Recurrent repeat expansions in human cancer genomes.

Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases1,2. However, repeat expansions are often not explored beyond neurological and neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs, a phenomenon termed microsatellite instability; however, larger repeat expansions have not been systematically analysed in cancer3-8. Here we identified TR expansions in 2,622 cancer genomes spanning 29 cancer types. In seven cancer types, we found 160 recurrent repeat expansions (rREs), most of which (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with enrichment near candidate cis-regulatory elements, suggesting a potential role in gene regulation. One rRE, a GAAA-repeat expansion, located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, in preliminary experiments, treating cells that harbour this rRE with a GAAA-targeting molecule led to a dose-dependent decrease in cell proliferation. Overall, our results suggest that rREs may be an important but unexplored source of genetic variation in human cancer, and we provide a comprehensive catalogue for further study.

Same-day post-therapy imaging with a new generation whole-body digital SPECT/CT in assessing treatment response to [177Lu]Lu-PSMA-617 in metastatic castration-resistant prostate cancer.

PURPOSE: Lutetium-177 [177Lu]Lu-PSMA-617 radioligand therapy (RLT) represents a significant advancement for metastatic castration-resistant prostate cancer (mCRPC), demonstrating improvements in radiographic progression free survival (rPFS) and overall survival (OS) with a low rate of associated side effects. Currently, most post-therapy SPECT/CT is conducted at 24 h after infusion. This study examines the clinical utility of a next-generation multi-detector Cadmium-Zinc-Telluride (CZT) SPECT/CT system (StarGuide) in same-day post-infusion assessment and early treatment response to [177Lu]Lu-PSMA-617. METHODS: In this retrospective study, 68 men with progressive mCRPC treated with [177Lu]Lu-PSMA-617 at our center from June 2022 to June 2023 were evaluated. Digital whole-body SPECT/CT imaging was performed after [177Lu]Lu-PSMA-617infusion (mean ± SD: 1.8 ± 0.6 h, range 1.1-4.9 h). Quantitative analysis of [177Lu]Lu-PSMA-617 positive lesions was performed in patients who underwent at least 2 post-therapy SPECT/CT, using liver parenchyma uptake as reference. Metrics including [177Lu]Lu-PSMA-617 positive total tumor volume (Lu-TTV), SUVmax and SUVmean were calculated. These quantitative metrics on post-infusion SPECT/CT images after cycles 1, 2 and 3 were correlated with overall survival (OS), prostate specific antigen-progression free survival (PSA-PFS) as defined by prostate cancer working group 3 (PCWG3), and PSA decrease over 50% (PSA50) response rates. RESULTS: 56 patients (means age 76.2 ± 8.1 years, range: 60-93) who underwent at least 2 post-therapy SPECT/CT were included in the image analysis. The whole-body SPECT/CT scans (~ 12 min per scan) were well tolerated, with 221 same-day scans performed (89%). At a median of 10-months follow-up, 33 (58.9%) patients achieved PSA50 after [177Lu]Lu-PSMA-617 treatment and median PSA-PFS was 5.0 months (range: 1.0-15 months) while median OS was not reached. Quantitative analysis of SPECT/CT images showed that 37 patients (66%) had > 30% reduction in Lu-TTV, associated with significantly improved overall survival (median not reached vs. 6 months, P = 0.008) and PSA-PFS (median 6 months vs. 1 months, P < 0.001). However, changes in SUVmax or SUVmean did not correlate with PSA-PFS or OS. CONCLUSION: We successfully implemented same-day post-therapy SPECT/CT after [177Lu]Lu-PSMA-617 infusions. Quantitation of 1-2 h post-therapy SPECT/CT images is a promising method for assessing treatment response. However, the approach is currently limited by its suboptimal detection of small tumor lesions and the necessity of incorporating a third-cycle SPECT/CT to mitigate the effects of any potential treatment-related flare-up. Further investigation in a larger patient cohort and prospective validation is essential to confirm these findings and to explore the role of SPECT/CT as a potential adjunct to PSMA PET/CT in managing mCRPC.

Detecting androgen receptor (AR), AR variant 7 (AR-V7), prostate-specific membrane antigen (PSMA), and prostate-specific antigen (PSA) gene expression in CTCs and plasma exosome-derived cfRNA in patients with metastatic castration-resistant prostate cancer (mCRPC) by integrating the VTX-1 CTC isolation system with the QIAGEN AdnaTest.

