Goetz Kloecker

Dectin-1 Activation by a Natural Product β-Glucan Converts Immunosuppressive Macrophages into an M1-like Phenotype.

Tumor-associated macrophages (TAM) with an alternatively activated phenotype have been linked to tumor-elicited inflammation, immunosuppression, and resistance to chemotherapies in cancer, thus representing an attractive target for an effective cancer immunotherapy. In this study, we demonstrate that particulate yeast-derived β-glucan, a natural polysaccharide compound, converts polarized alternatively activated macrophages or immunosuppressive TAM into a classically activated phenotype with potent immunostimulating activity. This process is associated with macrophage metabolic reprograming with enhanced glycolysis, Krebs cycle, and glutamine utilization. In addition, particulate β-glucan converts immunosuppressive TAM via the C-type lectin receptor dectin-1–induced spleen tyrosine kinase–Card9–Erk pathway. Further in vivo studies show that oral particulate β-glucan treatment significantly delays tumor growth, which is associated with in vivo TAM phenotype conversion and enhanced effector T cell activation. Mice injected with particulate β-glucan–treated TAM mixed with tumor cells have significantly reduced tumor burden with less blood vascular vessels compared with those with TAM plus tumor cell injection. In addition, macrophage depletion significantly reduced the therapeutic efficacy of particulate β-glucan in tumor-bearing mice. These findings have established a new paradigm for macrophage polarization and immunosuppressive TAM conversion and shed light on the action mode of β-glucan treatment in cancer.

Yeast-Derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-Derived Suppressor Cells (MDSC) by Inducing Polymorphonuclear MDSC Apoptosis and Monocytic MDSC Differentiation to APC in Cancer

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that promote tumor progression. In this study, we demonstrated that activation of a C-type lectin receptor, dectin-1, in MDSC differentially modulates the function of different MDSC subsets. Yeast-derived whole β-glucan particles (WGP; a ligand to engage and activate dectin-1, oral treatment in vivo) significantly decreased tumor weight and splenomegaly in tumor-bearing mice with reduced accumulation of polymorphonuclear MDSC but not monocytic MDSC (M-MDSC), and decreased polymorphonuclear MDSC suppression in vitro through the induction of respiratory burst and apoptosis. On a different axis, WGP-treated M-MDSC differentiated into F4/80+CD11c+ cells in vitro that served as potent APC to induce Ag-specific CD4+ and CD8+ T cell responses in a dectin-1–dependent manner. Additionally, Erk1/2 phosphorylation was required for the acquisition of APC properties in M-MDSC. Moreover, WGP-treated M-MDSC differentiated into CD11c+ cells in vivo with high MHC class II expression and induced decreased tumor burden when inoculated s.c. with Lewis lung carcinoma cells. This effect was dependent on the dectin-1 receptor. Strikingly, patients with non–small cell lung carcinoma that had received WGP treatment for 10–14 d prior to any other treatment had a decreased frequency of CD14−HLA-DR−CD11b+CD33+ MDSC in the peripheral blood. Overall, these data indicate that WGP may be a potent immune modulator of MDSC suppressive function and differentiation in cancer.

Stereotactic Body Radiation Therapy as Salvage for Intrathoracic Recurrence in Patients With Previously Irradiated Locally Advanced Non-Small Cell Lung Cancer.

Introduction:The purpose of this study is to provide data on the outcomes of using stereotactic body radiotherapy (SBRT) as a means of salvage for non–small cell lung cancer (NSCLC) relapses previously treated with radiation. Materials and Methods:The records of 128 consecutive patients treated with thoracic SBRT from 2009 through 2012 were retrospectively reviewed. Twenty-seven patients (29 lesions) treated with prior thoracic radiation for stage IIB-IIIB NSCLC with subsequent recurrences and retreated with SBRT were identified. Results:The median prior radiation dose was 64.8 Gy (range, 45 to 74 Gy) with a median retreatment dose of 50 Gy (range, 30 to 54 Gy), corresponding to a biological equivalent dose of 100 Gy (range, 48 to 151 Gy), at a median time of 13.4 months from prior radiation. The mean follow-up after salvage SBRT was 22 months. Local failure following salvage was 11%, nodal failure was 37%, and distant failure was 30%. The local recurrence-free survival at 2 years was 72%. Out-of-field failure was predictive for worse local control (hazard ratio, 47.38; 95% confidence interval, 5.795-64.899). Progression-free survival at 1 year was 55% and 38% at 2 years. Overall survival at 2 years from SBRT salvage was 79%. Salvage biological equivalent dose ≥100 Gy was predictive of improved progression-free survival (48% vs. 18%, P=0.021) and overall survival (91% vs. 52%, P=0.004) at 2 years. The rate of symptomatic pneumonitis was 63% and chest wall pain reported was 26%. Conclusions:We observed improved outcomes following SBRT as a means of salvage for locally advanced recurrent NSCLC over traditional radiation therapy options. The toxicities were greater than expected from naive lung irradiation, but the adverse effects remained controlled with medications.

