Whole-Slide Imaging, Mutual Information Registration for Multiplex Immunohistochemistry and Immunofluorescence.

Multiplex immunohistochemistry/immunofluorescence (mIHC/mIF) is a developing technology that facilitates the evaluation of multiple, simultaneous protein expressions at single-cell resolution while preserving tissue architecture. These approaches have shown great potential for biomarker discovery, yet many challenges remain. Importantly, streamlined cross-registration of multiplex immunofluorescence images with additional imaging modalities and immunohistochemistry (IHC) can help increase the plex and/or improve the quality of the data generated by potentiating downstream processes such as cell segmentation. To address this problem, a fully automated process was designed to perform a hierarchical, parallelizable, and deformable registration of multiplexed digital whole-slide images (WSIs). We generalized the calculation of mutual information as a registration criterion to an arbitrary number of dimensions, making it well suited for multiplexed imaging. We also used the self-information of a given IF channel as a criterion to select the optimal channels to use for registration. Additionally, as precise labeling of cellular membranes in situ is essential for robust cell segmentation, a pan-membrane immunohistochemical staining method was developed for incorporation into mIF panels or for use as an IHC followed by cross-registration. In this study, we demonstrate this process by registering whole-slide 6-plex/7-color mIF images with whole-slide brightfield mIHC images, including a CD3 and a pan-membrane stain. Our algorithm, WSI, mutual information registration (WSIMIR), performed highly accurate registration allowing the retrospective generation of an 8-plex/9-color, WSI, and outperformed 2 alternative automated methods for cross-registration by Jaccard index and Dice similarity coefficient (WSIMIR vs automated WARPY, P < .01 and P < .01, respectively, vs HALO + transformix, P = .083 and P = .049, respectively). Furthermore, the addition of a pan-membrane IHC stain cross-registered to an mIF panel facilitated improved automated cell segmentation across mIF WSIs, as measured by significantly increased correct detections, Jaccard index (0.78 vs 0.65), and Dice similarity coefficient (0.88 vs 0.79).

Immune cell subsets in interface cutaneous immune-related adverse events associated with anti-PD-1 therapy resemble acute graft versus host disease more than lichen planus.

BACKGROUND: Checkpoint immunotherapy is frequently associated with cutaneous immune-related adverse events (cirAEs), and among those, the most common subtype shows interface reaction patterns that have been likened to lichen planus (LP); however, cutaneous acute graft versus host disease (aGVHD) may be a closer histopathologic comparator. We used quantitative pathology to compare the immunologic composition of anti-PD-1-associated interface reactions to LP and aGVHD to assess for similarities and differences between these cutaneous eruptions. METHODS: Immunohistochemistry for CD4, CD8, CD68, PD-1, and PD-L1 was performed on formalin-fixed paraffin-embedded tissue from patients with anti-PD-1 interface cirAEs (n = 4), LP (n = 9), or aGVHD (n = 5). Densities of immune cell subsets expressing each marker were quantified using the HALO image analysis immune cell module. Plasma cell and eosinophil density were quantified on routine H&E slides. RESULTS: Specimens from patients with anti-PD-1 interface cirAEs showed equivalent total cell densities and immune cell composition to those with aGVHD. Patients with LP showed higher total immune cell infiltration, higher absolute T-cell densities, increased CD8 proportion, and reduced histiocytic component. The cases with the highest plasma cell counts were all anti-PD-1 interface cirAEs and aGVHD. CONCLUSION: The composition of immune cell subsets in anti-PD-1 interface cirAEs more closely resembles the immune response seen in aGVHD than LP within our cohort. This warrants a closer look via advanced analytics and may have implications for shared pathogenesis and potential treatment options.

Actinic Keratosis Color and Its Associations: A Retrospective Photographic, Dermoscopic, and Histologic Evaluation.

