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The hypothesis that microvesicle-mediated miRNA transfer converts noncancer stem cells into cancer stem cells (CSC) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how microvesicles derived from cancer-associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived microvesicles horizontally transferred miR-221 to tumor cells and, in combination with hormone therapy, activated an ERlo/Notchhi feed-forward loop responsible for the generation of CD133hi CSCs. Importantly, microvesicles from patients with HTR metastatic disease expressed high levels of miR-221. We further determined that the IL6–pStat3 pathway promoted the biogenesis of onco-miR-221hi CAF microvesicles and established stromal CSC niches in experimental and patient-derived breast cancer models. Coinjection of patient-derived CAFs from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated patient-derived xenograft (PDX) models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and microvesicles. Murine and human CAFs were enriched in HTR tumors expressing high levels of CD133hi cells. Depletion of murine CAFs from PDX restored sensitivity to HT, with a concurrent reduction of CD133hi CSCs. Conversely, in models of CD133neg, HT-sensitive cancer cells, both murine and human CAFs promoted de novo HT resistance via the generation of CD133hi CSCs that expressed low levels of estrogen receptor alpha. Overall, our results illuminate how microvesicle-mediated horizontal transfer of genetic material from host stromal cells to cancer cells triggers the evolution of therapy-resistant metastases, with potentially broad implications for their control. Cancer Res; 77(8); 1927–41. ©2017 AACR.

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With growing indications for Chimeric Antigen Receptor (CAR) T-cell therapy, toxicity profiles are evolving. There is an urgent and unmet need for approaches to optimally manage emerging adverse events that extend beyond the standard paradigm of cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS). While management guidelines exist for ICANS, there is little guidance on how to approach patients with neurologic comorbidities, and how to manage rare neurotoxicity presentations such as CAR T-cell therapy related cerebral edema, severe motor complications or late onset neurotoxicity. Here, we present three scenarios of patients treated with CAR T-cells who develop unique types of neurotoxicity, and we describe an approach to evaluation and management based on experience because objective data are limited. The goal of this manuscript is to develop awareness of emerging and unusual complications, discuss treatment approaches and help institutions and health care providers establish frameworks to navigate how to best address unusual neurotoxicities to ultimately improve patient outcomes.

BACKGROUND Despite favorable clinical outcomes, a subset of patients with thymic epithelial tumors (TETs) develop metastasis. The Cancer Genome Atlas (TCGA) provides genomic data on primary TETs (pTETs). This study assessed the molecular alterations and uncovered targetable pathways in metastatic TETs (mTETs). METHODS From 2015 to 2020, 49 patients with stage IV TETs underwent Clinical Laboratory Improvement Amendments–based sequencing using whole-exome sequencing (n = 33), panel-based testing (n = 12), and/or liquid biopsy (n = 24). Specimens were obtained from a metastatic organ (n = 36) or relapsed primary mediastinal mass (n = 10), whereas four patients underwent a liquid biopsy only. Data on pTETs were derived from the TCGA. RESULTS Compared with the pTET data set, patients with mTETs were younger (54 years v 60.5 years, P = .009) and had more aggressive histologies, with the most common tumor type being thymic carcinoma (n = 22, 40.7%) and B3 thymoma (n = 15, 27.8%). GTF2I was the most altered gene in primary thymomas (48.80%, n = 60). In metastatic thymoma and thymic carcinoma, TP53 was the most common genetic alteration (31% and 36%, respectively). In mTETs, the genomic alteration occurred in the TP53/CDK, EGFR/RAS, and PI3K/mTOR pathways. Biopsies obtained from distant metastasis were more commonly found to contain targetable mutations. There was an overlap of 61% (22 of 36) between tissue and liquid biopsy genomic alterations. CONCLUSION Clinically actionable genomic alterations are frequently observed in mTETs, indicating a value of repeat biopsy (preferably from a metastatic site of TETs for sequencing at the time of recurrence (TCGA data).

