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Rapid, sensitive, simultaneous quantification of multiple biomarkers in point-of-care (POC) settings could improve the diagnosis and management of sepsis, a common, potentially life-threatening condition. Compared to high-end commercial analytical systems, POC systems are often limited by low sensitivity, limited multiplexing capability, or low throughput. Here, we report an ultra-sensitive, multiplexed plasmonic sensing technology integrating chemifluorescence signal enhancement with plasmon-enhanced fluorescence detection. Using a portable imaging system, the dual chemical and plasmonic amplification enabled rapid analysis of multiple cytokine biomarkers in 1 hr with sub-pg/mL sensitivities. Furthermore, we also developed a plasmonic sensing chip based on nanoparticle-spiked gold nanodimple structures fabricated by wafer-scale batch processes. We used the system to detect six cytokines directly from clinical plasma samples (n = 20) and showed 100% accuracy for sepsis detection. The described technology could be employed in rapid, ultrasensitive, multiplexed plasmonic sensing in POC settings for myriad clinical conditions.

In the era of precision oncology, multicolor fluorescence imaging has become a core technology for multiplexed molecular analysis of cellular and tissue specimens. However, conventional solution-based staining is labor-intensive and time-consuming and requires considerable expertise to yield optimal results, which creates difficulties for employing this technology in resource-limited settings. Here, we report a new immunostaining method based on hydrogel stamping, which is simple, fast, easy to use, and reproducible. We showed that a hydrophilic hydrogel stamp could effectively transfer fluorescent antibodies to targets and withdraw an excess solution when the reaction is completed, obviating the need for extra washing. This unique property allows for quality immunostaining in 5 min for cells using one-eighth of antibody consumption compared to the conventional solution-based method. Furthermore, we implemented fluorescence quenching and immunocycling with hydrogel staining for multiplexed analysis of 9 protein markers at a single cell level. Finally, we applied the immunocycling method to human breast cancer tissue samples and showed quality immunostaining over a large area (∼2 cm

Background Poly (ADP-ribose) polymerase inhibitors (PARPi) have revolutionized the treatment of ovarian cancer; however, real-world data on kidney function among patients treated with PARPi are lacking. Methods We identified adults treated with olaparib or niraparib between 2015 and 2021 at a major cancer center in Boston, MA, USA. We determined the incidence of any acute kidney injury (AKI), defined as at least a 1.5-fold rise in serum creatinine from baseline in the first 12 months following PARPi initiation. We calculated the percentage of patients with any AKI and sustained AKI and adjudicated the etiologies by manual chart review. We compared trajectories in estimated glomerular filtration rate (eGFR) among PARPi-treated and carboplatin and paclitaxel-treated patients with ovarian cancer, matched by baseline eGFR. Results Of 269 patients, 60 (22.3%) developed AKI, including 43 of 194 (22.1%) olaparib-treated patients and 17 of 75 (22.7%) niraparib-treated patients. Only 9 of 269 (3.3%) had AKI attributable to the PARPi. Of the 60 patients with AKI, 21 (35%) had sustained AKI, of whom 6 had AKI attributable to the PARPi (2.2% of the whole cohort). eGFR declined within 30 days post-PARPi initiation by 9.61 (SD = 11.017) mL/min per 1.73 m2 but recovered by 8.39 (SD = 14.05) mL/min per 1.73 m2 within 90 days after therapy cessation. There was no difference in eGFR at 12 months post-therapy initiation in patients receiving PARPi or controls receiving carboplatin and paclitaxel (P = .29). Conclusions AKI is common following PARPi initiation as is a transient decline in eGFR; however, sustained AKI directly attributable to the PARPi and long-term eGFR decline are uncommon.

Bevacizumab; Folate receptor alpha; Platinum-resistant ovarian cancer Bevacizumab; Receptor de folato alfa; Cáncer de ovario resistente al platino Bevacizumab; Receptor de folat alfa; Càncer d'ovari resistent al platí Purpose Evaluate the antitumor activity and safety profile of the combination of mirvetuximab soravtansine and bevacizumab in patients with platinum-resistant ovarian cancer. Methods Patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer, whose most recent platinum-free interval was ≤6 months, were administered mirvetuximab soravtansine (6 mg/kg adjusted ideal body weight) and bevacizumab (15 mg/kg), intravenously, once every 3 weeks. Eligibility included FRα expression by immunohistochemistry (IHC; ≥25% of cells with ≥2+ intensity). Prior bevacizumab and/or PARP inhibitor (PARPi) treatment were permitted. The primary endpoint was confirmed objective response rate (ORR). Secondary endpoints included duration of response (DOR), progression-free survival (PFS), and safety. Results Ninety-four patients received combination treatment with mirvetuximab soravtansine and bevacizumab. Median age was 62 years (range, 39–81). Fifty-two percent had ≥3 prior therapies; 59% had prior bevacizumab; and 27% had prior PARPi. ORR was 44% (95% CI 33, 54) with 5 complete responses, median DOR 9.7 months (95% CI 6.9, 14.1), and median PFS 8.2 months (95% CI 6.8, 10.0). Treatment-related adverse events were consistent with the profiles of each agent, with the most common being blurred vision (all grades 57%; grade 3, 1%), diarrhea (54%; grade 3, 1%), and nausea (51%; grade 3, 1%). Conclusion The mirvetuximab soravtansine plus bevacizumab doublet is an active and well-tolerated regimen in patients with FRα-expressing platinum-resistant ovarian cancer. Promising activity was observed for patients regardless of level of FRα expression or prior bevacizumab. These data underscore the potential for mirvetuximab soravtansine as the combination partner of choice for bevacizumab in this setting. This study was supported by ImmunoGen, Inc.

