Laser Capture Microdissected Tumor Research Articles

Abstract ES8-2: Breast Cancer Stroma: A Predictor of Clinical Outcome and Tumour Heterogeneity

Abstract Breast cancer heterogeneity is one of the principal obstacles both to predicting outcome and to determining an effective course of treatment for this disease. Individual cases demonstrate heterogeneity at multiple levels, including those parameters assessed by studies of gene expression, chromosomal aberrations, and classical and immuno-pathology. Although genomic technologies have been used to gain a better understanding of the impact of gene expression heterogeneity on breast cancer outcome by identifying gene expression signatures associated with clinical outcome, histopathological breast cancer subtypes, and a variety of cancer–related pathways and processes, relatively little is known about the effects of heterogeneity in the tumor microenvironment. We have addressed changes in stroma by analyzing changes in gene expression in stromal tissue associated with breast tumors when compared to normal breast tissue. We have integrated gene expression data from laser capture microdissected breast tumor stroma with matched normal stroma. Using this approach we have identified that the microenvironment of a breast tumor can be classified into one of six distinct molecular phenotypes exhibiting distinct biological functions and carrying prognostic information independent of existing therapeutic biomarkers and tumor subtypes. A trained predictor of 23 genes was developed and contains new information to stratify breast cancer subtypes. This is independent of clinical parameters and published predictors of outcome and identifies patients with poor outcome in multiple breast cancer expression data generated using using whole tissue. The stromal predictor selects poor outcome patients from multiple clinical subtypes of breast cancer and contains genes representing distinct biological features, including differential immune response, angiogenic response, as well as a hypoxic response. Elements of this signature are present in murine models of breast cancer. These results highlight the complex relationship between the tumor and its microenvironment, and underline the role that the stroma plays in tumor progression. These results demonstrate an important role for the tumor microenvironment in defining breast cancer heterogeneity, with a consequent impact upon clinical outcome. Novel therapies could be targeted at the processes that define the stroma classes, suggesting new avenues for the development of individualized treatment regimens. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr ES8-2.

Molecular Analysis of HER2 Signaling in Human Breast Cancer by Functional Protein Pathway Activation Mapping

Targeting of the HER2 protein in human breast cancer represents a major advance in oncology but relies on measurements of total HER2 protein and not HER2 signaling network activation. We used reverse-phase protein microarrays (RPMA) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity. Three independent study sets, comprising a total of 415 individual patient samples from flash-frozen core biopsy samples and formalin-fixed and paraffin-embedded (FFPE) surgical and core samples, were analyzed via RPMA. The phosphorylation and total levels of the HER receptor family proteins and downstream signaling molecules were measured in laser capture microdissected (LCM) enriched tumor epithelium from 127 frozen pretreatment core biopsy samples and whole-tissue lysates from 288 FFPE samples and these results were compared with FISH and immunohistochemistry (IHC). RPMA measurements of total HER2 were highly concordant (>90% all sets) with FISH and/or IHC data, as was phosphorylation of HER2 in the FISH/IHC(+) population. Phosphorylation analysis of HER family signaling identified HER2 activation in some FISH/IHC(-) tumors and, identical to that seen with FISH/IHC(+) tumors, the HER2 activation was concordant with EGF receptor (EGFR) and HER3 phosphorylation and downstream signaling endpoint activation. Molecular profiling of HER2 signaling of a large cohort of human breast cancer specimens using a quantitative and sensitive functional pathway activation mapping technique reveals IHC(-)/FISH(-)/pHER2(+) tumors with HER2 pathway activation independent of total HER2 levels and functional signaling through HER3 and EGFR.

Retrospective EGFR mutation testing of clinical specimens from the EURTAC trial of erlotinib in non-small cell lung cancer (NSCLC) using a novel allele-specific PCR (AS-PCR) assay.