BACKGROUND: Therapies for metastatic castration-resistant prostate cancer (mCRPC) include targeting the androgen receptor (AR) with androgen receptor inhibitors (ARIs) and prostate-specific membrane antigen (PSMA). Having the ability to detect AR, AR splice variant 7 (AR-V7), or PSMA in circulating tumor cells (CTCs) or circulating exosomal cell-free RNA (cfRNA) could be helpful to guide selection of the appropriate therapy for each individual patient. The Vortex Biosciences VTX-1 system is a label-free CTC isolation system that enables the detection of the expression of multiple genes in both CTCs and exosomal cfRNA from the same blood sample in patients with mCRPC. Detection of both AR-V7 and PSMA gene expression in both CTCs and cfRNA simultaneously has not yet been reported. METHODS: To characterize the combined VTX-1-AdnaDetect workflow, 22Rv1 cancer cells were spiked into blood from healthy donors and processed with the VTX-1 to mimic patient samples and assess performances (capture efficiency, purity, AR and AR-V7 expression). Then, we collected 19 blood samples from 16 patients with mCRPC and therapeutic resistance to androgen receptor inhibitors (ARIs). Plasma was separated and the plasma-depleted blood was processed further with the VTX-1 to collect CTCs. Both plasma exosomal cfRNA and CTCs were subsequently analyzed for AR, AR-V7, PSMA, and prostate-specific antigen (PSA) mRNA expression using the AdnaTest ProstateCancerPanel AR-V7 assay. RESULTS: AR-V7 expression could be detected in 22Rv1 cells spiked into blood from healthy volunteers as well as in CTCs and plasma-derived exosomal cfRNA from patients with mCRPC by processing blood with the VTX-1 CTC isolation system followed by the AdnaTest ProstateCancerPanel AR-V7 assay. 94.7% of patient blood samples (18/19) had detectable AR expression in either CTCs or exosomal cfRNA (16 in CTCs, 12 in cfRNA). 15.8% of the 19 patient blood samples (3/19) were found to have AR-V7-positive (AR-V7+) CTCs, one of which was also AR-V7+ in the exosomal cfRNA analysis. 42.1% of patient blood samples (8/19) were found to be PSMA positive (PSMA+): 26.3% (5/19) were PSMA+ in the CTC analysis and 31.6% (6/19) were PSMA+ in the exosomal cfRNA analysis. Of those 8 PSMA+ samples, 2 had detectable PSMA only in CTCs, and 3 had detectable PSMA only in exosomal cfRNA. CONCLUSION: VTX-1 enables isolation of CTCs and plasma exosomes from a single blood draw and can be used for detecting AR-V7 and PSMA mRNA in both CTCs and cfRNA in patients with mCRPC and resistance to ARIs. This technology facilitates combining RNA measurements in CTCs and exosomal cfRNA for future studies to develop potentially clinically relevant cancer biomarker detection in blood.

Lay healthcare worker financial toxicity intervention: a pilot financial toxicity screening and referral program.

PURPOSE: Financial toxicity is a source of significant distress for patients with urologic cancers, yet few studies have addressed financial burden in this patient population. METHODS: We developed a financial toxicity screening program using a lay health worker (LHW) and social worker (SW) to assess and mitigate financial toxicity in a single academic medical clinic. As part of a quality improvement project, the LHW screened all newly diagnosed patients with advanced stages of prostate, kidney, or urothelial cancer for financial burden using three COST tool questions and referred patients who had significant financial burden to an SW who provided personalized recommendations. The primary outcome was feasibility defined as 80% of patients with financial burden completing the SW consult. Secondary outcomes were patient satisfaction, change in COST Tool responses, and qualitative assessment of financial resources utilized. RESULTS: The LHW screened a total of 185 patients for financial toxicity; 82% (n = 152) were male, 65% (n = 120) White, and 75% (n = 139) reported annual household income >$100,000 US Dollars; 60% (n = 114) had prostate cancer. A total of 18 (9.7%) participants screened positive for significant financial burden and were referred to the SW for consultation. All participants (100%) completed and reported satisfaction with the SW consultation and had 0.83 mean lower scores on the COST Tool post-intervention assessment compared to pre-intervention (95% confidence interval [0.26, 1.41]). CONCLUSION: This multidisciplinary financial toxicity intervention using an LHW and SW was feasible, acceptable, and associated with reduced financial burden among patients with advanced stages of urologic cancers. Future work should evaluate the effect of this intervention among cancer patients in diverse settings.