Targeting of Antigens to B Lymphocytes via CD19 as a Means for Tumor Vaccine Development

Ab therapy against surface Ags on tumor cells has demonstrated significant efficacy for some cancers. However, it is costly and patients frequently develop acquired resistance over time. In cases of Ab therapy resistance, T cell responses have been shown to be essential in controlling disease progression. Thus, vaccination that generates a sustained Ab response as well as a T cell response may be more effective and economical. In this article, we have developed a vaccination strategy by targeting protein Ags to B cells via a CD19 single-chain variable fragment miniAb. Using the tumor-associated Ag her-2/neu extracellular domain, we showed that the coengagement of CD19 and BCR induced full B cell activation to produce a high titer of Abs and enhanced CD4 Th2 response and CD8 T cell activation and differentiation. These Abs competitively inhibited humanized her-2/neu Ab binding and were capable of activating the complement and inhibiting human breast cancer growth in vitro. Therapeutic efficacy was demonstrated in vivo using murine mammary carcinoma models. Furthermore, four different extracellular domains of her-2/neu could be targeted to B cells to generate Abs against particular domains with different antitumor properties. This approach may offer a new avenue for vaccine development with significantly lower cost, which may be of use not only for cancer therapy but also for infectious agents.

Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays

We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the liquid biopsy was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ∼100% sensitivity, ∼91% specificity and ∼96% accuracy. In the blinded test, the signals were classified with ∼91% sensitivity, ∼82% specificity and ∼86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ∼1-17 cells per 5 μl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.

Static micro-array isolation, dynamic time series classification, capture and enumeration of spiked breast cancer cells in blood: the nanotube–CTC chip

We demonstrate the rapid and label-free capture of breast cancer cells spiked in blood using nanotube-antibody micro-arrays. 76-element single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (anti-EpCAM), Anti-human epithelial growth factor receptor 2 (anti-Her2) and non-specific IgG antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester. Following device functionalization, blood spiked with SKBR3, MCF7 and MCF10A cells (100/1000 cells per 5 μl per device, 170 elements totaling 0.85 ml of whole blood) were adsorbed on to the nanotube device arrays. Electrical signatures were recorded from each device to screen the samples for differences in interaction (specific or non-specific) between samples and devices. A zone classification scheme enabled the classification of all 170 elements in a single map. A kernel-based statistical classifier for the liquid biopsy was developed to create a predictive model based on dynamic time warping series to classify device electrical signals that corresponded to plain blood (control) or SKBR3 spiked blood (case) on anti-Her2 functionalized devices with ∼90% sensitivity, and 90% specificity in capture of 1000 SKBR3 breast cancer cells in blood using anti-Her2 functionalized devices. Screened devices that gave positive electrical signatures were confirmed using optical/confocal microscopy to hold spiked cancer cells. Confocal microscopic analysis of devices that were classified to hold spiked blood based on their electrical signatures confirmed the presence of cancer cells through staining for DAPI (nuclei), cytokeratin (cancer cells) and CD45 (hematologic cells) with single cell sensitivity. We report 55%-100% cancer cell capture yield depending on the active device area for blood adsorption with mean of 62% (∼12 500 captured off 20 000 spiked cells in 0.1 ml blood) in this first nanotube-CTC chip study.

The role of alectinib in the treatment of advanced ALK-rearranged non-small-cell lung cancer

ABSTRACT Introduction: The identification of anaplastic lymphoma kinase (ALK) gene rearrangements in subsets of non-small cell lung cancer patients has provided with unparalleled opportunities to hinder the progression of this disease through targeting the activity of these specific molecules. Unfortunately most patients develop disease progression in less than a year of treatment with crizotinib, the first-generation ALK-inhibitor. Areas covered: We review the resistance mechanisms to ALK inhibitors as well as an overview of the clinical activity of the alectinib, a second generation ALK inhibitor. Expert commentary: Second generation ALK inhibitors as alectinib and ceritinib can overcome crizotinib-resistant mutations and improve central nervous system control. Novel third-generation inhibitors and combination of agents give hope of achieving an even longer disease control in the next decade.

