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Comprehensive genomic profiling (CGP) allows for the detection of driver alterations at high resolution, but the limited number of approved targeted therapies and their high costs have contributed to its limited clinical utilization. We retrospectively compared data of 946 women with ovarian cancer (11.4% were referred to CGP, and 88.6% served as control) to examine whether CGP provides a prognosis benefit. Patient baseline parameters were similar between the groups. Cox regression analysis adjusted for age, disease stage at diagnosis, and recurrence status showed statistically significantly longer median overall survival (mOS) in the CGP group versus the control (73.4 versus 54.5 months, p < 0.001). Fifty-four patients (52.9%) had actionable mutations with potential treatments; twenty-six (48.2%) were treated with matched targeted therapy, showing a trend for longer mOS than the eighty-six women in the CGP group who were not given a suggested treatment (105.5 versus 63.6 months, p = 0.066). None of the genomic alterations predicted metastasis location. CCNE1 amplification and KRAS mutations were associated with shorter mOS. Patients with tumor mutation burden ≥4 mutations/megabase had longer mOS. High loss of heterozygosity was associated with longer mOS (99.0 versus 48.2 months, p = 0.004). CGP testing may provide both prognostic and predictive insights for treatment of patients with ovarian cancer. Prospective studies of larger cohorts are warranted.

We herein report a case of an altered immunocompetent patient with atypical monkeypox presentations after being vaccinated with JYNNOES vaccine. The initial presentation was of acute bacterial paronychia. This incident suggests the need for individuals at high-risk for monkeypox infection with altered immunity to receive 2 vaccination doses.

This work employs Adult Polyglucosan Body Disease (APBD) models to explore the efficacy and mechanism of action of 144DG11, a new polyglucosan-reducing lead compound discovered by a high-throughput screen (HTS). APBD is an adult onset glycogen storage disorder (GSD) manifesting as a debilitating progressive axonopathic leukodystrophy. APBD is caused by glycogen branching enzyme (GBE) deficiency leading to poorly branched and insoluble glycogen inclusions, which precipitate as neuropathogenic polyglucosans (PG). 144DG11 led to prolonged survival and improved motor parameters in a GBE knockin (Gbeys/ys) APBD mouse model. Histopathologically, 144DG11 reduced PG and glycogen levels in brain, liver, heart, and peripheral nerve. Indirect calorimetry experiments revealed that 144DG11 increases carbohydrate burn at the expense of fat burn, suggesting metabolic mobilization of pathogenic PG. These results were also reflected at the cellular level by increased glycolytic, mitochondrial and total ATP production. Mechanistically, we show that the molecular target of 144DG11 is the lysosomal membrane protein LAMP1, whose interaction with the compound, similar to LAMP1 knockdown, enhanced autolysosomal degradation of glycogen and lysosomal acidification. Enhanced mitochondrial activity and lysosomal modifications were also the most pronounced effects of 144DG11 in APBD patient fibroblasts as discovered by image-based multiparametric phenotyping analysis and corroborated by proteomics. In summary, this work presents a broad mechanistic and target-based characterization of 144DG11 in in vivo and cell models of the prototypical GSD APBD. This investigation warrants development of 144DG11 into a safe and efficacious GSD therapy.One Sentence SummaryA new compound, demonstrated to ameliorate APBD in vivo and ex vivo by autophagic catabolism of glycogen, may potentially become a universal drug for glycogen storage disorders.

HIV viremia can be controlled by chronic antiretroviral therapy. As a potentially single-shot alternative, B cells engineered by CRISPR/Cas9 to express anti-HIV broadly neutralizing antibodies (bNAbs) are capable of secreting high antibody titers. Here, we show that, upon immunization of mice, adoptively transferred engineered B cells home to germinal centers (GC) where they predominate over the endogenous response and differentiate into memory and plasma cells while undergoing class switch recombination (CSR). Immunization with a high affinity antigen increases accumulation in GCs and CSR rates. Boost immunization increases the rate of engineered B cells in GCs and antibody secretion, indicating memory retention. Finally, antibody sequences of engineered B cells in the spleen show patterns of clonal selection. Therefore, B cells can be engineered into what could be a living and evolving drug., Chronic antiretroviral therapy does not eradicate HIV infection. Here, the authors describe a potentially one-shot alternative by engineering B cells to express anti-HIV antibodies and undergo memory retention, isotype switching and clonal expansion

HIV viremia can be controlled by chronic antiretroviral therapy. As a potentially single-shot alternative, B cells engineered by CRISPR/Cas9 to express anti-HIV broadly neutralizing antibodies (bNAbs) were shown capable of secreting high antibody titers. Here, we demonstrate that, upon immunization of mice, adoptively transferred engineered B cells home to germinal centers (GC) where they predominate over the endogenous response and differentiate into memory and plasma cells while undergoing class switch recombination (CSR). Immunization with a higher affinity antigen increases accumulation in GCs and CSR rates. Boost immunization increases rates of engineered B cells in GCs and antibody secretion, indicating memory retention. Finally, antibody sequences of engineered B cells in the spleen show patterns of clonal selection. B cells may thus be engineered as a living and evolving drug.