10596 Background: Anti-EGFR inhibitors are superior to chemotherapy in first-line therapy of advanced EGFR-mutant NSCLC. The EURTAC trial was a randomized Phase III trial of erlotinib vs. chemotherapy in patients with EGFR-mutant NSCLC. Interim results showed significant improvement in progression-free survival (R. Rosell, ASCO 2011). An accurate rapid in vitro diagnostic for EGFR mutations is needed to select patients for this therapy. Methods: Prospective EGFR mutation testing for the trial was performed on laser-capture microdissected tumor cells using a combination of 3 lab-developed tests (LDTs), including a Length Analysis of Fluorescently-labeled PCR (Genescan) method for exon 19 deletions, a Taqman-based PCR assay for exon 21 mutation with laser-capture macrodissected tumor cells, and secondary Sanger sequencing. A subset of samples from the trial was retrospectively tested with an AS-PCR assay (cobas EGFR mutation test) which detects L858R and > 29 exon 19 deletions. The test provides automated results within 8 h; the DNA required can be isolated from one 5-micron tissue section. Four methods were compared: AS-PCR assay, LDT, direct Sanger sequencing and massively parallel sequencing (MPS; 454, Branford, CT). Results: LDT results were obtained for 1044 screened patients. Residual tumor blocks were available for 487 patients (47%), including 303 wild-type, 172 mutant (135 enrolled on the trial) and 12 inconclusive cases by the LDT. Comparison of AS-PCR and LDT results showed a positive percent agreement (PPA) – 93.7% (CI 88.8%, 96.5%), and negative percent agreement (NPA) – 97.5% (94.9%, 98.8%). Comparison of AS-PCR and Sanger results showed a PPA of 96.6% (91.7%, 98.7%) but an NPA of 88.3% (84.1%, 91.5%). Among 34 AS-PCR+/Sanger- case, MPS confirmed the presence of exon 19 deletions in 25 cases and L858R mutations in 7. Direct comparison of AS-PCR and MPS results showed a PPA of 93.1% (88.1%, 96.1%) and NPA of 97.7% (95.0%, 98.9%). Clinical outcomes for cases with mutations detected by the AS-PCR test will be presented. Conclusions: The AS-PCR assay was highly concordant with the LDT and MPS, and more sensitive than Sanger sequencing.

Abstract 1094: MicroRNA-30a inhibits vimentin expression and as a prognostic marker in breast cancer

Abstract Recent studies have suggested a significant role of microRNAs (miRNAs) in the regulation of cancer development. This study examined whether they play a role in breast cancer progression.A miRNA microarray analysis was performed on laser-capture microdissected breast tumors of different lymph-node metastasis status showing different progression signatures, indicated by overexpression of cyclin D1 and β-catenin genes, to identify specific miRNAs that exhibit significant differences in expression. Functional interaction between the candidate miRNA (i.e. miR-30a) and the gene (i.e. Vim gene, coding for vimentin, a protein involved in epithelial-mesenchymal transition) possibly regulated by miR-30a was verified. We further examined whether decreased expression of miR-30a was associated with breast cancer progression.miR-30a negatively regulated vimentin expression, by binding to the 3′-untranslated region of Vim. Ectopic expression of miR-30a was found to suppress the migration and invasiveness phenotypes of breast cancer cell lines. More importantly, breast cancer patients with decreased miR-30a level in primary cancerous sites were found to be associated with poor clinical features (late tumor stage and lymph node metastasis) and worse progression, demonstrating an increased hazard ratio (HR) for recurrence or recurrence plus mortality during the follow-up period (P<0.05). These findings provide a support of clinical importance of miR-30a in mediating breast tumor progression. Identification of miR-30a-mediated regulation of vimentin might provide a promising therapeutic target in treating breast cancer. 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 1094. doi:1538-7445.AM2012-1094

