Vamsi Parimi

A Novel Nanoparticulate Formulation of Arsenic Trioxide with Enhanced Therapeutic Efficacy in a Murine Model of Breast Cancer

Purpose: The clinical success of arsenic trioxide (As2O3) in hematologic malignancies has not been replicated in solid tumors due to poor pharmacokinetics and dose-limiting toxicity. We have developed a novel nanoparticulate formulation of As2O3 encapsulated in liposomal vesicles or “nanobins” [(NB(Ni,As)] to overcome these hurdles. We postulated that nanobin encapsulation of As2O3 would improve its therapeutic index against clinically aggressive solid tumors, such as triple-negative breast carcinomas. Experimental Design: The cytotoxicity of NB(Ni,As), the empty nanobin, and free As2O3 was evaluated against a panel of human breast cancer cell lines. The plasma pharmacokinetics of NB(Ni,As) and free As2O3 were compared in rats to measure drug exposure. In addition, the antitumor activity of these agents was evaluated in an orthotopic model of human triple-negative breast cancer. Results: The NB(Ni,As) agent was much less cytotoxic in vitro than free As2O3 against a panel of human breast cancer cell lines. In contrast, NB(Ni,As) dramatically potentiated the therapeutic efficacy of As2O3 in vivo in an orthotopic model of triple-negative breast cancer. Reduced plasma clearance, enhanced tumor uptake, and induction of tumor cell apoptosis were observed for NB(Ni,As). Conclusions: Nanobin encapsulation of As2O3 improves the pharmacokinetics and antitumor efficacy of this cytotoxic agent in vivo. Our findings demonstrate the therapeutic potential of this nanoscale agent and provide a foundation for future clinical studies in breast cancer and other solid tumors. Clin Cancer Res; 16(14); 3607–17. ©2010 AACR.

Hypoxia-Inducible Factor-1 α Expression Predicts Superior Survival in Patients With Diffuse Large B-Cell Lymphoma Treated With R-CHOP

PURPOSE Hypoxia-inducible factor (HIF) controls the expression of genes in response to hypoxia, as well as a wide range of other cellular processes. We previously showed constitutive stabilization of HIF-1alpha in the majority of patients with diffuse large B-cell lymphoma (DLBCL). To our knowledge, the prognostic significance of HIF in lymphoma has never been investigated. PATIENTS AND METHODS We studied the immunohistochemical protein expression of HIF-1alpha on tissue microarrays from 153 patients with DLBCL treated in sequential cohorts with cyclophosphamide, doxorubicin, oncovin, and prednisone (CHOP) or rituximab-CHOP (R-CHOP) from 1999 to 2002. Results were correlated with patient outcome. Results Median follow-up for all patients was 80 months. Among all patients, HIF-1alpha was expressed in 62% of germinal center and 59% of non-germinal center patients. With HIF-1alpha analyzed as a dependent variable, there were no survival differences in CHOP-treated patients. In the R-CHOP group, however, HIF-1alpha protein expression correlated with significantly improved progression-free survival (PFS) and overall survival (OS). Five-year PFS for HIF-1alpha-positive patients was 71% v 43% for HIF-1alpha-negative patients (P = .0187), whereas 5-year OS was 75% and 54%, respectively (P = .025). In multivariate analysis with International Prognostic Index criteria, HIF-1alpha remained a significant predictor for PFS (P = .026) and OS (P = .043). Compared with other biomarkers, HIF-1alpha correlated only with BCL6 (P = .004). In terms of gene expression, we found several common gene associations of HIF-1alpha and the stromal-1 signature with genes predominantly involved in regulation of the extracellular matrix (eg, BGN, COL1A2, COL5A1, and PLOD2). CONCLUSION The expression of HIF-1alpha protein is an important independent favorable prognostic factor for survival in patients with DLBCL treated with R-CHOP.

