The pattern dystrophies (PDs) are a group of primarily autosomal dominant inherited macular diseases that cause the deposition of lipofuscin in retinal pigment epithelium (RPE) and may lead to significant vision loss in later life. Patients can develop choroidal neovascularization (CNV) and/ or geographic atrophy (GA) and for this reason they are often misdiagnosed as age-related macular degeneration (AMD). We presented a case of a 66-year-old patient complaining of vision loss in the right eye (RE) for 8 months. At the initial examination, his best corrected visual acuity (BCVA) was 0.6 in the RE. Optical coherence tomography angiography (OCTA), fundus autofluorescence (FAF) and fundus fluorescein angiography (FFA) allowed to diagnose butterfly-shaped PD in both eyes with choroidal neovascularization (CNV) in the RE. The patient was treated with three intravitreal anti-vascular epithelial growth factor  injections during six weeks intervals, which improved and stabilized the BCVA of the RE to 0.7 during the over two-year observation period. Our report contributes to the still limited data regarding CNV associated with butterfly-shaped PDs and the results of treatment with ranibizumab.Abbreviations: AMD = age-related macular degeneration, anti-VEGF = anti-vascular epithelial growth factor, AOFVD = adult-onset foveomacular vitelliform dystrophy, BCVA = best corrected visual acuity, CNV = choroidal neovascularization, FAF = fundus autofluorescence, FFA = fundus fluorescein angiography, GA = geographic atrophy, LE = left eye, MIDD = maternally inherited diabetes and deafness, OCT = optical coherence tomography, OCTA = optical coherence tomography angiography, OU = oculus uterque, both eyes, PD = pattern dystrophy, PDSFF = pattern dystrophy simulating fundus flavimaculatus, PDT = photodynamic therapy, PRPH2 = peripherine-2, RE = right eye, RPE = retinal pigment epithelium, VA = visual acuity Moreover, PDs have been described in association with myotonic dystrophy, Kjellin syndrome, pseudoxanthoma elasticum, maternally inherited diabetes and deafness (MIDD) [2-5].The pattern dystrophies (PDs) are a heterogenous group of primarily autosomal dominantly inherited macular diseases characterized by an abnormal accumulation of lipofuscin material at the level of the retinal pigment epithelium (RPE) caused by mutations in the peripherin 2 gene on chromosome 6p21.1 [6]. PDs usually show bilateral and symmetrical involvement. However, the pattern distribution may be unilateral; each eye of the patient can have a distinct pattern, at different stages and the same mutation may correspond to various inter- and intrafamilial phenotypes [7]. PDs are divided into 5 types based on the pattern of pigment distribution: 1) adult-onset foveomacular vitelliform dystrophy (AOFVD); 2) butterfly-shaped pattern dystrophy; 3) pattern dystrophy simulating fundus flavimaculatus (PDSFF); 4) reticular dystrophy of the RPE; 5) fundus pulverulentus [1]. The onset of symptoms usually occurs in the fourth or fifth decade and involve moderate vision loss and metamorphopsia. Some patients may remain asymptomatic throughout life, but PDs may lead to severe visual impairment due to atrophy of the RPE-photoreceptor complex, development of choroidal neovascularization (CNV) or macular holes as well . Peripherine-2 (PRPH2) is a transmembrane glycoprotein crucial for the formation, stabilization and renewal of the photoreceptor outer segment discs by acting as an adhesive element [9]. The main feature in PDs is accumulation of extracellular material beneath the sensory retina at the fovea due to malfunction of photoreceptor outer segment metabolism [7]. Although various gene mutations have been shown in PDs, the diagnosis can be made mainly with the aid of clinical examination and conventional imaging methods [10]. PDs are a group of congenital disorders caused by the mutation of the peripherin 2. Both autosomal dominant and recessive transmission have been observed and cases of incomplete penetrance or variable expressivity have been reported as well [11]. Central serous chorioretinopathy, Stargardt disease and Best disease should be included in the differential diagnosis [12].The clinical features that distinguish PDs from AMD include a relative earlier age of symptoms onset, the fundoscopic absence of the typical drusen and the presence of yellow-gray pigment in various patterns in the macula that show distinct changes on FAF imaging. However, later stages of PDs may resemble AMD and pigment deposits can resemble drusen. What is more, patients can develop CNV and/ or geographic atrophy (GA) and for this reason these cases are often misdiagnosed as AMD [13]. The evolution of PD has been considered erroneously benign for a long time, probably due to frequent mistakes with more frequent and known macular diseases, above all AMD. It is postulated that 42% of patients with PDs will develop severe visual damage due to the atrophic (26%) or neovascular (18%) complications [14]. However, Casilo et al. [15] revealed that the PD subgroup showed thicker choroidal thickness both centrally and underneath the neovascular lesion in contradistinction to the AMD subgroup. Presumably, the thickening of the outer retinal vessels in the Haller layer could cause secondary damage to the overlying choriocapillaris network and induce ischemic phenomena responsible for neovascularization. Interestingly, one case of spontaneous regression of CNV in PD was observed [16].The precise mechanism of CNV development in PDs has not been established. Initially, it was hypothesized that the metabolic byproducts may accumulate after the RPE digests the mutant PRPH2 proteins, which may disrupt the extracellular matrix that helps maintain the barrier against CNV [17]. What is more, PDT has been used to treat PD-related subfoveal CNV. This approach provided temporary advantageous effect on VA, which was followed by a progressive deterioration in the long-term. Probably, the effect of laser photocoagulation or PDT on primarily impaired RPE cells may lead to greater damage with consequent apoptosis [18].Possible treatment options for CNV associated with PD include laser photocoagulation, photodynamic therapy (PDT) and anti-VEGF therapy. Previously described unexpected enlargement of the scar after laser photocoagulation of an extrafoveal CNV secondary to PD suggests that the PRE may be irreversibly damaged by the thermal insult . Parodi et al. [21] reported a series of 12 patients with PD and subfoveal CNV, who were treated with course of intravitreal bevacizumab and documented a good anatomical and functional outcome. Nangia et al. [22] reported a case of CNV secondary to PD simulating fundus flavimaculatus, whereas Gallego-Pianzo et al. [23] published a series of six patients diagnosed with CNV associated with AOFVD. Successful visual effects after intravitreal ranibizumab treatment were described in both studies. Empeslidis et al. [24] documented the satisfactory results of the use of intravitreal ranibizumab in the treatment of a patient with CNV due to butterfly-shaped PD of the macula.On the contrary, it is documented in literature that due to non-AMD conditions, CNV respond better to anti-VEGF treatment and sometimes even one intravitreal anti-VEGF injection was satisfactory to control the CNV and stabilize VA [15], a comparison between the long-term response to intravitreal ranibizumab among patients with PD-associated CNV and CNV in AMD, showed that eyes with PD had a poorer long-term response and greater tendency to atrophic evolution of the disease. Therefore, it is suggested that CNV associated with PD should not be “overtreated” to avoid further atrophic effects. One of the possible reasons for overtreatment lies in the hypothesis that part of the subretinal fluid could be due to RPE pump failure instead of CNV activity.However, in the pilot study performed by Casillo et al. [Our report contributes to the still limited data regarding the results of anti-VEGF treatment use in CNV associated with butterfly-shaped PD. We achieved improvement and stabilization of anatomical and functional status after three ranibizumab intravitreal injections. It may support the fact that anti-VEGF treatment offers significant benefits in patients with PD-associated CNV. Nevertheless, the further observation of long-term effects and larger study group are required to further improve the understanding of butterfly-shaped PDs and their treatment.Conflict of InterestThe authors declare that there are no conflicts of interests.Informed Consent and Human and Animal Rights statementsThe authors certify that they have obtained all appropriate patient consent forms. The patient has given his consent for his de-identified images and other clinical information to be used for teaching and research purposes. The patient understands that his name and initials will not be published and due efforts will be made to conceal the identity, but anonymity cannot be guaranteed. Authorization for the use of human subjectsEthical approval: The research related to human use complies with all the relevant national regulations, institutional policies, is in accordance with the tenets of the Helsinki Declaration and has been approved by the institutional review board of the Ophthalmology Department, Kornel Gibiński University Clinical Center, Medical University of Silesia, Katowice, Poland.AcknowledgementsNone. Sources of FundingNone. DisclosuresNone.