Optimized nLC-MS workflow for laser capture microdissected breast cancer tissue

Reliable sample preparation is of utmost importance for comparative proteome analysis, particularly when investigating minute amounts of clinical specimens, such as laser capture microdissected tumor tissue. In this study, we present an optimized nanoLC-MS workflow specifically for the analysis of laser capture microdissected breast cancer tissue. Analytical performance of different laser capture microdissection (LCM) functions available on the PALM system, time dependent trypsin digestion efficiency, effect of sample preparation and digestion time on peptide modification, semi-tryptic peptides and missed cleavages were evaluated. Our results show that microdissection from uncoated glass slides results in protein degradation; that protease and phosphatase inhibitors do not result in detectable improvement in number of peptides or semi-tryptic peptides; and that digestion time longer than four hours drastically reduces the number of missed cleavages, but also increases the number of unexpectedly modified peptides. Overalkylation was the most dominant side-reaction, which significantly increased overnight (P=0.05). The latter effect could almost completely be reverted by the use of a quenching agent (P=0.001). Taken together, our results show that it is of importance to carefully control sample handling steps so that reliable protein identification and quantitation can be performed within comparative proteomics studies using LCM. This article is part of a Special Issue entitled: Proteomics: The clinical link.

MiR-183-96-182 Cluster Is Overexpressed in Prostate Tissue and Regulates Zinc Homeostasis in Prostate Cells

Decreased zinc levels are a hallmark of prostate cancer tumors as zinc uniquely concentrates in healthy prostate tissue. Increased dietary zinc correlates with decreased risk of advanced prostate cancer and decreased mortality from prostate cancer. The mechanisms of prostatic zinc homeostasis are not known. Lower zinc levels in the tumor are correlated directly with decreased expression of the zinc transporter hZIP1. We report identification of a microRNA cluster that regulates multiple zinc transporters, including hZIP1. Screening in laser capture microdissected prostate cancer tumors identified miR-182 as a potential regulator of hZIP1. Regulation of hZIP1 by miR-182 via two binding sites was confirmed in primary prostate cell cultures. miR-96 and miR-183 are expressed as a cluster with miR-182 and share similar sequences. Array profiling of tissue showed that miR-183, -96, and -182 are higher in prostate cancer tissue compared with normal prostate. Overexpression of the entire miR-183-96-182 cluster suppressed five additional zinc transporters. Overexpression of miR-183, -96, and -182 individually or as a cluster diminished labile zinc pools and reduced zinc uptake, demonstrating this miR cluster as a regulator of zinc homeostasis. We observed regulation of zinc homeostasis by this cluster in prostate cells and HEK-293 cells, suggesting a universal mechanism that is not prostate-specific. To our knowledge, this is the first report of a miR cluster targeting a family of metal transport proteins. Individually or as a cluster, miR-183, -96, and -182 are overexpressed in other cancers too, implicating this miR cluster in carcinogenesis.

Protein Pathway Activation Mapping of Brain Metastasis from Lung and Breast Cancers Reveals Organ Type Specific Drug Target Activation

Brain metastases are the most common fatal complication of systemic cancer, especially of lung (40-50%) and breast (20-30%) cancers. In this era of personalized therapy, there is a critical need to uncover the signaling architecture of brain metastases; however, little is known about what signaling pathways are activated in the context of the brain microenvironment. Using a unique study set of 42 brain metastases from patients with breast or nonsmall cell lung cancer (NSCLC), the phosphorylation/activation states of 128 key signaling proteins involved in cancer signaling were measured in laser capture microdissected tumor epithelium using reverse phase protein microarray (RPMA) technology. Distinct pathway activation subgroups from both breast and lung metastases were underpinned by, among others, ERBB2, AKT, mTOR, EGFR, SMAD, and ERK-p38 signaling. Breast cancer metastases showed significantly (p < 0.05) higher activation of the c-ERBB2/IGFR-AKT pathway network compared to NSCLC metastases, whereas NSCLC metastases to the brain exhibited higher relative levels of many members of the EGFR-ERK signaling network. Protein pathway activation mapping using RPMA revealed both the heterogeneity of signaling networks in brain metastases that would require a prior stratification to targeted therapies as well as the requirement of direct analysis of the metastatic lesion.

