Abstract A065: Pooled Analysis of 7 Multicenter Prospective Studies (n=212) Using an Integrated Multi-Compartment Biomarker Ecosystem to Differentiate True Progression from Treatment Effect in High-Grade Glioma

Background: Tumor mutational burden (TMB) has emerged as a predictive biomarker of response to immune checkpoint inhibitor (ICI) therapy. Current panel-based TMB algorithms aggregate signal from certain types of somatic variants (e.g. non-synonymous coding SNVs); however, delineating the contributions of these and other types of mutations may refine TMB calculation from gene panels. Moreover, early studies suggest other possible genomic correlates of patient outcome to ICI which may be complementary to TMB. Here, we explore the landscape of mutations comprising TMB and other genomic features correlating with TMB on a subset of several thousand late-stage plasma samples run on GuardantOMNITM (OMNI), a highly sensitive 500-gene cfDNA sequencing platform. Methods: We developed a cfDNA-based TMB algorithm which is robust to variable tumor shedding levels and presence of clonal hematopoiesis. We assessed cfDNA-based TMB in over 1,000 plasma samples across six tumor types, including lung and prostate. We examine the contribution of nonsynonymous, synonymous, intronic SNVs, and indels to TMB score. We investigate correlations between TMB and additional genomic features, including chromosomal instability, loss of HLA-bearing chromosome 6p, microsatellite instability (MSI), and common oncogenic and resistance mutations. Results: We found that the distribution of WES-calibrated TMB scores across this cohort of samples is consistent with TCGA, with median 10 mutations/Mb and upper-tertile of 14 mutations/Mb across tumor types. The number of non-synonymous coding SNVs per sample correlated highly with synonymous coding SNV and intronic SNV counts (Pearson’s r > 0.7 for each). Including this additional signal in TMB calculation improves clinical sensitivity by up to 5%. In MSS samples, indels were highly correlated with SNVs, indicating that both likely arise from a similar underlying mechanism. We found no clear correlation between high TMB and chromosomal instability, with high TMB samples exemplifying a range of tumor ploidies. TMB association with oncogenic drivers is consistent with existing literature, with lower median TMB in EGFR-driven lung tumors (p < 0.01), but little to no correlation between TMB and KRAS or PIK3CA driver status, or STK11 loss of function (p > 0.05), suggesting these latter events could be independent clinical biomarkers to TMB. Conclusions: Panel-based TMB scores can leverage synonymous and non-coding mutations to strengthen the signal of exome-wide mutation load. As more patient outcome data becomes available, TMB algorithms and orthogonal biomarkers of tumor genome immunogenicity will evolve further for improved guidance of patient response to immunotherapy. Sequencing panels with high sensitivity for TMB, via large panel space, and the ability to detect copy-number variations and MSI-status, will be important for biomarker development and clinical applications. Citation Format: Katie Quinn, Elena Helman, Tracy Nance, Jennifer Yen, John Latham, Kristin Gleitsman, Ravi Vijaya-Satya, Carlo Artieri, Alex Artyomenko, Marcin Sikora, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. Landscape and genomic correlates of ctDNA-based tumor mutational burden across six solid tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3404.

Checkpoint inhibitor (CPI) therapy demonstrates a remarkable clinical benefit in many cancer types. However, the ability to successfully select patients who will benefit from CPIs is still limited. Tumor mutational burden (TMB), a measure of the number of somatic mutations per coding area of tumor genome, is a putative biomarker of response showing great promise in CPI and immunotherapy combination trials. The ability to measure TMB from tumor biopsies or plasma samples will be important for clinical adoption of this biomarker. Herein we report on initial performance evaluation of Illumina’s TruSight™ Oncology 500 gene (TSO500) NGS assay for the analysis of TMB in FFPE tissue and plasma cell-free (cf)DNA specimens. Illumina’s TSO500 assay employs hybrid-capture based approach for target enrichment coupled with unique molecular indices to enable low frequency variant detection of single nucleotide variants and indels. This comprehensive cancer panel interrogates relevant cancer biomarkers in >500 cancer genes (~2 Mb) from as little as 40 ng of FFPE DNA or 30 ng of cfDNA. In addition to variant calls, Illumina’s analysis pipeline reports a TMB score and microsatellite instability (MSI) status. Results obtained with the TSO500 TMB assay were compared to our validated whole exome sequencing (WES) TMB assessment for FFPE tissue specimens, with and without matched normal. As previously reported, the WES TMB tumor-only pipeline uses somatic variant classifications determined using a random forest model to generate TMB scores from analysis of tumor FFPE specimens. TSO500 TMB and MSI statues were evaluated in a set of 50 tumor FFPE specimens obtained from late-stage (III+) colorectal and bladder cancer patients. TSO500 TMB results correlated well with the WES-derived TMB results, although some differences were observed in the magnitude of high TMB scores. TSO500 TMB levels correlated strongly with the MSI status, with 100% of MSI-high tumors demonstrating high TMB scores (> 20), and greater than 85% of MSI-low tumors characterized as TMB-low (<20%). Additionally, correlations between tissue and blood-based TMB assessments in matched FFPE and plasma specimens are reported. Overall, our results demonstrate a strong correlation between TSO500 panel-based and whole exome sequencing-based TMB results in tissue and report comparisons to TMB calling in liquid biopsies. Our data further confirm that tumor mutational load is strongly connected to DNA damage repair deficiency in colorectal and bladder cancer patients. Citation Format: Stephanie B. Hastings, Gunjan Hariani, Victor J. Weigman, Tingting Jiang, Chen Zhao, Rajiv Raja, Traci Pawlowski, Philip Brohawn, Patrick Hurban. Panel-based tumor mutational burden (TMB) analysis of matched tumor and plasma specimens using Illumina’s TruSight Oncology 500 next-generation sequencing assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1346.

