Clinical FFPE Samples Research Articles

Defining tissue requirements for a reliable detection of tumor mutational burden (TMB) in clinical FFPE samples.

e14265 Background: In the context of immuno-oncology related cancer treatment, tumor mutational burden (TMB) is currently explored as a predictive biomarker for several human malignancies. Several sequencing assays are increasingly commercially available. Established methodologies require rather large amounts of DNA input (in the range of 100 ng) which, however, are frequently not available from small biopsies. We aim to investigate how tissue size and DNA input influence TMB scores. Methods: DNA from 20 specimens (12 biopsies of non-small cell lung cancer (NSCLC); 8 surgical resection specimens from NSCLC, colorectal cancer and endometrial carcinomas) was manually extracted by using the Qiagen GeneRead DNA FFPE kit. Cases were selected to provide a wide range of relative tumor cell content (from < 10% to > 50%) and to include microsatellite-stable and –instable (MSI) cases. Samples were analyzed in triplicates from predefined numbers of unstained sections of a standardized tissue size. DNA quantification was done fluorometrically and by qPCR. Up to 40 ng of DNA were analyzed with the QIASeq TMB Panel (incl. MSI primer boosters; Qiagen). Sequencing was done on an Illumina NextSeq platform, TMB scores and MSI status were determined by using the CLC workbench 5.0.1 (Qiagen). Results: Biopsy samples generated less numbers of DNA molecules (as detected by unique molecular identifiers, UMIs) and less overall reads compared to resection samples. UMI coverage was > 500x in all samples with > 15 ng DNA input. TMB scores were highly reproducible among all samples with > 15 ng DNA but differed significantly among samples with limited DNA input. Interestingly, TMB scores were inversely correlated with DNA input among those samples with < 15ng. Thus, valid TMB scores could also be obtained from only one slice of 1 cm2 tissue from tumor resections or 3 slices of an endoscopic biopsy (each of 5µm thickness). All pre-characterized MSI carcinomas could be detected correctly. Conclusions: We provide first evidence that TMB can be reliably determined also in small biopsies yielding limited DNA content. However, false high TMB scores can occur in samples with < 15ng DNA input. Our results contribute to the definition of minimum requirements (tissue size and DNA input) for valid TMB measurement on clinical FFPE samples.

Abstract P4-02-12: Comparison of RT-qPCR with consensus immunohistochemistry by three pathologists for ER, PR, HER2 and Ki-67 in Chinese breast cancer patients

Abstract Background During the diagnostic work-up of breast carcinomas, immunohistochemistry (IHC) is the currently used method for assessing the expression of estrogen- (ER) and progesterone-receptors (PR), human epidermal growth factor receptor 2 (HER2) as well as of Ki-67 as a marker of tumor cell proliferation. In this study, we analyzed the concordance of these four breast cancer biomarkers between the RT-qPCR- and IHC-based (evaluated by three independent pathologists) determinations. Methods The expression of ER/ESR1, PR/PGR, HER2/ERBB2 and Ki-67/MKI67 was determined in 269 FFPE breast cancer samples with tumor content >20% from Chinese patients. For IHC, the samples were freshly cut, stained and assessed by three independent pathologists using the same scoring methods in a blinded fashion (positivity defined as: ER/PR ≥1%, HER2 >2+ and Ki-67 ≥20%). Measurement of the markers on the mRNA level was done on total RNA extracts prepared from whole tissue sections from the same FFPE blocks using the CE-marked RT-qPCR based IVD MammaTyper® on a Cobas® z480 qPCR cycler. IHC assessments of the three pathologists were compared to each other with regard to concordance of positive/negative results. Subsequently, agreement of RT-qPCR and IHC results for each marker and in samples in which the three pathologists had a consensus positive/negative IHC result was determined. Furthermore, we compared the MammaTyper® assessments from a subset of whole FFPE sections to data obtained from paired samples enriched for invasive carcinoma via macrodissection. Results From the 269 samples, 256 were available for final analysis. When excluding cases with discordant IHC callings between the three pathologists (6.0% for ER; 7.4% PR; 4.1% Her2; 17.1% Ki-67)) the concordance to the RT-qPCR determination and consensus IHC-based analysis displayed an excellent agreement for ER (OPA: 95.4%, PPA: 97.5%, NPA: 91.5%, Kappa: 0.897), PR (OPA: 91.1%, PPA: 89.6%, NPA: 93.1%, Kappa: 0.820) and HER2 (OPA: 97.1%, PPA: 91.9%, NPA: 100.0%, Kappa: 0.936). For cancer MKI67 mRNA and Ki-67 protein expression, a lower but still good concordance was found (OPA: 90.1%, PPA: 91.8%, NPA: 83.3%, Kappa: 0.707). In addition, we could demonstrate an excellent agreement of quantitative RT-qPCR measurements between whole surface and paired tumor-enriched samples in 99 Chinese breast cancer patients with R2 of 0.927 for ER, 0.926 for PR, 0.923 for HER2 and 0.908 for KI67. Even under highly standardized IHC scoring conditions, the discordance rates in the RT-qPCR marker callings with 0.0% for ESR1, 5.0% for PGR, 3.0% for ERBB2, 13.1% for MKI67 were lower than disagreements by three pathologists on the identical slide. Conclusion Standardized determination of the breast cancer biomarkers ER, PR, HER2 and Ki-67 on the mRNA level shows high concordance to a consensus IHC determined by three experienced pathologists indicating that RT-qPCR may be a valid alternative for determining the four breast cancer biomarkers. In line with previous research we could show on a large set of samples that macrodissection is not required for reliable assessment of the four breast cancer markers in clinical FFPE samples. Citation Format: Teng X, Li X, Xu S, Zhang J, Hartmann K, Laible M, Hipfel R, Bai Y, Ba X, Wu Z, Wirtz RM, Liu S, Ugur S. Comparison of RT-qPCR with consensus immunohistochemistry by three pathologists for ER, PR, HER2 and Ki-67 in Chinese breast cancer patients [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 P4-02-12.

