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PDF file - 96K, ELISA data for individual markers in newly diagnosed samples separated based on stage at the time of diagnosis compared to matched controls. Only significant differences are demarcated. * indicates p

PDF file - 117K, ELISA data for individual markers in newly diagnosed samples separated by gender. Only significant differences are demarcated. * indicates p

PDF file - 70K, Correlations between different markers based on ELISA data of newly diagnosed samples.

PDF file - 112K, Workflow of experiments and sample cohorts described in the manuscript.

PDF file - 225K, Histograms for case-to-control ratios for each of the 9 MS experiments used for normalization.

PDF fiel - 144K, Peptide coverage for each of the 6 individual proteins in two colorectal cancer cell lines with different driver mutations.

Supplementary Figure and Table Legend from Detection of Elevated Plasma Levels of Epidermal Growth Factor Receptor Before Breast Cancer Diagnosis among Hormone Therapy Users

PDF file - 48K, Peptide sequences from proteins identified in immune complexes.

We assessed the autoantibody repertoire of a mouse model engineered to develop breast cancer and the repertoire of autoantibodies in human plasmas collected at a preclinical time point and at the time of clinical diagnosis of breast cancer. In seeking to identify common pathways, networks, and protein families associated with the humoral response, we elucidated the dynamic nature of tumor antigens and autoantibody interactions. Lysate proteins from an immortalized cell line from a MMTV-neu mouse model and from MCF7 human breast cancers were spotted onto nitrocellulose microarrays and hybridized with mouse and human plasma samples, respectively. Immunoglobulin-based plasma immunoreactivity against glycolysis and spliceosome proteins was a predominant feature observed both in tumor-bearing mice and in prediagnostic human samples. Interestingly, autoantibody reactivity was more pronounced further away than closer to diagnosis. We provide evidence for dynamic changes in autoantibody reactivity with tumor development and progression that may depend, in part, on the extent of antigen–antibody interactions. Cancer Res; 73(5); 1502–13. ©2012 AACR.

Supplementary Figure 1 from Detection of Elevated Plasma Levels of Epidermal Growth Factor Receptor Before Breast Cancer Diagnosis among Hormone Therapy Users

PDF file - 54K, Coefficients from linear regression models used for ROC analysis.

BACKGROUND Chronic graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality in transplant patients. We have previously shown that 3 doses of an anti-inducible costimulator (ICOS) mAb transiently ameliorated symptoms and extended survival of dogs affected by chronic GVHD over that of control dogs. The purpose of this study was to specifically correlate changes in T-cell populations in the peripheral blood with anti-ICOS treatment and chronic GVHD progression and regression to reach a better understanding of the mechanism of the disease and prioritize future studies. METHODS Peripheral blood cells from canines transplanted with DLA-mismatched bone marrow and peripheral blood mononuclear cells to generate chronic GVHD were analyzed by flow cytometry using a panel of antibodies specific to helper and cytolytic T cells. RESULTS Chronic GVHD was specifically associated with an increase in CD4+ICOS+ cells, ICOS+ cells expressing IL-17A, and CD8+ cells generating granzyme B. Treatment with anti-ICOS mAb at onset of chronic GVHD symptoms specifically targeted IL-17A+-expressing cells, transiently relieved symptoms, and lengthened survival but was unable to reduce the percentage of CD8+ T-cells expressing granzyme B. CONCLUSIONS These studies suggested a role for both CD4+ and CD8+ T cells in pathogenesis of chronic GVHD in the canine model. We propose that future studies should focus on further extending survival by developing a treatment that would control both CD4+ and CD8+ T cells.