BackgroundCurrently, BRAF inhibitors are primarily effective in LC patients with BRAF V600E mutation, while with limited benefit for patients with BRAF non-V600E mutation. Additionally, it is unclear whether immunotherapy is effective in LC patients with BRAF mutations, and the relationship between BRAF mutations and immune biomarkers, such as TMB, is also unclear. Here, we retrospectively investigated the relationship with BRAF mutation and TMB in Chinese LC patients.MethodsSamples were extracted from 3,136 our cohort samples of LC, which from OncoPanscan࣪ (Genetron Health) based sequencing of tissue. Because of the significantly different cohort characteristics, propensity score matching was used to resolve potential confounding by a nearest neighbor algorithm. The use of a multivariable logistic regression model to estimate the propensity score was based on age, sex, cancer type and detail panels. The samples after propensity score matching was used to investigate the relationship with TMB. ResultsIn our cohort, 171 samples had BRAF mutations and 2,965 samples had non-BRAF mutations. After propensity score analysis, 165 samples of BRAF mutations were screened with 165 paired samples of non-BRAF mutations.In the 165 samples of BRAF mutations, 83 samples of them had TMB information, while 83 samples of the 165 paired samples of non-BRAF mutations had TMB information. Patients with BRAF mutation had a higher median TMB than the patients with non-BRAF mutation (9.39 vs. 7.51 Muts/Mb, p=0.0545). Similarly, in these two groups, there was slightly higher ratio of samples with TMB ≥10muts/Mb (43.37% vs. 31.32%, P=0.0788). Among BRAF mutation group, when compared to BRAF V600E group (N=26), the median TMB was much higher in non-V600E group (N=57) (7.38 vs. 9.86 muts/Mb, P=0.0270). The proportion of TMB ≥10muts/Mb in BRAF non-V600E group was much higher than V600E group (49.12% vs. 30.77%, P=0.0094). Additionally, we analyzed the samples that had high TMB (TMB≥10 muts/Mb) and high PD-L1 (TPS≥50%). The BRAF mutation group had more proportion than non-BRAF mutation (14.46% vs.6.02%, p=0.0593), and the BRAF non-V600E group was higher than BRAF V600E group (15.79% vs. 11.54%, P=0.4150).ConclusionsCompared with non-BRAF mutation, BRAF mutation was associated with higher TMB, and it was also higher in BRAF non-V600E than BRAF V600E. These results suggested that patients with driver mutation of BRAF V600E had lower TMB and they always had good response to BRAF inhibitors. While LC patients with BRAF non-V600E always with higher TMB, thus, they may be more suitable for immunotherapy. However, more clinical research are needed to evaluate the effectiveness of immunotherapy. Citation Format: Jian Zhang, Yu Fang, Lei Sun, Hongling Yuan, Mao Shang, Xiaoyan Zhang, Honglin Zhu, Tonghui Ma. Investigating the potential relationship between BRAF mutations and tumor mutation burden (TMB) in lung cancer (LC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5292.

