Acquired Cross-Resistance in Small Cell Lung Cancer due to Extrachromosomal DNA Amplification of MYC Paralogs.

Small cell lung cancer (SCLC) presents as a highly chemosensitive malignancy but acquires cross-resistance after relapse. This transformation is nearly inevitable in patients but has been difficult to capture in laboratory models. Here, we present a preclinical system that recapitulates acquired cross-resistance, developed from 51 patient-derived xenograft (PDX) models. Each model was tested in vivo against three clinical regimens: cisplatin plus etoposide, olaparib plus temozolomide, and topotecan. These drug-response profiles captured hallmark clinical features of SCLC, such as the emergence of treatment-refractory disease after early relapse. For one patient, serial PDX models revealed that cross-resistance was acquired through MYC amplification on extrachromosomal DNA (ecDNA). Genomic and transcriptional profiles of the full PDX panel revealed that MYC paralog amplifications on ecDNAs were recurrent in relapsed cross-resistant SCLC, and this was corroborated in tumor biopsies from relapsed patients. We conclude that ecDNAs with MYC paralogs are recurrent drivers of cross-resistance in SCLC. SIGNIFICANCE: SCLC is initially chemosensitive, but acquired cross-resistance renders this disease refractory to further treatment and ultimately fatal. The genomic drivers of this transformation are unknown. We use a population of PDX models to discover that amplifications of MYC paralogs on ecDNA are recurrent drivers of acquired cross-resistance in SCLC. This article is featured in Selected Articles from This Issue, p. 695.

Acquired Cross-resistance in Small Cell Lung Cancer due to Extrachromosomal DNA Amplification of MYC paralogs.

Small cell lung cancer (SCLC) presents as a highly chemosensitive malignancy but acquires cross-resistance after relapse. This transformation is nearly inevitable in patients but has been difficult to capture in laboratory models. Here we present a pre-clinical system that recapitulates acquired cross-resistance in SCLC, developed from 51 patient-derived xenografts (PDXs). Each model was tested for in vivo sensitivity to three clinical regimens: cisplatin plus etoposide, olaparib plus temozolomide, and topotecan. These functional profiles captured hallmark clinical features, such as the emergence of treatment-refractory disease after early relapse. Serially derived PDX models from the same patient revealed that cross-resistance was acquired through a MYC amplification on extrachromosomal DNA (ecDNA). Genomic and transcriptional profiles of the full PDX panel revealed that this was not unique to one patient, as MYC paralog amplifications on ecDNAs were recurrent among cross-resistant models derived from patients after relapse. We conclude that ecDNAs with MYC paralogs are recurrent drivers of cross-resistance in SCLC. SIGNIFICANCE: SCLC is initially chemosensitive, but acquired cross-resistance renders this disease refractory to further treatment and ultimately fatal. The genomic drivers of this transformation are unknown. We use a population of PDX models to discover that amplifications of MYC paralogs on ecDNA are recurrent drivers of acquired cross-resistance in SCLC.

Early mobilization in coronavirus-19 patients treated with extracorporeal membrane oxygenation.

BACKGROUND: Coronavirus disease 2019 (COVID-19) pneumonia can be associated with refractory respiratory failure requiring extracorporeal membrane oxygenation(ECMO). Although ECMO has helped many COVID patients, optimal management strategies for these patients remain unknown. METHODS: We conducted a retrospective review of all COVID patients requiring ECMO at our hospital. Six months into the pandemic, we changed our management strategy to focus on early mobilization. The early mobilization effort included tracheostomy within 48 h of cannulation, decreasing sedation, and an aggressive physical and occupational therapy program progressing toward early ambulation while on ECMO. The primary outcome measured was survival to discharge. The primary stratification was based on the mobilization strategy. RESULTS: From 2020 to 2021, 47 COVID patients have been supported with ECMO at our institution. Five are still in the hospital on ECMO. 39 (83%) were supported with venovenous ECMO while 8 (17%) were supported with venoarterial or a right ventricular assist device type configuration. All 47 (100%) were cannulated at bedside with transesophageal echocardiographic guidance. The average age was 47 ± 9 years; 36(77%) were male; and 20 (43%) were Hispanic. The median duration of support was 22 (11-44) days. Excluding those who remain in the hospital and on support, overall survival to discharge was 24/42 (57%). When stratified by mobilization strategy, early tracheostomy and mobilization were associated with significantly improved survival (74% [17/23] vs. 37% [7/19], p = .02). There were no changes in patient acuity or duration of support throughout the study period. CONCLUSION: In conclusion, early tracheostomy, decreased sedation, and aggressive mobilization of COVID-19 ECMO patients is associated with improved survival.

