Fresh Tumor Tissue Research Articles

Achievements and challenges of adoptive T cell therapy with tumor-infiltrating or blood-derived lymphocytes for metastatic melanoma: what is needed to achieve standard of care?

Adoptive cell therapy (ACT) based on autologous T cell derived either from tumor as tumor-infiltrating lymphocytes (TILs) or from peripheral blood is developing as a key area of future personalized cancer therapy. TIL-based ACT is defined as the infusion of T cells harvested from autologous fresh tumor tissues after ex vivo activation and extensive expansion. TIL-based ACT has so far only been tested in smaller phase I/II studies, but these studies consistently confirm an impressive clinical response rate of up to 50 % in metastatic melanoma including a significant proportion of patients with durable complete tumor eradication. These remarkable results justify the need for a definitive phase III trial documenting the efficacy of this type of T cell-based Advanced Therapy Medicinal Product in order to pave the way for regulatory approval and implementation of TIL therapy as a new treatment standard in oncology practice. TIL-based ACT can, however, only be offered to a limited group of patients based on the need for accessible tumor tissue, the complexity of TIL production procedures, and the very intensive nature of this three-step treatment including both high-dose chemotherapy and interleukin-2 in addition to T cell infusion. To this end, adoptive T cell therapy using peripheral blood mononuclear cell-derived T cells could be a welcome alternative to circumvent these limitations and broaden up the applicability of ACT. Here, we discuss current initiatives in this focused research review.

A Translational Study of the Neoplastic Cells of Giant Cell Tumor of Bone Following Neoadjuvant Denosumab

Giant cell tumor of bone is a primary bone tumor that is treated surgically and is associated with high morbidity in many cases. This tumor consists of giant cells expressing RANK (receptor activator of nuclear factor-κB) and mesenchymal spindle-like stromal cells expressing RANKL (RANK ligand); the interaction of these cells leads to bone resorption. Denosumab is a monoclonal antibody that binds RANKL and directly inhibits osteoclastogenesis. Clinical studies have suggested clinical and histological improvement when denosumab was administered to patients with a giant cell tumor. However, no studies have yet examined the viability and functional characteristics of tumor cells following denosumab treatment. Specimens were obtained from six patients with a histologically confirmed giant cell tumor. Two of the patients had been treated with denosumab for six months. Primary cultures of stromal cells from fresh tumor tissue were established. Cell proliferation was measured over a two-day time course. The expression of RANKL and osteoprotegerin was analyzed with use of real-time PCR (polymerase chain reaction). Histological specimens from both patients who had completed denosumab treatment showed the absence of giant cells but persistence of stromal cells. Cell proliferation studies indicated that proliferation of stromal cells cultured from clinical specimens following denosumab treatment was approximately 50% slower than that of specimens from untreated patients. The expression of RANKL in the specimens from the treated patients was almost completely eliminated. Once the giant cell tumor tissue was no longer exposed to denosumab, the stromal cells continued to proliferate in vitro, albeit to a lesser degree. However, they also showed almost complete loss of RANKL expression. It is clear that treatment with denosumab only partially addresses the therapeutic need of patients with a giant cell tumor by wiping out the osteoclasts but leaving the neoplastic stromal cells proliferative.

Increased response of oxaliplatin or irinotecan on in vitro chemosensitivity to 5-fluorouracil in patients with colorectal cancer

Purpose: Five-fluorouracil (5-FU) is the basement drug of chemotherapy in patients with colorectal cancer. Recently, 5-FU and oxaliplatin or irinotecan is to be used by merging. This study investigated the chemosensitivity of 5-FU in patients with colorectal cancer, the additional effects of oxaliplatin or irinotecan to chemosensitivity when they are combined, and the effects of clinicopathologic factors to chemosensitivity. Methods: We performed in vitro chemosensitivity test for the fresh tumor tissue of 48 patients of colorectal cancer and evaluated the chemosensitivity to standard drugs (5-FU, 5-FU plus oxaliplatin: FOx, and 5-FU plus irinotecan: FIri) by using Histoculture drug response assay. Results: The average chemosensitiviy of FOx and FIri were significantly higher than that of 5-FU (32.3% and 36.0% vs. 21.8%, P<0.001). The chemosensitivity of FOx in the lymph node (LN) negative group was higher than LN positive group (35.3% vs. 19.3%, P=0.008). The group of under 70-year-old and the group of negative distant metastasis showed the trends of increased sensitivity to FOx (P=0.052, respectively). The chemosensitivity to FIri in the well or moderately differentiated group was significantly higher than the poorly differentiated goup (35.2% vs. 15.0%, P=0.038). Ki-67 positive group showed significantly increased chemosensitivity to FIri (P=0.021). Conclusion: This study demonstrates that combination of oxaliplatin or irinotecan to 5-FU can be more effective than 5-FU on in vitro chemosensitivity assay in colorectal cancer tissues. Oxaliplatin might be effective in LN negative group, and irinotecan might be effective in well differentiated group and Ki-67 positive group. Keywords: Anticancer drug sensitivity tests, Colorectal neoplasm, Fluorouracil, Oxaliplatin, Irinotecan

Establishment of an orthotopic transplantation model of tongue carcinoma.

