Matched Tumor Samples Research Articles

Abstract 2738: PD-L1/PD-1 checkpoint inhibition in anaplastic thyroid cancer and enhancement of ICAM1-targeted chimeric antigen receptor (CAR)-T cell tumor lysis

Abstract Poorly-differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) are associated with a rapidly progressive course and poor median survival. Recently we published promising results in a murine model showing efficient clearance of metastatic tumor burden of human ATC cell line 8505c and an anaplastic patient-derived cell line using healthy donor T cells transduced with a third-generation chimeric antigen receptor (CAR) construct targeting intercellular adhesion molecule-1 (ICAM1). However, patient-derived T cells using an identical ICAM1-targeted CAR construct demonstrated slower tumor-killing in vitro. To investigate the role of immune checkpoint proteins PD-L1 and PD-1 in thyroid cancer and as potential inhibitors of ICAM1-CAR T, we stained formalin-fixed paraffin-embedded archived tissue samples for PD-L1 (papillary thyroid cancer, PTC, n = 17; ATC, n = 13) using immunohistochemistry (IHC) and extracted peripheral blood mononuclear cells (PBMCs) from patient blood samples and healthy donors to evaluate PD-1 expression on CD3+/CD8+ T cells using flow cytometry (healthy donor, n = 2; PTC, n = 7; PDTC/ATC, n = 6). Twelve percent of PTC samples were PD-L1+ (threshold >1% positivity) versus 69% of PDTC/ATC samples (Chi-square, p = 0.012); PD-L1 positivity was associated with circumferential ICAM1 staining on IHC (Chi-square, p = 0.003). Similarly, median percentage of PD-1 expression on PBMC's was higher in PDTC/ATC than PTC (58.1% versus 5.2%, Wilcoxon rank sum test; p = 0.03). Interestingly, expression of elevated PD-1 was seen in PBMC samples from patients in the absence of PD-L1 positivity of the primary tumor (IHC) in 4 of 5 matched tumor samples. This suggests that PD-1 in circulating CD8+ cells may be an important predictor of response to checkpoint inhibitor therapy. Finally, effector:target assays with patient-derived ICAM1-CAR T effector cells (baseline PD-1 expression 96%) against 8505c (PD-L1 100%/ICAM1 68%) and matched patient-derived cell line (PD-L1 >50%/ICAM1 88%) showed enhanced tumor killing in the presence of anti-PD-1, which approached the level of tumor cell death seen with healthy donor ICAM1-CAR T. Our results show that PD-1 is increased in the peripheral blood of patients with poorly differentiated and anaplastic thyroid cancer and is a potential therapeutic target. Furthermore, anti-PD-1 may be used to counter PD-L1+ tumor-induced T cell suppression (or exhaustion) to increase the efficacy of CAR T cells in ATC. Citation Format: Katherine D. Gray, Yogindra Vedvyas, Olivia Kalloo, Enda Shevlin, Theresa Scognamiglio, Marjan Zaman, Andrew Tassler, Moonsoo M. Jin, Rasa Zarnegar, Thomas J. Fahey, Irene M. Min. PD-L1/PD-1 checkpoint inhibition in anaplastic thyroid cancer and enhancement of ICAM1-targeted chimeric antigen receptor (CAR)-T cell tumor lysis [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 2738.

Abstract 4578: Development of a rapid clinical grade assay to detect and monitor secondary KIT mutations in circulating free DNA (cfDNA) for personalization of targeted therapy for gastrointestinal stromal tumors (GIST)

Abstract Introduction: Genotype-directed therapies are revolutionizing care for gastrointestinal stromal tumors (GIST). Somatic mutations in KIT exons (ex) 11 and 9 induce constitutive kinase activity and account for ~95% of the primary oncogenic events in KIT-driven GIST; KIT mutations correlate with prognosis and predict clinical activity of tyrosine kinase inhibitors (TKI), such as imatinib. After a median of 24 months on imatinib, 85% of patients (pts) develop resistance and progress multifocally, mostly due to secondary KIT mutations in ex 13 and/or ex 17. We describe here a rapid, clinical grade droplet digital PCR (ddPCR) assay to detect and quantify KIT mutations associated with TKI resistance in cfDNA from plasma of metastatic (met) GIST pts. Unlike allele-specific ddPCR that requires splitting input DNA into many reactions, we designed pan-ex 17 KIT assay to capture the polyclonal mutational landscape of KIT in a single reaction. Methods: Tumor and plasma samples and clinical data were collected from pts with met GIST. ddPCR assays were developed for the detection in cfDNA of all common secondary KIT mutations associated with imatinib resistance (V654A, T670I, A829P and multiple ex 17 mutations). The pan-ex 17 mutation drop-off assay uses a FAM-labeled mutant (mut) probe that spans sequences encoding amino acids 819~824, and a VIC-labeled reference probe that is shared by both wild-type (wt) and mut alleles. Common ex 17 mutations were detected via cluster separation. V654A, T670I, A829P are detected with allele-specific assays that use a FAM-labeled mut probe and a VIC-labeled wt probe. To demonstrate analytical sensitivity/specificity of each assay, ddPCR cycling conditions were optimized to yield the maximum fluorescence signal with minimal noise signal. Results: The KIT ddPCR assays detect a mutation prevalence of 0.01 - 0.05%, with a sensitivity of 5 - 50 KIT mut copies in a background of 10,000 KIT wt copies, depending on the mutation assayed, with absolute specificity. Results were validated in a pilot observational cohort of pts who were treated with imatinib and progressed, for which matching plasma (1 ml) and tumor samples were analyzed to reveal identical mutations. In a separate cohort, ddPCR results were validated by sensitive plasma NGS. Conclusions: We have developed rapid, highly sensitive, and 100% specific quantitative assays to enable plasma-based tumor genotyping for secondary KIT resistance mutations, which has been technically optimized for translation into clinical practice. Analysis of cfDNA by ddPCR can successfully detect KIT secondary mutations in met GIST pts. In an ongoing study, serial monitoring of KIT mutations in cfDNA from pts with advanced GIST should allow for early detection of secondary mutations and optimization of genotype-targeted therapeutic management. Citation Format: Adrian Marino-Enriquez, Suzanne George, Julianna Supplee, Grace Heavey, Pasi A. Janne, Chandrajit Raut, Jonathan Fletcher, George D. Demetri, Cloud P. Paweletz, Yanan Kuang. Development of a rapid clinical grade assay to detect and monitor secondary KIT mutations in circulating free DNA (cfDNA) for personalization of targeted therapy for gastrointestinal stromal tumors (GIST) [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 4578.

