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The management of newly diagnosed primary central nervous system lymphoma (PCNSL) includes administration of high-dose methotrexate based regimens followed by consolidation therapy to minimize the risk of relapse. However, the best consolidation strategy (autologous hematopoietic cell transplant [auto-HCT] vs. whole-brain radiotherapy [WBRT]) is controversial. Hence, we performed a systematic review and meta-analysis of all randomized controlled trials that compared auto-HCT versus WBRT consolidation for patients with PCNSL after first-line treatment.The primary outcome was overall survival (OS), while the secondary outcomes included progression-free survival (PFS), response rates (overall response rate [ORR] and complete remission [CR]), relapse rate, treatment-related mortality (TRM), and neuropsychological adverse events. We performed a pooled analysis of the single-arm studies that incorporated auto-HCT or WBRT consolidation and evaluated neurocognitive outcomes. Only two studies met the inclusion criteria (n = 240). There was no significant difference in OS (HR = 1.50; 95% CI = 0.95-2.36), PFS (HR = 0.99; 95% CI = 0.44-2.22), ORR (RR = 1.48; 95% CI = 0.90-2.44), CR rate (RR = 1.21; 95% CI = 0.90-1.63), relapse rate (RR = 0.46; 95% CI = 0.05-4.28), and TRM (RR = 5.67; 95% CI = 1.01-31.91). The neuropsychological tests to assess neurocognitive domains were different and inconsistently reported in the two studies and therefore we were unable to perform a meta-analysis but provide a descriptive assessment. Both the studies showed a significant decline in the attention/executive function (based on the trail making test A and trail making test B) in those receiving WBRT compared to auto-HCT. We found 9 single-arm phase II studies that reported data on outcomes associated with either auto-HCT (5 studies) or WBRT (4 studies) consolidation. Of these, two studies (n = 43) reported data on neurocognitive decline following auto-HCT consolidation. Pooled proportion of patients with neurocognitive decline in these studies was 6% (95% CI, 0%-17%) for those receiving auto-HCT and there was no heterogeneity between studies (I

Progression of disease within 24 months (POD24) from diagnosis in marginal zone lymphoma (MZL) was shown to portend poor outcomes in prior studies. However, many patients with MZL do not require immediate therapy, and the time from diagnosis-to-treatment interval can be highly variable with no universal criteria to initiate systemic therapy. Hence, we sought to evaluate the prognostic relevance of early relapse or progression within 24 months from systemic therapy initiation in a large US cohort. The primary objective was to evaluate the overall survival (OS) in the two groups. The secondary objective included the evaluation of factors predictive of POD24 and the assessment of cumulative incidence of histologic transformation (HT) in POD24 versus non-POD24 groups. The study included 524 patients with 143 (27%) in POD24 and 381 (73%) in non-POD24 groups. Patients with POD24 had inferior OS compared to those without POD24, regardless of the type of systemic therapy received (rituximab monotherapy or immunochemotherapy) at diagnosis. After adjusting for factors associated with inferior OS in the univariate Cox model, POD24 remained associated with significantly inferior OS (HR = 2.50, 95% CI = 1.53–4.09, p = 0.0003) in multivariable analysis. The presence of monoclonal protein at diagnosis and those who received first-line rituximab monotherapy had higher odds of POD24 on logistic regression analysis. Patients with POD24 had a significantly higher risk for HT compared to those without POD24. POD24 in MZL might be associated with adverse biology and could be used as an additional information point in clinical trials and investigated as a marker for worse prognosis.

Most patients receiving CAR T-cell therapy (CAR-T) for aggressive B-cell lymphoma (B-NHL) will not experience a durable remission. There are several novel agents approved for the treatment relapsed, refractory aggressive B-NHL; however, it remains unclear how to sequence these therapies pre- and post-CAR-T (peri-CAR-T). We conducted a multicenter retrospective analysis for the purpose of describing peri-CAR-T practice patterns and survival predictors for patients receiving CD19-directed CAR-T. Patients (n=514) from thirteen centers treated with CAR-T for aggressive B-NHL between 2015-2021 were included. Clinical characteristics, CAR-T outcomes and treatment regimens administered pre- and post-CAR-T were collected. Survival curves were constructed using Kaplan Meier method. Multivariate Cox regression was used to determine the impact of variables on survival outcomes. For all patients receiving CAR-T, a greater number lines of therapy prior to CAR-T apheresis and receipt of bridging therapy were predictive of inferior progression-free survival (PFS) and overall survival (OS). From time of CAR-T infusion, median PFS and OS were 7.6 and 25.6 months (n=514). From time of progression post-CAR-T (n=254), median OS was 5.5 months. The median PFS of treatments given in the first-line post-CAR-T failure (n=167) was just 2.8 months. Patients with refractory disease at day 30 had inferior OS and were less likely to receive subsequent treatment(s) compared to other patients with CAR-T failure. AlloHCT for select patients at any time following CAR-T failure (n=16) led to durable responses in over half at one-year. These data provide a benchmark for future clinical trials in patients with progression post-CAR-T, an unmet clinical need.

Passive cavitation mapping (PCM) techniques typically utilize a time-exposure acoustic (TEA) approach, where the received radio-frequency data is beamformed, squared, and integrated over time. Such PCM-TEA cavitation maps typically suffer from long tail artifacts and poor axial resolution with pulse-echo diagnostic arrays. Here, we utilize a recently developed PCM technique based on cavitation source localization (CSL), which fits a hyperbolic function to the received cavitation wavefront. A filtering method based on the root-mean-squared error (rmse) of the hyperbolic fit is utilized to filter out spurious signals. We apply a wavefront correction technique to the signals with poor fit quality to recover additional cavitation signals and improve cavitation localization. Validation of the PCM-CSL technique with rmse filtering and wavefront correction was conducted in experiments with a tissue-mimicking flow phantom and an in vivo mouse model of cancer. It is shown that the quality of the hyperbolic fit, necessary for the PCM-CSL, requires an rmse

Ibrutinib is effective in the treatment of relapsed/refractory (R/R) marginal zone lymphoma (MZL) with an overall response rate (ORR) of 48%. However, factors associated with response (or lack thereof) to ibrutinib in R/R MZL in clinical practice are largely unknown. To answer this question, we performed a multicenter (25 US centers) cohort study and divided the study population into three groups: “ibrutinib responders”—patients who achieved complete or partial response (CR/PR) to ibrutinib; “stable disease (SD)”; and “primary progressors (PP)”—patients with progression of disease as their best response to ibrutinib. One hundred and nineteen patients met the eligibility criteria with 58%/17% ORR/CR, 29% with SD, and 13% with PP. The median PFS and OS were 29 and 71.4 months, respectively, with no difference in PFS or OS based on the ibrutinib line of therapy or type of therapy before ibrutinib. Patients with complex cytogenetics had an inferior PFS (HR = 3.08, 95% CI 1.23–7.67, p = 0.02), while those with both complex cytogenetics (HR = 3.00, 95% CI 1.03–8.68, p = 0.04) and PP (HR = 13.94, 95% CI 5.17–37.62, p p = 0.03). In this largest study to date evaluating outcomes of R/R MZL treated with ibrutinib, we show that patients with primary refractory disease and those with PP on ibrutinib are very high-risk subsets and need to be prioritized for experimental therapies.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma; 40% of patients relapse after a complete response or are refractory to therapy. To survive, the activated B-cell (ABC) subtype of DLBCL relies upon B-cell receptor signaling, which can be modulated by the activity of Bruton tyrosine kinase (BTK). Targeting BTK with ibrutinib, an inhibitor, provides a therapeutic approach for this subtype of DLBCL. However, non-Hodgkin lymphoma is often resistant to ibrutinib or acquires resistance soon after exposure. We explored how this resistance develops. We generated 3 isogenic ibrutinib-resistant DLBCL cell lines and investigated the deregulated pathways known to be associated with tumorigenic properties. Reduced levels of BTK and enhanced phosphatidylinositol 3-kinase (PI3K)/AKT signaling were hallmarks of these ibrutinib-resistant cells. Upregulation of PI3K-β expression was demonstrated to drive resistance in ibrutinib-resistant cells, and resistance was reversed by the blocking activity of PI3K-β/δ. Treatment with the selective PI3K-β/δ dual inhibitor KA2237 reduced both tumorigenic properties and survival-based PI3K/AKT/mTOR signaling of these ibrutinib-resistant cells. In addition, combining KA2237 with currently available chemotherapeutic agents synergistically inhibited metabolic growth. This study elucidates the compensatory upregulated PI3K/AKT axis that emerges in ibrutinib-resistant cells.

Ultrasound molecular imaging has been developed in the past two decades with the goal of non-invasively imaging disease phenotypes on a cellular level not depicted on anatomic imaging. Such techniques already play a role in pre-clinical research for the assessment of disease mechanisms and drug effects, and are thought to in the future contribute to earlier diagnosis of disease, assessment of therapeutic effects and patient-tailored therapy in the clinical field. In this review, we first describe the chemical composition and structure as well as the in vivo behavior of the ultrasound contrast agents that have been developed for molecular imaging. We then discuss the strategies that are used for targeting of contrast agents to specific cellular targets and protocols used for imaging. Next we describe pre-clinical data on imaging of thrombosis, atherosclerosis and microvascular inflammation and in oncology, including the pathophysiological principles underlying the selection of targets in each area. Where applicable, we also discuss efforts that are currently underway for translation of this technique into the clinical arena.

Beyond the emerging field of oncological ultrasound molecular imaging, the recent significant advancements in ultrasound and contrast agent technology have paved the way for therapeutic ultrasound mediated microbubble oscillation and has shown that this approach is capable of increasing the permeability of microvessel walls while also initiating enhanced extravasation and drug delivery into target tissues. In addition, a large number of preclinical studies have demonstrated that ultrasound alone or combined with microbubbles can efficiently increase cell membrane permeability resulting in enhanced tissue distribution and intracellular drug delivery of molecules, nanoparticles, and other therapeutic agents. The mechanism behind the enhanced permeability is the temporary creation of pores in cell membranes through a phenomenon called sonoporation by high-intensity ultrasound and microbubbles or cavitation agents. At low ultrasound intensities (0.3–3 W/cm2), sonoporation may be caused by microbubbles oscillating in a stable motion, also known as stable cavitation. In contrast, at higher ultrasound intensities (greater than 3 W/cm2), sonoporation usually occurs through inertial cavitation that accompanies explosive growth and collapse of the microbubbles. Sonoporation has been shown to be a highly effective method to improve drug uptake through microbubble potentiated enhancement of microvascular permeability. In this review, the therapeutic strategy of using ultrasound for improved drug delivery are summarized with the special focus on cancer therapy. Additionally, we discuss the progress, challenges, and future of ultrasound-mediated drug delivery towards clinical translation.