Giant Magnetoresistive Nanosensor Analysis of Circulating Tumor DNA Epidermal Growth Factor Receptor Mutations for Diagnosis and Therapy Response Monitoring.

BACKGROUND: Liquid biopsy circulating tumor DNA (ctDNA) mutational analysis holds great promises for precision medicine targeted therapy and more effective cancer management. However, its wide adoption is hampered by high cost and long turnaround time of sequencing assays, or by inadequate analytical sensitivity of existing portable nucleic acid tests to mutant allelic fraction in ctDNA. METHODS: We developed a ctDNA Epidermal Growth Factor Receptor (EGFR) mutational assay using giant magnetoresistive (GMR) nanosensors. This assay was validated in 36 plasma samples of non-small cell lung cancer patients with known EGFR mutations. We assessed therapy response through follow-up blood draws, determined concordance between the GMR assay and radiographic response, and ascertained progression-free survival of patients. RESULTS: The GMR assay achieved analytical sensitivities of 0.01% mutant allelic fraction. In clinical samples, the assay had 87.5% sensitivity (95% CI = 64.0-97.8%) for Exon19 deletion and 90% sensitivity (95% CI = 69.9-98.2%) for L858R mutation with 100% specificity; our assay detected T790M resistance with 96.3% specificity (95% CI = 81.7-99.8%) with 100% sensitivity. After 2 weeks of therapy, 10 patients showed disappearance of ctDNA by GMR (predicted responders), whereas 3 patients did not (predicted nonresponders). These predictions were 100% concordant with radiographic response. Kaplan-Meier analysis showed responders had significantly (P < 0.0001) longer PFS compared to nonresponders (N/A vs. 12 weeks, respectively). CONCLUSIONS: The GMR assay has high diagnostic sensitivity and specificity and is well suited for detecting EGFR mutations at diagnosis and noninvasively monitoring treatment response at the point-of-care.

18F-FPPRGD2 PET/CT in patients with metastatic renal cell cancer.

PURPOSE: The usefulness of positron emission tomography/computed tomography (PET/CT) using (18F)-2-fluoropropionyl-labeled PEGylated dimeric arginine-glycine-aspartic acid peptide [PEG3-E{c(RGDyk)}2] (18F-FPPRGD2) in patients with metastatic renal cell cancer (mRCC) has not been evaluated; therefore, we were prompted to conduct this pilot study. METHODS: Seven patients with mRCC were enrolled in this prospective study. 18F-FPPRGD2 and 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) PET/CT images were evaluated in a per-lesion analysis. Maximum standardized uptake value (SUVmax) and tumor-to-background ratio (T/B) were measured for all detected lesions, both before and after starting antiangiogenic therapy. RESULTS: Sixty lesions in total were detected in this cohort. SUVmax from 18F-FPPRGD2 PET/CT was lower than that from 18F-FDG PET/CT (4.4 ± 2.9 vs 7.8 ± 5.6, P < 0.001). Both SUVmax and T/B from 18F-FPPRGD2 PET/CT decreased after starting antiangiogenic therapy (SUVmax, 4.2 ± 3.2 vs 2.6 ± 1.4, P = 0.003; T/B, 3.7 ± 3.2 vs 1.5 ± 0.8, P < 0.001). Average changes in SUVmax and T/B were - 29.3 ± 23.6% and - 48.1 ± 28.3%, respectively. CONCLUSIONS: 18F-FPPRGD2 PET/CT may be an useful tool for monitoring early response to antiangiogenic therapy in patients with mRCC. These preliminary results need to be confirmed in larger cohorts.

Alteration of the lipid profile in lymphomas induced by MYC overexpression.

