Donna O. Butcher

Resolving conflicting data on expression of the Tn antigen and implications for clinical trials with cancer vaccines

The tumor-associated Tn antigen has been investigated extensively as a biomarker and therapeutic target. Cancer vaccines containing the Tn antigen as a single tumor antigen or as a component of a polyvalent vaccine have progressed into phase I and II clinical trials. One major focus of Tn-based vaccines is the treatment of prostate cancer patients. Although expression of the antigen on prostate tumors is a critical prerequisite, previous reports investigating Tn expression in prostate tumors have produced conflicting results. Using a combination of immunohistochemistry and carbohydrate microarray profiling, we show that only 4% to 26% of prostate tumors express the Tn antigen. Based on our results, the majority of prostate cancer patients do not express the appropriate antigen. Therefore, efforts to preselect the subset of prostate cancer patients with Tn-positive tumors or apply Tn vaccines to other cancers with higher rates of antigen expression could significantly improve clinical response rates. Because conflicting information on carbohydrate expression is a general problem for the field, the approach described in this article of analyzing antigen expression with multiple antibodies and using carbohydrate microarray profiles to interpret the results will be useful for the development of other carbohydrate-based cancer vaccines and diagnostics.[Mol Cancer Ther 2009;8(4):971–9)

Gene expression profiling of alveolar soft-part sarcoma (ASPS).

BackgroundAlveolar soft-part sarcoma (ASPS) is an extremely rare, highly vascular soft tissue sarcoma affecting predominantly adolescents and young adults. In an attempt to gain insight into the pathobiology of this enigmatic tumor, we performed the first genome-wide gene expression profiling study.MethodsFor seven patients with confirmed primary or metastatic ASPS, RNA samples were isolated immediately following surgery, reverse transcribed to cDNA and each sample hybridized to duplicate high-density human U133 plus 2.0 microarrays. Array data was then analyzed relative to arrays hybridized to universal RNA to generate an unbiased transcriptome. Subsequent gene ontology analysis was used to identify transcripts with therapeutic or diagnostic potential. A subset of the most interesting genes was then validated using quantitative RT-PCR and immunohistochemistry.ResultsAnalysis of patient array data versus universal RNA identified elevated expression of transcripts related to angiogenesis (ANGPTL2, HIF-1 alpha, MDK, c-MET, VEGF, TIMP-2), cell proliferation (PRL, IGFBP1, NTSR2, PCSK1), metastasis (ADAM9, ECM1, POSTN) and steroid biosynthesis (CYP17A1 and STS). A number of muscle-restricted transcripts (ITGB1BP3/MIBP, MYF5, MYF6 and TRIM63) were also identified, strengthening the case for a muscle cell progenitor as the origin of disease. Transcript differentials were validated using real-time PCR and subsequent immunohistochemical analysis confirmed protein expression for several of the most interesting changes (MDK, c-MET, VEGF, POSTN, CYP17A1, ITGB1BP3/MIBP and TRIM63).ConclusionResults from this first comprehensive study of ASPS gene expression identifies several targets involved in angiogenesis, metastasis and myogenic differentiation. These efforts represent the first step towards defining the cellular origin, pathogenesis and effective treatment strategies for this atypical malignancy.

Insulin-dependent diabetes induced by pancreatic beta cell expression of IL-15 and IL-15Rα

Increased serum levels of IL-15 are reported in type 1 diabetes (T1D). Here we report elevated serum soluble IL-15Rα levels in human T1D. To investigate the role of IL-15/IL-15Rα in the pathogenesis of T1D, we generated double transgenic mice with pancreatic β-cell expression of IL-15 and IL-15Rα. The mice developed hyperglycemia, marked mononuclear cell infiltration, β-cell destruction, and anti-insulin autoantibodies that mimic early human T1D. The diabetes in this model was reversed by inhibiting IL-15 signaling with anti-IL2/IL15Rβ (anti-CD122), which blocks IL-15 transpresentation. Furthermore, the diabetes could be reversed by administration of the Janus kinase 2/3 inhibitor tofacitinib, which blocks IL-15 signaling. In an alternative diabetes model, nonobese diabetic mice, IL15/IL-15Rα expression was increased in islet cells in the prediabetic stage, and inhibition of IL-15 signaling with anti-CD122 at the prediabetic stage delayed diabetes development. In support of the view that these observations reflect the conditions in humans, we demonstrated pancreatic islet expression of both IL-15 and IL-15Rα in human T1D. Taken together our data suggest that disordered IL-15 and IL-15Rα may be involved in T1D pathogenesis and the IL-15/IL15Rα system and its signaling pathway may be rational therapeutic targets for early T1D.