Phosphoproteomic Analysis of Signaling Pathways in Head and Neck Squamous Cell Carcinoma Patient Samples

Molecular targeted therapy represents a promising new strategy for treating cancers because many small-molecule inhibitors targeting protein kinases have recently become available. Reverse-phase protein microarrays (RPPAs) are a useful platform for identifying dysregulated signaling pathways in tumors and can provide insight into patient-specific differences. In the present study, RPPAs were used to examine 60 protein end points (predominantly phosphoproteins) in matched tumor and nonmalignant biopsy specimens from 23 patients with head and neck squamous cell carcinoma to characterize the cancer phosphoproteome. RPPA identified 18 of 60 analytes globally elevated in tumors versus healthy tissue and 17 of 60 analytes that were decreased. The most significantly elevated analytes in tumor were checkpoint kinase (Chk) 1 serine 345 (S345), Chk 2 S33/35, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) S65, protein kinase C (PKC) ζ/ι threonine 410/412 (T410/T412), LKB1 S334, inhibitor of kappaB alpha (IκB-α) S32, eukaryotic translation initiation factor 4E (eIF4E) S209, Smad2 S465/67, insulin receptor substrate 1 (IRS-1) S612, mitogen-activated ERK kinase 1/2 (MEK1/2) S217/221, and total PKC ι. To our knowledge, this is the first report of elevated PKC ι in head and neck squamous cell carcinoma that may have significance because PKC ι is an oncogene in several other tumor types, including lung cancer. The feasibility of using RPPA for developing theranostic tests to guide personalized therapy is discussed in the context of these data.

ADAM12 and ADAM17 Gene Expression in Laser-capture Microdissected and Non-microdissected Breast Tumors

ADAM (a disintegrin and metalloprotease)12 and ADAM17 are multidomain transmembrane proteins involved in ectodomain shedding of cytokines, growth factors and adhesion molecules, with pivotal activities in the tumor microenvironment. The aim of this study was to confirm the up-regulation of ADAM17 and ADAM12 gene splicing variants in breast tumors and to delineate their expression between laser-capture microdissected (LCM) and non-microdissected breast tumors. The gene expression was analyzed by quantitative-reverse transcription-PCR in a total sample of 109 breast tumors paired with corresponding non-neoplastic breast tissues. ADAM12 and 17 proteins expression for corresponding tissue samples was confirmed by immunohistochemistry. ADAM12S, 12L and 17 genes were significantly up-regulated in either malign or benign LCM samples when compared to non-tumor controls. For non-LCM samples, it was obtained also an increased expression for ADAM12 and 17 genes in cancers, while in benign tumors only ADAM12 variants were significantly up-regulated compared to controls. When benign versus malignant tumors were compared, in LCM samples all investigated genes displayed a higher expression in cancers, whereas in non-LCM, ADAM12 variants were overexpressed in benign samples. The increased expression of ADAM12 protein in the tumor cells and stroma of benign breast diseases was immunohistochemically confirmed. These differences between LCM and non-LCM samples were explained by the contribution of the stroma to the expression of this marker. This study underlines the accuracy conferred by homogenous LCM samples on gene expression profiles and confers further evidence regarding the role of ADAM12 and 17 in the breast tumorigenesis and progression.

Overexpression and Activation of the α9-Nicotinic Receptor During Tumorigenesis in Human Breast Epithelial Cells