This study aims to establish an orthotopic transplantation model of rabbit tongue carcinoma and study its biological characteristics. Tongue carcinoma was induced in purebred New Zealand white rabbits by exposure to 7,12-dimethylbenz[α]anthracene and mechanical stimulation. Fresh tumor tissues obtained from the induced tongue carcinoma model were then serially transplanted orthotopically into tongues of healthy rabbits. The tumor formation rate, invasion to surrounding tissues, regional lymph node metastases, and distant-organ metastases were investigated. Morphological observation by optical and electron microscopy, immunohistochemical examination, and chromosome analysis were performed. An orthotopic transplantation model of rabbit tongue carcinoma, designated as RSCC-2, was established. The tongue cancer was poorly differentiated squamous cell carcinoma. The tumor cell was hypotetraploid with a chromosome mode of 70. Immunohisto chemical examination showed positive staining for keratin. The tongue carcinoma survived in rabbits for 73 rounds of transplantation, using 465 rabbits in total. The average latent period was 12.5 days, and the average rabbit survival period was 37.5 days. The tumor formation rate was 10% to 20% in the first 20 rounds and increased gradually thereafter. After the 45th transplantation, the tumor formation rate and success rate of preservation in liquid nitrogen reached 100%. Regional lymph node metastases (35%) and lung metastases (20%) occurred after 50 rounds. In the advanced stage, tumors invaded the entire tongue. Animals suffered from weight loss and died of cachexia. RSCC-2 is the first animal model for orthotopical transplantation of primary tongue carcinoma. It successfully simulates the clinical pathological process of primary tongue cancer in human, provides invaluable insights into the pathogenesis and metastasis mechanisms, and can be useful for evaluating new therapeutics for the treatment of tongue cancer.

BRaf and MEK Inhibitors Differentially Regulate Cell Fate and Microenvironment in Human Hepatocellular Carcinoma

Small molecule inhibitors of the mitogen-activated protein kinase (MAPK) pathway, such as sorafenib, represent novel treatment options for advanced hepatocellular carcinoma. The aim of our study was to identify downstream targets as biomarker candidates that are directly linked to the oncogenic MAPK pathway in hepatocellular carcinoma and correlate with inhibition of this pathway by multikinase inhibitors. Hepatocellular carcinoma cell lines and fresh tumor and tumor-free liver tissues from patients with hepatocellular carcinoma were incubated with different BRaf or MEK inhibitors and analyzed for kinase phosphorylation, proliferation, induction of apoptosis, and chemokine secretion. Hepatocellular carcinoma cell lines responded differentially to these inhibitors in a dose-dependent manner, even those targeting the same kinase. Sorafenib inhibited both MEK1 and ERK1/2 phosphorylation at high but increased signaling at low concentrations. Similarly, PLX4720 increased MEK/ERK signaling independently from mutations in BRaf or NRas. MEK inhibitors decreased ERK1/2 phosphorylation in a dose-dependent manner. These signaling characteristics correlated with inhibition of proliferation, induction of apoptosis, and chemokine secretion. Fresh tissues derived from patients diagnosed with primary hepatocellular carcinoma responded to these inhibitors with changes in their microenvironment following the patterns observed in hepatocellular carcinoma cells. Oncogenic signaling of the MAPK pathway influences hepatocellular carcinoma sensitivity to treatment with BRaf and MEK inhibitors about cell fate independently from mutations in BRaf and NRas. MAPK inhibitors have a strong impact on chemokine secretion as a consequence of interference with oncogenic signaling. Therefore, novel biomarker candidates associated with the hepatocellular carcinoma microenvironment may be developed for prediction and monitoring of treatment response to small molecule inhibitors.