Purpose: To engineer a dual human and murine Thy1-binding single-chain-antibody ligand (Thy1-scFv) for contrast microbubble–enhanced ultrasound molecular imaging of pancreatic ductal adenocarcinoma (PDAC). Experimental Design: Thy1-scFv were engineered using yeast-surface-display techniques. Binding to soluble human and murine Thy1 and to Thy1-expressing cells was assessed by flow cytometry. Thy1-scFv was then attached to gas-filled microbubbles to create MBThy1-scFv. Thy1 binding of MBThy1-scFv to Thy1-expressing cells was evaluated under flow shear stress conditions in flow-chamber experiments. MBscFv-scrambled and MBNon-targeted were used as negative controls. All microbubble types were tested in both orthotopic human PDAC xenografts and transgenic PDAC mice in vivo. Results: Thy1-scFv had a KD of 3.4 ± 0.36 nmol/L for human and 9.2 ± 1.7 nmol/L for murine Thy1 and showed binding to both soluble and cellularly expressed Thy1. MBThy1-scFv was attached to Thy1 with high affinity compared with negative control microbubbles (P < 0.01) as assessed by flow cytometry. Similarly, flow-chamber studies showed significantly (P < 0.01) higher binding of MBThy1-scFv (3.0 ± 0.81 MB/cell) to Thy1-expressing cells than MBscFv-scrambled (0.57 ± 0.53) and MBNon-targeted (0.43 ± 0.53). In vivo ultrasound molecular imaging using MBThy1-scFv demonstrated significantly higher signal (P < 0.01) in both orthotopic (5.32 ± 1.59 a.u.) and transgenic PDAC (5.68 ± 2.5 a.u.) mice compared with chronic pancreatitis (0.84 ± 0.6 a.u.) and normal pancreas (0.67 ± 0.71 a.u.). Ex vivo immunofluorescence confirmed significantly (P < 0.01) increased Thy1 expression in PDAC compared with chronic pancreatitis and normal pancreas tissue. Conclusions: A dual human and murine Thy1-binding scFv was designed to generate contrast microbubbles to allow PDAC detection with ultrasound. Clin Cancer Res; 24(7); 1574–85. ©2018 AACR.

Ultrasound molecular imaging (USMI) is accomplished by detecting microbubble (MB) contrast agents that have bound to specific biomarkers, and can be used for a variety of imaging applications, such as the early detection of cancer. USMI has been widely utilized in preclinical imaging in mice; however, USMI in humans can be challenging because of the low concentration of bound MBs and the signal degradation caused by the presence of heterogenous soft tissue between the transducer and the lesion. Short-lag spatial coherence (SLSC) beamforming has been proposed as a robust technique that is less affected by poor signal quality than standard delay-and-sum (DAS) beamforming. In this paper, USMI performance was assessed using contrast-enhanced ultrasound imaging combined with DAS (conventional CEUS) and with SLSC (SLSC-CEUS). Each method was characterized by flow channel phantom experiments. In a USMI-mimicking phantom, SLSC-CEUS was found to be more robust to high levels of additive thermal noise than DAS, with a 6dB SNR improvement when the thermal noise level was +6dB or higher. However, SLSC-CEUS was also found to be insensitive to increases in MB concentration, making it a poor choice for perfusion imaging. USMI performance was also measured in vivo using VEGFR2-targeted MBs in mice with subcutaneous human hepatocellular carcinoma tumors, with clinical imaging conditions mimicked using a porcine tissue layer between the tumor and the transducer. SLSC-CEUS improved the SNR in each of ten tumors by an average of 41%, corresponding to 3.0dB SNR. These results indicate that the SLSC beamformer is well-suited for USMI applications because of its high sensitivity and robust properties under challenging imaging conditions.

Introduction The treatment of aggressive B-cell NHL has evolved rapidly over the last 5 years, owing to the FDA approval of 3 CD19 CAR T-cell constructs (CARTs) along with other novel targeted therapies. Real world practice data suggest that CARTs have been successfully administered in populations typically excluded from clinical trials. However, data on how to best utilize novel targeted agents as a pathway to CARTs and feasibility of CARTs in rare histologies remains limited. This retrospective multicenter study describes patient (pt) selection and practice patterns in pts treated with CD19 CARTs and provides insight on feasibility of CARTs in special populations. Methods Adult pts with R/R aggressive B-cell NHL treated with CD19 CARTs between 2015- 2020 across 12 US academic centers were identified. Data on demographic and clinical characteristics, disease and toxicity outcomes were collected. Univariate analyses (UVA) were performed to determine impact of demographic/clinical variables on survival. Survival curves were calculated using Kaplan-Meier method. Subgroup analysis was performed for pts with secondary central nervous system lymphoma (sCNSL). Results Clinical and demographic features were recorded from 400 pts (Table 1). Median age was 59 years (range 18-84). Of 271 pts with immunohistochemistry data, 79 (29%) had double-expressor lymphoma. Of 178 pts with FISH data captured, 62 (35%) had double/triple-hit lymphoma. Most common histological subtypes included 271 (68%) pts with de novo DLBCL, 81 (20%) with transformed FL, 13 (3%) with Richter's syndrome, and 8 (2%) with PMBCL. Rare histologies included 7 (2%) with transformed MZL, 5 (1%) with PTLD and 2 (0.5%) with grey zone lymphoma. 24 (6%) pts had sCNSL at time of CART apheresis. Two (0.5%) pts were HIV-positive. Median number of lines of therapy prior to CART was 2 (range 1-8); 182 (46%) pts received ≥ 3 lines. 114 (28%) pts previously had an autologous stem cell transplant. Targeted therapies used as salvage regimens at any point prior to CART are listed in Table 2: commonly used salvage targeted therapies included lenalidomide based therapy (imids, n=37, 9%), BTK inhibitors (BTKis, n=30, 8%), checkpoint inhibitors (CBIs, n=17, 4%) and polatuzumab-containing regimens (n=10, 3%). 2 (1%) pts received loncastuximab, and no pts received tafasitamab. Six (1.5%) pts proceeded to CART despite complete response to most recent pre-CART therapy. 191 (48%) pts received bridging between apheresis and CART infusion, choice of bridging noted in Table 2: the majority received chemotherapy (n=103, 54%); 28 (15%) received radiation (XRT); 25 (13%), 24 (13%) and 18 (9%) pts received imids, polatuzumab-containing regimens, or BTKis, respectively. With median follow-up of 22.4 months (mo) for the overall group, median (m) PFS was 11 mo (n=363); mOS, was 27 mo (n=397; Fig 1). Pts with sCNSL had a mPFS and mOS of 2 and 4 mo, respectively (Fig 1). On UVA, factors predicting poorer PFS and OS in the overall group included ≥3 pre-CART lines (p For outcomes according to bridging regimens: mPFS after CART for most commonly used systemic bridging therapies was 86 days (d) for platinum-based chemotherapy, 77 d for imids, 90 d for BTKis, 98 d for polatuzumab-bendamustine/rituximab, and 274 d for XRT. Median PFS for XRT bridging (274 d) was statistically better when compared to mPFS for listed systemic therapies combined (p Conclusion Survival outcomes with CARTs in our data set are consistent with those reported in clinical trial settings. CARTs are utilized in real world practice in rare subsets of aggressive R/R B-cell NHL not routinely included in clinical trials. Despite early data suggesting pts with sCNSL benefit from CART, our data suggest outcomes with CART are dismal in this group. Targeted therapies including imids, polatuzumab, BTKis and CBIs are feasible choices for salvage and/or bridging as a pathway to CARTs. Bridging with XRT resulted in improved mPFS post CART as compared to bridging with systemic therapies and suggests differences in pt selection for each with systemic therapies likely favored in those with more widespread disease burden. Minimal use of CD19-targeted agents pre-CART is attributed to later approval of these agents and concern for potential loss of CD19 antigen leading to CART resistance. Figure 1 Figure 1. Disclosures Cohen: Janssen, Adicet, Astra Zeneca, Genentech, Aptitude Health, Cellectar, Kite/Gilead, Loxo, BeiGene, Adaptive: Consultancy; Genentech, BMS/Celgene, LAM, BioINvent, LOXO, Astra Zeneca, Novartis, M2Gen, Takeda: Research Funding. Shouse: Kite Pharma: Speakers Bureau; Beigene: Honoraria. Hess: ADC Therapeutics: Consultancy; BMS: Speakers Bureau. Stephens: Beigene: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Epizyme: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Adaptive: Membership on an entity's Board of Directors or advisory committees; Arqule: Research Funding; Mingsight: Research Funding; JUNO: Research Funding; Celgene: Consultancy; CSL Behring: Consultancy; AstraZeneca: Consultancy; Abbvie: Consultancy; Innate Pharma: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ma: Loxo: Research Funding; AstraZeneca: Honoraria, Research Funding, Speakers Bureau; Juno: Research Funding; Beigene: Research Funding, Speakers Bureau; Abbvie: Honoraria, Research Funding; Janssen: Research Funding, Speakers Bureau; TG Therapeutics: Research Funding; Pharmacyclics: Research Funding, Speakers Bureau. Winter: BMS: Other: Husband: Data and Safety Monitoring Board; Actinium Pharma: Consultancy; Janssen: Other: Husband: Consultancy; Gilead: Other: Husband: Consultancy; Agios: Other: Husband: Consultancy; Epizyme: Other: Husband: Data and Safety Monitoring Board; Merck: Consultancy, Honoraria, Research Funding; Novartis: Other: Husband: Consultancy, Data and Safety Monitoring Board; Ariad/Takeda: Other: Husband: Data and Safety Monitoring Board; Karyopharm (Curio Science): Honoraria. Gordon: Zylem Biosciences: Patents & Royalties: Patents, No royalties; Bristol Myers Squibb: Honoraria, Research Funding. Danilov: Astra Zeneca: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy, Research Funding; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Rigel Pharm: Honoraria; Bayer Oncology: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding. Shah: Umoja: Consultancy; Legend: Consultancy; Kite: Consultancy; Miltenyi Biotec: Consultancy, Honoraria, Research Funding; Lily: Consultancy, Honoraria, Research Funding; Epizyme: Consultancy; Incyte: Consultancy. Barta: Seagen: Honoraria; Daiichi Sankyo: Honoraria; Acrotech: Honoraria; Kyowa Kirin: Honoraria. Epperla: Genzyme: Honoraria; Karyopharm: Other: Ad Board; Beigene: Speakers Bureau; Verastem: Speakers Bureau. Torka: TG Therapeutics: Membership on an entity's Board of Directors or advisory committees. Karmali: Epizyme: Consultancy; BeiGene: Consultancy, Speakers Bureau; EUSA: Consultancy; Roche: Consultancy; AstraZeneca: Speakers Bureau; Morphosys: Consultancy, Speakers Bureau; Karyopharm: Consultancy; Takeda: Research Funding; BMS/Celgene/Juno: Consultancy, Research Funding; Janssen/Pharmacyclics: Consultancy; Genentech: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding, Speakers Bureau.

