Evaluating User Experience and DNA Yield from Self-Collection Devices.

BACKGROUND: The COVID-19 pandemic emphasized an urgent need for devices used in the self-collection of biospecimens in an evolving patient care system. The mailing of biospecimen self-collection kits to patients, with samples returned via mail, provides a more convenient testing regimen, but could also impart patient sampling variabilities. User compliance with device directions is central to downstream testing of collected biospecimens and clear instructions are central to this goal. METHODS: Here, we performed an evaluation of 10 oral DNA collection devices involving either swab or saliva self-collection and analyzed ease of use and comfort level with a device, as well as DNA recovery quantity/quality and sample stability. RESULTS: We show that while these DNA quality/quantity metrics are comparable between devices, users prefer direct saliva collection over swab-based devices. CONCLUSIONS: This information is useful in guiding future experiments including their use in human RNA, microbial, or viral sample collection/recovery and their use in clinical testing.

SeekFusion - A Clinically Validated Fusion Transcript Detection Pipeline for PCR-Based Next-Generation Sequencing of RNA.

Detecting gene fusions involving driver oncogenes is pivotal in clinical diagnosis and treatment of cancer patients. Recent developments in next-generation sequencing (NGS) technologies have enabled improved assays for bioinformatics-based gene fusions detection. In clinical applications, where a small number of fusions are clinically actionable, targeted polymerase chain reaction (PCR)-based NGS chemistries, such as the QIAseq RNAscan assay, aim to improve accuracy compared to standard RNA sequencing. Existing informatics methods for gene fusion detection in NGS-based RNA sequencing assays traditionally use a transcriptome-based spliced alignment approach or a de-novo assembly approach. Transcriptome-based spliced alignment methods face challenges with short read mapping yielding low quality alignments. De-novo assembly-based methods yield longer contigs from short reads that can be more sensitive for genomic rearrangements, but face performance and scalability challenges. Consequently, there exists a need for a method to efficiently and accurately detect fusions in targeted PCR-based NGS chemistries. We describe SeekFusion, a highly accurate and computationally efficient pipeline enabling identification of gene fusions from PCR-based NGS chemistries. Utilizing biological samples processed with the QIAseq RNAscan assay and in-silico simulated data we demonstrate that SeekFusion gene fusion detection accuracy outperforms popular existing methods such as STAR-Fusion, TOPHAT-Fusion and JAFFA-hybrid. We also present results from 4,484 patient samples tested for neurological tumors and sarcoma, encompassing details on some novel fusions identified.

LPCAT1-TERT fusions are uniquely recurrent in epithelioid trophoblastic tumors and positively regulate cell growth.

Gestational trophoblastic disease (GTD) is a heterogeneous group of lesions arising from placental tissue. Epithelioid trophoblastic tumor (ETT), derived from chorionic-type trophoblast, is the rarest form of GTD with only approximately 130 cases described in the literature. Due to its morphologic mimicry of epithelioid smooth muscle tumors and carcinoma, ETT can be misdiagnosed. To date, molecular characterization of ETTs is lacking. Furthermore, ETT is difficult to treat when disease spreads beyond the uterus. Here using RNA-Seq analysis in a cohort of ETTs and other gestational trophoblastic lesions we describe the discovery of LPCAT1-TERT fusion transcripts that occur in ETTs and coincide with underlying genomic deletions. Through cell-growth assays we demonstrate that LPCAT1-TERT fusion proteins can positively modulate cell proliferation and therefore may represent future treatment targets. Furthermore, we demonstrate that TERT upregulation appears to be a characteristic of ETTs, even in the absence of LPCAT1-TERT fusions, and that it appears linked to copy number gains of chromosome 5. No evidence of TERT upregulation was identified in other trophoblastic lesions tested, including placental site trophoblastic tumors and placental site nodules, which are thought to be the benign chorionic-type trophoblast counterpart to ETT. These findings indicate that LPCAT1-TERT fusions and copy-number driven TERT activation may represent novel markers for ETT, with the potential to improve the diagnosis, treatment, and outcome for women with this rare form of GTD.