BACKGROUND AND OBJECTIVE: Actinic keratoses (AKs) have been described with varying color and morphology; however, no reports have demonstrated associations between color, vasculature, and inflammation. In this retrospective study, we analyze the clinical, dermoscopic, and histopathologic features of AKs to elucidate this relationship. METHODS: A retrospective search for patients diagnosed with AK between January 2018 and October 2019 was performed. Clinical and dermoscopic photographs and pathology slides for all included subjects were reviewed. RESULTS: Forty-nine images and histopathology slides were analyzed. Dermoscopy of white AKs demonstrated scale and absence of erythema with corresponding absence of inflammation on histopathology. Dermoscopy of brown AKs revealed pseudonetwork, absent scale, and a variable vessel pattern with pigment incontinence and absence of inflammation on histopathology. Red AKs had a distinct polymorphous vessel pattern and presence of erythema on dermoscopy. On histopathology, about half of samples showed increased vascularity and variable inflammation. Pink AK dermoscopy revealed a presence of erythema with corresponding presence of inflammation on histopathology. CONCLUSION: This report adds to our understanding of AKs and confirms that, in general, the pinker or redder the AK, the more prominent the inflammatory infiltrate and vasculature, respectively. Dermatologists should continue to use their diagnostic skills to successfully diagnose and triage AKs.

Clinical, morphologic, and genomic findings in ROS1 fusion Spitz neoplasms.

The presence of a characteristic chimeric fusion as the initiating genomic event is one defining feature of Spitz neoplasms. Characterization of specific subtypes of Spitz neoplasms allows for better recognition facilitating diagnosis. Data on clinical outcomes of the specific tumor types may help in predicting behavior. In this study we present the largest series to date on ROS1 fusion Spitz neoplasms. We present the clinical, morphologic, and genomic features of 17 cases. We compared the morphologic features of these 17 cases to a cohort of 99 other non-ROS1 Spitz neoplasms to assess for features that may have high specificity for ROS1 fusions. These tumors consisted of ten Spitz nevi and seven Spitz tumors. None of the cases met criteria for a diagnosis of Spitz melanoma. Morphologically, the ROS1 fusion tumors of this series were characterized by a plaque-like or nodular silhouette, often densely cellular intraepidermal melanocyte proliferation, frequent pagetosis, tendency toward spindle cell cytomorphology, low grade nuclear atypia, and floating nests with occasional transepidermal elimination. However, there was a significant range in microscopic appearances, including two cases with morphologic features of a desmoplastic Spitz nevus. Different binding partners to ROS1 were identified with PWWP2A and TPM3 being the most common. No case had a recurrence or metastasis. Our findings document that most ROS1 fusion Spitz neoplasms have some typical characteristic microscopic features, while a small proportion will have features overlapping with other genomic subtypes of Spitz neoplasms. Preliminary evidence suggests that they tend to be indolent or low grade neoplasms.

A Series of RET Fusion Spitz Neoplasms With Plaque-Like Silhouette and Dyscohesive Nesting of Epithelioid Melanocytes.

ABSTRACT: Two distinct studies have shown that RET fusions are found in 3%-4% of Spitz neoplasms. RET fusions have been well described in papillary thyroid cancer, non-small-cell lung cancer, breast cancer, and soft-tissue mesenchymal tumors as well as some other neoplasms. However, there are no comprehensive descriptions to date of the characteristic morphologic, clinical, or genomic findings in RET fusion Spitz neoplasms. In this study, we identified 5 cases of RET fusion Spitz neoplasms. These tumors showed characteristic morphologic features which included plaque-like silhouette and monotonous epithelioid cytology with expansile and dyscohesive nesting. Four of 5 patients including 1 diagnosed as Spitz melanoma had clinical follow-up all of which was uneventful. Furthermore, we describe the genomic sequences in 4 of these cases, 2 of which have previously described KIF5B-RET fusion and 2 of which had a novel LMNA-RET fusion. We believe this report significantly contributes to our current knowledge regarding Spitz neoplasms and describes characteristics features which can help with recognition of the RET subgroup of Spitz.

BRAF fusion Spitz neoplasms; clinical morphological, and genomic findings in six cases.

