Arati Desai

Transoral Robotic Surgery Alone for Oropharyngeal Cancer: An Analysis of Local Control

OBJECTIVE To evaluate local control following transoral robotic surgery (TORS) with the da Vinci Surgical System (Intuitive Surgical Inc) as a single treatment modality for oropharyngeal squamous cell carcinoma (OSCC). DESIGN Prospective, single-center, observational study. SETTING Academic university health system and tertiary referral center. PATIENTS Thirty adults with previously untreated OSCC. INTERVENTION Transoral robotic surgery with staged neck dissection as indicated. MAIN OUTCOME MEASURES Local control and margin status. RESULTS Thirty patients were enrolled with previously untreated OSCC and no prior head and neck radiation therapy. Follow-up duration was at least 18 months. At the time of diagnosis, 9 tumors were T1 (30%); 16 were T2 (53%); 4 were T3 (13%); and 1 was T4a (3%). The anatomic sites of these primary tumors were tonsil in 14 (47%), tongue base in 9 (30%), glossotonsillar sulcus in 3 (10%), soft palate in 3 (10%), and oropharyngeal wall in 1 (3%). There was only 1 patient (3%) who had a positive margin after primary resection; further resection achieved a final negative margin. Perineural invasion was noted in 3 tumors (10%). No patient received postoperative adjuvant therapy. At a mean follow-up of 2.7 years (range, 1.5-5.1 years), there was 1 patient with local failure (3%). CONCLUSION As the only modality used for treatment of pathologically low-risk OSCCs, TORS provides high local control and is associated with low surgical morbidity.

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

A trial of autologous T cells redirected to a specific mutation in glioblastoma patients illustrates mechanisms of resistance. Speeding toward CAR T cell therapy for glioblastoma Chimeric antigen receptor (CAR) T cells have been successfully implemented for treating leukemia and are now being investigated for solid tumors. O’Rourke et al. conducted a phase 1 safety study of autologous CAR T cells targeted to EGFR variant III in glioblastoma patients. Treatment seemed to be well tolerated, which is critical because other CAR T cell products have been implicated in devastating central nervous system complications. Of the 10 patients enrolled, 7 had surgical intervention, allowing for some analysis of the tumors and T cells in patients’ brains. The results of this trial indicate that CAR T cell therapy is a viable option for treating 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.

Tissue concentration of systemically administered antineoplastic agents in human brain tumors

The blood–brain-barrier (BBB) limits the penetration of many systemic antineoplastic therapies. Consequently, many agents may be used in clinical studies and clinical practice though they may not achieve therapeutic levels within the tumor. We sought to compile the currently available human data on antineoplastic drug concentrations in brain and tumor tissue according to BBB status. A review of the literature was conducted for human studies providing concentrations of antineoplastic agents in blood and metastatic brain tumors or high-grade gliomas. Studies were considered optimal if they reported simultaneous tissue and blood concentration, multiple sampling times and locations, MRI localization, BBB status at sampling site, tumor histology, and individual subject data. Twenty-Four studies of 19 compounds were included. These examined 18 agents in contrast-enhancing regions of high-grade gliomas, with optimal data for 2. For metastatic brain tumors, adequate data was found for 9 agents. Considerable heterogeneity was found in the measurement value, tumor type, measurement timing, and sampling location within and among studies, limiting the applicability of the results. Tissue to blood ratios ranged from 0.054 for carboplatin to 34 for mitoxantrone in high-grade gliomas, and were lowest for temozolomide (0.118) and etoposide (0.116), and highest for mitoxantrone (32.02) in metastatic tumors. The available data examining the concentration of antineoplastic agents in brain and tumor tissue is sparse and limited by considerable heterogeneity. More studies with careful quantification of antineoplastic agents in brain and tumor tissue is required for the rational development of therapeutic regimens.

Differentiating Tumor Progression from Pseudoprogression in Patients with Glioblastomas Using Diffusion Tensor Imaging and Dynamic Susceptibility Contrast MRI