RNA-Seq Reveals Differences in Expressed Tumor Mutation Burden in Colorectal and Endometrial Cancers with and without Defective DNA-Mismatch Repair.

Tumor mutation burden (TMB) is an emerging biomarker of immunotherapy response. RNA sequencing in FFPE tissue samples was used for determining TMB in microsatellite-stable (MSS) and microsatellite instability-high (MSI-H) tumors in patients with colorectal or endometrial cancer. Tissue from tumors and paired normal tissue from 46 MSI-H and 12 MSS cases were included. Of the MSI-H tumors, 29 had defective DNA mismatch-repair mutations, and 17 had MLH1 promoter hypermethylation. TMB was measured using the expressed somatic nucleotide variants (eTMB). A method of accurate measurement of eTMB was developed that removes FFPE-derived artifacts by leveraging mutation signatures. There was a significant difference in the median eTMB values observed between MSI-H and MSS cases: 27.3 versus 6.7 mutations/megabase (mut/Mb) (P = 3.5 × 10-9). Among tumors with defective DNA-mismatch repair, those with mismatch-repair mutations had a significantly higher median eTMB than those with hypermethylation: 28.1 versus 17.5 mut/Mb (P = 0.037). Multivariate analysis showed that MSI status, tumor type (endometrial or colorectal), and age were significantly associated with eTMB. Additionally, using whole-exome sequencing in a subset of these patients, it was determined that DNA TMB correlated well with eTMB (Spearman correlation coefficient, 0.83). These results demonstrate that RNA sequencing can be used for measuring eTMB in FFPE tumor specimens.

Chromosomal Junction Detection from Whole-Genome Sequencing on Formalin-Fixed, Paraffin-Embedded Tumors.

DNA junctions (DNAJs) frequently impact clinically relevant genes in tumors and are important for diagnostic and therapeutic purposes. Although routinely screened through fluorescence in situ hybridization assays, such testing only allows the interrogation of single-gene regions or known fusion partners. Comprehensive assessment of DNAJs present across the entire genome can only be determined from whole-genome sequencing. Structural variance analysis from whole-genome paired-end sequencing data is, however, frequently restricted to copy number changes without DNAJ detection. Through optimized whole-genome sequencing and specialized bioinformatics algorithms, complete structural variance analysis is reported, including DNAJs, from formalin-fixed DNA. Selective library assembly from larger fragments (>500 bp) and economical sequencing depths (300 to 400 million reads) provide representative genomic coverage profiles and increased allelic coverage to levels compatible with DNAJ calling (40× to 60×). Although consistently fragmented, more recently formalin-fixed, specimens (<2 years' storage) revealed consistent populations of larger DNA fragments. Optimized bioinformatics efficiently detected >90% of DNAJs in two prostate tumors (approximately 60% tumor) previously analyzed by mate-pair sequencing on fresh frozen tissue, with evidence of at least one spanning-read in 99% of DNAJs. Rigorous masking with data from unrelated formalin-fixed tissue progressively eliminated many false-positive DNAJs, without loss of true positives, resulting in low numbers of false-positive passing current filters. This methodology enables more comprehensive clinical genomics testing on formalin-fixed clinical specimens.

Molecular Genetic Landscape of Sclerosing Pneumocytomas.