Advancements in research have led to the steady decline of breast cancer mortality over the past thirty years. However, breast cancer incidence has continued to rise, resulting in an undue burden on healthcare costs and highlighting a great need for more effective breast cancer prevention strategies, including targeted chemo preventative agents. Efforts to understand the etiology of breast cancer have uncovered important roles for nuclear receptors in the development and progression of breast cancer. Targeted therapies to inhibit estrogen receptor (ER) and progesterone receptor (PR) signaling  have shown great promise for the treatment and prevention of hormone receptor (HR)-positive breast cancer. However, these drugs do not prevent HR-negative disease. Therefore, recent efforts have focused on novel targeted therapies with the potential to prevent both HR-positive and HR-negative breast cancer. Among these include drugs that target other nuclear receptors, such as retinoic acid receptor (RAR), retinoid X receptor (RXR) and vitamin D receptor (VDR). In this review we provide an overview of recent preclinical and clinical trials targeting members of the nuclear receptor superfamily for the prevention of breast cancer. In the last 10 years, the incidence of breast cancer in the United States has steadily increased, with more than 280,000 women expected to be diagnosed in 2023. Despite efficacious early detection measures and significant advances in treatment, breast cancer remains the most common cancer diagnosis and second leading cause of cancer death in women, with over 40,000 patients succumbing to the disease each year  appear more frequently in young women (<40 years of age), non-Hispanic black women and women carrying mutations in  disease –20. ThesAt present, the most effective primary prevention strategy for breast cancer is prophylactic surgery, consisting of both bilateral mastectomy and oophorectomy, which can reduce the risk of breast cancer by 90% –23. HoweThe human nuclear receptor superfamily includes 48 evolutionarily conserved transcription factors that recognize and respond to changes in physiological stimuli, such as steroid hormones, cholesterol metabolites and lipophilic vitamins . These rSteroid hormone receptors play a critical role in normal breast development as well as the initiation and progression of breast cancer. These receptors include the estrogen receptor (ER), progesterone receptor (PR), glucocorticoid receptor (GR), androgen receptor (AR), and mineralocorticoid receptor (MR) which primarily act as homodimers for transcription regulation. In the classical mode of genomic action, these receptors are inactive in the cytoplasm without ligand, bound to heat shock proteins for stability. Upon exposure to a ligand, the receptors dimerize and translocate to the nucleus where they interact with co-activators and co-repressors (often determined by the type of ligand) and bind specific responsive elements on DNA to activate or repress target gene transcription . HoweverDuring breast cancer carcinogenesis, steroid hormone receptors often become over or under expressed, resulting in the dysregulation of gene expression that can drive tumorigenesis , 30. It More than a century ago, it was first noted that advanced, inoperable breast tumors can shrink after the removal of the ovaries, becoming the first reported use of endocrine therapy . The mecSeveral generations of selective estrogen receptor modulators (SERMs) have been developed as breast specific ER antagonists with varying effects in other tissues, most notably as ER agonists in the bone and uterus . Tamoxifin situ (DCIS) is considered a non-invasive breast cancer that has been shown to increase the risk for invasive breast cancer , the second generation SERM raloxifene was found to reduce the risk of ER-positive breast cancer by 55–84%, without increasing the incidence of endometrial cancer –63. HoweSeveral phase III prevention trials have explored the use of third generation SERMs for osteoporosis risk reduction in postmenopausal women. In the PEARL trial, lasofoxifene was shown to reduce ER-positive breast cancer by 83% with even fewer reported toxicities than tamoxifen or raloxifene . HoweverBRCA1/2 mutation carriers with breast cancer risk of at least twice the average for age group by models of assessment), 6-months of treatment with Duavee significantly reduced mammographic density, proliferation (as assessed by staining for the Ki-67 proliferation marker) and additional breast cancer risk biomarkers while improving menopause-associated symptoms  has shown potential for breast cancer prevention. In a pilot study including 28 women at high-risk for breast cancer  can decrease tumor proliferation, as measured by Ki-67 expression, comparable to that of high dose tamoxifen (20 mg) . This fiLocalized treatment of tamoxifen via topical application is also being considered to overcome the adverse effects of systemic tamoxifen therapy. A randomized Phase II trial of 4-hydroxytamoxifen gel (Afimoxifen) versus oral tamoxifen, administered pre-surgery to women with DCIS, demonstrated that the antiproliferative effect of topical tamoxifen is similar to oral tamoxifen, but without the systemic endocrine effects, supporting the rationale for 4-hydroxytamoxifen gel in breast cancer prevention . A phaseTo date, clinical trials with SERMs have shown clear efficacy for the prevention of primary and recurrent ER-positive breast cancer, leading to the FDA approval of tamoxifen and raloxifene for high risk women. However, the uptake of SERMs for prevention has been low among high risk women, likely due to concerns about side effects. The field urgently needs a way to effectively prevent ER-positive breast cancer while minimizing adverse events to improve treatment uptake. Ongoing efforts to explore alternative dosing regimens and to develop newer SERMs with reduced toxicity may address these issues but it remains to be seen if patient acceptance will improve. Finally, although SERMs have shown great promise for the prevention of ER-positive breast cancer, they do not prevent ER-negative disease.An alternative strategy to reduce estrogen signaling in ER-positive breast cancer is through the inhibition of aromatase, an enzyme typically expressed in fat, stromal and muscle tissue but also breast cancer, responsible for converting androgens into estrogen . Since tAs with SERMs, several clinical trials have tested the ability of AIs to reduce invasive breast cancer incidence in women with DCIS . The results from this trial are expected in 2023.The AI letrozole has been effective as a first-line treatment for advanced stage ER-positive breast cancer in postmenopausal women and can also provide benefit as an adjuvant therapy for early-stage hormone responsive breast cancer , 89. EarLike SERMs, the major problems with the use of AIs for breast cancer prevention are the associated treatment toxicities  and the inability to prevent ER-negative breast cancer. Despite the promise of AIs for ER-positive breast cancer prevention, their use is largely restricted to postmenopausal women whom lack estrogen-producing ovaries. This unfortunately excludes premenopausal women from the benefits of AIs, specifically those at high-risk for breast cancer who are already in desperate need for effective preventative agents. To date, no AIs have been approved by the FDA for breast cancer prevention, however, they are often considered for breast cancer prevention in high-risk postmenopausal women as “off label” treatments.PGR (NR3C3), the gene encoding the many known isoforms of PR, is a direct target gene of ER and therefore depends on ER expression. For this reason, PR expression by IHC is also prognostic for breast cancer overall and disease-free survival. ER-positive/PR-negative tumors are less responsive to SERMS likely because the loss of PR indicates tumors with nonfunctional ER signaling (PR and its endogenous ligand progesterone (P4) are essential to normal and pregnancy associated mammary gland development. PR mainly exists as two functionally active isoforms; the full-length receptor PR-B preferentially binds with co-activators of gene transcription while the truncated receptor PR-A shows a greater binding affinity for co-repressors –94. In aignaling , 103.Although it is recognized that ER drives PR expression, it is also known that PR-A can inhibit ER transcriptional activity and plays an important role in the formation of breast cancer , 105. ReBecause of the known pro-tumorigenic mechanisms of PR activation, the use of antagonistic selective progesterone receptor modulators (SPRMs) are being investigated for the treatment and prevention of PR-positive breast cancer . These min vivo . In congruence with the mifepristone results, ulipristal acetate also reduced the normal breast tissue mitotic age  and are retained in the nucleus even in the inactive state, bound to DNA response elements with transcriptional co-repressors. Upon binding of a specific ligand, these nuclear receptors will dissociate with inhibitory factors and recruit co-regulators to modulate target gene transcription. There are many nuclear receptors known to dimerize with RXR for transcriptional regulation, including RXR itself as a homodimer . NuclearThe retinoic acid receptor (RAR) subfamily consists of three members: RARα, RARβ and RARγ. These nuclear receptors are activated by the major bioactive metabolite of vitamin A, retinoic acid (RA), and heterodimerize with the retinoid X receptor (RXR) to regulate the transcription of target genes involved in cell growth, differentiation and death. The first chemo-prevention trial with retinoids was in patients with head and neck cancers at high risk for recurrent and second primary tumors. This study demonstrated that treatment with 13-cis-RA (isotretinoin) could significantly reduce the formation of second primary tumors  and showin vitro and in vivo, similar to the effect of tamoxifen  while for others, the heterodimer partner ligand must be present for transcription modulation (non-permissive heterodimer). RXR can also form heterodimers with orphan nuclear receptors, lacking known endogenous ligands. Many synthetic agonists with high specificity for RXR (rexinoids), have been developed to mimic the growth inhibitory effects of RAR agonists while avoiding the toxicity associated with natural retinoids.BRCA1/2 mutations (NCT00055991). Pre- and post-menopausal women were treated with bexarotene or placebo for 4 weeks and proliferation associated biomarkers were compared between core needle biopsies at pre and post treatment timepoints. Results from this trial revealed no differences in Ki-67 or cyclin D1 expression between bexarotene and placebo treated groups. Though, in a subgroup analysis of postmenopausal women