Yangyang Wang

Targeting ALDHbright Human Carcinoma–Initiating Cells with ALDH1A1-Specific CD8+ T Cells

Purpose: Cancer-initiating cells (CIC) are considered to represent the subpopulation of tumor cells that is resistant to conventional cancer treatments, highly tumorigenic in immunodeficient mice, and responsible for tumor recurrence and metastasis. Based on an elevated aldehyde dehydrogenase (ALDH) activity attributable to ALDH1/3 isoforms, ALDHbright cells have been identified and isolated from tumors and shown to have characteristics of CIC. The ALDH1A1 isoform was previously identified as a tumor antigen recognized by CD8+ T cells. This study examines the ability of ALDH1A1-specific CD8+ T cells to eliminate ALDHbright cells and control tumor growth and metastases. Experimental Design: ALDHbright cells were isolated by flow cytometry using ALDEFLUOR from HLA-A2+ human head and neck, breast, and pancreas carcinoma cell lines and tested for their tumorigenicity in immunodeficient mice. ALDH1A1-specific CD8+ T cells were generated in vitro and tested for their ability to eliminate CICs in vitro and in vivo by adoptive transfer to immunodeficient mice bearing human tumor xenografts. Results: ALDHbright cells isolated by flow cytometry from HLA-A2+ breast, head and neck, and pancreas carcinoma cell lines at low numbers (500 cells) were tumorigenic in immunodeficient mice. ALDHbright cells present in these cell lines, xenografts, or surgically removed lesions were recognized by ALDH1A1-specific CD8+ T cells in vitro. Adoptive therapy with ALDH1A1-specific CD8+ T cells eliminated ALDHbright cells, inhibited tumor growth and metastases, or prolonged survival of xenograft-bearing immunodeficient mice. Conclusions: The results of this translational study strongly support the potential of ALDH1A1-based immunotherapy to selectively target CICs in human cancer. Clin Cancer Res; 17(19); 6174–84. ©2011 AACR.

CSPG4 in cancer: Multiple roles

Chondroitin sulfate proteoglycan 4 (CSPG4), also known as High Molecular Weight-Melanoma Associated Antigen, is a cell surface proteoglycan which has been recently shown to be expressed not only by melanoma cells, but also by various types of human carcinoma and sarcoma. Furthermore, at least in squamous cell carcinoma of head and neck and in basal breast carcinoma, CSPG4 is expressed by cancer stem cells. CSPG4 plays an important role in tumor cell growth and survival. These CSPG4-associated functional properties of tumor cells are inhibited by CSPG4-specific monoclonal antibodies (mAb) in vitro. Moreover, CSPG4-specific mAb can also inhibit tumor growth and metastasis in vivo. The anti-tumor effects of CSPG4-specific mAb are likely to reflect the blocking of important migratory, mitogenic and survival signaling pathways in tumor cells. These results indicate that CSPG4 is a promising new target to implement mAb-based immunotherapy of various types of cancer.

CSPG4 Protein as a New Target for the Antibody-Based Immunotherapy of Triple-Negative Breast Cancer