OBJECTIVE To assess predictors of prostate-specific membrane antigen (PSMA) expression in a genomic database; positron emission tomography with PSMA-targeted radiopharmaceuticals is increasingly being utilized. METHODS The de-identified Decipher Biosciences database, which includes expression for more than 46,000 coding and noncoding genes per patient, was queried for expression of FOLH1 (PSMA). Prostate cancer patients who underwent radical prostatectomy and received the Decipher Test were included in the analysis. PSMA expression was compared to the Gleason Grade Group, Decipher risk category (a validated 22 biomarker genomic score), basal versus luminal molecular subtype, and androgen receptor activity. Multivariable regression analyses were performed. RESULTS The Decipher de-identified Decipher Biosciences database contained 16,807 men who underwent prostatectomy with the average age being 65-year old and most being Gleason Grade Group 2 (35%) or 3 (27%). Higher Grade Group was associated with higher PSMA expression except in Grade Group 5 [Grade group: 1 (0.66), 2 (0.84), 3 (0.99), 4 (1.07), 5 (0.99), P

In murine model systems, inducible costimulator (ICOS) signaling has been implicated in the formation of chronic graft-versus-host disease (GVHD). Previously, we showed that chronic GVHD can be reproducibly produced in the dog hematopoietic cell transplantation (HCT) model and that ICOS expression is upregulated on T cells in dogs with chronic GVHD. The goal of the present study was to determine whether administration of a short course of anti-canine ICOS monoclonal antibody (mAb) could alter the rapid and progressive course of chronic GVHD. Five dogs underwent HCT from dog leukocyte antigen mismatched unrelated donors following total body irradiation. Post-grafting immunosuppression consisted of methotrexate (days 1, 3, 6, and 11) and cyclosporine (days -1 through 78). Anti-ICOS mAb (3 injections, 72 hours apart) was administered upon diagnosis of GVHD. One dog failed to respond to anti-ICOS mAb therapy and succumbed to chronic GVHD in a time course similar to control untreated dogs. Overall, anti-ICOS-treated dogs experienced a significant prolongation in survival from the time of diagnosis of chronic GVHD compared to control dogs. Within the limitations of the number of study dogs, we suggest that a short course of anti-ICOS mAb may be useful in the treatment of chronic canine GVHD.

Natural killer (NK) cells are non-T, non-B lymphocytes are part of the innate immune system and function without prior activation. The human NK cell surface determinant, CD94, plays a critical role in regulation of NK cell activity as a heterodimer with NKG2 subclasses. Canine NK cells are not as well defined as the human and murine equivalents, due in part to the paucity of reagents specific to cell surface markers. Canines possess NK/NKT cells that have similar morphological characteristics to those found in humans, yet little is known about their functional characteristics nor of cell surface expression of CD94. Here, we describe the development and function of a monoclonal antibody (mAb) to canine (ca) CD94. Freshly isolated canine CD94(+) cells were CD3(+/–), CD8(+/–), CD4(–), CD21(–), CD5(low), NKp46(+), and were cytotoxic against a canine target cell line. Anti-caCD94 mAb proved useful in enriching NK/NKT cells from PBMC for expansion on CTAC feeder cells in the presence of IL-2 and IL-15. The cultured cells were highly cytolytic with co-expression of NKp46 and reduced expression of CD3. Transmission electron microscopy revealed expanded CD94(+) lymphocytes were morphologically large granular lymphocytes with large electron dense granules. Anti-caCD94 (mAb) can serve to enrich NK/NKT cells from dog peripheral blood for ex vivo expansion for HCT and is a potentially valuable reagent for studying NK/NKT regulation in the dog.