Objective: PD-L1 expression and Tumor Mutation Burden (TMB) have independently emerged as prospective biomarkers of response to anti PD1-/PDL1 checkpoint inhibitors. However, TMB has not fully proven its value as a biomarker of Immunotherapy response in lung cancer. Moreover, FDA-approved CDx PD-L1 expression alone is not an optimal biomarker for checkpoint inhibitors and combined use of MSI, TMB and PD-L1 protein levels has been proposed. Here we present the correlation between genomic landscape, including TMB and MSI with PDL1 IHC in lung cancer specimens to help identify immunogenomic profiles for stratification of patients for check-point inhibitors therapies evaluation. Methods: 874 FFPE clinical samples across cancer types were characterized in our CLIA/CAP accredited clinical laboratory using a CLIA-validated NGS-based assay that interrogates SNVs, indels, TMB and Microsatellite Status (27 MS markers) using a 323 gene panel. TMB (mutations/Mb) is categorized as low (≤7), intermediate (7<TMB≤15) and high: (greater than 15). The de-identified aggregated results paired with PD-L1 IHC data from 424 lung cancer samples were analyzed and correlations between PD-L1 tumor proportion scores (TPS) and TMB results were made. In silico analyses were also performed on 5939 lung cancer samples from public databases. Results: The detected TMB median in lung cancer was 8.6 (range 0-126.4). Median PD-L1 TPS was 7%, with 26% of samples being PD-L1 negative (TPS<1%), 44% Low expressing (≥1-49%) and 31% High (≥50%). All samples were MSS. We found poor correlation between PD-L1 expression and TMB in NSCLC (r2=0.266). We classified samples based on TMB and PD-L1 TPS and found mutational correlations specific to in each of the following groups: PD-L1 High/TMB Low, PD-L1 High/TMB High, PD-L1 Low/TMB High, PD-L1 High/TMB High, and PD-L1 Neg/TMB High. Of note 67% (34/51) of PD-L1 High/TMB Low samples presented mutations either on EGFR (12%), KRAS (23.5%) or in genes from known driver TRK/MAPK pathways, whereas only KRAS was part of the frequently mutated gene signature with 36.5% (13/36) samples mutated on PD-L1 High/TMB High samples. Neither EGFR nor KRAS were found frequently mutated on PD-L1 Low/TMB High group (n=46). Strikingly the top 12 most frequently mutated genes on PD-L1 Neg/TMB High tumors were TP53, LRP1B, SPTA1, SMARCA4, GNAS, ALK, FGFR2, SLIT2, ROS1, AMER1, FAT1, and MED12. We found statistically significant co-occurrences between LRP1B and 7 other genes from this signature in additional 5939 lung cancer patients assessed. Conclusions: TMB and PD-L1 results do not correlate in lung cancer specimens. Common genomic alteration signatures might define subsets of lung cancer tumors with no PD-L1 expression to complement TMB and PD-L1 on the selection criteria for patients whom may benefit from checkpoint inhibitors. Citation Format: Fernando J. Lopez-Diaz, Lauryn Keeler, Forrest Blocker, Shiloh Sprankles, Ryan Bender, Sally Agersborg, Lawrence Weiss, Vincent Funari. Different genomic mutations signatures are associated to specific PD-L1/TMB states on lung cancer with potential value for patients screening for immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3170.

Background: Tumor mutational burden (TMB) is currently under investigation as a biomarker for predicting response to anti-cancer immunotherapy. TMB can be defined as the number of somatic nonsynonymous mutations per Mb in a cancer genome, therefore DNA artifacts could lead to inaccurate measurements. Cytosine deamination (C:G>T:A) is a well-known phenomenon in formalin-fixed, paraffin-embedded (FFPE) samples, producing errors in next-generation sequencing (NGS). Herein, we measured TMB in FFPE samples using two comprehensive NGS panels for routine diagnostics implementation. Methodology: TMB assessment was performed on FFPE samples by amplicon-based target enrichment on Thermo Fisher's Oncomine™ Tumor Mutation Load (OTML; n=9) and/or hybrid capture-based on Illumina's TruSight™ Oncology 500 Assay (TSO500; n=8), according to manufacturer's instructions. TMB values were compared for uracil-DNA glycosylase (UDG) treatment prior to PCR amplification and enrichment method. Samples were also compared according to their previous results in a clinically-certified NGS and an in-house microsatellite instability (MSI) assay. TMB >=10 mutations/Mb was considered high, and MSI >=20% was considered unstable. Results and Discussion: The concordance between TMB values for OTML and TSO500 was 75% (n=4) and 62.5% (n=8) with and without UDG treatment, respectively. UDG reduced artifactual C:G>T:A for both enrichment approaches, reinforcing its presence in FFPE DNA; however, there was no alterations on TMB results for both methods (TSO500 n=5; OTML n=7). C:G>T:A variants could be successfully filtered by low allelic frequency (<5%) or low quality (<q20) in non-treated samples. For OTML, a high baseline noise was observed in samples with high deamination scores (>60), leading to TMB overestimation (>350), which could not be corrected even after UDG treatment and manufacture's bioinformatic adjustment. Interestingly, the same samples presented low TMB values for TSO500. Samples with low deamination rates were not influenced by UDG treatment or bioinformatics adjustment regardless of enrichment method. TMB values from a certified NGS was completely concordant for TSO500 (n=4) but only 25% for OTML (n=4). MSI was fully concordant between certified NGS and TSO500 (n=4) and between in-house method and TSO500 (n=4) (value not analyzed by OTML assay). No QC parameter predicted differences between high and low deaminated samples. Conclusions: NGS enrichment methods strongly influenced TMB assessment. Hybrid-based capture showed better performance than amplicon-based. UDG treatment and higher allelic frequency cutoff could minimize false positives at variant calling level, although no significant effect could be observed for general TMB values. Future experiments should cover other alternatives to distinct FFPE DNA damages. Citation Format: Michele A. Pereira, Feliciana L. Marinho, Joice P. Silva, Renato Puga, Maíra C. Freire, Mariana R. Monteiro, Rafael L. Guedes, Luiz H. Araujo. Tumor mutational burden is affected by next-generation sequencing enrichment method in highly deaminated samples from solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1996.

