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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. We conducted a trial to assess whether a ctDNA-guided approach could reduce the use of adjuvant chemotherapy without compromising recurrence risk. Patients with stage II colon cancer were randomly assigned in a 2:1 ratio to have treatment decisions guided by either ctDNA results or standard clinicopathological features. For ctDNA-guided management, a ctDNA-positive result at 4 or 7 weeks after surgery prompted oxaliplatin-based or fluoropyrimidine chemotherapy. Patients who were ctDNA-negative were not treated. The primary efficacy end point was recurrence-free survival at 2 years. A key secondary end point was adjuvant chemotherapy use. Of the 455 patients who underwent randomization, 302 were assigned to ctDNA-guided management and 153 to standard management. The median follow-up was 37 months. A lower percentage of patients in the ctDNA-guided group than in the standard-management group received adjuvant chemotherapy (15% vs. 28%; relative risk, 1.82; 95% confidence interval [CI], 1.25 to 2.65). In the evaluation of 2-year recurrence-free survival, ctDNA-guided management was noninferior to standard management (93.5% and 92.4%, respectively; absolute difference, 1.1 percentage points; 95% CI, -4.1 to 6.2 [noninferiority margin, -8.5 percentage points]). Three-year recurrence-free survival was 86.4% among ctDNA-positive patients who received adjuvant chemotherapy and 92.5% among ctDNA-negative patients who did not. A ctDNA-guided approach to the treatment of stage II colon cancer reduced adjuvant chemotherapy use without compromising recurrence-free survival. (Supported by the Australian National Health and Medical Research Council and others; DYNAMIC Australian New Zealand Clinical Trials Registry number, ACTRN12615000381583.).

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 KRAS gene mutations with carefully thresholded protein biomarkers to determine whether the combination of these markers was superior to any single marker. The cohort tested included 221 patients with resectable pancreatic ductal adenocarcinomas and 182 control patients without known cancer. KRAS mutations were detected in the plasma of 66 patients (30%), and every mutation found in the plasma was identical to that subsequently found in the patient’s primary tumor (100% concordance). The use of KRAS in conjunction with four thresholded protein biomarkers increased the sensitivity to 64%. Only one of the 182 plasma samples from the control cohort was positive for any of the DNA or protein biomarkers (99.5% specificity). This combinatorial approach may prove useful for the earlier detection of many cancer types.

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 –7 mutants per base pair (bp). We demonstrate that it can evaluate mutations in a single amplicon or simultaneously in multiple amplicons, assess limited quantities of cell-free DNA with high recovery of both strands and reduce the error rate of existing PCR-based molecular barcoding approaches by >100-fold. Mutations present at a low frequency in a sample are detected with high sensitivity and a low error rate.

Mild periodic acid Schiff staining (mPAS) of human colonic tissue has been used to answer a variety of fundamental questions in germline and somatic genetics. mPAS stains sialic acids except when these glycans are modified by O-acetylation, but a full accounting of the genes contributing to sialoglycan acetylation is incomplete. Using haplotypes derived from whole genome sequencing, we identify a region on chromosome 11 that is associated with inherited differences in mPAS staining. Of the genes in this region, only haplotypes containing NXPE1 correlate perfectly with mPAS staining in the original cohort used for whole genome sequencing, as well as in a validation cohort. Transcriptomic analysis indicates that linked haplotypes are associated with altered expression of NXPE1 suggesting a possible genetic mechanism. Genetic manipulation of a common single nucleotide polymorphism observed in the haplotype region and located in NXPE1 ’s promoter alters expression and causes changes to modified sialic acid levels supporting this mechanism. Finally, high-performance liquid chromatography (HPLC) confirms that enzymatically active NXPE1 is capable of transferring an acetyl group from acetyl coenzyme A to sialic acid in vitro. These findings suggest that NXPE1 is the long-sought gene responsible for differences in colon mPAS staining and may be the prototype of a new family of sialic acid O-acetylation-modifying genes. Sialic acids are key cell-surface glycans that regulate immune signaling and cellular recognition. Here, the authors show that NXPE1 encodes a sialic acid O-acetyltransferase, revealing its role in modulating glycan-mediated signaling.

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. For a cellular target, we use paired HLA knockout cell lines to identify a panel of antibodies specific to HLA-A3. For a protein target, SLISY identifies 1279 clones that bound to the Receptor Binding Domain of the SARS-CoV-2 spike protein, with >40% of tested clones also neutralizing its interaction with ACE2 in in vitro assays. Using a multi-comparison SLISY against the Beta, Gamma, and Delta variants, we recovered clones that exhibited broad-spectrum neutralizing potential in vitro. By evaluating millions of scFvs simultaneously against multiple targets, SLISY allows the rapid identification of candidate scFvs with defined binding profiles facilitating the identification of antibodies with the desired biological activity. The covid pandemic has highlighted the need for rapid antibody development. Here, authors develop an approach called SLISY, which uses NGS with phage display to simultaneously assess millions of clones to rapidly isolate specific antibodies against SARS-CoV-2 and its evolving variants.