Large epidemiological cohort studies in the United States have indicated that active and passive smoking are associated with increased breast cancer risk. However, there was no direct evidence of an effect of tobacco carcinogens on the cellular molecules involved in breast tumorigenesis. Reverse transcription-polymerase chain reaction was used to determine the expression of all of the nicotinic acetylcholine receptor (nAChR) subunits in 50 human breast cancer samples and to determine the expression of the alpha9-nAChR subunit in 276 surgical and laser capture microdissected breast tumor vs normal tissue pairs. Stable MDA-MB-231 breast cancer cell lines were established in which expression of the alpha9-nAChR subunit was inhibited using short interfering RNA. MCF-10A normal human breast epithelial cells were established in which the alpha9-nAChR subunit could be conditionally overexpressed by removal of doxycycline from the culture fluid. Cell proliferation and soft agar assays and tumor growth in nude mice were used as measures of cell transformation. All statistical tests were two-sided. In 186 (67.3%) of the 276 paired samples, alpha9-nAChR mRNA was expressed at (mean 7.84-fold) higher levels in breast cancers than in surrounding normal tissue. Stable expression of alpha9-nAChR short interfering RNA in MDA-MB-231 cells attenuated nicotine-stimulated proliferation and growth in soft agar and reduced tumor volume when the cells were introduced as xenografts in SCID mice (n = 5 mice per group; mean tumor volume at 6 weeks treatment in mice injected with Si alpha9 cells = 995.6 mm(3), in mice injected with parental cells = 2993.2 mm(3), difference = 1997.6 mm(3), 95% confidence interval [CI] = 1705 to 2290.2 mm(3), P = .009). Long-term treatment of MCF-10A normal breast epithelial cells with either nicotine or its active metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, triggered precancerous transformation as defined by soft agar assay. Inducible overexpression of alpha9-nAChR in MCF-10A cell xenografts in nude mice substantially increased tumor growth (n = 5 mice per group; DOX+, mean tumor volume without nicotine vs with nicotine = 266.2 vs 501.6 mm(3), difference = 235.4 mm(3), 95% CI = 112.7 to 358 mm(3), P = .009; DOX-, mean tumor volume without nicotine vs with nicotine = 621.2 vs 898.6 mm(3), difference = 277.4 mm(3), 95% CI = 98.1 to 456.7 mm(3), P = .016; mean tumor volume in the presence of nicotine, DOX+ vs DOX- = 501.6 vs 898.6 mm(3), difference = 397 mm(3), 95% CI = 241.3 to 552.6 mm(3), P = .009). The alpha9-nAChR is important for nicotine-induced transformation of normal human breast epithelial cells.

Overexpression of C-MYC oncogene in prostate cancer predicts biochemical recurrence

Alterations of chromosome 8, including amplification at 8q24 harboring the C-MYC oncogene, have been noted as one of the most common chromosomal abnormalities in prostate cancer (CaP) progression. However, the frequency of C-MYC alterations in CaP has remained uncertain. A recent study, using a new anti-MYC antibody, described prevalent upregulation of nuclear C-MYC protein expression as an early oncogenic alteration in CaP. Further, we have recently reported regulation of C-MYC expression by ERG and a significant correlation between C-MYC overexpression and TMPRSS2-ERG fusion in early stage CaP. These emerging data suggest that increased C-MYC expression may be a critical and early oncogenic event driving CaP progression. In this study, we assessed whether C-MYC mRNA overexpression in primary prostate tumors was predictive of more aggressive tumor or disease progression. Our approach was to quantitatively determine C-MYC mRNA expression levels in laser capture micro-dissected tumor cells and matched benign epithelial cells in a radical prostatectomy cohort with long follow-up data available. On the basis of our results, we conclude that elevated C-MYC expression in primary prostate tumor is biologically relevant and may be a predictor of future biochemical recurrence.

Quantification of β-Catenin Signaling Components in Colon Cancer Cell Lines, Tissue Sections, and Microdissected Tumor Cells using Reaction Monitoring Mass Spectrometry

Reaction monitoring mass spectrometry has emerged as a powerful tool for targeted detection and quantification of proteins in clinical samples. Here, we report the use of gel electrophoresis for protein fractionation and liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM) screening for quantitative analysis of components from the Wnt/beta-catenin signaling pathway, which contributes to colon tumor formation and progression. In silico tools are used to design LC-MRM screens for each target protein. Following successful peptide detection, stable isotope labeled peptides are synthesized and developed as internal standards. Then, the assays are implemented in colon cancer cell lines to achieve detection in minimal amounts of cells, compatible with direct translation to clinical specimens. Selected assays are compared with qualitative results from immunoblotting (Westerns) and translated to individual frozen colon tissue sections and laser capture microdissected tumor cells. This LC-MRM platform has been translated from in vitro models to clinical specimens, forming the basis for future experiments in patient assessment.