Abstract LB-381: Effects of sequencing parameters and panel size on mutational burden calculations

Abstract Immunotherapies are an emerging approach for cancer treatment that leverage the power of the immune system to attack cancer cells. By targeting immune check-point pathways such as PD-1, these treatments have been highly effective for some patients diagnosed with skin, bladder, lung, or kidney cancer. However, response rates for these treatments vary, making it beneficial to identify patients who are likely to respond to the treatment. Qualitative methods for predicting immunotherapy response are available but are technically challenging and subjective. An emerging quantitative predictor of immunotherapy response, tumor mutational burden (TMB), measures the number of somatic mutations per Mb of the genome. TMB calculations using whole exome data have been shown to correlate with patient response to immunotherapies. However, derivation of this score is not well characterized, nor is the score universally performed across laboratories. Here we describe the results from calculating TMB scores varying cancer panel size and amount of sequencing generated. First, variants are called in matched tumor and normal samples within Exomes. Then low coverage variants are removed along with variants outside the targeted, high-confidence regions in each panel. The remaining variants in the normal samples constitute the germline variants and are subtracted from the number of variants identified in tumor samples to yield the TMB score. We then repeated this across multiple indications across TCGA to review robustness of score in indications across the spectra of burden. TMB scores from the our comprehensive cancer closely resembles scores calculated using whole exome data across indications. We propose rules for gene content and sequencing depth for providing a range of burden scores that could be used for determining thresholds needed for determining therapeutic implications. We suggest that only a small subset of genes are required for accurate TMB estimation, thus reducing costs associated with predicting patient response to immunotherapy. Citation Format: Scott Yourstone, Natalie Mola, Victor J. Weigman. Effects of sequencing parameters and panel size on mutational burden calculations [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 LB-381.

NKTR-214 (CD122-biased agonist) plus nivolumab in patients with advanced solid tumors: Preliminary phase 1/2 results of PIVOT.

3006Background: PIVOT is an ongoing, open-label, phase 1/2 study of NKTR-214 (214; CD122-biased agonist) plus PD-1 inhibitor nivolumab (N) in patients (pts) with advanced cancers (MEL, RCC, NSCLC, TNBC, and UC). 214 monotherapy increases newly proliferative CD8+ T cells in tumors and increases cell surface PD-1 and PD-L1 expression, demonstrating a potentially synergistic mechanism with anti-PD-1 therapy. Methods: In P1 dose escalation, pts received 214 (0.003, 0.006 or 0.009 mg/kg) with N (240 mg or 360 mg) administered IV as outpatient Q2W or Q3W; in P2 expansion, the RP2D of 214 (0.006 mg/kg) with N (360 mg) Q3W was administered concurrently. Response was assessed Q8W by RECIST v1.1. Matched tumor samples were evaluated for changes from baseline in immune cell populations, gene expression, and T cell receptor repertoire. Tumor baseline and on treatment PD-L1 protein expression was assessed (28-8 IHC assay). Results: As of 7FEB2018, 162 pts (P1, n = 38; P2, n = 124) were evaluable for safety. The most c...

Added Value of 50-Gene Panel Sequencing to Distinguish Multiple Primary Lung Cancers from Pulmonary Metastases: A Systematic Investigation

Differentiation between multiple primary lung cancers and pulmonary metastases (PM) has important implications in staging, prognosis, and treatment strategies. Clinical and immunohistopathologic criteria have been standardized; however, a substantial number of cases remain difficult to classify. Using next-generation sequencing, it is now possible to improve the classification of multiple lung cancer lesions. This study systematically investigated the value of routine morphologic and IHC characteristics, p53 protein expression, TP53 mutation analysis, and 50-gene panel sequencing (GPS) in 111 lesions from 50 patients with multiple lung lesions. Based on immunohistopathologic criteria, 32 paired lesions were classified as multiple primary lung cancer (MPLC) and 21 as PM. TP53 mutation analysis indicated MPLC in 23 and PM in 6 pairs, but in the majority of cases (n=28, 49%) no mutation was observed and no conclusion could be drawn. In contrast, only 2 pairs were not conclusive using GPS. In a significant number of matching tumor samples (n=19, 39%), sequencing results were contradictory to the initial immunohistopathology diagnosis. No separation in overall survival for classifications based on immunohistopathology was observed, while a clear but nonsignificant trend was observed concerning survival in MPLC patients (hazard ratio = 3.98) using 50-gene GPS. In about one-third of the patients, GPS provided additional information to improve the differentiation between MPLC and PM.

