cells. High tumor-infiltrating Tregs are thought to be an unfavorable prognostic indicator for HCC.

Human papillomavirus in oropharyngeal squamous cell carcinoma: Assessing virus presence in normal tissue and activity in cervical metastasis

Human papillomavirus (HPV) has been established as an etiologic and prognostic factor in oropharyngeal squamous cell carcinoma (OPSCC). HPV oncogenesis involves expression of E6/E7 oncoproteins, with downstream p53 degradation and pRb inhibition. Although much research has focused on HPV's oncogenic behavior in primary OPSCC, minimal information exists about HPV in adjacent normal and metastatic tissue. Retrospective cohort study Patient-matched tumor, normal, and metastatic tissue was gathered from 42 OPSCC patients and tested with real-time quantitative polymerase chain reaction (RT-qPCR), in situ hybridization (ISH), and immunohistochemistry (IHC). RT-qPCR was performed using total RNA from fresh-frozen tissues and primers for HPV16 E6, E7, and p16 transcripts. HPV ISH was performed to detect the presence of HPV DNA and IHC to detect p16 protein. Primary tumor, adjacent normal tissue, and tumor metastasis from 17 OPSCC patients were analyzed. When comparing the presence of HPV16 DNA in tumor, metastatic, and normal tissue by ISH, perfect correlation is found at all subsites (P < .0001). However, active infections determined by HPV16 E6 and E7 expression using quantitative polymerase chain reaction or p16 detection by IHC, were present only in primary and metastatic tissue (P = .0012, E6; P = .02, E7). No such correlation was found in normal tissue when compared to primary or metastatic tissue. There is a clear pattern of active HPV expression that correlates to disease course. In HPV-positive patients, all sites including primary, metastatic, and normal tissues are DNA positive. Transcriptionally active infections were detected in primary and metastatic tissues, whereas normal tissues appear to have latent infections.

Tumor-infiltrating FoxP3+ Tregs are associated with CD34 expression and prognosis of hepatocellular carcinoma

To investigate the correlation between FoxP3+ regulatory T lymphocytes (Tregs) in hepatocellular carcinomas (HCCs) and peritumoral tissues with CD34 expression and patient prognosis. Fifty-five sets of patient-matched tumors and peritumoral tissues were obtained during curative resection for HCC. In situ immunohistochemistry was used to assess and comparatively analyze Treg presence and CD34 expression in each specimen set. The relation between quantified Tregs values and various clinicopathologic factors were evaluated by the Spearman Rank Correlation test. Univariate (Log Rank test) and multivariate (Cox Regression model) analyses were used to determine the potential prognostic value of each factor. The average number of intratumoral Tregs was significantly higher than that in corresponding peritumoral tissues (10.8 (range: 4.4 to 19.4) vs. 1.4 (0.6 to 3.2), respectively; P less than 0.01). The presence of intratumoral Tregs correlated with up-regulated CD34 expression (r = 0.279, P less than 0.05). Increased number of intratumoral Tregs were significantly associated with decreased rates of overall survival (OS, P less than 0.05) and disease-free survival (DFS, P less than 0.05), and was identified as an independent prognostic factor (OS, hazard ratio (HR) = 3.310, 95% confidence interval (CI): 1.368-8.007, P less than 0.01; DFS, HR = 2.666, 95% CI: 1.321 to 6.394, P less than 0.01). Intratumoral infiltration by Tregs is a marker of poor prognosis in HCC patients.