BACKGROUND: Fusions involving the BRAF gene are responsible for 5% of Spitz neoplasms. To better characterize them, we report the clinical, morphological, and genomic findings of six BRAF fusion Spitz tumors. METHODS: The morphological, clinical, and molecular findings of six BRAF fusion Spitz neoplasms assessed by next generation sequencing (NGS) were compared to a control set of Spitz without BRAF fusions. RESULTS: BRAF fusion Spitz tumors had frequent predominance of epithelioid morphology (4/6 cases), frequent high-grade nuclear atypia and pleomorphism (5/6 cases), and a frequent desmoplastic base (3/6 cases). Five of six cases were diagnosed as atypical Spitz tumor and one as Spitz nevus. All cases had uneventful clinical follow-up. There were five different fusion partners, with CLIP2 being the most frequent. Secondary pathogenic mutations were frequent and chromosomal copy number changes were seen in three of six cases by an NGS platform. CONCLUSIONS: BRAF fusions Spitz usually have epithelioid morphology, high-grade nuclear atypia, and desmoplasia. Chromosomal copy number changes are not infrequent. While our cases had uneventful follow-up, a meta-analysis of the literature suggests that among the fusion subtypes associated with Spitz tumors, they are among the subgroups more likely to develop distant metastasis.

Melanocytic Neoplasms With MAP2K1 in Frame Deletions and Spitz Morphology.

With the advent of better molecular characterization of Spitz melanocytic neoplasms, there has been increasing effort to better understand and describe the relationships between specific driver fusion and/or mutations with the clinical and histomorphological characteristics of the lesions. Structural rearrangements in mitogen activated protein kinase genes have recently been noted to be important in Spitz neoplasms. Only very few reports, however, have described in detail melanocytic tumors with in frame deletions in MAP2K1. Cases in the literature with this aberration have been described as having a diagnosis of Spitz, deep penetrating nevi, or pigmented epithelioid melanocytoma. In this study, we describe a cohort of 6 cases with MAP2K1 activating in frame deletions. The morphologic spectrum of the cases was broad. Common features of these cases include Spitzoid cytomorphology (5/6) cases, prominent melanin pigmentation (4/6) cases, and deep penetrating nevi-like plexiform architecture (3/6) cases. The diagnoses at the time of clinical care of these cases included nevus of Reed (1/6), desmoplastic Spitz tumor (1/6), BAPoma (1/6), deep penetrating melanocytic nevus (2/6), and melanoma (1/6). Clinical follow-up was available in 3 of the 6 cases. None of the patients had a tumor recurrence. This builds on the growing literature to help expand the spectrum of changes associated with Spitzoid melanocytic neoplasms.

PD-1 Blockade with Pembrolizumab in Advanced Merkel-Cell Carcinoma.

BACKGROUND: Merkel-cell carcinoma is an aggressive skin cancer that is linked to exposure to ultraviolet light and the Merkel-cell polyomavirus (MCPyV). Advanced Merkel-cell carcinoma often responds to chemotherapy, but responses are transient. Blocking the programmed death 1 (PD-1) immune inhibitory pathway is of interest, because these tumors often express PD-L1, and MCPyV-specific T cells express PD-1. METHODS: In this multicenter, phase 2, noncontrolled study, we assigned adults with advanced Merkel-cell carcinoma who had received no previous systemic therapy to receive pembrolizumab (anti-PD-1) at a dose of 2 mg per kilogram of body weight every 3 weeks. The primary end point was the objective response rate according to Response Evaluation Criteria in Solid Tumors, version 1.1. Efficacy was correlated with tumor viral status, as assessed by serologic and immunohistochemical testing. RESULTS: A total of 26 patients received at least one dose of pembrolizumab. The objective response rate among the 25 patients with at least one evaluation during treatment was 56% (95% confidence interval [CI], 35 to 76); 4 patients had a complete response, and 10 had a partial response. With a median follow-up of 33 weeks (range, 7 to 53), relapses occurred in 2 of the 14 patients who had had a response (14%). The response duration ranged from at least 2.2 months to at least 9.7 months. The rate of progression-free survival at 6 months was 67% (95% CI, 49 to 86). A total of 17 of the 26 patients (65%) had virus-positive tumors. The response rate was 62% among patients with MCPyV-positive tumors (10 of 16 patients) and 44% among those with virus-negative tumors (4 of 9 patients). Drug-related grade 3 or 4 adverse events occurred in 15% of the patients. CONCLUSIONS: In this study, first-line therapy with pembrolizumab in patients with advanced Merkel-cell carcinoma was associated with an objective response rate of 56%. Responses were observed in patients with virus-positive tumors and those with virus-negative tumors. (Funded by the National Cancer Institute and Merck; ClinicalTrials.gov number, NCT02267603.).