Neoantigen peptides arising from genetic alterations may serve as targets for personalized cancer vaccines and as positive predictors of response to immune checkpoint therapy. Mutations in genes regulating RNA splicing are common in hematological malignancies leading to dysregulated splicing and intron retention (IR). In this study, we investigated IR as a potential source of tumor neoantigens in multiple myeloma (MM) patients and the relationship of IR-induced neoantigens (IR-neoAg) with clinical outcomes. MM-specific IR events were identified in RNA-sequencing data from the Multiple Myeloma Research Foundation CoMMpass study after removing IR events that also occurred in normal plasma cells. We quantified the IR-neoAg load by assessing IR-induced novel peptides that were predicted to bind to major histocompatibility complex (MHC) molecules. We found that high IR-neoAg load was associated with poor overall survival in both newly diagnosed and relapsed MM patients. Further analyses revealed that poor outcome in MM patients with high IR-neoAg load was associated with high expression levels of T-cell co-inhibitory molecules and elevated interferon signaling activity. We also found that MM cells exhibiting high IR levels had lower MHC-II protein abundance and treatment of MM cells with a spliceosome inhibitor resulted in increased MHC-I protein abundance. Our findings suggest that IR-neoAg may represent a novel biomarker of MM patient clinical outcome and further that targeting RNA splicing may serve as a potential therapeutic strategy to prevent MM immune escape and promote response to checkpoint blockade.

Objective To evaluate usability of and satisfaction with OrCam MyEye, a finger-size wearable assistive technology device for visually impaired during real-world tasks. Methods This prospective multicenter study was conducted on visually impaired people recruited from 5 vision rehabilitation centers. Patients performed real-world tasks such as near and distance reading, money handling, colour identification and faces recognition in 2 different scenarios: without using any low vision aid and with OrCam. System Usability Scale (SUS), Patient’s Global Impression of Change (PGIC), the Quebec User Evaluation of Satisfaction with Assistive Technology (QUEST 2.0) and the Psychosocial Impact of Assistive Devices Scale (PIADS) were administered after the use of the OrCam device. Results Among the 100 participants, use of OrCam MyEye device improved many daily-living tasks (F = 1.67, P 2 = 0.72, P

BACKGROUND: Central nervous system (CNS) mucormycosis is insidious and difficult to diagnose. It progresses rapidly and causes high mortality. Rare cases have been reported during ibrutinib use, which have poor prognosis. Through this case, we share the experience of successful diagnosis and treatment. We also emphasize the importance of focusing on high-risk groups, early diagnosis and prompt management. CASE DESCRIPTION: In this case, a 52-year-old patient was diagnosed with chronic lymphocytic leukemia (CLL) for more than 5 years. He was in remission after rituximab plus fludarabine and cyclophosphamide (RFC) regimen, and relapsed in the fourth year. During the ibrutinib monotherapy, the patient presented with sudden headache. Cranial imaging examination revealed a definite right occipitoparietal lobe mass with extensive edema. A rapid diagnosis of mucormycosis infection was made using metagenomic next-generation sequencing (mNGS). The patient at that time didn’t have neutropenia, but he had hypogammaglobulinemia. The infection was treated with amphotericin B cholesteryl sulfate complex, posaconazole, and interventional surgery, and the treatment was successful. At the same time, we considered the control of disease progression in this relapsed patient with, as well as to the drug interaction with posaconazole. We chose the next generation Bruton’s tyrosine kinase (BTK) inhibitor zanubrutinib as the treatment, whose safety has been identified. As of the submission date, the patient has been followed up for nearly 1 year, and his disease is stable. CONCLUSIONS: When new clinical problems arise in recurrent CLL patients, it is important to identify multiple factors, especially the insidious fungal infections. In particular, the immunocompromised patients should be concerned. CNS mucormycosis is extremely deadly, the early diagnosis will improve the prognosis. In clinical practice, the gold standard diagnosis of mucormycosis is difficult to obtain through pathology. In this case, mNGS was applied to quickly diagnose mucormycosis, enabling earlier treatment and ameliorating the prognosis. Thus, it will help us to early detect this group of people who may be potentially infected. Current guidelines do not recommend the prophylactic use of antifungal agents in treated CLL patients. However, in patients with prior severe infection or hypogammaglobulinemia, intravenous immunoglobulin is recommended to reduce the associated infection rate.