Translesion DNA synthesis mediates acquired resistance to olaparib plus temozolomide in small cell lung cancer.

In small cell lung cancer (SCLC), acquired resistance to DNA-damaging therapy is challenging to study because rebiopsy is rarely performed. We used patient-derived xenograft models, established before therapy and after progression, to dissect acquired resistance to olaparib plus temozolomide (OT), a promising experimental therapy for relapsed SCLC. These pairs of serial models reveal alterations in both cell cycle kinetics and DNA replication and demonstrate both inter- and intratumoral heterogeneity in mechanisms of resistance. In one model pair, up-regulation of translesion DNA synthesis (TLS) enabled tolerance of OT-induced damage during DNA replication. TLS inhibitors restored sensitivity to OT both in vitro and in vivo, and similar synergistic effects were seen in additional SCLC cell lines. This represents the first described mechanism of acquired resistance to DNA damage in a patient with SCLC and highlights the potential of the serial model approach to investigate and overcome resistance to therapy in SCLC.

Directed Hypercapnia as a Strategy to Wean Extracorporeal Membrane Oxygenation in COVID-19 Pneumonia.

Coronavirus disease 2019 pneumonia with respiratory failure refractory to maximum medical therapy has been successfully managed with venovenous extracorporeal membrane oxygenation. This report describes a process of using directed hypercapnia in 5 patients to wean them from prolonged extracorporeal support secondary to refractory hypercarbic respiratory failure.

Intermediate-term survival and functional outcomes of COVID-19 extracorporeal membrane oxygenation patients.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) has been associated with acceptable short-term survival in patients with refractory respiratory failure secondary to coronavirus disease 2019 (COVID-19) pneumonia. Previous studies have demonstrated acceptable long-term outcomes in patients supported with ECMO for respiratory failure of other etiologies. However, long-term survival and functional outcomes in COVID ECMO patients remain unknown. METHODS: We conducted a retrospective review of all COVID patients requiring ECMO at our hospital. The primary outcomes measured were survival to discharge and contemporary survival. Secondary outcomes included two simple functional assessments: the ongoing need for oxygen supplementation and the ability to return to work. Survival was calculated using the Kaplan-Meier method. Hazard ratios were calculated using Cox hazards regression models. RESULTS: From 2020 to 2021, 48 COVID patients have been supported with ECMO at our hospital. Four patients remain on support and were excluded from further analysis. The average age was 47 ± 8 years, 34 (77%) were males, and the plurality (19, 43%) were Hispanic. Median duration of support was 23 (12-51) days. Median follow-up was 106 (29-226) days. Survival to discharge was 59%. Kaplan-Meier 180-day survival was 51%. Long-term survival conditioned on survival to discharge was 89%. In evaluating functional outcomes, the overwhelming majority of patients no longer required oxygen supplementation (74%), and most had returned to work (52%). CONCLUSION: In conclusion, COVID ECMO patients have acceptable intermediate-term survival with adequate functional recovery.

Stiffness based enrichment of leukemia cells using microfluidics.

To improve the survival rate of cancer patients, new diagnosis strategies are necessary to detect lower levels of cancer cells before and after treatment regimens. The scarcity of diseased cells, particularly in residual disease after treatment, demands highly sensitive detection approaches or the ability to enrich the diseased cells in relation to normal cells. We report a label-free microfluidic approach to enrich leukemia cells from healthy cells using inherent differences in cell biophysical properties. The microfluidic device consists of a channel with an array of diagonal ridges that recurrently compress and translate flowing cells in proportion to cell stiffness. Using devices optimized for acute T cell leukemia model Jurkat, the stiffer white blood cells were translated orthogonally to the channel length, while softer leukemia cells followed hydrodynamic flow. The device enriched Jurkat leukemia cells from white blood cells with an enrichment factor of over 760. The sensitivity, specificity, and accuracy of the device were found to be > 0.8 . The values of sensitivity and specificity could be adjusted by selecting one or multiple outlets for analysis. We demonstrate that low levels of Jurkat leukemia cells (1 in 10 4 white blood cells) could be more quickly detected using flow cytometry by using the stiffness sorting pre-enrichment. In a second mode of operation, the device was implemented to sort resistive leukemia cells from both drug-sensitive leukemia cells and normal white blood cells. Therefore, microfluidic biomechanical sorting can be a useful tool to enrich leukemia cells that may improve downstream analyses.