BACKGROUND The cell surface proteoglycan, chondroitin sulfate proteoglycan 4 (CSPG4), is a potential target for monoclonal antibody (mAb)-based immunotherapy for many types of cancer. The lack of effective therapy for triple-negative breast cancer (TNBC) prompted us to examine whether CSPG4 is expressed in TNBC and can be targeted with CSPG4-specific mAb. METHODS CSPG4 protein expression was assessed in 44 primary TNBC lesions, in TNBC cell lines HS578T, MDA-MB-231, MDA-MB-435, and SUM149, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. The effect of CSPG4-specific mAb 225.28 on growth, adhesion, and migration of TNBC cells was tested in vitro. The ability of mAb 225.28 to induce regression of tumor metastases (n = 7 mice) and to inhibit spontaneous metastasis and tumor recurrence (n = 12 mice per group) was tested in breast cancer models in mice. The mechanisms responsible for the antitumor effect of mAb 225.28 were also investigated in the cell lines and in the mouse models. All statistical tests were two-sided. RESULTS CSPG4 protein was preferentially expressed in 32 of the 44 (72.7%) primary TNBC lesions tested, in TNBC cell lines, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. CSPG4-specific mAb 225.28 statistically significantly inhibited growth, adhesion, and migration of TNBC cells in vitro. mAb 225.28 induced 73.1% regression of tumor metastasis in a TNBC cell-derived experimental lung metastasis model (mAb 225.28 vs control, mean area of metastatic nodules = 44590.8 vs 165950.8 μm(2); difference of mean = 121360.0 μm(2), 95% confidence interval = 91010.7 to 151709.4 μm(2); P < .001). Additionally, mAb 225.28 statistically significantly reduced spontaneous lung metastases and tumor recurrences in an orthotopic xenograft mouse model. The mechanisms responsible for antitumor effect included increased apoptosis and reduced mitotic activity in tumor cells, decreased blood vessel density in the tumor microenvironment, and reduced activation of signaling pathways involved in cell survival, proliferation and metastasis. CONCLUSIONS This study identified CSPG4 as a new target for TNBC. The antitumor activity of CSPG4-specific mAb was mediated by multiple mechanisms, including the inhibition of signaling pathways crucial for TNBC cell survival, proliferation, and metastasis.

Programmed Cell Death Ligand 1 Expression in Osteosarcoma

Shen, Cote, and colleagues developed a quantitative RNA-based PD-L1 assay and report PD-L1 expression in 84% of human osteosarcomas, of which 24% were at high levels and correlated with TILs, indicating that this subset of patients may benefit from anti-PD-L1 immunotherapy. Programmed cell death ligand 1 (PDL1, also known as B7H1) is a cell-surface protein that suppresses the cytotoxic CD8+ T-cell–mediated immune response. PDL1 expression and its clinical relevance in sarcomas are not well understood. Therefore, we sought to measure RNA expression levels for PDL1 in 38 clinically annotated osteosarcoma tumor samples and aimed to determine if PDL1 expression correlates with clinical features and tumor-infiltrating lymphocytes (TIL). Quantitative real-time RT-PCR for PDL1 was optimized in 18 cell lines, of which 5 were osteosarcoma derived. qRT-PCR results were validated via flow cytometry and immunohistochemistry (IHC) in select cell lines. Total RNA was isolated from 38 human osteosarcoma samples for qRT-PCR analysis. Clinical data were sorted, and significance was determined by the Student t test. TILs were examined in patient samples by tissue microarray hematoxylin–eosin staining. We confirmed the constitutive PDL1 mRNA expression in cell lines by qRT-PCR, flow cytometry, and IHC. Across human osteosarcoma samples, PDL1 mRNA gene expression ranged over 4 log (>5,000-fold difference). Relative expression levels were evaluated against clinical factors such as age/gender, metastasis, recurrence, chemotherapy, percentage of necrosis, and survival; no significant associations were identified. The presence of TILs was associated with high PDL1 expression (R2 = 0.37; P = 0.01). In summary, we developed an RNA-based assay to determine PDL1 expression levels, and we show, for the first time, that high levels of PDL1 are expressed in a subset of osteosarcoma, and PDL1 expression is positively correlated with TILs. Multiple agents targeting PD1/PDL1 are in clinical development, and this may be a novel immunotherapeutic strategy for osteosarcoma clinical trials. Cancer Immunol Res; 2(7); 690–8. ©2014 AACR.