T cell activation and exhaustion are associated with highly coordinated changes in gene expression and chromatin accessibility. While the contribution of key, state-specific transcription factors to these processes has been extensively explored, the role for chromatin regulatory complexes remains less clear. The mammalian SWI/SNF (mSWI/SNF) complexes are a diverse family of ATP-dependent remodeling complexes that regulate DNA accessibility and hence facilitate timely and appropriate gene expression. Recent genome-scale CRISPR screens have highlighted the involvement of mSWI/SNF complexes in the acquisition of divergent T cell states; however, the mechanisms by which these complexes regulate T cell activation and exhaustion and how such activities may be leveraged for therapeutic benefit remain poorly understood. Here, we developed an in vitro system to map chromatin-level changes occurring during acute and chronic TCR stimulation in human T cells and to investigate the contribution of the mSWI/SNF complexes throughout these processes. By gene expression and chromatin profiling, we show that our in vitro model closely recapitulates the T cell activation and exhaustion phenotypes observed in human tumors. We demonstrate that the mSWI/SNF complexes contribute to shaping the epigenome of T cells at different activation and exhaustion stages, directing subsequent changes in gene expression. Specifically, deletion of components demarcating the cBAF subcomplex within the mSWI/SNF family improves T cell survival and proliferation and leads to the down-regulation of exhaustion hallmarks. Notably, by using four different compounds that either degrade or inhibit the catalytic activity of SMARCA2 and SMARCA4, we demonstrate that mSWI/SNF disruption significantly increases human T cell fitness, decreases exhaustion and is associated to memory-like hallmarks in chronically stimulated human T cells. Finally, pharmacologic mSWI/SNF inhibition improves CAR-T fitness and expansion and results in improved anti-tumor control in vivo. These findings reveal a central role for mSWI/SNF complexes in the processes of T cell activation and exhaustion, and suggest SMARCA4/2 ATPase inhibitors and degraders as potential strategies to enhance current T-cell based cancer immunotherapies. Citation Format: Elena Battistello, Kimberlee Hixon, Dawn E. Comstock, Clayton K. Collings, Xufeng Chen, Javier Rodriguez Hernaez, Soobeom Lee, Konstantinos Ntatsoulis, Annamaria Cesarano, Kathryn Hockemeyer, W. Nicholas Haining, Matthew T. Witkowski, Jun Qi, Aristotelis Tsirigos, Fabiana Perna, Ioannis Aifantis, Cigall Kadoch. Targeting mammalian SWI/SNF chromatin remodeling complexes to improve T cell-based cancer immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1794.

Acute myeloid leukemia (AML) is an aggressive, clonally heterogeneous, myeloid malignancy, with a 5-year overall survival of approximately 27%. It constitutes the most common acute leukemia in adults, with an incidence of 3-5 cases per 100,000 in the United States. Despite great advances in understanding the molecular mechanisms underpinning leukemogenesis, the past several decades had seen little change to the backbone of therapy, comprised of an anthracycline-based induction regimen for those who are fit enough to receive it, followed by risk-stratified post-remission therapy with consolidation cytarabine or allogeneic stem cell transplantation (allo-SCT). Allo-SCT is the most fundamental form of immunotherapy in which donor cytotoxic T and NK cells recognize and eradicate residual AML in the graft-versus-leukemia (GvL) effect. Building on that, several alternative or synergistic approaches to exploit both self and foreign immunity against AML have been developed. Checkpoint inhibitors, for example, CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors block proteins found on T cells or cancer cells that stop the immune system from attacking the cancer cells. They have been used with limited success in both the AML relapsed/refractory (R/R) and post SCT settings. AML tumor mutational burden is low compared to solid tumors and thus, it is less likely to generate neoantigens and respond to antibody-mediated checkpoint blockade that has shown unprecedented results in solid tumors. Therefore, alternative therapeutic strategies that work independently of the T cell receptor (TCR) specificity have been developed. They include bispecific antibodies, which recruit T cells through CD3 engagement, and in AML have shown an overall response rate ranging between 14 and 30% in early phase trials. Chimeric Antigen Receptor (CAR) T cell therapy is a type of treatment in which T cells are genetically engineered to produce a recombinant receptor that redirects the specificity and function of T lymphocytes. However, lack of cell surface targets exclusively expressed on AML cells including Leukemic Stem Cells (LSCs) combined with clonal heterogeneity represents the biggest challenge in developing CAR therapy for AML. Antibody-Drug Conjugates (ADC) constitute the only FDA-approved immunotherapy to treat AML with Gemtuzumab Ozogamicin, a CD33-specific ADC used in CEBPα-mutated AML. The identification of additional cell surface targets is critical for the development of other ADC's potentially useful in the induction and maintenance regimens, given the ease at which these reagents can be generated and managed. Here, we will review those immune-based therapeutic interventions and highlight active areas of research investigations toward fulfillment of the great promise of immunotherapy to AML.