Chronic liver injury leads to fibrosis and cirrhosis. Cirrhosis, the end stage of chronic liver disease, is a leading cause of death worldwide and increases the risk of developing hepatocellular carcinoma. Currently, there is a lack of effective antifibrotic therapies to treat fibrosis and cirrhosis. Development of antifibrotic therapies requires an in-depth understanding of the cellular and molecular mechanisms involved in inflammation and fibrosis after hepatic injury. Two growth factor signaling pathways that regulate liver fibrosis are transforming growth factor-β (TGFβ) and platelet-derived growth factor (PDGF). However, their specific contributions to fibrogenesis are not well understood. Using a genetic model of liver fibrosis, we investigated whether the canonical TGFβ signaling pathway was necessary for fibrogenesis. PDGF-C transgenic ( PDGF-C Tg) mice were intercrossed with mice that lack Smad3, and molecular and histological fibrosis was analyzed. PDGF-C Tg mice that also lacked Smad3 had less fibrosis and improved liver lobule architecture. Loss of Smad3 also reduced expression of collagen genes, which were induced by PDGF-C, but not the expression of genes frequently associated with hepatic stellate cell (HSC) activation. In vitro HSCs isolated from Smad3-null mice proliferated more slowly than cells from wild-type mice. Taken together, these findings indicate that PDGF-C activates TGFβ/Smad3 signaling pathways to regulate HSC proliferation, collagen production and ultimately fibrosis. In summary, these results suggest that inhibition of both PDGF and TGFβ signaling pathways may be required to effectively attenuate fibrogenesis in patients with chronic liver disease.

The repertoire of antigens associated with the development of an autoimmune response in breast cancer has relevance to detection and treatment strategies. We have investigated the occurrence of autoantibodies associated with the development of triple-negative breast cancer (TNBC) in the before diagnosis setting and in samples collected at the time of diagnosis of TNBC. Lysate arrays containing protein fractions from the TNBC MDA-MB-231 cell line were hybridized with TNBC plasmas from the Women's Health Initiative cohort, collected before clinical diagnosis and with plasmas from matched controls. An immune response directed against spliceosome and glycolysis proteins was observed with case plasmas as previously reported in estrogen receptor+ breast cancer. Importantly, autoantibodies directed against networks involving BRCA1, TP53, and cytokeratin proteins associated with a mesenchymal/basal phenotype were distinct to TNBC before diagnosis samples. Concordant autoantibody findings were observed with mouse plasma samples collected before occurrence of palpable tumors from a C3(1)-T triple negative mouse model. Plasma samples collected at the time of diagnosis of stage II TNBC and from matched healthy controls were subjected to proteomic analysis by mass spectrometry to identify Ig-bound proteins yielding a predominance of cytokeratins, including several associated with a mesenchymal/basal phenotype among cases compared with controls. Our data provide evidence indicative of a dynamic repertoire of antigens associated with a humoral immune response reflecting disease pathogenesis in TNBC. Cancer Res; 75(16); 3246–54. ©2015 AACR.

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet-derived growth factor C (PDGF-C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF-CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast-like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell-derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte-derived PDGF-C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF-CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF-C transgenic mice provide a unique model for the in vivo study of tumor–stromal interactions in the liver.

The T cell Ig and mucin domain 3 (Tim-3) receptor has been implicated as a negative regulator of adaptive immune responses. We have utilized a proteomic strategy to identify novel proteins associated with graft versus host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). Mass spectrometry analysis of plasma from subjects with mid-gut and upper-gut GVHD compared with those without GVHD identified increased levels of a protein identified with high confidence as Tim-3. A follow-up validation study using an immunoassay to measure Tim-3 levels in individual plasma samples from 127 patients demonstrated significantly higher plasma Tim-3 concentrations in patients with the more severe mid-gut GVHD, compared with those with upper-gut GVHD (P = .005), patients without GVHD (P = .002), and normal controls (P

Purpose Proteomics technologies are well suited for harnessing the immune response to tumor antigens for diagnostic applications as in the case of breast cancer. We previously reported a substantial impact of hormone therapy (HT) on the proteome. Here, we investigated the effect of HT on the immune response toward breast tumor antigens. Experimental design Plasmas collected 0–10 months prior to diagnosis of ER+ breast cancer from 190 postmenopausal women and 190 controls that participated in the Women's Health Initiative Observational Study were analyzed for the effect of HT on IgG reactivity against arrayed proteins from MCF-7 or SKBR3 breast cancer cell line lysates following extensive fractionation. Results HT user cases exhibited significantly reduced autoantibody reactivity against arrayed proteins compared to cases who were Not Current users. An associated reduced level of IL-6 and other immune-related cytokines was observed among HT users relative to nonusers. Conclusion and clinical relevance Our findings suggest occurrence of a global altered immune response to breast cancer-derived proteins associated with HT. Thus a full understanding of factors that modulate the immune response is necessary to translate autoantibody panels into clinical applications.

