Assessing the utility of molecular diagnostic classification for cancers of unknown primary.

BACKGROUND: Roughly 5% of metastatic cancers present with uncertain origin, for which molecular classification could influence subsequent management; however, prior studies of molecular diagnostic classifiers have reported mixed results with regard to clinical impact. In this retrospective study, we evaluated the utility of a novel molecular diagnostic classifier by assessing theoretical changes in treatment and additional testing recommendations from oncologists before and after the review of classifier predictions. METHODS: We retrospectively analyzed de-identified records from 289 patients with a consensus diagnosis of cancer of uncertain/unknown primary (CUP). Two (or three, if adjudication was required) independent oncologists separately reviewed patient clinical information to determine the course of treatment before they reviewed results from the molecular diagnostic classifier and subsequently evaluated whether the predicted diagnosis would alter their treatment plan. RESULTS: Results from the molecular diagnostic classifier changed the consensus oncologist-reported treatment recommendations for 235 out of 289 patients (81.3%). At the level of individual oncologist reviews (n = 414), 64.7% (n = 268) of treatment recommendations were based on CUP guidelines prior to review of results from the molecular diagnostic classifier. After seeing classifier results, 98.1% (n = 207) of the reviews, where treatment was specified (n = 211), were guided by the tissue of origin-specific guidelines. Overall, 89.9% of the 414 total reviews either expressed strong agreement (n = 242) or agreement (n = 130) that the molecular diagnostic classifier result increased confidence in selecting the most appropriate treatment regimen. CONCLUSIONS: A retrospective review of CUP cases demonstrates that a novel molecular diagnostic classifier could affect treatment in the majority of patients, supporting its clinical utility. Further studies are needed to prospectively evaluate whether the use of molecular diagnostic classifiers improves clinical outcomes in CUP patients.

Patient-derived micro-organospheres enable clinical precision oncology.

Patient-derived xenografts (PDXs) and patient-derived organoids (PDOs) have been shown to model clinical response to cancer therapy. However, it remains challenging to use these models to guide timely clinical decisions for cancer patients. Here, we used droplet emulsion microfluidics with temperature control and dead-volume minimization to rapidly generate thousands of micro-organospheres (MOSs) from low-volume patient tissues, which serve as an ideal patient-derived model for clinical precision oncology. A clinical study of recently diagnosed metastatic colorectal cancer (CRC) patients using an MOS-based precision oncology pipeline reliably assessed tumor drug response within 14 days, a timeline suitable for guiding treatment decisions in the clinic. Furthermore, MOSs capture original stromal cells and allow T cell penetration, providing a clinical assay for testing immuno-oncology (IO) therapies such as PD-1 blockade, bispecific antibodies, and T cell therapies on patient tumors.

Cabozantinib with or without Panitumumab for RAS wild-type metastatic colorectal cancer: impact of MET amplification on clinical outcomes and circulating biomarkers.

PURPOSE: Acquired resistance to EGFR inhibitors in metastatic colorectal cancer (mCRC) remains a hurdle for effective treatment. MET amplification has been indicated as a driver of acquired resistance. Clinical activity has been demonstrated for the combination of EGFR and MET inhibitors in mCRC. But the impact of this regimen on angiogenesis and inflammation remains largely unknown. METHODS: In this non-randomized, open-label phase Ib/II study, four patients were treated with cabozantinib alone and 25 patients received the combination of cabozantinib and panitumumab. MET amplification was detected in blood in all four patients treated with cabozantinib monotherapy and 5/25 patients treated with cabozantinib and panitumumab combination therapy. Plasma samples from 28 patients were available for biomarker analysis. RESULTS: A panel of circulating protein biomarkers was assessed in patient plasma at baseline and on-treatment. Baseline marker levels were analyzed for prognostic value for clinical outcomes, including MET amplification as a covariate. HGF and OPN were prognostic for both progression-free survival (PFS) and overall survival (OS), while six markers (IL-6, VCAM-1, VEGF-R1, TSP-2, TIMP-1, ICAM-1) were prognostic only for OS. In patients with MET amplification, baseline PDGF-AA, PDGF-BB, TGF-ß1, and VEGF-C levels were significantly higher, whereas baseline TGFß-R3 levels were significantly lower than MET non-amplified patients. On-treatment change of four markers (CD73, PlGF, PDGF-BB, VEGF) were significantly different between MET amplified and non-amplified subpopulations. CONCLUSION: This study identified circulating HGF and several inflammatory and angiogenic proteins as prognostic biomarkers. Furthermore, MET amplification status is associated with both baseline expression and on-treatment modulation of members of angiogenesis and TGF-ß pathway proteins. CLINICAL TRIALS REGISTRATION NUMBER: ClinicalTrials.gov identifier: NCT02008383.