Background: Tumor mutation burden (TMB) is emerging as a potential predictive biomarker for the efficacy of immune checkpoint inhibitors. TMB has traditionally been evaluated using whole exome sequencing (WES), an established method for analysis of genomic alterations that requires both tumor and germline DNA. In the phase 3 CheckMate 026 study (NCT02041533) of nivolumab vs chemotherapy in first-line non-small cell lung cancer (NSCLC), exploratory analyses were performed to determine the value of TMB as a predictive biomarker in 312 tumor samples using WES. Patients with baseline TMB in the upper tertile were associated with an increased objective response rate to nivolumab and longer median PFS, relative to patients with TMB in the lowest 2 tertiles and compared with chemotherapy-treated patients in the upper tertile (Carbone et al. N Engl J Med. 2017). Increasingly, targeted cancer gene panels, including the FoundationOne® (F1) assay, are being assessed as an alternative to WES for clinical use. To determine the feasibility of transitioning from WES to a potential in vitro diagnostic next-generation sequencing (NGS) platform for TMB analysis, we performed a study comparing values generated by WES with those generated by the F1 assay. Methods: Using the 2 hybridization-capture/NGS methods (WES and F1), TMB was assessed in formalin-fixed, paraffin-embedded NSCLC tumor samples. For WES, coding regions of 21,522 genes were analyzed, with TMB defined as the total number of missense mutations in the tumor exome. The F1 assay is a targeted gene panel of 315 cancer-related genes (Frampton et al. Nat Biotechnol. 2013), with TMB defined as the number of somatic mutations per megabase of sequenced tumor genome. Results: TMB data from 44 NSCLC samples were available on both F1 and WES platforms. We determined an empirical method to convert TMB values derived from WES into those derived from F1 to establish calibration between the 2 methodologies. TMB assessed by WES and by F1 was highly correlated (Spearman's r=0.9). As an additional test of concordance between the platforms, we examined agreement between the assays around the median WES value of 148 mutations. This value projects to 7.64 mutations per megabase on F1. The overall agreement between F1 and WES around these values was 86% (95% Wilson CI, 73-94). Positive and negative agreements between the 2 hybridization-capture/NGS methods were both 86% (95% Wilson CI, 67-95). Conclusion: Bridging TMB analysis by WES to F1 facilitates the transition of WES-derived biomarker data to the clinical in vitro diagnostic F1. This study demonstrates the feasibility of harmonization of TMB results across testing platforms, which provides alternative TMB testing options. Citation Format: Joseph D. Szustakowski, George Green, William J. Geese, Kim Zerba, Han Chang. Evaluation of tumor mutation burden as a biomarker for immune checkpoint inhibitor efficacy: A calibration study of whole exome sequencing with FoundationOne® [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5528.

To assess whether a new method of quantifying therapy-associated hemodynamic alterations may help to distinguish pseudoprogression from true progression in patients with high-grade glioma. Patients with high-grade glioma received concurrent chemoradiotherapy. Relative cerebral blood volume (rCBV) and blood flow (rCBF) maps were acquired before chemoradiotherapy and at week 3 during treatment on a prospective institutional review board-approved study. Pseudoprogression was defined as imaging changes 1 to 3 months after chemoradiotherapy that mimic tumor progression but stabilized or improved without change in treatment or for which resection revealed radiation effects only. Clinical and conventional magnetic resonance (MR) parameters, including average percent change of rCBV and CBF, were evaluated as potential predictors of pseudoprogression. Parametric response map (PRM), an innovative, voxel-by-voxel method of image analysis, was also performed. Median radiation dose was 72 Gy (range, 60 to 78 Gy). Of 27 patients, stable disease/partial response was noted in 13 patients and apparent progression was noted in 14 patients. Adjuvant temozolomide was continued in all patients. Pseudoprogression occurred in six patients. Based on PRM analysis, a significantly reduced blood volume (PRM(rCBV)) at week 3 was noted in patients with progressive disease as compared with those with pseudoprogression (P < .01). In contrast, change in average percent rCBV or rCBF, MR tumor volume changes, age, extent of resection, and Radiation Therapy Oncology Group recursive partitioning analysis classification did not distinguish progression from pseudoprogression. PRM(rCBV) at week 3 during chemoradiotherapy is a potential early imaging biomarker of response that may be helpful in distinguishing pseudoprogression from true progression in patients with high-grade glioma.