Background Primary bone diffuse large B cell lymphoma (DLBCL) is a variant of extranodal non-Hodgkin lymphoma (NHL) that is relatively rare, accounting for 3-15% of extranodal NHL and less than 1% of all NHL. Patients often present with pain and swelling or pathologic fracture of the affected area of the skeleton, and B symptoms are often less prevalent. Many cases are limited stage at presentation. Patients are usually treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) or similar induction therapy. Based on older studies showing improved outcomes associated with the addition of radiation (many completed prior to the rituximab era), patients often still receive combined-modality therapy today. However, it remains unknown whether consolidative radiation therapy (RT) confers additional benefit following rituximab-based chemoimmunotherapy (CIT) induction. We sought to address this question in a multicenter retrospective study. Methods We conducted a retrospective analysis of outcomes in a modern cohort of patients who underwent treatment for primary bone DLBCL using chemotherapy regimens in the rituximab era either with or without consolidative RT. Data was collected from 13 academic medical centers in the U.S., with patients treated between 2005 and 2019. Patients were identified using each institution's hematologic malignancy registries. Analysis was limited to patients with primary bone DLBCL, with stage I-II disease (primary site +/- locoregional lymph nodes) that could be encompassed in a radiation field. Patients who received CIT alone or CIT followed by RT were included. Treatment selection was at the treating physician's discretion. Electronic medical records were reviewed for: demographics, chemotherapy regimens, radiation treatments, and PET scan data. Local IRB approval was obtained at sites. All data was distributed in a de-identified fashion. The primary outcomes were overall survival (OS), and relapse-free survival (RFS). Secondary outcomes included: response after induction CIT induction and the need for additional therapies. Chi-square test analysis was used to compare categorical variables and the Wilcoxon rank-sum test was used for continuous and ordinal measures. Survival curves were estimated using the Kaplan-Meier method and compared between groups via the log-rank test. Multi-variable analysis (MVA) for OS and RFS was performed using RT vs no RT, PET status post CIT (negative vs positive) and rituximab in induction (Y/N) as variables. Results A total of 127 patients were included: 91 who received CIT and radiation (RT group), and 36 who received CIT alone (no RT group). The median age of patients at diagnosis in the RT group and no RT group was 58.5 and 57.0, respectively. For CIT, the majority of patients received R-CHOP (84%). Despite focusing on 2005-2019, a small percentage of patients (5 - 14%) in each group (RT and no RT) did not receive rituximab with their induction therapy. The OS at 10 years was 74.5% in the RT group and 86.5% in the no RT group (p = 0.29). The RFS at 10 years was 70.9% in the RT group and 78.2% in the no RT group (p = 0.85). Among the 91 patients who received RT, 67 (77.0%) received a dose of 36 Gy or higher. There was no difference in OS (p = 0.63) or RFS (p = 0.90) in patients who received > 36 Gy vs < 36 Gy of radiation. On MVA for OS, RT was not associated with improved OS, although rituximab as part of induction was (p = 0.048). On MVA for RFS, neither RT nor rituximab were associated with improved RFS. For rituximab the trend was in the direction of rituximab benefit (p = 0.11). Conclusion Our results demonstrate that, in the rituximab era, patients with limited stage primary bone DLBCL have excellent outcomes overall. In this study, neither RFS or OS appeared to be improved with the addition of consolidative RT. Rituximab, however, was associated with improved OS on MVA. Outcomes do not appear to differ based on the dose of RT (> 36 Gy vs < 36 Gy). Our data suggests that RT may have less benefit for primary bone DLBCL in the rituximab era, although it is possible that a subset of patients may benefit from consolidative radiation therapy. A larger data set would likely be needed to evaluate this. We plan to present data regarding PET responses at the meeting. Figure 1 Figure 1. Disclosures Lunning: Acrotech: Consultancy; AstraZeneca: Consultancy; Myeloid Therapeutics: Consultancy; Janssen: Consultancy; Morphosys: Consultancy; Novartis: Consultancy; Karyopharm: Consultancy; Kyowa Kirin: Consultancy; Verastem: Consultancy; Kite, a Gilead Company: Consultancy; Celgene, a Bristol Myers Squibb Co.: Consultancy; Spectrum: Consultancy; Daiichi-Sankyo: Consultancy; Beigene: Consultancy; Legend: Consultancy; ADC Therapeutics: Consultancy; TG Therapeutics: Consultancy; AbbVie: Consultancy. Cashen: Secura Bio, ADC Therapeutics: Consultancy. Bartlett: Autolus: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Forty Seven: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Kite, a Gilead Company: Research Funding; Merck: Research Funding; Millennium: Research Funding; Pharmacyclics: Research Funding; Seagen: Consultancy, Research Funding; Roche/Genentech: Consultancy; ADC Therapeutics: Consultancy, Research Funding. Caimi: XaTek: Patents & Royalties: Royalties from patents (wife); Amgen Therapeutics.: Consultancy; ADC Theraputics: Consultancy, Research Funding; Genentech: Research Funding; TG Therapeutics: Honoraria; Seattle Genetics: Consultancy; Verastem: Consultancy; Kite Pharmaceuticals: Consultancy. Rodgers: MJH Lifesciences: Consultancy. Barr: Morphosys: Consultancy; Gilead: Consultancy; Genentech: Consultancy; TG Therapeutics: Consultancy; Janssen: Consultancy; Beigene: Consultancy; Bristol Meyers Squibb: Consultancy; Seattle Genetics: Consultancy; AstraZeneca: Consultancy; Abbvie/Pharmacyclics: Consultancy. Epperla: Verastem: Speakers Bureau; Beigene: Speakers Bureau; Karyopharm: Other: Ad Board; Genzyme: Honoraria. Hill: AstraZenica: Consultancy, Honoraria; Celgene (BMS): Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Other: Travel Support, Research Funding; Pfizer: Consultancy, Honoraria; Gentenech: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Epizyme: Consultancy, Honoraria; Incyte/Morphysis: Consultancy, Honoraria, Research Funding; Beigene: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding. Karmali: BMS/Celgene/Juno: Consultancy, Research Funding; Epizyme: Consultancy; Karyopharm: Consultancy; Janssen/Pharmacyclics: Consultancy; Morphosys: Consultancy, Speakers Bureau; AstraZeneca: Speakers Bureau; BeiGene: Consultancy, Speakers Bureau; EUSA: Consultancy; Takeda: Research Funding; Genentech: Consultancy; Roche: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding, Speakers Bureau. Portell: Acerta/AstraZeneca: Research Funding; Xencor: Research Funding; Abbvie: Research Funding; Aptitude Health: Honoraria; Morphosys: Honoraria; Targeted Oncology: Honoraria; SeaGen: Research Funding; BeiGene: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Pharmacyclics: Honoraria; Kite: Honoraria, Research Funding; TG Therapeutics: Honoraria, Research Funding; Genentech: Research Funding; VelosBio: Research Funding. Fenske: Pharmacyclics: Consultancy; AstraZeneca: Speakers Bureau; Servier Pharmaceuticals: Consultancy; MorphoSys: Consultancy; Adaptive Biotechnologies: Consultancy; KaryoPharm: Consultancy; CSL Therapeutics: Consultancy; TG Therapeutics: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Sanofi: Speakers Bureau; Seattle Genetics: Speakers Bureau; Biogen: Consultancy; ADC Therapeutics: Consultancy; Kite (Gilead): Speakers Bureau; Beigene: Consultancy; AbbVie: Consultancy.

Background: Anti-CD19 chimeric antigen receptor T-cell therapy (CART) is a highly active therapy for relapsed/refractory (R/R) aggressive B-cell lymphoma. Nonetheless, most patients (pts) ultimately develop progressive disease (PD). There is little guidance on the optimal treatment approach(es) for these pts. We performed a multicenter retrospective analysis with a primary objective to assess treatment patterns and outcomes in pts with R/R aggressive B-cell lymphoma who develop PD after anti-CD19 CARTs. Methods: Pts with aggressive B-cell lymphoma treated with anti-CD19 CART between 2015 and 2020 across 12 US academic medical centers were included. Demographic and clinical characteristics were collected along with CART toxicities and response. Regimens administered as salvage post CART were assessed. Univariate analyses (UVA) were performed to determine impact of demographic and clinical variables on survival outcomes. All p-values were two-tailed. Survival curves were calculated using the Kaplan-Meier method. Results: A total of 400 pts received anti-CD19 CARTs and were included for analysis. For the entire cohort: median PFS and OS from time of CART infusion were 11 months [mo] and 27 mo respectively. On log-rank testing, pts who received ≥3 lines of pre-CART therapy and those with refractory disease pre-CART had significantly worse PFS (p=0.004 & 0.001) and OS (both p With median follow-up 22.4 mo, 190 pts (48%) had PD after CART; demographic and clinical variables of pts with and without PD are detailed in Table 1. Biopsy to confirm PD and assess CD19 status was done in 69 pts (36%) with CD19 negative relapse seen in 11 (16%). Of pts with PD, median PFS and OS from time of PD was 83 days (in pts who received salvage) and 174 days (for all PD pts) respectively. Pts with PD were more likely to have elevated LDH (p=0.001) and extranodal disease (p=0.003) at apheresis. For pts with PD after CART: 125 (65.5%) received further therapies. Pts were more likely to receive salvage therapies if their best response to CART was CR (p=0.026) or PR (p=0.015). Response rates of select first- and second-line therapies and PFS of first line therapies received after CART failure are detailed in figure 1. ORR and CRs were highest for polatuzumab, bendamustine, & rituximab (pola-BR; 73% & 40%), followed by BTK inhibitors (BTKi; 50% & 38%), and bispecific antibodies (bsAb) (50% & 25%). Five of 7 pts who received a BTKi had non-germinal center (GC) cell of origin (COO; 1 unknown COO). On log-rank testing, pts with elevated LDH (p=0.003) at time of apheresis and those with intermediate/high IPI (p=0.013) had inferior PFS with first salvage regimens. Median PFS was highest for pola-BR (4.5 mo, n=14), followed by bsAb (2.5 mo, n=8), lenalidomide +/- anti-CD20 antibody (1.8 mo, n=13), checkpoint inhibitors (CPI; 1.6 mo, n=10), BTKi (1.2 mo, n=8), radiation alone (1.2 mo; n=17), chemotherapy (1.1 mo, n=12), and tafasitamab + lenalidomide (0.9 mo, n=5). Median PFS for all treated pts was 1.8 mo. OS from start of first salvage regimen was highest for CPI (OS 12.4 mo, n=10), followed by pola-BR (8.9 mo, n=14), BTKi (8.8 mo, n=8), lenalidomide +/- anti-CD20 (8.7 mo, n=13), radiation alone (7.1 mo, n =17), bsAb (5.9 mo, n=8), chemotherapy (5.4 mo, n=12), and tafasitamab + lenalidomide (1.2 mo, n=5). 12 pts (6.3%) later received an allogeneic hematopoietic cell transplant (alloHCT). In alloHCT pts at last follow-up, 10 were evaluable for response: 7 had CR and 5 remain in CR. Clinical characteristics of pts who received alloHCT are detailed in table 2. Notably, median age was 59 years (41-68), 1 (8.3%) had a prior alloHCT, and 6 (50%) had prior autologous HCT. The majority had CR or PR as best response to CART (CR n=6, 50%; PR n=3, 25%), and only 1 pt (8.3%) with PD as best response to CART was salvaged with alloHCT. Conclusions: This is the largest reported analysis to date of pts with aggressive B-cell lymphoma who develop PD post-CART. The highest ORRs were with pola-BR, bsAb, and BTKi as first line of salvage. High response rates with BTKi may be attributed to non-GC COO in the majority of treated pts and perhaps a beneficial immunomodulatory effect on previously administered CARTs. AlloHCT remains a potential curative therapy for select pts with over half with durable remission; however, few ultimately received alloHCT. Despite increased use of novel therapies, survival in pts who progress after CART is still dismal warranting more effective therapies. Figure 1 Figure 1. Disclosures Epperla: Genzyme: Honoraria; Karyopharm: Other: Ad Board; Beigene: Speakers Bureau; Verastem: Speakers Bureau. Torka: TG Therapeutics: Membership on an entity's Board of Directors or advisory committees. Hess: ADC Therapeutics: Consultancy; BMS: Speakers Bureau. Cohen: Janssen, Adicet, Astra Zeneca, Genentech, Aptitude Health, Cellectar, Kite/Gilead, Loxo, BeiGene, Adaptive: Consultancy; Genentech, BMS/Celgene, LAM, BioINvent, LOXO, Astra Zeneca, Novartis, M2Gen, Takeda: Research Funding. Ma: Abbvie: Honoraria, Research Funding; Beigene: Research Funding, Speakers Bureau; Loxo: Research Funding; Juno: Research Funding; AstraZeneca: Honoraria, Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau; TG Therapeutics: Research Funding; Pharmacyclics: Research Funding, Speakers Bureau. Winter: Gilead: Other: Husband: Consultancy; Janssen: Other: Husband: Consultancy; Ariad/Takeda: Other: Husband: Data and Safety Monitoring Board; Epizyme: Other: Husband: Data and Safety Monitoring Board; Agios: Other: Husband: Consultancy; Actinium Pharma: Consultancy; BMS: Other: Husband: Data and Safety Monitoring Board; Merck: Consultancy, Honoraria, Research Funding; Novartis: Other: Husband: Consultancy, Data and Safety Monitoring Board; Karyopharm (Curio Science): Honoraria. Gordon: Zylem Biosciences: Patents & Royalties: Patents, No royalties; Bristol Myers Squibb: Honoraria, Research Funding. Danilov: Bayer Oncology: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Rigel Pharm: Honoraria; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy, Research Funding; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; SecuraBio: Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Gilead Sciences: Research Funding. Stephens: Adaptive: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Epizyme: Membership on an entity's Board of Directors or advisory committees; JUNO: Research Funding; Mingsight: Research Funding; CSL Behring: Consultancy; Novartis: Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Arqule: Research Funding; Celgene: Consultancy; Innate Pharma: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding. Shah: Miltenyi Biotec: Consultancy, Honoraria, Research Funding; Umoja: Consultancy; Incyte: Consultancy; Kite: Consultancy; Legend: Consultancy; Epizyme: Consultancy; Lily: Consultancy, Honoraria, Research Funding. Shouse: Beigene Pharmaceuticals: Honoraria; Kite Pharmaceuticals: Speakers Bureau. Barta: Acrotech: Honoraria; Daiichi Sankyo: Honoraria; Seagen: Honoraria; Kyowa Kirin: Honoraria. Karmali: Karyopharm: Consultancy; EUSA: Consultancy; Roche: Consultancy; Janssen/Pharmacyclics: Consultancy; Genentech: Consultancy; Morphosys: Consultancy, Speakers Bureau; Epizyme: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene/Juno: Consultancy, Research Funding; AstraZeneca: Speakers Bureau; Takeda: Research Funding; BeiGene: Consultancy, Speakers Bureau.

