A phase II study of a 5T4 oncofoetal antigen tumour-targeted superantigen (ABR-214936) therapy in patients with advanced renal cell carcinoma

In a phase II study, 43 renal cell carcinoma patients were treated with individualised doses of ABR-214936; a fusion of a Fab recognising the antigen 5T4, and Staphylococcal enterotoxin A. Drug was given intravenously on 4 consecutive days, treatment was repeated 1 month later. Treatment was associated with moderate fever and nausea, but well tolerated. Of 40 evaluable patients, 28 had disease control at 2 months, and at 4 months, one patient showed partial response (PR) and 16 patients stable disease. Median survival, with minimum follow-up of 26 months was 19.7 months with 13 patients alive to date. Stratification by the Motzer's prognostic criteria highlights prolonged survival compared to published expectation. Patients receiving higher drug exposure had greater disease control and lived almost twice as long as expected, whereas the low-exposure patients survived as expected. Sustained interleukin-2 (IL-2) production after a repeated injection appears to be a biomarker for clinical effect, as the induced-IL-2 level on the day 2 of treatment correlated with survival. The high degree of disease control and the prolonged survival suggest that this treatment can be effective. These findings will be used in the trial design for the next generation of drug, with reduced antigenicity and toxicity

Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen

Superantigens activate T-cells by linking the T-cell receptor to MHC class II on antigen-presenting cells, and novel reactivity can be introduced by fusing the superantigen to a targeting molecule. Thus, an antibody-targeted superantigen, which activates T cells to destroy tumour cells, might be used as cancer therapy. A suitable target is the 5T4 oncofetal antigen, which is expressed on many carcinomas. We constructed a fusion protein from a Fab of a monoclonal antibody recognizing the 5T4 antigen, and an engineered superantigen. The recombinant product 5T4FabV13-SEAD227A bound the 5T4 antigen expressed on the human non-small-cell lung cancer cell line Calu-1 with a Kd of 1.2 nM while the substitution of Asp227 to Ala in the superantigen moiety reduced binding activity to MHC class II. 5T4FabV13-SEAD227A tumour reactivity was demonstrated in 7/7 NSCLC samples by immunohistochemistry, while normal tissue reactivity was low to moderate. 5T4FabV13-SEAD227A induced significant T-cell-dependent in vitro killing of sensitive 5T4 bearing Calu-1 cells, with maximum lysis at 10−10M, while the capacity to lyse MHC class II expressing cells was approximately 1000 times less effective. Immunotherapy of 5T4FabV13-SEAD227A against human NSCLC was investigated in SCID mice reconstituted with human peripheral blood mononuclear cells. Mice carrying intreperitoneally growing Calu-1 cells showed significant reduction in tumour mass and number after intravenous therapy with 5T4FabV13-SEAD227A. Thus, 5T4FabV13-SEAD227A has highly attractive properties for therapy of human NSCLC. © 2001 Cancer Research Campaign http://www.bjcancer.co

Abstract PD05-03: What is the appropriate sample (s) on which to perform sequencing for mutational analysis to guide the selection of targeted therapy?