OBJECTIVES: Sclerosing pneumocytomas are rare pulmonary neoplasms that are typically benign. However, rare patients experience progressive disease, and therapy targeting specific genetic underpinnings could be an attractive therapeutic option. Recent studies have found recurrent AKT 1 mutations in sclerosing pneumocytoma, but little is known about whether oncogenic fusion genes may also be present. METHODS: To better understand the genetic background, 10 sclerosing pneumocytomas were subjected to next-generation sequencing cancer mutation panel testing (n = 9) and/or RNA sequencing (n = 3). The patients were all women (average age, 47 years; range, 17-74 years). RESULTS: Eight patients had solitary sclerosing pneumocytomas, while one had two tumors, and one had many bilateral tumors. Recurrent mutations were noted in genes involved in the mTOR pathway, including AKT1, PIK3R1, and PTEN. AKT1 alterations were particularly common, present in 78%. No recurrent genetic fusions were identified. The patient in our study with multiple bilateral lesions was treated with the mammalian target of rapamycin (mTOR) inhibitor everolimus, with no objective radiographic evidence of treatment response after 4 months. CONCLUSIONS: Our data further support that abnormal activation of the mTOR pathway is a consistent genetic event in sclerosing pneumocytoma. This warrants further exploration to determine if mTOR pathway inhibitors may be effective in patients with metastatic or recurrent disease.

Secondary renal neoplasia following chemotherapy or radiation in pediatric patients.

Renal neoplasia occurring as a second malignancy following childhood cancer has been most closely associated with neuroblastoma and Wilms tumor. While some cases have been associated with a genetic predisposition, nearly all are thought to result from "late effects" of therapy-related toxicity that involves chemotherapy or radiation. It is unclear if these tumors are enriched for specific molecular or morphologic characteristics. A query of our institutional nephrectomy registry of 8295 patients for renal neoplasia occurring post-treatment for childhood cancer revealed 6 patients with Wilms tumor, 4 with neuroblastoma, and 1 with acute lymphoblastic leukemia (ALL). Three additional cases of MiT family translocation renal cell carcinoma (RCC), from 2 patients, following chemotherapy for neuroblastoma and systemic lupus erythematosus and another of clear cell RCC post-ALL were included. The most common tumor type was clear cell RCC: 9/19 cases (47.4%), followed by metanephric adenoma and MiT family translocation RCC (3/19, 15.8%). There were no characteristic features to indicate a unique renal neoplasia subtype. Potential syndromic renal neoplasia occurred in 2 patients, metanephric adenomas and oncocytoma in a patient with hyperparathyroidism-jaw tumor syndrome post-treatment of Wilms tumor and a fumarate hydratase-deficient RCC in a patient post-treatment for ALL. The mean age at diagnosis of childhood neoplasia or treatment with chemotherapy or radiation was 4.7 years, and the average time to subsequent renal neoplasia was 31 years. Five (of 14) patients developed metastatic RCC, and there were 2 RCC-related deaths. These results indicate the need for extended clinical follow-up of these patients.

Collection and Handling of Thoracic Small Biopsy and Cytology Specimens for Ancillary Studies: Guideline From the College of American Pathologists in Collaboration With the American College of Chest Physicians, Association for Molecular Pathology, American Society of Cytopathology, American Thoracic Society, Pulmonary Pathology Society, Papanicolaou Society of Cytopathology, Society of Interventional Radiology, and Society of Thoracic Radiology.

CONTEXT.­: The need for appropriate specimen use for ancillary testing has become more commonplace in the practice of pathology. This, coupled with improvements in technology, often provides less invasive methods of testing, but presents new challenges to appropriate specimen collection and handling of these small specimens, including thoracic small biopsy and cytology samples. OBJECTIVE.­: To develop a clinical practice guideline including recommendations on how to obtain, handle, and process thoracic small biopsy and cytology tissue specimens for diagnostic testing and ancillary studies. METHODS.­: The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. Core needle biopsy, touch preparation, fine-needle aspiration, and effusion specimens with thoracic diseases including malignancy, granulomatous process/sarcoidosis, and infection (eg, tuberculosis) were deemed within scope. Ancillary studies included immunohistochemistry and immunocytochemistry, fluorescence in situ hybridization, mutational analysis, flow cytometry, cytogenetics, and microbiologic studies routinely performed in the clinical pathology laboratory. The use of rapid on-site evaluation was also covered. RESULTS.­: Sixteen guideline statements were developed to assist clinicians and pathologists in collecting and processing thoracic small biopsy and cytology tissue samples. CONCLUSIONS.­: Based on the systematic review and expert panel consensus, thoracic small specimens can be handled and processed to perform downstream testing (eg, molecular markers, immunohistochemical biomarkers), core needle and fine-needle techniques can provide appropriate cytologic and histologic specimens for ancillary studies, and rapid on-site cytologic evaluation remains helpful in appropriate triage, handling, and processing of specimens.