MP39-19 L-SELECTIN (CD62L) EXPRESSION IN HIGH GRADE UROTHELIAL CARCINOMA AS A POTENTIAL MARKER OF METASTATIC DISEASE

You have accessJournal of UrologyBladder Cancer: Basic Research IV1 Apr 2014MP39-19 L-SELECTIN (CD62L) EXPRESSION IN HIGH GRADE UROTHELIAL CARCINOMA AS A POTENTIAL MARKER OF METASTATIC DISEASE Dharamainder Choudhary, Poornima Hegde, Shilpa Choudhary, Kevin Claffey, Pramod Srivastava, Carol Pilbeam, and John Taylor Dharamainder ChoudharyDharamainder Choudhary More articles by this author , Poornima HegdePoornima Hegde More articles by this author , Shilpa ChoudharyShilpa Choudhary More articles by this author , Kevin ClaffeyKevin Claffey More articles by this author , Pramod SrivastavaPramod Srivastava More articles by this author , Carol PilbeamCarol Pilbeam More articles by this author , and John TaylorJohn Taylor More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1334AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES L-selectin (CD62L) is a vascular adhesion molecule constitutively expressed on leucocytes with a primary function of directing leucocyte migration and homing of lymphocytes to lymph nodes (LNs). Microarray data from laser captured, micro-dissected human specimens of high grade muscle invasive (MIBC) vs. low grade (LGBC) bladder cancers found CD62L to be the highest differentially expressed gene between the groups. We further characterized the mRNA and protein expression of CD62L in high grade cancer and its potential as a marker for metastatic disease. METHODS MIBC and LGBC fresh frozen, paraffin embedded and serum samples were obtained from the UCHC tumor bank. mRNA was evaluated by quantitative polymerase chain reaction (qPCR) and protein levels by immunohistochemistry (IHC) and enzyme linked immunosorbant assay (ELISA). Flow cytometry (FACS analysis) was used to identify the relative number of cells expressing CD62L in fresh tumor tissue. In silico studies were performed using the Oncomine Database. RESULTS Quantification of CD62L transcripts in high grade metastatic MIBC vs. LGBC frozen specimens(microarray; 3.81 ± 2.94 vs 0.06 ± 0.01 p=0.04 ,confirmed by qPCR 0.14 ± 0.04 vs. 23.31 ± 22.47) and immunohistochemistry on paraffin embedded high grade metastatic MIBC vs. LGBC specimens confirmed the elevated expression of CD62L in MIBC samples. Localization of CD62L was also confirmed in discrete foci of bladder tumor cells in LN specimens of high grade metastatic disease. Upregulated expression of CD62L (9.4-fold, p<0.01) was observed by FACS analysis of freshly isolated tumor cells from high grade cancers vs. LGBC specimens. Circulating CD62L levels were also found to be higher (640 ± 75 vs. 558 ± 34 ng/ml) in serum samples from patients with high grade metastatic vs. high grade non-metastatic MIBC. Furthermore, in silico analysis using the Oncomine microarray database showed an association of CD62L expression with tumor aggressiveness and clinical outcomes. CONCLUSIONS Our preliminary findings are the first report of increased expression of CD62L in MIBC and suggest a role in metastatic spread to LNs. CD62L could act as a potential marker for predicting patients who are at high risk of developing or harboring metastatic disease. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e433 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Dharamainder Choudhary More articles by this author Poornima Hegde More articles by this author Shilpa Choudhary More articles by this author Kevin Claffey More articles by this author Pramod Srivastava More articles by this author Carol Pilbeam More articles by this author John Taylor More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Stemness state regulators SALL4 and SOX2 are involved in progression and invasiveness of esophageal squamous cell carcinoma

Cancer stem cells, as a subgroup of tumor cells, resemble critical properties of embryonic stem cells (ESCs) such as self-renewal and maintenance of stemness state. SALL4 and SOX2 are two main transcription factors involving in maintenance of pluripotency, self-renewal and cell fate decision in ESCs. In this study, we aimed to elucidate the expression levels of these important transcription factors in esophageal squamous cell carcinoma (ESCC) and to reveal their probable roles in maintenance and progression of the disease. The expression level of SALL4 and SOX2 was analyzed in fresh tumoral tissues in comparison with distant tumor-free tissues of 50 ESCC patients by relative comparative real-time PCR. SALL4 and SOX2 were overexpressed in 64 and 32% of tumor samples, respectively, in significant correlation with each other (p = 0.028). There was a significantly inverse correlation between low level of SALL4 expression and metastasis of tumor cells into the lymph nodes (p = 0.035). Furthermore, co-overexpression of the genes was significantly correlated with the depth of tumor invasion (p = 0.045) and metastasis to the lymph nodes (p = 0.049). SALL4 and SOX2 are co-overexpressed in ESCC and have a significant correlation with invasion and metastasis of the disease. To the best of our knowledge, this is the first report of SALL4 clinical relevance in ESCC to date. The clinical consequences of SALL4-SOX2 association suggest a possible functional interaction between these factors in regulation of ESCC maintenance and aggressiveness and introduce these regulators of stemness state as potentially interesting therapeutic targets to bring new opportunities for onco-therapeutic modalities.