Introduction: A common initial treatment approach for PTLD includes reduction of immunosuppression (RIS) and single-agent rituximab, followed by sequential chemoimmunotherapy for non-responding patients (pts). However, in relapsed disease, there is less consensus on optimum therapy. Moreover, there are very limited data regarding the use of chimeric antigen receptor (CAR) T-cell therapy in relapsed/refractory SOT-related PTLD, despite its documented efficacy and tolerability in large B-cell lymphoma (LBCL). Methods: We conducted a multicenter, retrospective, RWE analysis of adults who had SOT-associated PTLD. We obtained baseline clinical features at diagnosis of PTLD including prior organ transplant, immunosuppression usage, international prognostic index (IPI), EBV status, treatment course, response to therapy, and clinical course with CD19 CAR-T. Post-CAR-T survival analyses were performed using Kaplan-Meier with comparison by response using log rank. Results: A total of 20 pts across 8 U.S. academic centers were included in the analysis. Pathology was monomorphic B-cell PTLD in all cases; most pts had diffuse LBCL NOS, with 3 high-grade BCL NOS cases. Prior SOT included 15 kidney transplants (75%), 3 liver (15%), 1 intestinal (5%), and 1 heart transplant (5%). Median time from SOT to PTLD diagnosis was 107 months (mos) (5-379). The median age at time of CART was 55 years (31-75 years) with 4 pts (22.2%) aged >70 years (see Table). The tumor EBV status was negative in 16 pts (80%), positive in 1 (5%), and unavailable in 3 pts (15%). The IPI score upon relapse prior to CAR-T therapy was 3-5 among 14 pts (70%) and 0-2 for the other 6 pts (30%). Most of the pts had stage III-IV disease (95%), and elevated LDH (80%) prior to CAR-T. There were also 5 pts (25%) with >1 site of extranodal involvement, and 1 pt (5%) had secondary central nervous system involvement. For initial therapy of PTLD, 4 pts (20%) received rituximab alone, 12 (60%) had R-CHOP, and 4 (20%) received dose-adjusted R-EPOCH. The median number of prior therapies prior to CAR-T was 2 (1-4); bridging therapy prior to CAR-T are listed in the Table. Immunosuppression was stopped completely prior to CAR-T infusion in 15 (75%) pts, with prednisone alone continued for 3. For CAR-T therapy, 13 pts (65%) received tisagenlecleucel and 7 pts (35%) axicabtagene ciloleucel; 18 received commercial product and 2 were treated on a clinical trial. For tolerability, 16 (80%) pts experienced cytokine release syndrome (CRS): 9 (45%) grade (G) 1, 5 (25%) G2, and 1 (5%) each for G3 and G4. The immune effector cell-associated neurotoxicity syndrome (ICANS) was observed in 14 (70%) pts: 2 (10%) G1, 5 (25%) G2, 5 (25%) G3, and 2 (10%) G4. There were 2 (10%) pts with treatment-related mortality (TRM) at 1.5 and 4.5 mos post-CAR-T (1 pneumonia/sepsis and 1 encephalopathy). The overall response rate was 65%, with 50% complete remission (CR) and 20% stable disease. 13 of 15 pts resumed immunosuppression post-CAR-T after a median of 2.2 mos (1-14). Of these pts, 8 were started on steroids; 2 on tacrolimus, 1 both steroid and tacrolimus; 1 everolimus; and 1 on sirolimus. 3 pts (15%) experienced allograft rejection after CAR-T, all of which were kidney SOTs occurring at 1, 4, and 14.6 mos after CAR-T infusion; 1 pt died due to disease progression (1.5 mos) and the 2 other pts required hemodialysis. CRS and ICANS scoring for these 3 pts were 0, 0, 2, and 1, 3, 1, respectively, and all were off immunosuppression at time of rejection. Overall, at a median follow-up of 25 mos (5-112), 13 pts (65%) had progression of disease or TRM, and 7 pts (35%) remained in remission (Figure A/B). Additionally, as highlighted in Figure C/D, all non-relapsing pts had achieved CR with CAR-T therapy (7/10 CR remain in remission), which was strongly associated with outcomes (PFS HR 0.03 (95% CI 0.00-0.29), P=0.002; and OS HR 0.06 (95% CI 0.01-0.57), P=0.014). Conclusions: This RWE provides the largest analysis to date of CD19 CAR-T therapy in relapsed/refractory SOT-related LBCL PTLD. We found that rates of CAR-T-related CRS and neurologic events appeared comparable with previous non-PTLD CAR-T data. Furthermore, 50% of SOT-PTLD pts obtained a CR, with approximately one-third of pts sustaining long-term remission, and achievement of CR was a critical factor associated with survival. Finally, with abrogation of immunosuppression prior to CAR-T, and careful resumption post-therapy, organ preservation was achieved in most pts. Figure 1 Figure 1. Disclosures Ghobadi: Wugen: Consultancy; Atara: Consultancy; Amgen: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Celgene: Consultancy. Lazaryan: Kadmon: Consultancy; Avrobio: Membership on an entity's Board of Directors or advisory committees; Humanigen: Membership on an entity's Board of Directors or advisory committees. Jacobson: Precision Biosciences: Consultancy, Honoraria, Other: Travel support; Kite, a Gilead Company: Consultancy, Honoraria, Other: Travel support; AbbVie: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Other: Travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: Travel support; Nkarta: Consultancy, Honoraria; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Other: Travel support; Lonza: Consultancy, Honoraria, Other: Travel support; Humanigen: Consultancy, Honoraria, Other: Travel support; Axis: Speakers Bureau; Clinical Care Options: Speakers Bureau. Naik: Sanofi: Other: Virtual Advisory Board Member ; Takeda: Other: Virtual Advisory Board Member ; Kite: Other: Virtual Advisory Board Member. Olszewski: TG Therapeutics: Research Funding; PrecisionBio: Research Funding; Celldex Therapeutics: Research Funding; Acrotech Pharma: Research Funding; Genentech, Inc.: Research Funding; Genmab: Research Funding. Nastoupil: TG Therapeutics: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead/Kite: Honoraria, Research Funding; ADC Therapeutics: Honoraria; Denovo Pharma: Other: DSMC; Epizyme: Honoraria, Research Funding; Bristol Myers Squibb/Celgene: Honoraria, Research Funding; Takeda: Honoraria, Other: DSMC, Research Funding; IGM Biosciences: Research Funding; Caribou Biosciences: Research Funding; Bayer: Honoraria; MorphoSys: Honoraria; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Ahmed: Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Seagen: Research Funding; Merck: Research Funding; Xencor: Research Funding.

Recent advancements in ultrasound and microbubble (USMB) mediated drug delivery technology has shown that this approach can improve spatially confined delivery of drugs and genes to target tissues while reducing systemic dose and toxicity. The mechanism behind enhanced delivery of therapeutics is sonoporation, the formation of openings in the vasculature, induced by ultrasound-triggered oscillations and destruction of microbubbles. In this review, progress and challenges of USMB mediated drug delivery are summarized, with special focus on cancer therapy.