Multi-institutional Analysis of Next Generation Sequencing of Cell Free Circulating Tumor DNA of Blood Samples from Recurrent and Metastatic Head and Neck Cancer Patients

Head and neck squamous cell carcinoma (HNSCC) is an increasingly prevalent disease but effective targeted therapy is lacking. The use of next-generation sequencing (NGS) in the identification of novel targets has been suggested as a way to potentially expand therapeutic options and thereby improve outcomes. We designed a retrospective study to further characterize the results of blood sample sequencing in recurrent and metastatic (R/M) head and neck cancer (HNC) patients, to determine its ability to identify actionable mutations, and also to further elucidate the utility of liquid biopsies and its role in patient management. Using Guardant360, a 70-gene circulating tumor DNA (ctDNA) NGS platform, molecular profiling of blood samples was obtained from patients with recurrent and metastatic (R/M) head and neck cancers. ctDNA sequencing data was compared to tumor NGS data, when available. Best response to therapy was assessed using RECIST. Eighty-eight HNSCC patients were evaluated from February 2015 to February 2017. The most common tumor type and histology was oropharyngeal squamous cell carcinoma (n=23), which was commonly human papillomavirus (HPV) positive (n=17). Other cancer types included nasopharyngeal, salivary gland, and thyroid cancers. The most common mutations identified by ctDNA analysis were TP53 (51%) PIK3CA (25%), NOTCH1 (14.8%), and ARID1A (14.8%). Of the 29 matched tumor samples, TP53 (48%) and PIK3CA (24%) were also reported with the highest frequency. Eighty-seven percent of HNSCC, 67% of NPC, 75% of thyroid, and 63% salivary gland cancer patients had actionable mutations. Among these, 10.2% (n=9) received matched targeted therapy (MTT): 1 (11%) had partial response (PR), 5 (56%) had stable disease (SD), and 3 (33%) had progressive disease (PD). Thirty-six (40.9%) patients received immunotherapy: 1 (2.8%) had complete response, 3 (8.3%) had partial response, 15 (41.7%) had stable disease, 16 (44.4%) had progressive disease, and 1 patient is not yet evaluable. Alterations identified by ctDNA may help inform management decisions in R/M HNSCC. The majority of patients had unique mutations identified on ctDNA. TP53 and PIK3CA mutations were seen at the highest frequency in both ctDNA and matched tumor samples. The utility of ctDNA NGS and its role in patient management should be explored in future studies.

Identification of novel prognosis-related genes associated with cancer using integrative network analysis

Prognosis identifies the seriousness and the chances of survival of a cancer patient. However, it remains a challenge to identify the key cancer genes in prognostic studies. In this study, we collected 2064 genes that were related to prognostic studies by using gene expression measurements curated from published literatures. Among them, 1820 genes were associated with copy number variations (CNVs). The further functional enrichment on 889 genes with frequent copy number gains (CNGs) revealed that these genes were significantly associated with cancer pathways including regulation of cell cycle, cell differentiation and mitogen-activated protein kinase (MAPK) cascade. We further conducted integrative analyses of CNV and their target genes expression using the data from matched tumour samples of The Cancer Genome Atlas (TCGA). Ultimately, 95 key prognosis-related genes were extracted, with concordant CNG events and increased up-regulation in at least 300 tumour samples. These genes, and the number of samples in which they were found, included: ACTL6A (399), ATP6V1C1 (425), EBAG9 (412), FADD (308), MTDH (377), and SENP5 (304). This study provides the first observation of CNV in prognosis-related genes across pan-cancer. The systematic concordance between CNG and up-regulation of gene expression in these novel prognosis-related genes may indicate their prognostic significance.

Genomic evolution and acquired resistance to preoperative chemoradiation therapy (CRT) in rectal cancer.

