Comparison of tumor-informed and tumor-naïve sequencing assays for ctDNA detection in breast cancer.

Analysis of circulating tumor DNA (ctDNA) to monitor cancer dynamics and detect minimal residual disease has been an area of increasing interest. Multiple methods have been proposed but few studies have compared the performance of different approaches. Here, we compare detection of ctDNA in serial plasma samples from patients with breast cancer using different tumor-informed and tumor-naïve assays designed to detect structural variants (SVs), single nucleotide variants (SNVs), and/or somatic copy-number aberrations, by multiplex PCR, hybrid capture, and different depths of whole-genome sequencing. Our results demonstrate that the ctDNA dynamics and allele fractions (AFs) were highly concordant when analyzing the same patient samples using different assays. Tumor-informed assays showed the highest sensitivity for detection of ctDNA at low concentrations. Hybrid capture sequencing targeting between 1,347 and 7,491 tumor-identified mutations at high depth was the most sensitive assay, detecting ctDNA down to an AF of 0.00024% (2.4 parts per million, ppm). Multiplex PCR targeting 21-47 tumor-identified SVs per patient detected ctDNA down to 0.00047% AF (4.7 ppm) and has potential as a clinical assay.

Synthesis of Sulfur-Substituted Bicyclo[1.1.1]pentanes by Iodo-Sulfenylation of [1.1.1]Propellane.

Thiols easily react with [1.1.1]propellane to give sulfur-substituted bicyclo[1.1.1]pentanes in radical reactions, but this reactivity is not replicated in the case of heterocyclic thiols. Herein, we address this issue by electrophilically activating [1.1.1]propellane to promote its iodo-sulfenylation with 10 classes of heterocyclic thiols in two protocols that can be conducted on a multigram scale without exclusion of air or moisture.

Characteristics, origin, and potential for cancer diagnostics of ultrashort plasma cell-free DNA.

Current evidence suggests that plasma cell-free DNA (cfDNA) is fragmented around a mode of 166 bp. Data supporting this view has been mainly acquired through the analysis of double-stranded cfDNA. The characteristics and diagnostic potential of single-stranded and damaged double-stranded cfDNA in healthy individuals and cancer patients remain unclear. Here, through a combination of high-affinity magnetic bead-based DNA extraction and single-stranded DNA sequencing library preparation (MB-ssDNA), we report the discovery of a large proportion of cfDNA fragments centered at ∼50 bp. We show that these "ultrashort" cfDNA fragments have a greater relative abundance in plasma of healthy individuals (median = 19.1% of all sequenced cfDNA fragments, n = 28) than in plasma of patients with cancer (median = 14.2%, n = 21, P < 0.0001). The ultrashort cfDNA fragments map to accessible chromatin regions of blood cells, particularly in promoter regions with the potential to adopt G-quadruplex (G4) DNA secondary structures. G4-positive promoter chromatin accessibility is significantly enriched in ultrashort plasma cfDNA fragments from healthy individuals relative to patients with cancers (P < 0.0001), in whom G4-cfDNA enrichment is inversely associated with copy number aberration-inferred tumor fractions. Our findings redraw the landscape of cfDNA fragmentation by identifying and characterizing a novel population of ultrashort plasma cfDNA fragments. Sequencing of MB-ssDNA libraries could facilitate the characterization of gene regulatory regions and DNA secondary structures via liquid biopsy. Our data underline the diagnostic potential of ultrashort cfDNA through classification for cancer patients.

Resistance to a nonselective 4-hydroxyphenylpyruvate dioxygenase-inhibiting herbicide via novel reduction-dehydration-glutathione conjugation in Amaranthus tuberculatus.

Metabolic resistance to 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides is a threat in controlling waterhemp (Amaranthus tuberculatus) in the USA. We investigated resistance mechanisms to syncarpic acid-3 (SA3), a nonselective, noncommercial HPPD-inhibiting herbicide metabolically robust to Phase I oxidation, in multiple-herbicide-resistant (MHR) waterhemp populations (SIR and NEB) and HPPD inhibitor-sensitive populations (ACR and SEN). Dose-response experiments with SA3 provided ED50 -based resistant : sensitive ratios of at least 18-fold. Metabolism experiments quantifying parent SA3 remaining in excised leaves during a time course indicated MHR populations displayed faster rates of SA3 metabolism compared to HPPD inhibitor-sensitive populations. SA3 metabolites were identified via LC-MS-based untargeted metabolomics in whole plants. A Phase I metabolite, likely generated by cytochrome P450-mediated alkyl hydroxylation, was detected but was not associated with resistance. A Phase I metabolite consistent with ketone reduction followed by water elimination was detected, creating a putative α,ß-unsaturated carbonyl resembling a Michael acceptor site. A Phase II glutathione-SA3 conjugate was associated with resistance. Our results revealed a novel reduction-dehydration-GSH conjugation detoxification mechanism. SA3 metabolism in MHR waterhemp is thus atypical compared to commercial HPPD-inhibiting herbicides. This previously uncharacterized detoxification mechanism presents a unique opportunity for future biorational design by blocking known sites of herbicide metabolism in weeds.

