Alterations In Tumor Microenvironment Research Articles

Essential roles of heat shock protein gp96 (Hsp90b1, grp94) in melanoma growth and melanosome development (46.1)

Abstract Heat shock protein gp96(HSP90b1,grp94) is an endoplasmic reticulum chaperone of the HSP90 family. It is a master molecular chaperone for Toll-like receptors and integrins. Accumulating clinical and pathological evidences indicate that enhanced gp96 expression in cancer tissue is a poor prognostic factor. However, the underlying mechanism is unclear. To address this question, we knocked down gp96 from melanoma cell line B16F1 (B16-KD) using lentivirus-mediated shRNA strategy. We found that knockdown of gp96 did not interfere with the in vitro growth of B16F1, but robustly suppressed the growth of orthotopically inoculated tumor in mice. Mechanistic study demonstrates that gp96 deletion compromises VEGF-A expression and inhibited tumor-associated angiogenesis. These results suggest that altered tumor microenvironment upon gp96 knockdown was responsible for the delayed tumor growth in vivo. Unexpectedly, we also observed that melanin production was significantly inhibited in B16-KD, which correlated with the reduced tyrosinase activity but not its expression in B16-KD cells. Thus, our study has uncovered two novel roles of gp96 in regulating tumor angiogenesis and in facilitating melanin synthesis. The molecular mechanisms of these findings are under active investigation.

P3-10-04: Halting Early Breast Cancer Progression with Omega-3 Ethyl Esters: Altering Tumor Microenvironment in 21T Series Cell Lines.

Abstract Observable infiltration of immune cells in stages of early breast cancer progression mirrors two recently conceptualized factors of carcinogenesis: tumor microenvironment and tumor-promoting inflammation. Clinical studies using immunohistochemical staining for CD45 in patient-derived breast tissue demonstrate the involvement of leukocytes in breast cancer development, but the lack of an appropriate in vitro model has hindered molecular studies thus far. However, the 21T series cell lines effectively mimic breast cancer progression. This cell line series was obtained from the same breast cancer patient and consists of three cell lines representing atypical hyperplasia (21PT), ductal carcinoma in situ (21NT) and invasive carcinoma (21MT-1). This unique cell line series is used in our molecular study of omega-3 ethyl esters as breast cancer preventive agents. Preliminary invasion assays have described varying degrees of monocyte recruitment according to stages of progression. 21PT cells when plated on the bottom chamber failed to mobilize monocytes as effectively as 21NT or 21MT-1 and the addition of omega-3 ethyl esters suppressed the baseline invasion capacity of monocytes by 30%. Epidemiologic studies suggest that omega-3 fatty acids can act as potent cancer preventive agents and are possibly more potent when used in an ethyl ester form. Based on this, we hypothesize that omega-3 ethyl esters may prohibit early breast cancer progression through inhibiting immune cell infiltration and disrupting the formation of a tumor-promoting inflammatory and oxidative microenvironment. Phenotypic studies aim to interrogate the effect of omega-3 ethyl esters on tumor microenvironment including chemokine secretion and MMP production whereas molecular experiments probe into the importance of the Wnt pathway as a key player in the transition from ADH to DCIS. This study will demonstrate how omega-3 ethyl esters may function as breast cancer preventive agents as well as provides a model of study to test the efficacy of cancer preventive agents in vitro. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-10-04.

Abstract C59: Antibody targeting of CD24 efficiently retards growth of experimental carcinomas.