A case report of microsatellite instability (MSI)-high, HER2 amplified pancreatic adenocarcinoma with central nervous system metastasis.

Pancreatic adenocarcinoma commonly presents as metastatic disease and harbors a dire prognosis due to its aggressive behavior, propensity for resistance to therapies, and lack of targetable driver mutations. Additionally, despite advances in other cancers, immunotherapy has been ineffective in this disease thus far and treatment remains centered around cytotoxic chemotherapy. Here, we present a case of a patient with pancreatic adenocarcinoma harboring both high microsatellite instability (MSI-H) and HER2 amplification. After an initial response to standard-of-care chemotherapy with FOLFIRINOX followed by progression, she was treated with dual immune checkpoint blockade, which resulted in a period of disease control. This was complicated by the development of autoimmune hypophysitis and an incidental finding of brain metastasis on magnetic resonance imaging (MRI). Her extracranial disease progressed while receiving stereotactic radiosurgery, with findings of lymphangitic spread in her lungs, and her treatment was changed to gemcitabine/nab-paclitaxel with trastuzumab. This resulted in a degree of extracranial disease control, though she experienced progressive brain metastases despite radiation and therapeutic switch to lapatinib and trastuzumab. Ultimately, the patient developed leptomeningeal disease which was not controlled by intrathecal trastuzumab. Given the rarity of central nervous system metastasis, HER2 amplification, and MSI in pancreatic cancer, this patient's presentation represents a confluence of multiple unique features. This case highlights the clinical value of up-front next-generation sequencing in metastatic pancreatic cancer and the ability of pancreatic cancer with actionable molecular variants to develop atypical sites of disease and adaptive resistance.

Cabozantinib and Panitumumab for RAS Wild-Type Metastatic Colorectal Cancer.

LESSONS LEARNED: Antitumor activity was observed in the study population. Dose modifications of cabozantinib improve long-term tolerability. Biomarkers are needed to identify patient populations most likely to benefit. Further study of cabozantinib with or without panitumumab in patients with metastatic colorectal cancer is warranted. BACKGROUND: The epidermal growth factor receptor (EGFR) antibody panitumumab is active in patients with RAS wild-type (WT) metastatic colorectal cancer (mCRC), but nearly all patients experience resistance. MET amplification is a driver of panitumumab resistance. Cabozantinib is an inhibitor of multiple kinases, including vascular endothelial growth factor receptor 2 (VEGFR2) and c-MET, and may delay or reverse anti-EGFR resistance. METHODS: In this phase Ib clinical trial, we established the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of cabozantinib and panitumumab. We then treated an expansion cohort to further describe the tolerability and clinical activity of the RP2D. Eligibility included patients with KRAS WT mCRC (later amended to include only RAS WT mCRC) who had received prior treatment with a fluoropyrimidine, oxaliplatin, irinotecan, and bevacizumab. RESULTS: Twenty-five patients were enrolled and treated. The MTD/RP2D was cabozantinib 60 mg p.o. daily and panitumumab 6 mg/kg I.V. every 2 weeks. The objective response rate (ORR) was 16%. Median progression free survival (PFS) was 3.7 months (90% confidence interval [CI], 2.3-7.1). Median overall survival (OS) was 12.1 months (90% CI, 7.5-14.3). Five patients (20%) discontinued treatment due to toxicity, and 18 patients (72%) required a dose reduction of cabozantinib. CONCLUSION: The combination of cabozantinib and panitumumab has activity. Dose reductions of cabozantinib improve tolerability.

A phase I study of gemcitabine + dasatinib (gd) or gemcitabine + dasatinib + cetuximab (GDC) in refractory solid tumors.

