Real-world comprehensive genomic and immune profiling reveals distinct age- and sex-based genomic and immune landscapes in tumors of patients with non-small cell lung cancer.

Introduction: Younger patients with non-small cell lung cancer (NSCLC) (<50 years) represent a significant patient population with distinct clinicopathological features and enriched targetable genomic alterations compared to older patients. However, previous studies of younger NSCLC suffer from inconsistent findings, few studies have incorporated sex into their analyses, and studies targeting age-related differences in the tumor immune microenvironment are lacking. Methods: We performed a retrospective analysis of 8,230 patients with NSCLC, comparing genomic alterations and immunogenic markers of younger and older patients while also considering differences between male and female patients. We defined older patients as those ≥65 years and used a 5-year sliding threshold from <45 to <65 years to define various groups of younger patients. Additionally, in an independent cohort of patients with NSCLC, we use our observations to inform testing of the combinatorial effect of age and sex on survival of patients given immunotherapy with or without chemotherapy. Results: We observed distinct genomic and immune microenvironment profiles for tumors of younger patients compared to tumors of older patients. Younger patient tumors were enriched in clinically relevant genomic alterations and had gene expression patterns indicative of reduced immune system activation, which was most evident when analyzing male patients. Further, we found younger male patients treated with immunotherapy alone had significantly worse survival compared to male patients ≥65 years, while the addition of chemotherapy reduced this disparity. Contrarily, we found younger female patients had significantly better survival compared to female patients ≥65 years when treated with immunotherapy plus chemotherapy, while treatment with immunotherapy alone resulted in similar outcomes. Discussion: These results show the value of comprehensive genomic and immune profiling (CGIP) for informing clinical treatment of younger patients with NSCLC and provides support for broader coverage of CGIP for younger patients with advanced NSCLC.

Comprehensive NGS profiling to enable detection of ALK gene rearrangements and MET amplifications in non-small cell lung cancer.

Introduction: Next-generation sequencing (NGS) is currently widely used for biomarker studies and molecular profiling to identify concurrent alterations that can lead to the better characterization of a tumor's molecular landscape. However, further evaluation of technical aspects related to the detection of gene rearrangements and copy number alterations is warranted. Methods: There were 12 ALK rearrangement-positive tumor specimens from patients with non-small cell lung cancer (NSCLC) previously detected via fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and an RNA-based NGS assay, and 26 MET high gene copy number (GCN) cases detected by FISH, selected for this retrospective study. All 38 pre-characterized cases were reassessed utilizing the PGDx™ elio™ tissue complete assay, a 505 gene targeted NGS panel, to evaluate concordance with these conventional diagnostic techniques. Results: The detection of ALK rearrangements using the DNA-based NGS assay demonstrated excellent sensitivity with the added benefit of characterizing gene fusion partners and genomic breakpoints. MET copy number alterations were also detected; however, some discordances were observed likely attributed to differences in algorithm, reporting thresholds and gene copy number state. TMB was also assessed by the assay and correlated to the presence of NSCLC driver alterations and was found to be significantly lower in cases with NGS-confirmed canonical driver mutations compared with those without (p=0.0019). Discussion: Overall, this study validates NGS as an accurate approach for detecting structural variants while also highlighting the need for further optimization to enable harmonization across methodologies for amplifications.

Validation of the Labcorp Plasma Focus Test to Facilitate Precision Oncology Through Cell-Free DNA Genomic Profiling of Solid Tumors.

Genomic profiling is critical for precision oncology to guide treatment decisions. Liquid biopsy testing is a complementary approach to tissue testing, particularly when tissue is not readily available. The Labcorp Plasma Focus test is a circulating cell-free DNA genomic profiling test that identifies actionable variants in solid cancers, including non-small-cell lung, colorectal, melanoma, breast, esophageal, gastroesophageal junction, and gastric cancers. This study highlights the analytical validation of the test, including accuracy compared with orthogonal methods, as well as sensitivity, specificity, precision, reproducibility, and repeatability. Concordance with orthogonal methods showed percent positive agreement of 98.7%, 89.3%, and 96.2% for single nucleotide variants (SNVs), insertion/deletions (indels), and copy number amplifications (CNAs), respectively, and 100.0% for translocations and microsatellite instability (MSI). Analytical sensitivity revealed a median limit of detection of 0.7% and 0.6% for SNVs and indels, 1.4-fold for CNAs, 0.5% variant allele frequency for translocations, and 0.6% for MSI. Specificity was >99% for SNVs/indels and 100% for CNAs, translocations, and MSI. Average positive agreement from precision, reproducibility, and repeatability experiments was 97.5% and 88.9% for SNVs/indels and CNAs, and 100% for translocations and MSI. Taken together, these data show that the Labcorp Plasma Focus test is a highly accurate, sensitive, and specific approach for cell-free DNA genomic profiling to supplement tissue testing and inform treatment decisions.

Next-Generation Sequencing Concordance Analysis of Comprehensive Solid Tumor Profiling between a Centralized Specialty Laboratory and the Decentralized Personal Genome Diagnostics elio Tissue Complete Kitted Solution.