Applying advanced proteomic technologies to prospectively collected specimens from large studies is one means of identifying preclinical changes in plasma proteins that are potentially relevant to the early detection of diseases such as breast cancer. We conducted 14 independent quantitative proteomics experiments comparing pooled plasma samples collected from 420 estrogen receptor–positive (ER+) breast cancer patients ≤17 months before their diagnosis and matched controls. Based on the more than 3.4 million tandem mass spectra collected in the discovery set, 503 proteins were quantified, of which 57 differentiated cases from controls with a P value of

Suppressor of cytokine signaling 3 (SOCS3) down-regulates several signaling pathways in multiple cell types, and previous data suggest that SOCS3 may shut off cytokine activation at the early stages of liver regeneration (Campbell, J.S., L. Prichard, F. Schaper, J. Schmitz, A. Stephenson-Famy, M.E. Rosenfeld, G.M. Argast, P.C. Heinrich, and N. Fausto. 2001.J. Clin. Invest. 107:1285–1292). We developed Socs3 hepatocyte-specific knockout (Socs3 h-KO) mice to directly study the role of SOCS3 during liver regeneration after a two-thirds partial hepatectomy (PH). Socs3 h-KO mice demonstrate marked enhancement of DNA replication and liver weight restoration after PH in comparison with littermate controls. Without SOCS3, signal transducer and activator of transcription 3 (STAT3) phosphorylation is prolonged, and activation of the mitogenic extracellular signal-regulated kinase 1/2 (ERK1/2) is enhanced after PH. In vitro, we show that SOCS3 deficiency enhances hepatocyte proliferation in association with enhanced STAT3 and ERK activation after epidermal growth factor or interleukin 6 stimulation. Microarray analyses show that SOCS3 modulates a distinct set of genes, which fall into diverse physiological categories, after PH. Using a model of chemical-induced carcinogenesis, we found that Socs3 h-KO mice develop hepatocellular carcinoma at an accelerated rate. By acting on cytokines and multiple proliferative pathways, SOCS3 modulates both physiological and neoplastic proliferative processes in the liver and may act as a tumor suppressor.

To facilitate selection of single-nucleotide polymorphisms (SNP) for molecular epidemiologic studies investigating the hormonal carcinogenesis hypothesis, we used two sequence homology–based tools [Sort Intolerant from Tolerant (SIFT) and Polymorphism Phenotype (PolyPhen)] to predict the potential impact a nonsynonymous SNP (nsSNP), which results in an amino acid substitution, may have on the activity of proteins encoded by genes involved in the steroid hormone metabolism and response pathway. We screened 137 variants. Of these, 28% were predicted by SIFT and PolyPhen as having a potentially damaging effect on protein function. Investigation into the association of these variant alleles with hormone-related cancers may prove to be fruitful.