Abstract 2106: Focal gene expression profiling of counterstained FFPE with correlation to morphology using TempO-Seq

Abstract We previously reported on use of the whole transcriptome TempO-Seq® assay to profile gene expression from 1 mm2 focal areas of 5 µm thick FFPE sections of normal and cancerous tissue to identify disease biomarkers and mechanistic pathways. We are now presenting data demonstrating that the TempO-Seq assay can be performed as an “in situ” assay on slides by an automated slide stainer, followed by counterstaining. Then, using an automated digital imaging platform, areas as small as 30 μm in diameter within the FFPE section can be profiled, permitting the gene expression data to be correlated directly to the specific morphology of that focal area. The pathologist uses a computer interface to select areas for profiling during the course of the histologic examination of the section, and then the TempO-Seq probes are automatically recovered from those region(s) of interest. After depositing them into PCR tubes, the assay is completed with PCR amplification and sample barcoding before pooling the samples into a library for sequencing. Analysis of the sequencing data is carried out automatically to report results. There is no use of laser capture or destruction of the tissue. The TempO-Seq processed slides can be archived and additional areas may be sampled at a later date. In this study, we sample replicate areas of matched normal vs. cancerous tissue, measuring gene biomarkers of clinical utility, gene fusions, and SNPs. We present gene expression profiles for replicate areas of pure stroma, single normal glands, single high-grade PIN glands, and single cancerous glands from prostate FFPE, demonstrating the statistical power of the TempO-Seq in situ assay as well as the correlation to focal histology and the use of focal differential expression to elucidate molecular pathways involved in the transition from normal tissue to cancer. We report measurements of clinically relevant biomarkers and signatures from clinical FFPE samples, plus therapeutic targets and drug resistance genes. This new approach demonstrates that complex molecular tests can be carried out by any pathologist in their own lab, and makes moot the issues of “% cancer” and amount of tissue required for testing. The approach we present brings extraction-free complex molecular testing of FFPE into the pathology lab and takes molecular pathology to a new level of simplicity, focal precision and correlation to morphology. Citation Format: Elliot Imler, Milos Babic, Deanna Adams, Peter Shepard, Joanne Yeakley, Raymond B. Nagle, Bruce Seligmann. Focal gene expression profiling of counterstained FFPE with correlation to morphology using TempO-Seq [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 2106.

Abstract 4331: High-throughput whole-genome sequencing of formalin fixed, paraffin-embedded tissues from colorectal cancer patients

Abstract Formalin-fixed, paraffin-embedded (FFPE) storage is a universally-adopted, cost-effective and long-term solution for tissue storage. Given the abundance of FFPE clinical samples, they provide a good source of tissue for genome sequencing studies. However, the extraction of nucleic acids from FFPE tissues yield highly fragmented DNA of variable quality. The poor quality and low yield make it difficult to generate adequate sequencing libraries for genomic studies. For a population cancer genome study, we optimized a procedure for whole-genome sequencing on FFPE tumor and normal tissue of colorectal cancer patients. This pilot study aimed to first identify changes in DNA copy number using low-pass sequencing, with the additional goal of later adding structural variant and gene fusion detection and single nucleotide variant analysis with deeper sequencing. We processed, sequenced and analyzed 193 patients. These FFPE samples had varying storage characteristics. We optimized the initial extraction quality thresholds, DNA extraction and library preparation to generate high quality sequencing data. We identified an initial sample quality and library threshold to hold the best correlation for sequencing quality. Optimal yield and DNA size from extracted FFPE was achieved by using Promega's LEV DNA FFPE Extraction Kit. Library prep optimization involved using mechanical shearing of DNA to increase overall library size for structural variant detection as well as using unique dual index adapters to identify index swapping artifacts during sequencing. An additional cleanup was also performed post-fragmentation to increase overall library size. Overall, we successfully developed a robust protocol for conducting whole genome sequencing on FFPE tumor samples. Citation Format: Matthew Kubit, Christina Wood-Bouwens, Sue Grimes, John Bell, GiWon Shin, Billy Lau, Mickey Miller, Kenneth Day, Helaman Escobar, Hanlee Ji, Lincoln Nadauld, Paul Van Hummelen. High-throughput whole-genome sequencing of formalin fixed, paraffin-embedded tissues from colorectal cancer patients [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 4331.

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.

A robust targeted sequencing approach for low input and variable quality DNA from clinical samples

Next-generation deep sequencing of gene panels is being adopted as a diagnostic test to identify actionable mutations in cancer patient samples. However, clinical samples, such as formalin-fixed, paraffin-embedded specimens, frequently provide low quantities of degraded, poor quality DNA. To overcome these issues, many sequencing assays rely on extensive PCR amplification leading to an accumulation of bias and artifacts. Thus, there is a need for a targeted sequencing assay that performs well with DNA of low quality and quantity without relying on extensive PCR amplification. We evaluate the performance of a targeted sequencing assay based on Oligonucleotide Selective Sequencing, which permits the enrichment of genes and regions of interest and the identification of sequence variants from low amounts of damaged DNA. This assay utilizes a repair process adapted to clinical FFPE samples, followed by adaptor ligation to single stranded DNA and a primer-based capture technique. Our approach generates sequence libraries of high fidelity with reduced reliance on extensive PCR amplification—this facilitates the accurate assessment of copy number alterations in addition to delivering accurate single nucleotide variant and insertion/deletion detection. We apply this method to capture and sequence the exons of a panel of 130 cancer-related genes, from which we obtain high read coverage uniformity across the targeted regions at starting input DNA amounts as low as 10 ng per sample. We demonstrate the performance using a series of reference DNA samples, and by identifying sequence variants in DNA from matched clinical samples originating from different tissue types.