PURPOSE: This study was conducted to define the maximum tolerated dose (MTD), recommended phase two dose (RPTD), and toxicities of gemcitabine + dasatinib (GD) and gemcitabine + dasatinib + cetuximab (GDC) in advanced solid tumor patients. METHODS: This study was a standard phase I 3 + 3 dose escalation study evaluating two combination regimens, GD and GDC. Patients with advanced solid tumors were enrolled in cohorts of 3-6 to either GD or GDC. Gemcitabine was dosed at 1000 mg/m2 weekly for 3 of 4 weeks, dasatinib was dosed in mg PO BID, and cetuximab was dosed at 250 mg/m2 weekly after a loading dose of cetuximab of 400 mg/m2. There were two dose levels for dasatinib: (1) gemcitabine + dasatinib 50 mg ± cetuximab, and (2) gemcitabine + dasatinib 70 mg ± cetuximab. Cycle length was 28 days. Standard cycle 1 dose-limiting toxicity (DLT) definitions were used. Eligible patients had advanced solid tumors, adequate organ and marrow function, and no co-morbidities that would increase the risk of toxicity. Serum, plasma, and skin biopsy biomarkers were obtained pre- and on-treatment. RESULTS: Twenty-five patients were enrolled, including 21 with pancreatic adenocarcinoma. Three patients received prior gemcitabine. Twenty-one patients were evaluable for toxicity and 16 for response. Four DLTs were observed: Grade (Gr) 3 neutropenia (GDC1, n = 1), Gr 3 ALT (GD2, n = 2), and Gr 5 pneumonitis (GDC2, n = 1). Possible treatment-emergent adverse events (TEAEs) in later cycles included: Gr 3-4 neutropenia (n = 7), Gr 4 colitis (n = 1), Gr 3 bilirubin (n = 2), Gr 3 anemia (n = 2), Gr 3 thrombocytopenia (n = 2), Gr 3 edema/fluid retention (n = 1), and Gr 3 vomiting (n = 3). Six of 16 patients (3 of whom were gemcitabine-refractory) had stable disease (SD) as best response, median duration = 5 months (range 1-7). One gemcitabine-refractory patient had a partial response (PR). Median PFS was 2.9 months (95% CI 2.1, 5.8). Median OS was 5.8 months (95% CI 4.1, 11.8). Dermal wound biopsies demonstrated that dasatinib resulted in a decrease of total and phospho-Src levels, and cetuximab resulted in a decrease of EGFR and ERBB2 levels. CONCLUSIONS: The MTD/RPTD of GD is gemcitabine 1000 mg/m2 weekly for 3 of 4 weeks and dasatinib 50 mg PO BID. The clinical activity of GD seen in this study was modest, and does not support its further investigation in pancreatic cancer.

Cell-Free DNA Profiling to Discover Mechanisms of Exceptional Response to Cabozantinib Plus Panitumumab in a Patient With Treatment Refractory Metastatic Colorectal Cancer.

MET amplification is rare in treatment-naïve metastatic colorectal cancer (CRC) tumors, but can emerge as a mechanism of resistance to anti-EGFR therapies. Preclinical and clinical data suggest that patients with MET amplified tumors benefit from MET-targeted therapy. Cabozantinib is an inhibitor of multiple tyrosine kinases, included c-MET. Panitumumab is an inhibitor of EGFR. This report describes a patient with KRAS, NRAS, and BRAF wild-type metastatic CRC who experienced disease progression on all standard chemotherapy and anti-EGFR antibody therapy. The patient was enrolled in a clinical trial evaluating the combination of cabozantinib plus panitumumab. After only 6 weeks of treatment, the patient experienced a significant anti-tumor response. Although tumor tissue was negative for MET amplification, molecular profiling of cell-free DNA (cfDNA) revealed MET amplification. This case represents the first report showing the activity of cabozantinib in combination with panitumumab in a patient with metastatic CRC, and suggests that MET amplification in cfDNA may be a biomarker of response. A clinical trial targeting MET amplified metastatic CRC is currently underway.

A Phase I Trial of the IGF-1R Antibody Ganitumab (AMG 479) in Combination with Everolimus (RAD001) and Panitumumab in Patients with Advanced Cancer.