Association of PD-1/PD-L axis expression with cytolytic activity, mutational load, and prognosis in melanoma and other solid tumors.

Programmed cell death protein-1 (PD-1)/programmed death ligand-1 (PD-L1) checkpoint blockade has led to remarkable and durable objective responses in a number of different tumor types. A better understanding of factors associated with the PD-1/PD-L axis expression is desirable, as it informs their potential role as prognostic and predictive biomarkers and may suggest rational treatment combinations. In the current study, we analyzed PD-L1, PD-L2, PD-1, and cytolytic activity (CYT) expression, as well as mutational density from melanoma and eight other solid tumor types using The Cancer Genome Atlas database. We found that in some tumor types, PD-L2 expression is more closely linked to Th1/IFNG expression and PD-1 and CD8 signaling than PD-L1 In contrast, mutational load was not correlated with a Th1/IFNG gene signature in any tumor type. PD-L1, PD-L2, PD-1, CYT expression, and mutational density are all positive prognostic features in melanoma, and conditional inference modeling revealed PD-1/CYT expression (i.e., an inflamed tumor microenvironment) as the most impactful feature, followed by mutational density. This study elucidates the highly interdependent nature of these parameters, and also indicates that future biomarkers for anti-PD-1/PD-L1 will benefit from tumor-type-specific, integrated, mRNA, protein, and genomic approaches.

Biodegradable STING agonist nanoparticles for enhanced cancer immunotherapy.

Therapeutic cancer vaccines require adjuvants leading to robust type I interferon and proinflammatory cytokine responses in the tumor microenvironment to induce an anti-tumor response. Cyclic dinucleotides (CDNs), a potent Stimulator of Interferon Receptor (STING) agonist, are currently in phase I trials. However, their efficacy may be limited to micromolar concentrations due to the cytosolic residence of STING in the ER membrane. Here we utilized biodegradable, poly(beta-amino ester) (PBAE) nanoparticles to deliver CDNs to the cytosol leading to robust immune response at >100-fold lower extracellular CDN concentrations in vitro. The leading CDN PBAE nanoparticle formulation induced a log-fold improvement in potency in treating established B16 melanoma tumors in vivo when combined with PD-1 blocking antibody in comparison to free CDN without nanoparticles. This nanoparticle-mediated cytosolic delivery method for STING agonists synergizes with checkpoint inhibitors and has strong potential for enhanced cancer immunotherapy.

Biodegradable nanoellipsoidal artificial antigen presenting cells for antigen specific T-cell activation.

Non-spherical nanodimensional artificial antigen presenting cells (naAPCs) offer the potential to systemically induce an effective antigen-specific immune response. In this report it is shown biodegradable ellipsoidal naAPCs mimic the T-Cell/APC interaction better than equivalent spherical naAPCs. In addition, it is demonstrated ellipsoidal naAPCs offer reduced non-specific cellular uptake and a superior pharmacokinetic profile compared to spherical naAPCs.

Differential polymer structure tunes mechanism of cellular uptake and transfection routes of poly(ß-amino ester) polyplexes in human breast cancer cells.