613 Background: Following pre-operative CRT for locally advanced rectal cancer, ~20% of patients achieve a pathologic complete response and 20-40% have little to no response (NR) at time of surgery. Pathologic downstaging after CRT is associated with improved outcomes but molecular predictors of response are poorly understood. The objective of this study was to characterize somatic mutations and derive genomic biomarkers distinguishing complete and excellent partial responders (R) vs NR through whole exome sequencing (WES) in pre- and post-CRT tumor samples. Methods: Rectal cancer patients treated with pre-operative CRT and having either a R or NR were identified. DNA extraction from clinical FFPE samples and WES were performed on pre/post-CRT tumor and matched germline DNA followed by genomic analysis to identify somatic mutations, insertion/deletions, and clonal/subclonal mutational evolution. RNA extraction/sequencing and GSEA was performed in parallel. Results: We identified 34 pre- and post-CRT matched tumor samples from 17 patients treated with pre-operative fluoropyrimidine-based CRT (50.4 Gy) followed by surgery within 8-10 weeks. Nine and 8 patients were classified as NR and R, respectively. All FFPE passed quality control metrics and were successfully sequenced. Mean somatic mutational burden pre-CRT was 3.78 mutations/Mb (rg, 2.25-8.43), which, unexpectedly, was not significantly increased in the post-CRT samples (4.23 mutations/Mb; rg, 1.74-8.96). NR cases had more concurrent KRAS/TP53 (KP) mutations than R cases (67% vs 13%, p < 0.05). Related to this finding, NR was associated with epithelial-to-mesenchymal transition (p < 0.001) and potentially linked to high intra-tumoral heterogeneity pre-CRT (p = 0.08). Conclusions: We have established feasibility and a pipeline for performing WES on pre- and post-CRT FFPE tumor samples. Our data suggest that evolution of CRT resistance is not through hypermutation. In conjunction with recent observations from our group (Hong, JNCI 2017; Wang, Cancer Res 2017), our findings strongly support the clinical utility of the KP genotype as a biomarker of CRT resistance and begin to offer insight into the underlying mechanisms on a genomic level.

Abstract B27: Analysis of the immune infiltrate in undifferentiated pleomorphic sarcoma of the extremity and trunk in response to radiotherapy

Abstract Introduction: Undifferentiated pleomorphic sarcoma of the extremity and trunk (ET UPS) presents a unique diagnostic and therapeutic challenge. Recently, immunotherapy has been employed in the treatment of patients with advanced UPS with preliminary evidence of benefit in 40% of a small initial cohort (SARC028). However, there is currently a paucity of data characterizing the immune infiltrate population in ET UPS. Radiotherapy (RT) is associated with improved locoregional control in combination with surgery. Localized RT has been shown in other tumor types to promote tumor antigen release and enhance tumor-specific targeting by the adaptive immune system. The aim of this study was two-fold: 1) to characterize the immune infiltrate in ET UPS and 2) to evaluate the effect of preoperative RT on immune cell infiltrates in ET UPS. Methods: We identified 17 matched ET UPS tumor samples before and after RT. Immunohistochemistry for CD8, CD4, PD-L1, PD1, CD3, CD163, and FOXP3 was performed on the matched pairs. Changes in immune infiltrate over time between the pairs were compared with a paired t-test. Results: CD8, CD4, PD-L1, PD1, CD3, CD163, and FoxP3 positive cells were identified in all samples. The effects were variable, but there was an overall trend towards an increase in the density of tumor-infiltrating immune cells of all assayed types in ET UPS following completion of RT, with increases in the median number of CD8 (55 vs. 111 cells/mm2, p=0.17), CD4 (3 vs. 13 cells/mm2, p=0.01), CD3 (158 vs. 219 cells/mm2, p=0.06), CD163 (1363 vs. 2001 cells/mm2, p=0.76), and FOXP3 (14 vs. 25 cells/mm2, p=0.23) positive cells in RT-treated tumor sections. Interestingly, although PD-L1 was not expressed in any pre-RT ET UPS tumors, positive PD-L1 expression was observed in 21% (3/14) of tumors after RT treatment (p=0.07). Conclusion: An immune infiltrate is present in ET UPS at the time of diagnosis, with a trend towards increased density of immune infiltrate and PD-L1 expression after completion of RT. These data support evaluating immune checkpoint inhibitors with standard of care RT in the treatment of ET UPS. Citation Format: Emily Z. Keung, Jen-Wei Tsai, Ali M. Ali, Janice N. Cormier, Andrew J. Bishop, B. Ashleigh Guadagnolo, Kelly K. Hunt, Keila E. Torres, Neeta Somaiah, Jennifer A. Wargo, Alexander Lazar, Wei-Lien Wang, Cristina L. Roland. Analysis of the immune infiltrate in undifferentiated pleomorphic sarcoma of the extremity and trunk in response to radiotherapy [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B27.

Family with sequence similarity 83, member B is a predictor of poor prognosis and a potential therapeutic target for lung adenocarcinoma expressing wild-type epidermal growth factor receptor.

Lung adenocarcinoma (ADC) patients with tumors that harbor no targetable driver gene mutation, such as epidermal growth factor receptor (EGFR) gene mutations, have unfavorable prognosis, and thus, novel therapeutic targets are required. Family with sequence similarity 83, member B (FAM83B) is a biomarker for squamous cell lung cancer. FAM83B has also recently been shown to serve an important role in the EGFR signaling pathway. In the present study, the molecular and clinical impact of FAM83B in lung ADC was investigated. Matched tumor and adjacent normal tissue samples were obtained from 216 patients who underwent complete lung resection for primary lung ADC and were examined for FAM83B expression using cDNA microarray analysis. The associations between FAM83B expression and clinicopathological parameters, including patient survival, were examined. FAM83B was highly expressed in tumors from males, smokers and in tumors with wild-type EGFR. Multivariate analyses further confirmed that wild-type EGFR tumors were significantly positively associated with FAM83B expression. In survival analysis, FAM83B expression was associated with poor outcomes in disease-free survival and overall survival, particularly when stratified against tumors with wild-type EGFR. Furthermore, FAM83B knockdown was performed to investigate its phenotypic effect on lung ADC cell lines. Gene silencing by FAM83B RNA interference induced growth suppression in the HLC-1 and H1975 lung ADC cell lines. FAM83B may be involved in lung ADC tumor proliferation and can be a predictor of poor survival. FAM83B is also a potential novel therapeutic target for ADC with wild-type EGFR.