Purpose:. Local recurrence (LR) accounts for &amp;gt;90% at the time of relapse in high grade glioma (HGG) following conventional therapy. Anti-angiogenic therapy with bevacizumab has shown impressive radiological and clinical responses in HGG. However, an apparent increase in diffuse invasive relapse (DIR) following therapy necessitated a detailed analysis of pattern of recurrence following therapy. Methods: One hundred and sixty five consecutive patients with HGG, either newly diagnosed (n=75) or recurrent (n= 90) received therapy with bevacizumab at 10 mg/kg every two weeks along with conventional chemotherapy ± involved field radiotherapy. The treatment was continued till the time of disease progression or the patient developed dose limiting toxicity. Treatment evaluation was done with neurological examination and magnetic resonance imaging at base line and every 8 weeks subsequently. Pattern of recurrence and time to relapse were the primary aims of the study. DIR was defined as involvement of multiple lobes ± crossing the midline. Survival was calculated from the start of bevacizumab therapy. Results: With a mean follow-up of 6.8 months (range 1-58), 100 of the 165 patients (60.6%) have relapsed with 76 as DIR and 70 have died. The median progression free survival (PFS) and overall survival (OS) were 5 and 9 months respectively. Of the 62 patients who failed initially as LR, 38 (61.3%) patients subsequently developed DIR. 38/75 (50.7%) patients with newly diagnosed and 38/90 (42.2%) of recurrent HGG failed ultimately as DIR. All 9 patients who presented with multicentric HGG relapsed as DIR. The median OS for the patients who failed as DIR was 9 months Vs 6 months for patients with LR alone (p=0.06). While the hazard risk for LR remained linear over time, the risk of DR increased exponentially with time (R2 = 0.933). Biopsies done on eight of these patients at the time of recurrence showed decreased expression of Olig-2 in 6, increased expression of SDF-1 in 5 and a variable expression of CXCR-4 in 6 patients. No change in expression of HIF-1, p53, PDGF or CD163 was seen. Conclusion: Invasion remains a dominant process in HGG as demonstrated by both imaging and pathology. Angiogenic blockade that results in increased invasiveness of HGG is worrisome and needs to be addressed in future clinical trials. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5336.

Purpose/Objective(s): TTFields are FDA-approved for the treatment of recurrent and newly diagnosed Glioblastoma (GBM) in the supratentorial brain. The EF-14 trial showed that combining TTFields with adjuvant chemo-radiation leads to a significant increase in overall survival compared to adjuvant chemo-radiation alone. High-grade gliomas also occur in the infratentorium and brainstem of pediatric and adult patients. Brainstem gliomas have a median survival of 9 to 11 months, necessitating the exploration of novel treatment combinations, such as delivering TTFields to the infratentorial brain. We recently showed in a simulation-based study that TTFields can be successfully delivered to the infratentorium. Effective delivery was achieved by placement of the arrays on the vertex, bilateral posterolateral occiput, and superior-posterior neck; the array placement results in TTFields at therapeutic intensities throughout the brainstem and cerebellum of realistic computational head models. The previous simulation-based study was performed using realistic computational models of healthy individuals; it did not provide detailed insight into field distributions within tumor tissue located in the infratentorium. Here, we aim to expand the results of this previous study, by simulating the delivery of TTFields to the infratentorium of adult and pediatric patients with high-grade brainstem gliomas. Materials/Methods: A realistic computational model was created using MRI data of adult and pediatric patients with high-grade brainstem gliomas. Transducer arrays were placed on the model and the delivery of TTFields to the infratentorial brain was then simulated using a commercial numerical solver. The electric field distribution in the supratentorium, infratentorium, and within the tumor were derived from the simulations, and the dose in the tumor analyzed. Results: The distribution of calculated isofield lines demonstrates effective delivery of TTFields to infratentorial brain tumors. Conclusion: Our results provides rationale for clinically investigating the utility of TTFields in treating infratentorial high-grade gliomas. Citation Format: Marigdalia K. Ramirez-Fort, Brittany Cross, Ariel Naveh, Shearwood McClelland III, Melissa Mendez, Roberto Santiago, Sean S. Mahase, Jaime Matta, Ze9ev Bomzon, Christopher S. Lange. Simulating delivery of TTFields to the infratentorium in patients with brainstem gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4430.

Purpose/Objective(s): TTFields are FDA-approved for the treatment of recurrent and newly diagnosed Glioblastoma (GBM) in the supratentorial brain. The EF-14 trial showed that combining TTFields with adjuvant chemo-radiation leads to a significant increase in overall survival compared to adjuvant chemo-radiation alone. High-grade gliomas also occur in the infratentorium and brainstem of pediatric and adult patients. Brainstem gliomas have a median survival of 9 to 11 months, necessitating the exploration of novel treatment combinations, such as delivering TTFields to the infratentorial brain. We recently showed in a simulation-based study that TTFields can be successfully delivered to the infratentorium. Effective delivery was achieved by placement of the arrays on the vertex, bilateral posterolateral occiput, and superior-posterior neck; the array placement results in TTFields at therapeutic intensities throughout the brainstem and cerebellum of realistic computational head models. The previous simulation-based study was performed using realistic computational models of healthy individuals; it did not provide detailed insight into field distributions within tumor tissue located in the infratentorium. Here, we aim to expand the results of this previous study, by simulating the delivery of TTFields to the infratentorium of adult and pediatric patients with high-grade brainstem gliomas. Materials/Methods: A realistic computational model was created using MRI data of adult and pediatric patients with high-grade brainstem gliomas. Transducer arrays were placed on the model and the delivery of TTFields to the infratentorial brain was then simulated using a commercial numerical solver. The electric field distribution in the supratentorium, infratentorium, and within the tumor were derived from the simulations, and the dose in the tumor analyzed. Results: The distribution of calculated isofield lines demonstrates effective delivery of TTFields to infratentorial brain tumors. Conclusion: Our results provides rationale for clinically investigating the utility of TTFields in treating infratentorial high-grade gliomas. Citation Format: Marigdalia K. Ramirez-Fort, Brittany Cross, Ariel Naveh, Shearwood McClelland III, Melissa Mendez, Roberto Santiago, Sean S. Mahase, Jaime Matta, Ze'ev Bomzon, Christopher S. Lange. Simulating delivery of TTFields to the infratentorium in patients with brainstem gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4430.