Abstract B44: Therapeutic response of primary glioblastoma cancer stem cells relative to patient-matched non-stem tumor cells

Abstract Background: Populations of cells within glioblastoma multiforme (GBM) tumors that have properties similar to neural stem cells are thought to be the source of tumor cell self-renewal. These cancer stem cells (CSCs) may also be particularly resistant to therapy, although support for this important concept is limited. We asked whether early passage primary CSC cultures respond differently than patient-matched non-CSC cultures to both standard-of-care (Temozolomide (TMZ) and Radiation (XRT)) and experimental epigenetic therapy targeting histone deactylases (HDACs). Experimental Design: We established primary cultures of CSCs as adherent glioma neural stem cell and patient-matched non-CSC monolayer cultures in serum from 10 primary glioblastomas. To test response to standard glioma therapy, we treated early passage cultures with single or multiple physiological doses of TMZ (5–50uM), with or without XRT (2Gy). To examine the relative efficacy of the HDAC inhibitor vorinostat, the paired cultures were treated with physiological doses (1–2uM). Response to therapy was assayed by cell cycle analysis on a BD FACSCalibur and for cell viability using CellTiter-Glo. MGMT methylation was assessed by methylation-specific PCR and genome-wide methylation analysis was performed on IIIumina Infinium arrays. Results: The majority of patient-derived primary cultures, including CSC and non-CSC cell lines, are resistant to clinically relevant doses of TMZ. This result is in line with the general lack of response in most but not all GBM patients. Furthermore, the in vitro response to TMZ correlated with the methylation status of MGMT, a robust prognostic marker of survival in GBM patients treated with TMZ. Surprisingly, we observed for two pairs of patient-matched cultures that the CSC cultures were more responsive to single and multi-fraction XRT treatment than the non-CSCs. To further understand this difference in response in patient-matched cultures, we performed genome-wide DNA methylation analysis and found over 1500 genes with differential methylation between CSC and non-CSC cultures, including PTEN and CDKN2A. We examined the status of CD133, a marker of stemness, for our CSC and non-CSC cultures and found that cultures that were enriched for CD133+ cells were not more resistant to either TMZ or XRT. Finally, we examined whether vorinostat, an HDAC inhibitor currently in clinical trials with TMZ/XRT for GBM patients, could sensitize cultures to standard of care therapy. Clinically relevant doses of vorinostat led to decreased cell viability but did not appear to have a synergistic effect when combined with either TMZ or XRT. Conclusions: Our study demonstrates that early passage primary gliomas cultures can be used to assess drug response in less time than the interval between surgery and onset of adjuvant therapy. Furthermore, we find that for our patient-matched cultures, the CSC population is not uniformly more resistant to therapy in vitro than the non-CSC population. If the in vitro response mirrors patient response in vivo, the in vitro cultures might be beneficial for making clinical decisions on a patient-specific basis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B44.

Highlights of This Issue

In this study, Trujillo and coworkers show that the field of histologically normal tissue up to 1 centimeter from human breast tumors contains a population of epithelial cellswith telomerase levels similar to the patient-matched tumor. This population has a mechanism for limitless replicative capacity and exists within tissue that has previously beenshown to exhibit genomic instability and a microenvironment that supports tumor initiation. Because adequate surgical margins are often no more than a few millimeters from the tumor margin, in many cases, this tissue is not removed during breast-conserving surgery and may have implications in local recurrence rates.The transmembrane serine protease hepsin is a highly upregulated gene in prostate cancer that promotes tumor progression and metastasis in preclinical models. Based on its substrate profile, Ganesan and colleagues investigated whether hepsin activates the precursor form of the plasminogen-like growth factor macrophage-stimulating protein (MSP). They show that both soluble and cell surface–expressed hepsin efficiently convert the inactive zymogen-like MSP precursor into a functionally competent heterodimeric form that induces phosphorylation of its cognate receptor RON and downstream signaling proteins promoting epithelial cell motility. These findings suggest that hepsin regulates the MSP/RON signaling pathway in cancer, immune disorders, and tissue homeostasis.FIP200 is a component of the ULK1/Atg13/FIP200 complex essential for autophagy induction in mammalian cells, which has been previously implicated in breast cancer. It is not clear, however, how autophagy function of FIP200 may contribute to its role in breast or other cancers. Here, Bae and Guan present data suggesting a novel function for FIP200 in the regulation of DNA damage response and cell survival through its activity in autophagy.These results suggest the possibility of FIP200 or other autophagy proteins as a potential treatment to enhance the efficiency of cancer therapy using DNA damage–inducing agents.To adapt to unfavorable growth conditions such as hypoxia, mTORC1, a critical regulator of cell growth, is inactivated. mTORC1 inhibition by hypoxia is mediated by REDD1, a HIF target gene. As a consequence of VHL inactivation, HIF and hypoxia-inducible genes are constitutively upregulated in clear-cell renal cell carcinoma (ccRCC), but whether this leads to REDD1 upregulation is unclear and mTORC1 is commonly activated in ccRCC. Kucejovaand colleagues show that REDD1 is consistently upregulated in ccRCC and that, whereas in some tumors REDD1 is engaged in mTORC1 inhibition, in others, strategies have evolved to uncouple mTORC1 from REDD1. Specifically, Kucejova and colleagues found somatic mutations in REDD1 and TSC1, which is required for mTORC1 inhibition by REDD1.