Despite exciting developments in cancer immunotherapy, its broad application is limited by the paucity of targetable antigens on the tumor cell surface. As an intrinsic cellular pathway, nonsense-mediated decay (NMD) conceals neoantigens through the destruction of the RNA products from genes harboring truncating mutations. We developed and conducted a high-throughput screen, based on the ratiometric analysis of transcripts, to identify critical mediators of NMD in human cells. This screen implicated disruption of kinase SMG1’s phosphorylation of UPF1 as a potential disruptor of NMD. This led us to design a novel SMG1 inhibitor, KVS0001, that elevates the expression of transcripts and proteins resulting from human and murine truncating mutations in vitro and murine cells in vivo. Most importantly, KVS0001 concomitantly increased the presentation of immune-targetable human leukocyte antigens (HLA) class I-associated peptides from NMD-downregulated proteins on the surface of human cancer cells. KVS0001 provides new opportunities for studying NMD and the diseases in which NMD plays a role, including cancer and inherited diseases. Immunotherapies are treatments that have revolutionized cancer care by helping a patient’s own immune system find and destroy cancer cells. Unfortunately, less than half of treated patients respond to these therapies, with tumors often learning to escape detection by the immune system. One way that cancer cells can evade the immune system is by preventing themselves from producing mutant proteins. By stopping these proteins from reaching the cell surface, the abnormal cell is less likely to be detected and killed by the immune system. One way cancer cells accomplish this is by destroying the RNA templates needed to make the proteins through a process called ‘nonsense-mediated decay’. Therefore, developing a therapy that can stop nonsense-mediated decay could help the immune system find and kill more tumor cells. Cook et al. screened thousands of drugs with the aim of finding one that blocks nonsense-mediated decay. Although one drug was identified that could inhibit a gene called SMG1 (which is known to activate nonsense-mediated decay), it was too toxic in animal models to be considered as a therapy. Therefore, Cook et al. developed a new drug targeting this gene that slowed tumor growth in mice without showing the same toxicity. Treating human cancer cells with the drug also increased the number of mutant proteins on the cell surface displayed to the immune system, suggesting the drug has the potential to prevent nonsense-mediated decay in humans. The findings suggest that the drug developed by Cook et al. may make it easier for the immune system to identify and destroy certain cancer cells. This might also be relevant for other conditions involving nonsense-mediated decay, such as cystic fibrosis, Alport’s disease, and Duchenne muscular dystrophy. If further studies confirm that the drug is safe and effective in humans, it could be used alongside cancer immunotherapies to improve patient response rates.

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 NF1 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. One of the most important aspects of clinical care for NF1 patients is monitoring PN for signs of malignant transformation to MPNST that occurs in 10–15% of patients. We perform aneuploidy analysis on ctDNA from 883 ostensibly healthy individuals and 28 patients with neurofibromas, including 7 patients with benign neurofibroma, 9 patients with PN and 12 patients with MPNST. Overall sensitivity for detecting MPNST using genome wide aneuploidy scoring was 33%, and analysis of sub-chromosomal copy number alterations (CNAs) improved sensitivity to 50% while retaining a high specificity of 97%. In addition, we performed mutation analysis on plasma cfDNA for a subset of patients and identified mutations in NF1 , NF2 , RB1 , TP53BP2 , and GOLGA2 . Given the high throughput and relatively low sequencing coverage required by our assay, liquid biopsy represents a promising technology to identify incipient MPNST.

Genetic analysis of endometriotic lesions from 39 patients revealed mutations in cancer driver genes in 26%. In some patients, the same mutation was present in lesions from different sites. The role of mutations in cancer-associated genes in endometriosis needs further study. Endometriosis is a relatively common disease, affecting up to 10% of women of reproductive age. 1 Its incidence is as high as 50% among adolescents with pelvic pain. 1 , 2 Clinical symptoms include dysmenorrhea, pelvic pain, and infertility. 1 , 3 , 4 Endometriotic lesions are considered to be benign (nonmalignant or nonneoplastic), inflammatory, estrogen-dependent lesions that are characterized by the ectopic presence of normal-appearing, functional endometrial tissue composed of glands and stroma outside of the uterus. 1 , 3 The disease is often associated with multiple lesions that can be distributed throughout the abdominal–pelvic peritoneum and visceral organs. There are three anatomical subtypes of endometriosis: superficial . . .

Many cancers can be cured by surgery and/or systemic therapies when detected before they have metastasized. This clinical reality, coupled with the growing appreciation that cancer's rapid genetic evolution limits its response to drugs, have fueled interest in methodologies for earlier detection of the disease. Cohen et al. developed a noninvasive blood test, called CancerSEEK that can detect eight common human cancer types (see the Perspective by Kalinich and Haber). The test assesses eight circulating protein biomarkers and tumor-specific mutations in circulating DNA. In a study of 1000 patients previously diagnosed with cancer and 850 healthy control individuals, CancerSEEK detected cancer with a sensitivity of 69 to 98% (depending on cancer type) and 99% specificity. Science , this issue p. 926 ; see also p. 866 A blood test that combines protein and DNA markers may allow earlier detection of eight common cancer types. 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. CancerSEEK tests were positive in a median of 70% of the eight cancer types. The sensitivities ranged from 69 to 98% for the detection of five cancer types (ovary, liver, stomach, pancreas, and esophagus) for which there are no screening tests available for average-risk individuals. The specificity of CancerSEEK was greater than 99%: only 7 of 812 healthy controls scored positive. In addition, CancerSEEK localized the cancer to a small number of anatomic sites in a median of 83% of the patients.

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. Combining aneuploidy with somatic mutation detection and eight standard protein biomarkers yielded a median sensitivity of 80% in these eight cancer types, while only 1% of 812 healthy controls scored positive.