Abstract It has been shown that CD24 drives lung metastasis formation of bladder cancer and might be a good candidate for mAb-dependent therapy. We tested whether mAb SWA11 to CD24 would be effective in preventing tumor growth in xenotransplanted mice. In an A549 lung carcinoma model cells were implanted s.c. into SCID/beige mice followed by multiple injections of mAb over 37 days. Therapy with anti-CD24 mAb SWA11 lead to substantially reduced tumor load. The final tumor mass of treated animals was significantly smaller compared to isotype-control mAb treated animals. Similar observations were made in therapy models of CD24-positive BxPC3 pancreatic adenocarcioma cells in NOD/SCID mice and SKOV-3ip ovarian carcinoma cells in CD1 mice. The therapeutic effects were associated with changes in tumor cell proliferation, apoptosis and angiogenesis. Surprisingly, we found that SWA11 mAb treatment significantly altered cytokine tumor microenvironment. Also the mAb treatment lead to the increased infiltration of tumor tissue with immune cells suggesting involvement of ADCC. Addition of SWA11 mAb to the gemcitabine treatment strongly potentiated its anti-cancer efficacy in A549 lung cancer model. Our data demonstrate that targeting of CD24 could be beneficial for the anti-cancer treatment using standard chemotherapy regimes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C59.

Abstract B12: Associations between invasive breast cancer with and without a DCIS component and breast cancer risk factors in African American and European American women

Abstract Introduction: Breast cancer presents as several clinically distinct forms, including invasive ductal carcinoma (IDC) with an associated ductal carcinoma in situ (DCIS) component, and pure IDC. The absence of concomitant DCIS was previously associated with a significantly worse prognosis for IDC, even after adjustment for stage and tumor size. Evidence from gene expression studies suggests that the absence of a DCIS component is associated with an altered tumor microenvironment, which may involve a distinct pathway of cancer development, with unique risk factors and prognoses. The absence of a DCIS component in IDC has not previously been examined in relation to the breast cancer disparities between African-American (AA) and European-American (EA) women. Methods: IDC breast cancer patients with or without a DCIS component were identified by examining pathological reports from the Women's Circle of Health Study, an on-going case-control study examining racial disparities in women diagnosed with breast cancer in NYC and NJ. Through in-person interviews information was collected on known and suspected breast cancer risk factors. In this case-case analysis, we examined these factors in relation to IDC with and without a DCIS component, adjusting for self-reported race and also stratified by race. Interactions were assessed with the Wald Chi-square. Odds ratios (ORs) and 95% confidence intervals (CIs) were derived to estimate the odds of having pure IDC compared to patients with a DCIS component. Results: Of 729 women with IDC, 19.5% were confirmed to have pure IDC and 80.5% IDC with a DCIS component. EA women were less likely than AA women to present with pure IDC without a DCIS component (OR=0.66; 95% CI 0.43–0.99). Women who breastfed were less likely to have pure IDC than IDC with a DCIS component (OR=0.65; 95% CI 0.40–1.04). Stratified analyses by race did not reveal major differences from unstratified analyses for breastfeeding (OR=0.64; 95% CI 0.35–1.16, OR=0.68; 95% CI 0.28–1.64 in AA and EA women, respectively). AA women with a body mass index (BMI) ≥30 compared to a BMI <25 were less likely to have pure IDC than IDC with DCIS (OR=0.52; 95% CI 0.25–0.97), whereas EA women with a high BMI were more likely to have pure IDC (OR=1.36; 95% CI 0.67–2.76, p for interaction=0.01). Pure IDC was directly and inversely associated with family history of breast cancer in EA and AA women, respectively (OR=1.89; 95% CI 1.01–3.55, OR=0.84; 95% CI 0.42–1.67, respectively, p for interaction=0.09). Conclusion: Compared to EA women, AA women are more likely to present with pure IDC without a DCIS component. Stratified analyses suggest that risk factors associated with pure IDC may be significantly different between AA and EA women. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B12.

Abstract B76: Synthetic triterpenoids as candidate drugs for cancer prevention and treatment