Background: Current standard of care for most patients (pts) with relapsed/refractory (RR) classical Hodgkin lymphoma (cHL) is systemic salvage chemotherapy followed by autologous stem cell transplant (ASCT). Given the treatment-related toxicities associated with ASCT and the expanding novel therapeutic options, it is desirable to identify pts with RR disease who may be cured by less toxic treatment approaches. We aimed to assess outcomes, evaluate treatment-related toxicities, and identify baseline characteristics for pts with RR cHL who were treated with radiation-based therapy as the initial salvage approach. Methods: We conducted a multicenter, retrospective study of pts with RR cHL who did not undergo consolidative ASCT as part of their first salvage therapy and instead were treated with either RT alone or in combination with systemic therapy. Pts who received systemic therapy as first salvage but did not proceed to ASCT and then received a RT-based salvage regimen were also included. We defined relapse as recurrence of disease after achievement of complete response (CR) and primary refractory disease as failure to achieve CR in response to frontline therapy. Responses were based on treating physician assessment using Revised Response Criteria for Malignant Lymphoma (Cheson et al, JCO 2014). Kaplan Meier survival analyses were performed using STATA 15.0 software. Toxicities were recorded using NCI Common Terminology Criteria for Adverse Events (CTCAE) v.5. Results: We identified 22 pts from 5 U.S centers with RR cHL who were treated between January 2009 and August 2019 with salvage RT alone or in combination with systemic therapy without consolidative ASCT. The pts' characteristics are summarized in Table 1. Both pts with relapsed (68%) and refractory (32%) disease were included. All pts with relapsed disease (N = 15) had localized relapse (stage I or II) and 67% had relapsed > 12 months after completion of frontline therapy. Three pts who received frontline RT had relapsed outside their prior RT volumes. The most common reason for not pursuing ASCT was that the relapsed or residual disease was localized (38%). Other reasons cited included age (11%), comorbidities (11%), and pt preference (11%). Pts with relapsed disease were more likely than pts with primary refractory disease to receive RT combined with systemic therapy as a salvage (60% vs 15%, p = 0.01). Out of 13, patients who received combined systemic therapy with RT, 77% (N = 10) received brentuximab vedotin (BV) and 23% received multi-agent chemotherapy. Median duration of BV treatment was 6 cycles. Four pts received multi-agent systemic salvage chemotherapy as first salvage but did not proceed to ASCT and then went on to receive a RT containing salvage regimen Among all 22 RR cHL pts, 82% achieved complete response (CR) and 14% had progressive disease after RT containing salvage regimen (Table 2). Of those achieving CR, 77% (95% CI: 44-92) remained disease free at 5 years. The 5-year PFS for the entire cohort was 68% (95% CI: 41-85). There was no difference in the 5-year PFS among those with primary refractory compared with relapsed disease as shown in Figure 1(c2=0.02, p=0.88). Furthermore, 5-year OS was 95% (95% CI: 69-99). In terms of toxicity, there were no deaths or hospitalizations attributed to complications from salvage therapy. The most common toxicities reported were cytopenias in 32% (N = 7) and peripheral neuropathy in 27% (N = 6). Only 3 pts experienced grade 3/4 adverse events. Systemic salvage therapy was discontinued in 3 pts due to toxicity. No long-term toxicities of RT were reported, but longer follow-up is needed. Conclusions: To the best of our knowledge, this is the first study to evaluate outcomes for patients with RR cHL in the United States who received RT-based salvage regimens without consolidative ASCT. In our study population of pts with localized relapse or refractory disease, 81% of pts achieved CR after this non-transplant approach and 5-year PFS was 68%. Our data provides evidence that RT alone or in combination with systemic therapy can be an effective and potentially less toxic treatment strategy for carefully selected pts. Further evaluation, including with prospective studies and long-term follow-up, is needed to corroborate these findings as well as to examine potential late effects. Disclosures Evens: Seattle Genetics: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Epizyme: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; MorphoSys: Consultancy, Honoraria; Research To Practice: Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria; Mylteni: Consultancy, Honoraria. Portell:Xencor: Research Funding; Roche/Genentech: Consultancy, Research Funding; Infinity: Research Funding; TG Therapeutics: Research Funding; AbbVie: Research Funding; Pharmacyclics: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Bayer: Consultancy; BeiGene: Consultancy, Research Funding; Kite: Consultancy, Research Funding; Acerta/AstraZeneca: Research Funding. Epperla:Pharmacyclics: Honoraria; Verastem Oncology: Speakers Bureau. Feldman:Seattle Genetics, Inc.: Consultancy, Honoraria, Other: Travel expenses, Research Funding, Speakers Bureau; Takeda: Honoraria, Other: Travel expenses; Celgene: Honoraria, Research Funding; Kite: Honoraria, Other: Travel expenses, Speakers Bureau; BMS: Consultancy, Honoraria, Research Funding; Corvus: Research Funding; Rhizen: Research Funding; Viracta: Research Funding; Trillium: Research Funding; Bayer: Consultancy, Honoraria; Abbvie: Honoraria; Pharmacyclics: Honoraria, Other, Speakers Bureau; Amgen: Research Funding; Cell Medica: Research Funding; Eisai: Research Funding; Kyowa Kirin: Consultancy, Research Funding; Pfizer: Research Funding; Portola: Research Funding; Janssen: Speakers Bureau; AstraZeneca: Consultancy. Landsburg:Triphase: Research Funding; Seattle Genetics: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Curis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding. Dwivedy Nasta:Roche: Research Funding; Rafael Pharmaceuticals: Research Funding; Incyte: Research Funding; Debiopharm: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Atara: Research Funding; Forty Seven: Research Funding. Gerson:Loxo: Research Funding; Genentech: Consultancy; Pharmacyclics: Consultancy; Abbvie: Consultancy. Barta:Pfizer: Honoraria; Janssen: Honoraria; Seattle Genetics: Honoraria, Research Funding; Atara: Honoraria; Monsanto: Consultancy. Chong:KITE Pharma: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Tessa: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Schuster:Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria. Svoboda:Atara: Consultancy; Genmab: Consultancy; TG: Research Funding; Seattle Genetics: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Merck: Research Funding; Incyte: Research Funding; Imbrium: Consultancy; BMS: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding; Adaptive: Consultancy.

Introduction: There is no standard induction therapy in extranodal marginal zone lymphoma (EMZL); current guidelines borrow from follicular lymphoma, where bendamustine and rituximab (BR) is an accepted standard. The data on BR in EMZL is limited (Rummel MJ et al. Lancet 2013 & Salar A et al. Blood 2017), so we explored BR activity as part of an international consortium. Methods: This retrospective analysis involved 11 cancer centers from US and Italy. We included patients with EMZL treated with frontline BR (1/2008 to 12/2019). Expert pathology review was performed by each participating institution following the 2016 WHO classification. Progression-free (PFS) and overall survival (OS) were estimated by Kaplan-Meier and associations with prognostic factors were assessed by log-rank test, univariable (UVA) and multivariable (MVA) Cox regression. MVA Cox models were constructed by selecting variables significant univariately. Results: 136 patients were identified; however, 18 patients with EMZL only located in bone marrow (BM) were excluded from this analysis to prevent possible inclusion of lymphoplasmacytic lymphoma. Thus, 118 patients were included in this study. Patient characteristics comprised median age of 61.5 years (range 21 to 85 years), women: 56.8%, ECOG performance status 0-1: 86%, stage III-IV: 79.7%, no B symptoms: 81.4%, normal LDH: 75.4%, BM involvement: 25.4%, and MALT-IPI score < 2: 63.6%. Most common extranodal (EN) sites were lung (22%), gastric (13.6%), ocular (11%), soft tissue (10.2%), salivary gland (10%), and gastrointestinal non-gastric (7.6%). Most patients presented with 1 or 2 EN sites (48.3% and 30.5%, respectively). Majority of patients (83.9%) had < 4 nodal sites. Paraprotein was positive in 27 of 80 (33.7%) patients with majority harboring IgM. The median number of BR cycles was 6 (range 1 to 6). Consolidation with radiation therapy was performed in 6% of the patients. Treatment response was determined by PET/CT in 70% and CT scans in the rest. The response to treatment was as follows: CR: 96 (81.4%), PR: 13 (11%), SD: 2 (1.7%), PD: 4 (3.4%), and unknown: 3 (2.5%) patients. No differences in response rate were observed by EN location. The incidence of infectious complications was 14% including herpes zoster (25%), pneumonia (18.7%) and influenza (18.7%). No treatment-related mortality was observed. Rituximab maintenance was implemented in 17% (n= 20) of the patients for a median duration of 11 (range 1 to 46) months. Biopsy-proven transformation to diffuse large B cell lymphoma occurred in 5.9% of the patients. Patients with lymphoma transformation had a higher mean SUV on diagnostic PET/CT (13.48 vs 7.77, P= 0.037, respectively). Secondary malignancies were observed in 6% of the patients with 1 case of acute myeloid leukemia. With a median follow up of 2.85 (range 0.08 to 9.45) years, the estimated 5-years PFS (Figure 1) and OS (Figure 2) were 72.3% (95%CI 59.3-81.8%) and 85.6% (95%CI 75.0-92.0%), respectively. No survival difference was observed between patients achieving CR or PR followed or not by rituximab maintenance, but the number of patients receiving maintenance was small. Similarly, no survival differences were observed in patients with gastric EMZL compared to non-gastric locations or by MALT-IPI score risk category. In both UVA and MVA analyses, variables associated with shorter survival were ECOG performance status ≥2 and failure to achieve CR (for MVA: ECOG PS ≥ 2 (HR: 6.56, P=0.006) and failure to achieve CR (HR: 5.35 P Conclusion: This study represents the largest analysis to date evaluating the activity and safety of BR in untreated EMZL. BR is a highly effective platform in upfront treatment of EMZL with majority of the patients achieving complete and durable remissions. ECOG PS ≥ 2 and failure to achieve CR were identified as prognostic factors associated with worse outcome in BR treated patients. High incidence of herpes zoster infection was observed in this study which has not been previously reported. In addition, increased median PFS and lower incidence of infectious complications was observed in this study compared to prior reports. Disclosures Alderuccio: ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Inovio Pharmaceuticals: Other: Family member; Forma Therapeutics: Other: Family member; Agios Pharmaceuticals: Other: Family member; Oncinfo: Honoraria; OncLive: Honoraria; Puma Biotechnology: Other: Family member; Foundation Medicine: Other: Family member. Beaven:Tessa Therapeutics: Research Funding; Seattle Genetics: Research Funding; Celgene: Research Funding; LoxoOncology: Research Funding; MorphoSysAb: Research Funding; Roche: Research Funding. Shouse:Kite Pharma: Honoraria, Speakers Bureau. Epperla:Verastem Oncology: Speakers Bureau; Pharmacyclics: Honoraria. Castillo:TG Therapeutics: Research Funding; Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding; Kymera: Consultancy; Abbvie: Research Funding; Janssen: Consultancy, Research Funding. Voorhees:AstraZeneca: Research Funding. Martin:Regeneron: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Karyopharm: Consultancy, Research Funding; Teneobio: Consultancy; Sandoz: Consultancy; I-MAB: Consultancy; Bayer: Consultancy; Beigene: Consultancy; Cellectar: Consultancy; Incyte: Consultancy; Kite: Consultancy; Morphosys: Consultancy. Kamdar:Roche: Research Funding. Herrera:AstraZeneca: Research Funding; Karyopharm: Consultancy; Immune Design: Research Funding; Gilead Sciences: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Pharmacyclics: Research Funding; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Other: Travel, Accomodations, Expenses, Research Funding. Friedberg:Roche: Other: Travel expenses; Acerta Pharma - A member of the AstraZeneca Group, Bayer HealthCare Pharmaceuticals.: Other; Portola Pharmaceuticals: Consultancy; Kite Pharmaceuticals: Research Funding; Bayer: Consultancy; Astellas: Consultancy; Seattle Genetics: Research Funding. Zinzani:Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Portola: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kirin Kyowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eusapharma: Consultancy, Speakers Bureau; Immune Design: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin: Consultancy, Speakers Bureau; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Therapeutics, Inc.: Honoraria, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immune Design: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lossos:Seattle Genetics: Consultancy, Other; Stanford University: Patents & Royalties; Janssen Scientific: Consultancy, Other; Verastem: Consultancy, Honoraria; Janssen Biotech: Honoraria; NCI: Research Funding.