Assessment of clinical outcomes with immune checkpoint inhibitor therapy in melanoma patients with CDKN2A and TP53 pathogenic mutations.

BACKGROUND: CDKN2A and TP53 mutations are recurrent events in melanoma, occurring in 13.3% and 15.1% of cases respectively and are associated with poorer outcomes. It is unclear what effect CDKN2A and TP53 mutations have on the clinical outcomes of patients treated with checkpoint inhibitors. METHODS: All patients with cutaneous melanoma or melanoma of unknown primary who received checkpoint inhibitor therapy and underwent genomic profiling with the 50-gene Mayo Clinic solid tumor targeted cancer gene panel were included. Patients were stratified according to the presence or absence of mutations in BRAF, NRAS, CDKN2A, and TP53. Patients without mutations in any of these genes were termed quadruple wild type (QuadWT). Clinical outcomes including median time to progression (TTP), median overall survival (OS), 6-month and 12-month OS, 6-month and 12-month without progression, ORR and disease control rate (DCR) were analyzed according to the mutational status of CDKN2A, TP53 and QuadWT. RESULTS: A total of 102 patients were included in this study of which 14 had mutations of CDKN2A (CDKN2Amut), 21 had TP53 mutations (TP53mut), and 12 were QuadWT. TP53mut, CDKN2Amut and QuadWT mutational status did not impact clinical outcomes including median TTP, median OS, 6-month and 12-month OS, 6-month and 12-month without progression, ORR and DCR. There was a trend towards improved median TTP and DCR in CDKN2Amut cohort and a trend towards worsened median TTP in the QuadWT cohort. CONCLUSION: Cell cycle regulators such as TP53 and CDKN2A do not appear to significantly alter clinical outcomes when immune checkpoint inhibitors are used.

Clinicopathologic, Immunohistochemical, and Molecular Characteristics of Ovarian Serous Carcinoma With Mixed Morphologic Features of High-grade and Low-grade Serous Carcinoma.

Despite the current classification of high-grade serous carcinoma (HGSCA) and low-grade serous carcinoma (LGSCA) as mutually exclusive diseases based on morphology and molecular pathogenesis, cases with mixed morphologic features of HGSCA and LGSCA have been reported. Herein we assess the clinicopathologic, immunohistochemical (IHC), and molecular genetic characteristics of a group of these cases, which we termed indeterminate grade serous carcinoma (IGSCA) in comparison with groups of HGSCA and LGSCA. Using the World Health Organization (WHO) classification criteria, we selected 27 LGSCA and 19 IGSCA for detailed morphologic study. Thirteen classic HGSCA, 19 classic LGSCA, and 19 IGSCA were selected for p53 and BRAF V600E IHC and molecular genetic testing by next-generation sequencing. IGSCA showed the architectural patterns of invasion of LGSCA, but with higher grade nuclear features focally and a mitotic index intermediate between LGSCA and HGSCA. Few cases in the IGSCA group showed mutant TP53 by IHC or sequencing (4/18, 22.2%), 1 case had mutant BRAF non-V600E by sequencing, and 1 had an NRAS mutation. When present, the mutations were identical in the low-grade and high-grade areas. The IGSCA group had a long-term survival similar to the classic HGSCA group. IGSCA with mixed morphologic features of HGSCA and LGSCA is a rare and potentially clinically aggressive variant of serous carcinoma. Their distinct morphologic, but heterogenous molecular features, including low frequency of TP53 and BRAF mutations suggest that these rare tumors may have a different pathogenesis pathway compared with classic HGSCA and classic LGSCA.

