TBCRC 002: a phase II, randomized, open-label trial of preoperative letrozole with or without bevacizumab in postmenopausal women with newly diagnosed stage 2/3 hormone receptor-positive and HER2-negative breast cancer.

BACKGROUND: In preclinical studies, the expression of vascular endothelial growth factor (VEGF) in hormone receptor-positive breast cancer is associated with estrogen-independent tumor growth and resistance to endocrine therapies. This study investigated whether the addition of bevacizumab, a monoclonal antibody against VEGF, to letrozole enhanced the antitumor activity of the letrozole in the preoperative setting. METHODS: Postmenopausal women with newly diagnosed stage 2 or 3 estrogen and/or progesterone receptor-positive, HER2-negative breast cancer were randomly assigned (2:1) between letrozole 2.5 mg PO daily plus bevacizumab 15 mg/kg IV every 3 weeks (Let/Bev) and letrozole 2.5 mg PO daily (Let) for 24 weeks prior to definitive surgery. Primary objective was within-arm pathologic complete remission (pCR) rate. Secondary objectives were safety, objective response, and downstaging rate. RESULTS: Seventy-five patients were randomized (Let/Bev n = 50, Let n = 25). Of the 45 patients evaluable for pathological response in the Let/Bev arm, 5 (11%; 95% CI, 3.7-24.1%) achieved pCR and 4 (9%; 95% CI, 2.5-21.2%) had microscopic residual disease; no pCRs or microscopic residual disease was seen in the Let arm (0%; 95% CI, 0-14.2%). The rates of downstaging were 44.4% (95% CI, 29.6-60.0%) and 37.5% (95% CI, 18.8-59.4%) in the Let/Bev and Let arms, respectively. Adverse events typically associated with letrozole (hot flashes, arthralgias, fatigue, myalgias) occurred in similar frequencies in the two arms. Hypertension, headache, and proteinuria were seen exclusively in the Let/Bev arm. The rates of grade 3 and 4 adverse events and discontinuation due to adverse events were 18% vs 8% and 16% vs none in the Let/Bev and Let arms, respectively. A small RNA-based classifier predictive of response to preoperative Let/Bev was developed and confirmed on an independent cohort. CONCLUSION: In the preoperative setting, the addition of bevacizumab to letrozole was associated with a pCR rate of 11%; no pCR was seen with letrozole alone. There was additive toxicity with the incorporation of bevacizumab. Responses to Let/Bev can be predicted from the levels of 5 small RNAs in a pretreatment biopsy. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov (Identifier: NCT00161291), first posted on September 12, 2005, and is completed.

PARP1 and phospho-p65 protein expression is increased in human HER2-positive breast cancers.

Previous studies have shown that basal breast cancers, which may have an inherent "BRCAness" phenotype and sensitivity to inhibitors of poly (ADP-Ribose) polymerase (PARP), express elevated levels of PARP1. Our lab recently reported that HER2+ breast cancers also exhibit sensitivity to PARP inhibitors (PARPi) by attenuating the NF-κB pathway. In this study, we assessed PARP1 and phospho-p65, a marker of activated NF-κB levels in human breast cancer tissues. PARP1 and PARP2 copy number, mRNA, and protein expression was assessed by interrogating the PAM-50 defined breast cancer patient set from the TCGA using cBioPortal. PARP1 and phospho-p65 immunohistochemistry and correlation to clinical parameters was conducted using 307 primary breast cancer specimens (132 basal, 82 luminal, 93 HER2+) through univariate and multivariate analyses. In the PAM50 breast cancer data set, PARP1 and 2 expression was altered in 24/58 (41 %) HER2+, 32/81 (40 %) basal, and 75/324 (23 %) luminal A/B breast cancer patients. This correlated with a statistically significant increase in PARP1 protein levels in HER2+ and basal but not luminal breast cancers (p = 0.003, p = 0.027, p = 0.289, respectively). No change in PARP2 protein level was observed. Interestingly, using breast cancer specimens from 307 patients, HER2 positivity correlated with elevated PARP1 expression (p < 0.0001) and was three times more likely than HER2 negative breast cancers to exhibit high PARP1 levels. No significant differences were noted between race, ER status, or PR status for PARP1 expression. Additionally, we found a significant correlation between HER2 status and phospho-p65 expression (p < 0.0001). Lastly, a direct correlation between PARP1 and phospho-p65 (p < 0.0001) was noted. These results indicate a potential connection between HER2, PARP1, and phospho-p65. Furthermore, these data suggest that the PARPi sensitivity we previously observed in HER2+ breast cancer cells may be due to elevated PARP1 expression.

Recurrent read-through fusion transcripts in breast cancer.