Extracellular vesicles (EVs) carry information inherited from parental cells, having significant potential for disease diagnosis. In blood, however, EVs are outnumbered10

PURPOSE We evaluated a clinical breast examination (CBE) screening program to determine the prevalence of breast abnormalities, number examined per cancer diagnosis, and clinical resources required for these diagnoses in a middle-income African setting. METHODS We performed a retrospective review of a CBE screening program (2015-2018) by Journey of Hope Botswana, a Botswana-based nongovernmental organization (NGO). Symptomatic and asymptomatic women were invited to attend. Screening events were held in communities throughout rural and periurban Botswana, with CBEs performed by volunteer nurses. Individuals who screened positive were referred to a private tertiary facility and were followed by the NGO. Data were obtained from NGO records. RESULTS Of 6,120 screened women (50 men excluded), 452 (7.4%) presented with a symptom and 357 (5.83%) were referred for further evaluation; 257 ultrasounds, 100 fine-needle aspirations (FNAs), 58 mammograms, and 31 biopsies were performed. In total, 6,031 were exonerated from cancer, 78 were lost to follow-up (67 for ≤ 50 years and 11 for > 50 years), and 11 were diagnosed with cancer (five for 41-50 years and six for > 50 years, 10 presented with symptoms). Overall breast cancer prevalence was calculated to be 18/10,000 (95% CI, 8 to 29/10,000). The number of women examined per breast cancer diagnosis was 237 (95% CI, 126 to 1910) for women of age 41-50 years and 196 (95% CI, 109 to 977) for women of age > 50 years. Median time to diagnosis for all women was 17.5 [1 to 32.5] days. CBE-detected tumors were not different than tumors presenting through standard care. CONCLUSION In a previously unscreened population, yield from community-based CBE screening was high, particularly among symptomatic women, and required modest diagnostic resources. This strategy has potential to reduce breast cancer mortality.

Assays for cancer diagnosis via the analysis of biomarkers on circulating extracellular vesicles (EVs) typically have lengthy sample workups, limited throughput or insufficient sensitivity, or do not use clinically validated biomarkers. Here we report the development and performance of a 96-well assay that integrates the enrichment of EVs by antibody-coated magnetic beads and the electrochemical detection, in less than one hour of total assay time, of EV-bound proteins after enzymatic amplification. By using the assay with a combination of antibodies for clinically relevant tumour biomarkers (EGFR, EpCAM, CD24 and GPA33) of colorectal cancer (CRC), we classified plasma samples from 102 patients with CRC and 40 non-CRC controls with accuracies of more than 96%, prospectively assessed a cohort of 90 patients, for whom the burden of tumour EVs was predictive of five-year disease-free survival, and longitudinally analysed plasma from 11 patients, for whom the EV burden declined after surgery and increased on relapse. Rapid assays for the detection of combinations of tumour biomarkers in plasma EVs may aid cancer detection and patient monitoring.

Extracellular vesicles (EVs), actively shed from a variety of neoplastic and host cells, are abundant in blood and carry molecular markers from parental cells. For these reasons, EVs have gained much interest as biomarkers of disease. Among a number of different analytical methods that have been developed, surface plasmon resonance (SPR) stands out as one of the ideal techniques given its sensitivity, robustness, and ability to miniaturize. In this Review, we compare different SPR platforms for EV analysis, including conventional SPR, nanoplasmonic sensors, surface-enhanced Raman spectroscopy, and plasmonic-enhanced fluorescence. We discuss different surface chemistries used to capture targeted EVs and molecularly profile their proteins and RNAs. We also highlight these plasmonic platforms' clinical applications, including cancers, neurodegenerative diseases, and cardiovascular diseases. Finally, we discuss the future perspective of plasmonic sensing for EVs and their potentials for commercialization and clinical translation. National Research Foundation (NRF) Accepted version This work was supported in part by the Singapore National Research Foundation Competitive Research Programme NRFCRP13-2014-01 (A.-Q.L.).

Purpose To evaluate the safety and clinical activity of mirvetuximab soravtansine, an antibody-drug conjugate comprising a humanized anti-folate receptor alpha (FRα) monoclonal antibody, cleavable linker, and the maytansinoid DM4, a potent tubulin-targeting agent, in combination with bevacizumab in patients with FRα-positive, platinum-resistant ovarian cancer. Methods Patients with platinum-resistant epithelial ovarian, fallopian tube, or primary peritoneal cancer were administered mirvetuximab soravtansine (6 mg/kg, adjusted ideal body weight) and bevacizumab (15 mg/kg) once every 3 weeks. Eligibility included FRα positivity by immunochemistry and prior bevacizumab exposure was permitted. Adverse events, tumor response, and progression-free survival (PFS) were determined. Results Sixty-six patients, with a median of 3 prior lines of therapy (range, 1–8), received the combination of mirvetuximab soravtansine and bevacizumab at full dosing during the escalation and expansion stages of the study. Adverse events were generally mild-to-moderate (≤grade 2) with diarrhea, blurred vision, nausea, and fatigue being the most common treatment-related toxicities. Six cases of pneumonitis (9%; all grade 1 or 2), an adverse event of special interest, were observed. The confirmed objective response rate (ORR) was 39%, including 5 complete responses and 21 partial responses, and the median PFS was 6.9 months. The combination was particularly active in the subset of patients (n = 16) who were bevacizumab-naive, less heavily pretreated (1–2 prior lines), and whose tumors exhibited medium/high FRα expression (ORR, 56% with a median duration of response of 12 months; PFS, 9.9 months). Conclusion The combination of mirvetuximab soravtansine with bevacizumab is well tolerated in patients with platinum-resistant, recurrent ovarian cancer. The encouraging efficacy measures compare favorably to reported outcomes for bevacizumab combined with standard chemotherapy in similar patient populations.

With the global burden of cancer on the rise, it is critical to developing new modalities that could detect cancer and guide targeted treatments in fast and inexpensive ways. The need for such technologies is vital, especially in underserved regions where severe diagnostic bottlenecks exist. Recently, we developed a low-cost digital diagnostic system for breast cancer using fine-needle aspirates (FNAs). Named, AIDA (artificial intelligence diffraction analysis), the system combines lens-free digital diffraction imaging with deep-learning algorithms to achieve automated, rapid, and high-throughput cellular analyses for breast cancer diagnosis of FNA and subtype classification for better-guided treatments (Min et al. ACS Nano 12:9081-9090, 2018). Although primarily validated for breast cancer and lymphoma (Min et al. ACS Nano 12:9081-9090, 2018; Im et al. Nat Biomed Eng 2:666-674, 2018), the system could be easily adapted to diagnosing other prevalent cancers and thus find widespread use for global health.