Phase I/II personalized therapy trial for metastatic colorectal cancer using functional pathway mapping: Stratification to imatinib therapy.

TPS194 Background: Profiling protein signaling pathways in human cancer is critical because proteins represent the targets for new molecular guided therapeutics. Pathway mapping of liver metastasis from patients with stage IV colorectal cancer (CRC) revealed the unexpected activation of the drug targets of imatinib: PDGFR, c-kit and c-abl. Based on these results, we are conducting a trial whereby patients with advanced CRC with WT kras are stratified for imatinib therapy in conjunction with panitumimab, based on the pathway activation status obtained from metastatic tumor biopsies using Reverse Phase Protein Microarray (RPMA) technology. This trial represents the first personalized therapy trial for stage IV CRC patients stratifying patients to targeted therapy based upon functional protein pathway activation of a metastatic lesion. The ability to perform pathway- based stratification in a clinical setting represents an important milestone for future personalized therapy trials. Further patient enrollment will be required to determine the clinical utility of this particular combination therapy for patients with stage IV CRC. Methods: Stage IV CRC patients with WT kras who progressed on prior therapy are eligible. A liver biopsy is taken for functional pathway activation analysis of PDGFR, c-kit and c-abl by RPMA analysis of the laser capture microdissected (LCM) tumor epithelium. A predefined cut-point, based on the activation of these proteins of an independent cohort of colorectal metastases, is used to stratify patients to: Arm 1: 28-day run-in with imatinib monotherapy (300-600 mg/kg/day) followed by a second biopsy and addition of panitumumab (6 mg/kg) or Arm 2: panitumimab alone. Evaluation is per RECIST. Further, comparisons are made with sequential biopsies of the liver metastasis obtained pre and post treatment with imatinib and downstream pathway activation of AKT, ERK and EGFR pathways is analyzed. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Theranostics Health Theranostics Health Theranostics Health

Protein pathway biomarker analysis of human cancer reveals requirement for upfront cellular-enrichment processing

Tissues are complex structures composed of different cell types, each of which present specific functions and characteristics. To better understand and measure the effect of tumor cell enrichment on protein pathway profiling and drug target activation measurements, the signaling activation portraits of laser capture microdissected (LCM) cancer epithelium and tumor stroma were compared with patient-matched whole-tissue specimens from 53 primary colorectal cancer samples. Microdissected material and whole-tissue lysate from contiguous cryostat sections were subjected to reverse-phase protein microarray analysis to determine the level of phopshorylation and expression of 75 different proteins known to be involved in cancer progression. The results revealed distinct differences in the protein activation portraits of cancer epithelium and stroma. Moreover, we found that the signaling activation profiles of the undissected whole-tissue specimens are profoundly different from the matched LCM material. Attempts to rescale the undissected pathway information based on percent endogenous tumor epithelium content were unsuccessful in recapitulating the LCM tumor epithelial signatures. Analysis of epidermal growth factor receptor phosphorylation and COX2 expression in these same sample sets revealed wholesale differences in the rank ordering of patient determination when LCM was compared with undissected samples. On the basis of these data, we conclude that accurate protein pathway activation status, which is under evaluation as a basis for patient selection and stratification for personalized therapy, must include upfront cellular-enrichment techniques such as LCM to generate accurate drug target activation status.