Abstract BACKGROUND: Success of targeted therapy requires expression of the protein. Tumor tissue source can include diagnostic biopsy, surgical samples from initial or follow-up surgeries, fluids e.g. pleural or ascites and circulating tumor cells (CTC). The goal of using CTCs was 1. To determine whether CTC can be used as a “liquid” tumor biopsy and enable gene sequence information at the single cell level and 2. To determine the heterogeneity represented in the circulation compared to that seen in solid tumor by examining single cells (or a small cluster of cells) for the presence of a specific mutation which was detected in tissue tumor source. METHODS: We performed sequencing for mutational analysis on tissue(s) from patients with inflammatory breast cancer (IBC). Tumor sources varied from mastectomy tissue, metastatic site(s) e.g. liver or skin from chest wall disease, pleural fluid and CTC isolated into pure single cell populations (or groups of cells) using Silicon Biosystems DEPArray. Ampli1™ WGA kit was used for CTC amplification. Of the 22 patients sequenced, mastectomy primary tumor was examined in 3, metastatic site skin chest wall disease in 15, other metastatic site in 4, pleural fluid in 2 and CTC collected to investigate p53 mutations in 8. RESULTS: To date 22 patients have had mutational data performed, 14/22 had mutations in p53, 4/22 in RB1, 2/22 in each PI3K and ERBB2, 1/22 in each of BRAC1, BRAC2, ATM, KRAS, Notch 1, MEN1 and ESR1. Numerous amplifications were noted including AKT1, RPTOR, MLC1, MYC, CCND1 and ERBB2. For one patient's chest wall biopsy compared to two single CTCs and a cluster of 10 CTCs the same TP53C229fs*10 mutation was detected revealing the same 2bp deletion. No 2bp deletion was found in single white blood cells. Whereas, another patient which showed a TP53 S215G mutation in her skin biopsy of chest wall disease, only amplifications of AURKA, CCND1, IGF1R, MDM2 and SRC in pleural tumor cells were detected and no mutations in three single CTC, two single pleural tumor cells and in single white blood cells were seen. Primary tumor tissue is being sort for both of these patients. Mutational data reviewed to date suggest that IBC is not one disease but a multiplicity of diseases, revealing a variety of target(s). Aberrations are not consistent across tissue source. CONCLUSIONS: Successful treatment outcomes using standard of care chemotherapy combined with target therapies will require not one, but a panel, of tissue sources for sequencing to guide the selection of appropriate targeted therapies. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD05-03.</jats:p

Abstract P6-02-05: A novel culturing 3-D model to evaluate the role of tumor microenvironment in IBC.

Abstract Introduction: Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer associated with extremely poor outcomes. The clinical and pathological characteristics of the disease are the peculiar invasion of the dermal lymphatics as tumor emboli and the development of early recurrences. We aimed to establish a 3D model to evaluate the role of tumor microenvironment. Methods: We used human tumor-associated fibroblasts (or fibroblasts derived from metastatic skin) from IBC patients to build a multilayer extracellular matrix structure which effectively mimics aspects of the mesenchymal microenvironment of IBCs. Using this in vivo-like microenvironment we proceeded to test both matrix effects upon IBC's phenotypes and IBC modifications upon the cell-derived 3D matrices. We seeded the IBC cells into the matrix and cultured for 3 days, then tested the characterization markers cancer cells and ECM e.g. Phalloidin, E-cadherin, Ki67, α5β1 integrin and fibronectin by immunofluorescence and the expression of E-cadherin and vimentin as marker of epithelial-mesenchymal transition (EMT) by western blot. Results: We divided six IBC cell lines into 2 groups depending on the phenotypes acquired when cultured in the IBC fibroblast-derived ECM. SUM149 (EGF receptor positive and aggressive phenotype), BR016 and LG018 (harvested from patient's pleural effusions) presented a single cell organization with a spindle-like or mesenchymal type (as opposed to cluster) morphology. In comparison, SUM190 (HER2 positive and non aggressive tumorigenesis), MDA-IBC-3 and FC-IBC-02 (abstracted from patient's pleural effusion) presented a phenotype resembling mammospheres or in vivo emboli. Moreover, this last group of cells showed a peculiar capability for ECM modifications which greatly differed from the ECM modifications that were apparent following 3 day culturing of the above mentioned group represented by SUM149. In addition, proliferation measurements by Ki67 expression demonstrated a significant increased in 3D culture for SUM149, BR016 and LG018 compared with that in 2D culture, while no differences in proliferation were observed in the other three cell lines. Moreover, the expression of E-cadherin known to be upregulated in IBC tumors was increased in all cancer cells when seeded into the human fibroblast-derived 3D matrix indicating a potential role of the microenvironment in promoting proliferation, growth and invasion. Conclusion: The present study demonstrated the establishment of a novel IBC stromal 3D model using extracellular matrix produced from human fibroblasts of patients with advanced IBC. We showed a dynamic interaction between cancer cells and the microenvironment and potential sorting of IBC cells into two discrete groups which also correlate with their aggressive behaviors in vivo. We believe that these system may serve to predict levels of IBC tumorigenesis. We will proceed to further study the two identified responsive phenotypes with the goal of uncovering mechanisms of IBC tumor-stromal interactions and better understand ECM influences upon IBC development and progression. The ultimate goal will be to use the system to study IBC biology and better design drugs that will specifically affect the newly identified phenotypes. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-02-05.</jats:p