Abstract LB-B11: Development and validation of ColoScape™: A new colorectal cancer mutation detection assay™

Abstract Introduction: Colorectal cancer is a highly preventable disease as early detection increases rates of patient survival to near 100%. Herein we report the development and validation of ColoScape™, a highly sensitive test powered by XNA technology to detect novel multigene mutation biomarkers in CRC by real-time PCR. The assay allows the sensitive detection of the presence or absence of mutations in the targeted regions of the genes interrogated in tissue biopsy (FFPE) and plasma samples.Methodology: The high sensitivity of this multigene biomarker assay is achieved due to xenonucleic acid (XNA) probe technology. XNA probes are novel backbone modified oligomers with natural nucleoside bases (A, T, C and G) that hybridize by Watson-Crick base pairing to natural DNA and RNA with much higher binding affinity. XNA probes are designed that bind to the selected wild-type sequences at the respective genetic loci in the target genes. These XNA probes cannot be extended by DNA polymerase thus suppress amplification of WT DNA templates and only allow amplification of the target mutant DNA templates in the sample. For each of the selected mutation sites, primers and FAM-labeled TaqMan probes were designed and tested together with the selected XNA oligomers. An internal PCR control selected in the Human -Actin (ACTB) gene was employed utilizing an HEX-labeled TaqMan probe.Results: The ColoScape™ kit was demonstrated to have robust analytical performance and clinical accuracy for FFPE, stool and plasma samples. The rapid, precise and sensitive molecular assay for mutation detection in colon cancer has key benefits listed below. The assays showed a sensitivity of as low as 0.1% mutation in 5-10 ng of WT DNA/well. No cross-reactivity was observed with wild-type up to 320ng purified gDNA and up to 20ng FFPE DNA per reaction demonstrating high specificity of the ColoScape™ assay. Intra-assay, inter-assay, lot-to-lot and operator variation comparison showed CV% between 3% and 8%. Excluding pre-cancer samples, the assay clinical specificity and sensitivity were 95% and 100%, respectively. Pre-cancer detection sensitivity was 60% and 62.5% for stool samples. For tested FFPE clinical samples, the assay specificity and sensitivity were 95% and 91% respectively while the assay clinical specificity and sensitivity were both 100% for plasma samples.Conclusion: The ColoScape™ Colorectal Cancer Mutation Detection qPCR assay is shown to be a sensitive tool intended to aide in the early detection of colorectal cancer, disease monitoring and therapeutic interventions. Citation Format: Michael J. Powell, Elena Peletskaya, Qing Sun, Larry Pastor, Aiguo Zhang, Kamila Koprowska, Walter Bodmer. Development and validation of ColoScape™: A new colorectal cancer mutation detection assay™ [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B11.

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.

Abstract 5095: Development of a pharmacodynamic biomarker assay for AZD4785, an antisense oligonucleotide targeting KRAS

Abstract AZD4785 is a potent and selective high affinity 2’-4’ constrained ethyl residues (cEt) containing therapeutic antisense oligonucleotide (ASO) targeting KRAS. In a patient-derived explant model of non-small cell lung cancer (NSCLC) carrying a KRAS G12C mutation systemic dosing of AZD4785 resulted in tumor regression. To determine the level of KRAS mRNA knockdown achieved by AZD4785 at a dose which causes regression (250 mg/kg/wk), tumor samples were collected and analyzed by RT-PCR. Mean levels of KRAS mRNA knockdown relative to the PBS control group were determined to be 61% and 78% from studies dosed for 2 and 4 weeks, respectively. However, by RT-PCR there was significant variability in KRAS mRNA levels within the PBS control group, and level of knockdown varied with different housekeeper normalization genes. In early clinical studies it will be essential to measure KRAS expression in tumor samples as a pharmacodynamic marker for AZD4785, hence we sought to develop a more robust assay compatible with FFPE clinical samples with low RNA yields. KRAS mRNA levels were quantified by the nanoString platform, using the panCancer pathways 770 gene codeset. This assay generated robust data from RNA extracted from 2 x 5µm sections of FFPE material from each sample, despite very limited yields of RNA. AZD4785 treatment resulted in comparable levels of KRAS knockdown to the RT-PCR assay, but assay variability was greatly reduced, from stdev >50% (RT-PCR) to 5% (nanoString) for PBS control group samples. Using the nanoString assay, mean KRAS mRNA knockdown was 52% and 68% (p<0.005). We also analyzed the effect of AZD4785 treatment on the wider 770 gene panel. Further evidence of KRAS mRNA knockdown was observed through significant downregulation of transcriptional targets of KRAS signalling (FOSL1, DUSP5, DUSP6) relative to the PBS control group. Neither HRAS nor NRAS levels were affected by AZD4785 treatment. This nanoString assay was applied to a panel of 50 clinical FFPE NSCLC samples and KRAS mRNA was detected above baseline in all samples. Furthermore the intrapatient variability of the KRAS mRNA signal was tested in sections >50μm apart within four NSCLC tissues and was found to be ≤21%. Taken together, these data validate the use of the KRAS nanoString assay for use in clinical FFPE tissues, and demonstrate the level of knockdown of KRAS mRNA to be >50% at a dose of AZD4785 that causes regression in a preclinical PDX model. Citation Format: Claire Rooney, Sarah Ross, Anna Staniszewska, Sabine Lennarz, Elaine Kilgour, Alexey S. Revenko, A. Robert Macleod, Andrew Pierce, Paul D. Lyne, J. Carl Barrett, Elizabeth A. Harrington. Development of a pharmacodynamic biomarker assay for AZD4785, an antisense oligonucleotide targeting KRAS [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 5095. doi:10.1158/1538-7445.AM2017-5095

