Purpose: We developed a method to monitor copy number variations (CNV) in plasma cell-free DNA (cfDNA) from patients with metastatic squamous non-small cell lung cancer (NSCLC). We aimed to explore the association between tumor-derived cfDNA and clinical outcomes, and sought CNVs that may suggest potential resistance mechanisms. Experimental Design: Sensitivity and specificity of low-pass whole-genome sequencing (LP-WGS) were first determined using cell line DNA and cfDNA. LP-WGS was performed on baseline and longitudinal cfDNA of 152 patients with squamous NSCLC treated with chemotherapy, or in combination with pictilisib, a pan-PI3K inhibitor. cfDNA tumor fraction and detected CNVs were analyzed in association with clinical outcomes. Results: LP-WGS successfully detected CNVs in cfDNA with tumor fraction !10%, which represented approximately 30% of the first-line NSCLC patients in this study. The most frequent CNVs were gains in chromosome 3q, which harbors the PIK3CA and SOX2 oncogenes. The CNV landscape in cfDNA with a high tumor fraction generally matched that of corresponding tumor tissue. Tumor fraction in cfDNA was dynamic during treatment, and increases in tumor fraction and corresponding CNVs could be detected before radiographic progression in 7 of 12 patients. Recurrent CNVs, such as MYC amplification, were enriched in cfDNA from posttreatment samples compared with the baseline, suggesting a potential resistance mechanism to pictilisib. Conclusions: LP-WGS offers an unbiased and highthroughput way to investigate CNVs and tumor fraction in cfDNA of patients with cancer. It may also be valuable for monitoring treatment response, detecting disease progression early, and identifying emergent clones associated with therapeutic resistance.

The 1986 Chernobyl nuclear power plant accident increased papillary thyroid carcinoma (PTC) incidence in surrounding regions, particularly for radioactive iodine (131I)–exposed children. We analyzed genomic, transcriptomic, and epigenomic characteristics of 440 PTCs from Ukraine (from 359 individuals with estimated childhood 131I exposure and 81 unexposed children born after 1986). PTCs displayed radiation dose–dependent enrichment of fusion drivers, nearly all in the mitogen-activated protein kinase pathway, and increases in small deletions and simple/balanced structural variants that were clonal and bore hallmarks of nonhomologous end-joining repair. Radiation-related genomic alterations were more pronounced for individuals who were younger at exposure. Transcriptomic and epigenomic features were strongly associated with driver events but not radiation dose. Our results point to DNA double-strand breaks as early carcinogenic events that subsequently enable PTC growth after environmental radiation exposure.

Many detection methods have been used or reported for the diagnosis and/or surveillance of SARS-CoV-2. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive, claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by RT-PCR, probably due to loss or degradation of virus RNA in the sampling process, or even mutation of the virus genome. Therefore, developing highly sensitive methods is imperative to ensure robust detection capabilities. With the goal of improving sensitivity and accommodate various application settings, we developed a multiplex-PCR-based method comprised of 172 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. In addition, optional sequencing can provide further confirmation as well as phylogenetic information of the identified virus(es) for specific strain discrimination, which will be of paramount importance for surveillance purposes that represent a global health imperative. Finally, we also developed in parallel a multiplex-PCR-based metagenomic method that is amenable to detect SARS-CoV-2, with the additional benefit of its potential for uncovering mutational diversity and novel pathogens at low sequencing depth.

Primary squamous cell carcinoma of the thyroid gland is rare. We report here a case of primary squamous cell carcinoma of the thyroid gland in a middle aged woman who had a thyroid nodule of 12 years duration with a recent rapid increase in size and associated with pressure symptoms. There was massive enlargement of the thyroid with retrosternal extension and fixity. Cervical nodes were also enlarged. The X-rays revealed calcification. A palliative thyroidectomy was done leaving the residual tumour behind. Endotracheal intubation and tracheostomy were required for respiratory distress in the postoperative period.

No abstract

Ex vivo human tumor models have emerged as promising, yet complex tools to study cancer immunotherapy response dynamics. Here, we present a strategy that integrates empirical data from an ex vivo human system with computational models to interpret the response dynamics of a clinically prescribed PD-1 inhibitor, nivolumab, in head and neck squamous cell carcinoma (HNSCC) biopsies (N = 50). Using biological assays, we show that drug-induced variance stratifies samples by T helper type 1 (Th1)-related pathways. We then built a systems biology network and mathematical framework of local and global sensitivity analyses to simulate and estimate antitumor phenotypes, which implicate a dynamic role for the induction of Th1-related cytokines and T cell proliferation patterns. Together, we describe a multi-disciplinary strategy to analyze and interpret the response dynamics of PD-1 blockade using heterogeneous ex vivo data and in silico simulations, which could provide researchers a powerful toolset to interrogate immune checkpoint inhibitors.