Mutational Profile in Vulvar, Vaginal, and Urethral Melanomas: Review of 37 Cases With Focus on Primary Tumor Site.

Melanomas of female genital tract are rare tumors with poor prognosis. While BRAF-V600E is the most common pathogenic mutation seen in cutaneous sun-exposed melanomas, mucosal and anogenital melanomas usually lack BRAF mutations and instead they harbor KIT alterations. The American Joint Committee on Cancer staging guideline (AJCC eighth edition) recommends using cutaneous melanoma guidelines for vulvar melanoma staging and does not provide any recommendations for vaginal melanoma staging. The aim of this study is to investigate the mutational status of invasive melanomas arising from different anatomic sites in lower female genital tract (vulvar hair-bearing skin, glabrous skin, vagina and urethra) in a group of 37 patients. Tumors were analyzed using a DNA targeted next-generation sequencing panel covering the 21 most common genes and mutation hotspots in melanomas. The most common genetic alterations in invasive melanomas of lower female genital tract are KIT (32%), TP53 (22%), and NF1 (19%). Overall 66% (21/32) of cases showed a pathogenic alteration in at least one of the MAPK pathway genes. No statistical significance seen between different primary tumor sites and the frequency of the oncogenic mutations, nor were any significant differences found by mutation status. Only one case of urethral melanoma showed a BRAF non-V600E mutation (D594G). Our results suggest a similar molecular pathogenesis and overall survival in melanomas arising from lower female genital tract, irrespective of their exact location in the urogenital area. Future classifications of melanoma should consider grouping vulvar melanomas with mucosal rather than cutaneous melanomas.

Combining copy number, methylation markers, and mutations as a panel for endometrial cancer detection via intravaginal tampon collection.

OBJECTIVE: We aimed to assess whether endometrial cancer (EC) can be detected in shed DNA collected with vaginal tampon by analyzing copy number, methylation markers, and mutations. METHODS: Tampons were collected prior to hysterectomy from 38 EC patients and 28 women with benign indications. Extracted tampon DNA underwent the following: 1) low-coverage whole genome sequencing (LC-WGS) to assess copy number, 2) pyrosequencing to measure percent promotor methylation of HOXA9, RASSF1, and CDH13 and 3) next generation sequencing (NGS) to identify mutations in 19 genes associated with EC identified through The Cancer Genome Atlas. Sensitivity and specificity for each test and test combinations were calculated. RESULTS: Methylation analysis yielded the highest specificities but lowest sensitivities (37-40% sensitivity; 100% specificity for HOXA9, RASSF1 and HTR1B) while mutation analysis had improved sensitivity (50% sensitivity; 83% specificity). Only one "false positive" result for copy number variants was identified among women with benign surgical indications, which was based on detection of copy number changes, and associated with a leiomyosarcoma that was only recognized at hysterectomy. Considering any of the 3 biomarker classes as a positive, resulted in a sensitivity of 92% and specificity of 86%. Mutation analysis did not add sensitivity to the combination of analysis of copy number and methylation. CONCLUSIONS: This study demonstrates a proof-of-principle for non-invasive yet precise detection of endometrial cancer. We propose that with improved biomarker testing, it may be possible to develop a clinically useful test for detecting EC.

Clinicopathological, immunophenotypic and genetic studies of mediastinal paragangliomas†.