Abstract 1626: Technical variability in NGS immune gene expression and mutation profiling has a nominal effect on tumor classification

Abstract Background: A custom NGS cancer immune gene expression assay was developed which measures the transcript level of >350 genes involved in T-cell receptor signaling (TCRS), tumor infiltrating lymphocyte (TILs) complement as well as other key targets expected to predict the likelihood of patient response to checkpoint inhibitors (CPI). In parallel to the gene expression assay, mutational profiling was carried out using the 409 gene Comprehensive Cancer Panel (ThermoFisher). As variability between runs is common when performing NGS assays a detailed comparison of specific technical variations were assessed for their ability to effect gene expression and mutation profiles of clinical FFPE samples. Methods: Studies were designed to characterize the analytical performance of the immune response NGS assay using RNA and DNA from a subset of 300 FFPE tissues representing NSCLC, melanoma, renal cell carcinoma and bladder cancer. As part of the study, we tested the impact of variability in RNA and DNA input quantity at the library preparation step, sample batch size which affects mapped reads/sample and depth of coverage, and linearity of expression and sensitivity of mutation profiling through serial dilutions of pico-molar (pM) input of normalized library. PCA and unsupervised clustering was performed on samples with checkpoint inhibition, TCRS and TILs genes as well as mutational profiling to reveal sample groups with three distinct immune signatures (low, indeterminate and high). Further correlation and over-representation analysis was performed to determine impact of technical characteristics on these three immune signatures. Results: Immune signatures including mutation profiles and gene expression levels were maintained throughout variable RNA/DNA input amounts at the library generation level as well as with diminution of pM levels of library pooled at the sequencing step. Increase in the number of mapped reads and sequencing depth through decreasing the number of batched samples per sequencing run also did not affect the gene expression and mutation profile signatures of the FFPE derived samples. Conclusion: The gene expression and mutation profiles responsible for classifying FFPE samples using NGS are not affected by variation normally introduced in the technical workflow commonly associated with these platforms. The analytical assessment of input at the nucleic acid, library, and sample size level has shown the plasticity available when using amplicon based NGS technologies for classifying the immune gene expression signature as well as mutational profiles of FFPE derived clinical tumor samples. This flexibility increases the strength and utility of NGS-base gene expression profiling and mutational analysis of tumor samples for both basic research and clinical applications. Citation Format: Sean Glenn, Jeffrey Conroy, Blake Burgher, Sarabjot Pabla, Maochun Qin, Jon Andreas, Vincent Giamo, Marc Ernstoff, Mary Nesline, Ji He, Mark Gardner, Carl Morrison. Technical variability in NGS immune gene expression and mutation profiling has a nominal effect on tumor classification [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 1626. doi:10.1158/1538-7445.AM2017-1626

Comprehensive genomic profiling to merge precision medicine with immunotherapy.

e23108 Background: Immunotherapy is rapidly emerging as one of the most promising therapeutic options in clinical oncology. However, not all patients will respond to immune-oncology drugs and diagnostic assays are urgently needed to identify biomarkers that predict response to these therapies. We developed an assay that utilizes next generation sequencing data to simultaneously determine different types of somatic changes in tumors. The assay was designed, in part, to facilitate identification of cancer patients most likely to respond to immunotherapies by detecting 1) CD274 (PD-L1) copy number alterations, 2) viral infections (HPV16/18 and EBV), 3) microsatellite instability (MSI), and 4) tumor mutation burden. Methods: We developed a 435-gene panel assay (CancerPlex) with the goal of identifying all the genomic changes that inform treatment decisions with the highest possible accuracy. We thoroughly evaluated the performance of the assay using reference samples that represent all classes of genetic variation, including SNPs, indels, copy number changes, rearrangements, HPV/EBV infection and MSI. FFPE clinical samples were also evaluated to assess the ability of the assay to detect genetic variation in complex, heterogeneous tumors. Results: The assay has excellent performance, with a mean sensitivity of 99.4% and specificity of 99.9% for detecting somatic mutations with an allele fraction as low as 10%. The assay identified the MSI status of colorectal tumors with the same sensitivity as immunohistochemistry but with greater sensitivity than PCR. We also showed that calculating tumor mutation burden using the 435-gene panel predicts response to pembrolizumab as effectively as using whole exome sequencing. Among 892 patients across all tumor types, 6.8% were identified as candidates for immunotherapy based upon high tumor mutation burden and/or MSI status. Conclusions: The capacity of the 435-gene panel to determine all of the critical genetic changes, tumor mutation burden, MSI status, CD274 (PD-L1) CNVs, and HPV/EBV status has important ramifications for patient treatment strategies, including identification of patients who are more likely to benefit from immune checkpoint inhibitor therapies.