Read-through fusion transcripts that result from the splicing of two adjacent genes in the same coding orientation are a recently discovered type of chimeric RNA. We sought to determine if read-through fusion transcripts exist in breast cancer. We performed paired-end RNA-seq of 168 breast samples, including 28 breast cancer cell lines, 42 triple negative breast cancer primary tumors, 42 estrogen receptor positive (ER+) breast cancer primary tumors, and 56 non-malignant breast tissue samples. We analyzed the sequencing data to identify breast cancer associated read-through fusion transcripts. We discovered two recurrent read-through fusion transcripts that were identified in breast cancer cell lines, confirmed across breast cancer primary tumors, and were not detected in normal tissues (SCNN1A-TNFRSF1A and CTSD-IFITM10). Both fusion transcripts use canonical splice sites to join the last splice donor of the 5' gene to the first splice acceptor of the 3' gene, creating an in-frame fusion transcript. Western blots indicated that the fusion transcripts are translated into fusion proteins in breast cancer cells. Custom small interfering RNAs targeting the CTSD-IFITM10 fusion junction reduced expression of the fusion transcript and reduced breast cancer cell proliferation. Read-through fusion transcripts between adjacent genes with different biochemical functions represent a new type of recurrent molecular defect in breast cancer that warrant further investigation as potential biomarkers and therapeutic targets. Both breast cancer associated fusion transcripts identified in this study involve membrane proteins (SCNN1A-TNFRSF1A and CTSD-IFITM10), which raises the possibility that they could be breast cancer-specific cell surface markers.

Basal-like breast cancer stem cells are sensitive to anti-DR5 mediated cytotoxicity.

Breast cancer stem cells (BrCSC) are resistant to common therapeutic modalities including chemotherapy, radiation, and hormonal agents. They are thought to contribute to treatment resistance, relapse, and metastases. This study examines the effect of a monoclonal anti-DR5 antibody (TRA-8) and chemotherapy (adriamycin, taxol) on BrCSC populations from basal-like breast cancer cell lines. Doubly enriched BrCSC (CD44(+), CD24(-), ALDH(+)) cells were exposed to TRA-8 and control reagents and examined for cytotoxicity, caspase activation, tumorsphere formation and tumorigenicity. Doubly enriched BrCSC populations expressed cell surface DR5 and were sensitive to TRA-8 mediated cytotoxicity with induction of caspase 8 and 3 activation. TRA-8 at sub-nanomolar concentrations inhibited 2LMP and SUM159 BrCSC tumorsphere formation and was more than 50-fold more inhibitory than TRAIL or anti-DR4 at equimolar concentrations. Chemotherapy treatment of 2LMP and SUM159 cell lines resulted in a relative increase of BrCSC, whereas TRA-8 produced a decrease in the percentage of BrCSC. TRA-8 exposure to 2LMP and SUM159 BrCSC preparations produced significant inhibition of tumorigenicity. DR5 maybe a therapeutic target on the surface of basal-like BrCSC which is amenable to agonistic monoclonal anti-DR5 therapy.

Effect of anti-DR5 and chemotherapy on basal-like breast cancer.

The purpose is to evaluate sensitivity of basal-like breast cancer to treatment with anti-DR5 alone and in combination with chemotherapy. Cytotoxicity of TRA-8 anti-DR5 alone and in combination with doxorubicin or paclitaxel was examined. The role of a DR5-associated molecule (DDX3) in the regulation of apoptosis by recruitment of cIAP1 to the DR5/DDX3 complex was studied. SUM159 and 2LMP orthotopic xenografts were treated with TRA-8 alone and in combination with Abraxane or doxorubicin, and tumor growth inhibition determined. Diffusion-weighted magnetic resonance imaging was used to monitor early tumor response. The majority (12/15) of basal-like cell lines were very sensitive to TRA-8-induced cytotoxicity (IC(50) values of 1.0-49 ng/ml). In contrast, 8/11 luminal or HER2-positive cell lines were resistant (IC(50) > 1,000 ng/ml). Enhanced killing of basal-like cell lines was produced by combination treatment with TRA-8 and doxorubicin. Majority of basal cell lines expressed lower levels of DR5-associated DDX3 and cIAP1 than luminal and HER2-positive cell lines. TRA-8 inhibited growth of basal xenografts and produced 20% complete 2LMP tumor regressions. TRA-8 and chemotherapy produced greater 2LMP growth inhibition than either alone. An increase in apparent diffusion coefficient in 2LMP tumors was measured in a week of therapy with TRA-8 and Abraxane. Basal-like cell lines were more sensitive to TRA-8-mediated cytotoxicity than HER2-over-expressing and luminal cell lines, and chemotherapy enhanced cytotoxicity. High sensitivity of basal cells to TRA-8 correlated with low expression of DR5/DDX3/cIAP1 complex. Treatment with TRA-8 and chemotherapy may be an effective therapy for basal-like breast cancer.

