Detection of human brain cancers using genomic and immune cell characterization of cerebrospinal fluid through CSF-BAM.

Unlabelled: Patients who have radiographically detectable lesions in their brain or other symptoms compatible with brain tumors pose challenges for diagnosis. The only definitive way to diagnose such patients is through brain biopsy, an obviously invasive and dangerous procedure. Here we present a new workflow termed "CSF-BAM" that simultaneously identifies cell or T cell receptor rearrangements, neuploidy, and using PCR-mediated amplification of both strands of the DNA from CSF samples.

Machine learning to detect the SINEs of cancer.

We previously described an approach called RealSeqS to evaluate aneuploidy in plasma cell-free DNA through the amplification of ~350,000 repeated elements with a single primer. We hypothesized that an unbiased evaluation of the large amount of sequencing data obtained with RealSeqS might reveal other differences between plasma samples from patients with and without cancer. This hypothesis was tested through the development of a machine learning approach called Alu Profile Learning Using Sequencing (A-PLUS) and its application to 7615 samples from 5178 individuals, 2073 with solid cancer and the remainder without cancer.

The Origin of Highly Elevated Cell-Free DNA in Healthy Individuals and Patients with Pancreatic, Colorectal, Lung, or Ovarian Cancer.

Unlabelled: Cell-free DNA (cfDNA) concentrations from patients with cancer are often elevated compared with those of healthy controls, but the sources of this extra cfDNA have never been determined. To address this issue, we assessed cfDNA methylation patterns in 178 patients with cancers of the colon, pancreas, lung, or ovary and 64 patients without cancer. Eighty-three of these individuals had cfDNA concentrations much greater than those generally observed in healthy subjects.

The rapid and highly parallel identification of antibodies with defined biological activities by SLISY.

The therapeutic applications of antibodies are manifold and the emergence of SARS-CoV-2 provides a cogent example of the value of rapidly identifying biologically active antibodies. We describe an approach called SLISY (Sequencing-Linked ImmunoSorbent assaY) that in a single experiment can assess the binding specificity of millions of clones, be applied to any screen that links DNA sequence to a potential binding moiety, and requires only a single round of biopanning. We demonstrate this approach using an scFv library applied to cellular and protein targets to identify specific or broadly reacting antibodies.

An isogenic cell line panel for sequence-based screening of targeted anticancer drugs.

We describe the creation of an isogenic cell line panel representing common cancer pathways, with features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cell lines were generated using CRISPR technologies. Surprisingly, most of these lines did not result in complete gene inactivation despite integration of sgRNA at the desired genomic site.

Circulating Tumor DNA Analysis Guiding Adjuvant Therapy in Stage II Colon Cancer.

Background: The role of adjuvant chemotherapy in stage II colon cancer continues to be debated. The presence of circulating tumor DNA (ctDNA) after surgery predicts very poor recurrence-free survival, whereas its absence predicts a low risk of recurrence. The benefit of adjuvant chemotherapy for ctDNA-positive patients is not well understood.

Detection of malignant peripheral nerve sheath tumors in patients with neurofibromatosis using aneuploidy and mutation identification in plasma.

Malignant peripheral nerve sheath tumors (MPNST) are the deadliest cancer that arises in individuals diagnosed with neurofibromatosis and account for nearly 5% of the 15,000 soft tissue sarcomas diagnosed in the United States each year. Comprised of neoplastic Schwann cells, primary risk factors for developing MPNST include existing plexiform neurofibromas (PN), prior radiotherapy treatment, and expansive germline mutations involving the entire gene and surrounding genes. PN develop in nearly 30-50% of patients with neurofibromatosis type 1 (NF1) and most often grow rapidly in the first decade of life.

Detection of low-frequency DNA variants by targeted sequencing of the Watson and Crick strands.

Identification and quantification of low-frequency mutations remain challenging despite improvements in the baseline error rate of next-generation sequencing technologies. Here, we describe a method, termed SaferSeqS, that addresses these challenges by (1) efficiently introducing identical molecular barcodes in the Watson and Crick strands of template molecules and (2) enriching target sequences with strand-specific PCR. The method achieves high sensitivity and specificity and detects variants at frequencies below 1 in 100,000 DNA template molecules with a background mutation rate of <5 × 10 mutants per base pair (bp).

Prognostic significance of postsurgery circulating tumor DNA in nonmetastatic colorectal cancer: Individual patient pooled analysis of three cohort studies.

Studies in multiple solid tumor types have demonstrated the prognostic significance of ctDNA analysis after curative intent surgery. A combined analysis of data across completed studies could further our understanding of circulating tumor DNA (ctDNA) as a prognostic marker and inform future trial design. We combined individual patient data from three independent cohort studies of nonmetastatic colorectal cancer (CRC).