only, the decrease in cyclin D1 expression was significantly reduced by 65% compared to placebo (The third-generation retinoid bexarotene (LGD1069) has been widely studied in the prevention of many cancers. In preclinical models of ER-negative BC, our lab has shown that 9-cis-RA and bexarotene can suppress mammary tumorigenesis and prevent the development of premalignant lesions –145. Thi placebo , suggest placebo . However placebo .To avoid the unwanted toxicity associated with weak RAR-binding, even more specific RXR agonists have been developed. Our group has previously shown that the rexinoid LG100268 is more effective for mammary tumor prevention with reduced toxicity compared to bexarotene in preclinical models of ER-negative breast cancer . Karen LThe highly specific rexinoid, 9cUAB30, has also been evaluated for chemoprevention in preclinical models of BC. Using the MNU induced mammary cancer model in female Sprague–Dawley rats, it was demonstrated that 9cUAB30 can delay the formation of tumors without signs of treatment toxicity . Based oIn the inactive state, vitamin D receptor (VDR) exists as a monomer in solution or homodimer bound to VDR response elements on DNA. When activated by its endogenous ligand, 1,25-dihydroxyvitamin D3 , VDR preferentially heterodimerizes with RXR to regulate the transcription of VDR target genes –165. In in vitro treatment with vitamin D inhibits the growth of breast cancer cell lines , there was no statistically significant difference in mammographic density between 20,000 IU per week and placebo treatment D, used in clinical trials to assess vitamin D uptake, may not reflect local concentrations of active metabolite in the breast tissue. It is also possible that the concentration of other nuclear receptor ligands can inhibit the activity of VDR. For example, RXR ligands are known to destabilize the VDR-RXR heterodimer  and coulSince endocrine targeted therapies can effectively prevent hormone receptor positive breast cancer and other nuclear receptor targets have been shown to prevent hormone receptor negative BC, it is reasonable that combination treatments for the prevention of breast cancer should be explored. Several pre-clinical trials have already investigated the effects of RXR agonists with ER modulators for the prevention of BC. Combination treatment of the rexinoid 9cUAB30 with the SERM tamoxifen was shown to inhibit the formation of mammary tumors in the MNU induced rat model more than single agent treatment . In the More recently, it has been demonstrated that retinoids can block PR binding at shared DNA response element regions and inhibit P4 stimulated growth of ER-positive breast cancer xenografts, suggesting a cross-talk between PR and RAR in regulating a subset of hormone responsive breast cancer . It has Despite promising preclinical results, very few combinations have been explored in clinical trials, likely due to toxicity concerns. In a randomized double-blind phase II trial of low-dose tamoxifen and the retinoid fenretinide alone or in combination in high-risk premenopausal women , most with intraepithelial neoplasia, it was found that both tamoxifen and fenretinide alone could reduce breast density and neoplastic events compared to placebo. However, the combination of low-dose tamoxifen with fenretinide did not show synergistic interaction, despite being well-tolerated by patients , 192.Targeting nuclear receptors for breast cancer prevention has been shown to be possible in multiple phase III trials. At present, only the anti-estrogen SERMs tamoxifen and raloxifene are FDA approved for breast cancer prevention. However, several other drugs have been found to reduce breast cancer risk in women without breast cancer including other SERMs (lasofoxifene) and aromatase inhibitors (anastrozole and exemestane). These drugs are FDA-approved for the treatment of breast cancer, so they can be used off-label, but are generally not used often. The most commonly used off-label drugs for breast cancer prevention are the aromatase inhibitors, anastrozole or exemestane, which can be considered for women who have contraindications for SERM use (such as having a prior deep venous thrombosis).There is no “predictive biomarker” in the normal breast tissue to predict a response to a preventive drug. Thus, breast cancer prevention drugs are chosen based on their overall efficacy and tolerability to individual women. Mild common toxicities (hot flushes) and rare more serious toxicities (blood clots and uterine cancer) have limited the use of SERMs for prevention among eligible high-risk women. AIs are more effective in treating and preventing breast cancer but are associated with a different spectrum of side effects that also limit the use of these agents for breast cancer prevention. Lower doses, modified treatment schedules, and local administration routes are currently being explored to determine if these efficacious drugs can be given more safely. Advancements in localized treatments are expected to overcome toxicity concerns and improve tolerability among high-risk women. But despite the success and specificity of SERMs and AIs for prevention, these endocrine therapies are only effective for the prevention of ER-positive breast cancer and have no effect on ER-negative disease.de novo and acquired resistance of ER-positive breast cancers to anti-estrogen therapy, it is common practice to add CDK4/6 inhibitors  to anti-estrogen therapy for the treatment of early and late stage breast cancers. In addition, anti-estrogen selective estrogen degraders (SERDS), such as fulvestrant, are also used to treat metastatic ER-positive breast cancers that have arisen after prior anti-estrogen therapy. These strategies have not yet been used in the prevention setting. However, for ER-positive breast cancers that arise after anti-estrogen preventive therapy, the combination of a different anti-estrogen drug plus a CDK4/6 inhibitor is often used for treatment.Another concern for the use of anti-estrogen therapy is the intrinsic resistance that can occur among a small subset of patients. In the treatment setting, to overcome The most common form of intrinsic resistance to anti-estrogen therapy in the prevention setting is the development of an ER-negative breast cancer, not prevented by anti-estrogen therapy. ER-negative breast cancers that arise after anti-estrogen preventive therapy are currently treated with standard chemotherapy. A major focus of the field has been to develop targeted preventive strategies (drug therapy or vaccines) to prevent these ER-negative breast cancers. While several agents reviewed here have shown promise in preclinical models for the prevention of ER-negative breast cancers, none have yet been approved for human use.Recent efforts to target AR have shown promising results in decreasing the growth of both HR-positive and HR-negative breast cancer. A preclinical treatment study using the antiandrogen enzalutamide demonstrated growth inhibition of AR-positive breast cancer including in a TNBC model . In addiBRCA mutant breast cancer , the Breast Cancer Research Foundation (PB) and NCI PREVENT Program .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Oral cancer is a disease primarily in older adults and extremely rare in young adults. Risk factors for oral cancer are irritants such as tobacco smoke and alcohol and chronic mechanical irritants but mechanisms involved in carcinogenesis in young adults are unclear because of less exposure to their risk factors. Herein, we report a rare case of gingival squamous cell carcinoma in a 19-year-old female patient, in whom the tumor predictably originated in the gingival sulcular epithelium. Histopathological examination of the resected tissue showed a cancer cell nest invading from the gingival sulcular epithelium without a breakdown of the basement membrane of the marginal gingival epithelium. Six years after the surgery, no recurrence or metastasis has been detected. The prevalence of oral cancers is greater in older adults. Its risk factors include irritants such as tobacco smoke, alcohol, chronic mechanical irritation, poor oral hygiene practice, chronic ulceration from an ill-fitting denture, and others . AlthougA 19-year-old female patient was referred to our department with 5 x 10 mm gingival swelling on the lingual aspect of the mandibular left second molar. The gingival swelling was accompanied by contact pain, and papillary hyperplasia was observed in the gingival crevice on the lingual aspect Figure . The adjPanoramic and dental radiographic images revealed alveolar bone resorption between the mandibular left first and second molars Figures , 2B.The presence of a dental prosthesis prevented contrast-enhanced CT scans from visualizing the tumor. Magnetic resonance imaging (MRI) captured a mass with a slight contrast enhancement effect at the lesion Figure .No signs of metastasis to lymph nodes or any other tissues or organs were detected. The result of the tissue biopsy showed a highly differentiated squamous cell carcinoma. The patient was diagnosed with squamous cell carcinoma (SCC) of the left mandibular gingiva (cT1N0M0). The patient was treated by excisional surgery to remove the entire tumor along with a safety margin of surrounding tissues above the mandibular canal, involving the mandibular bone, gingiva, and tooth. After marginal resection of the mandible, the wound had primary closure. Histopathological examination of the tissue resected during the procedure did not show any breakdown of the basement membrane of marginal gingival epithelium. However, it revealed an invasion of a cancer cell nest under the basement membrane extending into the gingival sulcular epithelium Figures -4C.The pathological diagnosis was mandibular gingival SCC (T1N0M0). As the resected specimen showed histologically negative margins, follow-up was performed without adjuvant therapy. After six years of the surgery, no sign of recurrence or metastasis is observed Figures , 5B.The rate of oral cancer is the highest among persons aged 60-80 years and slightly higher in males than females . AlthougPeriodontal disease, a chronic inflammatory disease, may be linked with developing cancers in the oral cavity ,15. In pThe tongue accounts for most oral cancer sites, followed by the gingiva . The locThis case report presented a rare case of squamous cell carcinoma originating from the gingival sulcular epithelium in a 19-year-old female. The patient in this report developed SCC despite no history of exposure to risk factors such as smoking, drinking, and periodontal disease. Furthermore, this patient is a rare case in which we may determine pathologically that it originated in the gingival sulcular epithelium. Further research is needed to learn more about the exact site and cause of carcinogenesis.