Treatment of human colon cancer xenografts with TRA-8 anti-death receptor 5 antibody alone or in combination with CPT-11.

PURPOSE: This study was designed to evaluate the in vitro cytotoxicity and in vivo efficacy of TRA-8, a mouse monoclonal antibody that binds to the DR5 death receptor for tumor necrosis factor-related apoptosis-inducing ligand (also called Apo2L), alone and in combination with CPT-11, against human colon cancer cells and xenografts. EXPERIMENTAL DESIGN: DR5 expression was assessed on human colon cancer cell lines using flow cytometry, and cellular cytotoxicity after TRA-8 treatment, alone and in combination with SN-38, was determined by measuring cellular ATP levels. Tumor growth inhibition and regression rates of well-established subcutaneous COLO 205, SW948, HCT116, and HT-29 colon cancer xenografts in athymic nude mice treated with TRA-8 or CPT-11 alone and in combination were determined. (99m)Tc-TRA-8 was used to examine tumor localization of TRA-8 in animals bearing each of the four xenografts. In addition, whole-body biodistribution and imaging was carried out in COLO 205-bearing animals using in vivo single-photon emission computed tomography imaging and tissue counting. RESULTS: DR5 expression was highest on HCT116, intermediate on SW948 and COLO 205 cells, and lowest on HT-29 cells. COLO 205 cells were the most sensitive to TRA-8-induced cytotoxicity in vitro, SW948 and HCT116 cell lines were moderately sensitive, and HT-29 cells were resistant. Combination treatment with TRA-8 and SN-38 produced additive to synergistic cytotoxicity against all cell lines compared with either single agent. The levels of apoptosis in all cell lines, including HT-29, were increased by combination treatment with SN-38. In vivo, combination therapy with TRA-8 and CPT-11 was superior to either single-agent regimen for three of the xenografts: COLO 205, SW948, and HCT116. COLO 205 tumors were most responsive to therapy with 73% complete regressions after combination therapy. HT-29 cells derived no antitumor efficacy from TRA-8 therapy. Tumor xenografts established from the four colon cancer cell lines had comparable specific localization of (99m)Tc-TRA-8. CONCLUSIONS: In vitro and in vivo effects of TRA-8 anti-DR5 monoclonal antibody on four different colon cancer cell lines and xenografts were quite variable. The HT-29 cell line had low surface DR5 expression and was resistant to TRA-8 both in vitro and in vivo. Three cell lines (COLO 205, SW948, and HCT116) exhibited moderate to high sensitivity to TRA-8-mediated cytotoxicity which was further enhanced by the addition of SN-38, the active metabolite of CPT-11. In vivo, the combination of TRA-8 and CPT-11 treatment produced the highest antitumor efficacy against xenografts established from the three TRA-8-sensitive tumor cell lines. All four colon cancer xenografts had comparable localization of (99m)Tc-TRA-8. These studies support the strategy of TRA-8/CPT-11 combined treatment in human colon cancer clinical trials.

Effect of TRA-8 anti-death receptor 5 antibody in combination with chemotherapy in an ex vivo human ovarian cancer model.

OBJECTIVES: To investigate the cytotoxicity of TRA-8, an antibody that specifically binds death receptor 5, alone and in combination with chemotherapy, using an ex vivo human ovarian cancer model. MATERIALS AND METHODS: Twenty-six ovarian cancer specimens were obtained during ovarian cancer debulking, and tumor slices were prepared with the Krumdieck tissue slicer. The tumor slices were exposed to varying concentrations of TRA-8, carboplatin/paclitaxel, or the combination of TRA-8 and chemotherapy. Using nonlinear modeling, dose-response curves and IC50 values were generated for specimens treated with TRA-8. The additive and synergistic cytotoxic effects of chemotherapy combination with TRA-8 were evaluated in specimens. In addition to adenosine triphosphate viability assays, the treated and untreated slices were assessed by immunohistochemistry to confirm apoptosis induction. RESULTS: Specimens from 13 patients yielded TRA-8-induced IC50 values. Of these specimens, 15% were found to be sensitive to TRA-8-induced cytotoxicity at IC50 doses less than 500 ng/mL. Specimens from 13 patients underwent combination treatment with TRA-8 and carboplatin/paclitaxel. Of these specimens, 77% exhibited additive cytotoxicity in comparison with those treated with either agent alone, whereas 15% exhibited synergistic cytotoxicity. Immunohistochemical analysis of terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling and cleaved caspase 3 staining demonstrated a dose-dependent increase in apoptosis with the combination treatment. CONCLUSIONS: This study demonstrates the efficacy of the death receptor monoclonal antibody TRA-8 in combination with conventional chemotherapy in an ex vivo human ovarian cancer model. This model can be used to assess cytotoxicity of novel agents in combination with chemotherapy in ovarian cancer.