Background: Highly selective targeted T cell therapies using chimeric antigen receptors (CAR) are emerging as safe and effective modalities for the treatment of cancer. Downregulation and/or mutation of targeted antigens is a common tactic used by cancer cells to create antigen loss escape variants; culminating in relapse. Hence, targeting two or more antigens on tumor cells simultaneously could result in better therapeutic efficacy. However, developing two separate cellular products for clinical use as combination therapy is difficult, owing to regulatory hurdles and cost. In contrast, rendering an individual T cell bispecific could increase target cell selectivity, improve T cell activation and offset tumor escape due to antigen loss. This approach could particularly be applicable in heterogeneous tumors like HGG. We have characterized and quantified the heterogeneity of antigen expression in HGG tissues and primary cell lines using immunofluorescent staining as well as flow cytometry. While dual targeting was clearly superior to single targeting, targeting of three antigens (or more) did not predict a significantly superior advantage in our patient cohort (Hegde et al. Mol Therapy 2013). Objective: The intent of this project is to develop an effective adoptive cell therapy for HGG using T cells genetically modified to express a bispecific CAR that incorporates two scFv antigen-recognition domains, in tandem (TanCAR), simultaneously targeting two glioma-restricted antigens, HER2 and IL-13Rα2. Methods: A model for the bispecific TanCAR was constructed using ModWeb as described previously by us (Grada and Hegde et al., Mol Ther Nucleic Acids 2013). Extracellular-domain was assembled on Clone Manager®, then cloned into the Gateway® entry vector pDONR™221 and sequence-verified. This antigen recognition domain was then cloned inline with a signaling domain of the co-stimulatory molecule, CD28 and T cell receptor ζ-chain. We used a retroviral system to graft T cells with HER2 and IL-13Rα2-specific TanCAR and confirmed their surface expression using flow cytometry. Their functionality was tested against plate-bound HER2 and IL-13Rα2 proteins as well as against HGG cells in co-cultures and cytotoxicity assays. Cytokine release was studied using ELISA. HER2-specific, IL-13Rα2-specific CAR T cells and non transduced (NT) T cells from the same donor served as controls. Results: The extracellular domain of the TanCAR includes an IL-13Rα2 scFv followed by a Gly-Ser linker, a HER2 specific scFv (FRP5) and another Gly-Ser tandem repeat hinge. The intracytoplasmic domain consists of a CD28/CD3ζ signaling-moiety. Fifty to 80% of T cells expressed HER2/IL-13Rα2 TanCAR on the cell surface. In Cr51 cytotoxicity assays, TanCAR-grafted bispecific T cells showed improved killing of IL-13Rα2 and HER2 positive HGG cell lines as well as autologous HGG cells over the control T cells from the same donor. In co-cultures with autologous HGG cells, HER2/IL-13Rα2 TanCAR T cells proliferated and released immunostimulatory cytokines. Though HER2/IL-13Rα2 TanCAR T cells were effectively activated and secreted IFN-γ and IL-2 on recognizing HER2 and IL-13Rα2 proteins individually, cytokine secretion was significantly higher on simultaneous exposure to both target antigens. Conclusion: The pattern of heterogeneity in HGG favors near complete targeting of tumor subpopulations using bispecific T cell approach. Simultaneous targeting of two glioma-restricted antigens using TanCAR modified T cells could improve T cell activation and can potentially be used to offset antigen escape variants and reduce tumor recurrence. Citation Format: Meenakshi Hegde, Amanda Corder, Zakaria Grada, Tiara T. Byrd, Vita S. Brawley, Helen E. Heslop, Stephen Gottschalk, Nabil Ahmed. Adoptive immunotherapy targeting tumor heterogeneity in high-grade glioma (HGG). [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B8.