A Pilot Study Assessing the Potential Role of non-CD133 Colorectal Cancer Stem Cells as Biomarkers

Introduction: Over 50% of patients with colorectal cancer (CRC) will progress and/or develop metastases. Biomarkers capable of predicting progression, risk stratification and therapeutic benefit are needed. Cancer stem cells are thought to be responsible for tumor initiation, dissemination and treatment failure. Therefore, we hypothesized that CRC cancer stem cell markers (CRCSC) will identify a group of patients at high risk for progression. Methods: Paraffin-embedded tissue cores of normal (n=8), and histopathologically well-defined primary (n= 30) and metastatic (n=10) CRC were arrayed in duplicate on tissue microarrays (TMAs). Expression profiles of non-CD133 CRCSC (CD29, CD44, ALDH1A1, ALDH1B1, EpCam, and CD166) were detected by immunohistochemistry and the association with clinicopathological data and patient outcomes was determined using standard statistical methodology. An independent pathologist, blinded to the clinical data scored the samples. Scoring included percent positive cells (0 to 4, 0 = <10%, 1 = 10 - 24%, 2 = 25 - 49%, 3 = 50 - 74%, 4 = 75 - 100%), and the intensity of positively stained cells (0 to 4; 0 = no staining, 1 = diminutive intensity, 2 = low intensity, 3 = intermediate intensity, 4 = high intensity). The pathologic score represents the sum of these two values, reported in this paper as a combined IHC staining score (CSS). Results: Of 30 patients 7 were AJCC stage IIA, 10 stage IIIB, 7 stage IIIC and 6 stage IV. Median follow-up was 113 months. DFI was 17 months. Median overall survival (OS) was not reached. Stage-specific OS was: II - not reached; III - not reached; IV - 11 months. In a univariate analysis, poor OS was associated with loss of CD29 expression; median OS, 32 months vs. not reached for CSS 3-7 vs. >7.5, respectively; p=0.052 comparing entire curves, after adjustment. In a Cox model analysis, loss of CD29 exhibited a trend toward association with survival (p=0.098) after adjusting for the effect of stage (p=0.0076). Greater expression of ALDH1A1 was associated with increasing stage (p=0.042 over stages 2, 3b, 3c, and 4) while loss of CD29 expression exhibited a trend toward being associated with stages 3 and 4 (p=0.08). Compared to normal colon tissue, primary tumors were associated with increased expression of ALDH1B1 (p=0.008). ALD1H1B1 expression level differed according to whether the tumor was moderately or poorly differentiated, well differentiated, or mucinous; the highest expression levels were associated with moderately or poorly differentiated tumors (p=0.011). Lymph node metastases were associated with a trend toward decreased expression of EpCAM (p = 0.06) when comparing 0 vs. 1 vs. 2+ positive lymph nodes, as was CD29 (p = 0.08) when comparing 0 vs. any positive lymph nodes. Compared to normal colon tissue metastatic colon cancers from different patients were associated with increased ALDH1B1 expression (p=0.001) whereas CD29 expression was higher in normal colonic tissue (p=0.014). Conclusion: CD29 may be associated with survival as well as clinical stage and number of lymph nodes. ALDH1B1 expression was associated with differentiation as well as type of tissue evaluated. ALDH1A1 was associated with clinical stage, and decreased EpCAM expression was found in patients with advanced lymph node stage. CRCSCs may be useful biomarkers to risk stratify, and estimate outcomes in CRC. Larger prospective studies are required to validate the current findings.

Therapeutic vulnerability of an in vivo model of alveolar soft part sarcoma (ASPS) to antiangiogenic therapy.

In vivo growth of alveolar soft part sarcoma (ASPS) was achieved using subcutaneous xenografts in sex-matched nonobese diabetic severe combined immunodeficiency mice. One tumor, currently at passage 6, has been maintained in vivo for 32 months and has maintained characteristics consistent with those of the original ASPS tumor including (1) tumor histology and staining with periodic acid Schiff/diastase, (2) the presence of the ASPL-TFE3 type 1 fusion transcript, (3) nuclear staining with antibodies to the ASPL-TFE3 type 1 fusion protein, (4) maintenance of the t(X;17)(p11;q25) translocation characteristic of ASPS, (5) stable expression of signature ASPS gene transcripts and finally, the development and maintenance of a functional vascular network, a hallmark of ASPS. The ASPS xenograft tumor vasculature encompassing nests of ASPS cells is highly reactive to antibodies against the endothelial antigen CD34 and is readily accessible to intravenously administered fluorescein isothiocyanate-dextran. The therapeutic vulnerability of this tumor model to antiangiogenic therapy, targeting vascular endothelial growth factor and hypoxia-inducible factor-1 alpha, was examined using bevacizumab and topotecan alone and in combination. Together, the 2 drugs produced a 70% growth delay accompanied by a 0.7 net log cell kill that was superior to the antitumor effect produced by either drug alone. In summary, this study describes a preclinical in vivo model for ASPS which will facilitate investigation into the biology of this slow growing soft tissue sarcoma and demonstrates the feasibility of using an antiangiogenic approach in the treatment of ASPS.