Background and Objectives: Vision impairments and related blindness are major public health problems. The prevalence of eye disease and barriers to optimal care markedly vary among different geographic areas. In the Abruzzo region (central Italy), an epidemiological surveillance on the state of ocular health in the population aged over 50 years was performed in 2019. Materials and Methods: Participants were sampled to be representative of the region’s inhabitants. Data were collected through a telephone interview and an eye examination. Prevalence of cataract, glaucoma, retinopathy, and maculopathy was assessed. The Cohen’s kappa (k) was used to measure the agreement between the presence of eye disease and awareness of the disease by the participants. Results: Overall, 983 people with a mean age of 66.0 ± 9.5 years were included in the study. The prevalence of cataracts, glaucoma, maculopathy, and retinopathy was 52.6%, 5.3%, 5.6%, and 29.1%, respectively. Among the total of the affected people, those aware of their condition were 21.8% (k = 0.12, slight agreement) for cataract, 65.4% (k = 0.78, substantial agreement) for glaucoma, 7.1% (k = 0.10, slight agreement) for maculopathy, and 0% for retinopathy (k = −0.004, agreement lower than that expected by chance). Refractive defects were corrected in the vast majority of participants. Conclusion: In the Abruzzo region, about two thirds of citizens aged 50 years or over suffer from cataract, glaucoma, retinopathy, or maculopathy, which are recognized as leading causes of blindness. Many people with eye disease do not know they have it. These data can be used by clinicians and policymakers to undertake clinical, political, and social actions.

Background/Objective: Every year in the United States, 32,000 individuals are diagnosed with Multiple Myeloma (MM) and 13,000 die from the disease. MM is a cancer of the plasma cells and while treatment exists, MM remains incurable. Our overall goal is to identify biologically and therapeutically relevant cell surface targets to develop MM immunotherapy. Methods: Given the genomic heterogeneity of MM, we performed surface analysis of seven MM cell lines by Mass-Spectrometry (MS), generating a pool of candidate proteins. Lacking specific tools for studying cell surface proteins, we developed an integrated computational tool with five distinct databases, and scored the likelihood of cell surface location for each molecule. One point was given for each database a protein appeared in. A protein receiving a score of three or more was considered a cell surface protein with high confidence. However, about one third of the candidates were not detected by the computational tool, thus requiring manual curation. We used UniProt and GeneCards to determine the subcellular locations of the unannotated IDs, and further confirmed their location with the computational tool. Results: Of the 5,454 UniProt IDs produced by MS, 2,026 were not annotated. Causes of an unannotated ID included having an obsolete ID, being an isoform, and/or not being located on the cell surface. Through manual annotation, 8 unique cell surface IDs were added to the existing 448. The 456 targets were further analyzed based on their expression profiles in both MM and normal tissues resulting in 94 of the most promising targets, some of which would have been missed without manual curation. Conclusion/Potential Impact: Public genomic databases are often incomplete and may contain errors making manual curation a necessary step in error reduction. Left unchecked, promising surface targets could have been overlooked hindering the creation of potentially curative immunotherapies.

