Evaluating the Cellular Targets of Anti-4-1BB Agonist Antibody during Immunotherapy of a Pre-Established Tumor in Mice

Manipulation of the immune system represents a promising avenue for cancer therapy. Rational advances in immunotherapy of cancer will require an understanding of the precise correlates of protection. Agonistic antibodies against the tumor necrosis factor receptor family member 4-1BB are emerging as a promising tool in cancer therapy, with evidence that these antibodies expand both T cells as well as innate immune cells. Depletion studies have suggested that several cell types can play a role in these immunotherapeutic regimens, but do not reveal which cells must directly receive the 4-1BB signals for effective therapy.We show that re-activated memory T cells are superior to resting memory T cells in control of an 8-day pre-established E.G7 tumor in mice. We find that ex vivo activation of the memory T cells allows the activated effectors to continue to divide and enter the tumor, regardless of antigen-specificity; however, only antigen-specific reactivated memory T cells show any efficacy in tumor control. When agonistic anti-4-1BB antibody is combined with this optimized adoptive T cell therapy, 80% of mice survive and are fully protected from tumor rechallenge. Using 4-1BB-deficient mice and mixed bone marrow chimeras, we find that it is sufficient to have 4-1BB only on the endogenous host alphabeta T cells or only on the transferred T cells for the effects of anti-4-1BB to be realized. Conversely, although multiple immune cell types express 4-1BB and both T cells and APC expand during anti-4-1BB therapy, 4-1BB on cells other than alphabeta T cells is neither necessary nor sufficient for the effect of anti-4-1BB in this adoptive immunotherapy model.This study establishes alphabeta T cells rather than innate immune cells as the critical target in anti-4-1BB therapy of a pre-established tumor. The study also demonstrates that ex vivo activation of memory T cells prior to infusion allows antigen-specific tumor control without the need for reactivation of the memory T cells in the tumor

An Interim Report Of CALGB 150607: Expression And Mutational Status Of c-MET, HGF, EGFR, KRAS, p53, c-CBL, And E-cadherin In Resected Lung Adenocarcinoma Specimens

Plenary Session: no. 190PURPOSE/OBJECTIVE(S): c-Met is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor plays a critical role in cancer growth, invasion, and metastasis. The main objective of this study is to evaluate the correla¬tion between c-MET mutation and expression with stage and overall survival in a large group of adenocarcinoma (AC) patients. The secondary aims are to determine the correlation between overall survival and the analysis of: 1) EGFR mutation & expression, 2) TP53 mutation & expression, 3)epithelial-to-mesenchymal transition, 4) KRAS mutation, and 5) CBL mutation, and to evaluate gene amplification of c-MET in AC patients and the sera levels of circulating c-Met and HGF. MATERIALS/METHODS: We evaluated 80 adenocarcinoma patients. Standard PCR and sequencing techniques for mutational analysis of MET, EGFR exons 18-21, TP53 exons 4-10, KRAS exon 2, and CBL exons 2-16. ELISA was used to quantify soluble c-Met and HGF in pre- and post-operative sera. c-MET, phosphorylated (pMET Y1003 and Y1230/34/35), p53, HGF, EGFR, and E-cadherin expression were evaluated by IHC. Staining inten¬sity was scored on four-point scale: 0, negative; 1+, weak; 2+, moderate; 3+, strong. The extent of staining was scored similarly: 0, negative; 1+, 1-10%; 2+, 11-50%; 3+, > 50%. The product of the intensity and extent of staining yielded final scores between 0 and 9. RESULTS: In 9 patients, six non-synonymous (NS) mutations were detected in MET (SEMA domain: E168D, M362T, N375S, and Q318K; JM domain: T992I and R970C). In EGFR, the NS mutation L858R was detected in two patients. We detected 11 NS mutations in TP53 (exon 4: E68*; exon 5: V157F, R175H, I162F, H193Y, Y163D; exon 8: R273L, R273C, V274L, A276F, and G266*). Five NS mutations were detected in exon 2 of KRAS (G12C, G12V, G12D, G12S and G13V). For both c-MET and HGF, there are 65 pairs pre- and post-operative sera with no missing values. There was a significant (p=0.008) increase of soluble c-MET in post- (1750 ng/ml ± 60.2) compared to pre-operative (1584 ng/ml ± 59.2) serum samples. HGF levels were significantly increased (p=0.028) in post- (1006 pg/ml ± 70.6) com¬pared to pre-operative samples (847 pg/ml ± 56.0). The mean expressions as determined by IHC: c-MET 3.9 (±0.3); pY1230/34/35 MET 2.3 (±0.3); pY1003MET 4.8 (±0.3); HGF 4.4 (±0.3); EGFR 4.3 (±0.4); TP53 3.8 (±0.3); and E-cadherin 5.4 (±0.4) CONCLUSION: Unique MET mutations were detected in key functional domains; the SEMA domain and the JM domain. Post-operative patient sera levels were increased dramatically both in HGF and soluble MET. MET, pMET (Y1003), EGFR, E-Cadherin, HGF and P53 were highly expressed. The expression and mutation data will be correlated with clinical outcomes to identify the prognostic role of c-Met

Characterizing cognitive control abilities in children with 16p11.2 deletion using adaptive 'video game' technology: a pilot study

Assessing cognitive abilities in children is challenging for two primary reasons: lack of testing engagement can lead to low testing sensitivity and inherent performance variability. Here we sought to explore whether an engaging, adaptive digital cognitive platform built to look and feel like a video game would reliably measure attention-based abilities in children with and without neurodevelopmental disabilities related to a known genetic condition, 16p11.2 deletion. We assessed 20 children with 16p11.2 deletion, a genetic variation implicated in attention deficit/hyperactivity disorder and autism, as well as 16 siblings without the deletion and 75 neurotypical age-matched children. Deletion carriers showed significantly slower response times and greater response variability when compared with all non-carriers; by comparison, traditional non-adaptive selective attention assessments were unable to discriminate group differences. This phenotypic characterization highlights the potential power of administering tools that integrate adaptive psychophysical mechanics into video-game-style mechanics to achieve robust, reliable measurements

Observer interpretation variability of peripapillary flow using the Heidelberg Retina Flowmeter.

To evaluate the intraobserver and interobserver reproducibility of the automatic full field perfusion image analysis (AFFPIA) program on Heidelberg Retina Flowmeter (HRF) derived perfusion images in a multicentre study group.A total of 10 subjects were consecutively recruited in the study. One eye was randomly selected for each patient. Blood flow was assessed by HRF and flow measurements were analyzed by using the AFFPIA program. AFFPIA calculates the Doppler frequency shift and the haemodynamic variables: flow for each pixel. Intraobserver and interobserver reproducibility was calculated for AFFPIA program. The retinal blood flow was calculated in the superior and inferior section, furthermore, each section was divided into three parts: the temporal area, the nasal, and the rim area, as for software, but only the temporal and nasal areas were considered in this study. The blood flow and the area considered were evaluated for each part.When the intraobserver and intraimage reproducibility was studied, the coefficient of variation ranged from 0.4 to 1.9\%. When the interobserver and intraimage reproducibility was studied, the retinal blood flow coefficient of variation ranged from 0.52 to 3.30\% for the supero-temporal area, from 0.13 to 2.67\% for the inferotemporal area, from 0.15 to 2.75\% for the supero-nasal area, and from 0.04 to 5.65\% for the infero-nasal area.Our results with AFFPIA showed an interobserver coefficient of variation of retinal blood flow measurements always less than 6\% in both temporal and nasal areas. No significant difference was found among the four observers for the flow measurements