Role of PAX8 in the regulation of MET and RON receptor tyrosine kinases in non-small cell lung cancer

BackgroundNon-small cell lung cancers (NSCLC) are highly heterogeneous at the molecular level and comprise 75% of all lung tumors. We have previously shown that the receptor tyrosine kinase (RTK) MET frequently suffers gain-of-function mutations that significantly promote lung tumorigenesis. Subsequent studies from our lab also revealed that PAX5 transcription factor is preferentially expressed in small cell lung cancer (SCLC) and promotes MET transcription. PAX8, however, is also expressed in NSCLC cell lines. We therefore investigated the role of PAX8 in NSCLC.MethodsUsing IHC analysis, PAX8 protein expression was determined in archival NSCLC tumor tissues (n = 254). In order to study the effects of PAX8 knockdown on NSCLC cellular functions such as apoptosis and motility, siRNA against PAX8 was used. Confocal fluorescence microscopy was used to monitor the localization of MET, RON and PAX8. The combinatorial effect of PAX8 knockdown and MET inhibition using SU11274 was investigated in NSCLC cell viability assay.ResultsRelative levels of PAX8 protein were elevated (≥ + 2 on a scale of 0–3) in adenocarcinoma (58/94), large cell carcinoma (50/85), squamous cell carcinoma (28/47), and metastatic NSCLC (17/28; lymph node). Utilizing early progenitors isolated from NSCLC cell lines and fresh tumor tissues, we observed robust overexpression of PAX8, MET, and RON. PAX8 knockdown A549 cells revealed abrogated PAX8 expression with a concomitant loss in MET and the related RON kinase expression. A dramatic colocalization between the active form of MET (also RON) and PAX8 upon challenging A549 cells with HGF was visualized. A similar colocalization of MET and EGL5 (PAX8 ortholog) proteins was found in embryos of C. elegans. Most importantly, knockdown of PAX8 in A549 cells resulted in enhanced apoptosis (~6 fold) and decreased cell motility (~45%), thereby making PAX8 a potential therapeutic target. However, the combinatorial approach of PAX8 knockdown and treatment with MET inhibitor, SU11274, had marginal additive effect on loss of NSCLC cell viability.ConclusionPAX8 provides signals for growth and motility of NSCLC cells and is necessary for MET and RON expression. Further investigations are necessary to investigate the therapeutic potential of PA8 in NSCLC.

T cells but not NK cells are associated with a favourable outcome for resected colorectal liver metastases

BackgroundThe adaptive immune response to colorectal cancer is important for survival. Less is understood about the role of innate lymphocytes, such as Natural Killer (NK) cells, which are abundant in human liver.MethodsSamples of fresh liver (n = 21) and tumour (n = 11) tissue were obtained from patients undergoing surgical resection of colorectal liver metastases. Flow cytometry was used to analyse the presence and phenotype of NK cells, as compared to T cells, in the tumour and liver tissue. Results were correlated with survival.ResultsNK cells were poorly recruited to the tumours (distant liver tissue 38.3%, peritumoural liver 34.2%, tumour 12.9%, p = 0.0068). Intrahepatic and intratumoural NK cells were KIR (killer immunoglobulin-like receptor)loNKG2Ahi whereas circulating NK cells were KIRhiNKG2Alo. By contrast T cells represented 65.7% of the tumour infiltrating lymphocytes. Overall survival was 43% at 5 years, with the 5-year survival for individuals with a T cell rich infiltrate being 60% (95% CI 17-93%) and for those with a low T cell infiltrate being 0% (95% CI 0-48%). Conversely individuals with higher levels of NK cells in the tumour had an inferior outcome, although there were insufficient numbers to reach significance (median survivals: NKHi 1.63 years vs NKLo 3.92 years).ConclusionsT cells, but not NK cells, are preferentially recruited to colorectal liver metastases. NK cells within colorectal metastases have an intrahepatic and potentially tolerogenic, rather than a peripheral, phenotype. Similar to primary tumours, the magnitude of the T cell infiltrate in colorectal metastases is positively associated with survival.

Distribution of endothelial progenitor cells in tissues from patients with gastric cancer.