Overexpression of the v-myc avian myelocytomatosis viral oncogene homolog (MYC) oncogene is one of the most commonly implicated causes of human tumorigenesis. MYC is known to regulate many aspects of cellular biology including glucose and glutamine metabolism. Little is known about the relationship between MYC and the appearance and disappearance of specific lipid species. We use desorption electrospray ionization mass spectrometry imaging (DESI-MSI), statistical analysis, and conditional transgenic animal models and cell samples to investigate changes in lipid profiles in MYC-induced lymphoma. We have detected a lipid signature distinct from that observed in normal tissue and in rat sarcoma-induced lymphoma cells. We found 104 distinct molecular ions that have an altered abundance in MYC lymphoma compared with normal control tissue by statistical analysis with a false discovery rate of less than 5%. Of these, 86 molecular ions were specifically identified as complex phospholipids. To evaluate whether the lipid signature could also be observed in human tissue, we examined 15 human lymphoma samples with varying expression levels of MYC oncoprotein. Distinct lipid profiles in lymphomas with high and low MYC expression were observed, including many of the lipid species identified as significant for MYC-induced animal lymphoma tissue. Our results suggest a relationship between the appearance of specific lipid species and the overexpression of MYC in lymphomas.

BCL-2 inhibition with ABT-737 prolongs survival in an NRAS/BCL-2 mouse model of AML by targeting primitive LSK and progenitor cells.

Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1(+)/c-Kit(+), and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS-AML transformation and survival of leukemic cells.

Coaches Activating, Reaching, and Engaging Patients to Engage in Advance Care Planning: A Randomized Clinical Trial.

Importance: Advance care planning (ACP) remains low among patients with advanced cancer. Multilevel interventions compared with clinician-level interventions may be more effective in improving ACP. Objective: To evaluate whether a multilevel intervention could improve clinician-documented ACP compared with a clinician-level intervention alone. Design, Setting, and Participants: This randomized clinical trial, performed from September 12, 2019, through May 12, 2021, included adults with advanced genitourinary cancers at an academic, tertiary hospital. Data analysis was performed by intention to treat from May 1 to August 10, 2023. Intervention: Participants were randomized 1:1 to a 6-month patient-level lay health worker structured ACP education along with a clinician-level intervention composed of 3-hour ACP training and integration of a structured electronic health record documentation template (intervention group) or to the clinician-level intervention alone (control group). Main Outcome and Measures: The primary outcome was ACP documentation in the electronic health record by the oncology clinician within 12 months after randomization. Secondary, exploratory outcomes included shared decision-making, palliative care use, hospice use, emergency department visits, and hospitalizations within 12 months after randomization. Results: Among 402 participants enrolled in the study, median age was 71 years (range, 21-102 years); 361 (89.8%) identified as male. More intervention group participants had oncology clinician-documented ACP than control group participants (82 [37.8%] vs 40 [21.6%]; odds ratio [OR], 2.29; 95% CI, 1.44-3.64). At 12-month follow-up, more intervention than control group participants had palliative care (72 [33.2%] vs 25 [13.5%]; OR, 3.18; 95% CI, 1.91-5.28) and hospice use (49 [22.6%] vs 19 [10.3%]; OR, 2.54; 95% CI, 1.44-4.51). There were no differences in the proportion of participants between groups with an emergency department visit (65 [30.0%] vs 61 [33.0%]; OR, 0.87; 95% CI, 0.57-1.33) or hospitalization (89 [41.0%] vs 85 [46.0%]; OR, 0.82; 95% CI, 0.55-1.22). Intervention group participants had fewer hospitalizations than control group participants (mean [SD] number of hospitalizations per year, 0.87 [1.60] vs 1.04 [1.77]) and a lower risk of hospitalization (incidence rate ratio, 0.80; 95% CI, 0.65-0.98). Conclusions and Relevance: In this randomized clinical trial, a multilevel intervention improved oncology clinician-documented ACP compared with a clinician-level intervention alone for patients with genitourinary cancer. The intervention is one approach to effectively increase ACP among patients with cancer. Trial Registration: ClinicalTrials.gov Identifier: NCT03856463.

Diverse and abundant phages exploit conjugative plasmids.