PURPOSE: This study evaluated the maximum tolerated dose or recommended phase II dose (RPTD) and safety and tolerability of the ganitumab and everolimus doublet regimen followed by the ganitumab, everolimus, and panitumumab triplet regimen. MATERIALS AND METHODS: This was a standard 3 + 3 dose escalation trial. Doublet therapy consisted of ganitumab at 12 mg/kg every 2 weeks; doses of everolimus were adjusted according to dose-limiting toxicities (DLTs). Panitumumab at 4.8 mg/kg every 2 weeks was added to the RPTD of ganitumab and everolimus. DLTs were assessed in cycle 1; toxicity evaluation was closely monitored throughout treatment. Treatment continued until disease progression or undesirable toxicity. Pretreatment and on-treatment skin biopsies were collected to assess insulin-like growth factor 1 receptor and mammalian target of rapamycin (mTOR) target modulation. RESULTS: Forty-three subjects were enrolled. In the doublet regimen, two DLTs were observed in cohort 1, no DLTs in cohort -1, and one in cohort -1B. The triplet combination was discontinued because of unacceptable toxicity. Common adverse events were thrombocytopenia/neutropenia, skin rash, mucositis, fatigue, and hyperglycemia. In the doublet regimen, two patients with refractory non-small cell lung cancer (NSCLC) achieved prolonged complete responses ranging from 18 to >60 months; one treatment-naïve patient with chondrosarcoma achieved prolonged stable disease >24 months. In dermal granulation tissue, the insulin-like growth factor receptor and mTOR pathways were potently and specifically inhibited by ganitumab and everolimus, respectively. CONCLUSION: The triplet regimen of ganitumab, everolimus, and panitumumab was associated with unacceptable toxicity. However, the doublet of ganitumab at 12 mg/kg every 2 weeks and everolimus five times weekly had an acceptable safety profile and demonstrated notable clinical activity in patients with refractory NSCLC and sarcoma. IMPLICATIONS FOR PRACTICE: This trial evaluated the maximum tolerated dose or recommended phase II dose and safety and tolerability of the ganitumab and everolimus doublet regimen followed by the ganitumab, everolimus, and panitumumab triplet regimen. Although the triplet regimen of ganitumab, everolimus, and panitumumab was associated with unacceptable toxicity, the doublet of ganitumab at 12 mg/kg every 2 weeks and everolimus at five times weekly had an acceptable safety profile and demonstrated notable clinical activity in patients with refractory non-small cell lung cancer and sarcoma.

Blood-based markers of efficacy and resistance to cetuximab treatment in metastatic colorectal cancer: results from CALGB 80203 (Alliance).

Circulating protein markers were assessed in patients with colorectal cancer (CRC) treated with cetuximab in CALGB 80203 to identify prognostic and predictive biomarkers. Patients with locally advanced or metastatic CRC received FOLFOX or FOLFIRI chemotherapy (chemo) or chemo in combination with cetuximab. Baseline plasma samples from 152 patients were analyzed for six candidate markers [epidermal growth factor (EGF), heparin-binding EGF (HBEGF), epidermal growth factor receptor (EGFR), HER2, HER3, and CD73]. Analyte levels were associated with survival endpoints using univariate Cox proportional hazards models. Predictive markers were identified using a treatment-by-marker interaction term in the Cox model. Plasma levels of EGF, HBEGF, HER3, and CD73 were prognostic for overall survival (OS) across all patients (KRAS mutant and wild-type). High levels of EGF predicted for lack of OS benefit from cetuximab in KRAS wild-type (WT) patients (chemo HR = 0.98, 95% CI = 0.74-1.29; chemo+cetuximab HR = 1.54, 95% CI = 1.05-2.25; interaction P = 0.045) and benefit from cetuximab in KRAS mutant patients (chemo HR = 1.72, 95% CI = 1.02-2.92; chemo+cetuximab HR = 0.90, 95% CI = 0.67-1.21; interaction P = 0.026). Across all patients, higher HER3 levels were associated with significant OS benefit from cetuximab treatment (chemo HR = 4.82, 95% CI = 1.68-13.84; chemo+cetuximab HR = 0.95, 95% CI = 0.31-2.95; interaction P = 0.046). CD73 was also identified as predictive of OS benefit in KRAS WT patients (chemo HR = 1.28, 95% CI = 0.88-1.84; chemo+cetuximab HR = 0.60, 95% CI = 0.32-1.13; interaction P = 0.049). Although these results are preliminary, and confirmatory studies are necessary before clinical application, the data suggest that HER3 and CD73 may play important roles in the biological response to cetuximab.

Acute Pancreatitis Associated With Ado-Trastuzumab Emtansine.