Brief overview of preclinical and clinical studies in the development of intraperitoneal radioimmunotherapy for ovarian cancer.

Due to the generally slow and incomplete transit of i.p. infused agents into the circulation, treating disease confined to the peritoneal cavity with chemotherapy, biologics, and/or radionuclides provides a pharmacologic advantage. A higher i.p. concentration can be achieved than could be tolerated by systemic administration. An advantage of i.p. versus i.v. administration for localization of radiolabeled antibodies to small peritoneal surface disease has been shown in animal model and human biopsy studies (1, 2). A recent phase III Gynecologic Oncology Group chemotherapy trial has confirmed a survival advantage for i.p. delivery among women undergoing initial therapy for advanced ovarian cancer (3). Although the therapy was more difficult to tolerate such that 60% of patients randomized to the i.p. arm did not complete the entire regimen, there was a 16-month survival advantage. I.p. radionuclide therapy has been used in treatment of ovarian cancer for more than three decades, but side effects have been problematic in non-tumor-targeted 32P therapy (4). Efforts to improve specificity have used a number of antigens expressed on ovarian cancer cells as targets for selective delivery of radionuclide-conjugates. Mouse models and cell culture have been prominent for preclinical study of agents and strategies in the development of i.p. targeted radionuclide therapy for ovarian cancer. Animal studies, which have directed clinical trials, have shown clear improvement in survival with various modifications including combination chemotherapy, pretargeting, and combination of antibodies over simply delivery of a radiolabeled antibody via i.p. route.

TRA-8 anti-DR5 monoclonal antibody and gemcitabine induce apoptosis and inhibit radiologically validated orthotopic pancreatic tumor growth.

PURPOSE: To evaluate agonistic TRA-8 monoclonal antibody to human death receptor 5 (DR5) and gemcitabine in vitro and in an orthotopic pancreatic cancer model. EXPERIMENTAL DESIGN: Pancreatic cancer cell lines were screened for DR5 expression, cytotoxicity, and apoptosis induced by TRA-8, gemcitabine, or gemcitabine and TRA-8. An orthotopic model of pancreatic cancer was established in severe combined immunodeficient mice. Mice were treated with TRA-8, gemcitabine, or a combination for one or two cycles of therapy. Tumor growth (ultrasound) and survival were analyzed. RESULTS: All five pancreatic cancer cell lines showed DR5 protein expression and varying sensitivity to TRA-8-mediated cytotoxicity. MIA PaCa-2 cells were very sensitive to TRA-8, moderately resistant to gemcitabine, with additive cytotoxicity to the combination. S2-VP10 cells were resistant to TRA-8 and sensitive to gemcitabine with synergistic sensitivity to the combination. Combination treatment in vitro produced enhanced caspase-3 and caspase-8 activation. A single cycle of therapy produced comparable efficacy for single-agent TRA-8 and the combination of TRA-8 and gemcitabine, with significant reduction in tumor size and prolonged survival compared with gemcitabine alone or control animals. With two cycles of therapy, TRA-8 and combination therapy produced enhanced inhibition of tumor growth compared with single-agent gemcitabine or untreated animals. However, the combination regimen showed enhanced survival as compared with single-agent TRA-8. CONCLUSIONS: Pancreatic cancer cell lines express varying levels of DR5 and differ in their sensitivity to TRA-8 and gemcitabine-induced cytotoxicity. TRA-8 with two cycles of gemcitabine therapy produced the best overall survival.

Trastuzumab-Resistant HER2+ Breast Cancer Cells Retain Sensitivity to Poly (ADP-Ribose) Polymerase (PARP) Inhibition.

HER2-targeted therapies, such as trastuzumab, have increased the survival rates of HER2+ breast cancer patients. However, despite these therapies, many tumors eventually develop resistance to these therapies. Our lab previously reported an unexpected sensitivity of HER2+ breast cancer cells to poly (ADP-ribose) polymerase inhibitors (PARPi), agents that target homologous recombination (HR)-deficient tumors, independent of a DNA repair deficiency. In this study, we investigated whether HER2+ trastuzumab-resistant (TR) breast cancer cells were susceptible to PARPi and the mechanism behind PARPi induced cytotoxicity. We demonstrate that the PARPi ABT-888 (veliparib) decreased cell survival in vitro and tumor growth in vivo of HER2+ TR breast cancer cells. PARP-1 siRNA confirmed that cytotoxicity was due, in part, to PARP-1 inhibition. Furthermore, PARP-1 silencing had variable effects on the expression of several NF-κB-regulated genes. In particular, silencing PARP-1 inhibited NF-κB activity and reduced p65 binding at the IL8 promoter, which resulted in a decrease in IL8 mRNA and protein expression. Our results provide insight in the potential mechanism by which PARPi induces cytotoxicity in HER2+ breast cancer cells and support the testing of PARPi in patients with HER2+ breast cancer resistant to trastuzumab. Mol Cancer Ther; 17(5); 921-30. ©2018 AACR.

