Abstract 4916: Development of a NGS-based method for EGFRvIII detection: sequence analysis of the junction

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary tumor in humans. One of the most common mutations in GBMs is an interstitial deletion in the epidermal growth factor receptor (EGFR), EGFRvIII, which occurs at a frequency of ∼30%. EGFR is a transmembrane tyrosine kinase receptor and the EGFRvIII mutant is characterized by a deletion of 267 amino acids in the extracellular domain leading to ligand independent constitutive activation. The deletion of exons 2-7 leads to an in-frame deletion in EGFR with a novel glycine residue at the junction. The amino acid at the junction of exons 1 and 2 is a valine, making the novel transcript an attractive target for immunotherapy. A custom next generation sequencing (NGS) based assay and bioinformatic pipeline have been developed in our laboratory to detect EGFRvIII from RNA extracted from formalin fixed paraffin embedded tissue. The targets include the exon 1-2 boundary (wild type), the exon 1-8 boundary (EGFRvIII), amplification of various sized RNA fragments to determine RNA degradation and bioavailability, and expression levels of three housekeeping genes. Following cDNA synthesis multiplex PCR of all targets are captured simultaneously for the sequencing library with NGS performed on the Illumina MiSeq. The output from the bioinformatics pipeline includes the sequence and number of reads from the wild-type and mutant, ratio of EGFRvIII reads with respect to total EGFR sequenced, expression of three housekeeping genes and relative amount of bioavailable EGFR RNA. This assay was validated through comparison of NGS sequence results with an established qRT-PCR to detect normal and mutant EGFR. Negative controls from normal brain (temporal lobe excisions from epilepsy patients) and adipose tissue (a tissue with high expression of EGFR) were used to determine whether low-level exon 1-8 fusions from mis-splicing were detectable in normal tissue (Figure 1). Twenty five GBM specimens were sequenced, with 8/25 positive for EGFRvIII (Figure 2), and confirmed by RT-PCR. In addition to detection of the EGFRvIII mutant, relative expression of EGFR is detected in this assay, and when taken together with EGFR amplifications detected by routine NGS panels, we can determine whether the EGFRvIII is present on the amplified or unamplified allele and whether additional mutations are detectable. Detection of EGFRvIII utilizing NGS improves the precision of mutant detection to better serve CART-EGFRvIII clinical trial to ensure the target is present. The NGS assay provides the EGFRvIII/wild-type ratio, relative expression levels for EGFR and EGFRvIII and evaluation of RNA degradation in a single assay. Figure 1A. Baseline in normal samples. EGFRvIII ratio in 18 “normal” brain and 11 adipose tissue samples, plotted without (top panel) and with (bottom panel) a EGFRvIII positive sample. Figure 2. EGFRvIII ratio in 25 GBM samples. Cutoff for EGFRvIII positive is EGFRvIII ratio of 0.3 (30%). Citation Format: Jianhua Zhao, Shrey Sukhadia, Alan Fox, David Lieberman, Barnett Li, Robert D. Daber, Matthew C. Hiemenz, David B. Roth, Maria Martinez-Large, Arati Desai, Donald M. O'Rourke, Marcela V. Maus, Jennifer JD Morrissette. Development of a NGS-based method for EGFRvIII detection: sequence analysis of the junction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4916. doi:10.1158/1538-7445.AM2015-4916</jats:p

A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma

We conducted a first-in-human study of intravenous delivery of a single dose of autologous T cells redirected to the epidermal growth factor receptor variant III (EGFRvIII) mutation by a chimeric antigen receptor (CAR). We report our findings on the first 10 recurrent glioblastoma (GBM) patients treated. We found that manufacturing and infusion of CAR-modified T cell (CART)-EGFRvIII cells are feasible and safe, without evidence of off-tumor toxicity or cytokine release syndrome. One patient has had residual stable disease for over 18 months of follow-up. All patients demonstrated detectable transient expansion of CART-EGFRvIII cells in peripheral blood. Seven patients had post-CART-EGFRvIII surgical intervention, which allowed for tissue-specific analysis of CART-EGFRvIII trafficking to the tumor, phenotyping of tumor-infiltrating T cells and the tumor microenvironment in situ, and analysis of post-therapy EGFRvIII target antigen expression. Imaging findings after CART immunotherapy were complex to interpret, further reinforcing the need for pathologic sampling in infused patients. We found trafficking of CART-EGFRvIII cells to regions of active GBM, with antigen decrease in five of these seven patients. In situ evaluation of the tumor environment demonstrated increased and robust expression of inhibitory molecules and infiltration by regulatory T cells after CART-EGFRvIII infusion, compared to pre-CART-EGFRvIII infusion tumor specimens. Our initial experience with CAR T cells in recurrent GBM suggests that although intravenous infusion results in on-target activity in the brain, overcoming the adaptive changes in the local tumor microenvironment and addressing the antigen heterogeneity may improve the efficacy of EGFRvIII-directed strategies in GBM

BRCA locus-specific loss of heterozygosity in germline BRCA1 and BRCA2 carriers

Complete loss of BRCA1 or BRCA2 function is associated with sensitivity to DNA damaging agents. However, not all BRCA1 and BRCA2 germline mutation-associated tumors respond. Herein we report analyses of 160 BRCA1 and BRCA2 germline mutation-associated breast and ovarian tumors. Retention of the normal BRCA1 or BRCA2 allele (absence of locus-specific loss of heterozygosity (LOH)) is observed in 7% of BRCA1 ovarian, 16% of BRCA2 ovarian, 10% of BRCA1 breast, and 46% of BRCA2 breast tumors. These tumors have equivalent homologous recombination deficiency scores to sporadic tumors, significantly lower than scores in tumors with locus-specific LOH (ovarian, P = 0.0004; breast P &lt; 0.0001, two-tailed Student's t-test). Absence of locus-specific LOH is associated with decreased overall survival in ovarian cancer patients treated with platinum chemotherapy (P = 0.01, log-rank test). Locus-specific LOH may be a clinically useful biomarker to predict primary resistance to DNA damaging agents in patients with germline BRCA1 and BRCA2 mutations.Most tumours associated with germline BRCA1/BRCA2 loss of function mutations respond to DNA damaging agents, however, some do not. Herein, the authors identify that a subset of breast/ovarian tumors retain a normal allele, which is associated with decreased overall survival after DNA damage-inducing platinum chemotherapy

Co‐targeting BET and MEK as salvage therapy for MAPK and checkpoint inhibitor‐resistant melanoma

Abstract Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses. NRAS‐mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance. We show that BET proteins are overexpressed in NRAS‐mutant melanoma and that high levels of the BET family member BRD4 are associated with poor patient survival. Combining BET and MEK inhibitors synergistically curbed the growth of NRAS‐mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy. Transcriptomic and proteomic analysis revealed that combining BET and MEK inhibitors mitigates a MAPK and checkpoint inhibitor resistance transcriptional signature, downregulates the transcription factor TCF19, and induces apoptosis. Our studies demonstrate that co‐targeting MEK and BET can offset therapy resistance, offering a salvage strategy for melanomas with no other therapeutic options, and possibly other treatment‐resistant tumor types