PUB079 EGFR and BRAF Mutation Detection in FFPE Samples of Lung Adenocarcinoma

EGFR-TKIs have been the standard first-line treatment for metastatic NSCLC patients with sensitive EGFR-mutants, meanwhile, other drugs target different driver mutants like BRAF-inhibitor dabrafenib has shown promising efficacy for metastatic BRAF-mutated NSCLC patients. Therefore, spotting patients carrying the targeted mutations is of great significance. However, molecular detection methods applied in clinical practice, especially detection of BRAF in NSCLC patients needs further exploration. Therefore, more sensitive and economic methods are required. We applied the CastPCR technology to the molecular detection of EGFR (del2235-2249, del2236-2250, T790M, L858R) and BRAF (V600E, G469A, D594G) mutations in 144 treatment-naive adenocarcinomas of the lung, and analyzed the association between the mutation rates and patients' clinicopathological features. EGFR mutation: 74 (51.4%, 74/144) cases were identified harboring EGFR mutations. 58 (40.3%, 58/144) carried sensitizing mutations (19Del or L858R) and 21 (14.6%, 21/144) carried resistant mutations (T790M). 10 (6.9%, 10/144) mutation-positive patients were double-mutated. EGFR mutation rate was significantly higher in female compared with that of males (60.9% v.s. 43.8%, P＜0.05), in non-smokers compared with that of smokers (62.8% v.s. 34.5%, P＜0.05). BRAF mutation:12 (8.3%, 12/144) patients were identified with BRAF mutations. 2 were V600E (16.7%, 2/12) and 10 (83.3%, 10/12) were non-V600E. Among the 10 non-V600E mutations, D594G accounted for 9 (90.0%，9/10) and G469A accounted for only 1 (10.0%, 1/10). Statistical analysis demonstrated that BRAF mutation rate was not associated with any of the clinicopathological features: age, gender, smoking status, differentiation degree, tumor size, regional lymph nodes metastasis and distant metastasis of (P≥0.05). The CastPCR technology is a robust method with high sensitivity for molecular detecting of EGFR and BRAF mutations in clinical FFPE samples.

Abstract 5274: A novel, statistical-based method to determine RNA expression by next-generation sequencing in clinical FFPE samples

Abstract Introduction: Next-generation sequencing (NGS) has become a critical diagnostic assay to identify pathologic SNVs, CNVs, and gene rearrangements. However, it has not been routinely used to assess expression levels of target genes that could indicate patient populations responsive to therapeutics. This is largely due to the absence of reliable bioinformatics tools for the assessment of expression levels within RNA assays. We have developed a novel within-sample distribution-based method that assesses the relative extremity of expression for individual genes. Our predominate focus has been on assessing the expression of NTRK1, NTRK2, NTRK3, ROS1, and ALK, however this method can be readily applied to other genes and NGS platforms. Methods: Within the kinase domain, the expression of a targeted region is represented by the number of unique deduplicated reads for NGS studies and normalized probe expression values (based on spike-in controls) for NanoString studies. A Poisson distribution is used to represent primer expression with the parameters estimated via maximum likelihood. The interquartile range (IQR) of the entirety of the sample's read counts is calculated and only those that do not exceed the third quartile bound by more than 150% of the IQR are considered during parameter estimation. A probability is then assigned to each of the primers based on their read counts. A geometric mean of the individual primer probabilities represents the probability value for the entire gene. Expression values are reported as -log10 (p-value) and cutoffs of 6 and 1 were used to call significant expression for NGS and NanoString platforms, respectively. Results: The NGS-based approach correctly identified significant expression in all gene rearrangement positive cell lines (n = 11). In cell lines not harboring gene rearrangements, the NGS and NanoString platforms showed 100% concordance in calling significant expression (n = 12). In a gene rearrangement negative FFPE cohort, concordance between NGS and NanoString platforms was 97%-99% for target genes (n = 102). ROS1 and ALK were most commonly found to be significantly expressed in FFPE samples (5% and 2%). Conclusions: We have developed a statistical-based approach to detecting expression levels for RNA-based NGS assays. This can be applied to cohort studies to not only identify clinical samples that may benefit from targeted kinase inhibitor therapeutics but also correlate with predicted outcome of disease. Citation Format: Robert Shoemaker, Aaron Berlin, Amy Diliberto, Heather Ely, Marissa Chen, Danielle Murphy, Jason Christiansen, Vince Reuter, Abel Licon. A novel, statistical-based method to determine RNA expression by next-generation sequencing in clinical FFPE samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5274.

Abstract 3626: Establishing a robust NGS laboratory workflow and analysis pipeline for FFPE specimen RNAseq to support biopharmaceutical translational research

Abstract Pharmaceutical R&D translational programs rely on analyzing DNA and RNA from patient specimens. High quality histopathology assessment is not possible from fresh and frozen tissues which are also difficult to store long term for follow up studies. FFPE preserves tissue architecture and allows easy archival storage and extracting genomic information from FFPE clinical trial samples is now commonly done, opening a variety of research possibilities. NGS is becoming the preferred approach to FFPE molecular profiling as it generates richer, more complete data than previously possible with microarray technologies. Our translational research objective was to quantify RNA isoforms for a number of genes requiring that we control for NGS coverage and analysis pipeline biases using embedded controls and comparing multiple calling algorithms. To optimize RNAseq library construction performance using FFPE samples we compared five commercially available rRNA depletion/mRNA selection methods for reducing rRNA abundance: Ovation® (Nugen), Ribo-Zero® (Epicentre), Ribo-Zero GOLD® (Epicentre), RiboGone® (Clonetech) and TruSeq® RNA Access (Illumina). We also evaluated DV200 as a performance metric for predicting NGS library quality for FFPE samples using TruSeq® (Illumina) and SMARTer® (Clonetech) library preparation methods. Finally, we compared RecoverAll® (Life Technologies) and Allprep® (Qiagen) RNA extraction method effects on NGS library quality. By performing the experimental comparisons using two NGS service providers, we were able to assess the impact of laboratory performance on NGS RNAseq results. Ultimately, we were able to define an NGS workflow based on sample QC assessment at multiple points in the process resulting in a highly reliable RNAseq workflow for clinical NGS specimens. Among the rRNA depletion methods, RiboGone and RNA Access outperformed alternative options. RiboGone virtually eliminated rRNA contamination from FFPE specimens with high reproducibility. RNA Access both eliminated rRNA and provided a high fraction of coding RNA sequences relative to the background of ncRNA allowing experimental designs with lower total read numbers. Depending on experimental requirements, either method is reliable; however, the two methods cannot be used interchangeably without computational normalization. The two library preparation and RNA extraction methods performed similarly in this experiment. DV200 was found to be an good metric for evaluating the quality of FFPE RNA and predicting success in generating RNAseq data. Our experience across NGS vendors showed that to get optimal results, vendor performance must be managed by using layers of controls with agreed on laboratory performance metrics and explicitly setting NGS experimental parameters in advance of performing the work. Citation Format: Konstantinos Mavrommatis, Lauren Intagliata, Garth McGrath, Daniel Civello, Maureen Cronin. Establishing a robust NGS laboratory workflow and analysis pipeline for FFPE specimen RNAseq to support biopharmaceutical translational research. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3626.