Retreatment with yttrium-90 ibritumomab tiuxetan in patients with B-cell non-Hodgkin's lymphoma.

There is no data on safety and efficacy of a second course of ibritumomab tiuxetan. In this work, data on patients with B-cell NHL who were treated with two courses of ibritumomab tiuxetan were analyzed. Eighteen such patients were analyzed (age: 58 years, 48 - 91), with a median of four prior regimens (1 - 7), stem cell transplantation (n = 5), and radiation therapy (n = 6). After the first course, G3/4 neutropenia and thrombocytopenia was 35% and 41%; overall response rate (ORR) was 89%; time between courses was 16.6 months (6.0 - 42.7). After the second course, the incidence of G3/4 neutropenia and thrombocytopenia was 28% and 44%; and ORR 77%. There were no infectious or bleeding complications, secondary myelodysplastic syndromes, or leukemias. Retreatment with the ibritumomab tiuxetan regimen was well tolerated, with a safety profile similar to that of the first course. To conclude, patients who benefited from the first course of ibritumomab tiuxetan can benefit from retreatment.

Phase I study of the intratumoral administration of recombinant canarypox viruses expressing B7.1 and interleukin 12 in patients with metastatic melanoma.

The objective of this study was to evaluate the safety and activity of the intratumoral administration of the immune costimulatory molecule, B7.1, encoded by a vector derived from the canarypox virus, ALVAC (ALVAC-B7.1), alone and with the intratumoral injection of ALVAC encoding the immune-stimulatory cytokine, interleukin 12 (ALVAC-IL-12). Fourteen patients with metastatic melanoma who had s.c. nodules received intratumoral injections on days 1, 4, 8, and 11. Nine patients were given escalating doses of up to 25 x 10(8) plaque-forming units of ALVAC-B7.1. Five patients were given 25 x 10(8) plaque-forming units of ALVAC-B7.1 combined with ALVAC-IL-12 50% tissue culture infective dose of 2 x 10(6). Toxicity was mild to moderate and consisted of inflammatory reactions at the injection site and fever, chills, myalgia, and fatigue. Higher levels of B7.1 mRNA were observed in ALVAC-B7.1-injected tumors compared with saline-injected control tumors. Higher levels of intratumoral vascular endothelial growth factor and IL-10, cytokines with immune suppressive activities, were also observed in ALVAC-B7.1- and ALVAC-IL-12-injected tumors compared with saline-injected controls. Serum levels of vascular endothelial growth factor increased at day 18 and returned to baseline at day 43. All patients developed antibody to ALVAC. Intratumoral IL-12 and IFN-gamma mRNA decreased. Changes in serum IL-12 and IFN-gamma levels were not observed. Tumor regressions were not observed. The intratumoral injections of ALVAC-B7.1 and ALVAC-IL-12 were well tolerated at these dose levels and at this schedule and resulted in measurable biological response. This response included the production of factors that may suppress the antitumor immunologic activity of these vectors.

Intraperitoneal pretarget radioimmunotherapy with CC49 fusion protein.

PURPOSE: This study examined a pretarget radioimmunotherapy strategy for treatment of an i.p. tumor model (LS174T). EXPERIMENTAL DESIGN: The strategy used regional administration (i.p.) of a novel targeting molecule composed of four CC49 anti-tumor-associated glycoprotein 72 (TAG-72) single-chain antibodies linked to streptavidin as a fusion protein (CC49 fusion protein); 24 hours later, a synthetic clearing agent was administered i.v. to produce hepatic clearance of unbound CC49 fusion protein/synthetic clearing agent complexes. Four hours later, a low molecular weight radiolabeled reagent composed of biotin conjugated to the chelating agent 7,10-tetra-azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) complexed with (111)In-, (90)Y-, or (177)Lu-DOTA-biotin was injected. RESULTS: Radiolocalization to tumor sites was superior with i.p. administration of radiolabeled DOTA-biotin as compared with i.v. administration. Imaging and biodistribution studies showed excellent tumor localization of radioactivity with (111)In- or (177)Lu-DOTA-biotin. Tumor localization of (111)In-DOTA-biotin was 43% ID/g and 44% ID/g at 4 and 24 hours with the highest normal tissue localization in the kidney with 6% ID/g at 48 and 72 hours. Therapy studies with (90)Y-DOTA-biotin at doses of 400 to 600 microCi or (177)Lu-DOTA-biotin at doses of 600 to 800 microCi produced significant prolongation of survival compared with controls (P = 0.03 and P < 0.01). CONCLUSIONS: Pretarget radioimmunotherapy using regional administration of CC49 fusion protein and i.p. (90)Y- or (177)Lu-DOTA-biotin represents a successful therapeutic strategy in the LS174T i.p. tumor model and this strategy may be applicable to human trials in patients with i.p. ovarian cancer.