Phages exert profound evolutionary pressure on bacteria by interacting with receptors on the cell surface to initiate infection. While the majority of phages use chromosomally encoded cell surface structures as receptors, plasmid-dependent phages exploit plasmid-encoded conjugation proteins, making their host range dependent on horizontal transfer of the plasmid. Despite their unique biology and biotechnological significance, only a small number of plasmid-dependent phages have been characterized. Here we systematically search for new plasmid-dependent phages targeting IncP and IncF plasmids using a targeted discovery platform, and find that they are common and abundant in wastewater, and largely unexplored in terms of their genetic diversity. Plasmid-dependent phages are enriched in non-canonical types of phages, and all but one of the 65 phages we isolated were non-tailed, and members of the lipid-containing tectiviruses, ssDNA filamentous phages or ssRNA phages. We show that plasmid-dependent tectiviruses exhibit profound differences in their host range which is associated with variation in the phage holin protein. Despite their relatively high abundance in wastewater, plasmid-dependent tectiviruses are missed by metaviromic analyses, underscoring the continued importance of culture-based phage discovery. Finally, we identify a tailed phage dependent on the IncF plasmid, and find related structural genes in phages that use the orthogonal type 4 pilus as a receptor, highlighting the evolutionarily promiscuous use of these distinct contractile structures by multiple groups of phages. Taken together, these results indicate plasmid-dependent phages play an under-appreciated evolutionary role in constraining horizontal gene transfer via conjugative plasmids.

Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR).

Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge and serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies of substrate-dependent target engagement remain sparse. Herein, we describe the Thermal Shift Assay with ATP and RNA (TSAR) as a template for proteome-wide discovery of substrate-dependent ligand binding. Using proteomic thermal shift assays, we show that simple biochemical additives can facilitate detection of target engagement in native cell lysates. We apply our approach to rocaglates, a family of molecules that specifically clamp RNA to eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), and potentially other members of the DEAD-box (DDX) family of RNA helicases. To identify unexpected interactions, we optimized a target class-specific thermal denaturation window and evaluated ATP analog and RNA probe dependencies for key rocaglate-DDX interactions. We report novel DDX targets of the rocaglate clamping spectrum, confirm that DDX3X is a common target of several widely studied analogs, and provide structural insights into divergent DDX3X affinities between synthetic rocaglates. We independently validate novel targets of high-profile rocaglates, including the clinical candidate Zotatifin (eFT226), using limited proteolysis-mass spectrometry and fluorescence polarization experiments. Taken together, our study provides a model for screening uncompetitive inhibitors using a systematic chemical-proteomics approach to uncover actionable DDX targets, clearing a path towards characterization of novel molecular clamps and associated RNA helicase targets.

THE IMPACT OF ROUTINE CARDIAC TROPONIN I-BASED CARDIOTOXICITY SCREENING ON CLINICAL OUTCOMES IN PATIENTS ON CANCER IMMUNOTHERAPY.

Purpose: Immune checkpoint inhibitors (ICI) used as cancer therapy have been associated with a range of cardiac immune-related adverse events (irAEs), including fulminant myocarditis with a high case fatality rate. Early detection through cardiotoxicity screening by biomarker monitoring can lead to prompt intervention and improved patient outcomes. In this study, we investigate the association between cardiotoxicity screening with routine serial troponin I monitoring in asymptomatic patients receiving ICI, cardiovascular adverse event (CV AE) detection, and overall survival (OS). Methods: We instituted a standardized troponin I screening protocol at baseline and with each ICI dose (every 2-4 weeks) in all patients receiving ICI at our center starting Jan 2019. We subsequently collected data in 825 patients receiving ICI at our institution from January 2018 to October 2021. Of these patients, 428 underwent cardiotoxicity screening with serial troponin I monitoring during ICI administration (Jan 2019-Oct 2021) and 397 patients were unmonitored (Jan 2018-Dec 2018). We followed patients for nine months following their first dose of ICI and compared outcomes of CV AEs and OS between monitored and unmonitored patients. Additionally, we investigated rates of CV AEs, all-cause mortality, and oncologic time-to-treatment failure (TTF) between patients with an elevated troponin I value during the monitoring period versus patients without elevated troponin I. Results: We found a lower rate of severe (grades 4-5) CV AEs, resulting in critical illness or death, in patients who underwent troponin monitoring (0.5%) compared to patients who did not undergo monitoring (1.8%), (HR 0.17, 95% CI 0.02-0.79, p = 0.04). There was no difference in overall CV AEs (grades 3-5) or OS between monitored and unmonitored patients. In the entire cohort, patients with at least one elevated troponin I during the follow up period, during routine monitoring or unmonitored, had a higher risk of overall CV AEs (HR 10.96, 95% CI 4.65-25.85, p<0.001) as well as overall mortality (HR 2.67, 95% CI 1.69 - 4.10, p<0.001) compared to those without elevated troponin. Oncologic time-to-treatment failure (TTF) was not significantly different in a sub-cohort of monitored vs. unmonitored patients. Conclusions: Patients undergoing cardiotoxicity screening with troponin I monitoring during ICI therapy had a lower rate of severe (grade 4-5) CV AEs compared patients who were not screened. Troponin I elevation in screened and unscreened patients was significantly associated with increased CV AEs as well as increased mortality. Troponin I monitoring did not impact oncologic time-to-treatment-failure in a sub-cohort analysis of patients treated with ICI. These results provide preliminary evidence for clinical utility of cardiotoxicity screening with troponin I monitoring in patients receiving ICI therapy.