In the prostate, the enzyme encoded by the SRD5A2 gene (5α-reductase) converts testosterone to dihydrotestosterone, a potent androgen that has been hypothesized to play a role in the genesis of prostate cancer. Several polymorphisms have been identified in the SRD5A2 gene, including a valine-to-leucine substitution (V89L) at codon 89, a variable number of TA dinucleotide repeats and a missense substitution at codon 49 resulting in an amino acid substitution of alanine with threonine (A49T). To investigate the influence of these polymorphisms on prostate cancer risk, we conducted a case-control study nested within the Beta-Carotene and Retinol Efficacy Trial. Genotypes were determined by PCR-based capillary electrophoresis using genomic DNA isolated from 300 cases and 300 controls matched on the basis of race, age at enrollment (within 5 years), enrollment study center and year of randomization. There was no association between V89L genotypes and prostate cancer risk. The age- and race-adjusted odds ratio (OR) associated with the VL and LL genotypes were 1.06 (95% confidence interval (CI) = 0.75-1.49) and 0.99 (95% CI = 0.57-1.73), respectively, as compared to the VV genotype. The age- and race-adjusted odds ratio for men having 1 TA(9) or TA(18) allele was 0.98 (95% CI = 0.64-1.48) when compared to men without TA repeats. The corresponding odds ratio for men without the TA(0) alleles was 0.68 (95% CI = 0.21-2.19). The age- and race-adjusted odds ratio associated with having at least 1 T allele at codon 49 was 1.11 (95% CI = 0.58-2.11), as compared to the AA genotype. Our results do not support the hypothesis that the V89L and A49T polymorphisms in the SRD5A2 gene are related to the risk of prostate cancer, but are compatible with the suggestion from earlier studies that men who are homozygous for the TA(9) or (18) alleles and men who have the TA(9)/TA(18) genotype are at a modestly reduced risk. © 2003 Wiley-Liss, Inc.

Background The use of autoantibodies for the early detection of breast cancer has generated much interest as antibodies can be readily assayed in serum when antigen levels are low. Ideally, diagnostic autoantibodies would be identified in individuals who harbored pre-invasive disease/high risk lesions leading to malignancy. Prospectively collected human serum samples from these individuals are rare and not often available for biomarker discovery. We questioned whether transgenic animals could be used to identify cancer-associated autoantibodies present at the earliest stages of the malignant transformation of breast cancer. Methods We collected sera from transgenic mice (TgMMTV-neu) from the time of birth to death by spontaneous mammary tumors. Using sera from a time point prior to the development of tumor, i.e. “pre-diagnostic”, we probed cDNA libraries derived from syngeneic tumors to identify proteins recognized by IgG antibodies. Once antigens were identified, selected proteins were evaluated via protein arrays, for autoantibody responses using plasma from women obtained prior to the development of breast cancer and matched controls. The ability of the antigens to discriminate cases from controls was assessed using receiver-operating-characteristic curve analyses and estimates of the area under the curve. Results We identified 6 autoantibodies that were present in mice prior to the development of mammary cancer: Pdhx, Otud6b, Stk39, Zpf238, Lgals8, and Vps35. In rodent validation cohorts, detecting both IgM and IgG antibody responses against a subset of the identified proteins could discriminate pre-diagnostic sera from non-transgenic control sera with an AUC of 0.924. IgG and IgM autoantibodies, specific for a subset of the identified antigens, could discriminate the samples of women who eventually developed breast cancer from case-matched controls who did not develop disease. The discriminatory potential of the pre-diagnostic autoantibodies was enhanced if plasma samples were collected greater than 5 months prior to a breast cancer diagnosis (AUC 0.68; CI 0.565-0.787, p = 0.0025). Conclusion Genetically engineered mouse models of cancer may provide a facile discovery tool for identifying autoantibodies useful for human cancer diagnostics.