e15029 Background: Gene fusions are usually caused by chromosomal rearrangements and frequently associated with various cancers. Detection of fusion mutations is an important part of cancer management, and are usually detected in RNA purified from biopsies of fresh tissue or FFPE sections. However, the amount of RNA from these samples is usually limited. We developed a new, PCR-based fusion detection method, which allows detection of unknown and novel fusion mutations. We demonstrate its consistent performance across various RNA inputs, and sequencing depths, in terms of technical performance and limit of detection. Methods: A multiplex PCR RNA fusion panel was designed to cover known RNA fusion targets and unknown fusion targets for target enrichment via single primer multiplex PCR. SeraSeq fusion Reference RNA was used to evaluate the limit of detection of the method. The amplicons were about 110 bp on average to allow for target amplification from challenging fragmented samples such as FFPE RNA. After reverse transcription, the panel was used in a multiplex PCR to amplify the targets, and a final PCR was used to add sequencing adapters and sample indexes. The resulting libraries were sequenced on Illumina sequencers. The data was analyzed for the detection of known fusion variants across varying initial inputs of targets, as well as the performance in terms of mapping and on-target rates, as well as the presence of human ribosomal RNA. Results: We found that the fragmented RNA length had little effect on the performance of the libraries. The required numbers of PCR cycles were optimized based on testing with various amounts of starting RNA input material. We observe a linear relationship between the library yields and RNA input amounts from 10 to 50ng, which had R1 mapping rates above 95% and 98%, respectively. We report the detection of all twelve fusion variants, which are targeted by our panel and present in the Seraseq Fusion RNA Mix v4 reference standard, and which are covered with an average sequencing depth of 0.2M reads, or 3600 reads/amplicon, across all assays. Even at inputs as low as 3 ng, all 12 fusions were typically detected. In general, the results for individual fusions among the different replicates were concordant, with limited observed variance in reads across some fusion junctions between assays and replicates. Over 90% of the reads supported the fusion calls from all inputs (ranging from 1.6 to 50 ng). Over 97% of the reads supported the fusion calls between 12 and 50 ng. Conclusions: A multi-lab validation confirmed the above results, and were also comparable when using an additional distinct CleanPlex fusion panel. To conclude, our CleanPlex fusion technology allows for reliable and simultaneous detection of 12 designed clinically relevant RNA fusions even at low input amounts.

Purpose: Blood-based candidate biomarkers of disease can be monitored by analyzing CTCs and/or circulating cfDNA isolated from the peripheral blood. Our primary objective is to understand the relative contributions of these circulating factors (i.e., CTCs and cfDNA) to the overall disease profile in MBC. Methods: Clinically archived FFPE tumor tissue and prospective blood samples are collected through a minimal risk protocol approved by the Mayo Clinic IRB (#16-001540) from patients with MBC and objective evidence of disease progression. Blood samples include 20 mL whole blood in Streck blood collection tubes (BCTs) for platelet poor plasma (PPP); 20 mL whole blood in AccuCyte BCTs for CTCs, WBCs, and PPP; and 10 mL whole blood in EDTA BCTs for PPP. Nucleated, EpCAM+/cytokeratin+/CD45- CTCs are identified, assessed for ER/HER2 status, and isolated using a centrifugation and direct imaging platform that allows for single cell retrieval (RareCyte). DNA is extracted from PPP, CTCs, WBCs, and FFPE tumor tissue using established methods. Targeted sequencing for SNVs/indels is performed on paired WBCs and CTC-DNA, AccuCyte-cfDNA, Streck-cfDNA, EDTA-cfDNA, and tumor tissue derived DNA using the same NGS panel and informatics pipeline (65 genes; CleanPlex OncoZoom; Paragon Genomics). Results: Tissue and blood samples were collected from 40 patients with metastatic breast cancer. 10 cases were selected for initial analyses on the basis of CTC yield (range 3-113 per 3.75 mL blood); up to 5 CTCs per subject were isolated and pooled for DNA extraction. Plasma cfDNA yields and variant allele frequencies were highly comparable between AccuCyte and Streck collected blood samples. Mutations (range 1-3) were identified in CTC-DNA and/or cfDNA in 9 of 10 cases for a total of 18 detected mutations: 10 in CTC-DNA and cfDNA (BRCA2 N372H, PIK3CA E542K, PIK3CA E545K (x3), PIK3CA H1047R, PTEN R130P, RET G691S, TP53 C135W, TP53 Q192*); 5 in CTC-DNA only (EGFR R521K, EGFR T790M, PIK3CA H1047R, SMAD4 C363Y, TP53 N263D); and 3 in cfDNA only (DNMT3A W893S, DNMT3A S714C, TP53 Q136E). Parallel analyses of samples from 10 more subjects are in progress. Analysis of tumor tissue for all 20 subjects and of EDTA-cfDNA and single CTCs for a subset of cases is ongoing. Updated results will be presented at the meeting. Conclusions: It is feasible to isolate high quality CTCs and cfDNA from the same blood collection tube to perform targeted sequencing; this streamlines specimen processing, decreases overall costs, and minimizes required blood volumes. Importantly, there is overlap in the majority of mutations identified in CTC-DNA and cfDNA, but actionable mutations (e.g., PIK3CA, EGFR) were detected in CTC-DNA only. The clinical and theranostic relevance of these findings is unclear and warrants further investigation. Citation Format: Minetta C. Liu, Karthik V. Giridhar, Roberto A. Leon Ferre, Jamie L. Carroll, Matthew P. Goetz, Tufia C. Haddad, Deanne R. Smith, Siddhartha Yadav, Vidushi Kapoor, Guoying Liu, Tad George, Nolan Ericson, Arturo B. Ramirez, Eric Kaldjian, Keegan E. Haselkorn. Comparison of circulating tumor cell (CTC) derived DNA and circulating cell-free DNA (cfDNA) from simultaneous blood sampling of patients with metastatic breast cancer (MBC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3119.