Repeat limited fasciectomy is a safe and effective treatment for recurrence of Dupuytren's disease.

AIMS: The results of surgery for Dupuytren's disease can be compromised by the potential for disease recurrence and loss of function. Selecting which patients will benefit from repeat surgery, when to operate, and what procedure to undertake requires judgement and an understanding of patient expectations and functional needs. We undertook this study to investigate patient outcomes and satisfaction following repeat limited fasciectomy for recurrent Dupuytren's disease. METHODS: We prospectively identified all patients presenting with recurrence of Dupuytren's disease who were selected for surgical treatment with repeat limited fasciectomy surgery between January 2013 and February 2015. Patients were assessed preoperatively, and again at a minimum of five years postoperatively. We identified 43 patients who were carefully selected for repeat fasciectomy involving 54 fingers. Patients with severe or aggressive disease with extensive skin involvement were not included; in our practice, these patients are instead counselled and preferentially treated with dermofasciectomy. The primary outcome measured was change in the Michigan Hand Outcomes Questionnaire (MHQ) score. Secondary outcomes were change in finger range of motion, flexion contracture, Semmes-Weinstein monofilament (SWM) values, and overall satisfaction. RESULTS: There was a significant improvement in MHQ scores, across all domains, with a mean overall score increase of 24 points (p < 0.001). The summed flexion contracture across the metacarpophalangeal joint (MCPJ) and the proximal interphalangeal joint (PIPJ) reduced from means of 72.0° (SD 15.9°) to 5.6° (SD 6.8°) (p < 0.001). A significant increase in maximal flexion was seen at the MCPJ (p < 0.001) but not the PIPJ (p = 0.550). The mean overall satisfaction score from the visual analogue scale was 8.9 (7.9 to 10.0). Complications were uncommon although five fingers showed reduced sensibility at final follow-up. CONCLUSION: Our study shows that repeat limited fasciectomy for selected patients presenting with recurrence of Dupuytren's disease can be an effective and safe treatment resulting in excellent patient-reported outcomes and levels of satisfaction. Cite this article: Bone Joint J 2021;103-B(5):946-950.

Day case locked anterior plating for distal radial fractures is safe with uninterrupted antithrombotic therapy.

We undertook a matched prospective cohort study over a 4-year period to examine the safety of continuing the administration of regular antithrombotic treatment with warfarin, clopidogrel or aspirin during day case surgical fixation of distal radial fractures. One hundred and one patients were identified and consented to participate in this study. There was only one reported complication: a superficial wound infection treated with antibiotics. No episodes of excessive bleeding were noted intraoperatively. All patients were discharged home on the day of surgery and there were no episodes of readmission, significant bleeding, haematoma requiring intervention, compartment syndrome or wound dehiscence. Complication rates were comparable with those of the matched cohort of patients undergoing the same procedure but who were not taking antithrombotic medications.Level of evidence: IV.

Optimise the microbial flora with milk and yoghurt to prevent disease.

Pathogenic bacteria, which are temporary or permanent members of our microbial flora, cause or contribute to a wide range of human disease at all ages. Conditions include Alzheimer's disease, atherosclerosis, diabetes mellitus, obesity, cancer, autoimmunity and psychosis, amongst others. The mechanism of damage is inflammation which can be chronic or acute. An optimal microbial flora includes a wide range of pathogenic bacteria in low dose. This allows specific immunity to be developed and maintained with minimal inflammatory damage. Human milk has evolved to deliver an optimal microbial flora to the infant. Cow's milk has the potential, following appropriate fortification, to maintain an optimal human microbial flora throughout life. Yoghurt is a fermented milk product in which bacteria normally present in milk convert sugars to lactic acid. The acid suppresses the growth of pathogens in the oral cavity, oropharynx and oesophagus. Thus yoghurt can restore an optimal flora in these regions in the short term. Since bacteria are transported between epithelial surfaces, yoghurt will also optimise the flora elsewhere. The judicious use of milk and yogurt could prevent a high proportion of human disease.