Recombinant human macrophage colony-stimulating factor in nonhuman primates: selective expansion of a CD16+ monocyte subset with phenotypic similarity to primate natural killer cells

The CD16 receptor (Fc gamma R-III) is found on many tissue macrophages (M phi s), but its expression on circulating monocytes is restricted to a small, phenotypically distinct subset. The number of these CD16+ monocytes may be markedly increased in response to sepsis, human immunodeficiency virus infection, or metastatic malignancy. We have recently shown that the CD16+ monocyte population is selectively expanded by administration of recombinant human macrophage colony- stimulating factor (rhM-CSF). In the current study, we used the highly rhM-CSF-responsive cynomolgus primate model to further characterize this novel monocyte population. Animals treated with rhM-CSF underwent a progressive and essentially complete conversion to the CD16+ monocyte phenotype, with up to a 50-fold increase in the number of CD16+ cells. This increase was paralleled by the emergence of a population of circulating cells that morphologically resembled large granular lymphocytes (LGLs). However, quantitatively, this population corresponded closely to the number of CD16+ monocytes, and fluorescence- activated cell sorting (FACS) confirmed that they were the same. In addition to their LGL-like morphology, many rhM-CSF-induced CD16+ monocytes showed a pattern of size, granularity, and quantitative cell surface marker expression that closely resembled the pretreatment LGL/natural killer (NK) cell population but that did not resemble the pretreatment monocyte population. However, rhM-CSF-induced CD16+ monocytes could be distinguished from LGL/ NK cells by fact that they all expressed cell surface receptors for rhM-CSF, and many of them showed reduced but detectable phagocytic and respiratory burst activity. Studies of human subjects treated with rhM-CSF also showed an analogous population of “LGL-appearing” CD16+ mononuclear cells. Thus, our studies reveal a previously unsuspected ability of cells in the monocyte lineage to adopt a phenotype similar to that of LGL/NK cells. The extent of this phenotypic convergence suggests that the two lineages retain access to elements of a similar developmental pathway.</jats:p

CD16+ monocytes in patients with cancer: spontaneous elevation and pharmacologic induction by recombinant human macrophage colony- stimulating factor

The small subset of circulating monocytes that express the maturation-associated CD16 antigen has recently been reported to be elevated in patients with bacterial sepsis. We now show that this novel CD16+ monocyte population is also spontaneously expanded in patients with cancer. We studied 14 patients with metastatic gastrointestinal carcinoma enrolled ina clinical trial of recombinant human macrophage colony-stimulating factor (rhMCSF) plus monoclonal antibody D612. We found that before any cytokine treatment, 12 of 14 patients constitutively displayed significant elevations in both the percentage and the absolute number of CD16+ monocytes, as compared with both normal subjects and ill patients with elevated monocyte counts but without malignancy. CD16+ monocytes accounted for 46% +/- 22% of total monocytes in the patients with cancer versus 5% +/- 3% for controls (P &lt; .01). The increase was not attributable to infection or intercurrent illness and appeared to be associated with the underlying malignancy itself. A similar spontaneous elevation of CD16+ monocytes was observed in 35 of 44 additional patients diagnosed with a variety of other solid tumors. When patients with gastrointestinal carcinoma were treated with rhMCSF, there was a marked further increase in the percentage of CD16+ monocytes (to 83% +/- 11%), as well as in the absolute number of CD16+ cells and the level of CD16 antigen expression. In every case, the patients with cancer showed a greater CD16+ monocyte response than the maximal response obtained in normal volunteer subjects treated witha similar regimen of rhMCSF (n = 5, P &lt; .001), suggesting that the presence of malignancy primed patients for enhanced responsiveness to rhMCSF. We hypothesize that spontaneous expansion of the CD16+ monocyte population may represent a novel biologic marker for a widespread and previously unsuspected host immune response to malignancy.</jats:p

Abstract P6-12-07: Prevalence of propionibacterium acnes and bartonella henselae DNA in patients with inflammatory breast cancer (IBC)

