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1.  Early Therapy Evaluation of Combined Cetuximab and Irinotecan in Orthotopic Pancreatic Tumor Xenografts by Dynamic Contrast-Enhanced Magnetic Resonance Imaging 
Molecular imaging  2011;10(3):153-167.
Early pancreatic cancer response following cetuximab and/or irinotecan therapies was measured by serial dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) before and during therapy. Groups 1 to 4 (n = 6/group) of SCID mice bearing orthotopic pancreatic adenocarcinoma xenografts expressing luciferase were treated with phosphate-buffered saline, cetuximab, irinotecan, or cetuximab combined with irinotecan, respectively, twice weekly for 3 weeks. DCE-MRI was performed on days 0, 1, 2, and 3 after therapy initiation, whereas anatomic magnetic resonance imaging was performed on days 0, 1, 2, 3, 6, and 13. Bioluminescence imaging was performed on days 0 and 21. At day 21, all tumors were collected for further histologic analyses (Ki-67 and CD31 staining), whereas tumor dimensions were measured by calipers. The Ktrans values in the 0.5 mm–thick peripheral tumor region were calculated, and the changes in Ktrans during the 3 days posttherapy were compared to tumor volume changes, bioluminescent signal changes, and histologic findings. The Ktrans changes in the peripheral tumor region after 3 days of therapy were linearly correlated with 21-day decreases in tumor volume (p < .001), bioluminescent signal (p = .050), microvessel densities (p = .002), and proliferating cell densities (p = .001). This study supports the clinical use of DCE-MRI for pancreatic cancer patients for early assessment of an anti–epidermal growth factor receptor therapy combined with chemotherapy.
PMCID: PMC3957340  PMID: 21496446
2.  New NSAID Targets and Derivatives for Colorectal Cancer Chemoprevention 
Clinical and preclinical studies provide strong evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) can prevent numerous types of cancers, especially colorectal cancer. Unfortunately, the depletion of physiologically important prostaglandins due to cyclooxygenase (COX) inhibition results in potentially fatal toxicities that preclude the long-term use of NSAIDs for cancer chemoprevention. While studies have shown an involvement of COX-2 in colorectal tumorigenesis, other studies suggest that a COX-independent target may be at least partially responsible for the antineoplastic activity of NSAIDs. For example, certain NSAID derivatives have been identified that do not inhibit COX-2 but have demonstrated efficacy to suppress carcinogenesis with potential for reduced toxicity. A number of alternative targets have also been reported to account for the tumor cell growth inhibitory activity of NSAIDs, including the inhibition of cyclic guanosine monophosphate phosphodiesterases (cGMP PDEs), generation of reactive oxygen species (ROS), the suppression of the apoptosis inhibitor protein, survivin, and others. Here, we review several promising mechanisms that are being targeted to develop safer and more efficacious NSAID derivatives for colon cancer chemoprevention.
PMCID: PMC3703626  PMID: 22893202
3.  The Effect of Antigen Retrieval on Cells Fixed in 10% Neutral Buffered Formalin Followed by Transfer to 70% Ethanol 
PLoS ONE  2013;8(12):e82405.
Fixation in 10% neutral buffered formalin prior to transfer to 70% ethanol for one week has been shown to adequately preserve immunorecognition of PCNA, cytokeratins AE1/AE3 and EGFr. This study investigated whether 12 hrs fixation in 10% NBF plus transfer to 70% ethanol for 4 weeks would similarly preserve immunorecognition to an extent where antigen retrieval (AR) used to reverse the masking effects of fixation on some antigens would not be necessary. Two cell lines, DU145 and SKOV3 were grown on coverslips and fixed either for 684 hrs in 10% NBF or for 12 hrs in 10% NBF which was then replaced with 70% ethanol for 672 hrs. The second experiment had the same design except an additional set of cells were subjected to heat-induced AR concomitantly. PCNA, cytokeratins AE1/AE3, and EGFr (membrane and cytoplasmic) were used to evaluate the effects of immunorecognition. Fixation in 10% NBF for 12 hrs plus transfer to 70% ethanol for 672 hrs did not preserve immunorecognition of PCNA adequately in either cell lines. Cytokeratins immunoreactivity was preserved by transfer to 70% ethanol. Cytoplasmic EGFr antigens were not adversely affected by 10% NBF fixation in either cell line and transfer to 70% ethanol had limited effects. With AR, there was little recovery of PCNA immunorecognition on cells fixed in only 10% NBF, but almost complete recovery for cells transferred to 70% ethanol. For cytokeratins there was complete recovery of immunorecognition either with only 10% NBF or 12 hrs plus transfer to 70% ethanol. For EGFr, AR resulted in complete loss of immunorecognition following either treatment. This study indicated that 12 hrs of fixation in 10% NBF plus transfer to 70% ethanol for 4 weeks with AR resulted in recovery of immunorecognition for PCNA and cytokeratins, but standard methods of AR caused loss of immunorecognition of EGFr.
PMCID: PMC3866107  PMID: 24358179
4.  Validation of a robust proteomic analysis carried out on formalin-fixed paraffin-embedded tissues of the pancreas obtained from mouse and human 
Proteomics  2012;12(22):3393-3402.