Restricted Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Pediatric B-Lineage Acute Lymphoblastic Leukemia Suggests Targetability with Therapeutic Monoclonal Antibodies

Background Despite high cure rates for pediatric B-lineage acute lymphoblastic leukemia (B-ALL), short-term and long-term toxicities and chemoresistance are shortcomings of standard chemotherapy. Immunotherapy and chemoimmunotherapy based on monoclonal antibodies (mAbs) that target cell surface antigens with restricted expression in pediatric B-ALL may offer the potential to reduce toxicities and prevent or overcome chemoresistance. The receptor tyrosine kinase ROR1 has emerged as a candidate for mAb targeting in select B-cell malignancies. Methodology and Principal Findings Using flow cytometry, Western blotting, immunohistochemistry, and confocal immunofluorescence microscopy, we analyzed the cell surface expression of ROR1 across major pediatric ALL subtypes represented by 14 cell lines and 56 primary blasts at diagnosis or relapse as well as in normal adult and pediatric tissues. Cell surface ROR1 expression was found in 45% of pediatric ALL patients, all of which were B-ALL, and was not limited to any particular genotype. All cell lines and primary blasts with E2A-PBX1 translocation and a portion of patients with other high risk genotypes, such as MLL rearrangement, expressed cell surface ROR1. Importantly, cell surface ROR1 expression was found in many of the pediatric B-ALL patients with multiply relapsed and refractory disease and normal karyotype or low risk cytogenetics, such as hyperdiploidy. Notably, cell surface ROR1 was virtually absent in normal adult and pediatric tissues. Conclusions and Significance Collectively, this study suggests that ROR1 merits preclinical and clinical investigations as a novel target for mAb-based therapies in pediatric B-ALL. We propose cell surface expression of ROR1 detected by flow cytometry as primary inclusion criterion for pediatric B-ALL patients in future clinical trials of ROR1-targeted therapies.

Activation of TLR4 Is Required for the Synergistic Induction of Dual Oxidase 2 and Dual Oxidase A2 by IFN-γ and Lipopolysaccharide in Human Pancreatic Cancer Cell Lines

Pancreatitis is associated with release of proinflammatory cytokines and reactive oxygen species and plays an important role in the development of pancreatic cancer. We recently demonstrated that dual oxidase (Duox)2, an NADPH oxidase essential for reactive oxygen species–related, gastrointestinal host defense, is regulated by IFN-γ–mediated Stat1 binding to the Duox2 promoter in pancreatic tumor lines. Because LPS enhances the development and invasiveness of pancreatic cancer in vivo following TLR4-related activation of NF-κB, we examined whether LPS, alone or combined with IFN-γ, regulated Duox2. We found that upregulation of TLR4 by IFN-γ in BxPC-3 and CFPAC-1 pancreatic cancer cells was augmented by LPS, resulting in activation of NF-κB, accumulation of NF-κB (p65) in the nucleus, and increased binding of p65 to the Duox2 promoter. TLR4 silencing with small interfering RNAs, as well as two independent NF-κB inhibitors, attenuated LPS- and IFN-γ–mediated Duox2 upregulation in BxPC-3 cells. Induction of Duox2 expression by IFN-γ and LPS may result from IFN-γ–related activation of Stat1 acting in concert with NF-κB–related upregulation of Duox2. Sustained extracellular accumulation of H2O2 generated by exposure to both LPS and IFN-γ was responsible for an ∼50% decrease in BxPC-3 cell proliferation associated with a G1 cell cycle block, apoptosis, and DNA damage. We also demonstrated upregulation of Duox expression in vivo in pancreatic cancer xenografts and in patients with chronic pancreatitis. These results suggest that inflammatory cytokines can interact to produce a Duox-dependent pro-oxidant milieu that could increase the pathologic potential of pancreatic inflammation and pancreatic cancer cells.