Prone or Left for Colonoscopy? A Randomized Controlled Trial of Prone Versus Left-sided Starting Position for Colonoscopy.

BACKGROUND AND AIMS: Data have emerged supporting the right-lateral starting position in reducing time to cecum and improving patient comfort for minimally sedated colonoscopy. We aimed to test whether prone starting position results in similar advantages in procedure time and patient tolerability in comparison to traditional left-sided starting position. MATERIALS AND METHODS: We conducted a randomized controlled trial in which patients were randomized to begin in either the prone or left-lateral (LL) position. A total of 181 adult patients undergoing scheduled colonoscopy were stratified by age, gender, body mass index, and experience of the endoscopist. Patients were then randomized 1:1 in permuted blocks. The primary outcome measure was time to cecal intubation and secondary outcome measures included patient comfort that was measured by visual analog scale. RESULTS: There was no benefit from prone starting positioning over conventional left-sided starting positioning. Further, prone starting position led to an increase in time to reach cecum (701 vs. 511 s; P=0.01). This could be in part explained by an increased time to reach transverse colon in patients positioned prone (332 vs. 258 s; P=0.06). Comfort levels were similar between patients positioned prone and LL (4 vs. 4 visual analog scale; P=0.6) although endoscopists found colonoscopies in which patients started prone more technically challenging than if started LL (5 vs. 4; P=0.002). CONCLUSIONS: Prone starting position did not improve time to reach cecum or patient comfort for minimally sedated patients undergoing colonoscopy. The ClinicalTrials.gov identifier is NCT02305706.

Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample throughput, and is well-suited for routine CTX monitoring programs.

The hierarchical model of stem cell genesis explains the man mouse paradox, Peto's paradox, the red cell paradox and Wright's enigma.

The central dogma of carcinogenesis is that deleterious mutations accumulate in regularly cycling stem cells and eventually one of the cells will acquire a specific set of mutations which leads to uncontrolled cell proliferation. Each mutation is rare and the specific set is extremely rare so that even though there are millions of stem cells in a small area of mucosa the specific set of mutations to initiate the process of malignancy will only arise in one stem cell at most; hence neoplasia is clonal. But this model predicts that men, who are 1000 times larger than mice and live 30 times as long, should have a vastly increased risk of cancer compared with mice, but they don't (man-mouse paradox). The model also predicts that the prevalence of cancer in men should rise as power function of age and mutagen dose, the former is correct but not the latter (Peto's paradox). Furthermore there are more mitotic divisions in red cell precursors than in all other stem cells combined and yet erythroleukaemia is rare (red cell paradox). The central dogma is also challenged by Wright's enigma; the observation that some gastro-intestinal neoplasms are polyclonal in origin. The problem with the central dogma is the concept of a regularly cycling stem cell. In fact it is possible to produce all the cells that arise in a human lifetime with fewer than 60 rounds of DNA replication separating the zygote from mature differentiated cells in extreme old age. This hierarchical model of stem cell genesis leads to a very low prevalence of cancer, unless the orderly progression of the hierarchy is disturbed by inflammation, ulceration or trauma. This model explains the paradoxes and Wright's enigma. It is suggested that the number of cell divisions that separate the zygote from stem cells is a key variable in carcinogenesis.

Phosphoinositide 3-kinase δ gene mutation predisposes to respiratory infection and airway damage.

Genetic mutations cause primary immunodeficiencies (PIDs) that predispose to infections. Here, we describe activated PI3K-δ syndrome (APDS), a PID associated with a dominant gain-of-function mutation in which lysine replaced glutamic acid at residue 1021 (E1021K) in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased immunoglobulin M, and reduced immunoglobulin G2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, which suggested a therapeutic approach for patients with APDS.

Low dose, early mucosal exposure will minimize the risk of microbial disease.

In the 21st century we will rediscover the germ theory of disease: germs not only cause infection as described in standard textbooks but also have a pathogenic role in autoimmunity, atherosclerosis, cancer and even acute psychiatric conditions. In order to reduce morbidity and mortality caused by common organisms we should ensure that exposure is early, often, by the mucosal route and in low dose. Micro-organisms should be delivered daily throughout life by respiratory mucosal spray or enteric coated pill, in precise dose and in a predetermined schedule.

Inter-individual differences in the ability of human milk-fat extracts to enhance the genotoxic potential of the procarcinogen benzo[a]pyrene in MCF-7 breast cells.