Platelet-derived growth factors (PDGFs) are critical for development; their over-expression is associated with fibrogenesis. Full-length PDGF-C is secreted as an inactive dimer, requiring cleavage to allow receptor binding. Previous studies indicate that tissue-type plasminogen activator (tPA) is the specific protease that performs this cleavage; in vivo confirmation is lacking. We demonstrate that primary hepatocytes from tpa KO mice produce less cleaved active PDGF-CC than do wild type hepatocytes, suggesting that tPA is critical for in vitro activation of this growth factor. We developed mice that over-express full-length human PDGF-C in the liver; these mice develop progressive liver fibrosis. To test whether tPA is important for cleavage and activation of PDGF-C in vivo, we intercrossed PDGF-C transgenic (Tg) and tpa knock-out (KO) mice, anticipating that lack of tPA would result in decreased fibrosis due to lack of hPDGF-C cleavage. To measure levels of cleaved, dimerized PDGF-CC in sera, we developed an ELISA that specifically detects cleaved PDGF-CC. We report that the absence of tpa does not affect the phenotype of `PDGF-C Tg mice. PDGF-C Tg mice lacking tPA have high serum levels of cleaved growth factor, significant liver fibrosis, and gene expression alterations similar to those of PDGF-C Tg mice with intact tPA. Furthermore, urokinase plasminogen activator and plasminogen activator inhibitor-1 expression are increased in PDGF-C Tg; tpa KO mice. Our ELISA data suggest a difference between in vitro and in vivo activation of this growth factor, and our mouse model confirms that multiple proteases cleave and activate PDGF-C in vivo.

We assessed the autoantibody repertoire of a mouse model engineered to develop breast cancer and the repertoire of autoantibodies in human plasmas collected at a preclinical time point and at the time of clinical diagnosis of breast cancer. In seeking to identify common pathways, networks, and protein families associated with the humoral response, we elucidated the dynamic nature of tumor antigens and autoantibody interactions. Lysate proteins from an immortalized cell line from a MMTV-neu mouse model and from MCF7 human breast cancers were spotted onto nitrocellulose microarrays and hybridized with mouse and human plasma samples, respectively. Immunoglobulin-based plasma immunoreactivity against glycolysis and spliceosome proteins was a predominant feature observed both in tumor-bearing mice and in prediagnostic human samples. Interestingly, autoantibody reactivity was more pronounced further away than closer to diagnosis. We provide evidence for dynamic changes in autoantibody reactivity with tumor development and progression that may depend, in part, on the extent of antigen–antibody interactions. Cancer Res; 73(5); 1502–13. ©2012 AACR.

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet-derived growth factor-c (PDGF-C) induces progressive fibrosis, chronic inflammation, neoangiogenesis, and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF-CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast-like cells that deposit extracellular matrix (ECM) and lead to production of growth factors and cytokines. We demonstrate induction of cytokines genes at two months, and stromal cell-derived hepatocyte growth factors that coincide with the onset of dysplasia at four months. Our results support a paracrine signaling model wherein hepatocyte-derived PDGF-C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury, and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF-CC levels were observed in livers of patients with non-alcoholic fatty steatohepatitis (NASH) and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF-C transgenic mice provide a unique model for the in vivo study of tumor-stromal interactions in the liver.

Longitudinal blood collections from cohort studies provide the means to search for proteins associated with disease before clinical diagnosis. We investigated plasma samples from the Women's Health Initiative (WHI) cohort to determine quantitative differences in plasma proteins between subjects subsequently diagnosed with colorectal cancer (CRC) and matched controls that remained cancer-free during the period of follow-up. Proteomic analysis of WHI samples collected before diagnosis of CRC resulted in the identification of six proteins with significantly (P < 0.05) elevated concentrations in cases compared with controls. Proteomic analysis of two CRC cell lines showed that five of the six proteins were produced by cancer cells. Microtubule-associated protein RP/EB family member 1 (MAPRE1), insulin-like growth factor–binding protein 2 (IGFBP2), leucine-rich alpha-2-glycoprotein (LRG1), and carcinoembryonic antigen (CEA) were individually assayed by enzyme linked immunosorbent assay (ELISA) in 58 pairs of newly diagnosed CRC samples and controls and yielded significant elevations (P < 0.05) among cases relative to controls. A combination of these four markers resulted in a receiver operating characteristics curve with an area under the curve value of 0.841 and 57% sensitivity at 95% specificity. This combination rule was tested in an independent set of WHI samples collected within 7 months before diagnosis from cases and matched controls resulting in 41% sensitivity at 95% specificity. A panel consisting of CEA, MAPRE1, IGFBP2, and LRG1 has predictive value in prediagnostic CRC plasmas. Cancer Prev Res; 5(4); 655–64. ©2012 AACR.