Automated Image Analysis for Characterization of Circulating Tumor Cells and Clusters Sorted by Magnetic Levitation.

Magnetic levitation-based sorting technologies have revolutionized the detection and isolation of rare cells, including circulating tumor cells (CTCs) and circulating tumor cell clusters (CTCCs). Manual counting and quantification of these cells are prone to time-consuming processes, human error, and inter-observer variability, particularly challenging when heterogeneous cell types in 3D clusters are present. To overcome these challenges, we developed "Fastcount," an in-house MATLAB-based algorithm for precise, automated quantification and phenotypic characterization of CTCs and CTCCs, in both 2D and 3D. Fastcount is 120 times faster than manual counting and produces reliable results with a ±7.3% deviation compared to a trained laboratory technician. By analyzing 400 GB of fluorescence imaging data, we showed that Fastcount outperforms manual counting and commercial software when cells are aggregated in 3D or staining artifacts are present, delivering more accurate results. We further employed Fastcount for automated analysis of 3D image stacks obtained from CTCCs isolated from colorectal adenocarcinoma and renal cell carcinoma blood samples. Interestingly, we observed a highly heterogeneous spatial cellular composition within CTCCs, even among clusters from the same patient. Overall, Fastcount can be employed for various applications with lab-chip devices, such as CTC detection, CTCC analysis in 3D and cell detection in biosensors.

Diverse and abundant phages exploit conjugative plasmids.

Phages exert profound evolutionary pressure on bacteria by interacting with receptors on the cell surface to initiate infection. While the majority of phages use chromosomally-encoded cell surface structures as receptors, plasmid-dependent phages exploit plasmid-encoded conjugation proteins, making their host range dependent on horizontal transfer of the plasmid. Despite their unique biology and biotechnological significance, only a small number of plasmid-dependent phages have been characterized. Here we systematically search for new plasmid-dependent phages targeting IncP and IncF plasmids using a targeted discovery platform, and find that they are common and abundant in wastewater, and largely unexplored in terms of their genetic diversity. Plasmid-dependent phages are enriched in non-canonical types of phages, and all but one of the 64 phages we isolated were non-tailed, and members of the lipid-containing tectiviruses, ssDNA filamentous phages or ssRNA phages. We show that plasmid-dependent tectiviruses exhibit profound differences in their host range which is associated with variation in the phage holin protein. Despite their relatively high abundance in wastewater, plasmid-dependent tectiviruses are missed by metaviromic analyses, underscoring the continued importance of culture-based phage discovery. Finally, we identify a tailed phage dependent on the IncF plasmid, and find related structural genes in phages that use the orthogonal type 4 pilus as a receptor, highlighting the promiscuous use of these distinct contractile structures by multiple groups of phages. Taken together, these results indicate plasmid-dependent phages play an under-appreciated evolutionary role in constraining horizontal gene transfer via conjugative plasmids.

Use of Machine Learning and Lay Care Coaches to Increase Advance Care Planning Conversations for Patients With Metastatic Cancer.