Abstract B38: Therapeutic response in primary glioblastoma neural stem cells relative to patient-matched non-stem tumor cells

Abstract Background: Glioblastoma multiforme (GBM) is the most common and most malignant form of primary brain cancer. The standard of care for GBM patients is surgical resection followed by radiation (XRT) and temozolomide (TMZ). Populations of cells within GBMs that have properties similar to neural stem cells are thought to be the source of tumor cell self-renewal. These cancer stem cells (CSCs) may also be particularly resistant to therapy, although support for this important concept is limited. Additionally, widespread epigenetic changes and their ensuing dysregulation of gene networks are known to contribute to GBM formation and to therapeutic response. In particular, the widespread deacetylation of histones occurs in cancers with poor prognosis. Histone deactylase inhibitors (HDACi) are in clinical trials for GBM patients and one particular agent, vorinostat, has modest effects as a single agent in recurrent GBM. We asked whether primary-derived CSC cultures might respond differently than patient-matched non-CSC cultures to both standard-of-care and experimental epigenetic therapy. Experimental Design: We established primary cultures of CSC as adherent glioma neural stem cell (GNS) and patient-matched non-CSC monolayer cultures in serum (ML) from each 10 primary glioblastomas. To test response to standard glioma therapy, we treated early passage cultures with physiological doses of TMZ (5-50 uM), with or without XRT (2Gy). To examine the relative efficacy of HDACi, the paired cultures were treated with physiological doses of vorinostat (1 −2 uM). Response to therapy was assayed by cell cycle analysis on a BD FACSCalibur. Results: The majority (90%) of patient-derived primary cultures, including GNS and ML cell lines, are resistant to physiological doses of TMZ, similar to the general lack of response in most but not all GBM patients. When treated with TMZ and XRT alone or in combination, one particular GNS exhibited a response to XRT and the combination with TMZ, but not TMZ alone, while the patient-matched ML culture did not exhibit sensitivity to XRT, contrary to expectations. Lastly, treatment with vorinostat resulted in G2 arrest and increased cell death for 90% of cultures. However, the response observed between patient-matched GNS and ML cultures was not uniform across the panel of GBMs, with some GNS lines being more sensitive and others more resistant compared to ML cultures. Conclusions: Most primary tumor-derived cultures are resistant to TMZ, irrespective of their enrichment for CSC. Furthermore, one GNS line was more sensitive to XRT than patient-matched non-CSC line suggesting that not all glioma CSCs are resistant to therapy as previously thought. The difference in response to vorinostat between GNS and non-CSC cultures reinforces this finding. Our study demonstrates that early passage primary gliomas cultures can be used to assess drug response in a timely manner. If the in vitro response mirrors in vivo response, this patient-specific model system might be beneficial for making clinical decisions on a patient-specific basis. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B38.