A number of reports have recently emerged with focus on extraction of proteins from formalin-fixed paraffin-embedded (FFPE) tissues for MS analysis; however, reproducibility and robustness as compared to flash frozen controls is generally overlooked. The goal of this study was to identify and validate a practical and highly robust approach for the proteomics analysis of FFPE tissues. FFPE and matched frozen pancreatic tissues obtained from mice (n = 8) were analyzed using 1D-nanoLC-MS(MS)2 following work up with commercially available kits. The chosen approach for FFPE tissues was found to be highly comparable to that of frozen. In addition, the total number of unique peptides identified between the two groups was highly similar, with 958 identified for FFPE and 1070 identified for frozen, with protein identifications that corresponded by approximately 80%. This approach was then applied to archived human FFPE pancreatic cancer specimens (n = 11) as compared to uninvolved tissues (n = 8), where 47 potential pancreatic ductal adenocarcinoma markers were identified as significantly increased, of which 28 were previously reported. Further, these proteins share strongly overlapping pathway associations to pancreatic cancer that include estrogen receptor α. Together, these data support the validation of an approach for the proteomic analysis of FFPE tissues that is straightforward and highly robust, which can also be effectively applied toward translational studies of disease.
PMCID: PMC3656494  PMID: 22997103
Animal proteomics; FPE; LC-MS; Nontagged proteomics; Pancreatic cancer; Systems biology
6.  Aurora Kinase-A Inactivates DNA Damage Induced Apoptosis and Spindle Assembly Checkpoint Response Functions of p73 
Cancer cell  2012;21(2):196-211.
Elevated Aurora kinase-A expression is correlated with abrogation of DNA damage induced apoptotic response and mitotic spindle assembly checkpoint (SAC) override in human tumor cells. We report that Aurora-A phosphorylation of p73 at serine235 abrogates its transactivation function and causes cytoplasmic sequestration in a complex with the chaperon protein mortalin. Aurora-A phosphorylated p73 also facilitates inactivation of SAC through dissociation of the MAD2-CDC20 complex in cells undergoing mitosis. Cells expressing phosphor-mimetic mutant (S235D) of p73 manifest altered growth properties, resistance to cisplatin induced apoptosis, as well as premature dissociation of the MAD2-CDC20 complex, and accelerated mitotic exit with SAC override in the presence of spindle damage. Elevated cytoplasmic p73 in Aurora-A overexpressing primary human tumors corroborates the experimental findings.
PMCID: PMC3760020  PMID: 22340593
7.  CCL25 mediates migration, invasion and matrix metalloproteinase expression by breast cancer cells in a CCR9-dependent fashion 
International journal of oncology  2011;38(5):1279-1285.
Breast cancer (BrCa) is one of the most frequently diagnosed cancers and the second leading cause of cancer-related deaths in North American women. Most deaths are caused by metastasis, and BrCa is characterized by a distinct metastatic pattern involving lymph nodes, bone marrow, lung, liver and brain. Migration of metastatic cells share many similarities with leukocyte trafficking, which are regulated by chemokines and their receptors. The current study evaluates the expression and functional role of CCR9, and its only known ligand, CCL25, in BrCa cell migration and invasion. Quantitative immunohistochemical analysis showed that both moderately and poorly differentiated BrCa tissue expressed significantly more (P<0.0001) CCR9 compared to non-neoplastic breast tissue. Interestingly, poorly differentiated BrCa tissue expressed significantly more (P<0.0001) CCR9 compared to moderately differentiated BrCa tissue. Similarly, CCR9 was highly expressed by the aggressive breast cancer cell line (MDA-MD-231) compared to the less aggressive MCF-7. Migration as well as invasion assays were used to evaluate the functional interaction between CCR9 and CCL25 in BrCa cell lines (MDA-MB-231 and MCF-7). Neutralizing CCR9-CCL25 interactions significantly impaired the migration and invasion of BrCa cells. Furthermore, CCL25 enhanced the expression of MMP-1, -9, -11 and -13 active proteins by BrCa cells in a CCR9-dependent fashion. These studies show CCR9 is functionally and significantly expressed by BrCa (poorly > moderately differentiated) tissue and cells as well as that CCL25 activation of this receptor promotes breast tumor cell migration, invasion and MMP expression, which are key components of BrCa metastasis.
PMCID: PMC3760515  PMID: 21344163
chemokine; immunobiology; matrix metalloproteinase
8.  Expression and histopathological correlation of CCR9 and CCL25 in ovarian cancer 
International journal of oncology  2011;39(2):373-381.