Actionable mutations in canine hemangiosarcoma.

BackgroundAngiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma.MethodsSplenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage.Results and conclusionsSequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

Genomic Evolution and Acquired Resistance to Pre-Operative Chemoradiation Therapy in Locally Advanced Rectal Cancer

Pre-operative CRT is the standard for locally advanced rectal cancers. However, at the time of surgery, patients have mixed responses with approximately 20% achieving a pathologic complete response (CR) and 20-40% having little to no response (NR). Pathologic downstaging after CRT is associated with improved outcomes but molecular predictors of response are incompletely understood. The long-term goal of this project is to characterize somatic mutations and derive mutational signatures distinguishing CR vs NR through whole exome sequencing analysis in pre- and post-CRT samples. In addition, we seek to describe the evolution of mutational load in tumors undergoing CRT, which may have implications for the role of immune checkpoint inhibitor therapies. Locally advanced rectal cancer patients treated with neoadjuvant CRT were identified. DNA extraction from clinical FFPE samples and whole exome sequencing was performed on pre- and post-treatment tumor and matched germline DNA followed by standard mutation analysis pipelines to identify somatic mutations, insertion/deletions, and copy number variations (CNV). We identified 40 pre- and post-CRT matched tumor samples from 20 patients. All were uniformly treated with pre-operative fluoropyrimidine-based CRT (50.4 Gy) followed by surgery within 8-10 weeks. Ten patients were classified as NR while 10 patients were partial or complete responders (PR/CR) at the time of surgery. In our initial cohort, all FFPE samples (n = 12) were successfully sequenced and passed quality control metrics. Mean somatic mutational burden pre-CRT was 7.26 mutations/Mb (range, 5.37-9.85), which is consistent with prior reports for colorectal tumors. Unexpectedly, mean somatic mutational burden in the post-CRT samples was not increased (mean, 6.40 mutations/Mb; range, 3.22-10.2) even though 83% of samples were from potentially aggressive tumors harboring mutations in the KRAS oncogene. Among the NR cases, 75% had concurrent KRAS/TP53 mutations. This observation is consistent with our recent findings on the radioresistance of this tumor genotype in lung and colon cancer patients, which confirms the usefulness of the current study cohort. However, our sample size is currently too small for meaningful statistical comparisons. Preliminary CNV analysis showed no major chromosomal changes between pre- and post-CRT samples. We have established feasibility and a pipeline for performing whole exome sequencing on FFPE pre- and post-CRT rectal samples. Our initial, KRAS mutation-enriched cohort demonstrates no significant change in mutational burden between pre- and post-CRT samples, suggesting that the evolution of treatment resistant subclones is not through hypermutation.

Use of Liquid Biopsy in Monitoring Colorectal Cancer Progression Shows Strong Clinical Correlation

BackgroundPatients with colorectal cancer (CRC) who are sensitive to epidermal growth factor antibodies inevitably acquire drug resistance. This study aimed to determine the usefulness of liquid biopsies for prognosis and clinical correlation. Materials and MethodsFor liquid biopsy tests, we extracted blood from 140 CRC patients with matched tumor samples. Circulating tumor cells (CTCs) and tumor DNA (ctDNA) were extracted before surgery and treatment. Samples were quantified and tested for mutations in KRAS, NRAS and BRAF. Kaplan-Meier analyses were performed for different groups of patients for association to overall survival. ResultsAmong the 140 CRC cases, we observed good agreement collectively in the molecular signatures of CTCs and ctDNA with matched tumor specimens (97% concordance). Patients who were subsequently refractory to either cetuximab or panitumumab showed changes in the molecular profiles and were positive for KRAS, NRAS or BRAF. Interestingly, we observed that most of these changes were detected in CTCs analyses first. Stratified analyses conducted by the change in molecular profiles showed this group of patients to have worse survival outcome compared with the wild type group. ConclusionsMonitoring CRC patients’ molecular changes in response to treatment via CTCs and ctDNA can provide real-time information to disease changes. The study demonstrated that the emergence of secondary mutations were strongly associated to poorer survival after treatment.

Gene expression profiling in uveal melanoma: technical reliability and correlation of molecular class with pathologic characteristics