PURPOSE: Patients with metastatic cancer benefit from advance care planning (ACP) conversations. We aimed to improve ACP using a computer model to select high-risk patients, with shorter predicted survival, for conversations with providers and lay care coaches. Outcomes included ACP documentation frequency and end-of-life quality measures. METHODS: In this study of a quality improvement initiative, providers in four medical oncology clinics received Serious Illness Care Program training. Two clinics (thoracic/genitourinary) participated in an intervention, and two (cutaneous/sarcoma) served as controls. ACP conversations were documented in a centralized form in the electronic medical record. In the intervention, providers and care coaches received weekly e-mails highlighting upcoming clinic patients with < 2 year computer-predicted survival and no prior prognosis documentation. Care coaches contacted these patients for an ACP conversation (excluding prognosis). Providers were asked to discuss and document prognosis. RESULTS: In the four clinics, 4,968 clinic visits by 1,251 patients met inclusion criteria (metastatic cancer with no prognosis previously documented). In their first visit, 28% of patients were high-risk (< 2 year predicted survival). Preintervention, 3% of both intervention and control clinic patients had ACP documentation during a visit. By intervention end (February 2021), 35% of intervention clinic patients had ACP documentation compared with 3% of control clinic patients. Providers' prognosis documentation rate also increased in intervention clinics after the intervention (2%-27% in intervention clinics, P < .0001; 0%-1% in control clinics). End-of-life care intensity was similar in intervention versus control clinics, but patients with ≥ 1 provider ACP edit met fewer high-intensity care measures (P = .04). CONCLUSION: Combining a computer prognosis model with care coaches increased ACP documentation.

Apoptosis-stimulating protein of p53 (ASPP2) heterozygous mice are tumor-prone and have attenuated cellular damage-response thresholds.

The expression of ASPP2 (53BP2L), a proapoptotic member of a family of p53-binding proteins, is frequently suppressed in many human cancers. Accumulating evidence suggests that ASPP2 inhibits tumor growth; however, the mechanisms by which ASPP2 suppresses tumor formation remain to be clarified. To study this, we targeted the ASPP2 allele in a mouse by replacing exons 10-17 with a neoR gene. ASPP2(-/-) mice were not viable because of an early embryonic lethal event. Although ASPP2(+/-) mice appeared developmentally normal, they displayed an increased incidence of a variety of spontaneous tumors as they aged. Moreover, gamma-irradiated 6-week-old ASPP2(+/-) mice developed an increased incidence of high-grade T cell lymphomas of thymic origin compared with ASPP2(+/+) mice. Primary thymocytes derived from ASPP2(+/-) mice exhibited an attenuated apoptotic response to gamma-irradiation compared with ASPP2(+/+) thymocytes. Additionally, ASPP2(+/-) primary mouse embryonic fibroblasts demonstrated a defective G(0)/G(1) cell cycle checkpoint after gamma-irradiation. Our results demonstrate that ASPP2 is a haploinsufficient tumor suppressor and, importantly, open new avenues for investigation into the mechanisms by which disruption of ASPP2 pathways could play a role in tumorigenesis and response to therapy.

Clinical laboratory tests associated with survival in patients with metastatic renal cell carcinoma: A Laboratory Wide Association Study (LWAS).

BACKGROUND: Prognostic models for patients with metastatic renal cell carcinoma (mRCC) include select laboratory values. These models have important limitations, including reliance on a limited array of laboratory tests, and use of dichotomous ("high-low") cutoffs. We applied a Laboratory-Wide Association Study (LWAS) framework to systematically evaluate common clinical laboratory results associated with survival for patients diagnosed with mRCC. METHODS: We used laboratory data for 3,385 patients diagnosed with mRCC from 2002 to 2017. We developed a LWAS framework, to examine the association with 53 common clinical laboratory tests results (641,712 measurements) and overall survival. We employed false-discovery rate to test the association of multiple laboratory tests with survival, and validated these results using 3 separate cohorts to generate a standardized hazard ratio (sHR), reported for a 1 standard deviation unit change in each laboratory test. RESULTS: The LWAS approach confirmed the association of laboratory values currently used in prognostic models with survival, including calcium (HR 1.35, 95%CI 1.24-1.48), leukocyte count (HR 1.40, 95%CI 1.30-1.51), platelet count (HR 1.36, 95%CI 1.27-1.51), and hemoglobin (HR 0.79, 95%CI 0.72-0.86). Use of these tests as continuous variables improved model performance. LWAS also identified acute phase reactants associated with survival not typically included in prognostic models, including serum albumin (HR 0.66, 95%CI 0.61-0.72), ferritin (HR 1.25, 95%CI 1.08-1.45), alkaline phosphatase (HR 1.31, 95%CI 1.23-1.40), and C-reactive protein (HR 1.70, 95%CI 1.14-2.53). CONCLUSIONS: Routinely measured laboratory tests can refine current prognostic models, facilitate comparisons across clinical trial cohorts, and match patients with specific systemic therapies.