Ovarian carcinoma is the most lethal gynecological malignancy among women and its poor prognosis is mainly due to metastasis. Chemokine receptor CCR9 is primarily expressed by a small subset of immune cells. The interactions between CCL25 and CCR9 have been implicated in leukocyte trafficking to the small bowel, a frequent metastatic site for ovarian cancer cells. We have previously shown that ovarian cancer cells express CCR9 and play an important role in cell migration, invasion and survival in the presence of its natural ligand in vitro. In this study, we have evaluated the expression of CCR9 and CCL25 in ovarian cancer cells and clinical samples. Ovarian cancer tissue microarrays from University of Alabama at Birmingham and AccuMax were stained for CCR9 and CCL25. Aperio ScanScope was used to acquire 80X digital images and expression analysis of CCR9 and CCL25. Flow cytometry and the Image stream system were used to conform the expression of CCR9 and CCL25 in ovarian cancer cells. Our results show significantly higher (p<0.001) expression of CCR9 and CCL25 in serous adenocarcinoma followed by serous papillary cystadenoma, endometrioid adeno-carcinoma, mucinous adenocarcinoma, cystadenoma, mucinous boderline adenocarcinoma, clear cell carcinoma, granulosa cell tumor, dysgerminoma, transitional cell carcinoma, Brenner tumor, yolk sac tumor, adenocarcinoma and fibroma cases, compared to non-neoplastic ovarian tissue. Similar to tissue expression, CCR9 was also significantly expressed by the ovarian cancer cell lines (OVCAR-3 and SK-OV-3) in comparison to normal adult ovarian epithelial cell. We provide the first evidence that CCR9 and its natural ligand CCL25 are highly expressed by ovarian cancer tissue and their expression correlates with histological subtypes. Expression of this chemokine receptor and its ligand CCL25 within primary tumor tissue further suggests a potential role of this chemokine-receptor axis in ovarian cancer progression.
PMCID: PMC3760589  PMID: 21637913
chemokine; ovarian cancer; CCR9 and CCl25
9.  Frequencies of mtDNA mutations in primary tissue of colorectal adenopolyps 
To investigate the potential role of mtDNA alterations during the onset of colorectal cancer, the occurrence of mtDNA variants in colorectal adenomatous (Tubular, Tubulovillous, and Villous) polyps, were studied. High resolution endonucleases and PCR-based sequence were applied to examine mtDNA variants in the ND and ATPase genes of 64 primary tissues of colorectal adenopolyps and their matched normal controls. Forty-two variants were observed and 57% (24/42) were not previously reported in the MITODAT reference. Fifty-eight percent of these variants were germline and homoplasmic transitions. The distribution of observed mtDNA variants includes: 31% (13/42) tubular, 52% (22/42) tubulovillous, 45% (19/42) villous, and 45% (19/42) cancer (including FAP and JVP). Notably, an unreported germline variant in the ATPase 8 gene at nucleotide position (np) G8573A was observed in tubulovillous adenomas tissues. The results suggest that some specific mtDNA variants may serve as a potential biomarker for colorectal adenomatous polyps.
PMCID: PMC3739296  PMID: 23747897
Colorectal; Adenomas; Cancer; Mitochondrial; DNA; Tubular; Tubulovillous; Villous
10.  Modern Age Pathology of Pulmonary Arterial Hypertension 
Rationale: The impact of modern treatments of pulmonary arterial hypertension (PAH) on pulmonary vascular pathology remains unknown.
Objectives: To assess the spectrum of pulmonary vascular remodeling in the modern era of PAH medication.
Methods: Assessment of pulmonary vascular remodeling and inflammation in 62 PAH and 28 control explanted lungs systematically sampled.
Measurements and Main Results: Intima and intima plus media fractional thicknesses of pulmonary arteries were increased in the PAH group versus the control lungs and correlated with pulmonary hemodynamic measurements. Despite a high variability of morphological measurements within a given PAH lung and among all PAH lungs, distinct pathological subphenotypes were detected in cohorts of PAH lungs. These included a subset of lungs lacking intima or, most prominently, media remodeling, which had similar numbers of profiles of plexiform lesions as those in lungs with more pronounced remodeling. Marked perivascular inflammation was present in a high number of PAH lungs and correlated with intima plus media remodeling. The number of profiles of plexiform lesions was significantly lower in lungs of male patients and those never treated with prostacyclin or its analogs.
Conclusions: Our results indicate that multiple features of pulmonary vascular remodeling are present in patients treated with modern PAH therapies. Perivascular inflammation may have an important role in the processes of vascular remodeling, all of which may ultimately lead to increased pulmonary artery pressure. Moreover, our study provides a framework to interpret and design translational studies in PAH.
PMCID: PMC3886716  PMID: 22679007
pulmonary circulation; vessel remodeling; angiogenesis; inflammation
11.  Translational pathology of neoplasia 
With the increasing use of individualized medical care (personalized medicine) in treating and managing patients with cancer, the utilization of biomarkers in selecting and tailoring such medical approaches also is increasing and becoming more important. Specifically, many therapies are effective against only a subgroup of a specific type of tumors and exposing patients with different non-responsive subgroups of the same tumor to ineffective therapies, not only exposes these patients needlessly to acute and chronic side effects of the therapy, but also adds to the costs of medical care. For example, the Oncotype Dx test for estrogen receptor positive tumors that are node negative has been used to identify low risk tumors for which surgery alone is an adequate therapy. Biomarkers may be used to aid in multiple aspects of medical care related to cancer, including early detection, diagnosis, risk assessment, as well as in predicting the aggressiveness of cancers (i.e., prognosis) and predicting the therapeutic efficacy of treatments (i.e., prediction). Biomarkers may be also used as surrogate endpoints to aid in evaluating therapies and preventive approaches. Types of biomarkers vary greatly and include histopathologic appearance, stage of the lesion, quantitative morphologic features, size of the lesion, metastatic pattern and extent of metastasis, as well as imaging and molecular features. The types of measurements of biomarkers also vary; for example, molecular features can be measured at the DNA, mRNA or protein levels as well as at regulatory levels (e.g., microRNA). The usefulness of each biomarker is limited by its sensitivity and specificity in fulfilling its role (e.g., in early detection) and the requirements of sensitivity and specificity to accomplish specific tasks are affected by multiple variables. For example, both very high specificity and sensitivity of a test are required to screen a population with a low prevalence of a specific tumor. The goal of this manuscript is to introduce the reader to how biomarkers may be used and the limitations on the uses of biomarkers in translational research.