Real-world treatment and survival of patients with advanced non-small cell lung Cancer: a German retrospective data analysis.

BACKGROUND: The objective of this study was to describe the real-world treatment and overall survival (OS) of German patients with a diagnosis of advanced non-small cell lung cancer (aNSCLC), and to explore factors associated with the real-world mortality risk. METHODS: This was a retrospective German claims data analysis of incident aNSCLC patients. Data were available from 01/01/2011 until 31/12/2016. Identification of eligible patients took place between 01/01/2012-31/12/2015, to allow for at least 1-year pre-index and follow-up periods. Inpatient and outpatient mutation test procedures after aNSCLC diagnosis were observed. Further, prescribed treatments and OS since first (incident) aNSCLC diagnosis and start of respective treatment lines were described both for all patients and presumed EGFR/ALK/ROS-1-positive patients. Factors associated with OS were analyzed in multivariable Cox regression analysis. RESULTS: Overall, 1741 aNSCLC patients were observed (mean age: 66·97 years, female: 29·87%). The mutation test rate within this population was 26·31% (n = 458), 26·6% of these patients (n = 122) received a targeted treatment and were assumed to have a positive EGFR/ALK/ROS-1 test result. Most often prescribed treatments were pemetrexed monotherapy as 1 L (21·23% for all and 11·11% for mutation-positive patients) and erlotinib monotherapy as 2 L (25·83%/38·54%). Median OS since incident diagnosis was 351 days in all and 571 days in mutation-positive patients. In a multivariable Cox regression analysis, higher age, a stage IV disease, a higher number of chronic drugs in the pre-index period and no systemic therapy increased the risk of early death since first aNSCLC diagnosis. On the other hand, female gender and treatment with therapies other than chemotherapy were associated with a lower risk of early death. CONCLUSIONS: Despite the introduction of new treatments, the real-world survival prognosis for aNSCLC patients remains poor if measured based on an unselected real-world population of patients. Still, the majority of German aNSCLC patients do not receive a mutation test.

A post-transcriptional program of chemoresistance by AU-rich elements and TTP in quiescent leukemic cells.

BACKGROUND: Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. RESULTS: We induce chemoresistant and G0 leukemic cells by serum starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the upregulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, Tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα prior to or along with chemotherapy substantially reduces chemoresistance in primary leukemic cells ex vivo and in vivo. CONCLUSIONS: These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE-bearing mRNAs that promote chemoresistance. By disrupting this pathway, we develop an effective combination therapy against chemosurvival.

Vertical Pathway Inhibition Overcomes Adaptive Feedback Resistance to KRASG12C Inhibition.

PURPOSE: Although KRAS represents the most commonly mutated oncogene, it has long been considered an "undruggable" target. Novel covalent inhibitors selective for the KRASG12C mutation offer the unprecedented opportunity to target KRAS directly. However, prior efforts to target the RAS-MAPK pathway have been hampered by adaptive feedback, which drives pathway reactivation and resistance. EXPERIMENTAL DESIGN: A panel of KRASG12C cell lines were treated with the KRASG12C inhibitors ARS-1620 and AMG 510 to assess effects on signaling and viability. Isoform-specific pulldown of activated GTP-bound RAS was performed to evaluate effects on the activity of specific RAS isoforms over time following treatment. RTK inhibitors, SHP2 inhibitors, and MEK/ERK inhibitors were assessed in combination with KRASG12C inhibitors in vitro and in vivo as potential strategies to overcome resistance and enhance efficacy. RESULTS: We observed rapid adaptive RAS pathway feedback reactivation following KRASG12C inhibition in the majority of KRASG12C models, driven by RTK-mediated activation of wild-type RAS, which cannot be inhibited by G12C-specific inhibitors. Importantly, multiple RTKs can mediate feedback, with no single RTK appearing critical across all KRASG12C models. However, coinhibition of SHP2, which mediates signaling from multiple RTKs to RAS, abrogated feedback reactivation more universally, and combined KRASG12C/SHP2 inhibition drove sustained RAS pathway suppression and improved efficacy in vitro and in vivo. CONCLUSIONS: These data identify feedback reactivation of wild-type RAS as a key mechanism of adaptive resistance to KRASG12C inhibitors and highlight the potential importance of vertical inhibition strategies to enhance the clinical efficacy of KRASG12C inhibitors.See related commentary by Yaeger and Solit, p. 1538.