BackgroundA 15-gene expression profile test has been clinically validated and is widely utilized in newly diagnosed uveal melanoma (UM) patients to assess metastatic potential of the tumor. As most patients are treated with eye-sparing radiotherapy, there is limited tumor tissue available for testing, and technical reliability and success of prognostic testing are critical. This study assessed the analytical performance of the 15-gene expression test for UM and the correlation of molecular class with pathologic characteristics.MethodsInter-assay, intra-assay, inter-instrument/operator, and inter-site experiments were conducted, and concordance of the 15-gene expression profile test results and associated discriminant scores for matched tumor samples were evaluated. Technical success was determined from de-identified clinical reports from January 2010 - May 2016. Pathologic characteristics of enucleated tumors were correlated with molecular class results.ResultsInter-assay concordance on 16 samples run on 3 consecutive days was 100%, and matched discriminant scores were strongly correlated (R2 = 0.9944). Inter-assay concordance of 46 samples assayed within a one year period was 100%, with an R2 value of 0.9747 for the discriminant scores. Intra-assay concordance of 12 samples run concurrently in duplicates was 100%; discriminant score correlation yielded an R2 of 0.9934. Concordance between two sites assessing the same tumors was 100% with an R2 of 0.9818 between discriminant scores. Inter-operator/instrument concordance was 96% for Class 1/2 calls and 90% for Class 1A/1B calls, and the discriminant scores had a correlation R2 of 0.9636. Technical success was 96.3% on 5516 samples tested since 2010. Increased largest basal diameter and thickness were significantly associated with Class 1B and Class 2 vs. Class 1A signatures.ConclusionsThese results show that the 15-gene expression profile test for UM has robust, reproducible performance characteristics. The technical success rate during clinical testing remains as high as first reported during validation. As molecular testing becomes more prevalent for supporting precision medicine efforts, high technical success and reliability are key characteristics when testing such limited and precious samples. The performance of the 15-gene expression profile test in this study should provide confidence to physicians who use the test’s molecular classification to inform patient management decisions.

Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in melanoma cells

A majority of patients with BRAF-mutated metastatic melanoma respond to therapy with BRAF inhibitors (BRAFi), but relapses are common owing to acquired resistance. To unravel BRAFi resistance mechanisms we have performed gene expression and mass spectrometry based proteome profiling of the sensitive parental A375 BRAF V600E-mutated human melanoma cell line and of daughter cell lines with induced BRAFi resistance. Increased expression of two novel resistance candidates, aminopeptidase-N (CD13/ANPEP) and ETS transcription factor FLI1 was observed in the BRAFi-resistant daughter cell lines. In addition, increased levels of the previously reported resistance mediators, receptor tyrosine kinase ephrine receptor A2 (EPHA2) and the hepatocyte growth factor receptor MET were also identified. The expression of these proteins was assessed in matched tumor samples from melanoma patients obtained before BRAFi and after disease progression. MET was overexpressed in all progression samples while the expression of the other candidates varied between the individual patients. Targeting CD13/ANPEP by a blocking antibody induced apoptosis in both parental A375- and BRAFi-resistant daughter cells as well as in melanoma cells with intrinsic BRAFi resistance and led to dephosphorylation of EPHA2 on S897, previously demonstrated to cause inhibition of the migratory capacity. AKT and RSK, both reported to induce EPHA2 S897 phosphorylation, were also dephosphorylated after inhibition of CD13/ANPEP. FLI1 silencing also caused decreases in EPHA2 S897 phosphorylation and in total MET protein expression. In addition, silencing of FLI1 sensitized the resistant cells to BRAFi. Furthermore, we show that BRAFi in combination with the multi kinase inhibitor dasatinib can abrogate BRAFi resistance and decrease both EPHA2 S897 phosphorylation and total FLI1 protein expression. This is the first report presenting CD13/ANPEP and FLI1 as important mediators of resistance to BRAF inhibition with potential as drug targets in BRAFi refractory melanoma.

Abstract 743: Detection of TERT C228T and C250T promoter mutations in melanoma tumor and plasma samples using novel mutation-specific droplet digital PCR assays

Abstract Purpose: Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. Mutations in 1 of 2 hot spots in the TERT promoter sequence are found in several cancers, including up to 85% of melanomas and the majority of cases that lack BRAF or NRAS mutations (about one-third of melanomas). Due to the high G-C content of the TERT promoter sequence these mutations can be difficult to detect using NGS approaches. We developed novel droplet digital PCR (ddPCR) assays to detect these 2 mutations with high sensitivity and specificity, and demonstrate the application of these assays in melanoma clinical samples. Methods: Assays were optimized using cell lines with Sanger sequencing-confirmed mutations: glioblastoma A172 (C228T), and melanoma NYU12-126 (C250T). We varied assay designs and amplification conditions to optimize probe-based detection using the Bio-Rad QX-200 ddPCR system. Assay sensitivities and specificities at various DNA input levels were determined using serial dilutions with 3 replicate wells for each condition. Sensitivity is defined as the lowest mutant allele dilution for which the confidence interval did not overlap with that of the 0% mutant wells. We used normal and cancer-derived DNA sources of different quality (e.g. normal human DNA (Promega), cancer cell lines, plasma and FFPE-derived DNAs) with and without the mutations, and compared the efficiency of detection of amplicons of 88, 113 and 163 base-pairs. We compared efficiencies to assays of similar size for RPP30, a housekeeping gene. Patient-matched metastatic melanoma tumors and plasma samples were analyzed to explore the clinical utility of these assays. Results: The assays showed greater sensitivity when higher amounts of DNA were analyzed. For C228T the limit of detection (LOD) of the mutant allele was 1%, 0.25% and 0.1% for 6.6ng/well, 33ng/well and 66ng/well respectively; for C250T the LODs were 0.25%, 0.05% and 0.05% respectively. Using normal human DNA, the efficiency of the TERT assays averaged approximately 90% of that for RPP30 across assays of similar size, and no decrease in assay efficiency was observed as amplicon length increased. In contrast, whereas amplicon size had only a modest effect on assay efficiency in plasma cfDNA, it gave a more pronounced effect on FFPE DNA’s, decreasing to 38% for the 163bp amplicon. We observed 100% concordance between TERT mutation detection by SNaPshot and ddPCR in 10 FFPE tumor samples, and in plasma samples from 4 metastatic melanoma patients with matching tumor samples. Conclusion: We developed robust ddPCR assays to detect TERT promoter mutations with high sensitivity and specificity. Mutated TERT DNA can be detected and quantitated in the plasma of patients with metastatic melanoma, and is likely to be present in the plasma of other cancer patients in whom TERT mutations occur. Citation Format: Broderick Corless, Gregory Chang, Samantha Cooper, Mahrukh Syeda, Iman Osman, George Karlin-Neumann, David Polsky. Detection of TERT C228T and C250T promoter mutations in melanoma tumor and plasma samples using novel mutation-specific droplet digital PCR assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 743. doi:10.1158/1538-7445.AM2017-743