Breakthroughs in deep molecular profiling of non-invasive clinical samples (“liquid biopsy”) promise to revolutionize how cancer is detected and monitored. Analyzing circulating biomarkers in patient blood has shown their diagnostic potential in advanced disease states. Early detection, however, remains challenging – most biomarkers either (i) arise in advanced disease and at low abundance or (ii) often suffer from poor specificity, thus creating false positives that are costly and stressful for patients. We have been exploring extracellular vesicles (EVs) as a new biomarker that may overcome these challenges. EVs are nanoscale membrane particles secreted by cells. Most types of cancer shed large numbers of EVs that carry molecular cargo from the parent tumor. Analyzing EVs thus can represent a new real-time window to detect, diagnose, and serially monitor tumor molecular status. This presentation will describe our recent advances that leverage EVs for early cancer detection. Specifically, we will discuss the single EV analysis (SEA) platform that characterizes individual EVs for multiple protein markers. This method helps us unveil EV heterogeneity, which maximizes biological information content (specificity and sensitivity) for tumor-derived vesicles. We will review the SEA’s development and pilot application to diagnose early cancers. Citation Format: Hakho Lee, Cesar M. Castro. Early cancer detection through the analyses of extracellular vesicles. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr IA020.

Tetrahydrocannabinol (THC), the primary psychoactive ingredient of cannabis, impairs cognitive and motor function in a concentration-dependent fashion. Drug testing is commonly performed for employment and law enforcement purposes; however, available tests produce low-sensitive binary results (lateral flow assays) or have long turnaround (gas chromatography-mass spectrometry). To enable on-site THC quantification in minutes, we developed a rapid assay for oral THC analysis called EPOCH, express probe for on-site cannabis inhalation. EPOCH features distinctive sensor design such as a radial membrane and transmission optics, all contained in a compact cartridge. This integrated approach permitted assay completion within 5 minutes with a detection limit of 0.17 ng/mL THC, which is below the regulatory guideline (1 ng/mL). As a proof of concept for field testing, we applied EPOCH to assess oral fluid samples from cannabis users (n = 43) and controls (n = 43). EPOCH detected oral THC in all specimens from cannabis smokers (median concentration, 478 ng/mL) and THC-infused food consumers. Longitudinal monitoring showed a fast drop in THC concentrations within the first 6 hours of cannabis smoking (half-life, 1.4 hours).

Most deaths (80%) from cervical cancer occur in regions lacking adequate screening infrastructures or ready access to them. In contrast, most developed countries now embrace human papillomavirus (HPV) analyses as standalone screening; this transition threatens to further widen the resource gap. Methods: We describe the development of a DNA-focused digital microholography platform for point-of-care HPV screening, with automated readouts driven by customized deep-learning algorithms. In the presence of high-risk HPV 16 or 18 DNA, microbeads were designed to bind the DNA targets and form microbead dimers. The resulting holographic signature of the microbeads was recorded and analyzed. Results: The HPV DNA assay showed excellent sensitivity (down to a single cell) and specificity (100% concordance) in detecting HPV 16 and 18 DNA from cell lines. Our deep learning approach was 120-folder faster than the traditional reconstruction method and completed the analysis in < 2 min using a single CPU. In a blinded clinical study using patient cervical brushings, we successfully benchmarked our platform's performance to an FDA-approved HPV assay. Conclusions: Reliable and decentralized HPV testing will facilitate cataloguing the high-risk HPV landscape in underserved populations, revealing HPV coverage gaps in existing vaccination strategies and informing future iterations.

We report a sensitive and versatile biosensing approach, LUCID (luminescence compact in vitro diagnostics), for quantitative molecular and cellular analyses. LUCID uses upconversion nanoparticles (UCNPs) as luminescent reporters in mutually exclusive photoexcitation and read-out sequences implemented on a smartphone. The strategy improves imaging signal-to-noise ratios, eliminating interference from excitation sources and minimizing autofluorescence, and thus enables filterless imaging. Here we developed a miniaturized detection system and optimized UCNPs for the system and biological applications. Nanoparticle luminescence lifetime was extended by controlling particle structure and composition. When tested with a range of biological targets, LUCID achieved high detection sensitivity (0.5 pM for protein and 0.1 pM for nucleic acids), differentiated bacterial samples, and allowed profiling of cells. In proof-of-concept clinical use, LUCID demonstrated effective screening of cancer cells in cervical brushing specimens, identifying patients at high risk for malignancy. These results suggest that LUCID could serve as a broadly applicable and inexpensive diagnostic platform.

ObjectiveTo evaluate correlations between rucaparib exposure and selected efficacy and safety endpoints in patients with recurrent ovarian carcinoma using pooled data from Study 10 and ARIEL2.MethodsEfficacy analyses were limited to patients with carcinomas harboring a deleterious BRCA1 or BRCA2 mutation who had received ≥2 prior lines of chemotherapy. Safety was evaluated in all patients who received ≥1 rucaparib dose. Steady-state daily area under the concentration-time curve (AUCss) and maximum concentration (Cmax,ss) for rucaparib were calculated for each patient and averaged by actual dose received over time (AUCavg,ss and Cmax,avg,ss) using a previously developed population pharmacokinetic model.ResultsRucaparib exposure was dose-proportional and not associated with baseline patient weight. In the exposure-efficacy analyses (n=121), AUCavg,ss was positively associated with independent radiology review-assessed RECIST response in the subgroup of patients with platinum-sensitive recurrent disease (n=75, p=0.017). In the exposure-safety analyses (n=393, 40mg once daily to 840mg twice daily [BID] starting doses), most patients received a 600mg BID rucaparib starting dose, with 27% and 21% receiving 1 or≥2 dose reductions, respectively. Cmax,ss was significantly correlated with grade≥2 serum creatinine increase, grade≥3 alanine transaminase/aspartate transaminase increase, platelet decrease, fatigue/asthenia, and maximal hemoglobin decrease (p&nbsp

The identification of patients with aggressive cancer who require immediate therapy is a health challenge in low- and middle-income countries. Limited pathology resources, high healthcare costs and large caseloads call for the development of advanced stand-alone diagnostics. Here, we report and validate an automated, low-cost point-of-care device for the molecular diagnosis of aggressive lymphomas. The device uses contrast-enhanced microholography and a deep learning algorithm to directly analyse percutaneously obtained fine-needle aspirates. We show the feasibility and high accuracy of the device in cells, as well as the prospective validation of the results in 40 patients clinically referred for image-guided aspiration of nodal mass lesions suspicious of lymphoma. Automated analysis of human samples with the portable device should allow for the accurate classification of patients with benign and malignant adenopathy. A low-cost point-of-care device that uses contrast-enhanced microholography and deep learning accurately detects aggressive lymphomas in patients referred for aspiration and biopsy of enlarged lymph nodes.