Abstract Angiogenesis is a limiting step for the switch from a dormant to malignant state in tumor progression. During tumor development tumor cells induce profound changes in the surrounding tissue, leading to the formation of an altered tumor microenvironment that is critical in permitting further tumor growth and tumor progression. Retinoblastoma (Rb) and colorectal cancer (CRC) display the complexity of the microenvironment constituted by different cell types and their extracellular matrix. Here we assessed the efficacy of two synthetic derivates of triterpenoid CDDO, CDDO-Im and CDDO-Me, on endothelial, retinoblastoma and colon cancer cells in order to evaluate their effect on angiogenesis, cancer progression and metastatic dissemination. We observed that CDDO-Im and CDDO-Me inhibited endothelial cells proliferation, migration and tube-formation, hampering the nuclear translocation of NF-kB and affecting the MAPK pathway. In vivo, in a matrigel sponge assay CDDO-Im interfered with angiogenesis when administrated into the pellets or by intraperitoneal injection. To assess whether these compounds could successfully impairs neuroectodermal-derived tumor growth, we performed in vitro experiments on Y79 and Weri-Rb1 retinoblastoma cells. We performed MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] and LDH (Lactate Dehydrogenase) assays to evaluate CDDO-Im effect on retinoblastoma cell viability and an Annexin V and 7-Amino-Actinomycin D staining to test apoptosis. We observed that CDDO-Im treatment induced a reduction of cell viability at nanomolar doses and early and late apoptosis at different time points. In order to overcome the side effects of standard chemotherapy drugs, such as vincristine sulphate, we evaluated if CDDO-Im could be used to lower chemotherapy doses. When we combined non-cytotoxic doses of CDDO-Im with vincristine sulphate we observed that after 72 hours of co-treatment, CDDO-Im was able to accomplish the same antiproliferative effect obtained using double doses of vincristine alone. We also tested the in vitro anti-tumour activity of CDDO-Me on human adenocarcinoma HT29 cell line. The triterpenoid was used alone or in combination with 5FU (5 Fluorouracil), the major chemotherapeutic agent used for CRC therapy. We performed an MTT assay to evaluate its effect on cell viability, an apoptosis test and an alkaline comet assay to assess the potential genotoxic damage. Preliminary results demonstrated that CDDO-Me induced an almost immediate inhibitory effect on HT29 cells, reducing cell viability already after 24 hours of treatment at 1000-fold lower concentration then 5FU. When HT29 were co-treated with both CDDO-Me and 5FU, no additive effects were observed. Respect to 5FU, CDDO-Me did not induce apoptosis even after long-term treatment, as well as genotoxicity, suggesting that it is able to block cell growth without exerting toxic effects. These data suggest that the two synthetic triterpenoid CDDO-Im and CDDO-Me could be promising compounds for cancer treatment, acting both on cancer cells and on endothelial cells that support tumor-associated angiogenesis. Our results open the possibility to use chemopreventive molecules in combination with current chemotherapic drugs, to improve their efficacy without cytotoxicity. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B76.

Tumor Microenvironment–Derived Proteins Dominate the Plasma Proteome Response during Breast Cancer Induction and Progression

Tumor development relies upon essential contributions from the tumor microenvironment and host immune alterations. These contributions may inform the plasma proteome in a manner that could be exploited for cancer diagnosis and prognosis. In this study, we employed a systems biology approach to characterize the plasma proteome response in the inducible HER2/neu mouse model of breast cancer during tumor induction, progression, and regression. Mass spectrometry data derived from approximately 1.6 million spectra identified protein networks involved in wound healing, microenvironment, and metabolism that coordinately changed during tumor development. The observed alterations developed prior to cancer detection, increased progressively with tumor growth and reverted toward baseline with tumor regression. Gene expression and immunohistochemical analyses suggested that the cancer-associated plasma proteome was derived from transcriptional responses in the noncancerous host tissues as well as the developing tumor. The proteomic signature was distinct from a nonspecific response to inflammation. Overall, the developing tumor simultaneously engaged a number of innate physiologic processes, including wound repair, immune response, coagulation and complement cascades, tissue remodeling, and metabolic homeostasis that were all detectable in plasma. Our findings offer an integrated view of tumor development relevant to plasma-based strategies to detect and diagnose cancer.

Is Cytomegalovirus a Therapeutic Target in Glioblastoma?

Several investigators have now demonstrated the expression of genes unique to cytomegalovirus (CMV) in malignant gliomas. Many of these genes promote oncogenesis, alter tumor microenvironment, and serve as immunologic targets. Is the level of CMV infection within tumor cells sufficient to drive important oncogenic or immunosuppressive processes? Can CMV serve as a target for therapeutic intervention?