It is accepted that endothelial progenitor cells (EPCs) are recruited into tumor sites and take part in the neovascularization of tumors. However, few articles have discussed the specific distribution of EPCs in vivo in tissues of gastric cancer patients. For this reason, the present study sought to elucidate EPC distribution in vivo in tissues of patients with gastric cancer. Fresh tumor tissues were collected from 26 newly diagnosed patients with histologically confirmed gastric cancer (mean age, 51 years; range, 21–81 years; 7 females, 19 males). All patients were treated surgically with curative intent. One portion of the fresh tissues was prepared for flow cytometric analysis and another was immediately snap frozen in liquid nitrogen and stored at −80°C for later use in quantitative polymerase chain reaction. The analysis was based on two groups of tissues, namely the cancer group and cancer-adjacent group. The presence of CD34/CD133 double-positive cells was determined in cancer-adjacent and cancer tissues by flow cytometry. The analysis revealed that the total number of EPCs in cancer tissue was slightly greater than the number in the cancer-adjacent tissue, but not to the point of statistical significance. The number of EPCs in cancer-adjacent and cancer tissues of patients with early-stage gastric cancer was higher than the EPC count in late-stage gastric cancer patients, and significant differences were identified in the number of EPCs in cancer tissue between patients of different tumor stages. Levels of cluster of differentiation (CD)34, CD133 and vascular endothelial growth factor receptor 2 were not significantly different in cancer-adjacent tissue compared with cancer tissue. These results suggest that cancer-adjacent and cancer tissue of gastric cancer patients may be used as a reference index in the clinical and pathological staging of tumors.

Leveraging RB status to define therapy for castrate-resistant prostate cancer.

96 Background: Loss of retinoblastoma (RB) tumor suppressor is overrepresented in castrate-resistant prostate cancer (CRPC) compared to primary PCa. We previously showed using analyses of human tissue and in vitro and in vivo modeling that RB constrains androgen receptor (AR) function, and that loss of RB is sufficient promote resistance to castration and AR antagonists. Thus, novel strategies are needed to treat RB-deficient tumor. By contrast, in tumors retaining RB, suppressing enhancing RB activity would be of therapeutic advantage, and may be accomplished through next-generation Cdk4/6 inhibitors. Methods: Stable isogenic pairs of prostate cancer cell lines either retaining RB or RB depleted (by shRNA) were assessed in vitro and in xenografts for response to Cdk4/6 kinase inhibitors or the cabazitaxel. In addition, using an ex vivo explant assay, fresh tumor tissue samples from radical prostatectomy were exposed to the Cdk4/6 inhibitor or cabazitaxel for up to 7 days, and evaluated by IHC for Ki67, Caspase-3, and AR. Results: Cdk4/6 inhibition blocks tumor cell proliferation dependent on RB status. This was further confirmed ex vivo, as evidenced by a marked reduction in Ki67 staining in Cdk4/6 inhibitor treated explant tissue from two prostate cancer patients. Conversely, in vitro studies revealed a modest sensitization of RB-depleted tumors to cabazitaxel that was dramatically enhanced in vivo and after castration. Cabazitaxel, like docetaxel, targets the cell architecture and induces cell death, but also induces a distinct gene expression profile that may partially explain efficacy in docetaxel-resistant tumors. Neither taxane showed affects on AR nuclear localization using in vivoor explant studies. Conclusions: These results strongly support our hypothesis that RB status can be used as a metric to define therapeutic response to cabazitaxel, as such that loss of RB function induces sensitization taxanes, whereas RB proficient tumors give an enhanced response to Cdk4/6 kinase inhibitors.

ZNF797 plays an oncogenic role in gastric cancer.

Human cancer cells resemble stem cells in expression signatures leading them to share some features, most notably, self-renewal. A complex network of transcription factors and signaling molecules are required for continuation of this trait. ZNF797 (SALL4) is a zinc finger transcriptional activator crucial for maintenance of self-renewal in stem cells; however, its expression level has not yet been elucidated in gastric tumor cells. Its expression was analyzed to determine this level and probable clinicopathological consequences. SALL4 expression in fresh tumor and distant tumor-free tissues from 46 colorectal samples was compared by real-time polymerase chain reaction. Greater than a 2-fold increase in SALL4 expression was detected in 89.5% of tumors vs normal related tissues. SALL4 expression was significantly correlated with tumor cell metastasis to lymph nodes, especially in moderately differentiated tumor samples (P < 0.05). Furthermore, higher levels of SALL4 mRNA expression were significantly associated with younger patients with tumor cells in stages I and II (P < 0.05). These results indicate a relationship between SALL4 expression and tumor cell metastasis to lymph nodes and consequent progression of tumors to advanced stages III and IV. Along with the promising evidence of its role in self-renewal in various cancers, SALL4 is introduced as a potentially interesting therapeutic target to reverse a number of aberrations that promote gastric tumor development and maintenance. This result may lead to new approaches for cancer therapy.