Death Induced by CD95 or CD95 Ligand Elimination

CD95 (Fas/APO-1), when bound by its cognate ligand CD95L, induces cells to die by apoptosis. We now show that elimination of CD95 or CD95L results in a form of cell death that is independent of caspase-8, RIPK1/MLKL, and p53, is not inhibited by Bcl-xL expression, and preferentially affects cancer cells. All tumors that formed in mouse models of low-grade serous ovarian cancer or chemically induced liver cancer with tissue-specific deletion of CD95 still expressed CD95, suggesting that cancer cannot form in the absence of CD95. Death induced by CD95R/L elimination (DICE) is characterized by an increase in cell size, production of mitochondrial ROS, and DNA damage. It resembles a necrotic form of mitotic catastrophe. No single drug was found to completely block this form of cell death, and it could also not be blocked by the knockdown of a single gene, making it a promising way to kill cancer cells.

A dose-ranging study of 0.5% bupivacaine or ropivacaine on the success and duration of the ultrasound-guided, nerve-stimulator-assisted sciatic nerve block: a double-blind, randomized clinical trial.

Background and Objectives Before bifurcation, the sciatic nerve is composed of 2 component nerves encased in a common investing extraneural layer (CIEL). We examined the effect of various volumes injected beneath the CIEL on the success and duration of sciatic nerve block. Methods Ultrasound-guided nerve-stimulator–assisted sciatic nerve blocks were performed on 142 subjects. Subjects were randomized into 14 groups (0.5% ropivacaine or bupivacaine) with epinephrine 1:300,000 in volumes ranging from 2.5 to 30 mL. Successful block was defined as a complete sensory and motor block at 60 minutes. The minimum threshold current, time to complete block, duration of sensory and motor block, postoperative pain, and analgesic requirements were recorded. Results The mean threshold current external to the CIEL was 0.52 (0.15) mA compared to 0.19 (0.09) mA beneath the CIEL (P < 0.001). Successful block was achieved in 30 of 40 subjects that received 5 mL or less of ropivacaine or bupivacaine compared with 97 of 99 that received 10 mL or greater volume (P = 0.006). Injection volumes greater than or equal to 10 mL produced complete sensory and motor block within 30 minutes. Volumes greater than 10 mL did not extend the duration of the sensory or motor block. Injection volumes of 2.5 and 5 mL were associated with delayed onset and decreased block duration and a greater fraction of subjects experiencing pain behind the knee. Conclusions Injecting 10 mL of 0.5% bupivacaine or ropivacaine below the CIEL produces comparable onset and duration of sensory and motor blockade as volumes as large as 30 mL.

Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells

Breast cancer onset and disease progression have been linked to members of the TGFβ superfamily and their downstream signaling components, the Smads. Alterations in Smad3 signaling are associated with the dichotomous role of TGFβ in malignancy, mediating both tumor suppressant and pro-metastatic behaviors. Overexpression of cell cycle regulators, cyclins D and E, renders cyclin-dependent kinases (CDKs) 4/2 hyperactive. Noncanonical phosphorylation of Smad3 by CDK4/2 inhibits tumor suppressant actions of Smad3. We hypothesized that CDK inhibition (CDKi) would restore Smad3 action and help promote cancer cell regression. Treatment of triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-436, Hs578T) with CDK2i or CDK4i resulted in increased Smad3 activity and decreased cell migration. Transfection with a 5M Smad3 construct containing inhibitory mutations in 5 CDK phosphorylation sites also resulted in decreased TNBC cell migration and invasion. MDA-MB-231 cells treated with CDK2i or CDK4i resulted in decreased Smad3 protein phosphorylation at the CDK phosphorylation T179 site, decreased MMP2 and c-myc expression, and increased p15 and p21 expression. Using a novel transfected cell array, we found that CDK2i treatment decreased activity of the epithelial-to-mesenchymal transition related transcription factors Snail and Twist. In vivo studies in an MDA-MB-231 tumor model showed that individual and combination treatment with paclitaxel and CDK2i resulted in decreased tumor volume and Ki67 staining. Collectively, these data support further investigation of targeted CDK inhibitors as a promising therapeutic strategy for TNBC, a breast cancer subtype with limited treatment options.