Abstract 4476: The effect of electrochemotherapy on metastatic potential of human melanoma cells SK-Mel28

Abstract Electrochemotherapy is a local treatment, combining chemotherapy and application of electric pulses to the tumour. Electrochemotherapy with bleomycin or cisplatin has demonstrated its effectiveness in controlling local tumour growth in the treatment of malignant melanoma and is proposed as one of the treatment options for in-transit melanoma metastases in Europe. An important aspect of successful treatment is prevention of metastasis, however, it is known that metastasis can be induced by different treatment modalities. Alterations in tumour microenvironment caused by surgery or radiation therapy, can lead to increased formation of metastasis. Furthermore, formation of metastases can be directly linked to migratory potential of the cells. Although electrochemotherapy is now routinely used for treatment of various cancers in many cancer centres, the effect of electrochemotherapy on the metastatic potential of tumour cells is not known. Therefore, the aim of this study was to evaluate the effect of electrochemotherapy with bleomycin and cisplatin on the metastatic potential of human malignant melanoma cells. Human malignant melanoma cells SK-MEL 28 were treated by electrochemotherapy with a range of bleomycin concentrations from 0.01 nM to 1μM and cisplatin concentrations from 2 to 80 μg/mL. SK-MEL 28 cells that were viable 48 h after electrochemotherapy were tested for their metastatic potential by ability to migrate and invade through Matrigel coated porous membrane. In addition, cell adhesion and proliferation using MTS assay were also tested. Furthemore, RNA was isolated from the cells 16 h after each treatment and differentially expressed genes were investigated by microarray analysis to evaluate the effect of electrochemotherapy with bleomycin or cisplatin on expression of genes involved in the development of cancer. The Gene Ontology Tree Machine program was used for gene enrichment analysis. Migration and invasion of melanoma cells that survived 48h after electrochemotherapy with bleomycin or cisplatin were not changed compared to control untreated cells. Also, there were no changes observed in cell adhesion on Matrigel. Gene expression analysis demonstrated that a very low number of genes were differentially expressed after electrochemotherapy with either bleomycin or cisplatin. Specifically, the expression of metastasis promoting genes was not increased after electrochemotherapy. Our data suggest that electrochemotherapy with bleomycin or cisplatin does not increase metastatic behaviour of human melanoma cells, having important clinical implication for safe use in treatment of melanoma metastases. 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 4476. doi:10.1158/1538-7445.AM2011-4476

Diffusion-Weighted MRI for Assessment of Early Cancer Treatment Response

Recent clinical practice for the management for cancer patients has begun to change from a statistical "one-size fits all" approach to medicine to more individualized care. Pre-treatment biomarkers (i.e. genetically and histologically based) have a growing role in providing guidance related to the appropriate therapy and likelihood of response; they do not take into account heterogeneity within the tumor mass. Thus, a biomarker which could be utilized to measure actual tumor response early following treatment initiation would provide an important opportunity to evaluate treatment effects on an individual patient basis. Diffusion weighted magnetic resonance imaging (DW-MRI) offers the opportunity to monitor treatment-associated alterations in tumor microenvironment using quantification of changes in tumor water diffusion values as a surrogate imaging biomarker. Results obtained thus far using DW-MRI have shown that changes in tumor diffusion values can be detected early following treatment initiation which correlate with traditional outcome measures. Sensitive imaging biomarkers are providing for the first time a means of assessing 3 dimensional tumor response early in the treatment cycle. This review highlights the development of DW-MRI and its proposed usefulness in the clinical management of cancer patients. The utility of DW-MRI for assessing therapeutic-induced response is further evaluated on tumors residing in the brain, head and neck and bone.

