Importance: Cell-free DNA (cfDNA) testing is increasingly used in the treatment of patients with advanced prostate cancer. Clonal hematopoiesis of indeterminate potential (CHIP) can interfere with cfDNA testing and cause incorrect interpretation of results. There is an urgent need to better understand this problem following recent US Food and Drug Administration approval of poly(ADP) ribose polymerase inhibitors (PARPi) for metastatic prostate cancer based on variants in DNA repair genes that can be affected by CHIP. Objective: To determine the prevalence of clinically relevant CHIP interference in prostate cancer cfDNA testing. Design, Setting, and Participants: We report a case series of 69 patients with advanced prostate cancer (metastatic disease or with rising PSA following localized therapy) who had cfDNA variant testing with a large panel cancer next generation sequencing assay (UW-OncoPlexCT). To determine the source of variants in plasma, we tested paired cfDNA and whole blood control samples. The study was carried out in an academic medical center system reference laboratory. Main Outcomes and Measures: Prevalence and gene spectrum of CHIP interference in patients with prostate cancer undergoing cfDNA testing. Results: We detected CHIP variants at 2% or more variant fraction in cfDNA from 13 of 69 men with prostate cancer (19%; 95% CI, 10%-30%). Seven men (10%; 95% CI, 4%-20%) had CHIP variants in DNA repair genes used to determine PARPi candidacy, including ATM (n=5), BRCA2 (n=1), and CHEK2 (n=1). Overall, CHIP variants accounted for almost half of the somatic DNA repair gene variants detected. Participant CHIP variants were exponentially correlated with older age (R2=0.82). CHIP interference variants could be distinguished from prostate cancer variants using a paired whole-blood control. Conclusions and Relevance: In this case series, approximately 10% of men with advanced prostate cancer had CHIP interference in plasma cfDNA in DNA repair genes that are used for eligibility of PARPi therapy, most frequently in ATM. Clinical cfDNA testing should include a paired whole-blood control to exclude CHIP variants and avoid misdiagnosis.
The chemotherapeutic enzyme asparaginase depletes systemic asparagine to kill cancers; however, its efficacy thus far is limited to a subset of leukemias. Hinze and colleagues identify that inhibiting proteasomal release of asparagine can sensitize colorectal cancers to asparagine depletion, providing a potential avenue to repurpose asparaginase for treatment of solid tumors.See related article by Hinze et al., p. 1690.
J Mammary Gland Biol Neoplasia
Tumor organoids mimic the architecture and heterogeneity of in vivo tumors and enable studies of collective interactions between tumor cells as well as with their surrounding microenvironment. Although tumor organoids hold significant promise as cancer models, they are also more costly and labor-intensive to cultivate than traditional 2D cell culture. We sought to identify critical factors regulating organoid growth ex vivo, and to use these observations to develop a more efficient organoid expansion method. Using time-lapse imaging of mouse mammary tumor organoids in 3D culture, we observed that outgrowth potential varies non-linearly with initial organoid size. Maximal outgrowth occurred in organoids with a starting size between ~10 to 1000 cells. Based on these observations, we developed a suspension culture method that maintains organoids in the ideal size range, enabling expansion from 1 million to over 100 million cells in less than 2 weeks and less than 3 hours of hands-on time. Our method facilitates the rapid, cost-effective expansion of organoids for CRISPR based studies and other assays requiring a large amount of organoid starting material.
The translation machinery is composed of a myriad of proteins and RNAs whose levels must be coordinated to efficiently produce proteins without wasting energy or substrate. However, protein synthesis is clearly not always perfectly tuned to its environment, as disruption of translation machinery components can lengthen lifespan and stress survival. While much has been learned from bacteria and yeast about translational regulation, much less is known in metazoans. In a screen for mutations protecting C. elegans from hypoxic stress, we isolated multiple genes impacting protein synthesis: a ribosomal RNA helicase gene, tRNA biosynthesis genes, and a gene controlling amino acid availability. To define better the mechanisms by which these genes impact protein synthesis, we performed a second screen for suppressors of the conditional developmental arrest phenotype of the RNA helicase mutant and identified genes involved in ribosome biogenesis. Surprisingly, these suppressor mutations restored normal hypoxic sensitivity and protein synthesis to the tRNA biogenesis mutants, but not to the mutant reducing amino acid uptake. Proteomic analysis demonstrated that reduced tRNA biosynthetic activity produces a selective homeostatic reduction in ribosomal subunits, thereby offering a mechanism for the suppression results. Our study uncovers an unrecognized higher-order-translation regulatory mechanism in a metazoan whereby ribosome biogenesis genes communicate with genes controlling tRNA abundance matching the global rate of protein synthesis with available resources.