Background: Tumor mutational burden (TMB) has been emerging as a relatively new biomarker that is independent of PDL1 for the prediction of response to the immune checkpoint inhibitor (ICI) treatment. A recent study has shown that whole exome sequencing (WES)-derived TMB correlates well with panel-derived TMB that is estimated using targeted sequencing. Here, we evaluate the correlation between panel-derived TMB with response to ICI treatment in urothelial cancers. Methods: FFPE tumor tissues from 30 patients with urothelial cancers who had previously received ICI as monotherapies or combination with chemotherapies at Chang Gung Memorial Hospital were retrospectively underwent targeted next-generation sequencing (ACTOncoTM) for the identification of nonsynonymous variants across 440 cancer- associated genes. Tumor mutational burden (TMB) was calculated by using the sequenced regions of ACTOncoTM to estimate the number of somatic nonsynonymous mutations per megabase of all protein-coding genes. The TMB calculation predicted somatic variants and applied a machine learning model with a cancer hotspot correction. RECIST criteria were used to categorize tumor response to the treatment. Results: Patients were defined as responders (CR/PR) and non-responders (SD/PD) according to the RECIST evaluation. In the cohort receiving ICI monotherapy, the responders (CR/PR, n=8) had significantly higher TMB than non-responders (SD/PD, n=15) (Median 16.2 muts/Mb vs. 6.5 muts/Mb, p=0.0035). One patient with moderate TMB (11.0 muts/Mb) who exhibited disease progression following ICI therapy harbored biallelic JAK2 inactivation. Three high-TMB tumors exhibited mutational signature of aristolochic acid exposure. Conclusions: Although the cohort size is small, this study showed that panel-derived TMB can serve as an independent predictive biomarker to identify urothelial cancer patients for immune checkpoint inhibitor therapy. Citation Format: Yi-Lin Hsieh, Pei-Ning Yu, Yi-Hua Jan, Meng-Shao Lai, Woei-Fuh Wang, De-Wei Zhuo, Shu-Jen Chen, Jen-Hao Cheng, Kien Thiam Tan, Yu-Li Su. Panel-derived tumor mutational burden (TMB) is associated with the response to the immune checkpoint inhibitors (ICIs) in urothelial cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3177.

Circulating tumour DNA in predicting and monitoring survival of patients with locally advanced rectal cancer undergoing multimodal treatment: long-term results from a prospective multicenter study.

Background: Increased tumor mutational burden (TMB) and inflammation are associated with improved clinical outcomes to immuno-oncology (I-O) therapy in many tumor types. Genomic correlates of response to nivolumab (N) vs dacarbazine (D) (CheckMate [CM] 066; NCT01721772) and N+ipilimumab (I) combination therapy or N vs I (CM 067; NCT01844505) were evaluated for association of TMB and an inflammatory gene signature with clinical outcomes. Methods: In pretreatment tumor samples from eligible patients (pts), TMB was analyzed by whole-exome sequencing using median number of total missense mutations to define high vs low TMB (TMB-H/TMB-L). Associations of TMB with progression-free survival (PFS) and overall survival (OS) were evaluated per protocol-defined exploratory analyses using 4-year follow-up data for both trials. PFS and OS associations with an inflammatory signature were assessed by RNAseq in CM 067 samples only and grouped into tertiles for analysis. HR and 95% CI were calculated using Cox modeling and P values were calculated using the Wald test. Results: In CM 066, TMB was evaluable in 122 of 411 pts. PFS and OS were significantly longer in pts treated with N, but not with D, for TMB-H vs TMB-L tumors. The benefit of PFS and OS for N vs D was greater in pts with TMB-H than TMB-L (Table). In CM 067, 583 of 937 pts were evaluable for TMB and 299 were evaluable using an inflammatory signature. PFS and OS were longer in each treatment (tx) arm in pts with TMB-H vs TMB-L (Table). Across tx arms, a numerical benefit was observed for PFS and OS in pts with high vs low inflammatory signature (Table). TMB-H and a high inflammatory signature score were independently associated with clinical outcomes to I-O. Conclusions: High TMB or high inflammatory signature was associated with benefit to I-O. TMB status did not differentiate between N+I and N, and higher inflammatory status increased the likelihood of benefit for N+I and N, suggesting further evaluation of these biomarkers to characterize the response to I-O regimens in melanoma. Association of TMB (CM 066) or TMB and inflammatory signature (CM 067) with PFS and OSCategorical comparisonClinical measureHR (95% CI; P value)CM 066NDPatients with evaluable TMB, n (high, low)53 (23, 30)69 (38, 31)TMB-H vs TMB-LPFSOS0.33 (0.16-0.69; 0.0031)0.43 (0.2-0.91; 0.027)0.67 (0.39-1.10; 0.13)0.66 (0.39-1.10; 0.13)N vs D in TMB-HPFSOS0.28 (0.14-0.59; 0.00083)0.35 (0.17-0.73; 0.0046)N vs D in TMB-LPFSOS0.57 (0.33-0.99; 0.047)0.55 (0.31-0.98; 0.044)Categorical comparisonClinical measureHR (95% CI; P value)CM 067N+INIPatients with evaluable TMB, n (high, low)197 (94, 103)192 (95, 97)194 (101, 93)TMB-H vs TMB-LPFSOS0.56 (0.39-0.81; 0.002)0.53 (0.34-0.81; 0.0032)0.45 (0.31-0.65; 0.00002)0.48 (0.31-0.72; 0.00046)0.64 (0.47-0.87; 0.005)0.59 (0.42-0.83; 0.0022)N+I vs N in TMB-HPFSOS0.95 (0.63-1.40; 0.81)0.97 (0.60-1.60; 0.88)-N+I vs N in TMB-LPFSOS0.77 (0.56-1.10; 0.12)0.87 (0.61-1.30; 0.46)-Patients with evaluable inflammatory signature, n (low, med, high)93 (33, 32, 28)106 (39, 31, 36)100 (28, 36, 36)Inflammatory signature, high vs lowPFSOS0.38 (0.18-0.80; 0.01)0.53 (0.24-1.18; 0.12)0.60 (0.34-1.00; 0.069)0.35 (0.18-0.69; 0.0025)0.48 (0.27-0.85; 0.012)0.52 (0.27-1.00; 0.049) Citation Format: F. Stephen Hodi, Jedd D. Wolchok, Dirk Schadendorf, James Larkin, Max Qian, Abdel Saci, Tina C. Young, Sujaya Srinivasan, Han Chang, Megan Wind-Rotolo, Jasmine I. Rizzo, Donald G. Jackson, Paolo A. Ascierto. Genomic analyses and immunotherapy in advanced melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT037.