Introduction Ibrutinib was FDA approved for relapsed or refractory (R/R) marginal zone lymphoma (MZL) based on a phase II clinical trial that showed an overall response rate of 48% (Noy et al, Blood 2017). However, factors associated with response to ibrutinib in R/R MZL and outcomes of patients after progression on ibrutinib are unknown. Given the poor survival in other B-cell lymphomas such as mantle cell lymphoma (MCL) after progression on ibrutinib (Martin P et al, Blood 2016), we sought to evaluate clinicopathologic characteristics predictive of ibrutinib failure in R/R MZL, and describe outcomes of patients who experienced progression on ibrutinib therapy. Methods We performed a multicenter retrospective study of MZL patients treated at 19 US medical centers. Eligible patients were ≥ 18 years diagnosed with MZL from 2010-2019, who received ibrutinib for R/R MZL. Patients achieving a complete response (CR) or partial response (PR) with ibrutinib were considered ibrutinib responders, while those who had stable disease (SD) or progression of disease (PD) were classified as non-responders. The primary endpoint was to evaluate factors predictive of primary progression (PD) on ibrutinib. Secondary endpoints include evaluation of predictors of overall survival (OS) and progression-free survival (PFS) following ibrutinib therapy, assessment of outcomes based on the sequencing of ibrutinib therapy, and evaluation of outcomes following ibrutinib failure. PFS was defined as time from the start of ibrutinib therapy until lymphoma relapse/progression or death from any cause, censoring at last clinical assessment if no progression or death. OS was defined as time from the start of ibrutinib treatment until death or last follow-up. A multivariable Poisson regression analysis was performed to model ibrutinib progression on the clinicopathologic factors (see Table). To identify significant predictors for OS and PFS, we used a multivariable Cox model. Results 101 patients with R/R MZL received ibrutinib, of whom 99 had sufficient data for inclusion in the analysis. Among these patients, 63% (n=62) had CR/PR to ibrutinib (ibrutinib responders, CR=17, PR=45) and 37% (n=37) had no response (ibrutinib non-responders, SD=25, PD=12). The median duration of follow-up was 1.8 years (range=0.1-5.4 years) and 2 years (range=0.2-6.3 years) for ibrutinib responders and non-responders, respectively. Baseline characteristics of the R/R MZL patients stratified by ibrutinib response are shown in the Table. Among all the baseline factors examined for association with ibrutinib progression, only primary refractory disease (refractory to frontline therapy, RR=3.78, 95%CI=1.36-10.45, p=0.01) was predictive of a higher probability of primary progression on ibrutinib on multivariable analysis. The median OS was significantly better for responders (NR [not reached], 95%CI=3.2-NR) compared to non-responders (3.4 years, 95%CI=1.4-NR) (Figure 1A). Achieving CR/PR with ibrutinib (HR=0.22, 95%CI=0.09-0.52) and lack of complex cytogenetics (HR=0.22, 95%CI=0.08-0.59) were predictors of superior PFS. Similarly, ibrutinib response (HR=0.13, 95%CI=0.03-0.53) and lack of complex cytogenetics (HR=0.19, 95%CI=0.04-0.87) were predictors of better OS. There was no difference in PFS or OS based on the timing of ibrutinib administration (second vs third vs fourth line and beyond, Figure 1B and 1C). The median post ibrutinib relapse/progression OS (PROS) for patients who initially responded then progressed on ibrutinib (secondary progression, n=19) was 4 years (Figure 1D). The median PROS for patients who had no response to ibrutinib were stratified according to SD vs PD. The median PROS for those who had SD was NR and those with PD was 0.1 year (Figure 1E). Conclusion This is the largest series of R/R MZL patients treated with ibrutinib. A history of primary refractory disease was predictive of primary progression on ibrutinib, while the presence of complex cytogenetics was associated with inferior PFS and OS. In contrast to MCL, the outcomes of patients who progress on ibrutinib in R/R MZL are not poor except for the primary progression cohort (those with PD as the best response to ibrutinib). Improving therapeutic options for patients who experience PD with ibrutinib treatment represents an urgent unmet need and these patients should be prioritized for evaluation of novel therapeutic approaches. Figure Disclosures Epperla: Verastem Oncology: Speakers Bureau; Pharmacyclics: Honoraria. Reddy:Genentech, BMS: Research Funding; KITE Pharma, Abbvie, BMS, Celgene: Consultancy. Caimi:Genentech: Research Funding; Celgene Corp: Other: Incyte Corporation - Ownership - Pharmacyclics, Inc. - Ownership - Celgene Corp. - Other, Speakers Bureau; ADC Therapeutics: Research Funding. Greenwell:Lymphoma Research Foundation: Research Funding; Acrotech Biopharma LLC, Kyowa Kirin: Consultancy. Janakiram:Takeda, Fate, Nektar: Research Funding. Olszewski:Genentech, Inc.: Research Funding; Spectrum Pharmaceuticals: Research Funding; TG Therapeutics: Research Funding; Adaptive Biotechnologies: Research Funding. Cohen:Genentech, BMS, Novartis, LAM, BioInvent, LRF, ASH, Astra Zeneca, Seattle Genetics: Research Funding; Janssen, Adicet, Astra Zeneca, Genentech, Aptitude Health, Cellectar, Kite/Gilead, Loxo: Consultancy. Palmisiano:AbbVie: Research Funding; Genentech: Research Funding. Awan:Genentech: Consultancy; Janssen: Consultancy; Astrazeneca: Consultancy; Abbvie: Consultancy; Pharmacyclics: Consultancy; Gilead Sciences: Consultancy; Kite Pharma: Consultancy; Dava Oncology: Consultancy; Celgene: Consultancy; Blueprint medicines: Consultancy; Sunesis: Consultancy; Karyopharm: Consultancy; MEI Pharma: Consultancy. Barta:Monsanto: Consultancy; Atara: Honoraria; Seattle Genetics: Honoraria, Research Funding; Janssen: Honoraria; Pfizer: Honoraria. Grover:Genentech: Research Funding; Tessa: Consultancy. Bartlett:Roche/Genentech: Consultancy, Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Affimed Therapeutics: Research Funding; Acerta: Consultancy; BTG: Consultancy; Seattle Genetics: Consultancy, Research Funding; Pharmacyclics: Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Research Funding; Merck: Research Funding; Forty Seven: Research Funding; Immune Design: Research Funding; Janssen: Research Funding; Kite, a Gilead Company: Research Funding; ADC Therapeutics: Consultancy; Autolus: Research Funding; BMS/Celgene: Research Funding. Christian:Acerta: Research Funding; Celgene: Research Funding; Genentech: Research Funding; Merck: Research Funding; Millenium: Research Funding; MorphoSys: Research Funding; F Hoffman-La Roche: Research Funding; Triphase: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; AstraZenica: Membership on an entity's Board of Directors or advisory committees. Herrera:Pharmacyclics: Research Funding; Immune Design: Research Funding; AstraZeneca: Research Funding; Seattle Genetics: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Other: Travel, Accomodations, Expenses, Research Funding; Karyopharm: Consultancy.

Hepatocellular carcinoma (HCC) is the most common cause of cancer-related mortality, and patients with HCC show poor response to currently available treatments, which demands new therapies. We recently developed a synthetic microRNA-based molecularly targeted therapy for improving HCC response to chemotherapy by eliminating drug resistance. We used ultrasound-targeted microbubble destruction (UTMD) to locally deliver microRNA-loaded nanoparticles to HCC. Since the immune microenvironment plays a crucial role in HCC disease development and response to treatment, and UTMD and microRNAs have the potential to interfere with the immune system, in this study we analyzed the immunomodulatory effects of UTMD and miRNAs in HCC. We used an immunocompetent syngeneic HCC mouse model for the study. We conducted cytokine profiling in tumor, lymph nodes, and serum of animals within the first 24 h of treatment to analyze changes in the level of pro- and antitumoral cytokines. The results showed: (1) Hepa1–6 syngeneic tumors expressed HCC-related cytokines, (2) UTMD-microRNA combination therapy triggered transient cytokine storms, and (3) delivery of microRNA-122 and anti-microRNA-21 affected the immune microenvironment by decreasing the level of GM-CSF in tumors while modulating protumoral IL-1α, IL-1β, IL-5, IL-6 and IL-17 and antitumoral IL-2 and IL-12 in tumor-proximal lymph nodes, and increasing IL-2 in the serum of tumor-bearing mice. Local delivery of targeted therapy by UTMD significantly reduced the concentration of IL-12 and IL-17 in lymph nodes of treated and contralateral tumors suggesting a systemic response. Conclusion UTMD-mediated delivery of microRNA-122 and anti-microRNA-21 modulated the immune microenvironment of Hepa1–6 tumors at the level of cytokine expressions. Exploiting antitumoral immune effects could enhance the therapeutic efficacy of the proposed combination therapy for HCC.

Cell-specific uptake of drug delivery systems (DDSs) are crucial to achieve optimal efficacy of many drugs. Widely employed strategies to facilitate targeted intracellular drug delivery involves attachment of targeting ligands (peptides or antibodies) to DDSs. Target receptors mutations can limit the effectiveness of this approach. Herein, we demonstrate, through in vitro inhibitory and drug delivery studies, that graphene nanoribbons (GNRs), water dispersed with the amphiphilic polymer called PEG-DSPE ((1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N [amino (polyethylene glycol)]) (induce membrane depolarization-mediated epidermal growth factor receptor (EGFR) activation. This phenomenon is ligand-independent and EGFR activation occurs via influx of Ca2+ ions from the extracellular space. We further provide evidence, through in vivo studies, that this mechanism could be exploited to facilitate efficacious drug delivery into tumors that overexpress EGFR. The results suggest that transient membrane depo...

Current efforts in the design and development of nonviral vectors for gene delivery and transfection have focused on the development of versatile agents that can load short or large sized genetic material, and are efficacious without eliciting toxicity in dividing and nondividing cells. Herein, we have investigated oxidized graphene nanoribbons (O-GNRs) as nonviral vectors for gene therapy and report in vitro studies that detail their cytotoxicity, intracellular and nuclear uptake, and gene delivery and transfection efficiencies. Our results indicate that, without additional functionalization with positively charged groups or other nonviral vectors, O-GNRs could load large amounts of small-sized single-stranded or large-sized double stranded genetic materials. O-GNRs at potential therapeutic doses (20-60 μg/mL) elicited lower cytotoxicity compared to widely used commercial nonviral gene delivery vectors (Polyethylenimine and Fugene 6). The O-GNR-plasmid DNA complexes showed uptake into vesicular structures of dividing Henrietta Lacks (HeLa) and nondividing Human umbilical vein endothelial cells (HUVEC), release into the cell's cytoplasm and entry into the nucleus. In these cells, O-GNRs loaded with enhanced green fluorescence protein (EGFP) plasmid or siRNA against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed a concentration- and time- dependent increase in gene delivery and gene transfection efficiencies up to 96-98%. The results suggest that O-GNRs are promising candidates as versatile and efficient nonviral vectors of small- or large-sized genetic material in primary and secondary cell types for gene therapy.