Objectives: Dickkopf-1 (DKK1) modulates Wnt signaling, promotes tumor growth through a CKAP4-AKT signaling pathway and contributes to an immune suppressive tumor microenvironment. DKN-01 (D), a neutralizing DKK1 antibody has demonstrated safety and clinical activity in advanced gynecologic malignancies. Prior literature has reported an association of CTNNB1 mutations and an aggressive biology and shorter survival in endometrioid endometrial cancer. We previously demonstrated Wnt activating mutations were associated with higher DKK1 tumoral expression in advanced gynecologic malignancies. Methods: Pts with recurrent gynecologic cancers [endometrial (EEC), ovarian (EOC) and carcinosarcoma (MMMT)] were treated with D as monotherapy (mono) or in combination with paclitaxel (pac) in a phase 2 basket trial (NCT03395080) whereby ≥50% must have had a Wnt signaling alteration. Here we report on the subgroup of pts with Wnt activating mutations (CTNNB1, RNF43, APC). Clopper-Pearson confidence intervals were used to study the association of Wnt activating mutations with clinical benefit (CR, PR or SD); Kaplan-Meier estimates/Cox-PH models were used for analyses of progression free survival (PFS) and overall survival (OS). Results: A total of 111 pts enrolled; 52 pts treated with D mono; 59 pts treated with D + pac. Mean number of prior therapies was 3.6 (range 1, 11) and 3.9 (range 1, 10) for D mono and D + pac, respectively. A total of 108 pts (97%) with available genetics; 23 pts (21%) had Wnt activating mutations [CTNNB1 (n=17), RNF43 (n=5), APC (n=3)]. Wnt activating mutations were more common in EEC (n=17/54, 31%) than EOC (n=2/33, 6%) and MMMT (n=4/24, 17%). Median duration of treatment with D was 6 cycles in pts with Wnt activating mutations compared with 3 cycles for pts without Wnt activating mutations. A total of 15 of 23 (65%) evaluable pts with Wnt activating mutations had clinical benefit [1 PR, 14 SD; durable SD (> 4 months) in 11 of 14 pts (48%)] compared with 34 of 76 (45%) evaluable pts [1 CR, 2 PR, 31 SD; durable SD (> 4 months) in 12 of 31 SD pts (16%)] without Wnt activating mutations. All 4 pts with an objective response had a PIK3CA mutation. Pts with Wnt activating mutations had longer PFS [median 5.5 mos; 95% CI (1.8, 6.0)] and OS [median 22.2 mos; 95% CI (7.3, NE)] compared with pts without Wnt activating mutations [median PFS 2.0 mos; 95% CI (1.8, 3.1)] and OS (median 9.9 mos; 95% CI (6.9, 12.5)]. Download : Download high-res image (65KB) Download : Download full-size image Conclusions: D has single agent activity in a subgroup of pts with Wnt activating mutations, historically identified as a poor prognostic group, whereby they experienced greater clinical benefit and longer survival compared with pts without Wnt activating mutations. A notable finding was that all responding pts had PIK3CA mutations. This study is ongoing and updated results will be presented.

Objectives: Immune checkpoint inhibitor (ICI) therapy is the standard-of-care in several solid tumor malignancies, including gynecologic cancers (GYN). Several recent approvals are based on histology-agonistic trials where the relative efficacy in GYN cancers is lacking. Also, real-world efficacy data on ICI therapy in GYN malignancies are limited. The response rates to ICI rechallenge and the histologies most likely to respond are unknown. This study evaluates and characterizes clinical response to ICI therapy in GYN cancers. Methods: An IRB approved retrospective review was performed on adult women with gynecologic malignancies treated with ICI, not under clinical trial, between January 2000 and June 2020 at the Mass General Brigham Integrated Health Care System and the Dana-Farber Cancer Institute. Variables analyzed included patient demographics, disease characteristics, overall response rates (ORR), time to response, duration of response (DOR), and progression free survival (PFS). Results: Database review identified 129 eligible patients. The primary sites of disease were uterus (40%), vulvovaginal (22%), ovary (21%), and cervix (17%). Most patients received PD-1 inhibitor monotherapy (80.6%), 16.3% of patients received combination CTLA-4 and PD-1 inhibitor therapy. Monotherapy with anti-PD-L1 and anti-CTLA4 represented 2.3% and 0.8%, respectively. The ORR across all disease sites was 45%. The complete response rate, partial response rate, and stable disease rates were 24%, 62%, and 14%, respectively. The ORR in cervical cancer was 54.5%, 51.9% in uterine cancer, 35% in vulvar cancer, and 33.3% in ovarian cancer. The overall median time to response and DOR was 2.5 months and 4.5 months, respectively. Median DOR/PFS (months) were 10/13 (uterine), 3/5 (ovary) 3.8/7 (cervix), 5/7.8 (vulvar). Twenty-eight patients received a subsequent line of ICI therapy, and of these, 11 responded, including 3 patients (all with vulvar or cervical cancer) who had not responded to 1st line therapy. Eight patients experienced responses in both lines. The only 2 patients with ovarian cancer who responded to second-line ICI had a previous response (and subsequent progression) to first-line ICI. Of nine patients receiving third-line ICI treatment, there was only 1 response in a patient with vulvar melanoma. There were no responders to the fourth rechallenge with immunotherapy. Download : Download high-res image (91KB) Download : Download full-size image Conclusions: Overall, 45% of patients responded to initial ICI therapy, with the majority experiencing a PR. Patients with uterine cancer benefited the longest from ICI therapy. Most patients who responded to second-line therapy were those who responded to initial therapy. All patients who responded to ICI rechallenge without an initial response had vulvar or cervical cancer. Patients with ovarian cancer who did not respond to initial ICI therapy were unlikely to respond to subsequent lines. The use and timing of ICI in GYN malignancies should consider primary site and patterns of prior response.