OBJECTIVES: Paragangliomas have unique features in the mediastinum, in part due to their location. Because of their paucity, they have not been thoroughly investigated. We studied the clinical, pathological, immunohistochemical and molecular features of mediastinal paragangliomas. METHODS: Immunohistochemistry, next-generation sequencing mutation panel and the Oncoscan assay were performed. RESULTS: Twenty-four patients with mediastinal paraganglioma (7 men, 29.2%) had a median age of 45.5 years (19.8-72.2). Twenty-one (87.5%) paragangliomas were completely resected. Six (of 24, 25.0%) tumours were considered metastatic. Mitotic activity occurred in 11 (of 24, 45.8%) paragangliomas. Programmed death-ligand 1 (PD-L1) (n = 23) was expressed in 6 (26%) patients in 10% (n = 2) and 1% (n = 4) of tumour cells, respectively. SDHB expression was lost in 19 (of 22, 86.4%) cases. ATRX expression was lost in 11 (of 23, 47.8%) cases. Next-generation sequencing revealed a single pathogenic mutation in 10 (of 19) specimens including SDHB (n = 4), SDHD (n = 6), SDHC (n = 1), ATRX (n = 1), and ≥2 mutations in 2 cases [SDHC and TERT (n = 1); SDHB, ATRX and TP53 (n = 1)]. Germline mutation analysis revealed the same succinate dehydrogenase mutation (or lack thereof) as identified in the paraganglioma in 11 (of 12) cases. During a median follow-up (n = 21) of 4.8 years (0.8-14.9), 3 patients developed metastases; 4 patients died, at least 1 of disease. CONCLUSIONS: Mediastinal paragangliomas can be associated with morbidity and mortality. Many mediastinal paragangliomas have been reported to be associated with syndromes such as multiple endocrine neoplasia, von Hippel-Lindau or succinate dehydrogenase syndrome with mutation profiles dominated by alterations in genes associated with these syndromes.

Molecular profiling of long-term IDH-wildtype glioblastoma survivors.

BACKGROUND: Glioblastoma (GBM) represents an aggressive cancer type with a median survival of only 14 months. With fewer than 5% of patients surviving 5 years, comprehensive profiling of these rare patients could elucidate prognostic biomarkers that may confer better patient outcomes. We utilized multiple molecular approaches to characterize the largest patient cohort of isocitrate dehydrogenase (IDH)-wildtype GBM long-term survivors (LTS) to date. METHODS: Retrospective analysis was performed on 49 archived formalin-fixed paraffin embedded tumor specimens from patients diagnosed with GBM at the Mayo Clinic between December 1995 and September 2013. These patient samples were subdivided into 2 groups based on survival (12 LTS, 37 short-term survivors [STS]) and subsequently examined by mutation sequencing, copy number analysis, methylation profiling, and gene expression. RESULTS: Of the 49 patients analyzed in this study, LTS were younger at diagnosis (P = 0.016), more likely to be female (P = 0.048), and MGMT promoter methylated (UniD, P = 0.01). IDH-wildtype STS and LTS demonstrated classic GBM mutations and copy number changes. Pathway analysis of differentially expressed genes showed LTS enrichment for sphingomyelin metabolism, which has been linked to decreased GBM growth, invasion, and angiogenesis. STS were enriched for DNA repair and cell cycle control networks. CONCLUSIONS: While our findings largely report remarkable similarity between these LTS and more typical STS, unique attributes were observed in regard to altered gene expression and pathway enrichment. These attributes may be valuable prognostic markers and are worth further examination. Importantly, this study also underscores the limitations of existing biomarkers and classification methods in predicting patient prognosis.

Recurrent MSC E116K mutations in ALK-negative anaplastic large cell lymphoma.

Anaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK)+ or ALK-, based on the presence or absence of ALK rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, MSC E116K, exclusively in ALK- ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of MSC E116K for ALK- ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting DUSP22 rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin-bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSCE116K acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of MYC and cell cycle inhibition. Chromatin immunoprecipitation-sequencing and transcriptome analysis identified the cell cycle regulatory gene E2F2 as a direct transcriptional target of musculin. MSCE116K reversed E2F2-induced cell cycle arrest and promoted expression of the CD30-IRF4-MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the MSC promoter. Finally, ALCL cells expressing MSC E116K were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent MSC mutation as a key driver of the CD30-IRF4-MYC axis and cell cycle progression in a unique subset of ALCLs.