Abstract 744: Cancer immunotherapy biomarker profiling assay

Abstract Selecting patients predisposed to respond to existing and novel immunotherapy treatments requires the development of novel prognostic and predictive biomarkers. Recent reports have identified several gene expression signatures specific for immunity status and immune contexture in solid tumor microenvironments, and these enable predictions of efficacy of a number of chemo- and immunotherapeutics. Robust methods for molecular characterizations of the immune mechanism in the tumor microenvironment are essential to meet the imminent need for diagnostic approaches that identify patient populations responsive to the growing number of immunotherapeutic treatments. Toward this goal, we developed the Cancer ImmuneNet 2500 (CIN2500) assay for profiling cellular composition in the tumor microenvironment and discovery of novel prognostic and predictive immune response biomarkers. The CIN2500 panel provides signatures for approximately 400 immunity-related genes from 16 predictive and prognostic core genes that have been validated in recent chemo- and immunotherapy clinical trials across several tumor types, including melanoma, colorectal, breast, and lung cancers. In addition, using computational analysis of an immunotherapy network model, we predicted and included in the assay approximately 300 key nodes in pathways specific for antigen presentation and recognition, inhibition, activation and motility of immune cells, and adhesion and apoptosis of cancer cells. The CIN2500 panel also includes a comprehensive set of 1,700 genes specific for detection and quantitative profiling in the tumor microenvironment of different types of activated immune cells of adaptive and innate immunity, and stromal, fibroblast, cancer, endothelial and adipose cell types. The CIN2500 assay quantitatively profiles the expression levels of approximately 2,500 key cancer genes from 10-100ng of total RNA using multiplex RT-PCR amplification followed by Next-Generation Sequencing (NGS). Built-in standards for each gene target enable sample-to-sample calibration of the NGS data and provide a reference to adjust for background noise that often depends on the quality of samples. Control studies have shown that the CIN2500 assay quantifiably measures 4 orders of magnitude variation in the expression levels of 2,500 immune-related genes from as few as 100 cells in whole lysate, and profiles from frozen biopsies, surgically-removed tumor samples, PBMCs, and FFPE clinical samples show a comparable range and sensitivity. We will present profiling data from infiltrating immune cells and key intact and deficient immune mechanisms in the tumor microenvironment of breast cancer samples to demonstrate the performance and utility of the CIN2500 assay. Citation Format: Alex Chenchik, Leonid Iakoubov, Mikhail Makhanov, Costa Frangou. Cancer immunotherapy biomarker profiling assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 744.

Abstract 1397: Oncomine focus assay: Simultaneous detection of clinically relevant hotspot mutations, CNVs, and gene fusions in 52 oncogenes relevant to solid tumors

Abstract Introduction: The Oncomine™ Focus Assay (OFA) is aimed to simultaneously detect and report hotspot mutations, Copy Number Variants (CNVs) and gene fusions in 52 oncogenes clinically relevant to solid tumors. With minimal DNA/RNA input, OFA employs AmpliSeq™ library preparation chemistry to enrich target regions for Ion Torrent™ Next Generation Sequencing. Variants are annotated for actionability by Oncomine® Knowledgebase. Here we report an analytical validation of OFA. Methods: DNA and RNA, extracted from the FFPE processed GM12878, was used as the negative control to evaluate the specificity of OFA. A RNA sample containing multiple oncogenic gene fusions and a DNA sample containing multiple hotspot SNV and indels were used as the positive controls. Fresh DNA from cancer cell lines (HCC1143 or NCI-H2122) along with DNA from matched normal cell lines, HorizonDx NGS standards TruQ-1, TruQ-2, several engineered FFPE samples with gene fusions or copy number changes, and 43 clinical FFPE samples of a variety of solid tumor types (non-small cell lung cancer, colorectal cancer, gastrointestinal stromal tumor, ovarian cancer, pancreatic cancer, and melanoma) were used to evaluate the OFA performance. The analysis of the sequencing data was primarily performed with the OFA pipeline integrated with the Oncomine® Knowledgebase. Only the genetic alterations with clinical utility were selected as the final output from the data pipeline. The SBS and indels detected by OFA were confirmed by Sanger sequencing. Any CNVs detected were confirmed by FISH, if possible, and detected fusions were confirmed by RT-PCR or FISH. Results: No clinically relevant genetic alterations were detected from the negative control FFPE-GM12878, indicating the high specificity of the OFA. The LOD for SBS and short indel detection was 5%, as assessed by TruQ-1 and TruQ-2, each containing 9-10 variants. The assay can detect gene fusion RNA present as 0.1%-1% of the total RNA, and gene amplifications are detected with a minimum of 50% tumor cell content. A known 12X CCND1 amplifications in HCC1143 and a 13X MYC amplification in NCI-H2122 were detected. In addition, a 13X EGFR amplification and 11X CDK4 amplification were detected in a lung cancer FFPE sample and confirmed by FISH. The analytical specificity for detection of SBS and indels was 100% (25/25). The analytical specificity for detection of CNV gain and gene fusions, and the assay sensitivity are currently under assessment. Conclusions: These results demonstrate the high sensitivity and specificity of OFA. With as little as 10 ng RNA and 10 ng DNA, OFA provides biomarker analysis of patient FFPE tumor specimens focusing on the detection of genetic alterations that have been targeted by oncology drugs or supported by published clinical evidence. Citation Format: Peng Fang, Zhenyu Yan, Paul Labrousse, Weihua Liu, Jennifer Biroschak, Jennifer Wright, Selby Weeks, Cindy Spittle, Chad Galderisi, Jin Li. Oncomine focus assay: Simultaneous detection of clinically relevant hotspot mutations, CNVs, and gene fusions in 52 oncogenes relevant to solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1397.