Identification of DHODH as a therapeutic target in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive lung cancer subtype with extremely poor prognosis. No targetable genetic driver events have been identified, and the treatment landscape for this disease has remained nearly unchanged for over 30 years. Here, we have taken a CRISPR-based screening approach to identify genetic vulnerabilities in SCLC that may serve as potential therapeutic targets. We used a single-guide RNA (sgRNA) library targeting ~5000 genes deemed to encode "druggable" proteins to perform loss-of-function genetic screens in a panel of cell lines derived from autochthonous genetically engineered mouse models (GEMMs) of SCLC, lung adenocarcinoma (LUAD), and pancreatic ductal adenocarcinoma (PDAC). Cross-cancer analyses allowed us to identify SCLC-selective vulnerabilities. In particular, we observed enhanced sensitivity of SCLC cells toward disruption of the pyrimidine biosynthesis pathway. Pharmacological inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in this pathway, reduced the viability of SCLC cells in vitro and strongly suppressed SCLC tumor growth in human patient-derived xenograft (PDX) models and in an autochthonous mouse model. These results indicate that DHODH inhibition may be an approach to treat SCLC.

Combination Olaparib and Temozolomide in Relapsed Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is an aggressive malignancy in which inhibitors of PARP have modest single-agent activity. We performed a phase I/II trial of combination olaparib tablets and temozolomide (OT) in patients with previously treated SCLC. We established a recommended phase II dose of olaparib 200 mg orally twice daily with temozolomide 75 mg/m2 daily, both on days 1 to 7 of a 21-day cycle, and expanded to a total of 50 patients. The confirmed overall response rate was 41.7% (20/48 evaluable); median progression-free survival was 4.2 months [95% confidence interval (CI), 2.8-5.7]; and median overall survival was 8.5 months (95% CI, 5.1-11.3). Patient-derived xenografts (PDX) from trial patients recapitulated clinical OT responses, enabling a 32-PDX coclinical trial. This revealed a correlation between low basal expression of inflammatory-response genes and cross-resistance to both OT and standard first-line chemotherapy (etoposide/platinum). These results demonstrate a promising new therapeutic strategy in SCLC and uncover a molecular signature of those tumors most likely to respond. SIGNIFICANCE: We demonstrate substantial clinical activity of combination olaparib/temozolomide in relapsed SCLC, revealing a promising new therapeutic strategy for this highly recalcitrant malignancy. Through an integrated coclinical trial in PDXs, we then identify a molecular signature predictive of response to OT, and describe the common molecular features of cross-resistant SCLC.See related commentary by Pacheco and Byers, p. 1340.This article is highlighted in the In This Issue feature, p. 1325.

Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish.

Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.

Feeling the Force: Measurements of Platelet Contraction and Their Diagnostic Implications.

In addition to the classical biological and biochemical framework, blood clots can also be considered as active biomaterials composed of dynamically contracting platelets, nascent polymeric fibrin that functions as a matrix scaffold, and entrapped blood cells. As platelets sense, rearrange, and apply forces to the surrounding microenvironment, they dramatically change the material properties of the nascent clot, increasing its stiffness by an order of magnitude. Hence, the mechanical properties of blood clots are intricately tied to the forces applied by individual platelets. Research has also shown that the pathophysiological changes in clot mechanical properties are associated with bleeding and clotting disorders, cancer, stroke, ischemic heart disease, and more. By approaching the study of hemostasis and thrombosis from a biophysical and mechanical perspective, important insights have been made into how the mechanics of clotting and the forces applied by platelets are linked to various diseases. This review will familiarize the reader with a mechanics framework that is contextualized with relevant biology. The review also includes a discussion of relevant tools used to study platelet forces either directly or indirectly, and finally, concludes with a summary of potential links between clotting forces and disease.