Phase II Trial on Extending the Maintenance Flushing Interval of Implanted Ports

PURPOSEnRetrospective studies suggest that it may be safe to extend the maintenance flushing interval of implanted ports from once every month, as recommended by the manufacturer, to once every 3 months, but no prospective cohort studies have been done specifically assessing the safety and feasibility of this intervention.nnnMETHODSnThis was a phase II study in oncologic patients who retained a functional port after completion of systemic chemotherapy. Patients enrolled in the study had their port flushed once every 3 months and were observed until completion of five scheduled flushes (one on enrollment and four additional flushes, one every 3 months) or development of any port-related complication, including infections, thrombosis, and occlusions. The primary end points were frequency of port-related complications and port failure requiring removal.nnnRESULTSnA total of 87 patients were enrolled in the study. The median follow-up time was 308 days, accounting for a total of 24,202 catheter-days. There were 10 port-related complications (11.49%; 95% CI, 4.85% to 18.14%). No infection or symptomatic thrombosis occurred. The mean time to port-related complication was 184 days. No patients developed port failure while on protocol, but on subsequent medical record review, four patients developed a complication that required port removal or port revision within 30 days of being removed from the trial (4.6%; 95% CI, 0.4% to 8.8%; 0.17/1,000 catheter-days).nnnCONCLUSIONnExtending the maintenance flushes of implanted ports in adult oncologic patients to once every 3 months is safe, effective, and likely to increase patient adherence and satisfaction while decreasing the associated cost.

Treatment and outcomes of non-small-cell lung cancer patients with high comorbidity

Background The life expectancy of untreated non-small-cell lung cancer (NSCLC) is dismal, while treatment for NSCLC improves survival. The presence of comorbidities is thought to play a significant role in the decision to treat or not treat a given patient. We aim to evaluate the association of comorbidities with the survival of patients treated for NSCLC. Methods We performed a retrospective study of patients aged ≥66 years with invasive NSCLC between the years 2007 and 2011 in the Surveillance, Epidemiology, and End Results Kentucky Cancer Registry. Comorbidity was measured using the Klabunde Comorbidity Index (KCI), and univariate and multivariate logistic regression models were used to measure association between receiving treatment and comorbidity. Kaplan–Meier plots were constructed to estimate time-to-event outcomes. Results A total of 4014 patients were identified; of this, 94.9% were white and 55.7% were male. The proportion of patients who did not receive any treatment was 8.7%, 3.9%, 19.1%, and 23.5% for stages I, II, III, and IV, respectively (p<0.0001). In multivariate analysis, older age, higher stage, and higher comorbidity (KCI ≥3) were associated with a lower likelihood of receiving any treatment. The median overall survival (OS) for untreated and KCI=0 was 17.7 months for stages I and II, 2.3 months for stage III, and 1.3 months for stage IV. The median OS for treated and KCI=0 was 58.9 months for stages I and II, 16.8 months for stage III, and 5.8 months for stage IV (p<0.01). Treatment was an independent predictor of OS in multivariate analysis that included KCI scores. Conclusion Our data suggest that lung cancer patients may derive a survival benefit from therapies, regardless of the presence of comorbidities, although the degree of benefit seems to decrease with higher KCI scores.

A phase 2, open-label, multi-center study of amuvatinib in combination with platinum etoposide chemotherapy in platinum-refractory small cell lung cancer patients

Background Amuvatinib (MP-470) is a multi-targeted kinase inhibitor with potent activity against c-Kit, synergistic with DNA-damaging agents. We evaluated amuvatinib in combination with platinum-etoposide (EP) chemotherapy by objective response rate, survival, and tolerability in platinum-refractory small cell lung cancer (SCLC) patients. Methods This study used a Simon 2-stage design requiring ≥3 centrally confirmed responses in the first 21 subjects. Subjects received EP with 300 mg amuvatinib orally three times daily in cycles of 21 days. A three-day amuvatinib run-in period before EP occurred in Cycle 1. Subjects received the same EP chemotherapy regimen given prior to progression/relapse. Results Among 23 subjects treated, we observed four PRs (17.4%) per RECIST 1.1, only two of which were centrally confirmed (8.7%, response duration 119, 151 days). Three subjects (13%) had confirmed stable disease. c-Kit H-score was ≥100 in two subjects whose respective durations of disease control were 151 and 256 days. Conclusions The addition of amuvatinib to EP chemotherapy in unselected, platinum-refractory SCLC did not meet the primary endpoint of ≥3 confirmed responses in stage 1. However, high c-Kit expression in two subjects with durable disease control suggests the potential for further study of amuvatinib in SCLC patients with high c-Kit expression.