Extracellular vesicles (EVs) are diverse, nanoscale membrane vesicles actively released by cells. Similar-sized vesicles can be further classified (e.g., exosomes, microvesicles) based on their biogenesis, size, and biophysical properties. Although initially thought to be cellular debris, and thus under-appreciated, EVs are now increasingly recognized as important vehicles of intercellular communication and circulating biomarkers for disease diagnoses and prognosis. Despite their clinical potential, the lack of sensitive preparatory and analytical technologies for EVs poses a barrier to clinical translation. New analytical platforms including molecular ones are thus actively being developed to address these challenges. Recent advances in the field are expected to have far-reaching impact in both basic and translational studies. This article aims to present a comprehensive and critical overview of emerging analytical technologies for EV detection and their clinical applications.

Kidney transplant patients require life-long surveillance to detect allograft rejection. Repeated biopsy, albeit the clinical gold standard, is an invasive procedure with the risk of complications and comparatively high cost. Conversely, serum creatinine or urinary proteins are noninvasive alternatives but are late markers with low specificity. We report a urine-based platform to detect kidney transplant rejection. Termed iKEA (integrated kidney exosome analysis), the approach detects extracellular vesicles (EVs) released by immune cells into urine; we reasoned that T cells, attacking kidney allografts, would shed EVs, which in turn can be used as a surrogate marker for inflammation. We optimized iKEA to detect T-cell-derived EVs and implemented a portable sensing system. When applied to clinical urine samples, iKEA revealed high level of CD3-positive EVs in kidney rejection patients and achieved high detection accuracy (91.1%). Fast, noninvasive, and cost-effective, iKEA could offer new opportunities in managing transplant recipients, perhaps even in a home setting.

Adverse food reactions, including food allergies, food sensitivities, and autoimmune reaction (e.g., celiac disease) affect 5-15% of the population and remain a considerable public health problem requiring stringent food avoidance and epinephrine availability for emergency events. Avoiding problematic foods is practically difficult, given current reliance on prepared foods and out-of-home meals. In response, we developed a portable, point-of-use detection technology, termed integrated exogenous antigen testing (iEAT). The system consists of a disposable antigen extraction device coupled with an electronic keychain reader for rapid sensing and communication. We optimized the prototype iEAT system to detect five major food antigens in peanuts, hazelnuts, wheat, milk, and eggs. Antigen extraction and detection with iEAT requires10 min and achieves high-detection sensitivities (e.g., 0.1 mg/kg for gluten, lower than regulatory limits of 20 mg/kg). When testing under restaurant conditions, we were able to detect hidden food antigens such as gluten within "gluten-free" food items. The small size and rapid, simple testing of the iEAT system should help not only consumers but also other key stakeholders such as clinicians, food industries, and regulators to enhance food safety.

Objectives Wnt/β-catenin signaling is frequently dysregulated in gynecologic malignancies. CTNNB1, APC and RNF43 mutations cause pathway activation; CTNNB1 stabilizing mutations lead to elevated DKK1 expression which promotes an immune suppressive tumor microenvironment. Neutralization by DKN-01 (D), a mAb against DKK1, is being tested in a phase 2 basket study. Methods Eligibility included recurrent endometrial cancer (EC) or platinum resistant/refractory ovarian cancer (OC) enriched (∼50%) for Wnt signaling-related genetic alterations. Subgroup analysis was done in pts with genetic alterations associated with activation of Wnt/β-catenin signaling (CTNNB1, APC or RNF43). Pts were assigned (MD discretion) to receive D (300 mg on Days 1 & 15) or D + paclitaxel (P) (80 mg/m2 on Days 1, 8 and 15) of a 28-day cycle. Primary endpoint is ORR; exploratory endpoints: DKK1 expression (serum/plasma/tumor), tumor genetics, infiltrating immune cells, and β-catenin IHC. Results 80 pts are enrolled: D (n=33, 19 EC, 14 OC); D + P (n=47; 28 EC, 19 OC); 18 pts with CTNNB1 (n=13), APC (n=2), RNF43 (n=2), or CTNNB1 + RNF43 (n=1). 54 pts evaluable for response (table 1). D and D + P were safe and well tolerated with no additive toxicities. The trial is ongoing; updated safety, efficacy and correlative work are pending. Conclusions D and D + P have activity in pts with recurrent gyn cancers; the role of Wnt/β-catenin pathway activation as a potential biomarker for response is currently under study. Clinical trial information: NCT03395080.

We report a sensitive and versatile biosensing approach, LUCID (luminescence compact in-vitro diagnostics), for quantitative molecular and cellular analyses. LUCID uses upconversion nanoparticles (UCNPs) as luminescent reporters in mutually exclusive photo-excitation and read-out sequences implemented on a smartphone. The strategy improves imaging signal-to-noise ratios, eliminating interference from excitation sources and minimizing autofluorescence, and thus enables filter-less imaging. Here we developed a miniaturized detection system and optimized UCNPs for the system and biological applications. Nanoparticle luminescence lifetime was extended by controlling particles’ structure and composition. When tested with a range of biological targets, LUCID achieved high detect sensitivity (0.5 pM for protein and 0.1 pM for nucleic acids), differentiated bacterial samples, and allowed profiling of cells. In proof-of-concept clinical use, LUICD demonstrated effective screening of cancer cells in cervical brushing specimens, identifying patients at high risk for malignancy. These results suggest that LUCID could serve as a broadly applicable and inexpensive diagnostic platform.

An inexpensive assay that uses thermophoresis to concentrate cancer extracellular vesicles bound to aptamers specific for target proteins on the vesicles’ surface, can classify cancer patients according to cancer type and stage.