PMCID: PMC3445029  PMID: 22112467
Sensitivity; specificity; early detection; prognosis; risk assessment; surrogate endpoints; diagnosis; receiver operating characteristic; prediction; biomarkers; prevalence; medical costs; side effects; histopathology; molecular features; imaging; prevention; treatment; personalized medicine; individualized medical care
12.  Issues in collecting, processing and storing human tissues and associated information to support biomedical research 
The availability of human tissues to support biomedical research is critical to advance translational research focused on identifying and characterizing approaches to individualized (personalized) medical care. Providing such tissues relies on three acceptable models – a tissue banking model, a prospective collection model and a combination of these two models. An unacceptable model is the “catch as catch can” model in which tissues are collected, processed and stored without goals or a plan or without standard operating procedures, i.e., portions of tissues are collected as available and processed and stored when time permits. In the tissue banking model, aliquots of tissues are collected according to SOPs. Usually specific sizes and types of tissues are collected and processed (e.g., 0.1 gm of breast cancer frozen in OCT). Using the banking model, tissues may be collected that may not be used and/or do not meet specific needs of investigators; however, at the time of an investigator request, tissues are readily available as is clinical information including clinical outcomes. In the model of prospective collection, tissues are collected based upon investigator requests including specific requirements of investigators. For example, the investigator may request that two 0.15 gm matching aliquots of breast cancer be minced while fresh, put in RPMI media with and without fetal calf serum, cooled to 4°C and shipped to the investigator on wet ice. Thus, the tissues collected prospectively meet investigator needs, all collected specimens are utilized and storage of specimens is minimized; however, investigators must wait until specimens are collected, and if needed, for clinical outcome. The operation of any tissue repository requires well trained and dedicated personnel. A quality assurance program is required which provides quality control information on the diagnosis of a specimen that is matched specifically to the specimen provided to an investigator instead of an overall diagnosis of the specimen via a surgical pathology report. This is necessary because a specific specimen may not match the diagnosis of the case due to many factors such as necrosis, unsuspected tumor invasion of apparently normal tissue, and areas of fibrosis which are mistaken grossly for tumor. Aliquots for quality control (QC) may or may not be collected at the time of collection and in some cases, QC may not occur until specimens are distributed to investigators. In establishing a tumor repository, multiple issues need to be considered. These include the available resources, long term support, space and equipment. The needs of the potential users need to be identified as to the types of tissues and services needed and the annotation expected. Other specific issues to be considered include collection of specimens potentially infected with blood borne pathogens (e.g., hepatitis B), charge back mechanisms, informatics needs and support, and investigator requirements (e.g., recognition of repository contributions in publications). In general, the repository should not perform the research of the investigators, but should provide the infrastructure necessary to support the research of the investigator. Thus, the goals of the repository must be established. Similarly, ethical and regulatory issues must be evaluated. In general, tissue repositories need ethical (e.g., IRB) and privacy (e.g., HIPAA) review. Also, safety issues need to be considered as well as how biohazards will be addressed by investigator-users. Considerations involving the transfer of specimens to other organization usually require a material transfer agreement (MTA). A MTA should address biohazards as well as indemnification. Thus, many issues must be considered and addressed in order to establish and operate successfully a biorepository.
PMCID: PMC3445033  PMID: 22112494
Tissue repositories; tissue banking; prospective collections; quality control; quality assurance; repository science; bias; clinical trials; epidemiology; annotation; material transfer agreement; shipping; informatics; safety; biohazards; security; difficult requests; caBIG; vocabulary; common data elements; chemical hazards; cost recovery; HIPAA; informed consent; demographics; clinical information; training; audits; good manufacturing practice; storage; specimen identification; services
13.  EMMPRIN as a novel target for pancreatic cancer therapy 
Anti-cancer drugs  2011;22(9):864-874.