Purpose: To date no biomarker has been identified that predicts response to ICI in mTNBC. This study aimed to explore if tumor genomic alterations correlate with efficacy of PD-1/PD-L1 inhibition in patients (pts) with mTNBC. Methods: Demographic, treatment response, and long-term outcome data were collected on patients with mTNBC treated at Dana-Farber Cancer Institute (DFCI) under several clinical trials incorporating PD-1/PD-L1 inhibitors, given as monotherapy or combined with chemotherapy (CT). Pts included in this analysis had available results of targeted exon sequencing performed using Oncopanel, our institutional gene sequencing panel, on archival tumor tissue. TMB was calculated by determining the number of non-synonymous somatic mutations that occur per megabase of exonic sequence data across all genes on the panel. High TMB was defined as 310 mutations/megabase. TMB and gene alterations were correlated with objective response rate (ORR) per RECIST 1.1, progression-free (PFS) and overall survival (OS). Results: A total of 50 pts with mTNBC were included in this analysis. At baseline, the median age was 55.9 years (31.8–75.9), 60% had ECOG 0 and 40% had ECOG 1, 72% had visceral metastasis, and 46% had received 31 prior lines of systemic therapy in the metastatic setting. While 26% of pts received monotherapy [pembrolizumab (n=7, NCT02447003); atezolizumab (n=6; NCT01375842)], 74% received combination with CT [pembrolizumab plus eribulin (n=31; NCT02513472); atezolizumab plus nab-paclitaxel (n=6; NCT01633970)]. PTEN alterations were present in 30% of pts (mutations = 7; one copy number loss = 7; two copy number loss = 1). Median follow-up was 14 months (1–40). Pts with tumors harboring PTEN alterations had lower ORR (7% vs 57%; P&amp;lt;0.001), shorter median PFS (2.3 vs 6.3 months; P=0.027), and shorter median OS (8.1 vs 20.1 months; P=0.012) compared to pts without PTEN alterations. The median TMB was 6.6 mut/Mb (1.2–50.8), and 23% of pts had a high TMB. While high TMB was not associated with higher ORR (P=0.56), it was associated with better median PFS (16.5 vs 2.4 months; P=0.017), and better median OS (not reached vs 13.5 months; P=0.026). Both PTEN status and TMB remained significantly associated with PFS in the multivariable model. Only PTEN status remained significantly associated with OS in the multivariable analysis with the same covariables. Ongoing analysis to better understand if these predictors are specific for predicting benefit to immunotherapy and/or a marker of chemotherapy resistance will be presented at the symposium. Conclusion: PTEN genomic alterations and TMB may impact benefit from PD-1/PD-L1 inhibitors largely administered with chemotherapy in mTNBC. These observations warrant prospective validation and may inform the importance of stratifying pts according to these characteristics in future randomized studies with ICI. Table 1.Multivariable analysis for PFS Hazard ratioConfidence Intervalp-valueCombination therapy0.420.16 – 1.130.009Visceral metastasis1.310.63 – 2.770.46Previous lines of therapy1.020.09 – 0.700.85ECOG 12.11.06 – 1.280.034PTEN altered3.741.65 – 8.440.002Hypermutated tumors0.850.75 – 0.970.011 Citation Format: Barroso-Sousa R, Tyekucheva S, Pernas-Simon S, Exman P, Jain E, Garrido-Castro AC, Hughes M, Bychkovsky B, Di Lascio S, Umeton R, Files J, Lindeman NI, MacConaill LE, Hodi FS, Krop IE, Dillon D, Winer EP, Wagle N, Lin NU, Mittendorf EA, Tolaney SM. PTEN alterations and tumor mutational burden (TMB) as potential predictors of resistance or response to immune checkpoint inhibitors (ICI) in metastatic triple-negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-02.