Successful gene delivery with nonviral particles has several barriers, including cellular uptake, endosomal escape, and nuclear transport. Understanding the mechanisms behind these steps is critical to enhancing the effectiveness of gene delivery. Polyplexes formed with poly(ß-amino ester)s (PBAEs) have been shown to effectively transfer DNA to various cell types, but the mechanism of their cellular uptake has not been identified. This is the first study to evaluate the uptake mechanism of PBAE polyplexes and the dependence of cellular uptake on the end group and molecular weight of the polymer. We synthesized three different analogues of PBAEs with the same base polymer poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) (B4S4) but with small changes in the end group or molecular weight. We quantified the uptake and transfection efficiencies of the pDNA polyplexes formulated from these polymers in hard-to-transfect triple negative human breast cancer cells (MDA-MB 231). All polymers formed positively charged (10-17 mV) nanoparticles of ∼200 nm in size. Cellular internalization of all three formulations was inhibited the most (60-90% decrease in cellular uptake) by blocking caveolae-mediated endocytosis. Greater inhibition was shown with polymers that had a 1-(3-aminopropyl)-4-methylpiperazine end group (E7) than the others with a 2-(3-aminopropylamino)-ethanol end group (E6) or higher molecular weight. However, caveolae-mediated endocytosis was generally not as efficient as clathrin-mediated endocytosis in leading to transfection. These findings indicate that PBAE polyplexes can be used to transfect triple negative human breast cancer cells and that small changes to the same base polymer can modulate their cellular uptake and transfection routes.

Association between pathologic response and survival after neoadjuvant therapy in lung cancer.

Neoadjuvant immunotherapy plus chemotherapy improves event-free survival (EFS) and pathologic complete response (0% residual viable tumor (RVT) in primary tumor (PT) and lymph nodes (LNs)), and is approved for treatment of resectable lung cancer. Pathologic response assessment after neoadjuvant therapy is the potential analog to radiographic response for advanced disease. However, %RVT thresholds beyond pathologic complete response and major pathologic response (≤10% RVT) have not been explored. Pathologic response was prospectively assessed in the randomized, phase 3 CheckMate 816 trial (NCT02998528), which evaluated neoadjuvant nivolumab (anti-programmed death protein 1) plus chemotherapy in patients with resectable lung cancer. RVT, regression and necrosis were quantified (0-100%) in PT and LNs using a pan-tumor scoring system and tested for association with EFS in a prespecified exploratory analysis. Regardless of LN involvement, EFS improved with 0% versus >0% RVT-PT (hazard ratio = 0.18). RVT-PT predicted EFS for nivolumab plus chemotherapy (area under the curve = 0.74); 2-year EFS rates were 90%, 60%, 57% and 39% for patients with 0-5%, >5-30%, >30-80% and >80% RVT, respectively. Each 1% RVT associated with a 0.017 hazard ratio increase for EFS. Combining pathologic response from PT and LNs helped differentiate outcomes. When compared with radiographic response and circulating tumor DNA clearance, %RVT best approximated EFS. These findings support pathologic response as an emerging survival surrogate. Further assessment of the full spectrum of %RVT in lung cancer and other tumor types is warranted. ClinicalTrials.gov registration: NCT02998528 .

The effect and role of carbon atoms in poly(ß-amino ester)s for DNA binding and gene delivery.

Polymeric vectors for gene delivery are a promising alternative for clinical applications, as they are generally safer than viral counterparts. Our objective was to further our mechanistic understanding of polymer structure-function relationships to allow the rational design of new biomaterials. Utilizing poly(ß-amino ester)s (PBAEs), we investigated polymer-DNA binding by systematically varying the polymer molecular weight, adding single carbons to the backbone and side chain of the monomers that constitute the polymers, and varying the type of polymer end group. We then sought to correlate how PBAE binding affects the polyplex diameter and ζ potential, the transfection efficacy, and its associated cytotoxicity in human breast and brain cancer cells in vitro. Among other trends, we observed in both cell lines that the PBAE-DNA binding constant is biphasic with the transfection efficacy and that the optimal values of the binding constant with respect to the transfection efficacy are in the range (1-6) × 10(4) M(-1). A binding constant in this range is necessary but not sufficient for effective transfection.