The objective of this study was to evaluate extracelluar matrix metalloproteinase (EMMPRIN) as a novel target in orthotopic pancreatic-cancer murine models. MIA PaCa-2 human pancreatic tumor cells were implanted in groups 1 and 3-7, while MIA PaCa-2 EMMPRIN knockdown cells were implanted in group 2. Dosing with anti-EMMPRIN antibody started immediately after implantation for groups 1-3 (residual tumor model) and at 21 days after cell implantation for groups 4-7 (established tumor model). Groups 3, 5, and 7 were treated with anti-EMMRPIN antibody (0.2-1.0 mg) twice weekly for 2-3 weeks, while the other groups served as the control. In residual tumor model, tumor growth of anti-EMMPRIN treated group was successfully arrested for 21 days (15±4 mm3), significantly lower than that of EMMPRIN knockdown group (80±15 mm3; p=0.001) or control group (240±41 mm3; p<0.001). In established tumor model, anti-EMMPRIN therapy lowered tumor-volume increase about 40% compared with control regardless of dose amount. Ki67-expressed cell densities of group 5 was 939±150 mm−2, significantly lower than that of group 4 (1709±145 mm−2; p=0.006). Microvessel density of group 5 (30±6 mm−2) was also significantly lower than that of group 4 (53±5 mm−2; p=0.014), while the microvessel size of group 5 (191±22 μm2) was significantly larger than that of group 4 (113±26 μm2; p=0.049). These data show the high potential of anti-EMMPRIN therapy for pancreatic cancer, and support its clinical translation.
PMCID: PMC3587034  PMID: 21730821
EMMPRIN; Targeted therapy; Pancreatic cancer
14.  The biology of incipient, pre-invasive or intraepithelial neoplasia 
Invasive tumors (cancers or malignant lesions) typically develop in the setting in which there is the presence of putative non-invasive lesions and the development of these non-invasive lesions frequently precedes the development of cancers. For some organs, such as the oral cavity, cervix and skin, the respective putative pre-invasive lesions can be observed over time and documented to progress to invasive lesions. However, for less readily observable lesions, such as those of the prostate, the progression of the pre-invasive lesions, e.g., prostatic intraepithelial neoplasia (PIN) and prostatic proliferative inflammatory atrophy (PIA) to prostatic cancer are more difficult to document. Thus, for most organ systems, specific pre-invasive neoplastic lesions have been proposed based upon the apparent observations of one or more of the following: 1) microinvasive disease developing from a pre-invasive neoplastic lesion, 2) the general association of the pre-invasive lesion with invasive lesions, 3) the subsequent development of invasive lesions following diagnosis of the pre-invasive lesion, 4) correlations of the molecular features of the putative pre-invasive lesion with the matching invasive lesions, and 5) reductions in the rate of cancer following removal of the pre-invasive lesion. When there are mixtures of pre-invasive lesions with actual cancers in the same case, some of the above specific associations are more difficult to make. Several terms have been used to describe pre-invasive lesions, many of which are now less useful as our knowledge of these lesions increases. It is now commonly accepted that these lesions are a features of the spectrum of neoplastic development and most are accepted as “neoplastic lesions” with associated molecular features, even though they may be reversible even if they have mutations in suppressor genes (e.g., p53) or are associated with viral etiologies (e.g., cervical intraepithelial neoplasia). The overall term, “pre-invasive neoplasia”, seems to best describe these putative pre-invasive lesions. Thus, terms such as incipient neoplasia should be abandoned. The term “intra-epithelial neoplasia” with an associated grade, which has been developed for pre-invasive neoplastic lesions of the cervix, i.e. cervical intraepithelial neoplasia (CIN), seems to be a terminology that adds consistency across epithelial organs. Thus, adoption of these terms for the additional organ sites of pancreas (PanIN) and prostate (PIN) seems accepted. Less descriptive terms such as the degrees of dysplasia of the oral cavity and bronchopulmonary system and actinic keratosis and Bowen's disease of the skin might be better designated as oral intraepithelial neoplasia (OIN), pulmonary intraepithelial neoplasia (PulIN) and dermal intraepithelial neoplasia (DIN). The etiology of pre-invasive neoplasia is the etiology of the matching cancers. Some obvious initiating factors include exposure to the whole range of ionizing and non-ionizing radiation, tobacco abuse and a broad range of other carcinogens (e.g., benzene). A frequent initiation factor is the setting of long standing continuing damage, inflammation and repair (LOCDIR) which leads to early molecular features associated with neoplasia after about one year. An excellent example of this is ulcerative colitis (UC) in which dysregulation of microsatellite repair enzymes have been documented one year following diagnosis of UC. While the nomenclature, description, diagnosis and etiology of pre-invasive neoplasia has advanced, approaches to therapy of such lesions have not progressed adequately even though it has been identified that, for example, removal of polyps periodically from the colorectum, DCIS from the breast, and high grade CIN from the cervix, results in a reduction in the development of cancers of the colorectum, breast, and cervix, respectively. With the development of more molecularly targeted therapy with fewer side effects, preventive therapies may be more successfully targeted to pre-invasive neoplastic lesions.