PD-L1 and HLA Class I Antigen Expression and Clinical Course of the Disease in Intrahepatic Cholangiocarcinoma

Purpose: More effective therapy is needed for intrahepatic cholangiocarcinoma (ICC). The encouraging clinical results obtained with checkpoint molecule-specific monoclonal antibodies (mAb) have prompted us to investigate whether this type of immunotherapy may be applicable to ICC. The aims of this study were to determine whether (i) patients mount a T-cell immune response to their ICC, (ii) checkpoint molecules are expressed on both T cells and tumor cells, and (iii) tumor cells are susceptible to recognition by cognate T cells. Experimental Design: Twenty-seven ICC tumors were analyzed for (i) lymphocyte infiltrate, (ii) HLA class I and HLA class II expression, and (iii) PD-1 and PD-L1 expression by T cells and ICC cells, respectively. The results of this analysis were correlated with the clinicopathologic characteristics of the patients investigated. Results: Lymphocyte infiltrates were identified in all tumors. PD-L1 expression and HLA class I antigen expression by ICC cells was observed in 8 and 11, respectively, of the 27 tumors analyzed. HLA class I antigen expression correlated with CD8+ T-cell infiltrate. Furthermore, positive HLA class I antigen expression in combination with negative/rare PD-L1 expression was associated with favorable clinical course of the disease. Conclusions: ICC patients are likely to mount a T-cell immune response against their own tumors. Defects in HLA class I antigen expression in combination with PD-L1 expression by ICC cells provide them with an immune escape mechanism. This mechanism justifies the implementation of immunotherapy with checkpoint molecule-specific mAbs in patients bearing ICC tumors without defects in HLA class I antigen expression. Clin Cancer Res; 22(2); 470–8. ©2015 AACR.

Aldehyde dehydrogenase 1A1 expression in breast cancer is associated with stage, triple negativity, and outcome to neoadjuvant chemotherapy

Studies have shown that ALDH1A1 expression in the breast is associated with worse clinical outcome. ALDH1A1 inactivates cyclophosphamide, which is an integral agent in breast cancer chemotherapy regimens. The purposes of this study were to verify these results, to correlate ALDH1A1 expression with clinical outcome in patients treated with cyclophosphamide as part of the chemotherapy (adjuvant or neoadjuvant), and to evaluate ALDH1A1 as a useful marker to predict the clinical outcome of breast cancer subsets. A total of 513 primary breast cancers were studied. Tissue microarrays of the studied cases were stained with ALDH1A1. Key clinicopathological information was obtained. Disease-free survival and overall survival were calculated. Patients with neoadjuvant therapy who had substantial residual cancer burden (RCB) were included in the study. Fishers exact test and Kaplan–Meier methods were used for statistical analysis. ALDH1A1 was expressed in 53 (10%) patients, with a higher frequency in triple negative, followed by HER2+, and finally hormonal receptor+/HER2− (P<0.0001). Tumors with advanced stage, node-positive, or larger tumor size were correlated with ALDH1A1 expression (P=0.006, P<0.0001, and P=0.05, respectively). ALDH1A1 expression was also correlated with worse disease-free survival (P<0.006) and overall survival (P<0.01) in patients who were treated with neoadjuvant chemotherapy. In all, 8 of 22 (36%) received neoadjuvant chemotherapy and died of disease-expressed ALDH1A1 (P=0.008). Similarly, 8 of 23 (35%) who received neoadjuvant chemotherapy and had tumor recurrence expressed this marker (P=0.002). The risk of recurrence was fivefold greater than negative ALDH1A1 tumors. The risk of recurrence became 11-fold greater when cyclophosphamide but not trastuzumab was part of the regimen. Our results are consistent with previous studies. Moreover, we found that ALDH1A1 could be a useful marker to predict worse clinical outcome after chemotherapy in the neoadjuvant setting with substantial RCB. However, a larger cohort is required to verify our results.

Functional Characterization of an scFv-Fc Antibody that Immunotherapeutically Targets the Common Cancer Cell Surface Proteoglycan CSPG4

Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human single-chain antibody termed scFv-FcC21 and characterized its specificity and antitumor activity. Viable CSPG4(+) melanoma cells were used in a screen of a human scFv phage display library that included CDR3 engineered to optimize antibody binding sites. The scFv antibody isolated was then recombinantly engineered with a human immunoglobulin G1 Fc region to construct the fully human antibody scFv-FcC21, which recognized tumors of neuroectodermal origin, various types of carcinomas, mesotheliomas, and sarcomas as well as myeloid leukemias. scFv-FcC21 inhibited in vitro growth and migration of tumor cells and in vivo growth of human tumor xenografts. These effects were mediated by inhibition of the activation of extracellular signal-regulated kinase and focal adhesion kinase signaling pathways that are critical for tumor cell growth and migration, respectively. Our findings define the CSPG4-specific fully human scFv-FcC21 antibody as a candidate therapeutic agent to target the many types of tumors that express CSPG4.