BACKGROUND It is estimated that approximately 1.3 billion people live with some form of distance or near visual impairment. Numerous studies have been carried out to evaluate the effects of biofeedback (BF) and establish if it could be a useful tool in vision rehabilitation for various eye diseases. OBJECTIVE This systematic review aimed: 1) to examine the current evidence of BF efficacy for the rehabilitation of the visually impaired and 2) to describe methodological variations used in previous BF studies to provide recommendations for vision rehabilitation interventions. METHODS A systematic review was conducted in the Medline, PubMed, Cochrane Library and Web of Science databases to collect documents published between January 2000 and May 2020. Of the 1,960 studies identified, 43 met the criteria for inclusion. The following information was collected from each study: sample size, control group, any eye disease, apparatus used, frequency and number of sessions of BF, main outcomes of training and whether a follow-up was conducted. The first group included studies published as scientific articles in peer-reviewed journals. The second group included abstracts of studies presented at peer-reviewed conferences. Publications were also grouped according to the eye disease treated. RESULTS 25 articles and 18 peer-reviewed conference abstracts (PRCAs) were included in this review. BF stimulation is a commonly used technique for the treatment of visual impairment caused by macular disease. Most BF studies evaluate the effect of training on the preferred retinal locus (PRL), particularly with regard to fixation location and stability. Across these studies, participants who received BF intervention improved fixation stability and reading speed. High variability in the number of sessions and the duration of BF training was found. Most studies did not use a control group. CONCLUSIONS The findings of this review present evidence for biofeedback treatment in vision rehabilitation, with improved oculomotor abilities. Currently, it is not possible to formulate evidence-based recommendations for a standard training procedure due to the poor quality of existing randomised controlled trials. High-quality studies are needed to develop standard protocols for a range of eye diseases.

PURPOSE The purpose of this study was to investigate the tear meniscus in medically controlled glaucoma patients (MCGP) using anterior segment-optical coherence tomography (AS-OCT). MATERIALS AND METHODS Fifty-six MCGP, 24 patients with evaporative dry eye (EDE), and 30 healthy subjects (controls), were enrolled. MCGP were divided into group 1 (14 eyes): β-blockers; group 2 (14 eyes): prostaglandin analogs; group 3 (28 eyes) ≥2 drugs. Ocular Surface Disease Index (OSDI) questionnaire, tear film break-up time, corneal fluorescein staining, Schirmer Test I, and tear meniscus height (lower and upper: L-TMH, U-TMH) and area (L-TMA, U-TMA) using AS-OCT, were performed. RESULTS OSDI score was higher (P

Targeted immunotherapy in acute myeloid leukemia (AML) is challenged by the lack of AML-specific target antigens and clonal heterogeneity, leading to unwanted on-target off-leukemia toxicity and risk of relapse from minor clones. We hypothesize that combinatorial targeting of AML cells can enhance therapeutic efficacy without increasing toxicity. To identify target antigen combinations specific for AML and leukemic stem cells, we generated a detailed protein expression profile based on flow cytometry of primary AML (n = 356) and normal bone marrow samples (n = 34), and a recently reported integrated normal tissue proteomic data set. We analyzed antigen expression levels of CD33, CD123, CLL1, TIM3, CD244 and CD7 on AML bulk and leukemic stem cells at initial diagnosis (n = 302) and relapse (n = 54). CD33, CD123, CLL1, TIM3 and CD244 were ubiquitously expressed on AML bulk cells at initial diagnosis and relapse, irrespective of genetic characteristics. For each analyzed target, we found additional expression in different populations of normal hematopoiesis. Analyzing the coexpression of our six targets in all dual combinations (n = 15), we found CD33/TIM3 and CLL1/TIM3 to be highly positive in AML compared with normal hematopoiesis and non-hematopoietic tissues. Our findings indicate that combinatorial targeting of CD33/TIM3 or CLL1/TIM3 may enhance therapeutic efficacy without aggravating toxicity in immunotherapy of AML.