Non-invasive therapies for the treatment of hepatocellular carcinoma (HCC) would be of great benefit to public health. To this end, we have developed a platelet-derived growth factor-C (PDGF-C) transgenic (Tg) mouse model, which mimics many aspects of human liver carcinogenesis. Specifically, overexpression of PDGF-C results in liver fibrosis, which is preceded by activation and proliferation of hepatic stellate cells, and is followed by the development of dysplastic lesions and angiogenesis, and progression to HCCs by 8 months of age. Here, we show that PDGF-C overexpression induces the proliferation of endothelial-like cells that are present in tumors and adjacent non-neoplastic parenchyma. The protein tyrosine kinase inhibitor, imatinib (Gleevec), decreases the proliferation of non-parenchymal cells (NPC) in vitro and in vivo, with concomitant inhibition of Akt. In vivo treatment with imatinib also blocks the expression of CD34 in PDGF-C Tg mice. Decreased NPC proliferation and CD34 expression correlated with lower levels of active ERK1/2 and total levels of PDGF receptor alpha (PDGFRalpha). In summary, the small molecule inhibitor imatinib attenuates stromal cell proliferation in PDGF-C-induced HCC, which coincides with decreased expression of both CD34 and PDGFRalpha, and activated Akt. Our findings suggest that imatinib may be efficacious in the treatment of hepatocarcinogenesis, particularly when neovascularization is present.

The MYC oncogene induces both cell proliferation and apoptosis. The apoptotic function of MYC is thought to inhibit carcinogenesis; thus, when disrupted, tumorigenic potential is increased. Both MYC and transforming growth factor alpha (TGFalpha) are commonly over-expressed in hepatocellular carcinomas, and transgenic mice expressing these genes rapidly develop tumors via the suppression of MYC-induced apoptosis by the growth factor. However, the nature of the interactions between MYC and TGFalpha are not well understood. Specifically, it is unclear whether TGFalpha acts only as an anti-apoptotic factor in its interactions with MYC or whether it has substantial effects on cell growth. We investigated whether TGFalpha can provide additional mitogenic signals if it is not required to act as an anti-apoptotic factor. We demonstrate that expression of MYC and TGFalpha in liver progenitor cells (known as oval cells) results in enhanced cell proliferation in culture and the generation of poorly differentiated tumors after inoculation into nude mice. We further demonstrate that while the apoptosis-deficient T58A and S71F alleles of MYC retain their ability to promote oval cell proliferation, they have opposite growth interactions with TGFalpha. The T58A allele has a stimulatory effect on both proliferation and tumorigenicity. In contrast, co-expression of the S71F allele reduces proliferation and slows tumor development. We conclude that the tumorigenic growth effects of MYC in TGFalpha-expressing liver progenitor cells are not solely dependent on its apoptotic activity.

Longitudinal blood collections from cohort studies provide the means to search for proteins associated with disease processes prior to diagnosis. We have investigated pre-diagnostic plasma samples to determine quantitative differences in plasma protein between subjects subsequently diagnosed with colorectal cancer (CRC) and matched controls that remain cancer free during an 18 month period of active follow-up. In-depth quantitative proteomic profiling was performed on 90 pre-diagnostic plasma samples. Experiments compared nine pools each consisting of plasma aliquots from 10 CRC subjects diagnosed within 18 months after blood draw and 10 matched controls. MAPRE1 was confirmed in a set of 58 pairs of plasma from newly diagnosed CRC cases and controls using enzyme linked immunosorbent assay (ELISA). MAPRE1 was further tested by ELISA in a second independent set of 100 pre-diagnostic CRC plasma samples diagnosed within 18 months after blood draw and 100 matched controls. Proteomic analysis of pre-diagnostic plasma samples collected prior to diagnosis of CRC yielded a set of proteins with elevated concentrations (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5102. doi:10.1158/1538-7445.AM2011-5102