Abstract 3835: High-purity patient-derived cells: An advanced cell culture model for precision cancer medicine

Abstract Purpose: Patient-derived cells (PDCs) established from malignant effusions can represent an excellent tool for the study of non-small cell lung cancer (NSCLC) with oncogenic driver mutations. However, because of cellular paucity and low tumor purity, PDCs have a limited utility for in-depth molecular analysis. In this study, we developed a high-purity patient-derived cell (HP-PDC) method for primary culture of malignant effusions. Experimental design: HP-PDCs were established by a multistep tumor cell isolation protocol as follows. 1) Initial cultures of malignant effusions were observed under a light microscope to identify spheroid-forming cells. 2) Tumor purity of PDCs was analyzed by Immunofluorescence (IF) and flow cytometry. 3) Mutation status of EGFR or ALK in PDCs was screened by direct or PCR-based sequencing and RT-PCR and then was compared with driver mutations detected in matching patient tumor samples. 4) HP-PDCs were tested for drug efficacies by a cell viability assay, a colony-forming assay, and western blot. 5) HP-PDCs were screened with a panel of 84 kinase inhibitors to identify effective drugs. Results: A consecutive series of 45 malignant effusion samples was collected from 36 patients with advanced NSCLC. Spheroid-forming cells were detected in 19 samples (42.2%) which were cultured in AR5 media to establish PDCs. IF analysis revealed that TTF-1 was upregulated in 9 PDCs. These 9 PDCs with high TTF-1 expression also showed EpCAM-positive cell population in flow cytometry with high purity of over 70%. Using Sanger sequencing, EGFR exon 19 deletion was detected 3 cases, EGFR T790M with L858R in 2 cases, and ALK rearrangements in 4 cases. Two of PDCs established from patients, who carried EGFR mutations and progressed on gefitinib, showed resistance to gefitinib in vitro. One of gefitinib-resistant PDCs was sensitive to AZD9291 with IC50 value of 47 nM. Three of PDCs established from patients, who carried ALK fusion genes and progressed on ALK inhibitors (crizotinib, alectinib, and ceritinib respectively), also recapitulated patient’s response to the ALK inhibitors. In an alectinib-resistant PDC, crizotinib and ceritinib exhibited potency with IC50 values of 321 nM and 215 nM, respectively. Colony-forming assay and western blot results were correlated with the cell viability assay data. Time spent for establishment of HP-PDCs ranged from 26 days to 132 days. Conclusions: Nine HP-PDCs established by multistep tumor cell isolation protocol had high tumor purity, and recapitulated patient’s genomic profile and drug responses. HP-PDC is a powerful preclinical model which predicts responses to targeted drugs in a short period of time and may provide an excellent platform for personalized therapies in patients with advanced NSCLC. Citation Format: Seok-Young Kim, Hwan Kim, Sung-Moo Kim, Hye Ryun Kim, Mi Ran Yun, Ji Min Lee, Hun Mi Choi, Byoung Chul Cho. High-purity patient-derived cells: An advanced cell culture model for precision cancer medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3835. doi:10.1158/1538-7445.AM2017-3835

Abstract 2920: Characterizing human melanoma treatment response in vivo using single-cell mass cytometry analysis of longitudinal tumor biopsies