KRAS G12C NSCLC Models Are Sensitive to Direct Targeting of KRAS in Combination with PI3K Inhibition.

PURPOSE: KRAS-mutant lung cancers have been recalcitrant to treatments including those targeting the MAPK pathway. Covalent inhibitors of KRAS p.G12C allele allow for direct and specific inhibition of mutant KRAS in cancer cells. However, as for other targeted therapies, the therapeutic potential of these inhibitors can be impaired by intrinsic resistance mechanisms. Therefore, combination strategies are likely needed to improve efficacy.Experimental Design: To identify strategies to maximally leverage direct KRAS inhibition we defined the response of a panel of NSCLC models bearing the KRAS G12C-activating mutation in vitro and in vivo. We used a second-generation KRAS G12C inhibitor, ARS1620 with improved bioavailability over the first generation. We analyzed KRAS downstream effectors signaling to identify mechanisms underlying differential response. To identify candidate combination strategies, we performed a high-throughput drug screening across 112 drugs in combination with ARS1620. We validated the top hits in vitro and in vivo including patient-derived xenograft models. RESULTS: Response to direct KRAS G12C inhibition was heterogeneous across models. Adaptive resistance mechanisms involving reactivation of MAPK pathway and failure to induce PI3K-AKT pathway inactivation were identified as likely resistance events. We identified several model-specific effective combinations as well as a broad-sensitizing effect of PI3K-AKT-mTOR pathway inhibitors. The G12Ci+PI3Ki combination was effective in vitro and in vivo on models resistant to single-agent ARS1620 including patient-derived xenografts models. CONCLUSIONS: Our findings suggest that signaling adaptation can in some instances limit the efficacy of ARS1620 but combination with PI3K inhibitors can overcome this resistance.

SHP2 inhibition restores sensitivity in ALK-rearranged non-small-cell lung cancer resistant to ALK inhibitors.

Most anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung tumors initially respond to small-molecule ALK inhibitors, but drug resistance often develops. Of tumors that develop resistance to highly potent second-generation ALK inhibitors, approximately half harbor resistance mutations in ALK, while the other half have other mechanisms underlying resistance. Members of the latter group often have activation of at least one of several different tyrosine kinases driving resistance. Such tumors are not expected to respond to lorlatinib-a third-generation inhibitor targeting ALK that is able to overcome all clinically identified resistant mutations in ALK-and further therapeutic options are limited. Herein, we deployed a shRNA screen of 1,000 genes in multiple ALK-inhibitor-resistant patient-derived cells (PDCs) to discover those that confer sensitivity to ALK inhibition. This approach identified SHP2, a nonreceptor protein tyrosine phosphatase, as a common targetable resistance node in multiple PDCs. SHP2 provides a parallel survival input downstream of multiple tyrosine kinases that promote resistance to ALK inhibitors. Treatment with SHP099, the recently discovered small-molecule inhibitor of SHP2, in combination with the ALK tyrosine kinase inhibitor (TKI) ceritinib halted the growth of resistant PDCs through preventing compensatory RAS and ERK1 and ERK2 (ERK1/2) reactivation. These findings suggest that combined ALK and SHP2 inhibition may be a promising therapeutic strategy for resistant cancers driven by several different ALK-independent mechanisms underlying resistance.

Genomic and Functional Fidelity of Small Cell Lung Cancer Patient-Derived Xenografts.