GalNAcα1-3Gal, a new prognostic marker for cervical cancer

Cancer cells undergo significant changes in carbohydrate expression, and these alterations can be useful as biomarkers and therapeutic targets. In this study, we investigated the expression of carbohydrate antigens containing a terminal GalNAcα1‐3Gal or GalNAcα1‐6Gal on human cervix and cervical carcinoma. Monoclonal antibodies to each of these carbohydrates were generated by immunizing rabbits with the corresponding antigen conjugated to KLH followed by hybridoma production. Antibodies were screened and evaluated using a combination of carbohydrate microarray profiling, ELISA, dot blot and immunohistochemical staining to verify specificity. Antibody 132‐3 was found to selectively recognize GalNAcα1‐3Gal with little cross‐reactivity to other structurally similar antigens such as GalNAcα1‐6Gal, blood group A, Forssman antigen and the Tn antigen on both solution assays and human tissue. Although GalNAcα1‐6Gal expression was not detected, GalNAcα1‐3Gal expression was found on 55% of squamous cell carcinomas. Expression in normal tissue was observed but was restricted to the suprabasal epithelial layer. Importantly, we found expression of the antigen on cervical cancer had a statistically significant correlation with the 5‐year survival rate of the patients (48 vs. 85% for antigen negative vs. positive, p = 0.017). Expression of GalNAcα1‐3Gal did not correlate with other clinical factors including tumor stage, size and lymph node metastasis, indicating the antigen is a new, independent biomarker for the prognosis of cervical cancer. Published 2009 UICC.

Dual oxidase 2 and pancreatic adenocarcinoma: IFN-γ-mediated dual oxidase 2 overexpression results in H 2 O 2 -induced, ERK-associated up-regulation of HIF-1α and VEGF-A

Several NADPH oxidase family members, including dual oxidase 2 [DUOX2], are expressed in human tumors, particularly gastrointestinal cancers associated with long-standing chronic inflammation. We found previously that exposure of pancreatic ductal adenocarcinoma cells to the pro-inflammatory cytokine IFN-γ increased DUOX2 expression (but not other NADPH oxidases) leading to long-lived H2O2 production. To elucidate the pathophysiology of DUOX2-mediated H2O2 formation in the pancreas further, we demonstrate here that IFN-γ-treated BxPC-3 and CFPAC-1 pancreatic cancer cells (known to increase DUOX2 expression) produce significant levels of intracellular oxidants and extracellular H2O2 which correlate with concomitant up-regulation of VEGF-A and HIF-1α transcription. These changes are not observed in the PANC-1 line that does not increase DUOX2 expression following IFN-γ treatment. DUOX2 knockdown with short interfering RNA significantly decreased IFN-γ-induced VEGF-A or HIF-1α up-regulation, as did treatment of pancreatic cancer cells with the NADPH oxidase inhibitor diphenylene iodonium, the multifunctional reduced thiol N-acetylcysteine, and the polyethylene glycol-modified form of the hydrogen peroxide detoxifying enzyme catalase. Increased DUOX2-related VEGF-A expression appears to result from reactive oxygen-mediated activation of ERK signaling that is responsible for AP-1-related transcriptional effects on the VEGF-A promoter. To clarify the relevance of these observations in vivo, we demonstrate that many human pre-malignant pancreatic intraepithelial neoplasms and frank pancreatic cancers express substantial levels of DUOX protein compared to histologically normal pancreatic tissues, and that expression of both DUOX2 and VEGF-A mRNAs is significantly increased in surgically-resected pancreatic cancers compared to the adjacent normal pancreas.

ASPS-1, a novel cell line manifesting key features of alveolar soft part sarcoma.

In vitro growth of alveolar soft part sarcoma (ASPS) and establishment of an ASPS cell line, ASPS-1, are described in this study. Using a recently developed xenograft model of ASPS derived from a lymph node metastasis, organoid nests consisting of 15 to 25 ASPS cells were isolated from ASPS xenograft tumors by capture on 70 &mgr;m filters and plated in vitro. After attachment to the substratum, these nests deposited small aggregates of ASPS cells. These cells grew slowly and were expanded over a period of 3 years and have maintained characteristics consistent with those of both the original ASPS tumor from the patient and the xenograft tumor including (1) presence of the alveolar soft part locus-transcription factor E3 type 1 fusion transcript and nuclear expression of the alveolar soft part locus-transcription factor E3 type 1 fusion protein; (2) maintenance of the t(X;17)(p11;q25) translocation characteristic of ASPS; and (3) expression of upregulated ASPS transcripts involved in angiogenesis (ANGPTL2, HIF-1-&agr;, MDK, c-MET, VEGF, and TIMP-2), cell proliferation (PRL, PCSK1), metastasis (ADAM9), as well as the transcription factor BHLHB3 and the muscle-specific transcripts TRIM63 and ITG&bgr;1BP3. This ASPS cell line forms colonies in soft agar and retains the ability to produce highly vascularized ASPS tumors in NOD.SCID/NCr mice. Immunohistochemistry of selected ASPS markers on these tumors indicated similarity to those of the original patient tumor as well as to the xenografted ASPS tumor. We anticipate that this ASPS cell line will accelerate investigations into the biology of ASPS including identification of new therapeutic approaches for treatment of this slow growing soft tissue sarcoma.