Comparative analysis of thymidylate synthase at the protein, mRNA, and DNA levels as prognostic markers in colorectal adenocarcinoma

To determine the best routinely available molecular methodology for assessing thymidylate synthase (TS) as a prognostic marker in colorectal adenocarcinoma, TS was examined at the protein, mRNA, and DNA levels. Direct comparison of these routinely available assays has not been systematically studied across a large colon cancer patient cohort with long-term follow-up. We studied 150 surgically resected colorectal adenocarcinoma patients who received postoperative 5-Fluorouracil (5-FU) chemotherapy. TS immuunohistochemistry and real-time quantitative RT-PCR and PCR genotyping on patient-matched tumor and normal tissues were performed. Surprisingly, mRNA values in normal tissue varied from 0.11 to 62.0 and significantly correlated with mRNA values of matched tumor tissues. Although higher tumor/normal ratios of mRNA expression trended toward poorer patient survival, neither this nor TS immunohistochemistry results were statistically significant predictors. TS tumor genotype was generally concordant with matched normal tissues. Further, the 2R/3R genotype of 5'-TSER was significantly correlated with poorer patient survival (P = 0.0249) and was also an independent prognostic marker on multivariate analysis. TS genotyping on paraffin-embedded fixed tissues proved to be the most useful method for prediction of outcome of 5-FU treatment in patients with colorectal adenocarcinoma.

Genome-wide expression patterns of invasion front, inner tumor mass and surrounding normal epithelium of colorectal tumors

Colorectal tumors have characteristic genome-wide expression patterns that allow their distinction from normal colon epithelia and facilitate clinical prognosis. The expression heterogeneity within a primary colorectal tumor has not been studied on a genome scale yet. Here we investigated three compartments of colorectal tumors, the invasion front, the inner tumor mass, and surrounding normal epithelial tissue by microdissection and microarray-based expression profiling. In both tumor compartments many genes were differentially expressed when compared to normal epithelium. The sets of significantly deregulated genes in both compartments overlapped to a large extent and revealed various interesting known and novel pathways that could have contributed to tumorigenesis. Cells from the invasion front and inner tumor mass, however, did not show significant differences in their expression profile, neither on the single gene level nor on the pathway level. Instead, gene expression differences between individuals are more pronounced as all patient-matched tumor samples clustered in close proximity to each other. With respect to invasion front and inner tumor mass we conclude that the specific tumor cell micro-environment does not have a strong influence on expression patterns: largely similar genome-wide expression programs operate in the invasion front and interior compartment of a colorectal tumor.

Rapid protein display profiling of cancer progression directly from human tissue using a protein biochip

The complicated, changing pattern of protein expression should contain important information about the pathologic process taking place in the cells of actual tissue. Utilization of this information for the selection of druggable targets could be possible if a means existed to rapidly analyze and display changes in protein expression in defined microscopic cellular subpopulations. As a demonstration of feasibility, we show the generation of sensitive, rapid, and reproducible molecular weight protein profiles of patient-matched normal, premalignant, malignant, and metastatic microdissected cell populations from stained human esophageal, prostate, breast, ovary, colon, and hepatic tissue sections through the application of an affinity-based biochip. Reproducible, discriminatory protein biomarker profiles can be obtained from as few as 25 cells in less than 5 min from dissection to the generation of the protein fingerprint. Furthermore, these protein pattern profiles reveal reproducible changes in expression as cells undergo malignant transformation, and are discriminatory for different tumor types. Consistent protein changes were identified in the microdissected cells from patient-matched tumor and normal epithelium from eight out of eight different malignant esophageal tissue sets and three out of three malignant prostate tissue sets. A means to rapidly generate a display of expressed proteins from microscopic cellular populations sampled from tissue could be an important enabling technology for pharmacoproteomics, molecular pathology, drug intervention strategies, therapeutic assessment of drug entities, disease diagnosis, toxicity, and gene therapy monitoring. Drug Dev. Res. 49:34–42, 2000. Published 2000 Wiley-Liss, Inc.