Abstract Introduction: Metastatic melanoma that harbor BRAFV600E mutations are treated with inhibitors that inhibit MAPK signaling downstream of BRAF. Although these treatments improve patient survival, the majority of tumors develop resistance. It is increasingly clear that the presence and emergence of cell subsets in the tumor microenvironment shapes treatment responses. Mass cytometry is a single cell platform capable of measuring >35 proteins on millions of tumor cells. Here, mass cytometry was used to characterize matched tumor samples obtained before, during, and after therapy in order to quantify the impact of therapy on tumor cell populations. Methods: Melanoma tumors from adults with metastases were biopsied or surgically resected from 12 patients with consent. Patients received two weeks of BRAFV600E inhibitor dabrafenib followed by two weeks of dabrafenib and MEK inhibitor trametinib. Tumors were enzymatically digested and cryopreserved with protocols for mass cytometry (Leelatian and Doxie et al., Cytometry B 2016). Samples were stained with a 30+ antibody panel focused on melanoma identity and markers of lineage and trafficking for leukocytes, fibroblast, and endothelial cells. Viable nucleated cells were identified by total histone H3 and rhodium dye exclusion. Expert biaxial gating and viSNE analysis were used to identify, computationally map, and track populations and their subsets over time. Tissue microarrays were also analyzed by histology to confirm findings observed by mass cytometry. Results: More than 99% of cells were characterized and fell into one of the four major populations. Melanoma phenotypes included loss of MHC class I and/or expression of one or more markers of identity including SOX10, SOX2, nestin, S100β, and MCAM. Significant decreases in nestin, MCAM, MHC I and S100β (p < 0.01, p < 0.05, p < 0.05, and p<0.05, respectively) were observed in cells after treatment. Decreases in nestin and S100β populations identified by mass cytometry were observed in histology analysis of TMAs from matched tumors. Analysis of pre- and post-therapy melanoma cells and BRAF mutated cell lines indicated pre-therapy tumor cells had phenotypes most similar to cell lines. Strikingly, 6hrs or 1 day of treatment of cell lines with dabrafenib and trametinib did not result in decrease in nestin expression. The nestin low post-therapy phenotype of melanoma tumor cells was present in all melanoma cell lines at a low abundance (average of 7.0% +/- 3.9% in cell lines, N = 7). Conclusions: Single cell analysis and longitudinal monitoring of patient’s revealed tumor ecosystems experience rapid changes in cellular diversity during targeted therapy. Lower nestin expression distinguished treated melanoma tumor cells. The in vitro results suggest that in vivo remodeling of the tumor microenvironment results from a shift in host-tumor cellular interactions and not a direct response to inhibitors. Citation Format: Deon B. Doxie, Allison R. Greenplate, Kirsten E. Diggins, Caroline E. Maier, Jeffrey A. Sosman, Mark C. Kelley, Jonathan M. Irish. Characterizing human melanoma treatment response in vivo using single-cell mass cytometry analysis of longitudinal tumor biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2920. doi:10.1158/1538-7445.AM2017-2920

Association of mutant EGFR L858R and exon 19 concentration in circulating cell-free DNA using droplet digital PCR with response to EGFR-TKIs in NSCLC.

The present study aimed to determine the diagnostic concordance of plasma epidermal growth factor receptor (EGFR) mutation using droplet digital polymerase chain reaction (ddPCR) with tumor tissue samples and the predictive clinical significance of plasma EGFR mutation concentration. Plasma DNA samples from patients with non-small cell lung cancer (NSCLC) were analyzed for EGFR exon 21 codon 858 (L858R) mutation, deletion of exon 19 (ex19del) and exon 20 codon 790 (T790M) mutation using ddPCR. Firstly, the mutations in the plasma samples were compared with the matched tumor samples to determine the concordance. Secondly, image examination follow-ups were analyzed to assess the association between plasma EGFR mutation concentration and patients' response to EGFR-tyrosine kinase inhibitors (TKIs). A total of 51 patients with NSCLC were enrolled, including 48 newly diagnosed patients. Compared with tumor tissue samples, the sensitivity and specificity of ddPCR were 76.19% (16/21) and 96.55% (28/29) for mutant L858R, and 88.89% (8/9) and 100% (41/41) for ex19del, respectively. No patient exhibited the T790M mutation in the tumor tissue or plasma samples. Furthermore, 5 patients with the L858R mutation and 4 patients with ex19del in plasma and tumor tissue samples had been followed up with image examination for ≥3 months following EGFR-TKI treatment. The baseline mutant EGFR concentrations were positively correlated with a reduction in tumor burden (Spearman's r=0.7000, P=0.0358). When analyzed separately, ex19del concentrations (Spearman's r=1.0000, P<0.0001) were also positively correlated with the reduction, while mutant L858R concentrations were not (Spearman's r=0.7000, P=0.1881). In the present study, detection of plasma EGFR mutations using ddPCR exhibited sufficient concordance with tumor tissue sample results. Baseline plasma mutant EGFR and ex19del concentrations were significantly and positively correlated with response to EGFR-TKIs.

A prospective analysis of germline alterations (GA) in biliary tract cancer (BTC).

4085 Background: The incidence of hereditary cancer predisposition syndromes in patients (pts) with BTC is unknown. Cholangiocarcinoma has been reported in pts with germline mutations in BAP1, BRCA1/2, and mismatch repair genes. These associations are poorly characterized to date and the majority of pts do not undergo clinical germline analysis (CGA). Methods: Pts with BTC were offered consent to CGA between 01/2016 and 01/2017 under an IRB approved protocol (NCT01775072). Using the MSK-IMPACT platform, 76 genes associated with hereditary cancer predisposition were analyzed for germline variants and matched tumor samples were analysed for somatic alterations in &gt; 340 genes. Demographic and clinical data were collected. Results: 78 patients were accrued: Intrahepatic = 52, extrahepatic = 13, gallbladder = 13. Median age at diagnosis was 57 years (range 21-80), 45 (58%) had a positive family history of cancer in at least one 1st degree or two 2nd degree relatives. 7 patients had a personal history of cancer. A pathogenic or likely pathogenic GA was identified in 16 pts (20%). (See table). Conclusions: Prospective analysis of GAs in pts with BTC, unselected by family history or age, revealed potentially actionable findings in 20% of pts. CGA in pts with BTC may benefit patients and their families in view of screening and therapeutic implications. [Table: see text]