Objectives: Immune checkpoint inhibitors (ICI) are becoming standard of care in several advanced/recurrent gynecologic (GYN) malignancies. Yet, reported toxicities derive from basket and histology-specific clinical trials. Aggregate reports of real-world ICI toxicity, specifically in GYN cancers, are lacking. This study assesses and describes immune-related adverse events (irAE) in women receiving ICI. Methods: An IRB-approved retrospective review was performed on adult women with GYN malignancies treated with ICI, not under clinical trial, between January 2000 and June 2020 at the Mass General Brigham Integrated Health Care System and the Dana Farber Cancer Institute. Variables analyzed included patient demographics, ICI class, duration of therapy, time to first toxicity, subsequent toxicity with ICI retreatment, hospitalization rate, and therapy discontinuation. Toxicity was assessed by using CTCAE v5 criteria. Results: A total of 129 patients met inclusion criteria. The median age at the time of ICI treatment was 62 years. Fifty-two patients (40%) had uterine cancer, vulvar/vaginal; 28 (22%), ovarian; 27 (21%), and 22 (17%) had cervical cancer. Eighty percent of patients received anti-PD-1 monotherapy, 16.3% received combination anti-CTLA-4 and PD-1, 2.3% received anti-PD-L1 monotherapy, and 0.78% received anti-CTLA-4 monotherapy. Twenty-two percent of patients received a second line of ICI after responding or progressing on a previous ICI, and 7% received a third line of ICI. Overall, 55 patients (42.6%) experienced an irAE. The median time to first toxicity was 42 days. Of the 50 patients with assessable toxicity, 25 (50%) experienced grade 1 toxicity, 17 (34%) grade 2, 18 (36%) grade 3, and 2 (4%) grade 4. There were 15 (25%) hospitalizations, and there were no treatment-related deaths. Twenty-one patients (38%) experienced GI toxicity, including colitis and hepatitis. Thirteen patients (23.6%) developed a rash, and 16 (29%) patients had endocrine toxicity, predominantly hypothyroidism. Pneumonitis occurred in 5.5% of patients. In total, 109 patients (84.4%) discontinued ICI therapy. Only 18 patients (16.5%) discontinued due to toxicity, and 58 patients (53.2%) discontinued for progressive disease. On rechallenge, 32% of patients developed new or recurrent toxicity. Download : Download high-res image (85KB) Download : Download full-size image Conclusions: ICI therapy is well-tolerated in patients with GYN cancer. Less than half of our cohort experienced autoimmune-related side effects, and most of these toxicities were grade 1. A very small percentage of patients discontinue treatment for toxicity, and on rechallenge, most patients do not develop new or worsening toxicity. This information may help guide patient counseling when initiating ICI therapy or in the development of combination clinical trials involving ICI.

TPS5609 Background: Despite significant strides in understanding the molecular pathogenesis of EC, there remain few effective therapies for recurrent disease. Deeper insight into the roles of disordered tumor vasculature and HIF1α- and VEGF-mediated immunosuppressive effects on myeloid-derived suppressor cells, T-cells, and PD-L1 expression contributed to the development of new targeted regimens. Activity of pembrolizumab and lenvatinib was demonstrated in a phase 2 trial in MMR-P EC (NCT02501096). By inhibiting VEGF receptor (VEGFR) and PD-L1 signaling, immunologically “cold” tumors may become inflamed. However, there are concerns regarding the toxicity of pembrolizumab/lenvatinib and alternatives are sought. The combination of the anti-PD-L1 antibody avelumab with axitinib, an inhibitor of VEGFR 1-3 and PDGFR with more potent IC50 inhibitory activity than lenvatinib, has also shown synergistic activity and is FDA approved as first line treatment for patients with renal cell cancer. We therefore hypothesized that this combination would be well tolerated and efficacious in recurrent MMR-P EC. Methods: This is an investigator-initiated, phase 2, two-stage single cohort trial evaluating avelumab with axitinib in recurrent or persistent EC. Participants must have MMR-P EC of any histology and have received at least one chemotherapeutic regimen, with no upper limit on the number of prior lines received. Prior use of immune checkpoint (IC) inhibitors is excluded. Treatment consists of avelumab 800mg IV every 2 weeks and axitinib 5mg orally twice daily. Co-primary endpoints are progression-free survival at 6 months (PFS6) and objective response rate by RECIST 1.1. Translational objectives include characterization of tumor-infiltrating lymphocytes, infiltrating myeloid cells, expression of IC markers, and whole exome sequencing to evaluate mutations in genes related to DNA repair and immunologic response. This is a two-stage design in the method of Sill et al, with 16 participants anticipated in stage 1 and 19 participants in stage 2, for a total of 35 participants. Accrual is ongoing. Clinical trial information: NCT02912572.

Almost all cases of cervical cancer are caused by the human papilloma virus (HPV). Detection of pre-cancerous cervical changes provides a window of opportunity for cure of an otherwise lethal disease when metastatic. With a greater understanding of the biology and natural course of high-risk HPV infections, screening methods have shifted beyond subjective Pap smears toward more sophisticated and objective tactics. This has led to a substantial growth in the breadth and depth of HPV-based cervical cancer screening tests, especially in developed countries without constrained resources. Many low- and middle-income countries (LMICs) have less access to advanced laboratories and healthcare resources, so new point-of-care (POC) technologies have been developed to provide test results in real time, improve the efficiency of techniques, and increase screening adoption. In this Review, we will discuss how novel decentralized screening technologies and computational strategies improve upon traditional methods and how their realized promise could further democratize cervical cancer screening and promote greater disease prevention.

Exosomes have recently emerged as highly promising cancer biomarkers because they are abundant in biofluids, carry proteins and RNA reflecting their originating cells and are stable over weeks. Beyond abundance and stability, detailed exosome analyses could be clinically useful for diagnosing and profiling cancers. Despite their clinical potential, simple, reliable and sensitive approaches for rapidly quantifying exosomes and their molecular information has been challenging. Therefore, there is a clear need to develop next-generation sensing technologies for exosome detection and analysis. In this critical review, we will describe three nanotechnology sensing platforms developed for analysis of exosomal proteins and RNAs directly from clinical specimens and discuss future development to facilitate their translation into routine clinical use.