Up‐regulation of the inflammatory‐reparative phenotype in human prostate carcinoma

Recent studies have underlined the role of tumor cells in the endogenous synthesis of pro-inflammatory molecules. We tested whether malignant progression in prostate cancer was associated with the activation of a phenotype typical of the innate immune system. The expression of a set of molecules involved in tissue inflammation and repair was measured by real-time PCR and Western blot analysis in prostate samples in the absence or slight presence of a detectable leukocyte infiltrate. Whole tumor and non-tumor samples were analyzed in addition to laser-capture microdissected tumor and host epithelium. Receptor for advanced glycation end products, purine receptor, inducible enzymes cyclooxygenase-2 and nitric oxide synthase-2, pentraxin-3 and growth-survival factor receptors such as epithelial growth factor and estrogen alpha and beta receptors were all studied. A global survey approach showed an up-regulation in tumor samples of all of the studied genes, with the exception of ERbeta. A laser-capture microdissection approach highlighted over-expression of pro-inflammatory molecules in each tumor sample examined. Nuclear translocation of nuclear factor-kB subunit p65 was observed in tumor tissues. These data support the evidence that molecules typical of the innate immune system, similar to that of activated leukocytes, are produced by prostate epithelial cells and that their expression is up-regulated in malignant cells. We suggest that the observed pro-inflammatory and repair process activation may represent an important molecular mechanism in the progression of prostate cancer.

Multiplexed Cell Signaling Analysis of Metastatic and Nonmetastatic Colorectal Cancer Reveals COX2-EGFR Signaling Activation as a Potential Prognostic Pathway Biomarker

The identification of prognostic determinants of colorectal cancer (CRC), including prediction of occult metastasis, is of urgent consideration, based on the tremendous differences in outcome and survival between patients who present with metastasis or develop metastasis versus those patients with organ-confined or nonrecurrent disease. Currently, a great deal of attention has been focused on using gene expression profiles of tumor specimens as a launch point for prognostic biomarker discovery. In our study, we chose to focus on functional protein-based pathway biomarkers as a new information archive because it is these proteins that form the functional signaling networks that control cell growth, motility, apoptosis, survival, and differentiation. We used reverse-phase protein microarray analysis of laser capture microdissected CRC tumor specimens to profile broad cell signaling pathways from patients who presented with liver metastasis versus patients who remained recurrence free after follow-up. Our results indicate that members of the EGFR and COX2 signaling pathways appear differentially activated in the primary tumors of patients with synchronous metastatic disease. If validated in larger study sets, this pathway defect might be useful as a prognostic clinical tool as well as a guide to potential therapeutic intervention strategies that target occult disease and/or preventative measure.

Decreased expression of thymidine phosphorylase/platelet‐derived endothelial cell growth factor in basal cell carcinomas

Thymidine phosphorylase (TP)/platelet-derived endothelial cell growth factor is associated with tumor angiogenesis. We evaluated the TP mRNA and protein expression in basal cell carcinomas (BCC) and in various skin tumors including numerous BCC histological simulants. Immunohistochemistry was performed on 99 paraffin sections of formalin-fixed skin tumors using monoclonal antibodies (mAb) against TP. TP mRNA levels were measured by real time RT-PCR in whole BCCs (wBCC) and laser capture microdissected (LCM) BCC tumor cells. TP immunostaining was negative in all BCC variants and in most of the benign trichogeneic tumors studied. By contrast, TP was constantly immunodetected in actinic keratosis (AK), squamous cell carcinomas (SCC), syringomatous carcinomas (SC), basosquamous carcinomas (BSC) and melanomas. TP mRNA levels were low and statistically not different in wBCC and normal skin but were strongly downregulated in LCM-BCC as compared with LCM-normal epidermis. We concluded that (i) anti-TP mAb is an useful marker to differentiate BCC from AK, SCC, BSC and SC but not from trichoblastic tumors, (ii) the lack of TP protein expression in BCC tumoral cells is linked to transcriptional regulatory mechanisms, (iii) the low TP mRNA levels in whole BCC may be related to the low intra-tumoral microvessel density, the slow growth and the very low metastatic potential of these tumors.