Graphene is a multifunctional carbon nanomaterial and could be utilized to develop platform technologies for cancer therapies. Its surface can be covalently and noncovalently functionalized with anticancer drugs and functional groups that target cancer cells and tissue to improve treatment efficacies. Furthermore, its physicochemical properties can be harnessed to facilitate stimulus responsive therapeutics and drug delivery. This review article summarizes the recent literature specifically focused on development of graphene technologies to treat cancer. We will focus on advances at the interface of graphene based drug/gene delivery, photothermal/photodynamic therapy and combinations of these techniques. We also discuss the current understanding in cytocompatibility and biocompatibility issues related to graphene formulations and their implications pertinent to clinical cancer management.

Hepatocellular carcinoma (HCC) is the second-leading cause of cancer related deaths worldwide and new strategies to efficiently treat HCC are critically needed. The aim of this study was to assess the longitudinal treatment effects of two complementary miRNAs (miRNA-122 and antimiR-21) encapsulated in biodegradable poly lactic-co-glycolic acid (PLGA) - poly ethylene glycol (PEG) nanoparticles (PLGA-PEG-NPs), administered by an ultrasound-guided and microbubble-mediated delivery approach in doxorubicin-resistant and non-resistant human HCC xenografts. Using in vitro assays, we show that repeated miRNA treatments resulted in gradual reduction of HCC cell proliferation and reversal of doxorubicin resistance. Optimized US parameters resulted in a 9-16 fold increase (p = 0.03) in miRNA delivery in vivo in HCC tumors after two US treatments compared to tumors without US treatment. Furthermore, when combined with doxorubicin (10 mg/kg), longitudinal miRNA delivery showed a significant inhibition of tumor growth in both resistant and non-resistant tumors compared to non-treated, and doxorubicin treated controls. We also found that ultrasound-guided miRNA therapy was not only effective in inhibiting HCC tumor growth but also allowed lowering the dose of doxorubicin needed to induce apoptosis. In conclusion, the results of this study suggest that ultrasound-guided and MB-mediated delivery of miRNA-122 and antimiR-21, when combined with doxorubicin, is a highly effective approach to treat resistant HCC while reducing doxorubicin doses needed for treating non-resistant HCC in longitudinal treatment experiments. Further refinement of this strategy could potentially lead to better treatment outcomes for patients with HCC.

As the only FDA-approved near-infrared fluorophore, indocyanine green (ICG) is commonly used to image vasculature in vivo. ICG degrades rapidly in solution, which limits its usefulness in certain applications, including time-sensitive surgical procedures. We propose formulations that address this shortcoming via complexation with β-cyclodextrin derivatives (β-CyD), which are known to create stabilizing inclusion complexes with hydrophobic molecules. Here, we complexed ICG with highly soluble methyl β-CyD and FDA-approved sulfobutyl ether β-CyD (Captisol(®) ) in aqueous solution. We measured the fluorescence of the complexes over 24 h. We found that both CyD+ICG complexes exhibit sustained fluorescence increases of >2.0× versus ICG in water and >20.0× in PBS. Using transmission electron microscopy, we found evidence of reduced aggregation in complexes versus ICG alone. We thus conclude that this reduction in aggregation helps mitigate fluorescence autoquenching of CyD+ICG complexes compared in ICG alone. We also found that while ICG complexed with methyl β-CyD severely reduced the viability of MRC-5 fibroblasts, ICG complexed with sulfobutyl ether β-CyD had no effect on viability. These results represent an important first step toward enhancing the utility of aqueous ICG by reducing aggregation-dependent fluorescence degradation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1457-1464, 2016.

Ultrasound induced microbubble cavitation can cause enhanced permeability across natural barriers of tumors such as vessel walls or cellular membranes, allowing for enhanced therapeutic delivery into the target tissues. While enhanced delivery of small (100 nm) therapeutic carriers into cancer remains unclear and may require a higher pressure for sufficient delivery. Enhanced delivery of larger therapeutic carriers such as FDA approved pegylated poly(lactic-co-glycolic acid) nanoparticles (PLGA-PEG-NP) has significant clinical value because these nanoparticles have been shown to protect encapsulated drugs from degradation in the blood circulation and allow for slow and prolonged release of encapsulated drugs at the target location. In this study, various acoustic parameters were investigated to facilitate the successful delivery of two nanocarriers, a fluorescent semiconducting polymer model drug nanoparticle as well as PLGA-PEG-NP into human colon cancer xenografts in mice. We first measured the cavitation dose produced by various acoustic parameters (pressure, pulse length, and pulse repetition frequency) and microbubble concentration in a tissue mimicking phantom. Next, in vivo studies were performed to evaluate the penetration depth of nanocarriers using various acoustic pressures, ranging between 1.7 and 6.9 MPa. Finally, a therapeutic microRNA, miR-122, was loaded into PLGA-PEG-NP and the amount of delivered miR-122 was assessed using quantitative RT-PCR. Our results show that acoustic pressures had the strongest effect on cavitation. An increase of the pressure from 0.8 to 6.9 MPa resulted in a nearly 50-fold increase in cavitation in phantom experiments. In vivo, as the pressures increased from 1.7 to 6.9 MPa, the amount of nanoparticles deposited in cancer xenografts was increased from 4- to 14-fold, and the median penetration depth of extravasated nanoparticles was increased from 1.3-fold to 3-fold, compared to control conditions without ultrasound, as examined on 3D confocal microscopy. When delivering miR-122 loaded PLGA-PEG-NP using optimal acoustic settings with minimum tissue damage, miR-122 delivery into tumors with ultrasound and microbubbles was 7.9-fold higher compared to treatment without ultrasound. This study demonstrates that ultrasound induced microbubble cavitation can be a useful tool for delivery of therapeutic miR loaded nanocarriers into cancer in vivo.

During Leishmania donovani (LD) infection Interleukin (IL)-10 favors parasite replication and plays a central role as a target for immune-based therapy. Glycogen synthase kinase 3 (GSK3)β differentially regulates TLR-mediated cytokine production. CREB, an important transcription factor that induces IL-10 production is negatively regulated by GSK3β. However, down regulation of IL-10 via CREB suppression has not been well explored in controlling LD infection. Here we demonstrate that, the TLR4 agonist 29 KDa β 1,4-galactose terminal glycoprotein (GP29) of LD activated GSK3β through TLR4 to induce IL-12-mediated Nitric oxide (NO) production that resulted in effective parasite clearance from macrophages. GSK3β activation abrogated both CREB phosphorylation and IL-10 production. Two subcutaneous injections of GP29 at fortnightly intervals in a 4-week infected mouse model of LD resulted in a dominant IL-12-mediated NO production and 100% animals were protected against a subsequent challenge with virulent LD parasites. Complete absence of GP29 mediated protection with down regulated NO and IL-12 production and dominant IL-10 production in presence of the GSK3β inhibitor, Lithium chloride reiterated the role of GSK3β in disease resolution in the murine model of visceral leishmaniasis.

Ultrasound/microbubble (US/MB)-mediated drug delivery is a promising approach to effectively deliver therapeutic agents to target tumors by increasing vascular permeability through sonoporation. Equipped with focused ultrasound, drug delivery platforms can increase site-specificity. To move toward clinical translation, such a platform requires both image guidance for accurate location of tumor targets and quantitative mapping of the therapeutic dose for effective treatment.

Monitoring cavitation is important for therapeutic applications relying on cavitation of microbubbles. Passive cavitation mapping (PCM) can provide quantitative and spatial information of cavitation activities; however, PCM based on time exposure acoustics (PCM-TEA) suffers from poor axial resolution. We propose a novel cavitation mapping algorithm with improved axial resolution based on localization of cavitation events from the individual element signals of the transducer array, even when the timing of the cavitation event is unknown.

The increased utilization of graphene nanoribbons (GNRs) for biomedical and material science applications necessitates the thorough evaluation of potential toxicity of these materials under both intentional and accidental exposure scenarios. We here investigated the effects of structural disruption of GNRs (induced by low-energy bath and high-energy probe sonication) to in vitro (human cell lines), and in vivo (Oryzias latipes embryo) biological systems. Our results demonstrate that low concentration (20 µg ml−1) suspensions of GNRs prepared by as little as 1 min of probe sonication can cause significant decreases in the overall metabolic state of cells in vitro, and increased embryo/larval mortality in vivo, as compared to bath sonicated or unsonicated suspensions. Structural analysis indicates that probe sonication leads to disruption in GNR structure and production of smaller carbonaceous debris, which may be the cause of the toxicity observed. These results point out the importance of assessing post-production structural modifications for any application using nanomaterials. Copyright © 2014 John Wiley & Sons, Ltd.

Single-walled carbon nanotubes synthesized with iron (Fe-SWCNT) or gadolinium (Gd-SWCNT) show promise as high performance multimodal contrast and drug-delivery agents. Our purpose was to evaluate potential vasoactive effects of SWCNT. Stable aqueous solutions of Fe-SWCNTs or Gd-SWCNTs were made using the biocompatible amphiphilic polymer N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoylsn-glycero-3- phosphoethanolamine (PEG-DSPE). Both aggregated and non-aggregated (sonicated) formulations were tested. The initial vasoactivity of the formulations and their potential for inducing pro-inflammatory endothelial dysfunction were investigated in the hamster cheek pouch and murine cremaster muscle intravital microscopy models. These models provide an assay to test several formulations/dosages in a paired fashion. Abluminal exposure to small arterioles exposes both endothelial and vascular smooth muscle cells. Using abluminal exposures of dosages that would approximate the first pass of an i.v. bolus injection, both Fe-SWCNTs and Gd-SWCNTs were immediately vasoactive. Aggregated formulations induced dilation and non-aggregated formulations induced constriction in both hamsters and mice. Endothelial dysfunction was evident after exposure to either aggregated or non-aggregated forms. General loss of dilator capability was seen after exposure to non-aggregated but not aggregated forms. Thus concentrations mimicking bolus dosing of PEG-DSPE coated SWCNT induce both acute and chronic vascular responses.

For the development of potent novel antileishmanial agents, 3,3'-(arylmethylene)bis(2-hydroxynaphthalene-1,4 dione) derivatives were synthesized from lawsone and evaluated for cytotoxicity on Leishmania donovani promastigotes as well as on leishmanial DNA topoisomerase-I. Enzyme inhibition studies were conducted with simultaneous and preincubation conditions. Total inhibition is compared to camptothecin (CPT), which was taken as positive control on both the systems of enzyme inhibition. The range of activity varied from 37.5 to 70 µM in simultaneous assay and 13-16 µM in preincubation assay. Furthermore, when evaluated against L. donovani promastigotes, the synthesized compounds exhibited the activity ranging from 2 to 14 µM. The results revealed that all the compounds exhibit promising antileishmanial activity.