5553 Background: Ovarian cancer (OC) is the second most common gynecologic malignancy and the most common cause of gynecologic cancer mortality in the United States. Homologous recombination deficiency (HRD), including the BRCA mutations, are found in 50% of OC tumors. Next generation sequencing (NGS) provides understanding the underlying molecular and genetic patterns to improve OC treatment. This study examines the prognostic and predictive biomarkers identified with NGS in hopes to improve OC patients outcomes. Methods: The patient cohort included 890 consecutive OC patients treated between 2002 and 2020,at the Tel-Aviv Medical Center. We retrospectively evaluated patients with histopathologically confirmed OC. Cox models were used to analyze the clinical impact of various mutations and biomarkers among OC patients with and without FoundationOne CDx NGS testing, by assessing overall survival (OS), progression free survival (PFS), and physicians' timing preferences for referral to NGS testing. Results: Among the 890 OC patients, 103 (11.57%) completed NGS molecular testing. The median OS among patients with and without NGS testing, adjusted for age, stage and recurrence status, was 73.36 and 68.50 months, respectively (P =.02). The median PFS was 17.23 and 17.43 months, respectively (P =.77). We also evaluated physicians' preferences regarding timing of molecular profiling, upon diagnosis, after first recurrence and at advanced line of treatment in 31.95%, 36.08% and 26.8% of practitioners, respectively. Of the patients who completed NGS, 48 (52.75%) harbored actionable mutations, and 21 patients (43.75%) received matched targeted therapy. Forty-five patients were microsatellite stable (MSS) (45%), 55 with undetermined status (55%) and 0 patients with MSI-H. Forty-one (71.93%) patients had low ( < 5) tumor mutation burden status (TMB), 16 (28.07%) intermediate (5-15) and none with high ( > 15) TMB. There was no noticeable survival difference when comparing low with intermediate TMB (P = 0.3). Loss of heterozygosity (LOH) was a significant prognostic biomarker. Patients with high LOH (hLOH > = 16%) had longer OS compared to low LOH (lLOH < 16%), 99.02 vs. 50.23 months, respectively (P

Many tumors are hierarchically organized, with a minor population of cancer stem cells (CSCs)/tumor-initiating cells at the apex of the hierarchy. These CSCs are highly enriched for the ability to drive tumor initiation, metastasis, and disease recurrence after therapy. Thus, a better understanding of the CSC population will be critical to the design of more effective anticancer therapeutics. To address this challenge, we have generated a lentiviral-based cancer stem cell sensor that reports with high temporal resolution on the cancer stem cell phenotype as a dynamic state. Expression of a destabilized fluorescent protein is driven by activation of an artificial enhancer element (SORE6) that responds to the master stem cell transcription factors Oct4 and Sox2 or their paralogs. We have extensively validated this reporter as marking cells with the expected characteristics of cancer stem cells, including the ability to self-renew, initiate, and sustain tumorigenesis and metastasis and exhibit chemoresistance (Tang et al., Stem Cell Reports 4:155-159). Intravital videomicroscopy and ex vivo imaging of orthotopically-implanted MDAMB231 breast tumors carrying the SORE6 stem cell sensor revealed that 60% of the CSCs in the primary tumor are found in association with macrophages. Macrophage depletion with clodronate liposomes in vivo reduced the number of CSCs in the tumor, indicating a role for macrophages in supporting or expanding the CSC population. Importantly, intravital imaging showed instances in which contact with intratumoral macrophages appeared to cause an induction of the stem phenotype in non-stem tumor cells and that this induction is concentrated at TMEM intravasation doorways where macrophages are enriched. Modeling this phenomenon in heterotypic cocultures in vitro, we showed that coculture of tumor cells with M2-like macrophages but not endothelial cells could increase the proportion of CSCs. This increase was contact dependent and involved Notch signaling. Single cell fate-mapping experiments in the coculture model revealed that the macrophages caused a >4x increase in the rate of direct conversion of non-stem to stem cells, without affecting CSC proliferation. The induced CSCs were capable of initiating tumorsphere formation in vitro, thus confirming their functionality. In summary, this novel imaging approach allowed us to make the important finding that macrophage contact can induce phenotypic plasticity in more differentiated tumor cells, thereby allowing them to acquire a stem phenotype during intravasation and dissemination. These results suggest that targeting the interaction between macrophages and tumor cells may be a new strategy to reduce the cancer stem cell population both locally and systemically. Citation Format: Bingwu Tang, Ved Sharma, Yarong Wang, Yuval Raviv, David Entenberg, Maja Oktay, John Condeelis, Lalage Wakefield. A fluorescent cancer stem cell sensor reveals dynamic induction of a stem phenotype in non-stem tumor cells on contact with macrophages in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B10.