Abstract Introduction: Inflammatory breast cancer (IBC) is a very aggressive variant of breast cancer with a poor prognosis.. Mouse Mammary Tumor-associated Virus (MMTV) and other infectious agents have been considered as possible etiological agents of IBC particularly related to the initial description of higher incidence in women living in rural areas in North Africa. The etiological role of bacteria in this disease has never been explored in spite of the evidence that chronic infections with certain bacteria can facilitate tumors development. We retrospectively evaluated tissue samples from patients with recurrent IBC to identify potential bacterial agents that could play a role in the development and progression of the disease. Methods: DNA was isolated from formalin-fixed paraffin embedded samples of 24 Inflammatory Breast Cancer (IBC) patients whose specimen had been submitted for genomic analysis using next-genomic sequencing (Foundation One™). An additional 3 non-IBC patients (lymph node, lung metastatic lesions) were included in the study. Unselected DNA libraries from these samples were pooled and sequenced on a HiSeq-2000 sequencer. Comparing the sequence data to a reference of 5,569 bacterial and viral genomic sequences identified the presence of Propionibacterium acnes, Ralstonia pickettii and Methylobacterium consequently, we enriched the DNA libraries for the presence of these species as well as bacterial rRNA genes using hybrid capture with synthesized oligonucleotide baits and sequenced the enriched libraries. Results: Twenty three IBC patients and 3 non-IBC breast cancer patients were included in the study. Tissue specimens included, 14 chest wall and skin;6 breast, 2 lymph nodes; 1 liver, 1 lung, 1 pleural fluid, 1 brain. In 16 out of the 23 IBC (70%) of specimens we detected bacteria DNA. Propionibacterium acnes were detected in 12 cases. Bartonella henselae was detected in 1 out of the 23 IBC specimens. Furthermore, additional detected species included Ralstonia pickettii (3 cases) and Pseudomonas aeruginosa (2). No bacteria were detected in samples from non-IBC breast cancer patients. Conclusions: In this study, we identified Propionibacterium acnes and Bartonella henselae in samples from IBC patients. The anaerobic Gram-positive bacterium P. acnes is ubiquitously found in sebaceous follicles of the human skin. Recent reports showed that P. acnes was present in a high number of cancerous prostate tissue samples. It has been suggested that P. acnes may be a contributing factor to the initiation or progression of prostate cancer. Bartonella is a Gram-negative bacteria usually associated with cat-scratch disease, urban trench fever, bacillary angiomatosis-peliosis and endocarditis. Some reports that have showed some similarities between cat scratch disease and inflammatory breast cancer. Our results suggested that P. acnes or Bartonella henselae infections might contribute to the clinical and pathological characteristics of IBC, associated with rapid spread of the breast tumor cells through the lymphatic system. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-07.</jats:p

P2-05-04: Mapping the Specific Gene Families Activated in the Lymphangiogenesis and Vasculogenic Mimicry Exhibited by Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. Although IBC is diagnosed less commonly than other types of breast cancer, it is extremely aggressive, and accounts for a disproportionate number of breast cancer related deaths annually. IBC exhibits very specific patterns of lymphangiogenesis and vasculogenic mimicry, however detailed studies of the genes and proteins involved in these angiogenic processes are lacking. This study performed whole unbiased gene transcription studies with validation by protein arrays using all available pre-clinical cell lines and in vivo xenograft models of IBC, including a new model of IBC, FC-IBC01, which exhibits lymphovascular invasion, to identify the specific pathways involved in the distinctive angiogenesis observed in IBC. Materials and Methods: Real-time quantitative RT-PCR, cDNA microarray gene profiling, immunofluorescence with confocal imaging and protein arrays were used to examine differential expression of specific angiogenic gene families including VEGFA,B,C,D, VEGF Receptor genes, and ANG/TIE genes linked to angiogenesis and lymphangiogenesis. Results: Activity of the matrix metalloproteinase, MMP-2, is required for IBC tumor cells to undergo vasculogenic mimicry (VM), which is associated with a loss of TIMP-2, a well known inhibitor of angiogenesis. Therapeutics that target MMP activity can successfully inhibit this VM. Furthermore, pre-clinical models of IBC that form IBC tumor emboli exhibit lymphovascular invasion that is associated with distinct patterns of expression of genes that encode for distinct receptor tyrosine kinases that may represent important therapeutic targets for IBC. Discussion: Identification of the distinct angiogenic pathways that are activated in IBC provides insight into the therapeutic targets that may abrogate the distinct lymphovascular invasion and vasculogenic mimicry that are linked to the aggressive metastasis of IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-04.</jats:p