Outcomes following a false positive multi-cancer early detection (MCED) test: Results from DETECT-A, the first large, prospective, interventional MCED study.

Guideline recommended standard of care (SoC) screening is available for four cancer types; most cancer-related deaths are caused by cancers without SoC screening. DETECT-A is the first prospective interventional trial evaluating an MCED blood test (CancerSEEK) in women without a history of cancer, providing the first opportunity to assess the long-term outcomes of individuals with false positive (FP) MCED results. This prospective analysis of DETECT-A participants with FP results evaluates the performance of an imaging-based diagnostic workflow and examines cancer risk following a FP result. This analysis included all DETECT-A participants with a positive CancerSEEK test and subsequent flourine-18 fluorodeoxyglucose positron emission tomography-IV contrast enhanced computed tomography (18-F-FDG PET-CT) imaging and clinical workup indicating no evidence of cancer within one year of enrollment (n=98). Medical records, study interactions, and study surveys were used to assess cancer incidence, treatments, and clinical outcomes through August 2023. Ninety-five of 98 participants with a FP result remained cancer-free with a median follow-up of 3.6 years (IQR: 2.5-4.1) from determination of FP status. Three incident cancers were observed over the follow-up period. One bilateral stage IIIC ovarian cancer was diagnosed 1.9 years after determination of FP status; two stage I breast cancers were diagnosed 0.1 and 1.6 years from determination of FP status. The annual incidence rate of cancer during follow-up from FP determination was 1.0% (95% CI: 0.2%-2.8%). Participants with a positive CancerSEEK test who underwent 18-F-FDG PET-CT and clinical workup without cancer findings had low risk for cancer over the following several years.

Cancer therapy with antibodies.

The greatest challenge in cancer therapy is to eradicate cancer cells with minimal damage to normal cells. Targeted therapy has been developed to meet that challenge, showing a substantially increased therapeutic index compared with conventional cancer therapies. Antibodies are important members of the family of targeted therapeutic agents because of their extraordinarily high specificity to the target antigens. Therapeutic antibodies use a range of mechanisms that directly or indirectly kill the cancer cells. Early antibodies were developed to directly antagonize targets on cancer cells. This was followed by advancements in linker technologies that allowed the production of antibody-drug conjugates (ADCs) that guide cytotoxic payloads to the cancer cells. Improvement in our understanding of the biology of T cells led to the production of immune checkpoint-inhibiting antibodies that indirectly kill the cancer cells through activation of the T cells. Even more recently, bispecific antibodies were synthetically designed to redirect the T cells of a patient to kill the cancer cells. In this Review, we summarize the different approaches used by therapeutic antibodies to target cancer cells. We discuss their mechanisms of action, the structural basis for target specificity, clinical applications and the ongoing research to improve efficacy and reduce toxicity.

TRBC1-targeting antibody-drug conjugates for the treatment of T cell cancers.

Antibody and chimeric antigen receptor (CAR) T cell-mediated targeted therapies have improved survival in patients with solid and haematologic malignancies1-9. Adults with T cell leukaemias and lymphomas, collectively called T cell cancers, have short survival10,11 and lack such targeted therapies. Thus, T cell cancers particularly warrant the development of CAR T cells and antibodies to improve patient outcomes. Preclinical studies showed that targeting T cell receptor ß-chain constant region 1 (TRBC1) can kill cancerous T cells while preserving sufficient healthy T cells to maintain immunity12, making TRBC1 an attractive target to treat T cell cancers. However, the first-in-human clinical trial of anti-TRBC1 CAR T cells reported a low response rate and unexplained loss of anti-TRBC1 CAR T cells13,14. Here we demonstrate that CAR T cells are lost due to killing by the patient's normal T cells, reducing their efficacy. To circumvent this issue, we developed an antibody-drug conjugate that could kill TRBC1+ cancer cells in vitro and cure human T cell cancers in mouse models. The anti-TRBC1 antibody-drug conjugate may provide an optimal format for TRBC1 targeting and produce superior responses in patients with T cell cancers.

Real-time evaluation and adaptation to facilitate rapid recruitment in a large, prospective cohort study.