Anti-GD3 monoclonal antibody effects on lymphocytes and antibody-dependent cellular cytotoxicity.

PURPOSE: Antibodies targeting GD3 gangliosides highly expressed on melanomas mediate immune effector functions in vitro and inhibit animal model melanoma tumor growth in vivo. Because GD3 is expressed also on a subpopulation of human lymphocytes, we characterized the in vitro immune effects of murine R24 and a chimeric anti-GD3 antibody (KW-2871). DESIGN: Anti-GD3 complement-mediated (CMC) and antibody-dependent cellular cytotoxicity (ADCC) were tested against cell line Mel-624. Antibody-mediated lymphocyte expression of interleukin (IL)-2, IL-4, IL-10, and interferon-gamma (IFN-gamma) was quantified. The effect of antibody and antibody-treated lymphocyte supernates on effector cell ADCC and Fc receptor expression were evaluated. RESULTS: R24 and KW-2871 antibodies mediated CMC and ADCC to the Mel-624 cell line. R24 induced potent lymphocyte proliferation and enhanced lymphocyte RNA expression of IL-4 (2-4 logs), IL-10, and IFN-gamma (> 10-fold). KW-2871 induced no lymphocyte proliferation and had minimal effects on lymphokine expression (< 5-fold). Preincubation of effector cells with either antibody inhibited ADCC and reduced monocyte expression of FcgammaRI and II. Supernates of effector cells preincubated with either antibody were able to inhibit ADCC. CONCLUSIONS: R24 and KW-2871 antibody differ in their lymphocyte proliferation and lymphokine release activity but have similar inhibition of lymphocyte ADCC and FcgammaR expression in vitro.

A phase I study of an anti-GD3 monoclonal antibody, KW-2871, in patients with metastatic melanoma.

PURPOSE: KW-2871 (IgG1 kappa chimeric antibody) targets GD3, which is upregulated in melanomas. We conducted a phase I trial of KW-2871 in patients with metastatic melanoma. METHODS: Seventeen (17) patients were enrolled and received an initial test dose (10 mg/m2) intravenously. Two 2 weeks later, patients were stratified into 4 cohorts to receive 4 doses of KW-2871 (infused over 1 hour) at 2-week intervals (20, 40, 60, and 80 mg/m2). No premedications were administered for the test or first therapeutic doses. RESULTS: Dose-limiting toxicities were Grade 3 laryngospasm and chest tightness with the initial therapeutic infusion at doses of 80 and 60 mg/m2. The maximum tolerated dose (MTD) was established at 40 mg/m2 of KW2871. The most common side-effect was urticaria (Grades 1-3) in 16 of 16 patients during an initial therapeutic infusion without premedication. The mean terminal half-life, clearance, and area under the concentration-time curve (AUC(0-t)) at a dose of 40 mg/m2 for course 1 were 146 +/- 31 hours, 28 +/- 6 ml/hour, and 1922 +/- 491 mcg*hour/mL, respectively. Anti-human chimeric antibody was not detected. Two (2) patients in the 40 mg/m2 cohort had stable disease. CONCLUSIONS: An MTD of 40 mg/m2 without premedication was established for KW-2871, with urticaria being the most common side-effect and dose-limiting anaphylactoid infusion reactions.

Expression of the MHC Class II Pathway in Triple-Negative Breast Cancer Tumor Cells Is Associated with a Good Prognosis and Infiltrating Lymphocytes.

Triple-negative breast cancer (TNBC) is a subtype with heterogeneous patient outcomes. Approximately 40% of patients experience rapid relapse, while the remaining patients have long-term disease-free survival. To determine if there are molecular differences between primary tumors that predict prognosis, we performed RNA-seq on 47 macrodissected tumors from newly diagnosed patients with TNBC (n = 47; 22 relapse, 25 no relapse; follow-up median, 8 years; range, 2-11 years). We discovered that expression of the MHC class II (MHC II) antigen presentation pathway in tumor tissue was the most significant pathway associated with progression-free survival (HR, 0.36; log-rank P = 0.0098). The association between MHC II pathway expression and good prognosis was confirmed in a public gene expression database of 199 TNBC cases (HR, 0.28; log-rank P = 4.5 × 10(-8)). Further analysis of immunohistochemistry, laser-capture microdissected tumors, and TNBC cell lines demonstrated that tumor cells, in addition to immune cells, aberrantly express the MHC II pathway. MHC II pathway expression was also associated with B-cell and T-cell infiltration in the tumor. Together, these data support the model that aberrant expression of the MHC II pathway in TNBC tumor cells may trigger an antitumor immune response that reduces the rate of relapse and enhances progression-free survival. Cancer Immunol Res; 4(5); 390-9. ©2016 AACR.