A phenotypic hallmark of cancer is aberrant transcriptional regulation. Transcriptional regulation is controlled by a complicated array of molecular factors, including the presence of transcription factors, the deposition of histone post-translational modifications, and long-range DNA interactions. Determining the molecular identity and function of these various factors is necessary to understand specific aspects of cancer biology and reveal potential therapeutic targets. Regulation of the genome by specific factors is typically studied using chromatin immunoprecipitation followed by sequencing (ChIP-Seq) that identifies genome-wide binding interactions through the use of factor-specific antibodies. A long-standing goal in many laboratories has been the development of a ‘reverse-ChIP’ approach to identify unknown binding partners at loci of interest. A variety of strategies have been employed to enable the selective biochemical purification of sequence-defined chromatin regions, including single-copy loci, and the subsequent analytical detection of associated proteins. This review covers mass spectrometry techniques that enable quantitative proteomics before providing a survey of approaches toward the development of strategies for the purification of sequence-specific chromatin as a ‘reverse-ChIP’ technique. A fully realized reverse-ChIP technique holds great potential for identifying cancer-specific targets and the development of personalized therapeutic regimens. Among the many remarkable phenotypes differentiating cancer from healthy states, transcriptional dysregulation is one of the most marked . Indeed,cis- and trans-acting factors. Promoters proximal to gene bodies and enhancers distal from gene bodies are cis elements that contain recognition elements for transcription factors to bind to sequence-specific elements. Transcription factors in turn recruit other trans-acting factors involved in transcription and chromatin remodeling. Chromatin remodelers can install epigenetic marks, such as histone posttranslational modifications and DNA methylation, altering the standard ‘histone code’ ..29].A reliable reverse-ChIP approach would prove as powerful for reverse genomics as ChIP-Seq has been for forward genomics. Understanding how genomic differences give rise to phenotypic diversity requires identifying the molecular mediators that are present in experimentally identified regions. An ideal reverse-ChIP technique would involve the enrichment of an experimenter-defined genomic region to homogeneity from background chromatin followed by identification of the molecular composition of the region. Identifying the molecular composition of an isolated locus would involve determining the presence of protein and nucleic acid factors, their relative stoichiometry, and any associated posttranslational or posttranscriptional modifications. Achieving this ambitious goal is limited in practice by experimental realities. For example, histone proteins are ubiquitously present in the background and the experimenter-defined locus. Though histones with a specific posttranslational modification may be detected in a reverse-ChIP experiment, identifying statistically significantly enriched histone modifications is much more difficult as a result of the high background signal due to the structural importance of histones in chromatin architecture. False negatives can result from proteins truly present at a locus of interest that are also expressed at high levels elsewhere. A more tractable experimental goal is the identification of uniquely bound molecular factors in the region, such as the presence of transcription factors that specifically regulate the region of interest and nowhere else. Contemporary reverse-ChIP experiments tend to focus on identifying molecular regulators uniquely present at the chosen locus and demonstrating the importance of those factors in regulating genomic expression.Several demanding experimental difficulties not present in ChIP-Seq must be surmounted to develop a successful reverse-ChIP approach . The issAnother issue in reverse-ChIP experiments is the high background. Unlike ChIP-Seq, which can include a PCR amplification step to increase signal, a reverse-ChIP technique must directly detect proteins present at the chosen locus. For some regulatory regions of interest , there are only two copies of the locus per diploid cell , while the remaining billions of base pairs in the genome and their associated protein complexes are sources of nonspecific background signal. A typical promoter is ~1 kilobase in length, meaning it comprises approximately 0.0001% of the 3 billion base pair human genome. However, a 1 kb promoter represents approximately 0.0083% of the 12 Mb yeast genome, meaning that there is more signal relative to the background for an individual, single-copy regulatory region in yeast. Accordingly, yeast genetics has been a testing ground for locus-specific chromatin isolation. Complicating matters further is the significant biochemical similarity that exists across all genomic regions: negatively charged DNA tightly winds around positively charged nucleosomes composed of histone proteins. Distinguishing proteins present at a specific locus from ubiquitously expressed chromatin structural proteins is a nontrivial challenge.One strategy to overcome the difficulties associated with the high background is to target repetitive elements such as telomeres, which comprise ~0.01% of the human genome. Telomeres are repetitive regions that protect the ends of chromosomes during cell division and are critical to cancer biology. In healthy states, telomeres shorten as cells divide until they reach replicative senescence. Telomeres play a key role in cancer biology since cancer cells must activate mechanisms to maintain telomere length to allow for perpetual cell division ,38,39,40Nonrepetitive genomic elements require high input volumes to generate enough signal for reliable detection above the background. Detecting the proteins associated with single-copy loci is the ultimate goal of reverse-ChIP experiments. The widespread utilization of alternative promoters is a common feature across many types of cancer ,42,43,44−15 moles) level. Therefore, at least several hundred million cells are required to detect a singly-bound protein present at a unique genetic locus (~108 molecules). Fortunately, transcription factor binding sites are distributed periodically near gene promoters to increase the local concentration of regulatory factors, reducing the technical detection requirements beyond that demanding limit . . 114]. Tbacteria . Protein2 plates per condition to address this problem. By contrast, protein-based purification can have higher sensitivity. Rapid immunoprecipitation of endogenous proteins (RIME), an antibody-based ChIP strategy, can detect novel proteins involved in chromatin complexes with as few as 106 cells . . 118]. Sin yeast . Purificin yeast . The aut6 cells by using a sequential immunoprecipitation strategy. After purifying the tagged region with an anti-FLAG antibody, the chromatin was purified with an antibody against Stat1, a known protein binding to the locus. Stat1 was detected by Western blotting, demonstrating the feasibility of using iChIP as a reverse-ChIP strategy. A key step in any iChIP experiment is performing control experiments to demonstrate that the introduction of the DNA recognition sequence does not disrupt the regulatory function of the region under study.An advancement in protein-based purification strategies came with the development of insertional chromatin immunoprecipitation (iChIP) . BuildinDrosophila embryos correlated chromatin conformation with gene expression in a cell type-specific manner . . IRF-1 lma cells . The trama cells . After itrometry . The dattrometry . The proSome researchers have utilized CRISPR-enChIP to interrogate repetitive genomic elements beyond telomeres and centromeres. More than half the genome is composed of repetitive elements, and these regions are a large source of human genetic variation and have contributions to health and disease ,157. In GAL1 promoter in yeast in a CRISPR-ChAP-MS approach . . 164]. Cem cells . Many RNSequence-defined chromatin purification by the CAPTURE technique can also reveal long-range DNA contacts by tying to a sequencing readout. These experiments are analogous to chromatin conformation capture (3C)  and relaResearchers have utilized proximity labeling to study chromatin in order to take advantage of the uniquely strong interaction between biotin and streptavidin . These t7 cells cultured for 24 h). Newly identified proteins were confirmed to interact with the region targeted by the sgRNA via super-resolution microscopy in live cells, orthogonally confirming the results from the CasID experiments. In an extension of the CasID work by a separate research group, the protein desmoplakin, previously known only for its role in cell-cell adhesion, was identified as a telomere-associated protein and confirmed through functional experiments to play a role in maintaining telomere length [A BirA biotin ligase mutated to promiscuously attach biotin to all nearby lysine residues was fused to dCas9 for the CasID technique . In the e length . Desmople length .An alternative proximity labeling strategy leverages the engineered peroxidase APEX2 . This apThe direct fusion of APEX2 to dCas9 has also been utilized for genetically targeted proximity labeling. In a technique termed dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging (C-BERST), U2OS cells were engineered to contain a dCas9-APEX2 fusion protein under the control of a Tet-On promoter . The conhTERT or MYC promoters in separate experiments. hTERT encodes the human telomerase protein whose function has been implicated in a multitude of cancers [MYC encodes an oncogene commonly expressed in cancers [In a concurrent report, a dCas9-APEX2 approach termed genetic locus proteomics (GLoPro) demonstrated the ability to target non-repetitive, single-copy loci . In this cancers ,190,191. cancers ,195,196.Control GLoPro experiments showed a labeling radius of ~400 bp (~3 nucleosomes). Key to success for targeting single-copy loci was the tiling approach used for sgRNAs—multiple separate engineered sgRNA cell lines targeting the promoter region allowed samples to be pooled to increase signal. Additionally, any interactions disrupted by the binding of dCas9-APEX2 in any individual sgRNA cell line can be picked up by the other cell lines. A TMT-based quantitative proteomics approach used four to five individual sgRNA cell lines targeting each promoter along with a no-sgRNA control. Highly abundant proteins, such as histones and subunits of RNA polymerase, were identified but not enriched (presumably due to high background), but several classic sequence-specific transcription factors were enriched and confirmed to interact with each locus through conventional ChIP experiments. This observation highlights an important consideration in reverse-ChIP experiments: proteins with high levels of background expression are more difficult to identify as locus-specific binders. In other words, necessary attempts to minimize false positives can often result in false negatives.Ripk3 locus in mouse epithelial cells by a separate research group [Ripk3 promoter by ChIP-qPCR. Knockdown of the factors also prevented the increase in RIPK3 transcription typically observed upon TNFα stimulation. Confirmation of putative transcriptional regulators by both ChIP and functional assays gives high confidence that GLoPro is a powerful tool for identifying novel contributors to locus-specific proteomes.The GLoPro technique was adapted to study the regulation of the ch group . RIPK3 ich group ,201,202.ch group . Three sSeveral key considerations in designing a dCas9-based reverse-ChIP experiment emerge from the numerous reports leveraging the modular sgRNA-targeted CRISPR system. As with all reverse-ChIP approaches, control experiments are necessary to demonstrate that expression and/or binding of dCas9 does not disrupt the regulatory function of the region under study. Unique to dCas9-based experiments is the need to carefully design sgRNAs to minimize off-target binding and the associated control experiments to determine the degree of enrichment for predicted off-target sites. Minimizing expression levels of dCas9 is crucial when sgRNAs target non-repetitive regions. For single-copy loci, only two dCas9 molecules can specifically bind per diploid cell; increased expression of dCas9 provides more background, which necessitates larger input volumes to increase signal. Tiling sgRNAs to target single-copy loci can increase the relevant signal and identify proteins for which binding is disrupted in any individual sgRNA cell line. Appropriate negative controls must be included to filter out nonspecific interactions. Important negative controls include not expressing dCas9 to identify background binders, expression of dCas9 with no sgRNA or a nonspecific sgRNA to identify off-target effects, and exclusion of chemicals necessary to induce biotinylation in proximity labeling experiments. Using a nonspecific sgRNA provides a cleaner control than not expressing a sgRNA since the dynamics of dCas9 interrogating the genome varies with the presence or absence of a sgRNA. In some cases, a control sgRNA targeting a specific region far from the gene under study can be used . Proximity labeling-based experiments also have special considerations. There is a differential abundance of surface-exposed lysine residues vs. surface-exposed tyrosine residues, so BirA vs. APEX2 experiments will have different biases for what proteins can be identified. Another important consideration is the difference in time scales for each method (hours for BirA vs. minutes or less for APEX2). Last, as with all reverse-ChIP approaches, any proteins identified must be confirmed to be functionally relevant via orthogonal experiments and/or confirmation from the literature.Understanding the vast complexity of gene regulation necessitates tools that enable the unbiased quantitative identification of molecular factors interacting with any arbitrarily selected genomic locus. Many labs have devoted an enormous amount of effort to developing strategies for the selective purification of any chosen sequence-defined region. Though these efforts have uncovered novel biological insights, they are often only performed by specialist laboratories that have the expertise to effectively engage in labor-intensive processes. The emergence of dCas9-based reverse-ChIP strategies provides a modular tool that shows high potential for more widespread adoption. Increasingly powerful and sophisticated mass spectrometry protocols coupled with chromatin purification construct a list of candidate regulators that must be confirmed through orthogonal experiments. Advancements in mass spectrometry techniques will continue to provide opportunities to advance locus-specific chromatin isolation techniques . ChromatBox 19–1011 cells in some protocols). The signal-boosting strategy from single cell proteomics provides a possible experimental paradigm to lessen the labor-intensive workflow in reverse-ChIP strategies. Individual cell lines used to isolate locus-specific chromatin could be labeled with TMTs using a smaller input volume, while the parent cell line can have ~109 or more cells labeled to serve as the signal-boosting channel. The large number of proteins present in the parent cell line could serve as a platform for ‘amplification’ of the proteins present in the purified target region in an analogy to PCR.A current frontier in quantitative mass spectrometry research is the development of techniques for single-cell proteomics ,205. TheReverse-ChIP strategies have evolved from proof-of-concept experiments targeting repetitive genetic elements requiring specialist knowledge to strategies that allow for the study of single-copy loci by laboratories that have not devoted their careers to developing the necessary technology. These approaches have matured to the point that identification of functionally relevant molecular regulators at an arbitrarily selected locus of interest can be achieved. A longer-term goal will be to advance the techniques to the point that the entire proteome of a specific locus, including the stoichiometry of all components and identification of protein post-translational modifications, becomes routine. As adoption of reverse-ChIP protocols becomes more widespread, especially with the use of dCas9, the fields of transcriptional regulation and epigenetics can be expected to tackle long-standing questions with unprecedented molecular resolution. Development of an allele-specific reverse-ChIP strategy has the potential to provide a crucial tool to study genomic imprinting . Expert Adoption of reverse-ChIP techniques by cancer biologists will be invaluable in developing personalized cancer therapeutics. The phenotype of excessive cell growth embodied by cancer provides a technical advantage for contemporary reverse-ChIP experiments, which require large initial sample inputs. A future aim for clinical application of these technologies would be to create patient-specific, tumor-derived cell lines. Genomic and transcriptomic analysis can identify utilization of cancer specific promoters and other aspects of the cancer ‘regulome’ that can then be targeted for locus-specific chromatin purification. Identification of the molecular players controlling regulation at these key cancer genes in a patient-centered way will provide an avenue to develop personalized therapeutic regimens.