Tumor microenvironment modifications induced by soluble VEGF receptor expression in a rat liver metastasis model

Vascular endothelial growth factor is a potent pro-angiogenic growth factor which is also known to alter tumor microenvironment by inhibiting dendritic cell differentiation and promoting accumulation of myeloid-derived suppressor cells. In the present study, we analyzed the modifications induced by intratumoral expression of sFLT-1, a soluble VEGF receptor, in a rat metastatic colon carcinoma model. We generated colon cancer cell lines stably expressing sFLT-1 or a mock construct. Human umbilical vein endothelial cells cultured with conditioned medium from sFLT-1-expressing tumor cells exhibit a significantly decreased survival, demonstrating the functionality of the secreted sFLT-1. In vivo, sFLT-1 expression induced a 30% decrease in microvessel density in 15-day old experimental liver metastasis from colon carcinoma. Tumor growth was inhibited by 63% and 52% in left and right liver lobes respectively within 25 days. In these tumors, sFLT-1 expression was associated with a decreased myeloid cell infiltration and a modification in the expression of several cytokines/chemokines. Altogether, these results suggest that VEGF trapping by sFLT-1 intratumoral expression results in reduced vascularization, tumor growth inhibition and modification of immune tumor microenvironment.

Irradiation-Dependent Effects on Tumor Perfusion and Endogenous and Exogenous Hypoxia Markers in an A549 Xenograft Model

Hypoxia is a major determinant of tumor radiosensitivity, and microenvironmental changes in response to ionizing radiation (IR) are often heterogenous. We analyzed IR-dependent changes in hypoxia and perfusion in A549 human lung adenocarcinoma xenografts. Immunohistological analysis of two exogenously added chemical hypoxic markers, pimonidazole and CCI-103F, and of the endogenous marker Glut-1 was performed time dependently after IR. Tumor vessels and apoptosis were analyzed using CD31 and caspase-3 antibodies. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and fluorescent beads (Hoechst 33342) were used to monitor vascular perfusion. CCI-103F signals measuring the fraction of hypoxic areas after IR were significantly decreased by approximately 50% when compared with pimonidazole signals, representing the fraction of hypoxic areas from the same tumors before IR. Interestingly, Glut-1 signals were significantly decreased at early time point (6.5 h) after IR returning to the initial levels at 30.5 h. Vascular density showed no difference between irradiated and control groups, whereas apoptosis was significantly induced at 10.5 h post-IR. DCE-MRI indicated increased perfusion 1 h post-IR. The discrepancy between the hypoxic fractions of CCI-103F and Glut-1 forces us to consider the possibility that both markers reflect different metabolic alterations of tumor microenvironment. The reliability of endogenous markers such as Glut-1 to measure reoxygenation in irradiated tumors needs further consideration. Monitoring tumor microvascular response to IR by DCE-MRI and measuring tumor volume alterations should be encouraged.

Oncoprotein metastasis and its suppression revisited

The past two decades have witnessed an increasing appreciation of the role of the tumor microenvironment, of genetic and epigenetic alterations in normal cells adjacent to tumors and of the migration of normal cells with aberrant intrinsic properties in cancer pathophysiology. Aside from these insights, a novel concept termed "oncoprotein metastasis" (OPM) has recently been advanced and proposed to reflect protein-based neoplastic phenomena that might occur even before any modifications relating to the morphology, location or (epi)genetic outfit of cells during the malignant process. Here, evidence is presented that supports the OPM perception and thus should contribute not only to further rethink the definition of a normal cell, but also the treatment of cancer disease in the years to come.

Serum cytokines in follicular lymphoma. Correlation of TGF-β and VEGF with survival

The prognosis of follicular lymphoma could vary with the tumor immune microenvironment. We evaluated the prognostic value of serum levels of ten cytokines. Our study cohort included 60 follicular lymphoma patients and 20 controls. Serum was available at diagnosis in 31 patients, at first relapse in 18, and complete remission in 11. Bioplex technology was used for determination of nine cytokines [interleukin (IL)-1Ra, IL-6, IL-7, IL-10, IL-13, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (b-FGF)]. Transforming growth factor beta (TGF-β) was measured by sandwich enzyme-linked immunosorbent assay. IL-1Ra, IL-6, IL-7, IL-10, IL-13, TNF-α, VEGF, and PDGF levels were found increased in follicular lymphoma patients compared to controls. Multivariate analysis identified early stage and high TGF-β levels as independent predictors of overall survival associated with improved outcome. High lactate dehydrogenase and VEGF levels were independently associated with poorer progression-free survival. These results show the prognostic value of TGF-β and VEGF in follicular lymphoma and suggest their contribution to tumor microenvironment alterations.