Ado-trastuzumab emtansine (T-DM1) is a novel antibody-drug conjugate with current FDA recommendation for second-line treatment of HER-2-positive metastatic breast cancer. It is a human epidermal growth factor receptor (HER-2)-targeted antibody-drug conjugate composed of trastuzumab, a stable thioether linker, and the potent cytotoxic agent DM1 (derivative of maytansine). Ado-trastuzumab emtansine improved both progression-free and overall survival as reported in EMILIA trial. With ongoing clinical trials in adjuvant and first-line setting for HER-2-positive early and metastatic breast cancer, it is prudent to recognize, report, and treat any adverse events related to T-DM1. We report a case of acute pancreatitis in a 54-year-old woman with metastatic breast cancer after she received her first dose of ado-trastuzumab emtansine. To the best of our knowledge, this is the first reported case of acute pancreatitis with probable association with ado-trastuzumab emtansine.

Direct Evidence of Target Inhibition with Anti-VEGF, EGFR, and mTOR Therapies in a Clinical Model of Wound Healing.

PURPOSE: In early clinical testing, most novel targeted anticancer therapies have limited toxicities and limited efficacy, which complicates dose and schedule selection for these agents. Confirmation of target inhibition is critical for rational drug development; however, repeated tumor biopsies are often impractical and peripheral blood mononuclear cells and normal skin are often inadequate surrogates for tumor tissue. Based upon the similarities of tumor and wound stroma, we have developed a clinical dermal granulation tissue model to evaluate novel targeted therapies. EXPERIMENTAL DESIGN: A 4-mm skin punch biopsy was used to stimulate wound healing and a repeat 5-mm punch biopsy was used to harvest the resulting granulation tissue. This assay was performed at pretreatment and on-treatment evaluating four targeted therapies, bevacizumab, everolimus, erlotinib, and panitumumab, in the context of three different clinical trials. Total and phosphorylated levels VEGFR2, S6RP, and EGFR were evaluated using ELISA-based methodologies. RESULTS: Significant and consistent inhibition of the VEGF pathway (using VEGFR2 as the readout) was observed in granulation tissue biopsies from patients treated with bevacizumab and everolimus. In addition, significant and consistent inhibition of the mTOR pathway (using S6RP as the readout) was observed in patients treated with everolimus. Finally, significant inhibition of the EGFR pathway (using EGFR as the readout) was observed in patients treated with panitumumab, but this was not observed in patients treated with erlotinib. CONCLUSIONS: Molecular analyses of dermal granulation tissue can be used as a convenient and quantitative pharmacodynamic biomarker platform for multiple classes of targeted therapies.

A Phase I/biomarker study of bevacizumab in combination with CNTO 95 in patients with advanced solid tumors.

PURPOSE: Inhibition of tumor angiogenesis is an effective mechanism to limit tumor growth; dual inhibition may result in additional benefit. Bevacizumab is a monoclonal antibody directed against vascular endothelial growth factor (VEGF), and intetumumab is a fully humanized monoclonal antibody that blocks αv integrins when complexed with ß integrins. We evaluated the safety, tolerability, and efficacy of the combination of bevacizumab plus intetumumab in patients with refractory solid tumors. We also explored the effects of these agents on plasma-based biomarkers and wound angiogenesis. METHODS: Patients with refractory solid tumors, Karnofsky performance status ≥70%, and adequate organ function were eligible. Plasma samples and wound biopsies were obtained at baseline and on-treatment. RESULTS: Twelve patients were enrolled and received study drug. No tumor responses were noted. Observed toxicities included three cases of transient uveitis likely related to intetumumab and one case of reversible posterior leukoencephalopathy syndrome likely related to bevacizumab. Biomarker analysis revealed changes in soluble endoglin, soluble E-cadherin, and soluble E-selectin as well as PlGF and VEGF-D while on treatment. There was no observed impact of bevacizumab plus intetumumab on the phosphorylated or total levels of paxillin in wound tissue; however, an increase in the ratio of phospho/total paxillin levels was noted. CONCLUSIONS: Bevacizumab and intetumumab can be administered safely in combination. Bevacizumab plus intetumumab treatment resulted in changes in the plasma levels of several extracellular matrix interacting proteins and angiogenic factors.

Dasatinib (BMS-35482) interacts synergistically with docetaxel, gemcitabine, topotecan, and doxorubicin in ovarian cancer cells with high SRC pathway activation and protein expression.