Cancer stem cells (CSCs) are a subpopulation of tumor cells that are uniquely capable of initiating and sustaining tumorigenesis, and they have also been implicated in driving disease recurrence after cancer therapy. We have previously developed a novel lentiviral-based reporter system for direct visualization, quantitation and isolation of cells with CSC properties. The construct consists of a tandemly-repeated composite SOX2-OCT4 response element (“SORE6”) driving expression of a destabilized fluorescent protein reporter (Tang et al. Stem Cell Reports, 2015). TGF-βs are pleiotropic regulatory factors with complex roles in tumorigenesis. We and others have hypothesized that TGF-β can regulate the CSC population in breast cancer with stimulatory or inhibitory effects depending the model and stage of cancer development. Using the MCF10Ca1h and MCF7 breast cancer models, which retain tumor suppressor responses to TGF-β, here we show that TGF-β treatment significantly reduces the size of the CSC population in vitro. Although TGF-β had relatively little effect on invasion and migration of the bulk population in these models, it clearly inhibited migration and invasion of the CSCs, suggesting that biological responses to TGF-β can vary depending on the position of the cell in the differentiation hierarchy. Combining our stem cell reporter with a TGF-β pathway reporter, we further show that CSCs in the MCF10Ca1h model have higher endogenous activation of the TGF-β pathway than does the bulk tumor cell population, and by time-lapse video microscopy we find that CSCs with active TGF-β signaling are relatively quiescent. Furthermore, neutralization of TGF-β in vivo leads to an increased representation of CSCs in MCF10Ca1h and MCF7 tumors. Thus our preliminary results suggest that in breast cancer models where TGF-β acts as a tumor suppressor, TGF-β signaling is preferentially activated in the CSC compartment and keeps a subpopulation of CSCs in a quiescent and stationary state. These findings have important implications for the clinical use of TGF-β pathway antagonists. In contrast, in the metastatic breast cancer MDA-MB231 model, in which TGF-β acts as a pro-progression factor, we show that TGF-β can increase the size of the CSC population and enhance invasion. In this model, the CSCs are enriched for the ability to form macroscopic lung metastases when compared with non-CSCs, and we show that this effect is due in part to a selective survival advantage of the CSCs in the first 24 hours after arrival at the metastatic site. The role of TGF-β in this process is currently under investigation. Citation Format: Binwu Tang, George Baker, Joshua Zent, Yuval Raviv, Lalage Wakefield. Transforming growth factor-beta regulation of cancer stem cell dynamics at different stages of breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4782. doi:10.1158/1538-7445.AM2017-4782

Bootstrap samples with noise are shown to be an effective smoothness and capacity control technique for training feedforward networks and for other statistical methods such as generalized additive models. It is shown that noisy bootstrap performs best in conjunction with weight-decay regularization and ensemble averaging. The two-spiral problem, a highly non-linear, noise-free data, is used to demonstrate these findings. The combination of noisy bootstrap and ensemble averaging is also shown useful for generalized additive modelling, and is also demonstrated on the well-known Cleveland heart data.

Transplantation of B cells engineered ex vivo to secrete broadly neutralizing antibodies (bNAbs) has shown efficacy in disease models. However, clinical translation of this approach would require specialized medical centers, technically demanding protocols and major histocompatibility complex compatibility of donor cells and recipients. Here we report in vivo B cell engineering using two adeno-associated viral vectors, with one coding for Staphylococcus aureus Cas9 (saCas9) and the other for 3BNC117, an anti-HIV bNAb. After intravenously injecting the vectors into mice, we observe successful editing of B cells leading to memory retention and bNAb secretion at neutralizing titers of up to 6.8 µg ml