Multiple myeloma (MM) is an incurable malignancy of mature plasma cells. Despite major advances in the therapeutic armamentarium of MM, only 50% of patients survive more than 5 years after diagnosis, with significantly lower rates (21%) for high-risk patients. Chimeric Antigen Receptor (CAR) T-cell therapy targeting BCMA (B-cell maturation antigen) shows high response rates in relapsed/refractory patients. However, most patients have disease remission that lasts less than 18 months, prompting the search for additional and synergistic therapeutic approaches. We unbiasedly mapped the cell surface proteome of MM by integrating Mass-Spectrometry (MS) and RNA-seq analyses from 7 MM cell lines and 904 primary MM patient samples bearing high-risk cytogenetics. To identify cell surface proteins, we ran a pool of 4,761 proteins and 16,000+ transcripts through five repositories. An integrated scoring database was developed by scoring each ID based on the number of databases (0-5) it was identified in, with 0 if the molecule was not found in any and 5 if the protein was found in all five. We identified 402 proteins with a surface score of 3 or higher in MM cell lines and patient samples by transcriptomics and proteomics. We prioritized the 326 candidates that were more highly expressed in patients. Based on functional enrichment analyses, we found the proteins formed three main networks with immune mechanisms representing the largest cluster (227 out of 326 cell surface proteins) followed by transporters and adhesion proteins.Based on a pipeline we previously established (1), we further selected 97 candidates minimally expressed in normal tissues. This list included current therapeutic targets such as BCMA, SLAMF7, ITGB7 and LY9. Validation in primary patient samples by western blot and flow-cytometric analyses, enabled the identification of 10 top candidates (CCR1, CD320, FCRL3, IL12RB1, ITGA4, LAX1, LILRB4, LRRC8D, SEMA4A, SLAMF6) that resulted most frequently and highly expressed. We found that LAX1, LILRB4 and SEMA4A significantly impact myeloma patient overall survival based on Kaplan-Meier analysis in the MM Research Foundation (MMRF) cohort (2). CCR1, IL12RB1, LILRB4 and SEMA4A were upregulated by the treatment with Bortezomib or Venetoclax that conversely, decreased BCMA expression in MM U266 cells. By stratifying the patient population, we found that the SEMA4A and LAX1 were up-regulated in patients with t(4;14) compared to patients with no cytogenetic abnormality; LILRB4 in patients with t(14;16) and CCR1 patients with t(14;16) and t(14;20). By calculating co-expression levels CCR1-LILRB4 and CCR1-FCRL3 resulted co-expressed in 100% of patients. For safety purposes (3), we excluded candidates with high (>55%) protein abundance in highly-purified normal hematopoietic stem cells and activated T-cells, narrowing down the list to 6 top candidates (CCR1, FCRL3, IL12RB1, LILRB4, LRRC8D, SEMA4A). To define the function of this group of promising cell surface targets, we used a CRISPR/Cas9 inducible system in KMS11 MM cells. We found that knock-out of CCR1, LRRC8D and SEMA4A individually reduces the MM cell growth by ~60%, 50% and 50% respectively, and almost completely abrogates MM cell migration through porous chambers by >80%. By co-culturing irradiated KO and control MM cells with healthy donor T-cells we also found that lack of CCR1 increased T-cell proliferation by 50% compared to controls and enhanced killing of MM cells, suggesting that CCR1 may suppress T-cell mediated immune responses in addition to play a role in MM cell survival and migration. This study suggests the contribution of an altered MM surfaceome to disease development and may lead to potential novel immunotherapeutic approaches for high-risk MM. References 1. Perna F et al., Cancer Cell 2017 3. Dong C et al., in press Oncogene 2021 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The hypothesis that microvesicle-mediated miRNA transfer converts noncancer stem cells into cancer stem cells (CSC) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how microvesicles derived from cancer-associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived microvesicles horizontally transferred miR-221 to tumor cells and, in combination with hormone therapy, activated an ERlo/Notchhi feed-forward loop responsible for the generation of CD133hi CSCs. Importantly, microvesicles from patients with HTR metastatic disease expressed high levels of miR-221. We further determined that the IL6–pStat3 pathway promoted the biogenesis of onco-miR-221hi CAF microvesicles and established stromal CSC niches in experimental and patient-derived breast cancer models. Coinjection of patient-derived CAFs from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated patient-derived xenograft (PDX) models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and microvesicles. Murine and human CAFs were enriched in HTR tumors expressing high levels of CD133hi cells. Depletion of murine CAFs from PDX restored sensitivity to HT, with a concurrent reduction of CD133hi CSCs. Conversely, in models of CD133neg, HT-sensitive cancer cells, both murine and human CAFs promoted de novo HT resistance via the generation of CD133hi CSCs that expressed low levels of estrogen receptor alpha. Overall, our results illuminate how microvesicle-mediated horizontal transfer of genetic material from host stromal cells to cancer cells triggers the evolution of therapy-resistant metastases, with potentially broad implications for their control. Cancer Res; 77(8); 1927–41. ©2017 AACR.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies. However, graft-versus-host disease (GVHD) and relapse after allo-HSCT remain major impediments to the success of allo-HSCT. Chimeric antigen receptors (CARs) direct tumor cell recognition of adoptively transferred T cells. CD19 is an attractive CAR target, which is expressed in most B cell malignancies, as well as in healthy B cells. Clinical trials using autologous CD19-targeted T cells have shown remarkable promise in various B cell malignancies. However, the use of allogeneic CAR T cells poses a concern in that it may increase risk of the occurrence of GVHD, although this has not been reported in selected patients infused with donor-derived CD19 CAR T cells after allo-HSCT. To understand the mechanism whereby allogeneic CD19 CAR T cells may mediate anti-lymphoma activity without causing a significant increase in the incidence of GVHD, we studied donor-derived CD19 CAR T cells in allo-HSCT and lymphoma models in mice. We demonstrate that alloreactive T cells expressing CD28-costimulated CD19 CARs experience enhanced stimulation, resulting in the progressive loss of both their effector function and proliferative potential, clonal deletion, and significantly decreased occurrence of GVHD. Concurrently, the other CAR T cells that were present in bulk donor T cell populations retained their anti-lymphoma activity in accordance with the requirement that both the T cell receptor (TCR) and CAR be engaged to accelerate T cell exhaustion. In contrast, first-generation and 4-1BB-costimulated CAR T cells increased the occurrence of GVHD. These findings could explain the reduced risk of GVHD occurring with cumulative TCR and CAR signaling.