Correlation between the methylation of SULF2 and WRN promoter and the irinotecan chemosensitivity in gastric cancer

BackgroundAt present, no study has compared the correlation between SULF2, WRN promoter methylation and clinicopathological parameters of patients with gastric cancer and the sensitivity to irinotecan (CPT-11).MethodsWe collected 102 fresh tumor tissues from pathologically diagnosed gastric carcinoma patients. Methylation specific PCR was used to detect the promoter methylation of SULF2 and WRN. The chemosensitivity of irinotecan to gastric tomor was tested by MTT. Then we compared the chemosensitivity difference of the methylated group with unmethylated group.ResultsThe rates of SULF2, WRN methylation were 28.3% (29/102) and 23.6% (24/102), separately. Patients with SULF2 methylation were more sensitive to CPT-11 than those without SULF2 methylation (P < 0.01). Patients with both SULF2 and WRN methylation were also more sensitive to CPT-11 than others ( P < 0.05).ConclusionSULF2 and WRN promoter methylation detection indicates potential predictive biomarkers to identify and target the most sensitive gastric cancer subpopulation for personalized CPT-11 therapy.

A preliminary study of genes related to concomitant chemoradiotherapy resistance in advanced uterine cervical squamous cell carcinoma

Background Tumor intrinsic chemoradiotherapy resistance is the primary factor in concomitant chemoradiotherapy failure in advanced uterine cervical squamous cell carcinoma. This study aims to identify a set of genes and molecular pathways related to this condition. Methods Forty patients with uterine cervical squamous cell carcinoma in International Federation of Gynecology and Obstetrics stage IIb or IIIb, treated with platinum-based concomitant chemoradiotherapy between May 2007 and December 2012, were enrolled in this trial. Patients included chemoradiotherapy resistant (n=20) and sensitive (n=20) groups. Total RNA was extracted from fresh tumor tissues obtained by biopsy before treatment and microarray analysis was performed to identify genes differentially expressed between the two groups. Results Microarray analysis identified 108 genes differentially expressed between concomitant chemoradiotherapy resistant and sensitive patients. Functional pathway cluster analysis of these genes revealed that DNA damage repair, apoptosis, cell cycle, Map kinase signal transduction, anaerobic glycolysis and glutathione metabolism were the most relevant pathways. Platelet-derived growth factor receptor alpha (PDGFRA) and protein kinase A type 1A (PRKAR1A) were significantly upregulated in the chemoradiosensitive group, while lactate dehydrogenase A (LDHA), bcl2 antagonist/killer 1 (BAK1), bcl2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and cyclin-dependent kinase 7 (CDK7) were upregulated in the chemoradiotherapy resistant group. Conclusion We have identified seven genes that are differentially expressed in concomitant chemoradiotherapy resistant and sensitive uterine cervical squamous cell carcinomas, which may represent primary predictors for this condition.

Abstract C30: Four dimensional molecular analysis of pediatric diffuse intrinsic pontine glioma

Abstract Introduction: Diffuse Intrinsic Pontine Glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Molecular characterization is limited due to lack of tissue. Recent investigations suggest possible molecular subtypes may account for the historical poor response to therapy. We previously generated protein profiles of CSF and formalin fixed DIPG tumor specimens to characterize patterns of protein expression. Here, we present the first comprehensive tissue proteome of fresh frozen DIPG tumor specimens (n=16) and normal brain tissue (n=10). We characterize differential protein expression in DIPG tumor specimens, and compare these to gene expression and DNA methylation profiles of the same tissue. Methods: Normal brain and tumor tissue was collected intraoperatively or post-mortem. Extracted total tissue protein was quantified by mass spectrometry (MS/MS) via LTQ-Orbitrap-XL and database search using the Sequest algorithm. Gene expression profiles were detected using whole-genome Human HT-4 v12 Gene Expression Bead Chips. DNA methylation profiles were characterized after bisulphite conversion using Infinium HumanMethylation 450K BeadChip arrays. Quantitative and statistical analysis was performed with Genome Studio, ProteoIQ, and Partek Genomics Suite. Functional analysis was performed using Ingenuity Pathways Analysis. Gene and protein expression was validated via western blot and immunohistochemical staining of tumor and normal brain tissue. Results: 1,918 differentially expressed genes were detected in DIPG tumor tissue (ANOVA, p&amp;lt;0.05, FC &amp;gt;2 or &amp;lt;-2). Unsupervised clustering revealed two distinct subgroups with differential ShH activity (GLI1 z-score -2.000 vs 2.000) and expression of GLI1, GLIPR1, PTCHD1 and SMO. Protein profiling revealed high expression of TLN1, CLU, EEF2 (FC &amp;gt;2), with differential ShH pathway (GLI1 z-score -0.626 vs. 2.254) and protein expression COL1A2, LMNA, MAP4, NES, NRCAM, STMN1, and TNC between subgroups (ANOVA, p&amp;lt;0.05, FC &amp;gt;2 or &amp;lt;-2). Concordant differences in DNA methylation were detected in related genes, including GLI1, FOXF1, SMO, SHH, and SUFU (ANOVA, p&amp;lt;0.05, FC&amp;lt;-2 or &amp;gt;2). Conclusions: We present the first comprehensive protein profile of DIPG fresh frozen tumor tissue and correlate to tissue gene expression profiles, which suggest differential activity of SHh pathway. This may in part be explained by differential methylation patterns of key genes. Proteomic analysis of DIPG tumor tissue reveals protein expression profiles reflective of this differential pathway activation, and hence may be useful tool for elucidating mechanisms of brainstem gliomagenesis in what likely is a heterogeneous tumor population. Biomarkers identified through proteomic analysis may in turn serve to more accurately diagnose patients with DIPG and measure response to therapy. Citation Format: Amanda Saratsis, Sridevi Yadavilli, Madhuri Kambhampati, Eric Raabe, Suresh Magge, Javad Nazarian. Four dimensional molecular analysis of pediatric diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C30.