Background: Proteomic analyses of mouse lung cancer models have yielded circulating markers that can translate to human disease. Analysis of angiopoietin-like protein 3 (ANGPTL3) in pre-diagnostic human lung cancer sera identified two types of complementary, circulating markers: protein concentration and the occurrence of autoantibodies to ANGPTL3. Methods: Sera collected up to a year prior to diagnosis of lung cancer from 26 patients and 26 matched healthy controls were analyzed to determine both the circulating concentration of and autoantibody reactivity to ANGPTL3. Circulating levels were measured by commercial enzyme-linked immunosorbent assay (ELISA) and autoantibody reactivity was measured by fluorescent microarray. Additionally, sera from 38 newly diagnosed patients with lung cancer and 38 healthy individuals were also analyzed for ANGPTL3 circulating levels and autoantibody reactivity. Results: Both ANGPTL3 concentration and autoantibody reactivity were significantly elevated in sera of non-small cell lung cancer (NSCLC) patients collected 0-12 months prior to diagnosis compared to control sera (p=0.12 and p=0.035, respectively). The circulating concentration of ANGPTL3 and autoantibody reactivity were also significantly elevated in the sera of newly diagnosed NSCLC patients compared to control sera (p=0.0039 and p=0.0013, respectively). Autoantibody reactivity to ANGPTL3 and protein concentration in individual patient sera was inversely correlated. A combined analysis of autoantibody reactivity and circulating concentration improved predictive performance by receiver operating characteristic (ROC) analysis compared to individual markers, achieving 53% sensitivity at 90% specificity and an area under the curve of 81%. Western blot analysis showed expression of ANGPTL3 in NSCLC cell lines, but not in an immortalized bronchioepithelial cell line. Immunohistochemical analysis of ANGPTL3 in lung tumor tissue arrays revealed robust staining in the cytoplasm of neoplastic epithelial cells in both adenocarcinomas and squamous cell carcinomas. The addition of ANGPTL3 to a previously published panel of three autoantibodies (i.e. ANXA1, YWHAQ, and LAMR1) for early lung cancer detection improves the predictive performance of the panel to discriminate cases from controls in sera collected prior to the onset of symptoms. Conclusions: Circulating levels of ANGPTL3 and autoantibody reactivity were significantly elevated in newly diagnosed NSCLC and an independent set of pre-diagnostic samples compared to controls. A combination of circulating levels and autoantibody reactivity for ANGPTL3 improves the predictive performance. ANGPTL3 is strongly expressed in NSCLC cell lines and tissue. Our findings indicate that the occurrence of autoantibodies and the concentration of ANGPTL3 in serum may have utility for the early diagnosis of NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2814. doi:10.1158/1538-7445.AM2011-2814

Background Women's Health Initiative randomized trials of postmenopausal hormone therapy reported intervention effects on several clinical outcomes, with some important differences between estrogen alone and estrogen plus progestin. The biologic mechanisms underlying these effects, and these differences, have yet to be fully elucidated. Methods Baseline serum samples were compared with samples drawn 1 year later for 50 women assigned to active hormone therapy in both the estrogen-plus-progestin and estrogen-alone randomized trials, by applying an in-depth proteomic discovery platform to serum pools from 10 women per pool. Results In total, 378 proteins were quantified in two or more of the 10 pooled serum comparisons, by using strict identification criteria. Of these, 169 (44.7%) showed evidence (nominal P < 0.05) of change in concentration between baseline and 1 year for one or both of estrogen-plus-progestin and estrogen-alone groups. Quantitative changes were highly correlated between the two hormone-therapy preparations. A total of 98 proteins had false discovery rates < 0.05 for change with estrogen plus progestin, compared with 94 for estrogen alone. Of these, 84 had false discovery rates