BACKGROUND: Recruiting large cohorts efficiently can speed the translation of findings into care across a range of scientific disciplines and medical specialties. Recruitment can be hampered by factors such as financial barriers, logistical concerns, and lack of resources for patients and clinicians. These and other challenges can lead to underrepresentation in groups such as rural residents and racial and ethnic minorities. Here we discuss the implementation of various recruitment strategies for enrolling participants into a large, prospective cohort study, assessing the need for adaptations and making them in real-time, while maintaining high adherence to the protocol and high participant satisfaction. METHODS: While conducting a large, prospective trial of a multi-cancer early detection blood test at Geisinger, an integrated health system in central Pennsylvania, we monitored recruitment progress, adherence to the protocol, and participants' satisfaction. Tracking mechanisms such as paper records, electronic health records, research databases, dashboards, and electronic files were utilized to measure each outcome. We then reviewed study procedures and timelines to list the implementation strategies that were used to address barriers to recruitment, protocol adherence and participant satisfaction. RESULTS: Adaptations to methods that contributed to achieving the enrollment goal included offering multiple recruitment options, adopting group consenting, improving visit convenience, increasing the use of electronic capture and the tracking of data and source documents, staffing optimization via leveraging resources external to the study team when appropriate, and integrating the disclosure of study results into routine clinical care without adding unfunded work for clinicians. We maintained high protocol adherence and positive participant experience as exhibited by a very low rate of protocol deviations and participant complaints. CONCLUSION: Recruiting rapidly for large studies - and thereby facilitating clinical translation - requires a nimble, creative approach that marshals available resources and changes course according to data. Planning a rigorous assessment of a study's implementation outcomes prior to study recruitment can further ground study adaptations and facilitate translation into practice. This can be accomplished by proactively and continuously assessing and revising implementation strategies.

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. Samples from patients with cancer and controls were prespecified into four cohorts used for model training, analyte integration, and threshold determination, validation, and reproducibility. A-PLUS alone provided a sensitivity of 40.5% across 11 different cancer types in the validation cohort, at a specificity of 98.5%. Combining A-PLUS with aneuploidy and eight common protein biomarkers detected 51% of the cancers at 98.9% specificity. We found that part of the power of A-PLUS could be ascribed to a single feature-the global reduction of AluS subfamily elements in the circulating DNA of patients with solid cancer. We confirmed this reduction through the analysis of another independent dataset obtained with a different approach (whole-genome sequencing). The evaluation of Alu elements may therefore have the potential to enhance the performance of several methods designed for the earlier detection of cancer.

Aneuploidy Landscape in Precursors of Ovarian Cancer.

PURPOSE: Serous tubal intraepithelial carcinoma (STIC) is now recognized as the main precursor of ovarian high-grade serous carcinoma (HGSC). Other potential tubal lesions include p53 signatures and tubal intraepithelial lesions. We aimed to investigate the extent and pattern of aneuploidy in these epithelial lesions and HGSC to define the features that characterize stages of tumor initiation and progression. EXPERIMENTAL DESIGN: We applied RealSeqS to compare genome-wide aneuploidy patterns among the precursors, HGSC (cases, n = 85), and histologically unremarkable fallopian tube epithelium (HU-FTE; control, n = 65). On the basis of a discovery set (n = 67), we developed an aneuploidy-based algorithm, REAL-FAST (Repetitive Element AneupLoidy Sequencing Fallopian Tube Aneuploidy in STIC), to correlate the molecular data with pathology diagnoses. We validated the result in an independent validation set (n = 83) to determine its performance. We correlated the molecularly defined precursor subgroups with proliferative activity and histology. RESULTS: We found that nearly all p53 signatures lost the entire Chr17, offering a "two-hit" mechanism involving both TP53 and BRCA1 in BRCA1 germline mutation carriers. Proliferatively active STICs harbor gains of 19q12 (CCNE1), 19q13.2, 8q24 (MYC), or 8q arm, whereas proliferatively dormant STICs show 22q loss. REAL-FAST classified HU-FTE and STICs into 5 clusters and identified a STIC subgroup harboring unique aneuploidy that is associated with increased proliferation and discohesive growth. On the basis of a validation set, REAL-FAST showed 95.8% sensitivity and 97.1% specificity in detecting STIC/HGSC. CONCLUSIONS: Morphologically similar STICs are molecularly distinct. The REAL-FAST assay identifies a potentially "aggressive" STIC subgroup harboring unique DNA aneuploidy that is associated with increased cellular proliferation and discohesive growth. REAL-FAST offers a highly reproducible adjunct technique to assist the diagnosis of STIC lesions.

The Liquid Biopsy Consortium: Challenges and opportunities for early cancer detection and monitoring.