Applying Standard Clinical Chemistry Assay Validation to Droplet Digital PCR Quantitative Liquid Biopsy Testing.

BACKGROUND: Droplet digital PCR (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, assay performance criteria must be established in a standardized manner to harness this potential. We reasoned that standard protocols used in clinical chemistry assay validation should be able to fill this need. METHODS: We validated KRAS, EGFR, and BRAF quantitative ddPCR assays based on the Clinical Laboratory Improvement Act regulations for laboratory-developed tests in clinical chemistry and the matching Clinical and Laboratory Standards Institute guidelines. This included evaluation of limit of the blank (LOB), limit of detection (LOD), limit of quantification (LOQ), intraassay and interassay imprecision, analytical range, dilution linearity, accuracy (including comparison with orthogonal platforms), reference range study, interference, and stability studies. RESULTS: For the ddPCR assays, the LOB was 4 mutant copies, LODs were 12 to 22 copies, and LOQs were 35 to 64 copies. The upper limit of the dynamic range was 30000 copies, and dilutions were linear down to the LOQs with good accuracy of spike recovery of Horizon reference material. Method comparisons with next-generation sequencing and an alternative ddPCR platform showed complete qualitative agreement and quantitative concordance, with slopes of 0.73 to 0.97 and R 2s of 0.83 to 0.99. No substantial interferences were discovered. Wild-type copy numbers in plasma ranged from 462 to 6169/mL in healthy individuals. CONCLUSIONS: Standard clinical chemistry assay validation protocols can be applied to quantitative ddPCR assays. This should facilitate comparison of the performance of different assays and allow establishment of minimal significant change thresholds in monitoring applications.

Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors.

We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion-positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration-approved and investigational targeted therapies.

Analysis of Cell-Free DNA to Assess Risk of Tumoremia Following Endoscopic Ultrasound Fine-Needle Aspiration of Pancreatic Adenocarcinomas.

BACKGROUND & AIMS: Cellular and nuclear material from tumors disseminates into the bloodstream (tumoremia), but it is not clear whether medical procedures cause release of this material or contribute to formation of metastases. We performed a prospective study of blood samples from patients with pancreatic adenocarcinoma (PDAC) to determine whether endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) associates with markers of tumoremia. METHODS: We obtained peripheral blood from 104 patients (35 with PDAC) before and after EUS-FNA of primary tumors; blood samples from 69 healthy individuals were used as controls. Plasma concentrations of cell-free DNA (cfDNA) were measured, and cfDNA and primary tumor samples were analyzed to detect activating mutations in KRAS. Potential development of tumoremia was defined by an increase in cfDNA of 2-fold or more, and/or detection of mutant KRAS in samples collected after FNA from patients whose blood samples did not contain detectable mutant KRAS before FNA. RESULTS: Peripheral blood concentrations of cfDNA were 1200 ng/ml (500-3300 ng/ml) before FNA vs 1400 ng/ml (900-4000 ng/ml) after FNA (P = .391). Tumoremia was detected in 10/35 patients (28.6%): 7 patients had a ≥2-fold increase in cfDNA concentration (20.6%) and 3 patients had circulating tumor DNA with KRAS mutations after FNA that were not detected before FNA (8.8%). New distant metastases were detected in 1.3 ± 0.82 patients with tumoremia vs 0.64 ± 0.81 without (P = .0375). Overall mortality did not differ significantly between patients with tumoremia (10/10 deaths, 100%) vs those without (19/25 deaths, 76%) nor did survival times of deceased patients (13.3 months for patients with tumoremia; range, 5.8-14.9 months vs 11.1 months for patients without tumoremia; range, 5.5-14.5 months). However, 6 patients without tumoremia were alive at a mean 23.9 months after EUS-FNA (range, 19.9-25 months after EUS-FNA) vs none of the patients with tumoremia. CONCLUSION: In patients with PDAC, EUS-FNA associates with increased plasma concentration of cfDNA and increased detection of mutant KRAS after the procedure (markers of tumoremia and possible new distant metastasis). Although levels of cfDNA and activating mutations in KRAS are logical markers of tumoremia, they may not serve as the ideal biomarkers of this process. These findings are preliminary and do not indicate a need to modify current practice, yet further studies are needed.