Molecularly-targeted microbubbles (MBs) are increasingly being recognized as promising contrast agents for oncological molecular imaging with ultrasound. With the detection and validation of new molecular imaging targets, novel binding ligands are needed that bind to molecular imaging targets with high affinity and specificity. In this study we assessed a novel class of potentially clinically translatable MBs using an engineered 10(th) type III domain of human-fibronectin (MB-FN3VEGFR2) scaffold-ligand to image VEGFR2 on the neovasculature of cancer. The in vitro binding of MB-FN3VEGFR2 to a soluble VEGFR2 was assessed by flow-cytometry (FACS) and binding to VEGFR2-expressing cells was assessed by flow-chamber cell attachment studies under flow shear stress conditions. In vivo binding of MB-FN3VEGFR2 was tested in a transgenic mouse model (FVB/N Tg(MMTV/PyMT634Mul) of breast cancer and control litter mates with normal mammary glands. In vitro FACS and flow-chamber cell attachment studies showed significantly (P

Leishmania donovaniare the causative agents of visceral leishmaniasis worldwide. Lack of vaccines and emergence of drug resistance warrants the need for improved drug therapy and newer therapeutic intervention strategies against leishmaniasis. In the present study, we have investigated the effect of the natural indoloquinoline alkaloid cryptolepine onL. donovaniAG83 promastigotes. Our results show that cryptolepine induces cellular dysfunction inL. donovanipromastigotes, which leads to the death of this unicellular parasite. Interestingly, our study suggest that cryptolepine-induced cell death ofL. donovaniis counteracted by initial autophagic features elicited by the cells. For the first time, we show that autophagy serves as a survival mechanism in response to cryptolepine treatment inL. donovanipromastigotes and inhibition of autophagy causes an early increase in the amount of cell death. This study can be exploited for designing better drugs and better therapeutic strategies against leishmaniasis in future.

Aim: The systemic administration of graphene nanoribbons for a variety of in vivo biomedical applications will result in their interaction with cellular and protein components of the circulatory system. The aim of this study was to assess the in vitro effects of graphene nanoribbons (O-GNR) noncovalently functionalized with PEG-DSPE (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N [amino (polyethylene glycol)]) on some of the key hematological and vascular components of the circulatory system. Methods: Transmission electron microscopy was used to characterize the nanoparticles. ELISA-based assays, bright-field microscopy, transmission electron microscopy and colorimetric assays were used to assess toxicological effects. Results: Our findings taken together indicate that low concentrations of O-GNR-PEG-DSPE (, Graphene nanoribbons are a class of carbon-based nanostructures derived from multiwalled carbon nanotubes that have been shown to have unique properties and high potential for drug-delivery applications in recent studies from our group. However, further development of this nanoparticle for biomedical applications will be possible only after its interactions with components of the circulatory system are suitably characterized. Toward that goal, this study is aimed at identifying potential toxicities of graphene nanoribbons in the circulatory system. Results from this study will give us indications about safe dosages and lay the foundation toward further animal studies.

As the only FDA-approved near-infrared fluorophore, indocyanine green (ICG) is commonly used to image vasculature in vivo. ICG degrades rapidly in solution, which limits its usefulness in certain applications, including time-sensitive surgical procedures. We propose formulations that address this shortcoming via complexation with β-cyclodextrin derivatives (β-CyD), which are known to create stabilizing inclusion complexes with hydrophobic molecules. Here, we complexed ICG with highly soluble methyl β-CyD and FDA-approved sulfobutyl ether β-CyD (Captisol(®) ) in aqueous solution. We measured the fluorescence of the complexes over 24 h. We found that both CyD+ICG complexes exhibit sustained fluorescence increases of2.0× versus ICG in water and20.0× in PBS. Using transmission electron microscopy, we found evidence of reduced aggregation in complexes versus ICG alone. We thus conclude that this reduction in aggregation helps mitigate fluorescence autoquenching of CyD+ICG complexes compared in ICG alone. We also found that while ICG complexed with methyl β-CyD severely reduced the viability of MRC-5 fibroblasts, ICG complexed with sulfobutyl ether β-CyD had no effect on viability. These results represent an important first step toward enhancing the utility of aqueous ICG by reducing aggregation-dependent fluorescence degradation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1457-1464, 2016.

In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB(25)R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB(25)R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance.

The increased utilization of graphene nanoribbons (GNRs) for biomedical and material science applications necessitates the thorough evaluation of potential toxicity of these materials under both intentional and accidental exposure scenarios. We here investigated the effects of structural disruption of GNRs (induced by low-energy bath and high-energy probe sonication) to in vitro (human cell lines), and in vivo (Oryzias latipes embryo) biological systems. Our results demonstrate that low concentration (20 µg ml(-1) ) suspensions of GNRs prepared by as little as 1 min of probe sonication can cause significant decreases in the overall metabolic state of cells in vitro, and increased embryo/larval mortality in vivo, as compared to bath sonicated or unsonicated suspensions. Structural analysis indicates that probe sonication leads to disruption in GNR structure and production of smaller carbonaceous debris, which may be the cause of the toxicity observed. These results point out the importance of assessing post-production structural modifications for any application using nanomaterials.

Graphene nanoparticles dispersions show immense potential as multifunctional agents for in vivo biomedical applications. Herein, we follow regulatory guidelines for pharmaceuticals that recommend safety pharmacology assessment at least 10 – 100 times higher than the projected therapeutic dose, and present comprehensive single dose response, expanded acute toxicology, toxicokinetics, and respiratory/cardiovascular safety pharmacology results for intravenously administered dextran-coated graphene oxide nanoplatelet (GNP-Dex) formulations to rats at doses between 1–500 mg/kg. Our results indicate that the maximum tolerable dose (MTD) of GNP-Dex is between 50 mg/kg ≤ MTD < 125 mg/kg, blood half-life < 30 minutes, and majority of nanoparticles excreted within 24 hours through feces. Histopathology changes were noted at ≥ 250 mg/kg in the heart, liver, lung, spleen, and kidney; we found no changes in the brain and no GNP-Dex related effects in the cardiovascular parameters or hematological factors (blood, lipid, and metabolic panels) at doses < 125 mg/kg. The results open avenues for pivotal preclinical single and repeat dose safety studies following good laboratory practices (GLP) as required by regulatory agencies for investigational new drug (IND) application.

Leishmania, a protozoan parasite, causes a wide range of human diseases ranging from the localized self-healing cutaneous lesions to fatal visceral leishmaniasis. Toxicity of traditional first line drugs and emergence of drug-resistant strains have worsened the situation. DNA topoisomerase II in kinetoplastid protozoan parasites are of immense interest as drug target because they take part in replication of unusual kinetoplast DNA network. In this study, we have taken target-based therapeutic approaches to combat leishmaniasis. Two isobenzofuranone compounds, viz., (1) 3,5-bis(4-chlorophenyl)-7-hydroxyisobenzofuran-1(3H)-one (JVPH3) and (2) (4-bromo)-3'-hydroxy-5'-(4-bromophenyl)-benzophenone(JVPH4) were synthesized chemically and characterized by NMR and mass spectrometry analysis. Activity of type II DNA topoisomerase of leishmania (LdTOPII) was monitored by decatenation assay and plasmid cleavage assay. The antiparasitic activity of these compounds was checked in experimental BALB/c mice model of visceral leishmaniasis. Isobenzofuranone derivatives exhibited potent antileishmanial effect on both antimony (Sb) sensitive and resistant parasites. Treatment with isobenzofuranone derivatives on promastigotes caused induction of reactive oxygen species (ROS)-mediated apoptosis like cell death in leishmania. Both the compounds inhibited the decatenation activity of LdTOPII but have no effect on bi-subunit topoisomerase IB. Treatment of LdTOPII with isobenzofuranone derivatives did not stabilize cleavage complex formation both in vitro and in vivo. Moreover, treatment with isobenzofuranone derivatives on Leishmania donovani-infected mice resulted in clearance of parasites in liver and spleen by induction of Th1 cytokines. Taken together, our data suggest that these compounds can be exploited as potential antileishmanial agents targeted to DNA topoisomerase II of the parasite.

The unicellular organism Leishmania undergoes apoptosis-like cell death in response to external stress or exposure to antileishmanial agents. Here, we showed that 3- O ,28- O -disuccinyl betulin (DiSB), a potent topoisomerase type IB inhibitor, induced parasitic cell death by generating oxidative stress. The characteristic feature of the death process resembled the programmed cell death (PCD) seen in higher eukaryotes. In the current study, the generation of reactive oxygen species (ROS), followed by the depolarization of mitochondrial membrane potential (ΔΨ m ), caused a loss in ATP production in Leishmania parasites. This further gave positive feedback to produce a large amount of ROS, which in turn caused oxidative DNA lesions and genomic DNA fragmentation. The treatment of promastigotes with DiSB induced high expression levels of metacaspase protein that led to cell death in this unicellular organism. The PCD was insensitive to benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), suggesting that the death process was not associated with the activation of caspases. DiSB treatment translocated Leishmania donovani endonuclease G (LdEndoG) from mitochondria to the nucleus, which was responsible for the DNA degradation process. Conditional antisense knockdown of L. donovani metacaspase (LdMC), as well as EndoG, -subverted death of the parasite and rescued cell cycle arrest in G 1 phase. The present study on the effector molecules associated with the PCD pathway of the parasite should help to manifest the mechanisms of PCD and also might be exploited in antileishmanial chemotherapy.

Lignans are diphenyl propanoids with vast range of biological activities. The present study provides an important insight into the anti-leishmanial activities of two lignan glycosides, viz. lyoniside and saracoside. These compounds inhibit catalytic activities of topoisomerase IB (LdTopIB) of Leishmania donovani in non-competitive manner and stabilize the LdTopIB mediated cleavage complex formation both in vitro and in Leishmania promastigotes and subsequently inhibit the religation of cleaved strand. These two compounds not only poison LdTopIB but also can interact with the free enzyme LdTopIB. We have also shown that lyoniside and saracoside are cytotoxic to promastigotes and intracellular amastigotes. The protein–DNA complex formation leads to double strand breaks in DNA which ultimately triggers apoptosis-like cell death in the parasite. Along with their cytotoxicity towards sodium antimony gluconate (SAG) sensitive AG83 strain, their ability to kill SAG resistant GE1 strain makes these two compounds potential anti-leishmanial candidates. Not only they effectively kill L. donovani amastigotes inside macrophages in vitro , lyoniside and saracoside demonstrated strong anti-leishmanial efficacies in BALB/c mice model of leishmaniasis . Treatment with these lignan glycosides produce nitric oxide and reactive oxygen species which result in almost complete clearance of the liver and splenic parasite burden. These compounds do not inhibit human topoisomerase IB upto 200 μM concentrations and had poor cytotoxic effect on uninfected cultured murine peritoneal macrophages upto 100 μM concentrations. Taken together it can be concluded that these compounds can be developed into excellent therapeutic agent against deadly disease leishmaniasis.

We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24 h. However, their uptake was ~ 38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251 but showed little/no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. Cell death in U251 was necrotic, probably due to oxidative degradation of APE-1. From the Clinical Editor This study reports on the utility of PEG-DSPE coated oxidized graphene nanoribbons as anti-tumor drug delivery agents of Lucanthone into Glioblastoma Multiformae cells targeting base excision repair enzyme APE-1, demonstrating promising anti-tumor effects with good preservation of healthy cells.