The emerging field of liquid biopsy stands at the forefront of novel diagnostic strategies for cancer and other diseases. Liquid biopsy allows minimally invasive molecular characterization of cancers for diagnosis, patient stratification to therapy, and longitudinal monitoring. Liquid biopsy strategies include detection and monitoring of circulating tumor cells, cell-free DNA, and extracellular vesicles. In this review, we address the current understanding and the role of existing liquid-biopsy-based modalities in cancer diagnostics and monitoring. We specifically focus on the technical and clinical challenges associated with liquid biopsy and biomarker development being addressed by the Liquid Biopsy Consortium, established through the National Cancer Institute. The Liquid Biopsy Consortium has developed new methods/assays and validated existing methods/technologies to capture and characterize tumor-derived circulating cargo, as well as addressed existing challenges and provided recommendations for advancing biomarker assays.

Extrinsic and intrinsic preanalytical variables affecting liquid biopsy in cancer.

Liquid biopsy, through isolation and analysis of disease-specific analytes, has evolved as a promising tool for safe and minimally invasive diagnosis and monitoring of tumors. It also has tremendous utility as a companion diagnostic allowing detection of biomarkers in a range of cancers (lung, breast, colon, ovarian, brain). However, clinical implementation and validation remains a challenge. Among other stages of development, preanalytical variables are critical in influencing the downstream cellular and molecular analysis of different analytes. Although considerable progress has been made to address these challenges, a comprehensive assessment of the impact on diagnostic parameters and consensus on standardized and optimized protocols is still lacking. Here, we summarize and critically evaluate key variables in the preanalytical stage, including study population selection, choice of biofluid, sample handling and collection, processing, and storage. There is an unmet need to develop and implement comprehensive preanalytical guidelines on the optimal practices and methodologies.

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

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 major contributor of cfDNA in all samples was leukocytes, accounting for ∼76% of cfDNA, with neutrophils predominating. This was true regardless of whether the samples were derived from patients with cancer or the total plasma cfDNA concentration. High levels of cfDNA observed in patients with cancer did not come from either neoplastic cells or surrounding normal epithelial cells from the tumor's tissue of origin. These data suggest that cancers may have a systemic effect on cell turnover or DNA clearance. SIGNIFICANCE: The origin of excess cfDNA in patients with cancer is unknown. Using cfDNA methylation patterns, we determined that neither the tumor nor the surrounding normal tissue contributes this excess cfDNA-rather it comes from leukocytes. This finding suggests that cancers have a systemic impact on cell turnover or DNA clearance. See related commentary by Thierry and Pisareva, p. 2122. This article is featured in Selected Articles from This Issue, p. 2109.

Hydrophobic interactions dominate the recognition of a KRAS G12V neoantigen.

Specificity remains a major challenge to current therapeutic strategies for cancer. Mutation associated neoantigens (MANAs) are products of genetic alterations, making them highly specific therapeutic targets. MANAs are HLA-presented (pHLA) peptides derived from intracellular mutant proteins that are otherwise inaccessible to antibody-based therapeutics. Here, we describe the cryo-EM structure of an antibody-MANA pHLA complex. Specifically, we determine a TCR mimic (TCRm) antibody bound to its MANA target, the KRASG12V peptide presented by HLA-A*03:01. Hydrophobic residues appear to account for the specificity of the mutant G12V residue. We also determine the structure of the wild-type G12 peptide bound to HLA-A*03:01, using X-ray crystallography. Based on these structures, we perform screens to validate the key residues required for peptide specificity. These experiments led us to a model for discrimination between the mutant and the wild-type peptides presented on HLA-A*03:01 based exclusively on hydrophobic interactions.

ME3BP-7 is a targeted cytotoxic agent that rapidly kills pancreatic cancer cells expressing high levels of monocarboxylate transporter MCT1.

Nearly 30% of Pancreatic ductal adenocarcinoma (PDAC)s exhibit a marked overexpression of Monocarboxylate Transporter 1 (MCT1) offering a unique opportunity for therapy. However, biochemical inhibitors of MCT1 have proven unsuccessful in clinical trials. In this study we present an alternative approach using 3-Bromopyruvate (3BP) to target MCT1 overexpressing PDACs. 3BP is a cytotoxic agent that is known to be transported into cells via MCT1, but its clinical usefulness has been hampered by difficulties in delivering the drug systemically. We describe here a novel microencapsulated formulation of 3BP (ME3BP-7), that is effective against a variety of PDAC cells in vitro and remains stable in serum. Furthermore, systemically administered ME3BP-7 significantly reduces pancreatic cancer growth and metastatic spread in multiple orthotopic models of pancreatic cancer with manageable toxicity. ME3BP-7 is, therefore, a prototype of a promising new drug, in which the targeting moiety and the cytotoxic moiety are both contained within the same single small molecule. One Sentence Summary: ME3BP-7 is a novel formulation of 3BP that resists serum degradation and rapidly kills pancreatic cancer cells expressing high levels of MCT1 with tolerable toxicity in mice.