Pembrolizumab-Induced Lichen Planus Pemphigoides in a Patient with Metastatic Adrenocortical Cancer: A Case Report and Literature Review.

While the advent of immune-checkpoint inhibitors has revolutionized cancer therapy, immune-related adverse effects (irAEs) have also been on the rise. Cutaneous toxicities are among the most common irAEs, especially in the context of programmed cell death protein-1 (PD-1) inhibitors like pembrolizumab. Herein, we report a case of anti-PD-1-induced lichen planus pemphigoides (LPP)-a rare autoimmune blistering disorder with characteristics of both lichen planus and bullous pemphigoid. To our knowledge, this is the first reported case of LPP following anti-PD-1 therapy for metastatic adrenocortical cancer. Recognizing that LPP is within the spectrum of irAEs is important, especially as the indications for immunotherapy grow to include rarer malignancies like adrenocortical cancer. In addition to our case presentation, we also provide a comprehensive review of the literature surrounding immunotherapy-induced LPP-highlighting key characteristics towards the early recognition and clinical management of this cutaneous irAE.

Biomaterial-Mediated Genetic Reprogramming of Merkel Cell Carcinoma and Melanoma Leads to Targeted Cancer Cell Killing In Vitro and In Vivo.

Tumor immunotherapy is a promising anticancer strategy; however, tumor cells may employ resistance mechanisms, including downregulation of major histocompatibility complex (MHC) molecules to avoid immune recognition. Here, we investigate reprogramming nanoparticles (NPs) that deliver immunostimulatory genes to enhance immunotherapy and address defective antigen presentation in skin cancer in vitro and in vivo. We use a modular poly(beta-amino ester) (PBAE)-based NP to deliver DNA encoding 4-1BBL, IL-12, and IFNγ to reprogram human Merkel cell carcinoma (MCC) cells in vitro and mouse melanoma tumors in vivo to drive adaptive antitumor immune responses. Optimized NP formulations delivering 4-1BBL/IL-12 or 4-1BBL/IL-12/IFNγ DNA successfully transfect MCC and melanoma cells in vitro and in vivo, respectively, resulting in IFNγ-driven upregulation of MHC class I and II molecules on cancer cells. These NPs reprogram the tumor immune microenvironment (TIME) and elicit strong T-cell-driven immune responses, leading to cancer cell killing and T-cell proliferation in vitro and slowing tumor growth and improving survival rates in vivo. Based on expected changes to the tumor immune microenvironment, particularly the importance of IFNγ to the immune response and driving both T-cell function and exhaustion, next-generation NPs codelivering IFNγ were designed. These offered mixed benefits, exchanging improved polyfunctionality for increased T-cell exhaustion and demonstrating higher systemic toxicity in vivo. Further profiling of the immune response with these NPs provides insight into T-cell exhaustion and polyfunctionality induced by different formulations, providing a greater understanding of this immunotherapeutic strategy.

Uptake and transfection with polymeric nanoparticles are dependent on polymer end-group structure, but largely independent of nanoparticle physical and chemical properties.