Progressive tumor growth-associated altered tumor microenvironment: implications in a tumor stage-dependent modulation in survival of a murine T cell lymphoma

In the present study using a transplantable murine T cell lymphoma designated as Dalton's lymphoma, we investigated the role of oxygen, glucose and other environmental factors in evolution of altered survival responses in tumor cells during the late tumor-bearing stages. Tumor progression was observed to be associated with an improvement in the survival ability of tumor cells. Moreover, tumor serum and ascitic fluid obtained from the late tumor bearing stage was found to augment tumor cell survival in vitro, indicating that these humoral components of tumor bearing host contain factors capable of modulating tumor survival. Progressive tumor growth was also shown to be associated with depletion in glucose and oxygen content in the fluids of tumor microenvironment along with a concomitant augmentation in the production of lactate and lactate dehydrogenase. Moreover, tumor cells expressed higher amount of mRNA for inducible nitric oxide synthase, Hypoxia inducible factor-1 and 2 and Hsp70 and VEGF proteins during the late tumor bearing stages. Splenic macrophages and non-adherent splenic lymphocytes from tumor-bearing mice showed an increased production of IL-6, TGF-beta, IFN-gamma, IL-2R and VEGF during the late tumor-bearing stage, which could be implicated in the differential regulation of tumor growth in a tumor stage dependent manner. In conclusion, the observations of the present study suggest that factors contained in altered tumor microenvironment may act in concert to cause behavioral alterations in tumor cells, with respect to survival, during the course of the progression of a nonsolid T cell lymphoma.

Abstract B105: Inhibition of angiogenesis by oleanoleic acid-derived triterpenoids: mechanisms of action and potential prostate cancer prevention

Abstract B105 Angiogenesis is a limiting step for the switch from a dormant to malignant state in tumor progression. During tumor development tumor cells induce profound changes in the surrounding tissue, leading to the formation of an altered tumor microenvironment that is critical in permitting further tumor growth and tumor progression.. Angiogenesis and inflammation are common targets of many chemopreventive molecules that suppress the antiangiogenic switch in premalignant tumors.We evaluated in vitro and in vivo the antiangiogenic activity of two synthetic oleanoleic acid-derived triterpenoids under study for their chemopreventive and therapeutic potential, CDDO -Me and -Im. In vitro both compounds exhibited a strong inhibitory activity on endothelial cells isolated from umbilical veins (HUVEC). Futher, these compounds inhibited inflammatory cell chemotaxis towards specific chemokine stimului. The triterpenoids interfered with activation of the PI3K/Akt pathway following VEGF stimulation and activation of the NF-kappaB pathway subsequent to TNFalpha stimulation. Moreover CDDO-Me induces cell death in androgen-responsive and unresponsive human prostate cancer cell lines at very low concentrations, by acting agains GS3K pathway (Venè R, Larghero P, Arena G, Sporn MB, Albini A, Tosetti F. Cancer Res. 2008).In vivo CDDO-Im and especially CDDO-Me significantly inhibit neoangiogenesis in the matrigel sponge assay in C57BL mice, reducing both CD31+ cell number and Hb content in the sponges. We tested different schedules of treatment, by administering compounds as prevention before matrigel injection, as therapy after injection and as combination of prevention and therapy. All three protocols reduced angiogenesis when compared to controls the combinationof prevention and intervention was the most effective and at very low dosages. Since currently CDDO-Me is being assessed phase I trials in the US, the potential anti-angiogenic activity of these drugs should be taken into consideration in evaluating efficacy in these trials. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B105.