Low-cost, rapid and accurate detection technologies are key requisites to cope with the growing global cancer challenges. The need is particularly pronounced in resource-limited settings where treatment opportunities are often missed due to the absence of timely diagnoses. We herein describe a Holographic Assessment of Lymphoma Tissue (HALT) system that adopts a smartphone as the basis for molecular cancer diagnostics. The system detects malignant lymphoma cells labeled with marker-specific microbeads that produce unique holographic signatures. Importantly, we optimized HALT to detect lymphomas in fine-needle aspirates from superficial lymph nodes, procedures that align with the minimally invasive biopsy needs of resource-constrained regions. We equipped the platform to directly address the practical needs of employing novel technologies for "real world" use. The HALT assay generated readouts in

Background: Dickkopf-1 (DKK1) modulates Wnt signaling, promotes tumor growth through a CKAP4-AKT signaling pathway and contributes to an immune suppressive tumor microenvironment. DKN-01, a neutralizing DKK1 antibody has demonstrated safety and clinical activity in advanced gynecologic malignancies. We report response and survival outcomes in EEC patients (pts) treated with DKN-01 monotherapy by tumor genetics (Wnt signaling alterations and PI3K/AKT mutations). Methods: Pts with recurrent EEC were treated with DKN-01 as monotherapy (mono) or in combination with paclitaxel in a phase 2 basket trial (NCT03395080) whereby ≥50% must have had a Wnt signaling alteration. Here we report on EEC mono group only. Clopper-Pearson confidence intervals and logistic regression were used to study the association of Wnt signaling alterations with clinical benefit (CR, PR or SD) following DKN-01 monotherapy; Kaplan-Meier estimates/Cox-PH models were used for analyses of progression free survival (PFS) and overall survival (OS). Results: As of 30 December 2019, 29 EEC pts enrolled; 21 pts (72%) had Wnt signaling alterations [ARID1A most common (11 pts, 38%)] and 25 (86%) had PI3K/AKT mutations [PIK3CA in 13 pts (45%)]. Median number of prior therapies was 4 (range 1, 9); 20 pts (69%) had endometrioid histology and 19 pts (66%) had G2/G3 tumor grade at diagnosis. 26 pts were evaluable, 20 pts Wnt altered and 6 pts non-Wnt altered; 24 pts with and 2 pts without PI3K/AKT mutations. In the Wnt altered group, there was 1 cCR, 1 cPR, 8 SD (6 durable SD >120 days) and 10 PD compared with 0 responders, 1 SD and 5 PD in the non-Wnt altered group. In the 24 pts with PI3K/AKT mutations there was 1 cCR, 1 cPR, 8 SD (6 durable SD >120 days) and 14 PD compared with 1 SD and 1 PD in the 2 pts without PI3K/AKT mutations. The 2 responding pts had PI3KCA mutations in common. Probability of 6 month PFS/OS was 30% (95% CI:12.3, 50.1) and 70% (95% CI: 45.1, 85.3) for Wnt altered pts vs 18% (95% CI: 0.8, 53.8) and 50% (95% CI: 11.1, 80.4) for non-Wnt altered, respectively. Conclusions: DKN-01 has single agent activity in heavily pretreated EEC pts. EEC pts frequently had Wnt signaling alterations and PIK3/AKT mutations; greater clinical benefit was demonstrated in pts whose tumors had Wnt signaling alterations. This study is ongoing and updated results will be presented. Citation Format: Rebecca Arend, Cesar Castro, Erica Hamilton, Kristopher LyBarger, Linda Duska, Michael Kagey, William Bradley, Jasgit Sachdev, Cynthia A. Sirard, David M. O'Malley. Patients with recurrent epithelial endometrial cancers (EEC) and Wnt signaling alterations demonstrated greater clinical benefit when treated with DKN-01 monotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO036.

Extracellular vesicles (EVs) represent promising circulating biomarkers for cancers, but their high-throughput analyses in clinical settings prove challenging due to lack of simple, fast, and robust EV assays. Here, we describe a bead-based EV assay detected by flow cytometry, which integrates EV capture using microbeads with EV protein analyses by flow cytometry. The assay is fast (< 4 hrs for 48 samples), robust and compatible with conventional flow cytometry instruments for high-throughput EV analysis. With the method, we successfully analyzed a panel of pancreatic cancer biomarkers in EVs from plasma samples of pancreatic cancer patients. The assay is readily translatable to other biomarkers or cancer types and can be run with standard materials on conventional flow cytometers, making it highly flexible and adaptable to diverse research and clinical needs.

6004 Background: Mirvetuximab soravtansine (MIRV) is an ADC comprising a FRα-binding antibody, cleavable linker, and the maytansinoid DM4, a potent tubulin-targeting agent. As part of the Phase 1b FORWARD II trial (NCT02606305), the combination of MIRV with bevacizumab (BEV) was evaluated in pts with FRα-positive (medium/high expression; ≥ 50%/ ≥75% of cells with PS2+ staining intensity), platinum agnostic ovarian cancer, defined as pts with either platinum resistant (PR) (recurrence within 6 months after last platinum dose) or platinum sensitive (PS) responded to the last platinum therapy received before study entry and did not progress within 6 months) disease for whom a non-platinum based doublet would be appropriate. Methods: Pts received MIRV (6 mg/kg; adjusted ideal body weight) and BEV (15 mg/kg) on Day 1 of a 21-day cycle. Responses were assessed by investigator according to RECIST 1.1 and adverse events (AEs) evaluated by CTCAE v4.03. Results: In total, 60 pts received the combination, with a median age of 60 years, and a median of 2 prior lines of systemic therapy (range 1-4). Platinum status was determined for 56 pts, with 30 (50%) considered PR and 26 (43%) considered PS; platinum status data were incomplete for 4 pts. The most common treatment related AEs (percent all grade/grade 3+) were diarrhea (65/2), blurred vision (62/3), nausea (55/0), and fatigue (55/5). The most common treatment related grade 3+ AEs were hypertension and neutropenia, (10% each); all other grade 3+ events occurred in ≤ 5% of pts. Serious AEs regardless of relationship to study drug were infrequent, with the most common events being small intestinal obstruction in 3 pts, 5% (grade 3) and pneumonitis in 3 pts, 5% (2 grade 1; 1 grade 2). Objective responses were seen in 26 pts for a confirmed overall response rate (ORR) of 43% (95% CI, 31, 57). In a subset analysis of pts with high FRα expressing tumors (n = 33), the confirmed ORR was 61% (95% CI, 42, 77), with an ORR of at least 50% in each of the PR and PS subsets. With a median follow-up of 5.5 months, the duration of response and progression free survival data are immature. Conclusions: The combination of MIRV with BEV demonstrates an encouraging ORR with a favorable tolerability profile in pts with recurrent ovarian cancer regardless of platinum sensitivity, particularly in those with tumors that express high levels of FRα. Clinical trial information: NCT02606305.