An accurate understanding of wildfire impacts is critical to the success of any post-fire management framework. Fire severity maps are typically created from satellite-derived imagery that are capable of mapping fires across large spatial extents, but cannot detect damage to individual trees. In recent years, higher resolution fire severity maps have been created from orthomosaics collected from remotely piloted aerial systems (RPAS). Digital aerial photogrammetric (DAP) point clouds can be derived from these same systems, allowing for spectral and structural features to be collected concurrently. In this note, a methodology was developed to analyze fire impacts within individual trees using these two synergistic data types. The novel methodology presented here uses RPAS-acquired orthomosaics to classify trees based on a binary presence of fire damage. Crown scorch heights and volumes are then extracted from fire-damaged trees using RPAS-acquired DAP point clouds. Such an analysis allows for crown scorch heights and volumes to be estimated across much broader spatial scales than is possible from field data.There was a distinct difference in the spectral values for burned and unburned trees, which allowed the developed methodology to correctly classify 92.1% of trees as either burned or unburned. Following a correct classification, the crown scorch heights of burned trees were extracted at high accuracies that when regressed against field-measured heights yielded a slope of 0.85, an R-squared value of 0.78, and an RMSE value of 2.2 m. When converted to crown volume scorched, 83.3% of the DAP-derived values were within ± 10% of field-measured values.This research presents a novel post-fire methodology that utilizes cost-effective RPAS-acquired data to accurately characterize individual tree-level fire severity through an estimation of crown scorch heights and volumes. Though the results were favorable, improvements can be made. Specifically, through the addition of processing steps that would remove shadows and better calibrate the spectral data used in this study. Additionally, the utility of this approach would be made more apparent through a detailed cost analysis comparing these methods with more conventional field-based approaches. The ability to rapidly evaluate the spatial extent and severity of a forest fire is critical to understanding and predicting its short- and long-term impacts on a given ecosystem  to acquire imagery at sub-meter resolutions. RPAS-acquired imagery, in particular has seen a rapid uptake across multiple disciples including ecology . The average horizontal and vertical root-mean-square errors (RMSEs) of the GPS points were 1.2 and 1.9 m. More detailed RPAS camera specifications and flight parameters can be found in Arkin et al. .Field data was collected at eighteen, 11.28-m fixed radius plots, randomly located within these RPAS acquisition areas using a stratified sampling methodology to ensure that a range of fire severities  were equally sampled. Fire severity was classified using relativized differenced Normalized Burn Ratios (RdNBR) derived from pre- and post-fire Landsat scenes according to the methods described by  were stem-mapped and had a series of measurements taken. Stem-mapping was completed by collecting the distance and azimuth from plot center to each tree bole at breast height using a tripod-mounted TruPulse Laser Rangefinder. Each stem-mapped tree had the following information recorded: species, pre- and post-fire state , crown class , DBH, bole char height (lowest and highest), total height, height of live crown, and height of crown scorch. Dead trees were distinguished from dying trees based on the absence of any green foliage.Pseudotsuga menziesii var. glauca), hybrid white spruce (14%) (Picea engelmannii X Picea glauca), and lodgepole pine (6%) (Pinus contorta), with small components of subalpine fir (Abies lasiocarpa), trembling aspen (Populus tremuloides), and paper birch (Betula papyrifera). Approximately 66% of sampled trees had some amount of visible crown damage, with almost 50% of sampled trees having 100% crown scorch or consumption.Crown scorch height was defined as the lowest height of living foliage and later used to calculate crown length scorched. Crown volume scorched was then calculated using the total and scorched crown lengths using the methods described in Hood et al. 2 and orthomosaics at an average spatial resolution of 4.41 cm. Following the photogrammetric processing, the generated DAP point clouds were clipped to 30-m radius plot buffers and normalized to ground level using the previously acquired LiDAR point cloud. The normalized point clouds were then clipped to 15-m radius plot buffers and had ground points classified and removed. Points less than 2 m above ground level were also removed. All point cloud processing steps described in this section were completed using LAStools , false negative (FN), and false positive (FP) trees, as well as through the calculation of recall  All segmented trees were visually analyzed and those outside the 11.28-m radius plot or less than 0.3 mG) − (R − B))/((2 × G) + (R + B))  was estimated using the same methods described in Hood et al. n = 161) and codominant  trees, and lowest for trees that were suppressed  or dead pre-fire . The intermediate trees likely had the largest impact on the relatively poor overall recall due to their abundance in the field data (n = 216) and low recall rate (0.49).In total, 698 trees were measured in the field, of which 405 (TP) were successfully matched to a segmented tree and 293 (FN) were not. One hundred thirty-eight (FP) segmented trees were not matched to any field-measured tree. Respectively, the TP, FN, and FP values resulted in an overall recall of 0.58 and an overall precision of 0.75. The proportion of TP trees and recall rates for each tree class can be seen below in Fig. The classification accuracy was favorable for both unburned (88.4%) and burned (94.0%) trees, with an overall accuracy of 92.1% Fig. . In addiThe first two example trees shown in Fig. When applied to the 251 trees that were correctly classified as burned, the crown scorch height estimation predicted crown scorch heights well Fig. . The strResults from the crown volume scorched calculations are shown in Fig. The novel classification routine was able to correctly classify the majority of trees based on a presence or absence of fire damage. The success of this routine was largely due to the more consistent spectral signatures observed for the orthomosaics as opposed to the spectral values in the DAP point cloud. The inconsistency of DAP spectral values were most pronounced in lower portions of tree crowns due to the effect that neighboring trees have on relative illumination (Wallace et al. In many cases, the negative GLI pixels in these trees represented bare ground pixels. The amount of bare ground pixels within crown boundaries was reduced based on the point thinning method described in section “Classification of burned and unburned trees”, but some ground pixels remained due to the DAP point clouds’ inability to accurately detect ground features near tree crowns (Graham et al. Conversely, the misclassified burned trees all had low proportions of negative GLI values, similar to those of most unburned trees. The relative absence of negative GLI pixels was primarily due to these misclassified trees not having any crown scorch visible from the orthomosaics. Most of these trees had field-measured crown scorch recorded only in lower portions of the crown. These scorched portions were obscured by upper crowns or by dominant neighboring trees that occluded portions of misclassified trees.Once a tree was correctly classified as burned, the crown scorch height and volume estimations were highly accurate. Errors in prediction were primarily a result of differences in how the field data were collected compared to how the segmented DAP point clouds were analyzed. The field measurements recorded the lowest crown scorch height on a particular tree, whereas the point clouds required a majority of DAP points to have negative values within a 10-cm slice of a tree. In cases where the crown was scorched along a horizontal line, these differing methodologies resulted in nearly identical measures. These differences would have been reduced, but not eliminated, if crown scorch height was estimated as the highest point of scorched foliage. The largest differences in extracted and measured heights occurred where the crown was scorched more on one side of the tree than the other. In these cases, the field data suggested a much lower crown scorch height than was derived from the DAP point cloud. These differences were particularly pronounced in the crown volume scorched results, with 14 trees having scorched volumes estimated between 30 and 64% higher than the field-measured values.While 698 trees were sampled in the field, only 405 were successively matched to a DAP-derived tree. Of these 171 had crown scorch volumes equal to 100%, and another 138 were unburned. This only left 96 trees that had scorch volumes between 0 and 100%. As these trees are the most difficult to characterize, future studies should seek to selectively sample a higher proportion of trees that had moderate crown scorch volumes. Additionally, field measurements that more directly quantify the percent of needles that were scorched may have revealed more nuanced results that could further inform this methodology. Crown volume scorched was estimated from total crown length and scorched crown length (Hood et al. The ability of DAP point clouds to record spectral information in addition to structural information represents a key strength of the technology. However, the application of DAP is fundamentally limited by its inability to derive subcanopy points to the same degree as LiDAR (White et al. The RPAS images used to derive the orthomosaics and DAP point clouds were collected around solar noon under optimum light conditions in order to ensure the spectral quality of the data. Despite these precautions, there was still considerable shadowing present within the DAP point clouds and, to a lesser degree, the RPAS-derived orthomosaics, especially for more obscured features. As previously mentioned, this shadowing likely contributed to the amount of negative GLI values within unburned crowns. Radiometrically calibrating these data would likely have minimized these unexpected negative GLI values and is increasingly being seen as a necessary processing step within any image-based RPAS methodology (Assmann et al. The focus of this research is on the development and use of RPAS-based remote sensing methods to estimate crown scorch heights and volumes within individual conifer trees. This type of analysis has value in estimating fire intensity (Van Wagner The novel methods presented in this research successfully analyzed fire impacts across individual trees segmented from DAP point clouds. A large majority of trees were correctly classified using an orthomosaic-based approach, including 88.4% of unburned trees and 94.0% of burned trees. For successfully classified burned trees, crown scorch heights and volumes were reliably estimated. Field-measured and extracted crown scorch height values corresponded well, with an R-squared value of 0.78 and an RMSE value of 2.2 m. Crown volume scorched estimates were also carried out very accurately, with 83.3% of DAP-derived values being with ±10% of field-derived values. These favorable results indicate that this method can reliably assess and map crown scorch height across large numbers of individually segmented trees. Such estimations of crown scorch height have value in estimating maximum fire intensity, predicting delayed mortality, and identifying tree refugia. While the potential application of these methods is promising, there are certain limitations associated with the data and methods used in this study. Primary among these is related to an inability to reliably detect and characterize understory trees from the RPAS-acquired orthomosaics and DAP point clouds. The methods used in this study also did not include any additional steps to radiometrically calibrate or to remove shadows from the data. Such steps would likely increase the accuracy and reliability of these methods. We recommend the future inclusion of acquisition and processing steps to address these limitations. Additionally, we recommend the development of a more integrated remote sensing framework that would allow for fire severity to be analyzed and mapped using systems of various spatial scales and resolutions depending on particular management needs.