Characterization of FGFR1 Locus in sqNSCLC Reveals a Broad and Heterogeneous Amplicon.

FGFR1 amplification occurs in ~20% of sqNSCLC and trials with FGFR inhibitors have selected FGFR1 amplified patients by FISH. Lung cancer cell lines were profiled for sensitivity to AZD4547, a potent, selective inhibitor of FGFRs 1–3. Sensitivity to FGFR inhibition was associated with but not wholly predicted by increased FGFR1 gene copy number. Additional biomarker assays evaluating expression of FGFRs and correlation between amplification and expression in clinical tissues are therefore warranted. We validated nanoString for mRNA expression analysis of 194 genes, including FGFRs, from clinical tumour tissue. In a panel of sqNSCLC tumours 14.4% (13/90) were FGFR1 amplified by FISH. Although mean FGFR1 expression was significantly higher in amplified samples, there was significant overlap in the range of expression levels between the amplified and non-amplified cohorts with several non-amplified samples expressing FGFR1 to levels equivalent to amplified samples. Statistical analysis revealed increased expression of FGFR1 neighboring genes on the 8p12 amplicon (BAG4, LSM1 and WHSC1L1) in FGFR1 amplified tumours, suggesting a broad rather than focal amplicon and raises the potential for codependencies. High resolution aCGH analysis of pre-clinical and clinical samples supported the presence of a broad and heterogeneous amplicon around the FGFR1 locus. In conclusion, the range of FGFR1 expression levels in both FGFR1 amplified and non-amplified NSCLC tissues, together with the breadth and intra-patient heterogeneity of the 8p amplicon highlights the need for gene expression analysis of clinical samples to inform the understanding of determinants of response to FGFR inhibitors. In this respect the nanoString platform provides an attractive option for RNA analysis of FFPE clinical samples.

Sensitive, specific, and modular KRAS, NRAS, and BRAF assays for simultaneous detection of 30 important point mutations in colorectal cancer specimens.

547 Background: Point mutations in KRAS, NRAS, and BRAF are implicated in the oncogenic RAS/RAF/MEK/ERK pathway and are prognostic and predictive markers in treating metastatic colorectal cancer patients. Here, we report the performance of three single tube highly multiplex and modular KRAS/NRAS/BRAF Assays capable of detecting 30 point mutations from FFPE-extracted DNA on the fully automated Modaplex System. Methods: Primers for multiplex KRAS/NRAS/BRAF Assays to detect 13, 13, and 4 point mutations for KRAS, NRAS, and BRAF genes, respectively, were designed using proprietary technology. Analytical sensitivity of each mutation was assessed using mutation specific ultramer oligos or Horizon Dx Mockblock specimens. Analytical specificity was determined on known wild-type FFPE DNAs. The assays were tested on DNA extracted from clinical FFPE specimens that were characterized by pyrosequencing. Modaplex data on these specimens were compared to pyrosequencing for concordance. Results: The modular Modaplex KRAS/NRAS/BRAF point mutation panels detect and differentiate 30 mutations in the KRAS, NRAS, and BRAF genes in three wells. Each assay requires just 5 µL of clinical sample extract (10-50ng DNA per assay) with a setup time of 15-30 minutes. The total run time including data analysis and setup is 4 hours. The kit includes an internal control to determine mutation status; and calibration controls to determine amplicon size. Analytical studies demonstrate the assays are sensitive (number of copies detected in one reaction), selective (mutant to wild-type ratio), and specific (relative to wild-type genomic DNA background). Nearly 110 clinical FFPE samples were tested, and the assays showed more than 95% concordance with pyrosequencing method (98% for BRAF, 97% for KRAS, and 100% for NRAS). Conclusions: Modaplex KRAS/NRAS/BRAF assays provide an accurate and sensitive method of mutation detection. These automated multiplexed assays to detect key mutations in three genes can be performed on a single platform facilitating clinical or translational research. *Modaplex KRAS/NRAS/BRAF Assays are for Research Use Only. Not for clinical diagnostic use.