CpG Oligonucleotide (1018 ISS) May Overcome Rituximab (R) Resistance in Follicular Lymphoma (FL) through Enhanced ADCC, T Cell Expansion, and Alteration of Tumor Microenvironment.

Immunostimulatory oligonucleotides containing nonmethylated CpG motifs (ISS) have multiple actions on adaptive and innate immune responses, including enhanced antigen presentation and costimulatory molecule expression, stimulation of dendritic cell maturation, and induction of cytokines that result in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and Th1 immune responses. Through these mechanisms, we hypothesized ISS might be able to overcome R resistance. Our completed phase I study combining an ISS (1018 ISS, Dynavax Technologies) with R in patients (pts) with relapsed or refractory, advanced stage NHL, demonstrated that the combination is safe, and causes a dose-related increase in the expression of several interferon (IFN)-inducible genes.(Blood 105:489). We have subsequently enrolled 16 pts (median age 60; 9 female) with R-naïve or sensitive FL in a phase II trial to further investigate the efficacy and biological activity of this rational combination. Pts received 4 injections of 1018 ISS (0.2 mg/kg sc) at weekly intervals, beginning at the time of the second of 4 weekly standard R infusions. No grade III/IV toxicities occurred. Genotyping of 13 evaluable pts for the FcγRIII-A 158 polymorphism revealed VF (n=6) and FF (n=7). Flow cytometry of peripheral blood demonstrated a significant expansion of CD3+ cells in 9 pts following 4 weeks of 1018 ISS. When tested with allogeneic stimulation using an EBV transformed B cell line (Mann) in ELISPOT assay, T cell secretion of IFN-γ was increased following 1018 ISS (mean count pre 1018 ISS: 362; mean count post 1018 ISS: 565; p = 0.05), whereas T cell secretion of IL-2 did not change, both consistent with an emerging Th1 immune response. Using a standard chromium release assay, 7/13 pts (54%) demonstrated increases in ADCC of peripheral blood mononuclear cells following ISS compared to baseline (effector:target ratio of 25:1); including 5 pts with FCγRIIIA genotype 158 FF. Skin biopsies were obtained in all patients at baseline, and following ISS therapy at the injection site and a distant site. Following 1018 ISS, 5 pts (42%) had significant skin infiltration with CD8+ lymphocytes; and 5 pts had increased numbers of CD68+ cells at injection sites. Biopsies of tumor were obtained at baseline, and post 1018 ISS treatment when possible, and evidence of CD8+ T cell infiltration within residual FL nodules was observed following R and 1018 ISS therapy. As of 08/05, at a median follow-up of 9 months, only 3 pts have experienced disease progression, one of whom subsequently converted to CRu without further therapeutic intervention. We conclude that the combination of 1018 ISS and rituximab is safe, and demonstrates significant local and systemic biological activity. Moreover, the demonstration of enhanced ADCC in pts with an FCγRIIIA 158 FF genotype (expected poor prognosis following R in pts with FL), the associated development of a Th1 response, and measurable alteration of the tumor microenvironment all suggest ISS could overcome both innate and acquired R resistance mechanisms in pts with FL.

The challenge of pathway and environment-mediated drug resistance.

New insights into cancer biology have allowed the definition of pathways critical to the success of the cancer cell. These include enhanced activity of positive regulators of cell proliferation, including oncogenes, and loss of tumor suppressor gene function; inactivation of cell death or enhancement of srvival functions; activation of telomerase, and enhanced ability of tumor cells to invade and reorganize host stroma. It follows then that these pathways may also mediate drug resistance, in a way that is fundamentally different from classically defined mechanisms for drug resistance which focus on altered handling of the drug or the drug's target by the resistant tumor cell. It is expected that resistance will occur that is predicated on altered pathway predominance in a tumor and altered tumor microenvironment. Cases which exemplify this possibility are presented, including up-regulation of angiogenesis-related regulators in relation to inhibition of EGF-related proliferative and angiogenesis-promoting functions. The implications of this thinking in relation to development of combination regimens targeting distinct pathways are considered.