Small cell lung cancer (SCLC) patient-derived xenografts (PDX) can be generated from biopsies or circulating tumor cells (CTC), though scarcity of tissue and low efficiency of tumor growth have previously limited these approaches. Applying an established clinical-translational pipeline for tissue collection and an automated microfluidic platform for CTC enrichment, we generated 17 biopsy-derived PDXs and 17 CTC-derived PDXs in a 2-year timeframe, at 89% and 38% efficiency, respectively. Whole-exome sequencing showed that somatic alterations are stably maintained between patient tumors and PDXs. Early-passage PDXs maintain the genomic and transcriptional profiles of the founder PDX. In vivo treatment with etoposide and platinum (EP) in 30 PDX models demonstrated greater sensitivity in PDXs from EP-naïve patients, and resistance to EP corresponded to increased expression of a MYC gene signature. Finally, serial CTC-derived PDXs generated from an individual patient at multiple time points accurately recapitulated the evolving drug sensitivities of that patient's disease. Collectively, this work highlights the translational potential of this strategy.Significance: Effective translational research utilizing SCLC PDX models requires both efficient generation of models from patients and fidelity of those models in representing patient tumor characteristics. We present approaches for efficient generation of PDXs from both biopsies and CTCs, and demonstrate that these models capture the mutational landscape and functional features of the donor tumors. Cancer Discov; 8(5); 600-15. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

Towards remote assessment and screening of acute abdominal pain using only a smartphone with native accelerometers.

Smartphone-based telehealth holds the promise of shifting healthcare from the clinic to the home, but the inability for clinicians to conduct remote palpation, or touching, a key component of the physical exam, remains a major limitation. This is exemplified in the assessment of acute abdominal pain, in which a physician's palpation determines if a patient's pain is life-threatening requiring emergency intervention/surgery or due to some less-urgent cause. In a step towards virtual physical examinations, we developed and report for the first time a "touch-capable" mHealth technology that enables a patient's own hands to serve as remote surrogates for the physician's in the screening of acute abdominal pain. Leveraging only a smartphone with its native accelerometers, our system guides a patient through an exact probing motion that precisely matches the palpation motion set by the physician. An integrated feedback algorithm, with 95% sensitivity and specificity, enabled 81% of tested patients to match a physician abdominal palpation curve with <20% error after 6 attempts. Overall, this work addresses a key issue in telehealth that will vastly improve its capabilities and adoption worldwide.

Microfluidic Transduction Harnesses Mass Transport Principles to Enhance Gene Transfer Efficiency.

Ex vivo gene therapy using lentiviral vectors (LVs) is a proven approach to treat and potentially cure many hematologic disorders and malignancies but remains stymied by cumbersome, cost-prohibitive, and scale-limited production processes that cannot meet the demands of current clinical protocols for widespread clinical utilization. However, limitations in LV manufacture coupled with inefficient transduction protocols requiring significant excess amounts of vector currently limit widespread implementation. Herein, we describe a microfluidic, mass transport-based approach that overcomes the diffusion limitations of current transduction platforms to enhance LV gene transfer kinetics and efficiency. This novel ex vivo LV transduction platform is flexible in design, easy to use, scalable, and compatible with standard cell transduction reagents and LV preparations. Using hematopoietic cell lines, primary human T cells, primary hematopoietic stem and progenitor cells (HSPCs) of both murine (Sca-1+) and human (CD34+) origin, microfluidic transduction using clinically processed LVs occurs up to 5-fold faster and requires as little as one-twentieth of LV. As an in vivo validation of the microfluidic-based transduction technology, HSPC gene therapy was performed in hemophilia A mice using limiting amounts of LV. Compared to the standard static well-based transduction protocols, only animals transplanted with microfluidic-transduced cells displayed clotting levels restored to normal.

Kilogram-scale prexasertib monolactate monohydrate synthesis under continuous-flow CGMP conditions.

Advances in drug potency and tailored therapeutics are promoting pharmaceutical manufacturing to transition from a traditional batch paradigm to more flexible continuous processing. Here we report the development of a multistep continuous-flow CGMP (current good manufacturing practices) process that produced 24 kilograms of prexasertib monolactate monohydrate suitable for use in human clinical trials. Eight continuous unit operations were conducted to produce the target at roughly 3 kilograms per day using small continuous reactors, extractors, evaporators, crystallizers, and filters in laboratory fume hoods. Success was enabled by advances in chemistry, engineering, analytical science, process modeling, and equipment design. Substantial technical and business drivers were identified, which merited the continuous process. The continuous process afforded improved performance and safety relative to batch processes and also improved containment of a highly potent compound.