PMCID: PMC3430522  PMID: 22112468
Intraepithelial neoplasia; pre-invasive neoplasia; prostatic intraepithelial neoplasia; pancreatic intraepithelial neoplasia; cervical intraepithelial neoplasia; adenomatous polyps; ductal carcinoma in situ; lobular carcinoma in situ; inflammation; radiation; viral infections; carcinogens; dysplasia; actinic keratosis; repair; angiogenesis; LOCDIR
15.  microRNAs are Stable in Formalin-Fixed Paraffin-Embedded Archival Tissue Specimens of Colorectal Cancers Stored for up to 28 Years 
MicroRNAs (miRNAs) have prognostic and therapeutic value for colorectal cancers (CRCs). Although formalin-fixed paraffin-embedded (FFPE) tissues are available for biomarker studies, the stability of miRNAs in these tissues stored for long periods (>20 years) is unknown. The present effort involved analysis of 345 FFPE CRC tissues, stored for 6 to 28 years (1982-2004), for the expression of six miRNAs (miR-20a, miR-21, miR-106a, miR-181b, miR-203, and miR-324-5p) using TaqMan® microRNA assays and quantitative real-time PCR (qRT-PCR). Evaluation, by linear regression analysis, of miRNA expression among archived CRC tissues found similar levels of all six miRNAs in tissues stored over this period (correlation coefficients, R2, ranged from <0.0001-0.009; and t-test p-values were ≥ 0.05). Thus, miRNAs are stable in FFPE tissues stored for long periods of time, and such samples can be used for discovery of biomarkers.
PMCID: PMC3681606  PMID: 22202009
miRNA; Colorectal Cancer; FFPE; Stability; Biomarkers
16.  Increased expression of activation-induced cytidine deaminase is associated with anti-CCP and rheumatoid factor in rheumatoid arthritis 
Rheumatoid arthritis (RA) is associated with higher levels of autoantibodies and IL-17. Here, we investigated if ectopic lymphoid follicles and peripheral blood mononuclear cells (PBMCs) from RA patients exhibit increased activation-induced cytidine deaminase (AID), and if increased AID is correlated with serum levels of autoantibodies and IL-17. The results of immunohistochemical staining showed that organized germinal centers were observed in 6 of the 12 RA synovial samples, and AID+ cells were found almost exclusively in the B-cell areas of these follicles. Aggregated but not organized lymphoid follicles were found in only one OA synovial sample without AID+ cells. Significantly higher levels of AID mRNA (Aicda) detected by RT-PCR were found in the PBMCs from RA patients than PBMCs from normal controls (p<0.01). In the PBMCs from RA patients, AID was expressed predominately by the CD10+IgM+CD20+ B-cell population and the percentage of these cells that expressed AID was significantly higher than in normal controls (p < 0.01). Aicda expression in the PBMCs correlated significantly and positively with the serum levels of rheumatoid factor (RF) (p ≤ 0.0001) and anti-cyclic citrullinated peptide (CCP) (p = 0.0005). Serum levels of IFN-γ (p = 0.0005) and IL-17 (p = 0.007), but not IL-4, also exhibited positive correlation with the expression of AID. These results suggest that the higher levels of AID expression in B cells of RA patients correlate with, and may be associated with the higher levels of T helper cell cytokines IFN-γ and IL-17, leading to the development of anti-CCP and RF.
PMCID: PMC3674772  PMID: 19703021
17.  Myb overexpression overrides androgen depletion–induced cell cycle arrest and apoptosis in prostate cancer cells, and confers aggressive malignant traits: potential role in castration resistance 
Carcinogenesis  2012;33(6):1149-1157.
Myb, a cellular progenitor of v-Myb oncogenes, is amplified in prostate cancer and exhibits greater amplification frequency in hormone-refractory disease. Here, we have investigated the functional significance of Myb in prostate cancer. Our studies demonstrate Myb expression in all prostate cancer cell lines (LNCaP, C4-2, PC3 and DU145) examined, whereas it is negligibly expressed in normal/benign prostate epithelial cells (RWPE1 and RWPE2). Notably, Myb is significantly upregulated, both at transcript (>60-fold) and protein (>15-fold) levels, in castration-resistant (C4-2) cells as compared with androgen-dependent (LNCaP) prostate cancer cells of the same genotypic lineage. Using loss and gain of function approaches, we demonstrate that Myb promotes and sustains cell cycle progression and survival under androgen-supplemented and -deprived conditions, respectively, through induction of cyclins (A1, D1 and E1), Bcl-xL and Bcl2 and downregulation of p27 and Bax. Interestingly, Myb overexpression is also associated with enhanced prostate-specific antigen expression. Furthermore, our data show a role of Myb in enhanced motility and invasion and decreased homotypic interactions of prostate cancer cells. Myb overexpression is also associated with actin reorganization leading to the formation of filopodia-like cellular protrusions. Immunoblot analyses demonstrate gain of mesenchymal and loss of epithelial markers and vice versa, in Myb-overexpressing LNCaP and -silenced C4-2 cells, respectively, indicating a role of Myb in epithelial to mesenchymal transition. Altogether, our studies provide first experimental evidence for a functional role of Myb in growth and malignant behavior of prostate cancer cells and suggest a novel mechanism for castration resistance.