The aim of this work was to characterize the choriocapillaris (CC) in patients with Stargardt disease (STGD) using the swept source widefield optical coherence tomography angiography (SS WF OCTA) and to compare CC perfusion density to retinal sensitivity, analyzed using microperimetry (MP). This cross-sectional study included 9 patients (18 eyes) with STGD and central CC atrophy (stage 3 STGD). The CC was analyzed using SS WF OCTA and areas of different CC impairment were quantified and correlated with retinal sensitivity analyzed using MP. The main outcome measures were the percent perfused choriocapillaris area (PPCA), retinal sensitivity, and correlation between PPCA and retinal sensitivity. Seventeen eyes of 9 patients suffering from stage 3 STGD were analyzed. SS WF OCTA revealed a vascular rarefaction in central atrophic zones and a near atrophy halo of choriocapillaris impairment. In all eyes were noticed a central atrophy (CA) area with absolute absence of CC that corresponded to 0 dB points at MP, a near atrophy (NA) zone of PPCA impairment that included points with decreased sensitivity at MP and a distant from atrophy (DA) zone with higher PPCA and retinal sensitivity values. The mean difference of PPCA and retinal sensitivity between NA and CA and DA and CA was statistical significantly different (p &lt, 0.01), the latter showing higher values. A direct relationship between PPCA and retinal sensitivity was found (p &lt, 0.001). Choriocapillaris damage evaluated using SS WF OCTA correlates with MP, these data suggest that CC impairment may be a predictor of retinal function in patients with STGD.

We investigated the ocular surface (OS) system modifications after completely successful glaucoma surgery in thirty-eight patients undergoing trabeculectomy (surgical group), using laser scanning confocal microscopy (LSCM) and impression cytology (IC). Twenty-six medically controlled glaucomatous patients served as controls (medical group). LSCM, IC, and the ocular surface disease index (OSDI) and National Eye Institute Visual Function Questionnaire-25 (NEI VFQ-25) questionnaires, were performed at baseline and after six months. The main outcomes were: goblet cell density (GCD), limbal dendritic cell density (LDCD), subbasal corneal nerve inhomogeneity (SCNI), Meibomian gland density and inhomogeneity (MGD, MGI), and HLA-DR positivity. There were no significant baseline differences between groups. At the sixth month, the surgical group showed a GCD increase (p