Assessing aneuploidy with repetitive element sequencing.

We report a sensitive PCR-based assay called Repetitive Element AneupLoidy Sequencing System (RealSeqS) that can detect aneuploidy in samples containing as little as 3 pg of DNA. Using a single primer pair, we amplified ∼350,000 amplicons distributed throughout the genome. Aneuploidy was detected in 49% of liquid biopsies from a total of 883 nonmetastatic, clinically detected cancers of the colorectum, esophagus, liver, lung, ovary, pancreas, breast, or stomach.

A multimodality test to guide the management of patients with a pancreatic cyst.

Pancreatic cysts are common and often pose a management dilemma, because some cysts are precancerous, whereas others have little risk of developing into invasive cancers. We used supervised machine learning techniques to develop a comprehensive test, CompCyst, to guide the management of patients with pancreatic cysts. The test is based on selected clinical features, imaging characteristics, and cyst fluid genetic and biochemical markers.

Prognostic Potential of Circulating Tumor DNA Measurement in Postoperative Surveillance of Nonmetastatic Colorectal Cancer.

Importance: For patients with resected, nonmetastatic colorectal cancer (CRC), the optimal surveillance protocol remains unclear.

Objective: To evaluate whether serial circulating tumor DNA (ctDNA) levels detected disease recurrence earlier, compared with conventional postoperative surveillance, in patients with resected CRC.

Design, Setting, And Participants: This study included patients (n = 58) with stage I, II, or III CRC who underwent radical surgical resection at 4 Swedish hospitals from February 2, 2007, to May 8, 2013.

Evaluation of liquid from the Papanicolaou test and other liquid biopsies for the detection of endometrial and ovarian cancers.

We report the detection of endometrial and ovarian cancers based on genetic analyses of DNA recovered from the fluids obtained during a routine Papanicolaou (Pap) test. The new test, called PapSEEK, incorporates assays for mutations in 18 genes as well as an assay for aneuploidy. In Pap brush samples from 382 endometrial cancer patients, 81% [95% confidence interval (CI), 77 to 85%] were positive, including 78% of patients with early-stage disease.

Detection and localization of surgically resectable cancers with a multi-analyte blood test.

Earlier detection is key to reducing cancer deaths. Here, we describe a blood test that can detect eight common cancer types through assessment of the levels of circulating proteins and mutations in cell-free DNA. We applied this test, called CancerSEEK, to 1005 patients with nonmetastatic, clinically detected cancers of the ovary, liver, stomach, pancreas, esophagus, colorectum, lung, or breast.

Combined circulating tumor DNA and protein biomarker-based liquid biopsy for the earlier detection of pancreatic cancers.

The earlier diagnosis of cancer is one of the keys to reducing cancer deaths in the future. Here we describe our efforts to develop a noninvasive blood test for the detection of pancreatic ductal adenocarcinoma. We combined blood tests for gene mutations with carefully thresholded protein biomarkers to determine whether the combination of these markers was superior to any single marker.

Cancer-Associated Mutations in Endometriosis without Cancer.

Background: Endometriosis, defined as the presence of ectopic endometrial stroma and epithelium, affects approximately 10% of reproductive-age women and can cause pelvic pain and infertility. Endometriotic lesions are considered to be benign inflammatory lesions but have cancerlike features such as local invasion and resistance to apoptosis.

Methods: We analyzed deeply infiltrating endometriotic lesions from 27 patients by means of exomewide sequencing (24 patients) or cancer-driver targeted sequencing (3 patients).

The Effect of Preservative and Temperature on the Analysis of Circulating Tumor DNA.

Analysis of genomic alterations in cell-free DNA (cfDNA) is evolving as an approach to detect, monitor, and genotype malignancies. Methods to separate the liquid from the cellular fraction of whole blood for circulating tumor DNA (ctDNA) analyses have been largely unstudied, although these may be a critical consideration for assay performance. To evaluate the influence of blood processing on cfDNA and ctDNA quality and yield, we compared the cfDNA levels in serum with those in plasma.

Whole-Exome Sequencing Analyses of Inflammatory Bowel Disease-Associated Colorectal Cancers.

Background & Aims: A long duration of inflammatory bowel disease (IBD) increases the risk for colorectal cancer. Mutation analysis of limited numbers of genes has indicated that colorectal tumors that develop in patients with IBD differ from those of patients without IBD. We performed whole-exome sequencing analyses to characterize the genetic landscape of these tumors.