Genomic tumor profiling by next-generation sequencing (NGS) allows for large-scale tumor testing to inform targeted cancer therapies and immunotherapies, and to identify patients for clinical trials. These tests are often underutilized in patients with late-stage solid tumors and are typically performed in centralized specialty laboratories, thereby limiting access to these complex tests. Personal Genome Diagnostics Inc., elio tissue complete NGS solution is a comprehensive DNA-to-report kitted assay and bioinformatics solution. Comparison of 147 unique specimens from >20 tumor types was performed using the elio tissue complete solution and Foundation Medicine's FoundationOne test, which is of similar size and gene content. The analytical performance of all genomic variant types was evaluated. In general, the overall mutational profile is highly concordant between the two assays, with agreement in sequence variants reported between panels demonstrating >95% positive percentage agreement for single-nucleotide variants and insertions/deletions in clinically actionable genes. Both copy number alterations and gene translocations showed 80% to 83% positive percentage agreement, whereas tumor mutation burden and microsatellite status showed a high level of concordance across a range of mutation loads and tumor types. The Personal Genome Diagnostics Inc., elio tissue complete assay is comparable to the FoundationOne test and will allow more laboratories to offer a diagnostic NGS assay in house, which will ultimately reduce time to result and increase the number of patients receiving molecular genomic profiling and personalized treatment.

Non-equivalence of nuclear import among nuclei in multinucleated skeletal muscle cells.

Skeletal muscle is primarily composed of large myofibers containing thousands of post-mitotic nuclei distributed throughout a common cytoplasm. Protein production and localization in specialized myofiber regions is crucial for muscle function. Myonuclei differ in transcriptional activity and protein accumulation, but how these differences among nuclei sharing a cytoplasm are achieved is unknown. Regulated nuclear import of proteins is one potential mechanism for regulating transcription spatially and temporally in individual myonuclei. The best-characterized nuclear localization signal (NLS) in proteins is the classical NLS (cNLS), but many other NLS motifs exist. We examined cNLS and non-cNLS reporter protein import using multinucleated muscle cells generated in vitro, revealing that cNLS and non-cNLS nuclear import differs among nuclei in the same cell. Investigation of cNLS nuclear import rates in isolated myofibers ex vivo confirmed differences in nuclear import rates among myonuclei. Analyzing nuclear import throughout myogenesis revealed that cNLS and non-cNLS import varies during differentiation. Taken together, our results suggest that both spatial and temporal regulation of nuclear import pathways are important in muscle cell differentiation and protein regionalization in myofibers.

Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants.

Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR.

Tumor-specific mutations can be identified in circulating, cell-free DNA in plasma or serum and may serve as a clinically relevant alternative to biopsy. Detection of tumor-specific mutations in the plasma, however, is technically challenging. First, mutant allele fractions are typically low in a large background of wild-type circulating, cell-free DNA. Second, the amount of circulating, cell-free DNA acquired from plasma is also low. Even when using digital PCR (dPCR), rare mutation detection is challenging because there is not enough circulating, cell-free DNA to run technical replicates and assay or instrument noise does not easily allow for mutation detection <0.1%. This study was undertaken to improve on the robustness of dPCR for mutation detection. A multiplexed, preamplification step using a high-fidelity polymerase before dPCR was developed to increase total DNA and the number of targets and technical replicates that can be assayed from a single sample. We were able to detect multiple cancer-relevant mutations within tumor-derived samples down to 0.01%. Importantly, the signal/noise ratio was improved for all preamplified targets, allowing for easier discrimination of low-abundance mutations against false-positive signal. Furthermore, we used this protocol on clinical samples to detect known, tumor-specific mutations in patient sera. This study provides a protocol for robust, sensitive detection of circulating tumor DNA for future clinical applications.

Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner.

Heterotrimeric protein phosphatase 2A (PP2A) consists of catalytic C (PP2Ac), structural A, and regulatory B-type subunits, and its dysfunction has been linked to cancer. Reversible methylation of PP2Ac by leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1 (PME-1) differentially regulates B-type subunit binding and thus PP2A function. Polyomavirus middle (PyMT) and small (PyST) tumor antigens and SV40 small tumor antigen (SVST) are oncoproteins that block PP2A function by replacing certain B-type subunits, resulting in cellular transformation. Whereas the B-type subunits replaced by these oncoproteins seem to exhibit a binding preference for methylated PP2Ac, PyMT does not. We hypothesize that circumventing the normal cellular control of PP2A by PP2Ac methylation is a general strategy for ST- and MT-mediated transformation. Two predictions of this hypothesis are (1) that PyST and SVST also bind PP2A in a methylation-insensitive manner and (2) that down-regulation of PP2Ac methylation will activate progrowth and prosurvival signaling and promote transformation. We found that SVST and PyST, like PyMT, indeed form PP2A heterotrimers independently of PP2Ac methylation. In addition, reducing PP2Ac methylation through LCMT-1 knockdown or PME-1 overexpression enhanced transformation by activating the Akt and p70/p85 S6 kinase (S6K) pathways, pathways also activated by MT and ST oncoproteins. These results support the hypothesis that MT and ST oncoproteins circumvent cellular control of PP2A by methylation to promote transformation. They also implicate LCMT-1 as a negative regulator of Akt and p70/p85 S6K. Therefore, disruption of PP2Ac methylation may contribute to cancer, and modulation of this methylation may serve as an anticancer target.