Multiple Myeloma (MM) is an incurable disease, with a particularly poor prognosis for patients with refractory/relapsed MM (RRMM) or high-risk cytogenetics. Chimeric Antigen Receptor (CAR) T-cell therapy targeting BCMA can induce deep responses in highly pretreated RRMM; however, remissions are not sustained, and the majority of patients eventually relapse. We hypothesized that genomic determinants of MM dictate the expression of surface targets that can be of use for immune-targeting. We analyzed the gene expression of 24 immunotherapeutic targets in 1900 MM patients. In patients with t(14;16), we found that IGF1R, ITGB7, GPRC5D, CD70, TACI and ICAM1 were overexpressed compared to the t(11;14) group. In patients with t(14;20), ITGB7, GPRC5D and TACI were overexpressed, and in patients with t(4;14), IGF1R, GPRC5D, CD70, CD44, BCMA, CD138, FUT3, SLAMF7, CD56 and CD200 were overexpressed compared to the standard risk group. We found that ITGB7, CD86, CD81 and IGF1R were overexpressed in non-hyperdiploid (NHRD) and IGKC, CD138, BCMA, CD74, CD47, BST2, LY9, CD200, CD56, CD1D, ICAM1 and CD19 were overexpressed in hyperdiploid (HRD) patient samples. By stratifying patients using mutated genes, copy number events and chromosomal level changes, we found that GPRC5D, ITGB7, IGF1R and CD70 expression were increased in patients bearing 5-9 and/or 10+ drivers compared to 0-4 drivers. A gene co-expression network analysis enabled the identification of 30 gene modules highly correlating with our targets, suggesting mechanistic insights. Our results provide a means for target selection for precision CAR therapy, by considering both the patient's genomic backgrounds and cancer cell surface profiles. The quest for novel MM targets for immunotherapies remains open, and CAR target discovery driven by specific genetic events remains an active area of investigation.

Epigenetic regulators play critical roles in normal hematopoiesis, and the activity of these enzymes is frequently altered in hematopoietic cancers. The major type II protein arginine methyltransferase PRMT5 catalyzes the formation of symmetric dimethyl arginine and has been implicated in various cellular processes, including pluripotency and tumorigenesis. Here, we generated Prmt5 conditional KO mice to evaluate the contribution of PRMT5 to adult hematopoiesis. Loss of PRMT5 triggered an initial but transient expansion of hematopoietic stem cells (HSCs); however, Prmt5 deletion resulted in a concurrent loss of hematopoietic progenitor cells (HPCs), leading to fatal BM aplasia. PRMT5-specific effects on hematopoiesis were cell intrinsic and depended on PRMT5 methyltransferase activity. We found that PRMT5-deficient hematopoietic stem and progenitor cells exhibited severely impaired cytokine signaling as well as upregulation of p53 and expression of its downstream targets. Together, our results demonstrate that PRMT5 plays distinct roles in the behavior of HSCs compared with HPCs and is essential for the maintenance of adult hematopoietic cells.

Chromosomal deletions associated with human diseases, such as cancer, are common, but synteny issues complicate modeling of these deletions in mice. We use cellular reprogramming and genome engineering to functionally dissect the loss of chromosome 7q (del(7q)), a somatic cytogenetic abnormality present in myelodysplastic syndromes (MDS). We derive del(7q)- and isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from hematopoietic cells of MDS patients and show that the del(7q) iPSCs recapitulate disease-associated phenotypes, including impaired hematopoietic differentiation. These disease phenotypes are rescued by spontaneous dosage correction and can be reproduced in karyotypically normal cells by engineering hemizygosity of defined chr7q segments in a 20-Mb region. We use a phenotype-rescue screen to identify candidate haploinsufficient genes that might mediate the del(7q)- hematopoietic defect. Our approach highlights the utility of human iPSCs both for functional mapping of disease-associated large-scale chromosomal deletions and for discovery of haploinsufficient genes.

Gardner et al. recently reported on two cases of mixed lineage leukemia (MLL) + acute myeloid leukemia (AML) occurring after CD19 chimeric antigen receptor (CAR) therapy for MLL + B-acute lymphoblastic leukemia (ALL) (1). This report provides new insights into mechanisms of immune escape from targeted immunotherapy and strategies to prevent their occurrence with adapted immune interventions.

Summary Chimeric antigen receptor (CAR) therapy targeting CD19 has yielded remarkable outcomes in patients with acute lymphoblastic leukemia. To identify potential CAR targets in acute myeloid leukemia (AML), we probed the AML surfaceome for overexpressed molecules with tolerable systemic expression. We integrated large transcriptomics and proteomics datasets from malignant and normal tissues, and developed an algorithm to identify potential targets expressed in leukemia stem cells, but not in normal CD34 + CD38 − hematopoietic cells, T cells, or vital tissues. As these investigations did not uncover candidate targets with a profile as favorable as CD19, we developed a generalizable combinatorial targeting strategy fulfilling stringent efficacy and safety criteria. Our findings indicate that several target pairings hold great promise for CAR therapy of AML.