The application of esophageal stent closure of fistulas is limited in application to thoracogastric airway fistulas due to changes in anatomical structure after surgery. Additionally, the unique location of three-branch orificium fistulas and large orificium fistulas can cause complications such as stent displacement and lax sealingThe patient in this case, a 56-year-old man, experienced a thoracogastric airway fistula after radical esophagectomy. Despite receiving prior esophageal stent treatment, the patient experienced a recurrence of symptoms 5 months post-surgery, worsening his nutritional status and quality of life. Given the patientʼs large fistula and the potential for incomplete sealing with airway stenting, a therapeutic regimen involving coils combined with occlusive devices was proposed.After determining the location of the fistula , occlusVideo 1 Thoracogastric airway fistula closed by occluder devices and coils under fluoroscopic guidance.Re-examination demonstrated the successful closure of the thoracogastrotracheal fistula . The deEndoscopy_UCTN_Code_CPL_1AH_2AG


What are the utilization rates and comparative safety outcomes of robotic-assisted cholecystectomy vs laparoscopic cholecystectomy across the United States?This cohort study of claims data for Medicare beneficiaries identified 1 026 088 who underwent a cholecystectomy from 2010 through 2019. Robotic-assisted cholecystectomy rates increased 37-fold, and this procedure was associated with a higher rate of bile duct injury compared with laparoscopic cholecystectomy (0.7% vs 0.2%).In the absence of other advantages over an already minimally invasive procedure, the findings of this study call into question the role of the robotic platform for cholecystectomy. Robotic-assisted cholecystectomy is rapidly being adopted into practice, partly based on the belief that it offers specific technical and safety advantages over traditional laparoscopic surgery. Whether robotic-assisted cholecystectomy is safer than laparoscopic cholecystectomy remains unclear.To determine the uptake of robotic-assisted cholecystectomy and to analyze its comparative safety vs laparoscopic cholecystectomy.This retrospective cohort study used Medicare administrative claims data for nonfederal acute care hospitals from January 1, 2010, to December 31, 2019. Participants included 1 026 088 fee-for-service Medicare beneficiaries 66 to 99 years of age who underwent cholecystectomy with continuous Medicare coverage for 3 months before and 12 months after surgery. Data were analyzed August 17, 2022, to June 1, 2023.Surgical technique used to perform cholecystectomy: robotic-assisted vs laparoscopic approaches.The primary outcome was rate of bile duct injury requiring definitive surgical reconstruction within 1 year after cholecystectomy. Secondary outcomes were composite outcome of bile duct injury requiring less-invasive postoperative surgical or endoscopic biliary interventions, and overall incidence of 30-day complications. Multivariable logistic analysis was performed adjusting for patient factors and clustered within hospital referral regions. An instrumental variable analysis was performed, leveraging regional variation in the adoption of robotic-assisted cholecystectomy within hospital referral regions over time, to account for potential confounding from unmeasured differences between treatment groups.A total of 1 026 088 patients  were included in the study. The use of robotic-assisted cholecystectomy increased 37-fold from 211 of 147 341 patients (0.1%) in 2010 to 6507 of 125 211 patients (5.2%) in 2019. Compared with laparoscopic cholecystectomy, robotic-assisted cholecystectomy was associated with a higher rate of bile duct injury necessitating a definitive operative repair within 1 year . Robotic-assisted cholecystectomy was also associated with a higher rate of postoperative biliary interventions, such as endoscopic stenting . There was no significant difference in overall 30-day complication rates between the 2 procedures. The instrumental variable analysis, which was designed to account for potential unmeasured differences in treatment groups, also showed that robotic-assisted cholecystectomy was associated with a higher rate of bile duct injury .This cohort study’s finding of significantly higher rates of bile duct injury with robotic-assisted cholecystectomy compared with laparoscopic cholecystectomy suggests that the utility of robotic-assisted cholecystectomy should be reconsidered, given the existence of an already minimally invasive, predictably safe laparoscopic approach. This cohort study compares the rates and safety outcomes of robotic-assisted cholecystectomy with laparoscopic cholecystectomy in a nationally representative population of older adults. Evidence in general surgery, the field currently experiencing the fastest adoption of robotic-assisted procedures,1 suggests that these patterns may expose patients to harm as individual surgeons learn the new surgical approach.9 For example, while cholecystectomy remains one of the most routine operations performed by general surgeons, a recent several-fold increase in the use of robotic-assisted surgical procedures has driven the ongoing debate over the potential safety implications of this change in practice.10The US is a global outlier in the adoption of robotic-assisted surgery.11 and potentially increases a surgeon’s ability to perform difficult procedures in a minimally invasive fashion.12 Studies15 comparing the safety of these approaches found equivalency, but are limited to single-center case series inclusive of surgeons with the most robotic-assistance experience. Whether those outcomes reflect current surgical practice, especially as robotic-assisted cholecystectomy is adopted by a larger and potentially more novice group of surgeons, represents crucial information for surgeons, referring physicians, and patients.Whether robotic-assisted cholecystectomy leads to even safer outcomes than minimally invasive laparoscopic cholecystectomy remains unclear. Some contend that robotic-assisted cholecystectomy may be safer because it offers 3-dimensional visualization, enhanced instrument articulation to allow for more complex maneuvers, novel ways to visualize biliary anatomy,We performed a study of US Medicare beneficiaries to evaluate the comparative safety of robotic-assisted vs laparoscopic cholecystectomy among a diverse group of patients and surgeons. We leveraged regional variation in the adoption of robotic-assisted cholecystectomy over time as an instrumental variable, accounting for both measured and potentially important but unmeasured sources of bias, to compare outcomes following both procedures.STROBE) reporting guideline.This cohort study was deemed exempt from review by the University of Michigan Institutional Review Board, which also waived the requirement to obtain informed consent because data were deidentified. The study followed the Strengthening the Reporting of Observational Studies in Epidemiology , International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10), and Current Procedural Technology codes  within 1 year following cholecystectomy. These 2 surgical procedures are performed as definitive treatments for severe bile duct injury, which was defined using ICD-9 or ICD-10 code for bile duct injury and ICD-9 or ICD-10 and Current Procedural Technology codes for surgical or endoscopic biliary interventions, which included endoscopic retrograde cholangiopancreatography and common bile duct exploration  were abstracted from Medicare's enrollment database, which is derived from patient self-report and submitted to the Social Security Administration. Race and ethnicity were assessed here to provide additional clarity of the demographics of this patient population. All models accounted for clustering of outcomes within hospital referral regions with robust standard errors.We used univariable statistical tests to compare baseline differences among patients who underwent each operation. We used multivariable logistic regression models to evaluate the association between robotic surgery and postoperative complications. All models included the following covariates: age, race and ethnicity, sex, 29 Elixhauser18 The directionality of selection bias in this case was not clear. On one hand, patients undergoing robotic-assisted surgery may be healthier at baseline or have less complex anatomy, as surgeons exploring a new operative approach may wish to learn on more straightforward cases. On the other hand, patients undergoing robotic-assisted surgery may be more complex and surgeons may prefer to make use of the potentially advantageous technologies inherent to the robotic platform. An instrumental variable approach allowed us to address the association of selection bias to explicitly compare outcomes among patients who would be considered candidates for either approach, that is, laparoscopic or robotic-assisted procedures.We performed an instrumental variable analysis to address potential confounding from unmeasured differences among patients who underwent robotic-assisted vs laparoscopic surgery.19 This instrumental variable met several prespecified criteria for being considered a suitable instrument. First, it was correlated with the exposure (receipt of robotic-assisted cholecystectomy) but not associated with the outcome (bile duct injury) except through its correlation with the exposure. Second, we evaluated the strength of the instrument’s association with receiving robotic-assisted cholecystectomy by calculating the Kleibergen-Paap Wald F statistic (F = 2.2 × 106). An F statistic higher than 10 is typically considered to be a strong instrument.20Our instrumental variable was the proportional use of robotic-assisted cholecystectomy across the hospital referral region in the year prior to a patient’s operation. Prior studies have similar variables exploiting regional and temporal variation in the adoption of a new operation or operative approach to answer comparative safety or effectiveness questions.21 Our first-stage logistic regression modeled a patient’s odds of undergoing robotic-assisted cholecystectomy, adjusted for the instrumental variable  and other patient-level factors . We calculated the raw residuals from this first-stage model and included them as covariates in the second-stage model, which estimated the relative risk of bile duct injury adjusted for patient characteristics in a manner identical to our primary analysis. This instrumental variable analysis was also repeated for our secondary outcomes .We used a 2-stage residual inclusion estimation method for our instrumental variable analysis, which has been shown to lead to more consistent estimates than that of other instrumental variable estimation methods, particularly with nonlinear outcomes.ICD-9 and ICD-10 diagnosis codes, we conducted separate analyses for the ICD-9 and ICD-10 coding periods.We performed several sensitivity analyses to provide greater context