Development of nonviral particles for gene delivery requires a greater understanding of the properties that enable gene delivery particles to overcome the numerous barriers to intracellular DNA delivery. Linear poly(beta-amino) esters (PBAE) have shown substantial promise for gene delivery, but the mechanism behind their effectiveness is not well quantified with respect to these barriers. In this study, we synthesized, characterized, and evaluated for gene delivery an array of linear PBAEs that differed by small changes along the backbone, side chain, and end group of the polymers. We examined particle size and surface charge, polymer molecular weight, polymer degradation rate, buffering capacity, cellular uptake, transfection, and cytotoxicity of nanoparticles formulated with these polymers. Significantly, this is the first study that has quantified how small differential structural changes to polymers of this class modulate buffering capacity and polymer degradation rate and relates these findings to gene delivery efficacy. All polymers formed positively charged (zeta potential 21-29 mV) nanosized particles (∼150 nm). The polymers hydrolytically degraded quickly in physiological conditions, with half-lives ranging from 90 min to 6 h depending on polymer structure. The PBAE buffering capacities in the relevant pH range (pH 5.1-7.4) varied from 34% to 95% protonatable amines, and on a per mass basis, PBAEs buffered 1.4-4.6 mmol of H(+)/g. When compared to 25 kDa branched polyethyleneimine (PEI), PBAEs buffer significantly fewer protons/mass, as PEI buffers 6.2 mmol of H(+)/g over the same range. However, due to the relatively low cytotoxicity of PBAEs, higher polymer mass can be used to form particles than with PEI and total buffering capacity of PBAE-based particles significantly exceeds that of PEI. Uptake into COS-7 cells ranged from 0% to 95% of cells and transfection ranged from 0% to 93% of cells, depending on the base polymer structure and the end modifications examined. Five polymers achieved higher uptake and transfection efficacy with less toxicity than branched-PEI control. Surprisingly, acrylate-terminated base polymers were dramatically less efficacious than their end-capped versions, in terms of both uptake (1-3% for acrylate, 75-94% for end-capped) and transfection efficacy (0-1% vs 20-89%), even though there are minimal differences between acrylate and end-capped polymers in terms of DNA retardation in gel electrophoresis, particle size, zeta potential, and cytotoxicity. These studies further elucidate the role of polymer structure for gene delivery and highlight that small molecule end-group modification of a linear polymer can be critical for cellular uptake in a manner that is largely independent of polymer/DNA binding, particle size, and particle surface charge.

Gene delivery nanoparticles specific for human microvasculature and macrovasculature.

Endothelial cell dysfunction is a critical component of ocular diseases such as age-related macular degeneration and diabetic retinopathy. An important limitation in endothelial cell research is the difficulty in achieving efficient transfection of these cells. A new polymer library was here synthesized and utilized to find polymeric nanoparticles that can transfect macrovascular (human umbilical vein, HUVECs) and microvascular (human retinal, HRECs) endothelial cells. Nanoparticles were synthesized that can achieve transfection efficiency of up to 85% for HRECs and 65% for HUVECs. These nanoparticle systems enable high levels of expression while avoiding problems associated with viral gene delivery. The polymeric nanoparticles also show cell-specific behavior, with a high correlation between microvascular and macrovascular transfection (R(2) = 0.81) but low correlation between retinal endothelial and retinal epithelial transfection (R(2) = 0.21). These polymeric nanoparticles can be used in vitro as experimental tools and potentially in vivo to target and treat vascular-specific diseases. FROM THE CLINICAL EDITOR: Polymeric nanoparticles were synthesized with the goal of transfecting endothelial cells, which are commonly considered difficult targets. The authors report excellent transfection efficiency of up to 85% for human retinal and 65% for human umbilical vein endothelial cells. These NPs can be used in vitro as experimental tools and potentially in vivo to target and treat vascular-specific diseases.

Calciphylaxis Cutis Associated With Fibroblast Growth Factor Receptor (FGFR) Inhibitor Therapy: A New Challenge.

Specific fibroblast growth factor receptor (FGFR) inhibitors have been developed to treat malignancies harboring fusions or rearrangements in FGFR2 or FGFR3. Here, we report a case of calciphylaxis cutis in association with FGFR inhibitor therapy in a patient with FGFR2 rearranged cholangiocarcinoma. Although calciphylaxis cutis typically arises in the setting of hyperphosphatemia and end-stage renal disease, this patient had preserved renal function, normal serum calcium, and only modestly elevated serum phosphorus levels, which is similar to other recent reports of calciphylaxis in patients receiving FGFR inhibitor therapy. Calciphylaxis cutis is a possible adverse event observed with FGFR inhibitor therapy, and the mechanism of calciphylaxis cutis in association with FGFR inhibitor therapy warrants further investigation.