The CSPG4-specific monoclonal antibody enhances and prolongs the effects of the BRAF inhibitor in melanoma cells.

PLX4032 is a BRAF-selective inhibitor shown to be efficacious in the treatment of melanomas presenting with the BRAFV600E mutation. However, favorable responses to treatment are short-lived, and complete remission is rarely observed. Therefore, it is important to identify novel therapies designed to enhance treatment responses and to increase the longevity of initial response to BRAF inhibitors. To this end, we characterized the effects of the 225.28 chondroitin sulfate proteoglycan 4 (CSPG4)–specific monoclonal antibody (mAb) capable of blocking multiple signaling pathways important to cell growth, migration, and survival. Addition of 225.28 to the treatment regimen enhanced the in vitro response magnitude and the duration efficacy of PLX4032 in treating CSPG4+, BRAFV600E melanoma cells (melanomaBRAF(V600E)/CSPG4+ cells). Data presented in this report demonstrated that (1) treatments comprised of PLX4032 and mAb 225.28 were more effective at inhibiting melanomaBRAF(V600E)/CSPG4+ cell growth than either agent alone, (2) mAb 225.28 prevented/delayed the development of resistance in melanomaBRAF(V600E)/CSPG4+ cells to PLX4032, and (3) the mechanism of action of the combination therapy caused a down-regulation in multiple signaling pathways. This study provides a foundation for future investigations designed to improve BRAF inhibitor effectiveness in vitro and in vivo for treating melanomaBRAF(V600E)/CSPG4+ cells in combination with a CSPG4-specific mAb.

Pathologic and gene expression features of metastatic melanomas to the brain

The prognosis of metastatic melanomas to the brain (MBM) is variable with prolonged survival in a subset. It is unclear whether MBM differ from extracranial metastases (EcM) and primary melanomas (PrM).

CSPG4 as a prognostic biomarker in chordoma

BACKGROUND There are currently no generally accepted biomarkers used in the clinical treatment of chordoma tumors. CSPG4 has been associated with disease severity in other tumors. PURPOSE This study aimed to characterize the frequency of CSPG4 expression in chordoma tumors and to correlate it with disease severity and clinical outcome. STUDY DESIGN A retrospective review of clinical outcomes and immunohistochemical staining using tissue micro-array was carried out. PATIENT SAMPLE The sample comprised 86 patients treated for chordoma at a single center (1985-2007). OUTCOME MEASURES Survival and incidence of metastases were the outcome measures. METHODS Pathologic specimens of chordoma tumors were evaluated for the expression of CSPG4 by immunohistochemical staining with mAbs. Chi-square testing and Cox proportional hazard regression analysis were used to evaluate the impact of CSPG4 expression on survival and incidence of metastases, while controlling for patient age, sex, and surgical margins. RESULTS Average patient age at the time of presentation was 59.8 years (standard deviation [SD] 13.7). Average follow-up was 6.5 years (SD 4.8). Twenty (23%) patients developed metastatic disease. At the time of final follow-up, 57 patients (66%) had died. Chordoma tumors from 62 patients (72%) stained positive for CSPG4. CSPG4 expression more than doubled the risk of death (hazard ratio [HR] 2.3; 95% CI 1.04, 5.17). CSPG4 positive tumors were also associated with an increased risk of metastatic disease (31% for CSPG4 positive tumors vs. 0% in CSPG4 negative, p=.02). CONCLUSIONS Results presented here support the consideration of using CSPG4 as a biomarker establishing the prognosis for chordoma tumors. A positive CSPG4 stain may be associated with an increased risk of metastasis and mortality from disease.