Understanding the antibody response is critical to developing vaccine and antibody-based therapies and has inspired the recent development of new methods to isolate antibodies. Methods to define the antibody-antigen interactions that determine specificity or allow escape have not kept pace. We developed Phage-DMS, a method that combines two powerful approaches-immunoprecipitation of phage peptide libraries and deep mutational scanning (DMS)-to enable high-throughput fine mapping of antibody epitopes. As an example, we designed sequences encoding all possible amino acid variants of HIV Envelope to create phage libraries. Using Phage-DMS, we identified sites of escape predicted using other approaches for four well-characterized HIV monoclonal antibodies with known linear epitopes. In some cases, the results of Phage-DMS refined the epitope beyond what was determined in previous studies. This method has the potential to rapidly and comprehensively screen many antibodies in a single experiment to define sites essential for binding interactions.
Proc Natl Acad Sci U S A
Head and neck squamous cell carcinoma (HNSCC) associated with high-risk human papilloma virus (HPV) infection is a growing clinical problem. The WEE1 kinase inhibitor AZD1775 (WEE1i) overrides cell cycle checkpoints and is being studied in HNSCC regimens. We show that the HPV16 E6/E7 oncoproteins sensitize HNSCC cells to single-agent WEE1i treatment through activation of a FOXM1-CDK1 circuit that drives mitotic gene expression and DNA damage. An isogenic cell system indicated that E6 largely accounts for these phenotypes in ways that extend beyond p53 inactivation. A targeted genomic analysis implicated FOXM1 signaling downstream of E6/E7 expression and analyses of primary tumors and The Cancer Genome Atlas (TCGA) data revealed an activated FOXM1-directed promitotic transcriptional signature in HPV+ versus HPV- HNSCCs. Finally, we demonstrate the causality of FOXM1 in driving WEE1i sensitivity. These data suggest that elevated basal FOXM1 activity predisposes HPV+ HNSCC to WEE1i-induced toxicity and provide mechanistic insights into WEE1i and HPV+ HNSCC therapies.
Work identifying how stalk morphogenesis in a species of Alphaproteobacteria is controlled unveils an interesting mechanism that other bacteria may utilize to generate the variety of bacterial cell morphologies found across the bacterial domain.
J Immunol Methods
The isolation of human monoclonal antibodies (mAbs) arising from natural infection with human pathogens has proven to be a powerful technology, facilitating the understanding of the host response to infection at a molecular level. mAbs can reveal sites of vulnerability on pathogens and illuminate the biological function of the antigenic targets. Moreover, mAbs have the potential to be used directly for therapeutic applications such as passive delivery to prevent infection in susceptible target populations, and as treatment of established infection. The isolation of antigen-specific B cells from vaccine trials can also assist in deciphering whether the desired B cells are being targeted by a given vaccine. Several different processes have been developed to isolate mAbs, but all are generally labor-intensive and result in varying degrees of efficiency. Here, we describe the development of a cost-effective feeder cell line that stably expresses CD40-ligand, interleukin-2 and interleukin-21. Sorting of single B cells onto a layer of irradiated feeder cells sustained antibody production that permits functional screening of secreted antibodies in a manner that enables subsequent recovery of B cells for recombinant antibody cloning. As a proof of concept, we show that this approach can be used to isolate B cells that secrete antibodies that neutralize human papilloma virus (HPV) from participants of an HPV vaccine study.
Clin Genitourin Cancer
BACKGROUND: Micropapillary urothelial carcinoma (MPC) is a rare urothelial carcinoma variant with conflicting data guiding clinical practice. In this study, we explored oncologic outcomes in relation to neoadjuvant chemotherapy (NAC) in a retrospective cohort of patients with MPC, alongside data from Surveillance, Epidemiology, and End Results (SEER)-Medicare. PATIENTS AND METHODS: We retrospectively identified patients with MPC or conventional urothelial carcinoma (CUC) without any variant histology undergoing radical cystectomy (RC) in our institution (2003-2018). SEER-Medicare was also queried to identify patients diagnosed with MPC (2004-2015). Clinicopathologic data and treatment modalities were extracted. Overall survival (OS) was estimated with the Kaplan-Meier method. Mann-Whitney-Wilcoxon and chi-square tests were used for comparative analysis and Cox regression for identifying clinical covariates associated with OS. RESULTS: Our institutional database yielded 46 patients with MPC and 457 with CUC. In SEER-Medicare, 183 patients with MPC were identified, and 63 (34%) underwent RC. In the institutional cohort, patients with MPC had significantly higher incidence of cN+ (17% vs. 8%), pN+ stage (30% vs. 17%), carcinoma-in-situ (43% vs. 25%), and lymphovascular invasion (30% vs. 16%) at RC versus those with CUC (all P< .05). Pathologic complete response (ypT0N0) to NAC was 33% for MPC and 35% for CUC (P= .899). Median OS was lower for institutional MPC versus CUC in univariate analysis (43.6 vs. 105.3 months, P= .006); however, MPC was not independently associated with OS in the multivariate model. Median OS was 25 months in the SEER MPC cohort for patients undergoing RC, while NAC was not associated with improved OS in that group. CONCLUSION: Pathologic response to NAC was not significantly different between MPC and CUC, while MPC histology was not an independent predictor of OS. Further studies are needed to better understand biological mechanisms behind its aggressive features as well as the role of NAC in this histology variant.