BACKGROUND AND PURPOSE: Early assessment of treatment response is critical in patients with glioblastomas. A combination of DTI and DSC perfusion imaging parameters was evaluated to distinguish glioblastomas with true progression from mixed response and pseudoprogression. MATERIALS AND METHODS: Forty-one patients with glioblastomas exhibiting enhancing lesions within 6 months after completion of chemoradiation therapy were retrospectively studied. All patients underwent surgery after MR imaging and were histologically classified as having true progression (>75% tumor), mixed response (25%–75% tumor), or pseudoprogression (<25% tumor). Mean diffusivity, fractional anisotropy, linear anisotropy coefficient, planar anisotropy coefficient, spheric anisotropy coefficient, and maximum relative cerebral blood volume values were measured from the enhancing tissue. A multivariate logistic regression analysis was used to determine the best model for classification of true progression from mixed response or pseudoprogression. RESULTS: Significantly elevated maximum relative cerebral blood volume, fractional anisotropy, linear anisotropy coefficient, and planar anisotropy coefficient and decreased spheric anisotropy coefficient were observed in true progression compared with pseudoprogression (P < .05). There were also significant differences in maximum relative cerebral blood volume, fractional anisotropy, planar anisotropy coefficient, and spheric anisotropy coefficient measurements between mixed response and true progression groups. The best model to distinguish true progression from non–true progression (pseudoprogression and mixed) consisted of fractional anisotropy, linear anisotropy coefficient, and maximum relative cerebral blood volume, resulting in an area under the curve of 0.905. This model also differentiated true progression from mixed response with an area under the curve of 0.901. A combination of fractional anisotropy and maximum relative cerebral blood volume differentiated pseudoprogression from nonpseudoprogression (true progression and mixed) with an area under the curve of 0.807. CONCLUSIONS: DTI and DSC perfusion imaging can improve accuracy in assessing treatment response and may aid in individualized treatment of patients with glioblastomas.

Induction chemotherapy with cetuximab, carboplatin and paclitaxel for the treatment of locally advanced squamous cell carcinoma of the head and neck.

Although controversy exists in the management of locally advanced squamous cell carcinoma of the head and neck (LA-SCCHN), clinicians often use induction chemotherapy for treatment of the most advanced cases. One promising regimen combines weekly cetuximab (400 mg/m2 loading dose followed by 250 mg/m2) with carboplatin (AUC of 2) and paclitaxel (90 mg/m2). We retrospectively evaluated patients treated with this regimen prior to definitive chemoradiation or surgery between May 2008 and December 2011. The primary endpoint used for this retrospective analysis was feasibility. Thirty consecutive, unselected patients were included. Median follow-up was 13.7 months (range, 5.0–38.7 months). All but one patient had stage IV SCCHN. Dose intensity was high for carboplatin (92%), paclitaxel (93%) and cetuximab (85%). Grade 3–4 toxicities occurred in <7% of the study population and were limited to rash, neutropenia and infusion reactions. Response rate (RR) to induction chemotherapy was 97% (30% complete response, 67% partial response). All patients completed subsequent chemoradiotherapy or surgery. Nineteen patients (63%) demonstrated a complete response and 11 patients (37%) demonstrated a partial response. Median overall survival and progression-free survival data are not yet mature. The RR to therapy in our off-protocol experience is at least comparable to that observed in the two phase II studies of this regimen and appears superior to that observed with docetaxel, cisplatin and fluorouracil (TPF).

Repeatability of 18F-FLT PET in a Multi-Center Study of Patients with High Grade Glioma

Quantitative 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) PET has potential as a noninvasive tumor biomarker for the objective assessment of response to treatment. To guide interpretation of these quantitative data, we evaluated the repeatability of 18F-FLT PET as part of a multicenter trial involving patients with high-grade glioma. Methods: 18F-FLT PET was performed on 10 patients with recurrent high-grade glioma at 5 different institutions within the Adult Brain Tumor Consortium trial ABTC1101. Data were acquired according to a double baseline protocol in which PET examinations were repeated within 2 d of each other with no intervening treatment. On each of the 2 imaging days, dedicated brain PET was performed at 2 time points, 1 and 3 h after 18F-FLT administration. Tumor SUVs and related parameters were measured at a central laboratory using various volumes of interest: isocontour at 30% of the maximum pixel (SUVmean_30%), gradient-based segmentation (SUVmean_gradient), the maximum pixel (SUVmax), and a 1-mL sphere at the region of highest uptake (SUVpeak). Repeatability coefficients (RCs) were calculated from the relative differences between corresponding SUV measurements obtained on the 2 d. Results: RCs for tumor SUVs were 22.5% (SUVmean_30%), 23.8% (SUVmean_gradient), 23.2% (SUVmax), and 18.5% (SUVpeak) at 1 h after injection. Corresponding data at 3 h were 22.4%, 25.0%, 27.3%, and 23.6%. Normalizing the tumor SUV data with reference to a background region improved repeatability, and the most stable parameter was the tumor-to-background ratio derived using SUVpeak (RC, 16.5%). Conclusion: SUV quantification of 18F-FLT uptake in glioma had an RC in the range of 18%–24% when imaging began 1 h after 18F-FLT administration. The volume-of-interest methodology had a small but not negligible influence on repeatability, with the best performance obtained using SUVpeak. Although changes in 18F-FLT SUV after treatment cannot be directly interpreted as a change in tumor proliferation, we have established ranges beyond which SUV differences are likely due to legitimate biologic effects.