Seq-ing the SINEs of central nervous system tumors in cerebrospinal fluid.

It is often challenging to distinguish cancerous from non-cancerous lesions in the brain using conventional diagnostic approaches. We introduce an analytic technique called Real-CSF (repetitive element aneuploidy sequencing in CSF) to detect cancers of the central nervous system from evaluation of DNA in the cerebrospinal fluid (CSF). Short interspersed nuclear elements (SINEs) are PCR amplified with a single primer pair, and the PCR products are evaluated by next-generation sequencing. Real-CSF assesses genome-wide copy-number alterations as well as focal amplifications of selected oncogenes. Real-CSF was applied to 280 CSF samples and correctly identified 67% of 184 cancerous and 96% of 96 non-cancerous brain lesions. CSF analysis was considerably more sensitive than standard-of-care cytology and plasma cell-free DNA analysis in the same patients. Real-CSF therefore has the capacity to be used in combination with other clinical, radiologic, and laboratory-based data to inform the diagnosis and management of patients with suspected cancers of the brain.

Detection of rare mutations, copy number alterations, and methylation in the same template DNA molecules.

The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To further increase the sensitivity of such assays with limited amounts of sample, it would be useful to be able to evaluate the same template molecules for all these changes. Here, we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The epigenetic and genetic alterations present in the DNA molecules can then be obtained from the original and copied strands, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung, and colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics.

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. 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.

Identification of nonsense-mediated decay inhibitors that alter the tumor immune landscape.

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. This screen revealed disruption of kinase SMG1's phosphorylation of UPF1 as a potent disruptor of NMD. This led us to design a novel SMG1 inhibitor, KVS0001, that elevates the expression of transcripts and proteins resulting from truncating mutations in vivo and in vitro . Most importantly, KVS0001 concomitantly increased the presentation of immune-targetable HLA class I-associated peptides from NMD-downregulated proteins on the surface of cancer cells. KVS0001 provides new opportunities for studying NMD and the diseases in which NMD plays a role, including cancer and inherited diseases. One Sentence Summary: Disruption of the nonsense-mediated decay pathway with a newly developed SMG1 inhibitor with in-vivo activity increases the expression of T-cell targetable cancer neoantigens resulting from truncating mutations.

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. A subset of the lines harbored biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterized lines covering 19 frequently lost cancer pathways. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of TP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.

Inpatient Administration of Alpha-1-Adrenergic Receptor Blocking Agents Reduces Mortality in Male COVID-19 Patients.

Apha-1-adrenergic receptor antagonists (α1-blockers) can suppress pro-inflammatory cytokines, thereby potentially improving outcomes among patients with COVID-19. Accordingly, we evaluated the association between α1-blocker exposure (before or during hospitalization) and COVID-19 in-hospital mortality. We identified 2,627 men aged 45 or older who were admitted to Mount Sinai hospitals with COVID-19 between February 24 and May 31, 2020, in New York. Men exposed to α1-blockers (N = 436) were older (median age 73 vs. 64 years, P < 0.001) and more likely to have comorbidities than unexposed men (N = 2,191). Overall, 777 (29.6%) patients died in hospital, and 1,850 (70.4%) were discharged. Notably, we found that α1-blocker exposure was independently associated with improved in-hospital mortality in a multivariable logistic analysis (OR 0.699; 95% CI, 0.498-0.982; P = 0.039) after adjusting for patient demographics, comorbidities, and baseline vitals and labs. The protective effect of α1-blockers was stronger among patients with documented inpatient exposure to α1-blockers (OR 0.624; 95% CI 0.431-0.903; P = 0.012). Finally, age-stratified analyses suggested variable benefit from inpatient α1-blocker across age groups: Age 45-65 OR 0.483, 95% CI 0.216-1.081 (P = 0.077); Age 55-75 OR 0.535, 95% CI 0.323-0.885 (P = 0.015); Age 65-89 OR 0.727, 95% CI 0.484-1.092 (P = 0.124). Taken together, clinical trials to assess the therapeutic value of α1-blockers for COVID-19 complications are warranted.

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 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.

Multicancer early detection test: Preclinical, translational, and clinical evidence-generation plan and provocative questions.

Minimally invasive molecular biomarkers have been applied to the early detection of multiple cancers in large scale case-control and cohort studies. These demonstrations of feasibility herald the potential for permanent transformation of current cancer screening paradigms. This commentary discusses the major opportunities and challenges facing the preclinical development and clinical validation of multicancer early detection test strategies. From a diverse set of early detection research perspectives, the authors recommend specific approaches and highlight important questions for future investigation.