to our primary findings. We examined any potential association that a hospital’s experience with robotics may have with comparative safety by calculating their annual volume of robotic-assisted cholecystectomy procedures, and then stratified hospitals into quartiles of volume. We also conducted stratified analyses based on clinical urgency (elective vs urgent or emergent) and diagnosis  as well as patient factors (with vs without obesity). Finally, as our study period spanned both The statistical analysis was performed using Stata, version 17 (StataCorp LLC). We used a 2-sided approach at the .05 significance level, or 95% CIs excluding 1, for all hypothesis testing.A total of 1 026 088 fee-for-service Medicare beneficiaries  underwent cholecystectomy during the study period. The use of robotic-assisted cholecystectomy increased 37-fold from 211 of 147 341 patients (0.1%) in 2010 to 6507 of 125 211 patients (5.2%) in 2019 . The useP < .001) and more likely to be African American or Black  or Hispanic  (P < .001). These patient were also more likely to have an elective cholecystectomy  and less likely to undergo a cholecystectomy for cholecystitis . Baseline characteristics of the cohort by minimally invasive procedure type is given in Of 1 001 004 patients who underwent robotic-assisted vs 25 084 who underwent laparoscopic surgery, those who underwent robotic-assisted surgery were younger  . PatientRobotic-assisted cholecystectomy was associated with higher risk of bile duct injury necessitating a definitive operative repair within 1 year (0.7%) compared with laparoscopic cholecystectomy . Compared with laparoscopic cholecystectomy, robotic-assisted cholecystectomy was also associated with higher rates of biliary interventions  and serious complications . There was no significant difference between the 2 procedures in 30-day overall complication rates  .The instrumental variable balanced measured patient characteristics stratified about the median of the instrumental variable , which was confirmed in our instrumental variable analysis. Overall complication rates were, on the other hand, similar between laparoscopic and robotic-assisted surgery. Taken together, our data suggest that the utility of robotic-assisted cholecystectomy should be reconsidered, given the availability of an already minimally invasive, predictably safe laparoscopic approach.24 Single-center retrospective cohort studies14 comparing laparoscopic and robotic-assisted cholecystectomy have previously suggested comparable rates of postoperative complications, while acknowledging higher total hospital costs attributable to robotics . One study25 of surgical procedures included in the national inpatient sample found higher overall complication rates for robotic-assisted cholecystectomy compared with laparoscopic cholecystectomy (15.5% vs 11.7%), but this was perhaps attributable to the higher comorbidity burden of patients who underwent robotic-assisted surgery .26 Our work fills 2 key gaps in knowledge around the comparative outcomes of robotic-assisted surgery. First, we specifically provided estimates for rates of technical complications, such as bile duct injury, in a nationally representative sample of patients and surgeons. Second, we leveraged variations in how robotic-assisted surgery was adopted to address issues such as selection bias that may lead to incorrect conclusions about why robotic-assisted cholecystectomy may or may not be safer than laparoscopic cholecystectomy. In other words, we provided a specific mechanism by which robotic-assisted cholecystectomy was less safe despite the purported benefits inherent to the technology.The overall rates of bile duct injury in this study are consistent with prior population-based analyses, particularly for patients who underwent inpatient laparoscopic cholecystectomy.27 When laparoscopic cholecystectomy was introduced approximately 30 years ago as a minimally invasive alternative to open surgery, it was appropriately criticized for higher rates of technical complications, such as common bile duct injury.29 This complication results in a cascade of endoscopic or highly morbid surgical interventions 31 that decrease long-term survival.32 Increasing familiarity with laparoscopy along with deliberate continuing education programs, such as the SAGES Safe Cholecystectomy Program,35 have rendered laparoscopic cholecystectomy the standard of care, with lower complication rates and faster recovery compared with open surgery.27 The present study further supports the success of those initiatives, as the rate of bile duct injury for laparoscopic cholecystectomy in 2010 was 0.3% and dropped to 0.1% by 2019. On the other hand, robotic-assisted cholecystectomy had a 0.4% bile duct injury rate by 2019 for the 5.2% of Medicare beneficiaries who underwent cholecystectomy that year. Unfortunately, the clinical justification to devote time and resources to expose patients to increased risk as surgeons progress along the procedural learning curve from laparoscopic to robot-assisted cholecystectomy remains theoretical.Our results reinforce an important longitudinal pattern that globally affects the quality of care for numerous patients undergoing cholecystectomy each year: the rates of open cholecystectomy continue to decline.9 Beyond clinical justifications, industry marketing and patient preferences  have also been implicated in the expansion of robotic-assisted surgery across a range of clinical specialties.37The extent to which the pattern of moving away from open surgery is further driven by the adoption of robotic-assisted surgery, with the potential to allow more difficult operations to be performed in a minimally invasive fashion, is unclear. Our findings do not suggest that the growth of robotic-assisted cholecystectomy is being driven by these more complex cases that would have otherwise been performed by an open approach. First, we found that robotic-assisted cholecystectomy was more likely to be elective and performed for biliary colic or gallstone pancreatitis rather than for acute or chronic cholecystitis. This finding may suggest that surgeons are using this technique for lower-risk cholecystectomy procedures while they are learning the technique. Furthermore, if robotic-assisted cholecystectomy was being performed on candidates who were not laparoscopic cholecystectomy candidates, the rates of bile duct injury in the open group would increase over time as only the highest-risk patients would remain. Instead, rates of bile duct injury continued to decrease for both laparoscopic and open cholecystectomy approaches. Finally, overall complication rates between robotic-assisted and laparoscopic surgical procedures were similar. As a result, it is plausible that at least some of the robotic-assisted cholecystectomy procedures are replacing laparoscopic cholecystectomy. This has been observed in other clinical domains. For example, the adoption of robotic-assisted colectomy was associated with a 2:1 replacement of laparoscopic surgical procedures  over open ones.25 Even though some randomized controlled trials comparing robotic-assisted and laparoscopic surgery for rectal cancer resection or inguinal hernia repair did not demonstrate differences in outcomes, many studies contend that there are benefits from enabling surgeons to perform these technically complex operations in a minimally invasive fashion.39 In other words, the technical challenges of performing those operations laparoscopically may drive surgeons to pursue mastery of the potentially more accommodating robotic-assisted alternative.3Surgeons remain optimistic that the robotic platform will increase patients’ access to minimally invasive operations even if there are some practical trade-offs to consider. For example, there is a large body of evidence suggesting that robotic-assisted surgery is more expensive than laparoscopy, has longer operative times, and requires more specialized operating room staff.15 But this justification and others related to expanding clinical applications for robotic-assisted surgery may need to be more carefully balanced against any appreciable disadvantages related to patient safety.For cholecystectomy, however, the traditional laparoscopic approach is a core competency for practicing surgeons and general surgical trainees. In the absence of a clear safety advantage, it is not clear what problem the robotic technology is addressing. Because cholecystectomy is such a familiar operation, there may be other reasons behind the adoption of robotic-assisted cholecystectomy. For example, surgeons may leverage their familiarity with cholecystectomy to practice their robotic-assisted surgical skills.23 In addition, we were unable to quantify using claims data whether the robotic platform was increasing the number of patients who could receive a minimally invasive operation or whether the robotic platform was simply replacing the laparoscopic approach. However, we attempted to control for these issues in multiple ways. First, we used diagnoses and outcomes reliably coded in claims data. Second, we specifically focused on a primary outcome that was previously used in similar studies32 and is less subject to coding discrepancies. Finally, we performed an instrumental variable analysis to control for unmeasured factors that may bias our comparisons between both procedure approaches. This study also focused on the Medicare population that underwent inpatient or observation status procedures, which may limit the generalizability of our findings. However, cholecystectomy remains one of the most common general surgical operations performed in individuals of all ages, and we would not expect the results to differ purely on age .This study has limitations. Because this was a retrospective study using administrative claims data, our results relied on accurate coding of diagnoses, comorbidities, and postoperative complications. Limitations associated with unmeasured variables, such as sociodemographic factors, comorbidities, and surgical complexity, may also have biased our results. We were unable to capture procedural factors that have been shown to play a crucial role in the development of bile duct injuries, such as the severity and chronicity of gallbladder inflammation. Furthermore, we were unable to identify intraoperative conversions in the data, which limits our ability to comment on the outcomes associated with converting from one procedure to another. While rare, intraoperative conversions have been associated with significantly higher rates of bile duct injury in a previous study.This cohort study found that, among Medicare beneficiaries, the use of robotic-assisted cholecystectomy increased from 2010 through 2019 and was associated with higher rates of bile duct injury compared with laparoscopic cholecystectomy. In the absence of other advantages over an already minimally invasive procedure, these data call into question the practice of robotic-assisted cholecystectomy.