Strong expression of EZH2 and accumulation of trimethylated H3K27 in diffuse large B-cell lymphoma independent of cell of origin and EZH2 codon 641 mutation

Gain-of-function EZH2 mutation promotes H3K27 trimethylation (H3K27me3) and lymphoid transformation of germinal center (GC) derived B-cell lymphoma, such as GCB diffuse large B-cell lymphoma (DLBCL), but not activated B-cell (ABC) DLBCL. It is unclear whether expression levels of EZH2 and consequential H3K27me3 vary by EZH2 mutation and/or cell-of-origin in DLBCL. Ninety lymphoma samples including 40 DLBCLs were studied by immunohistochemistry. EZH2 Y641 mutations were detected in three of 20 (15%) GCB and none of 20 ABC types. All 40 DLBCLs showed strong EZH2, expression with high-level H3K27me3 in 90% GCBs and 95% ABCs. In 50 other B-cell lymphomas except for follicular lymphoma, strong EZH2 expression correlated with high-grade features. Immunoblot of DLBCL cell lines and microarray gene expression study of EZH2 in B-cell lymphomas were consistent with the immunohistochemistry findings. High-level EZH2 and H3K27me3 were common in DLBCL independent of cell-of-origin and EZH2 mutation. High-level EZH2 in lymphoma of aggressive features suggests additional therapeutic targets.

A Phase I Trial of Aminolevulinic Acid-Photodynamic Therapy for Treatment of Oral Leukoplakia

BACKGROUND Photodynamic therapy with aminolevulinic acid (ALA PDT) for oral leukoplakia has shown promising effects in regression of oral leukoplakia. Although ALA has been extensively studied and is an ideal photosensitizer, the optimal light dose for treatment of oral leukoplakia has not been determined. We conducted a phase I study to determine MTD and DLT of PDT in patients treated with ALA for leukoplakia. METHODS Patients with histologically confirmed oral leukoplakia received a single treatment of ALA PDT in cohorts with escalating doses of light (585nm). Clinical, histologic, and biologic markers were assessed. RESULTS Analysis of 11 participants is reported. No significant toxicity from ALA PDT was observed in patients who received ALA with a light dose of up to 4J/cm(2). One participant experienced transient grade 3 transaminase elevation due to ALA. One participant had a partial clinical response 3months after treatment. Biologic mucosal risk markers showed no significant associations. Determination of MTD could not be accomplished within a feasible timeframe for completion of the study. CONCLUSIONS ALA PDT could be safely administered with a light dose up to 4J/cm(2) and demonstrated activity. Larger studies are needed to fully elucidate the MTD and efficacy of ALA-PDT.

Stable luciferase expression does not alter immunologic or in vivo growth properties of GL261 murine glioma cells

BackgroundGL261 cells are murine glioma cells that demonstrate proliferation, invasion, and angiogenesis when implanted in syngeneic C57BL/6 mice, providing a highly useful immunocompetent animal model of glioblastoma. Modification of tumor cells for luciferase expression enables non-invasive monitoring of orthotopic tumor growth, and has proven useful for studying glioblastoma response to novel therapeutics. However, tumor modification for luciferase has the potential for evoking host immune response against otherwise syngeneic tumor cells, thereby mitigating the tumor cells’ value for tumor immunology and immunotherapy studies.MethodsGL261 cells were infected with lentivirus containing a gene encoding firefly luciferase (GL261.luc). In vitro proliferation of parental (unmodified) GL261 and GL261.luc was measured on days 0, 1, 2, 4, and 7 following plating, and the expression of 82 mouse cytokines and chemokines were analyzed by RT-PCR array. Cell lines were also evaluated for differences in invasion and migration in modified Boyden chambers. GL261 and GL261.luc cells were then implanted intracranially in C57BL/6 mice, with GL261.luc tumor growth monitored by quantitative bioluminescence imaging, and all mice were followed for survival to compare relative malignancy of tumor cells.ResultsNo difference in proliferation was indicated for GL261 vs. GL261.luc cells (p>0.05). Of the 82 genes examined by RT-PCR array, seven (9%) exhibited statistically significant change after luciferase modification. Of these, only three changed by greater than 2-fold: BMP-2, IL-13, and TGF-β2. No difference in invasion (p=0.67) or migration (p=0.26) was evident between modified vs. unmodified cells. GL261.luc cell luminescence was detectable in the brains of C57BL/6 mice at day 5 post-implantation, and tumor bioluminescence increased exponentially to day 19. Median overall survival was 20.2 days versus 19.7 days for mice receiving implantation with GL261 and GL261.luc, respectively (p=0.62). Histopathologic analysis revealed no morphological difference between tumors, and immunohistochemical analysis showed no significant difference for staining of CD3, Ki67, or CD31 (p>0.05 for all).ConclusionsLuciferase expression in GL261 murine glioma cells does not affect GL261 proliferation, invasion, cytokine expression, or in vivo growth. Luciferase modification increases their utility for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

c-Met Overexpression in Cervical Cancer, a Prognostic Factor and a Potential Molecular Therapeutic Target.