PURPOSE: This study aimed to explore the activity of dasatinib in combination with docetaxel, gemcitabine, topotecan, and doxorubicin in ovarian cancer cells. METHODS: Cells with previously determined SRC pathway and protein expression (SRC pathway/SRC protein IGROV1, both high; SKOV3, both low) were treated with dasatinib in combination with the cytotoxic agents. SRC and paxillin protein expression were determined pretreatment and posttreatment. Dose-response curves were constructed, and the combination index (CI) for drug interaction was calculated. RESULTS: In the IGROV1 cells, dasatinib alone reduced phospho-SRC/total SRC 71% and p-paxillin/t-paxillin ratios 77%. Phospho-SRC (3%-33%; P = 0.002 to 0.04) and p-paxicillin (6%-19%; P = 0.01 to 0.05) levels were significantly reduced with dasatinib in combination with each cytotoxic agent. The combination of dasatinib and docetaxel, gemcitabine, or topotecan had a synergistic antiproliferative effect (CI, 0.49-0.68), whereas dasatinib combined with doxorubicin had an additive effect (CI, 1.08).In SKOV3 cells, dasatinib resulted in less pronounced reductions of phospho-SRC/total SRC (49%) and p-paxillin/t-paxillin (62%). Phospho-SRC (18%; P < 0.001) and p-paxillin levels (18%; P = 0.001; 9%; P = 0.007) were significantly decreased when dasatinib was combined with docetaxel and topotecan (p-paxillin only). Furthermore, dasatinib combined with the cytotoxics in the SKOV3 cells produced an antagonistic interaction on the proliferation of these cells (CI, 1.49-2.27). CONCLUSIONS: Dasatinib in combination with relapse chemotherapeutic agents seems to interact in a synergistic or additive manner in cells with high SRC pathway activation and protein expression. Further evaluation of dasatinib in combination with chemotherapy in ovarian cancer animal models and exploration of the use of biomarkers to direct therapy are warranted.

Dasatinib (BMS-35482) potentiates the activity of gemcitabine and docetaxel in uterine leiomyosarcoma cell lines.

BACKGROUND: To explore the activity of dasatinib alone and in combination with gemcitabine and docetaxel in uterine leiomyosarcoma (uLMS) cell lines, and determine if dasatinib inhibits the SRC pathway. METHODS: SK-UT-1 and SK-UT-1B uLMS cells were treated with gemcitabine, docetaxel and dasatinib individually and in combination. SRC and paxcillin protein expression were determined pre- and post-dasatinib treatment using Meso Scale Discovery (MSD) multi-array immunogenicity assay. Dose-response curves were constructed and the coefficient of drug interaction (CDI) and combination index (CI) for drug interaction calculated. RESULTS: Activated phosphorylated levels of SRC and paxillin were decreased after treatment with dasatinib in both cell lines (p < 0.001). The addition of a minimally active concentration of dasatinib (IC25) decreased the IC50 of each cytotoxic agent by 2-4 fold. The combination of gemcitabine-docetaxel yielded a synergistic effect in SK-UT-1 (CI = 0.59) and an antagonistic effect in SK-UT-1B (CI = 1.36). Dasatinib combined with gemcitabine or docetaxel revealed a synergistic anti-tumor effect (CDI < 1) in both cell lines. The triple drug combination and sequencing revealed conflicting results with a synergistic effect in SK-UT-1B and antagonistic in SK-UT-1. CONCLUSION: Dasatinib inhibits the SRC pathway and yields a synergistic effect with the two-drug combination with either gemcitabine or docetaxel. The value of adding dasatinib to gemcitabine and docetaxel in a triple drug combination is uncertain, but may be beneficial in select uLMS cell lines. Based on our pre-clinical data and known activity of gemcitabine and docetaxel, further evaluation of dasatinib in combination with these agents for the treatment of uLMS is warranted.

Dual inhibition of αV integrins and Src kinase activity as a combination therapy strategy for colorectal cancer.