Intratumoral administration of a recombinant canarypox virus expressing interleukin 12 in patients with metastatic melanoma.

The aim of this study was to evaluate the tolerability and activity of intratumoral administered human interleukin 12 encoded by a vector derived from the canarypox virus (ALVAC-IL-12). Nine patients with surgically incurable metastatic melanoma who had subcutaneous nodules available for injection were enrolled. ALVAC-IL-12 was administered by intratumoral injection on days 1, 4, 8, and 11. Tumor nodules greater than 2 cm in diameter were injected with 2 x 10(6) median tissue culture infectious doses (TCID(50)), and smaller tumors were injected with 1 x 10(6) TCID(50). The total dose per patient per time point ranged from 1 x 10(6) to 4 x 10(6) TCID(50). Toxicity was mild to moderate and consisted of inflammatory reactions at the injection site and fever associated with chills, myalgia, and fatigue. No dose-limiting toxicities occurred. Increases in IL-12 mRNA, and also increases in interferon gamma mRNA, were observed in ALVAC-IL-12-injected tumors compared with saline-injected control tumors in four of the nine patients. ALVAC-IL-12-injected tumors were also characterized by T cell infiltration. Three patients demonstrated increases in serum IL-12 and in interferon gamma levels. All patients developed neutralizing IgG antibody to the canarypox vector. One patient manifested a complete response of injected subcutaneous metastases and uninjected in-transit metastases. The intratumoral injection of ALVAC-IL-12 at these dose levels and according to this schedule was well tolerated and resulted in measurable biologic response in patients with metastatic melanoma.

Patient-specific dosimetry of pretargeted radioimmunotherapy using CC49 fusion protein in patients with gastrointestinal malignancies.

UNLABELLED: Pretargeted radioimmunotherapy (RIT) using CC49 fusion protein, comprised of CC49-(scFv)4 and streptavidin, in conjunction with 90Y/111In-DOTA-biotin (DOTA = dodecanetetraacetic acid) provides a new opportunity to improve efficacy by increasing the tumor-to-normal tissue dose ratio. To our knowledge, the patient-specific dosimetry of pretargeted 90Y/111In-DOTA-biotin after CC49 fusion protein in patients has not been reported previously. METHODS: Nine patients received 3-step pretargeted RIT: (a) 160 mg/m2 of CC49 fusion protein, (b) synthetic clearing agent (sCA) at 48 or 72 h later, and (c) 90Y/111In-DOTA-biotin 24 h after the sCA administration. Sequential whole-body 111In images were acquired immediately and at 2-144 h after injection of 90Y/111In-DOTA-biotin. Geometric-mean quantification with background and attenuation correction was used for liver and lung dosimetry. Effective point source quantification was used for spleen, kidneys, and tumors. Organ and tumor 90Y doses were calculated based on 111In imaging data and the MIRD formalism using patient-specific organ masses determined from CT images. Patient-specific marrow doses were determined based on radioactivity concentration in the blood. RESULTS: The 90Y/111In-DOTA-biotin had a rapid plasma clearance, which was biphasic with <10% residual at 8 h. Organ masses ranged from 1,263 to 3,855 g for liver, 95 to 1,009 g for spleen, and 309 to 578 g for kidneys. The patient-specific mean 90Y dose (cGy/37 MBq, or rad/mCi) was 0.53 (0.32-0.78) to whole body, 3.75 (0.63-6.89) to liver, 2.32 (0.58-4.46) to spleen, 7.02 (3.36-11.2) to kidneys, 0.30 (0.09-0.44) to lungs, 0.22 (0.12-0.34) to marrow, and 28.9 (4.18-121.6) to tumors. CONCLUSION: Radiation dose to normal organs from circulating radionuclide is substantially reduced using pretargeted RIT. Tumor-to-normal organ dose ratios were increased about 8- to 11-fold compared with reported patient-specific mean dose to liver, spleen, marrow, and tumors from 90Y-CC49.

A phase I study of anti-kinase insert domain-containing receptor antibody, IMC-1C11, in patients with liver metastases from colorectal carcinoma.