Purpose: This study aimed to assess the association between pretreatment c-Met overexpression in local-regional advanced cervical cancer patients treated definitively with concurrent chemoradiation therapy (CRT) and treatment outcomes including overall survival (OS), progression-free survival (PFS), distant metastases (DM) control, and local-regional control (LC). Patients and Methods: This Institutional Review Board–approved study included cervical cancer patients treated definitively and consecutively with CRT. Evaluation of cytoplasmic immunoreactivity for c-Met was performed and scored semiquantitatively by 3 pathologists, blinded to the treatment outcomes, and incorporated both the intensity and percentage of immunoreactivity in invasive carcinoma (H score). Treatment outcomes were reviewed and reported. Outcomes were stratified by c-Met overexpression and tumor characteristics. OS, PFS, LC, and DC rates were obtained via the Kaplan-Meier method and differences between groups were evaluated by the log-rank test. Hazard ratios were obtained via Cox regression for both univariate and multivariate analyses. Results: The 5-year OS, PFS, LC, and DC were 57.18%, 48.07%, 72.11%, and 62.85%, respectively. Ten (35.7%) and 18 patients (64.3%) had c-Met H index >30 and<30, respectively. c-Met overexpression was significantly associated with worse 3- and 5-year OS (P=0.003), PFS (P=0.002), LC (P=0.01), and DC (P=0.0003). Patients with c-Met overexpression had a hazard ratio of 6.297, 5.782, 6.28, and 18.173 for the risks of death, disease progression, local recurrence, and DM, respectively. Conclusion: c-Met overexpression could be a potential predictive marker and therapeutic target for local-regional advanced cervical cancer patients treated definitively with CRT.

Overexpression of lipid metabolism genes and PBX1 in the contralateral breasts of women with estrogen receptor-negative breast cancer

Risk biomarkers for estrogen receptor (ER)‐negative breast cancer have clear value for breast cancer prevention. We previously reported a set of lipid metabolism (LiMe) genes with high expression in the contralateral unaffected breasts (CUBs) of ER‐negative cancer cases. We now further examine LiMe gene expression in both tumor and CUB, and investigate the role of Pre‐B‐cell leukemia homeobox‐1 (PBX1) as a candidate common transcription factor for LiMe gene expression. mRNA was extracted from laser‐capture microdissected epithelium from tumor and CUB of 84 subjects (28 ER‐positive cases, 28 ER‐negative cases, 28 healthy controls). Gene expression was quantitated by qRT‐PCR. Logistic regression models were generated to predict ER status of the contralateral cancer. Protein expression of HMGCS2 and PBX1 was measured using immunohistochemistry. The effect of PBX1 on LiMe gene expression was examined by overexpressing PBX1 in MCF10A cells with or without ER, and by suppressing PBX1 in MDA‐MB‐453 cells. The expression of DHRS2, HMGCS2, UGT2B7, UGT2B11, ALOX15B, HPGD, UGT2B28 and GLYATL1 was significantly higher in ER‐negative versus ER‐positive CUBs, and predicted ER status of the tumor in test and validation sets. In contrast, LiMe gene expression was significantly lower in ER‐negative than ER‐positive tumors. PBX1 overexpression in MCF10A cells up‐regulated most LiMe genes, but not in MCF10A cells overexpressing ER. Suppressing PBX1 in MDA‐MB‐453 cells resulted in decrease of LiMe gene expression. Four binding sites of PBX1 and cofactor were identified in three lipid metabolism genes using ChIP‐qPCR. These data suggest a novel role for PBX1 in the regulation of lipid metabolism genes in benign breast, which may contribute to ER‐negative tumorigenesis.