Both Src and αV integrins are important for tumor growth and angiogenesis. They are interconnected and responsible for important features of the tumor phenotype including invasiveness, metastasis, angiogenesis, and resistance to apoptosis. This study examines whether combinational inhibition of both integrin and Src pathways would exert greater antiangiogenesis and antitumor effects than either pathway alone. Using in-vitro cell culture systems, the activity of CNTO95 (Intetumumab), an αV integrin inhibitor, and dasatinib, an Src inhibitor, on proliferation, adhesion, and migration was evaluated in colon cancer cell lines, HCT-116 and RKO, as well as HUVEC cells. The antiangiogenic effect of this combinatory regimen was also tested using an in-vitro tubular network formation assay. The effects of CNTO95 and dasatinib on the activation of Src and integrin pathway signal transduction were also determined by western blotting. The combination of CNTO95 plus dasatinib inhibited adhesion, migration, and paxillin phosphorylation in both HCT-116 and RKO cells. CNTO95 and dasatinib also led to increased apoptosis of HCT-116 cells; however, similar effects were not observed in RKO cells. In addition, dual treatment of CNTO95 and dasatinib exerted enhanced effects on HUVEC cell proliferation, invasion, tubular network formation, and paxillin phosphorylation. In conclusion, our results suggest that concurrent inhibition of both the integrin and the Src pathways exert more pronounced antiangiogenic and antitumor effects than with either pathway being inhibited alone.

Phase I study of bevacizumab, everolimus, and panobinostat (LBH-589) in advanced solid tumors.

PURPOSE: To define the maximum tolerated dose, clinical toxicities, and pharmacodynamics of bevacizumab, everolimus, and panobinostat (LBH-589) when administered in combination to patients with advanced solid tumor malignancies. EXPERIMENT DESIGN: Subjects received 10 mg of panobinostat three times weekly, 5 or 10 mg everolimus daily, and bevacizumab at 10 mg/kg every 2 weeks. Dose-limiting toxicities (DLTs) were assessed in cycle 1; toxicity evaluation was closely monitored throughout treatment. Treatment continued until disease progression or undesirable toxicity. Protein acetylation was assessed in peripheral blood mononuclear cells (PBMC) both at baseline and on treatment. RESULTS: Twelve subjects were evaluable for toxicity and nine subjects for response. DLTs in cohort 1 included grade 2 esophagitis and grade 3 oral mucositis; DLTs in cohort -1 were grade 2 ventricular arrhythmia and grade 2 intolerable skin rash. Common adverse events were diarrhea (50 %), headache (33 %), mucositis/stomatitis (25 %), hyperlipidemia (25 %), and thrombocytopenia (25 %). There was 1 partial response; an additional 2 subjects had stable disease as best response. No consistent changes in protein acetylation in PBMC were observed in samples available from eight patients on treatment compared with baseline. CONCLUSIONS: Bevacizumab, everolimus, and panobinostat in combination at the lowest proposed doses did not have an acceptable safety and tolerability profile and did not consistently inhibit HDAC activity; therefore, we do not recommend further evaluation.

E proteins are required to activate germline transcription of the TCR Vbeta8.2 gene.

Each TCR Vbeta gene is regulated by an individual Vbeta promoter, which becomes active prior to V(D) J recombination and drives germline transcription. It has been shown that Vbeta gene locus activation and recombination are dependent on the Vbeta promoter. However, transcription factors that regulate Vbeta germline transcription remain largely undefined. A major challenge in studying Vbeta gene germline transcription is the quantitative assessment of relatively low-level transcripts in T-cell progenitors. Here we used the established Vbeta8.2(CD2) knock-in mouse model to assess functions of E-protein transcription factors in Vbeta8.2 germline transcription. We show that E proteins are required for the activation but not the maintenance of the Vbeta8.2 germline transcription during thymocyte development. The activation of Vbeta8.2 germline transcription depends more on the E proteins encoded by the E2A gene than by the HEB gene. We further show that IL-7 receptor (IL-7R)-mediated signals are essential for Vbeta8.2 germline transcription. We provide evidence that IL-7R expression is only partially controlled by E2A, suggesting a role for E2A in driving Vbeta8.2 germline transcription independent of IL-7R activation.

Germline transcription from T-cell receptor Vbeta gene is uncoupled from allelic exclusion.

Allelic exclusion operates in B and T lymphocytes to ensure clonal expression of antigen receptors after V(D)J recombination. Germline transcription, which proceeds V(D)J recombination, has been postulated to provide an instructive signal for allelic exclusion. Here, we use a genetic marker to track germline transcription from a Vbeta gene within the TCRbeta locus. We find that developing thymocytes exhibit uniformed, bi-allelic activation of the Vbeta gene before V-DJ recombination, a process subject to allelic exclusion. We further show that V-DJ rearrangement promotes activation rather than silencing of germline transcription from the remaining Vbeta genes on either the functionally or non-functionally rearranged chromosome. Results presented here suggest that germline transcription, although necessary for V(D)J recombination, is not sufficient to instruct allelic exclusion.