Microvesicles as a Biomarker for Tumor Progression versus Treatment Effect in Radiation/Temozolomide-Treated Glioblastoma Patients

The standard of care for glioblastoma (GB) is surgery followed by concurrent radiation therapy (RT) and temozolomide (TMZ) and then adjuvant TMZ. This regime is associated with increased survival but also increased occurrence of equivocal imaging findings, e.g., tumor progression (TP) versus treatment effect (TE), which is also referred to as pseudoprogression (PsP). Equivocal findings make decisions regarding further treatment difficult and often delayed. Because none of the current imaging assays have proven sensitive and specific for differentiation of TP versus TE/PsP, we investigated whether blood-derived microvesicles (MVs) would be a relevant assay. METHODS: 2.8 ml of citrated blood was collected from patients with GB at the time of their RT simulation, at the end of chemoradiation therapy (CRT), and multiple times following treatment. MVs were collected following multiple centrifugations (300g, 2500g, and 15,000g). The pellet from the final spin was analyzed using flow cytometry. A diameter of approximately 300 nm or greater and Pacific Blue–labeled Annexin V positivity were used to identify the MVs reported herein. RESULTS: We analyzed 19 blood samples from 11 patients with GB. MV counts in the patients with stable disease or TE/PsP were significantly lower than patients who developed TP (P = .014). CONCLUSION: These preliminary data suggest that blood analysis for MVs from GB patients receiving CRT may be useful to distinguish TE/PsP from TP. MVs may add clarity to standard imaging for decision making in patients with equivocal imaging findings.

CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges

In patients with certain hematologic malignancies, the use of autologous T cells genetically modified to express chimeric antigen receptors (CARs) has led to unprecedented clinical responses. Although progress in solid tumors has been elusive, recent clinical studies have demonstrated the feasibility and safety of CAR T-cell therapy for glioblastoma. In addition, despite formidable barriers to T-cell localization and effector function in glioblastoma, signs of efficacy have been observed in select patients. In this review, we begin with a discussion of established obstacles to systemic therapy in glioblastoma and how these may be overcome by CAR T cells. We continue with a summary of previously published CAR T-cell trials in GBM, and end by outlining the key therapeutic challenges associated with the use of CAR T cells in this disease.

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

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. O9Rourke, 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

RNA-seq for identification of therapeutically targetable determinants of immune activation in human glioblastoma

IntroductionWe sought to determine which therapeutically targetable immune checkpoints, costimulatory signals, and other tumor microenvironment (TME) factors are independently associated with immune cytolytic activity (CYT), a gene expression signature of activated effector T cells, in human glioblastoma (GBM).MethodsGlioVis was accessed for RNA-seq data from The Cancer Genome Atlas (TCGA). For subjects with treatment-naïve, primary GBM, we quantified mRNA expression of 28 therapeutically targetable TME factors. CYT (geometric mean of GZMA and PRF1 expression) was calculated for each tumor. Multiple linear regression was performed to determine the relationship between the dependent variable (CYT) and mRNA expression of each of the 28 factors. Variables associated with CYT in multivariate analysis were subsequently evaluated for this association in an independent cohort of newly diagnosed GBMs from the Chinese Glioma Cooperative Group (CGCG).Results109 TCGA tumors were analyzed. The final multiple linear regression model included the following variables, each positively associated with CYT except VEGF-A (negative association): CSF-1 (p = 0.003), CD137 (p = 0.042), VEGF-A (p < 0.001), CTLA4 (p = 0.028), CD40 (p = 0.023), GITR (p = 0.020), IL6 (p = 0.02), and OX40 (p < 0.001). In CGCG (n = 52), each of these variables remained significantly associated with CYT in univariate analysis except for VEGF-A. In multivariate analysis, only CTLA4 and CD40 remained statistically significant.ConclusionsUsing multivariate modeling of RNA-seq gene expression data, we identified therapeutically targetable TME factors that are independently associated with intratumoral cytolytic T-cell activity in human GBM. As a myriad of systemic immunotherapies are now available for investigation, our results could inform rational combinations for evaluation in GBM.