Pulmonary invasive mucinous adenocarcinoma and mixed invasive mucinous/nonmucinous adenocarcinoma-a clinicopathological and molecular genetic study with survival analysis.

Invasive mucinous adenocarcinoma is a variant of lung adenocarcinoma, which may be mixed with nonmucinous adenocarcinoma. KRAS mutations are common, but other clinical and genetic features are not clearly established. Lung adenocarcinomas (n=760) with ≥5 years of follow-up comprised 3 nonoverlapping cohorts for survival analysis. Mucinous tumors were evaluated with Ion AmpliSeq Cancer Hotspot Panel v2. Cases without detected mutations were tested for ALK and ROS1 and by OncoScan array. Fifty-seven invasive mucinous adenocarcinomas and 54 mixed mucinous/nonmucinous adenocarcinomas were identified. Mucinous tumors constituted 27 of 218 nonselected patients (12.4%), 23 of 268 never-smokers (8.6%), and 61 of 274 in a smokers cohort enriched for lepidic growth (22.3%). In the lepidic-enriched smokers, patients with mucinous tumors experienced worse overall survival (P=.006) and progression-free survival (P=.024), which persisted on multivariable analysis. No survival differences were observed in the other cohorts. KRAS mutations were common (76% of invasive mucinous adenocarcinomas, 68% of mixed mucinous/nonmucinous), and 38% of KRAS mutations occurred with other mutations, especially STK11. Six cases had potentially targetable mutations (3 ALK, 2 EGFR, 1 BRAF V600E). All ALK-rearranged tumors were mixed mucinous/nonmucinous. Four of 6 cases without hotspot mutations showed complex copy number/structural abnormalities. Pulmonary invasive mucinous adenocarcinomas and mixed nonmucinous/mucinous adenocarcinomas are clinically and genetically similar, except for a higher rate of ALK rearrangement in mixed tumors. Survival for mucinous tumors is similar to that for nonmucinous tumors in a nonselected cohort, although worse survival was seen in a cohort of smokers enriched for lepidic growth.

Computer-Aided Nodule Assessment and Risk Yield (CANARY) may facilitate non-invasive prediction of EGFR mutation status in lung adenocarcinomas.

Computer-Aided Nodule Assessment and Risk Yield (CANARY) is quantitative imaging analysis software that predicts the histopathological classification and post-treatment disease-free survival of patients with adenocarcinoma of the lung. CANARY characterizes nodules by the distribution of nine color-coded texture-based exemplars. We hypothesize that quantitative computed tomography (CT) analysis of the tumor and tumor-free surrounding lung facilitates non-invasive identification of clinically-relevant mutations in lung adenocarcinoma. Comprehensive analysis of targetable mutations (50-gene-panel) and CANARY analysis of the preoperative (≤3 months) high resolution CT (HRCT) was performed for 118 pulmonary nodules of the adenocarcinoma spectrum surgically resected between 2006-2010. Logistic regression with stepwise variable selection was used to determine predictors of mutations. We identified 140 mutations in 106 of 118 nodules. TP53 (n = 48), KRAS (n = 47) and EGFR (n = 15) were the most prevalent. The combination of Y (Yellow) and G (Green) exemplars, fibrosis within the surrounding lung and smoking status were the best discriminators for an EGFR mutation (AUC 0.77 and 0.87, respectively). None of the EGFR mutants expressing TP53 (n = 5) had a good prognosis based on CANARY features. No quantitative features were significantly associated with KRAS mutations. Our exploratory analysis indicates that quantitative CT analysis of a nodule and surrounding lung may noninvasively predict the presence of EGFR mutations in pulmonary nodules of the adenocarcinoma spectrum.