Extracorporeal cardiopulmonary resuscitation (ECPR) is an emerging treatment for refractory cardiac arrest. In recent years, several randomized controlled trials have been published that aimed to address the efficacy and effectiveness of ECPR for out-of-hospital cardiac arrest (OHCA). Despite the lack of high-quality evidence concerning clinical effectiveness and cost-effectiveness, ECPR is increasingly implemented throughout the world. In this review, we aim to provide an overview of the current status of ECPR for OHCA.Randomized controlled trials showed diverging results, largely due to differences in selection criteria and study design. Single-center studies, performed in centers with extraordinary expertise and dedication consistently achieve a low-flow time of around 60 min, but such achievements are rarely reproduced outside these centers. Strict patient selection can improve outcome but simultaneously limits the caseload. Preliminary data suggest that outcome may also be improved by avoiding hyperoxia postresuscitation.The potential of ECPR to increase survival in selected patients in highly dedicated systems seems to be proven, the question remains whether ECPR for OHCA can be widely implemented successfully and can develop into a sustainable, commonplace resource-effective treatment. The incidence of EMS-treated out-of-hospital cardiac arrest (OHCA) is approximately 30–90 per 100 000 persons per year in the developed world with an overall mortality of 84-95% . Patient▪▪]. The ARREST trial randomized 30 adult patients with an initial shockable rhythm, who were presented at the Emergency Department (ED) without return of spontaneous circulation (ROSC) after at least three defibrillation attempts. Patients with an unfavorable metabolic profile were excluded from ECPR despite being allocated to ECPR, but retained in the intention-to-treat analysis. This single-center study which was terminated early for overwhelmingly positive outcomes showed a 36.2 absolute risk reduction for its primary outcome, hospital survival, in favor of ECPR and demonstrated the efficacy of ECPR within a fully dedicated multidisciplinary, transmural system.Until 2020 all available evidence on ECPR for OHCA has been derived from observational data, particularly originating from southeast Asia, Germany, and France . In 20206▪▪]. This single-center RCT differed from the ARREST trial in several aspects of the study design. Most importantly, patients were randomized prehospital and became eligible after 5 min of unsuccessful advanced cardiac life support. Moreover, patients with all presenting rhythms were eligible, providing that OHCA was of presumed cardiac origin. Patients were either transported to an ECPR-capable hospital using mechanical chest compression devices or remained on scene. Patients arriving at the hospital without ROSC received ECPR. The trial was discontinued after 256 patients when a prespecified between-group difference of 15% could no longer be achieved. The absolute risk reduction for the primary endpoint  was 9%. The lower risk reduction in the Prague OHCA study in comparison with the ARREST trial can be ascribed to the inclusion of patients with nonshockable rhythms . The absolute risk reduction for the primary endpoint, survival with favorable neurology after 30 days was 4%. This difference was not statistically significant. A major point of criticism that was raised concerning the external validity of the INCEPTION trial is the time from cardiac arrest to ECMO flow which was 74 min median, which is substantially longer than in the ARREST trial and the Prague OHCA study . However, the low flow times achieved during the INCEPTION trial are comparable to those typically achieved by other large centers presenting their experience with ECPR . However, such a selection criterion imposes selection bias, when centers that failed to successfully implement an effective ECPR program are excluded. This may be overcome by selecting ECMO-ready centers without an active ECPR program who are willing to go through a structured time-limited implementation program before start of enrollment. In addition, sample size should be adequate to detect a clinically important difference .The authors of this paper were members of the Trial Steering Committee of the INCEPTION trial.


Lacunar strokes are the hallmark of cerebral small vessel disease. There are several well-established mechanisms for the pathogenesis of lacunar stroke, but the cardioembolic mechanism is not well-established. Three cases of acute ischemic stroke following elective cardiac and cerebral catheterization are reported. These cases had typical lacunar-looking infarcts on neuroimaging despite strong evidence of an embolic source with temporal correlation. Awareness of such findings and pathogenesis may help investigational workup and management of these patients. Lacunar strokes account for up to a quarter of all ischemic strokes and measure less than 20 mm in diameter. They are usually caused by the occlusion of a perforator of an intracranial artery . There aCase 1A 58-year-old Caucasian woman with multiple vascular risk factors, including hypertension and hyperlipidemia, presented to the hospital for an elective diagnostic cardiac catheterization after she was found to have unstable angina a few days earlier. After the diagnostic part, angioplasty of the left anterior descending artery was performed with full-dose heparin anticoagulation. She began endorsing diplopia immediately post-procedure. A neurological exam showed impaired adduction of the right eye with preserved convergence, consistent with right medial rectus palsy. Her National Institutes of Health Stroke Scale (NIHSS) score was 1. CT head without contrast showed an Alberta stroke program early CT score (ASPECTS) of 10. CT angiograms of the head and neck were notable for mild atherosclerotic disease in bilateral cavernous segments of internal carotid arteries without flow-limiting stenosis or large vessel occlusion. She was not deemed a candidate for systemic thrombolysis due to heparin use during the procedure with an aPTT of 62 at the time of evaluation and mild deficit. MRI brain without contrast showed acute small infarcts in the right posterior midbrain suggestive of lacunar small vessel stroke and right cerebellum infarct (confirms catheter-related embolic etiology)  to work up coronary artery disease. LHC was performed with right radial access and noted severe atherosclerotic aortic arch disease and three-vessel coronary artery disease. Systemic anticoagulation was not administered, and no intraprocedural complications were noted. The patient was then transferred to the recovery area, where she was observed to have an inability to raise her left arm about 20 minutes after the procedure. A code stroke was activated, and the patient was found to have left-sided weakness, ataxia, and dysarthria with an NIHSS score of 5 and blood pressure of 180/67 mmHg. A CT brain showed an ASPECTS score of 10. The CTA of the head and neck showed mild to moderate extracranial and intracranial atherosclerotic disease without flow-limiting stenosis or large vessel occlusion. The patient received intravenous t-PA. MRI brain showed a moderate area of diffusion restriction in the right hemi-pons. Additionally, there were supratentorial chronic microvascular ischemic changes without brainstem involvement. Transthoracic echocardiogram with a bubble study showed no interatrial shunt with impaired relaxation and an estimated EF of 60-65%. At the three-month follow-up, the patient’s NIHSS score improved to 3, and the modified Rankin score was 2 (Figure Lacunar strokes are largely due to the pathologic consequences of underlying cerebral small vessel disease -9. Even Three cases of cerebrovascular events after elective diagnostic cardiac and cerebral angiograms are reported. Patients developed symptoms that were recognized post-operatively, and an MRI brain showed findings of acute stroke located in typical lacunar areas involving white matter and brainstem supplied by small penetrating arteries. The proposed mechanism of ischemic stroke in the first case was embolic from artery-to-artery embolism to perforator branches involving vertebrobasilar circulation. In the second case, a paramedian pontine area infarct is found. The patient was found to have a large atrial myxoma extended to the left ventricle, a known high-risk cardioembolic source, and the patient was also found to have chronic strokes on the contralateral territory. In this case, two potential causes of stroke were secondary to the cardioembolic source from the atrial myxoma and periprocedural cerebral angiography. The third patient received t-PA with clinical improvement, but the MRI brain showed a right hemi-pons infarct. The proposed cause was embolic material from extensive aortic arch disease, which got dislodged during cardiac catheterization. Therefore, acute infarcts were observed on MRI images, which would typically represent small vessel stroke patterns but with a known history of embolic etiology. Despite well-defined clinical and temporal causative factors for cardioembolic in these series, these associations may be due to shared risk factors of small vessel disease, such as hypertension, dyslipidemia, diabetes mellitus, and cigarette smoking. It is important to recognize cardioembolic etiology in lacunar-appearing infarcts, as this was diagnostic and prognostic implications. In addition, their responses to treatment with an antithrombotic or anticoagulant drug may be distinct and may involve careful clinical decision-making.Neuroimaging findings of small vessel lacunar strokes typically involve periventricular deep white matter, deep grey matter nuclei, and brainstem. Thromboembolic strokes typically appear cortical and usually multiple within the same arterial territory or multiple territories. The three cases have shown embolic proven infarcts looking like lacunar infarcts on neuroimaging. Awareness of such findings on imaging may help during investigational stroke workup and subsequent treatment.