Higher Tumor to Benign Ratio of the Androgen Receptor mRNA Expression Associates with Prostate Cancer Progression after Radical Prostatectomy

Alterations of androgen receptor (AR) functions caused by overexpression, amplification, or mutation have been described in a significant subset of advanced prostate cancer (CaP). Because AR mutations or amplification are rare in early stage CaP, we hypothesized that altered AR expression in prostate tumor cells may provide a prognostic indicator of disease progression. RNA from laser capture microdissected (LCM) tumor and benign epithelial cells from radical prostatectomy specimens of 115 hormone-naive patients were studied. Expression of AR and GAPDH genes were measured by duplex quantitative real-time polymerase chain reaction (RT-PCR) in 230 specimens. A ratio of the expression of AR gene, normalized to GAPDH gene expression in the same specimens, was compared in tumor and benign epithelial cells (tumor-to-benign ratio) and correlated with clinicopathologic features. Paired t test analysis revealed a 62% lower AR expression in tumor tissue compared with benign tissue (P = 0.0005). However, multivariate Cox proportional hazards regression analysis of time to PSA recurrence revealed that higher tumor cell associated AR expression (continuous, log-transformed), significantly increases odds of prostate-specific antigen (PSA) recurrence (P = 0.0139) when controlling for age at surgery, race, time from diagnosis to surgery, risk stratification, pathologic T stage, Gleason sum, and margin status. Quantitative determination of AR gene expression levels in prostate epithelial cells may be useful for predicting PSA recurrence. This study supports the accumulating data suggesting that gain of AR function may contribute to CaP progression.

Contaminating cells alter gene signatures in whole organ versus laser capture microdissected tumors: a comparison of experimental breast cancers and their lymph node metastases

Genome-wide expression profiling has expedited our molecular understanding of the different subtypes of breast cancers, as well as defined the differences among genes expressed in primary tumors and their metastases. Laser-capture microdissection (LCM) coupled to gene expression analysis allows us to understand how specific cell types contribute to the total cancer gene expression signature. Expression profiling was used to define genes that contribute to breast cancer spread into and/or growth within draining lymph nodes (LN). Whole tumor xenografts and their matched whole LN metastases were compared to LCM captured cancer cells from the same tumors and matched LN metastases. One-thousand nine-hundred thirty genes were identified by the whole organ method alone, and 1,281 genes by the LCM method alone. However, less than 1% (30 genes) of genes that changed between tumors and LN metastases were common to both methods. Several of these genes have previously been implicated in cancer aggressiveness. Our data show that whole-organ and LCM based gene expression profiling yield distinctly different lists of metastasis-promoting genes. Contamination of the tumor cells, and cross reactivity of mouse RNA to human-specific chips may explain these differences, and suggests that LCM-derived data may be more accurate.

Low-level genomic instability is a feature of papillary thyroid carcinoma: an array comparative genomic hybridization study of laser capture microdissected papillary thyroid carcinoma tumors and clonal cell lines.

Abstract Context.—Previous chromosomal comparative genomic hybridization (CGH) studies of papillary thyroid carcinoma (PTC) have demonstrated a low prevalence of aberrations, with the majority of tumors showing no evidence of chromosomal instability. The technique of CGH can be optimized, however, using array CGH and laser capture microdissection to ensure pure cell populations for analysis. Objective.—To assess PTC using array CGH applied to laser capture microdissected tumor cells and pure cell cultures. Design.—Well-characterized PTC (known ret/PTC and BRAF mutation status), including samples from 5 tumors with classic morphology, 3 follicular variant tumors, and 3 clonal PTC cell lines, were analyzed. Results.—Copy gain and loss occurred in all of the tumor cases and cell lines examined. The most common recurrent aberrations involved gains on chromosomes 1, 5, 7, 11, 15, 17, and 22, with recurrent deletions occurring on chromosomes 4, 18, and 19. Analysis of the data from the 8 tumor samples showed th...