Detection of a novel RET gene fusion in a non-small cell lung cancer patient using AMP chemistry

In lung cancer, several driver Receptor Tyrosine Kinases (RTKs) have been identified in gene fusions, including ALK, RET, ROS1, NTRK1, NRG1 and FGFR1/2/3. These oncogenic gene fusions have become either direct targets or biomarkers for molecular therapies for patients with lung cancer. Screening patients for the presence of these gene fusions would be critical for oncologists to select appropriate therapeutic intervention or for drug developers to design novel therapeutics and conduct clinical trials. However, detection of fusions, especially unknown fusions, from FFPE samples is challenging for many diagnostic techniques. Archer™ FusionPlex™ Lung Thyroid Panel is built on Anchored Multiplex PCR (AMP™) Chemistry, allowing detection of both novel and known fusions. Here we report the results from our study using the Archer Lung-Thyroid Panel. We identified and verified the presence of a novel fusion between Myosin Phosphatase Rho Interacting Protein (MPRIP) and RET oncogene, in an FFPE sample from a non-small cell lung cancer (NSCLC) patient. FFPE samples from 20 NSCLC patients were analyzed in the study. An RNA sample containing an ALK and ROS1 fusion was used as the positive control, and FFPE processed GM12878 was used as the negative control. In addition, FFPE controls containing TMP3-NTRK1 and CCDC6-RET were also used to evaluate the performance of the panel to detect fusions. Total nucleic acid (TNA) was extracted from the FFPE sections using the Agencourt™ FORMAPURE™ kit. For each sample, 200 ng of TNA was used for library preparation. The barcoded libraries from 14 samples were pooled and sequenced on an Illumina MiSeq using a v2 flow cell. The FASTA files from the MiSeq were then analyzed by the ArcherDX data analysis pipeline to determine the presence of gene fusions with driver genes including ALK, FGFR3, MET, NTRK1, NTRK3, NTRK3, PPARG, RET, and ROS1. The detected fusions in clinical samples were further confirmed by Sanger sequencing and FISH. The known fusions, including EM4-ALK, SLC34A2-ROS1, TMP3-NTRK1, and CCDC6-RET were successfully detected in the positive RNA and FFPE control samples. For all of the clinical FFPE samples, one 10 micron section was sufficient to yield more than the required 200 ng TNA for library preparation. Although three of the twenty (15%) FFPE samples failed the initial sample quality metrics, libraries were prepared for all 20 NCSLC FFPE samples and sequenced on MiSeq. Among the three samples that did not pass the initial sample QC, two of them also failed the QC metrics for fusion detection. From the seventeen samples that did pass the initial QC, one sample failed the fusion QC. Overall, the success rate for the clinical samples tested with this panel was 85%. Among the 20 NSCLC samples, one sample was found to harbor a novel, in-frame fusion between exon 19 of MPRIP and exon 12 of RET. This type of fusion would retain an intact kinase domain (exons 12-18) of RET, and would presumably be oncogenic. Using the RNA portion of the TNA extracted, cDNA was synthesized using an independent method, and sequenced by Sanger. The results confirmed the presence of an RNA transcript spanning exon 19 of MPRIP and exon 12 of RET. Furthermore, FISH was performed with the RET (10q11.2) break-apart probe, and confirmed the rearrangement involving the RET region of chromosome 10. These results show the Archer™ FusionPlex™ Lung Thyroid Panel can accurately detect gene fusions with both known and unknown partner and driver genes. Further investigation is needed to better understand the clinical significance and prevalence of the novel MPRIP and RET fusion.

Abstract A010: Cancer immunotherapy biomarker profiling assay

Abstract Introduction: Selecting patients predisposed to respond to existing and novel immunotherapy treatments requires the development of novel prognostic and predictive biomarkers. Recent reports have identified several gene expression signatures specific for immunity status and immune contexture in solid tumor microenvironments, and enable predictions of efficacy of a number of chemo- and immunotherapeutics. We used these expression signatures to develop a robust standardized assay—the Cancer Immunotherapy 2000 (CI2000) panel—that would dissect immunosurveillance mechanisms and discover novel prognostic and predictive immune response biomarkers. Material and Method: The CI2000 panel provides signatures for approximately 400 immunity-related genes from 16 predictive and prognostic core genes that have been validated in recent chemo- and immunotherapy clinical trials across several tumor types, including melanoma, colorectal, breast, and lung cancers. In addition, using computation analysis of immunotherapy network model, we predicted key nodes in pathways specific for antigen presentation and recognition, inhibition, activation and motility of immune cells, adhesion, and apoptosis of cancer cells. This computational analysis produced approximately 400 more targets beyond the core panel. The CI2000 panel also includes a comprehensive set of 1,200 genes specific for detection and quantitative profiling in the tumor microenvironment of different types of activated immune cells of adaptive and innate immunity. Further, an additional set of housekeeping genes with constant expression between different cancer types was included in the assay for data normalization. Results and Discussion: The CI2000 assay quantitatively profiles the expression levels of approximately 2,000 key cancer genes from 10-100ng of total RNA using multiplex RT-PCR amplification followed by next-generation HT sequencing (NGS). Built-in standards for each gene target enable sample-to-sample calibration of the NGS data and provide a reference to adjust for background noise which often depends on the quality of samples. Control studies have shown that the CI2000 assay quantifiably measures 4 orders of magnitude variation in the expression levels of 2,000 key cancer immune-related genes from as few as 100 cells in whole lysate, and profiles from frozen biopsies, surgically-removed tumor samples, PBMCs, and FFPE clinical samples show a comparable range and sensitivity. We will present profiling data from infiltrating immune cells and key intact and deficient immune mechanisms in the tumor microenvironment of breast cancer samples to demonstrate the performance and utility of the CI2000 assay. Conclusions: Comprehensive profiling of tumor-associated immune cells with the CI2000 gene panel provides a sensitive, standardized, and quantitative method to investigate cancer immunoediting mechanisms. Robust methods for molecular characterizations of the immune mechanism in the tumor microenvironment are essential to meet the imminent need for diagnostic approaches that identify patient population responsive to the growing number of immunotherapeutic treatments. Toward this goal, the described CI2000 assay provides a platform for the discovery prognostic and predictive immune response biomarker signatures. Citation Format: Alex Chenchik, Leonid Iakoubov, Michael Makhanov, Costa Frangou. Cancer immunotherapy biomarker profiling assay. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A010.