Telaglenastat plus Everolimus in Advanced Renal Cell Carcinoma: A Randomized, Double-Blinded, Placebo-Controlled, Phase II ENTRATA Trial.

PURPOSE: Glutaminase is a key enzyme, which supports elevated dependency of tumors on glutamine-dependent biosynthesis of metabolic intermediates. Dual targeting of glucose and glutamine metabolism by the mTOR inhibitor everolimus plus the oral glutaminase inhibitor telaglenastat showed preclinical synergistic anticancer effects, which translated to encouraging safety and efficacy findings in a phase I trial of 2L+ renal cell carcinoma (RCC). This study evaluated telaglenastat plus everolimus (TelaE) versus placebo plus everolimus (PboE) in patients with advanced/metastatic RCC (mRCC) in the 3L+ setting (NCT03163667). PATIENTS AND METHODS: Eligible patients with mRCC, previously treated with at least two prior lines of therapy [including ≥1 VEGFR-targeted tyrosine kinase inhibitor (TKI)] were randomized 2:1 to receive E, plus Tela or Pbo, until disease progression or unacceptable toxicity. Primary endpoint was investigator-assessed progression-free survival (PFS; one-sided α <0.2). RESULTS: Sixty-nine patients were randomized (46 TelaE, 23 PboE). Patients had a median three prior lines of therapy, including TKIs (100%) and checkpoint inhibitors (88%). At median follow-up of 7.5 months, median PFS was 3.8 months for TelaE versus 1.9 months for PboE [HR, 0.64; 95% confidence interval (CI), 0.34-1.20; one-sided P = 0.079]. One TelaE patient had a partial response and 26 had stable disease (SD). Eleven patients on PboE had SD. Treatment-emergent adverse events included fatigue, anemia, cough, dyspnea, elevated serum creatinine, and diarrhea; grade 3 to 4 events occurred in 74% TelaE patients versus 61% PboE. CONCLUSIONS: TelaE was well tolerated and improved PFS versus PboE in patients with mRCC previously treated with TKIs and checkpoint inhibitors.

Telaglenastat Plus Cabozantinib or Everolimus for Advanced or Metastatic Renal Cell Carcinoma: An Open-Label Phase I Trial.

PURPOSE: Dual inhibition of glucose and glutamine metabolism results in synergistic anticancer effects in solid tumor models. Telaglenastat, an investigational, small-molecule, glutaminase inhibitor, exhibits modest single-agent activity in renal cell carcinoma (RCC) patients. This phase Ib trial evaluated telaglenastat plus cabozantinib or everolimus, agents known to impair glucose metabolism in patients with metastatic RCC (mRCC). PATIENTS AND METHODS: mRCC patients received escalating doses of telaglenastat [400-800 mg per os (p.o.) twice daily] in a 3 + 3 design, plus either everolimus (10 mg daily p.o.; TelaE) or cabozantinib (60 mg daily p.o.; TelaC). Tumor response (RECISTv1.1) was assessed every 8 weeks. Endpoints included safety (primary) and antitumor activity. RESULTS: Twenty-seven patients received TelaE, 13 received TelaC, with median 2 and 3 prior therapies, respectively. Treatment-related adverse events were mostly grades 1 to 2, most common including decreased appetite, anemia, elevated transaminases, and diarrhea with TelaE, and diarrhea, decreased appetite, elevated transaminases, and fatigue with TelaC. One dose-limiting toxicity occurred per cohort: grade 3 pruritic rash with TelaE and thrombocytopenia with TelaC. No maximum tolerated dose (MTD) was reached for either combination, leading to a recommended phase II dose of 800-mg telaglenastat twice daily with standard doses of E or C. TelaE disease control rate (DCR; response rate + stable disease) was 95.2% [20/21, including 1 partial response (PR)] among 21 patients with clear cell histology and 66.7% (2/3) for papillary. TelaC DCR was 100% (12/12) for both histologies [5/10 PRs as best response (3 confirmed) in clear cell]. CONCLUSIONS: TelaE and TelaC showed encouraging clinical activity and tolerability in heavily pretreated mRCC patients.