PMCID: PMC3514863  PMID: 22431717
18.  A Novel Sulindac Derivative that Potently Suppresses Colon Tumor Cell Growth by Inhibiting cGMP Phosphodiesterase and β-Catenin Transcriptional Activity 
Nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely reported to inhibit tumor growth by a COX-independent mechanism, although alternative targets have not been well defined or used to develop improved drugs for cancer chemoprevention. Here, we characterize a novel sulindac derivative referred to as sulindac benzylamine (SBA) that does not inhibit COX-1 or COX-2, yet potently inhibits the growth and induces the apoptosis of human colon tumor cells. The basis for this activity appears to involve cyclic guanosine 3′,5′,-monophosphate phosphodiesterase (cGMP PDE) inhibition as evident by its ability to inhibit cGMP hydrolysis in colon tumor cell lysates and purified cGMP-specific PDE5, increase intracellular cGMP levels, and activate cGMP-dependent protein kinase G at concentrations that suppress tumor cell growth. PDE5 was found to be essential for colon tumor cell growth as determined by siRNA knockdown studies, elevated in colon tumor cells as compared with normal colonocytes, and associated with the tumor selectivity of SBA. SBA activation of PKG may suppress the oncogenic activity of β-catenin as evident by its ability to reduce β-catenin nuclear levels, Tcf (T-cell factor) transcriptional activity, and survivin levels. These events preceded apoptosis induction and appear to result from a rapid elevation of intracellular cGMP levels following cGMP PDE inhibition. We conclude that PDE5 and possibly other cGMP degrading isozymes can be targeted to develop safer and more efficacious NSAID derivatives for colorectal cancer chemoprevention.
PMCID: PMC3546530  PMID: 22556201
19.  An Extensive Targeted Proteomic Analysis of Disease-Related Protein Biomarkers in Urine from Healthy Donors 
PLoS ONE  2013;8(5):e63368.
The analysis of protein biomarkers in urine is expected to lead to advances in a variety of clinical settings. Several characteristics of urine including a low-protein matrix, ease of testing and a demonstrated proteomic stability offer distinct advantages over current widely used biofluids, serum and plasma. Improvements in our understanding of the urine proteome and in methods used in its evaluation will facilitate the clinical development of urinary protein biomarkers. Multiplexed bead-based immunoassays were utilized to evaluate 211 proteins in urines from 103 healthy donors. An additional 25 healthy donors provided serial urine samples over the course of two days in order to assess temporal variation in selected biomarkers. Nearly one-third of the evaluated biomarkers were detected in urine at levels greater than 1ng/ml, representing a diverse panel of proteins with respect to structure, function and biological role. The presence of several biomarkers in urine was confirmed by western blot. Several methods of data normalization were employed to assess impact on biomarker variability. A complex pattern of correlations with urine creatinine, albumin and beta-2-microglobulin was observed indicating the presence of highly specific mechanisms of renal filtration. Further investigation of the urinary protein biomarkers identified in this preliminary study along with a consideration of the underlying proteomic trends suggested by these findings should lead to an improved capability to identify candidate biomarkers for clinical development.
PMCID: PMC3665773  PMID: 23723977
20.  Comparison of the predictive qualities of three prognostic models of colorectal cancer 
Most discoveries of cancer biomarkers involve construction of a single model to determine predictions of survival. ‘Data-mining’ techniques, such as artificial neural networks (ANNs), perform better than traditional methods, such as logistic regression. In this study, the quality of multiple predictive models built on a molecular data set for colorectal cancer (CRC) was evaluated. Predictive models (logistic regressions, ANNs, and decision trees) were compared, and the effect of techniques for variable selection on the predictive quality of these models was investigated. The Kolmogorov-Smirnoff (KS) statistic was used to compare the models. Overall, the logistic regression and ANN methods outperformed use of a decision tree. In some instances (e.g., for a model that included ‘all variables without tumor stage’ and use of a decision tree for variable selection), the ANN marginally outperformed logistic regression, although the difference between the accuracy of the KS statistic was minimal (0.80 versus 0.82). Regardless of the variable(s) and the methods for variable selection, all three predictive models identified survivors and non-survivors with the same level of statistical accuracy.
PMCID: PMC3658118  PMID: 20515758
Artificial neural networks; Colorectal cancer; Decision trees; Kolmogorov-Smirnoff statistic; Logistic regression; Predictive models
21.  Anti-Inflammatory Mechanisms of Apolipoprotein A-I Mimetic Peptide in Acute Respiratory Distress Syndrome Secondary to Sepsis 
PLoS ONE  2013;8(5):e64486.
Acute respiratory distress syndrome (ARDS) due to sepsis has a high mortality rate with limited treatment options. High density lipoprotein (HDL) exerts innate protective effects in systemic inflammation. However, its role in ARDS has not been well studied. Peptides such as L-4F mimic the secondary structural features and functions of apolipoprotein (apo)A-I, the major protein component of HDL. We set out to measure changes in HDL in sepsis-mediated ARDS patients, and to study the potential of L-4F to prevent sepsis-mediated ARDS in a rodent model of lipopolysaccharide (LPS)-mediated acute lung injury, and a combination of primary human leukocytes and human ARDS serum. We also analyzed serum from non-lung disease intubated patients (controls) and sepsis-mediated ARDS patients. Compared to controls, ARDS demonstrates increased serum endotoxin and IL-6 levels, and decreased HDL, apoA-I and activity of anti-oxidant HDL-associated paraoxanase-1. L-4F inhibits the activation of isolated human leukocytes and neutrophils by ARDS serum and LPS in vitro. Further, L-4F decreased endotoxin activity and preserved anti-oxidant properties of HDL both in vitro and in vivo. In a rat model of severe endotoxemia, L-4F significantly decreased mortality and reduces lung and liver injury, even when administered 1 hour post LPS. Our study suggests the protective role of the apoA-I mimetic peptide L-4F in ARDS and gram-negative endotoxemia and warrant further clinical evaluation. The main protective mechanisms of L-4F are due to direct inhibition of endotoxin activity and preservation of HDL anti-oxidant activity.