PURPOSE: Angiogenesis plays an important role in colorectal cancer progression. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in endothelial mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain-containing receptor (KDR) appear to be principally up-regulated during tumorigenesis. A chimeric anti-KDR antibody, IMC-1C11, blocks VEGFR-KDR interaction and inhibits VEGFR-induced endothelial cell proliferation. This trial seeks to assess the safety, tolerability and feasibility of targeting an important pathway in tumorigenesis. EXPERIMENTAL DESIGN: In a dose-escalation, single-agent study of IMC-1C11, we enrolled 14 patients with colorectal carcinoma and hepatic metastases. Safety-, pharmacokinetic-, immunogenicity-, and magnetic resonance imaging-assessed alteration of vascular effects of IMC-1C11 were evaluated in this trial. IMC-1C11 was infused weekly at 0.2 mg/kg (n = 3), 0.6 mg/kg (n = 4), 2.0 mg/kg (n = 3), and 4.0 mg/kg (n = 4) for 4 weeks, which constituted a cycle. RESULTS: No grade-3 or -4 IMC-1C11-related toxicities were observed. Minor grade-1 bleeding events were observed in four patients [0.2 mg/kg (n = 1) and 0.6 mg/kg (n = 3)]. Each resolved quickly and required no intervention. The starting dose of IMC-1C11 was selected to achieve a C(max) of approximately 5 micro g/ml. This concentration prevented KDR phosphorylation in vitro. Pharmacokinetic analysis demonstrated that the plasma t(1/2) and C(max) were dose dependent with a plasma t(1/2) of 67 +/- 3 h at the 4-mg/kg dose level. Human antichimeric antibodies were detected in 7 of 14 patients. The antibodies to IMC-1C11 inhibited the circulation of the agent in two patients. One patient had prolonged stable disease for seven cycles (28 weeks). The mean changes in tumor-influx volume-transfer constant k(in) (min(-1)) and enhancement factor after 4 weeks of therapy were significantly decreased compared with pretreatment values in 11 patients. CONCLUSION: IMC-1C11 was both safe and well tolerated. Drug levels of IMC-1C11 were reliably predicted. Further clinical investigation of anti-VEGFR/KDR agents is warranted.

Antitumor efficacy of TRA-8 anti-DR5 monoclonal antibody alone or in combination with chemotherapy and/or radiation therapy in a human breast cancer model.

PURPOSE: A monoclonal antibody (TRA-8) has been developed that binds to death receptor 5 (DR5), one of two death receptors bound by tumor necrosis factor-related apoptosis-inducing ligand. The purpose of this study was to evaluate in vitro the binding and cytotoxicity of TRA-8 to human breast cancer cell lines. The antitumor efficacy of TRA-8 was evaluated in a xenograft human breast cancer murine model, as a single agent and in combination with chemotherapy or radiation therapy. Anti: The binding of TRA-8 to a panel of nine human breast cancer cell lines was evaluated by indirect immunofluorescence and flow cytometry. Cytotoxicity of TRA-8 alone and in the presence of Adriamycin or paclitaxel was measured in vitro using the ATP-lite assay. Antitumor efficacy was determined by treatment of nude mice bearing well-established s.c. DR5-positive 2LMP human breast cancer xenografts with TRA-8 alone or in combination with Adriamycin or paclitaxel. Tumor size and regression rates were determined. In addition, a study was carried out with TRA-8 and Adriamycin in combination with 3 Gy (60)Co irradiation of 2LMP xenografts on days 9 and 17. RESULTS: All nine human breast cancer cell lines expressed DR5 with TRA-8 reactivity varying from strongly to weakly positive. Four cell lines were sensitive to TRA-8 cytotoxicity with IC(50) of 17-299 ng/ml, whereas other cell lines had weak cytotoxicity or were resistant. In vivo studies demonstrated significant inhibition of growth of 2LMP xenografts by TRA-8 treatment alone. The combination of TRA-8 + Adriamycin or paclitaxel produced significant inhibition of tumor growth as compared with controls or either agent alone. An aggregate analysis of all 166 animals studied demonstrated that TRA-8 alone or in combination with Adriamycin, paclitaxel, or radiation produced a significant increase in tumor doubling time compared with any modality alone with mean doubling time in days of 12 (untreated), 14 (radiation), 17 (Adriamycin), 25 (paclitaxel), 39 (Adriamycin + radiation), 47 (TRA-8), 65 (TRA-8 + radiation), 71 (TRA-8 + paclitaxel), 81 (TRA-8 + Adriamycin), and >140 (TRA-8 + Adriamycin and radiation). Complete tumor regressions occurred in 1 of 42 untreated animals, 1 of 54 animals receiving chemotherapy and/or radiation, and 28 of 68 animals receiving TRA-8 alone or TRA-8 combination regimens. Fourteen of those 28 complete regressions did not relapse over periods of follow-up between 99 and 171 days, with a mean of 146 +/- 24 days. CONCLUSIONS: The TRA-8 anti-DR5 antibody alone or in combination with chemotherapy and/or radiation has striking antitumor efficacy in breast cancer xenograft models. Additional studies with other tumor types and chemotherapy agents are warranted. These studies support the generation of a humanized TRA-8 for introduction into early clinical trials.