Abstract A107: Elevated hinge cleavage of immunoglobulin G correlates with immune suppression and increased matrix metalloproteinases in breast tumor tissues

Abstract Growing evidence indicates that the tumor microenvironment has suppressive immune system and immune evasion is emerging as one of the cancer hallmarks. Immunoglobulin G (IgG) is an important component of human adaptive immunity in removing pathological cells including tumor cells. Recently, we reported that the cancer therapeutic antibody trastuzumab, a humanized IgG1, can be cleaved at the hinge region by matrix metalloproteinases (MMPs) in vitro, and the hinge cleaved trastuzumab (scIgG-T) showed significant loss of immune effector functions such as antibody dependent cellular cytotoxicity (ADCC) and anti-tumor efficacy in vivo (Fan et al., Breast Cancer Research 2012). The objectives of this study are 1) to investigate if the Fc hinge cleavage of IgG occurs in tumor tissues of breast cancer patients; 2) to understand the relationship among IgG Fc hinge cleavage, MMP expression, and anticancer immunity. Methods: Surgically removed fresh tumor tissues (n=23, 11 with upfront surgery and 12 with neoadjuvant therapies) from breast cancer patients with informed consent (protocol # HSC-MS-10-0580) were snap frozen in liquid nitrogen and stored in a -80oC freezer until analysis. Normal breast tissues (n=20) were obtained from an outside vendor (Cureline Inc., CA) as snap frozen tissues. Peripheral blood mononuclear cells (PBMCs) were freshly prepared from patient blood samples collected before surgery and were preserved in liquid nitrogen storage tank until analysis. Hinge cleaved IgGs were quantified by ELISA and immuno-histochemistry (IHC) detection with a panel of anti-hinge specific antibodies developed at Johnson &amp; Johnson. MMP expression was profiled using a reverse phase protein array method (Ray Biotech Inc., CA) and populations of immune effector cells in patient PBMCs were determined by multi-color flow cytometry. Results: Levels of single hinge cleaved IgGs (scIgGs) were significantly higher in breast cancer tumor tissue than in normal breast tissue. The elevated Fc hinge cleavage of IgGs in breast tumor tissues was positively correlated with high levels of MMPs and expression of MMP-9 showed linear correlation (R=0.78) with the level of scIgGs in the tumor tissues. Notably, Fc hinge cleavage of IgGs in residual tumor tissues from patients treated with neoadjuvant therapies was similar to the levels in the normal breast tissues, while tumor tissues from patients with up-front surgery had significantly higher Fc hinge cleavage than both normal and neoadjuvant treated tumor tissues. In comparison with PBMCs from patients with the up-front surgery, PBMCs from patients with neoadjuvant therapies had an increased percentage of NK cells (CD56+) and immune cells with Fc gamma receptor IIa (CD32a) expression. In addition, percentages of immune effector cells with CD56+/CD16+ (Fc gamma receptor RIII), CD56+/CD64+ (Fc gamma receptor RI), and CD14+/CD16+ were also higher in PBMCs from patients treated with neoadjuvant therapies than those from patients with up-front surgery. Taken together, our results suggest that the reduced scIgG levels in neoadjuvant treated tumor tissue correlated with the better profiles of immune effector cells, while the increased Fc hinge cleavage in breast tumor tissue before therapeutic intervention is linked with immune suppression in the tumor microenvironment. Citation Format: Ningyan zhang, Anneliese Gonzalez, Hui Deng, Xuejun Fan, Luis Baez Vallecillo, songlin zhang, Randall Brezski, Emily Roberson, Robert Jorden, William Strohl, Zhiqiang An. Elevated hinge cleavage of immunoglobulin G correlates with immune suppression and increased matrix metalloproteinases in breast tumor tissues. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A107.