PMCID: PMC3653907  PMID: 23691230
22.  Exosomes and Cancer: A Newly Described Pathway of Immune Suppression 
Exosomes are small (30 to 100 nm) membrane-bound particles that are released from normal, diseased, and neoplastic cells and are present in blood and other bodily fluids. Exosomes contain a variety of molecules including signal peptides, mRNA, microRNA, and lipids. Exosomes can function to export from cells unneeded endogenous molecules and therapeutic drugs. When exosomes are taken up by specific cells, they may act locally to provide autocrine or paracrine signals or, at a distance, as a newly described nanoparticle-based endocrine system. Specifically, mRNA transferred to cells by exosomes can result in the production of new proteins. In cancer, signals via exosomes affect the immune system by inhibiting the functions of T cells and normal killer (NK) cells and by inhibiting the differentiation of precursors to mature antigen-presenting cells. Also, exosomes increase the number and/or activity of immune suppressor cells, including myeloid-derived suppressor cells, T-regulatory cells, and CD14+, HLA-DR–/low cells. The effects of exosomes on the development and progression of cancers, with an emphasis on suppression of immune surveillance, is described. Also discussed are potential uses of exosomes clinically, in the development of vaccines, in targeting tumors, and in diagnosis and/or early detection.
PMCID: PMC3155407  PMID: 21224375
23.  Prognostic Significance of p53 Codon 72 Polymorphism Differs with Race in Colorectal Adenocarcinoma 
Several studies have examined the prognostic value of the codon 72 polymorphism of the p53 gene in colorectal adenocarcinoma, but none have addressed patient race/ethnicity. Therefore, this study assessed the prognostic value of this polymorphism in African American and Caucasian colorectal adenocarcinoma patients separately.
Experimental Design
Colorectal adenocarcinomas from137 African Americans and 236 non-Hispanic Caucasians were assessed for p53 mutations and genotyped for the codon 72 polymorphism. The phenotypes were correlated with p53 mutational status, clinicopathologic features, and patient survival using the χ2 test and Kaplan-Meier and Cox regression models.
The incidence of p53 mutations was similar in African American and Caucasian patients (50% versus 54%, respectively); however, the homozygous Pro72 allele frequency was higher in African Americans (17%) as compared with Caucasians (7%). In contrast, the homozygous Arg72 allele frequency was higher in Caucasians (36%) than in African Americans (19%). In African Americans but not Caucasians, the Pro/Pro phenotype significantly correlated with a higher incidence of missense p53 mutations and with nodal metastasis. African Americans, but not Caucasians, with the Pro/Pro phenotype had significantly higher mortality (log-rank P = 0.005 versus. P = 0.886) and risk of death due to colorectal adenocarcinoma (hazard ratio, 2.15; 95% confidence interval, 1.02-4.53 versus hazard ratio, 1.60; 95% confidence interval, 0.69-3.18) than those with the phenotype Arg/Arg orArg/Pro.
The higher frequency of the Pro/Pro phenotype of p53 in African American patients with colorectal adenocarcinoma is associated with an increased incidence of p53 mutations, with advanced tumor stage, and with short survival.
PMCID: PMC3635077  PMID: 19339276
24.  A Migration Signature and Plasma Biomarker Panel for Pancreatic Adenocarcinoma 
Pancreatic ductal adenocarcinoma is a disease of extremely poor prognosis for which there are no reliable markers of asymptomatic disease. To identify pancreatic cancer biomarkers, we focused on a genomic interval proximal to the most common fragile site in the human genome, chromosome 3p12, which undergoes smoking-related breakage, loss of heterozygosity, and homozygous deletion as an early event in many epithelial tumors, including pancreatic cancers. Using a functional genomic approach, we identified a seven-gene panel (TNC, TFPI, TGFBI, SEL-1L, L1CAM, WWTR1, and CDC42BPA) that was differentially expressed across three different expression platforms, including pancreatic tumor/normal samples. In addition, Ingenuity Pathways Analysis (IPA) and literature searches indicated that this seven-gene panel functions in one network associated with cellular movement/morphology/development, indicative of a “migration signature” of the 3p pathway. We tested whether two secreted proteins from this panel, tenascin C (TNC) and tissue factor pathway inhibitor (TFPI), could serve as plasma biomarkers. Plasma ELISA assays for TFPI/TNC resulted in a combined area under the curve (AUC) of 0.88 and, with addition of CA19-9, a combined AUC for the three-gene panel (TNC/TFPI/CA19-9), of 0.99 with 100% specificity at 90% sensitivity and 97.22% sensitivity at 90% specificity. Validation studies using TFPI only in a blinded sample set increased the performance of CA19-9 from an AUC of 0.84 to 0.94 with the two-gene panel. Results identify a novel 3p pathway–associated migration signature and plasma biomarker panel that has utility for discrimination of pancreatic cancer from normal controls and promise for clinical application.
PMCID: PMC3635082  PMID: 21071578

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