Background Intraperitoneal (IP) chemotherapy can improve outcomes for women with optimally cytoreduced epithelial ovarian cancer but toxicities are a concern. We conducted 2 phase 2 trials of an IV/IP regimen using carboplatin and paclitaxel without (Trial A) and with bevacizumab (Trial B). Methods Both trials consisted of carboplatin AUC 6 day 1, and paclitaxel 60 mg/m2 on days 1,8, 15 of a 21-day cycle; in Trial B, patients received IV bevacizumab 15 mg/kg every cycle starting cycle 2. Chemotherapy was administered IV for cycle 1 and then IP for all subsequent cycles. Primary objectives included safety and tolerability, pathologic CR rate (Trial A), and the rate of completion of IP cycles of therapy (Trial B). Progression-free (PFS), overall survival (OS), and pharmacokinetic analysis were secondary endpoints. Results 81 patients were treated on both trials (n = 40 and 41 in trials A and B, respectively). Median age for trials A and B was 59 (range, 36–76) and 55 (range, 19–69) years, respectively. 68% and 85% of patients, respectively for A and B, completed at least 4 cycles of treatment in both trials. Treatment with bevacizumab resulted in higher rates of grade 3 fatigue (37 versus 33%) and grade 3–4 diarrhea (22 versus 8%). Median PFS was 23.5 (95%CI 16.2–35.3) and 25 (95%CI 16.4–42.7) months, respectively; median OS was 68 (95%CI 49.5-NR) and 79.7 (95%CI 59.0–79.7) months, respectively for Trial A and B. Conclusions Weekly administered IP carboplatin and IP paclitaxel is tolerable and safe with similar activity with and without concommittant bevacizumab in these 2 trials.

Lens-free digital in-line holography (LDIH) is a promising microscopic tool that overcomes several drawbacks (e.g., limited field of view) of traditional lens-based microcopy. However, extensive computation is required to reconstruct object images from the complex diffraction patterns produced by LDIH. This limits LDIH utility for point-of-care applications, particularly in resource limited settings. We describe a deep transfer learning (DTL) based approach to process LDIH images in the context of cellular analyses. Specifically, we captured holograms of cells labeled with molecular-specific microbeads and trained neural networks to classify these holograms without reconstruction. Using raw holograms as input, the trained networks were able to classify individual cells according to the number of cell-bound microbeads. The DTL-based approach including a VGG19 pretrained network showed robust performance with experimental data. Combined with the developed DTL approach, LDIH could be realized as a low-cost, portable tool for point-of-care diagnostics.

Low-field, mobile NMR systems are increasingly used across diverse fields, including medical diagnostics, food quality control, and forensics. The throughput and functionality of these systems, however, are limited due to their conventional single-channel detection: one NMR probe exclusively uses an NMR console at any given time. Under this design, multi-channel detection could only be accomplished by either serially accessing individual probes or stacking up multiple copies of NMR electronics; this approach still retains limitations such as long assay times and increased system complexity. Here we present a new scalable architecture, HERMES (hetero-nuclear resonance multichannel electronic system), for versatile, high-throughput NMR analyses. HERMES exploits the concept of software-defined radio by virtualizing NMR electronics in the digital domain. This strategy i) creates multiple NMR consoles without adding extra hardware; ii) acquires signals from multiple NMR channels in parallel; and iii) operates in wide frequency ranges. All of these functions could be realized on-demand in a single compact device. We interfaced HERMES with an array of NMR probes; the combined system simultaneously measured NMR relaxation from multiple samples and resolved spectra of hetero-nuclear spins ((1)H, (19)F, (13)C). For potential diagnostic uses, we applied the system to detect dengue fever and molecularly profile cancer cells through multi-channel protein assays. HERMES holds promise as a powerful analytical tool that enables rapid, reconfigurable, and parallel detection.

The global burden of cancer, severe diagnostic bottlenecks in underserved regions, and underfunded health care systems are fueling the need for inexpensive, rapid, and treatment-informative diagnostics. On the basis of advances in computational optics and deep learning, we have developed a low-cost digital system, termed AIDA (artificial intelligence diffraction analysis), for breast cancer diagnosis of fine needle aspirates. Here, we show high accuracy (>90%) in (i) recognizing cells directly from diffraction patterns and (ii) classifying breast cancer types using deep-learning-based analysis of sample aspirates. The image algorithm is fast, enabling cellular analyses at high throughput (∼3 s per 1000 cells), and the unsupervised processing allows use by lower skill health care workers. AIDA can perform quantitative molecular profiling on individual cells, revealing intratumor molecular heterogeneity, and has the potential to improve cancer diagnosis and treatment. The system could be further developed for other cancers and thus find widespread use in global health.

Sepsis is an often fatal condition that arises when the immune response to an infection causes widespread systemic organ injury. A critical unmet need in combating sepsis is the lack of accurate early biomarkers that produce actionable results in busy clinical settings. Here, we report the development of a point-of-care platform for rapid sepsis detection. Termed IBS (integrated biosensor for sepsis), our approach leverages (i) the pathophysiological role of cytokine interleukin-3 (IL-3) in early sepsis and (ii) a hybrid magneto-electrochemical sensor for IL-3 detection. The developed platform produces test results within 1 h from native blood samples and detects IL-3 at a sensitivity of10 pg/mL; this performance is5-times faster and10-times more sensitive than conventional enzyme-linked immunoadsorbent assays, the current gold standard. Using clinical samples, we show that elevated plasma IL-3 levels are associated with high organ failure rate and thus greater risk of mortality, confirming the potential of IL-3 as a sepsis diagnostic biomarker. With further system development ( e. g., full automation, data security measures) and rigorous validation studies, the compact and fast IBS could be a practical clinical tool for timely diagnosis and proactive treatment of sepsis.