Isolation of Human Melanoma Stem Cells Using ALDH as a Marker

This unit describes a protocol for establishing a patient-derived tumor xenograft model (PDTX model) of human melanoma and isolating human melanoma stem cells from human melanoma specimens using aldehyde dehydrogenase (ALDH) as a marker. One major limitation of analyzing a small fraction of cancer stem cells from patient tumor samples is that substantial quantities of fresh tumor tissues are not available. To overcome this challenge, we have established a PDTX model of human melanoma. In this model, human tumor tissues obtained from melanoma patients are dissected into small pieces and subsequently implanted into immunocompromised mice. The PDTX tumors retain fundamental genotypic and phenotypic features of the original tumors and are suitable for further biological analyses. Using the PDTX model, we have analyzed ALDH-labeled human melanoma stem cells. This unit will describe how to establish a PDTX model using human tumor samples. We will also describe how to isolate and analyze ALDH-labeled human melanoma stem cells using this model.

The preliminary establishment and application of a new method for evaluating K-ras mutations based fluid chip

Objective A new method for detecting K-ras mutations based liquid chip was used to evaluate K-ras mutations associated with non-small cell lung cancer (NSCLC) patients,to direct the personalized treatment and prognosis evaluation. Methods Take the diagnosis technology research methods,the sensitivity and repeatability of the liquid chip K-ras gene mutation detection method were assessed.A total of 100 NSCLC patients from Nov 2011 to Feb 2012 in Shanghai Chest hospital were included in this study,the fresh tumor tissues were collected for DNA extraction.The 2nd exon 12 and 13 codons,containing 8 K-ras mutations occuring in high frequency were amplified by polymerase chain reaction (PCR),followed by ligation of the PCR products to a series of special probes using ligase detection reaction (LDR),then the PCR-LDR products were analyzed by liquid chip platform.Direct sequencing was applied to compare with the detection results. Results The sensitivity of liquid chip technology detection was 10%-20%,higher than the traditional sequencing method by 1%.Average CV value was 4%-15% and showed good repeatability.5 K-ras mutations in 100 patients (5%) were detected using multiplex PCR-LDR combined fluid chip methods,including 3 Gly12Val and 2 Gly12 Asp mutations in exon 2.The 5 K-ras mutations were verified accurately by direct sequencing. Conclusions The novel detection method of K-ras mutations based PCR-LDR and fluid chip shows high throughput,high sensitivity,good repeatability and the results are reliable and accurate.This method can be used to accurately identified K-ras mutations for NSCLC patients prior to their targeted therapy with TKIs.(Chin J Lab Med,2013,36:704-707) Key words: Microfluidic analytical techniques; Polymerase chain reaction; Ligase chain reaction; Genes; ras; Genotype; Carcinoma; non-small-cell lung

Human papillomavirus genotypes distribution in 175 invasive cervical cancer cases from Brazil

BackgroundInvasive cervical cancer is the second most common malignant tumor affecting Brazilian women. Knowledge on Human Papillomavirus (HPV) genotypes in invasive cervical cancer cases is crucial to guide the introduction and further evaluate the impact of new preventive strategies based on HPV. We aimed to provide updated comprehensive data about the HPV types’ distribution in patients with invasive cervical cancer.MethodsFresh tumor tissue samples of histologically confirmed invasive cervical cancer were collected from 175 women attending two cancer reference hospitals from São Paulo State: ICESP and Hospital de Câncer de Barretos. HPV detection and genotyping were performed by the Linear Array HPV Genotyping Test (Roche Molecular Diagnostics, Pleasanton,USA).Results170 out of 172 valid samples (99%) were HPV DNA positive. The most frequent types were HPV16 (77.6%), HPV18 (12.3%), HPV31 (8.8%), HPV33 (7.1%) and HPV35 (5.9%). Most infections (75%) were caused by individual HPV types. Women with adenocarcinoma were not younger than those with squamous cell carcinoma, as well, as women infected with HPV33 were older than those infected by other HPV types. Some differences between results obtained in the two hospitals were observed: higher overall prevalence of HPV16, absence of single infection by HPV31 and HPV45 was verified in HC-Barretos in comparison to ICESP patients.ConclusionsTo our knowledge, this is one of the largest studies made with fresh tumor tissues of invasive cervical cancer cases in Brazil. This study depicted a distinct HPV genotype distribution between two centers that may reflect the local epidemiology of HPV transmission among these populations. Due to the impact of these findings on cervical cancer preventive strategies, extension of this investigation to routine screening populations is warranted.

The &lt;em&gt;In ovo&lt;/em&gt; CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions. This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.