Environmental factors are believed to play an important role in cancer aetiology. Whether environmental pollutants act in isolation or in combination within mixtures remains unclear. Four human milk-fat extracts (from resident U.K. women) were screened for levels of organochlorinated and brominated compounds prior to being tested (1-50 mg-equiv) for micronucleus (MN)-forming activity in MCF-7 cells. Using the cytokinesis-block micronucleus assay, micronuclei (MNi) were scored in 1000 binucleate cells per treatment. Cell viability (% plating efficiency) and immunohistochemical detection of p53 induction were also measured. The effects of treatment with 1 mg-equiv of extract in combination with benzo[a]pyrene (BP) were also examined. BP-DNA adducts were detected and quantified by 32P-postlabeling analysis. Dose-related increases in MNi independent of pollutant concentrations were induced by milk-fat extracts. All four extracts elevated the percentage of p53 positive cells, although not always in a dose-related fashion. Some combinations resulted in profound low-dose-induced increases in MNi and significant elevations in the percentage of p53 positive cells, which occurred without further reduction in cell viability or mitotic rate. When one particular extract was combined with BP, a 100-fold increase in BP-DNA adducts was detected as compared with the levels induced by BP alone; an effect not induced by other extracts. This adduct-enhancing extract was fractionated into 14 fractions that were subsequently tested (1 mg-equiv of original extract) in combination with 0.01 microM BP. Fraction 1, into which nonpolar pollutants mostly eluted, enhanced MN-forming activity with BP. Surprisingly, the more polar and less likely to contain fat-soluble pollutants fractions 5 and 8 also enhanced MN-forming activity. No identifiable pollutants were present in these fractions. The results suggest that different environmental pollutants present in human tissue may influence the susceptibility of target cells to initiating events.

Low dose induction of micronuclei by lindane.

Environmental contaminants possessing hormonal activity have long been suspected of playing a role in cancer causation. What is unclear is whether such agents elicit their effects through genotoxic and/or epigenetic mechanisms. gamma-Hexachlorocyclohexane (gamma-HCH, lindane) was tested in the 10(-12)-10(-4) M range. Chromosomal damage in MCF-7 breast cells and PC-3 prostate cells was assessed using the cytokinesis block micronucleus assay. Micronuclei (MNi) were scored in 1000 binucleate cells per treatment. Cell viability and cell cycle kinetics were also assessed, along with immunocytochemical and quantitative gene expression analyses of CDKN1A (P21WAF1/CIP1), BCL-2 and BAX. Following 24 h treatment, lindane (10(-12)-10(-10) M) induced increases (up to 5-fold) in MNi in both cell lines. Increases in MNi occurred in the absence of DNA single-strand breaks or cytotoxicity and, compared with benzo[a]pyrene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, at low concentrations. Lindane induced more MNi than the alpha or beta stereoisomers of HCH. Low dose lindane (10(-12)-10(-10) M) significantly elevated the percentage of MCF-7 cells staining positive for Bcl-2 and of PC-3 cells staining positive for Bax. Only high dose lindane (10(-4) M) disrupted cell cycle kinetics with increases in percentage of cells in G1 and decreases in percentage of cells in G2/M. Despite a comparable high dose lindane induction of cell cycle arrest, marked increases in expression of P21WAF1/CIP1 were observed only in MCF-7 cells, although in PC-3 cells a significant increase (P < 0.0005) in the percentage of cells staining positive for p21Waf1/Cip1 was seen. These results suggest that 'environmental' concentrations of lindane can induce a number of subtle alterations in breast and prostate cells in the absence of cytotoxicity.

UV-A induces persistent genomic instability in human keratinocytes through an oxidative stress mechanism.

Ultraviolet-A (UV-A, 320 to 400 nm) radiation comprises 95% of the solar ultraviolet radiation (UVR) reaching the earth's surface. It has been associated experimentally and epidemiologically with malignant melanoma. In this study we investigated whether UV-A radiation can induce a persistent, heritable hypermutability in mammalian cells similar to that observed following ionising radiation (IR). Using the immortalized human skin keratinocyte cell line HaCaT we found that UV-A radiation does lead to a continuing reduction in plating efficiency, an increased "spontaneous" mutant fraction, and an increase in micronucleus formation up to 21 d after initial exposure. Reversal of these effects using catalase may indicate a role for hydrogen peroxide in this phenomenon. These results add to the significance of UV-A radiation as a risk factor in skin carcinogenesis.