Stricturing Crohn's Disease Single-Cell RNA Sequencing Reveals Fibroblast Heterogeneity and Intercellular Interactions.

BACKGROUND & AIMS: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full-thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single-cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. METHODS: We performed scRNAseq of 13 fresh full-thickness CD resections containing noninvolved, inflamed nonstrictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next-generation sequencing, proteomics, and animal models. RESULTS: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and up-regulated, and its profibrotic function was validated using NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and knock-out and antibody-mediated CDH11 blockade in experimental colitis. CONCLUSIONS: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and open potential therapeutic developments. This work has been posted as a preprint on Biorxiv under doi: 10.1101/2023.04.03.534781.

A Different Way to Think About Syndromes of Hereditary Colorectal Cancer.

BACKGROUND: Hereditary colorectal cancer is an increasingly complex field in which the commoner syndromes are being augmented by rarer genetic presentations contributing to familial polyposis and colorectal cancer. Coming to grips with the complexity is difficult because of the phenotypic and genotypic overlap between syndromes. OBJECTIVE: This study aimed to describe a new way of thinking about syndromes of hereditary colorectal cancer based on their embryonic tissue of origin. DATA SOURCES: Articles were searched through PubMed and MEDLINE. STUDY SELECTION: The terms "hereditary colorectal cancer," "syndromes of hereditary colorectal cancer," and "hereditary polyposis" were used to direct the search. RESULTS: Primarily endoderm-derived syndromes were different from mesoderm-derived syndromes in their genetics, molecular biology, histology, and clinical course. LIMITATIONS: There is considerable phenotypic and genotypic overlap between syndromes, even when considering embryonic tissue of origin. CONCLUSIONS: Thinking about hereditary syndromes of colorectal cancer from the perspective of embryonic tissue of origin provides a fresh look at phenotype and genotype that opens new areas of exploration. UNA FORMA DIFERENTE DE PENSAR SOBRE LOS SNDROMES DEL CNCER COLORRECTAL HEREDITARIO: ANTECEDENTES:El cáncer colorrectal hereditario es un campo cada vez más complejo donde los síndromes más comunes se ven aumentados por presentaciones genéticas más raras que contribuyen a la poliposis familiar y al cáncer colorrectal. Hacer frente a esta complejidad resulta difícil debido a la superposición fenotípica y genotípica entre los síndromes.OBJETIVO:En este artículo, describimos una nueva forma de pensar sobre los síndromes de cáncer colorrectal hereditario en función del origen de su tejido embrionario.FUENTES DE DATOS:Se realizaron búsquedas de artículos en Pubmed y Medline.SELECCIÓN DE ESTUDIOS:Se utilizaron los términos "cáncer colorrectal hereditario", "síndromes de cáncer colorrectal hereditario", "poliposis hereditaria" para dirigir la búsqueda.RESULTADOS:Principalmente los síndromes derivados del endodermo fueron diferentes a los síndromes derivados del mesodermo en su genética, biología molecular, histología y curso clínico.LIMITACIONES:Existe una superposición fenotípica y genotípica considerable entre los síndromes, incluso cuando se considera el tejido de origen embrionario.CONCLUSIÓN:Pensar en los síndromes hereditarios del cáncer colorrectal desde la perspectiva del tejido embrionario de origen proporciona una nueva mirada al fenotipo y al genotipo que abre nuevas áreas de exploración. (Traducción-Dr Osvaldo Gauto ).

A Review of Autoimmune Enteropathy and Its Associated Syndromes.

Autoimmune enteropathy is an extremely rare condition characterized by an abnormal intestinal immune response which typically manifests within the first 6 months of life as severe, intractable diarrhea that does not respond to dietary modification. Affected individuals frequently present with other signs of autoimmunity. The diagnosis is made based on a characteristic combination of clinical symptoms, laboratory studies, and histological features on small bowel biopsy. Autoimmune enteropathy is associated with a number of other conditions and syndromes, most notably immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome and autoimmune polyglandular syndrome type 1 (APS-1). Diagnosis and treatment is challenging, and further research is needed to better understand the pathogenesis, disease progression, and long-term outcomes of these conditions.

Histopathologic Patterns of Recurrent Choroidal Melanoma Following I-125 Plaque Brachytherapy.

PURPOSE: Histologic correlation of clinical patterns of recurrent choroidal melanoma following I-125 plaque brachytherapy was performed to identify pathologic mechanisms of recurrence. METHODS: We reviewed 7 cases of recurrent choroidal melanoma following I-125 plaque brachytherapy managed with enucleation. Clinical characteristics included tumor dimensions, radiation dose, time to local recurrence, and clinical pattern of recurrence. Histopathology (hematoxylin and eosin and periodic acid - Schiff) and immunohistochemistry (Ki-67, CD-163, HMB45, and SOX10) were performed. RESULTS: Mean follow-up time and time to local recurrence were 42 and 21 months after brachytherapy, respectively. Tumor recurrences were described clinically as marginal in 43%, diffuse in 29%, and extraocular extension (EOE) in 29%. Eighty-six percent were classified as mixed cell type and 14% were epithelioid type. Tumor zonation (histologic demarcation between zones of recurrent and nonrecurrent tumor cells by immunohistochemistry) was present in marginal and EOE cases (n = 6) and absent in the diffuse cases (n = 2). Ki-67 proliferative index was higher in marginal and EOE recurrences, while diffuse cases showed uniform -Ki-67 staining. CD-163 staining was found to be greater in nonrecurrent tumor. HMB45 correlated with SOX10 with a greater staining in recurrent tumor. CONCLUSION: Our observations provide a correlation between histopathologic and clinical patterns of local recurrence of choroidal melanoma after brachytherapy.

Patterns of polyp histology: predictors of peril in the mucosa.

BACKGROUND: Precursor colonic polyps of varied subtypes correlate with the known neoplastic pathways. When patients present with synchronous pre-malignant polyps of multiple histologies, multiple genetic mechanisms are likely to be active, potentially resulting in a more unstable, tumourigenic mucosa. METHODS: We hypothesized that patients with a combination of sessile serrated adenomas/polyps (SSA/Ps), hyperplastic (HP) polyps and adenomas would be at highest risk of developing dysplasia/cancer compared to SSA/Ps alone, due to the synergistic effect of multiple active carcinogenic pathways. A prospective colonoscopy database was examined for patients with a history of SSA/P. Patients were placed into four groups based on patterns of polyp histology as follows: (i) only SSA/Ps; (ii) SSA/P + HP; (iii) SSA/Ps + adenomas; and (iv) SSA/Ps + HP + adenomas. These groups were compared in terms of the numbers, size, location and histology of polyps and personal or family history of colorectal cancer. RESULTS: A total of 374 patients were included. The average age was 70 years (range 21-88), and 43% were male. There was a trend towards the most aggressive neoplastic pattern in group 4, associated with a tendency to larger SSA/Ps, more villous architecture in the adenomas and more high-grade dysplasia in both types of polyps. It was also associated with multiplicity of both SSA/Ps and adenomas. No SSA/Ps existing in the absence of adenomas had cytological dysplasia. CONCLUSION: The combination of SSA/Ps, HP and adenomas in the colorectal epithelium seems to be a marker for aggressive carcinogenesis and suggests that accurate and effective surveillance is important to manage this risk.

The location of premalignant colorectal polyps under age 50: a further rationale for screening sigmoidoscopy.

PURPOSE: The incidence of colorectal cancer (CRC) among young adults has been dramatically rising, with guidelines for screening recently adjusted to start at age 45. However, knowledge of the precursor lesions is limited. We recently reported that 83% of CRC diagnosed under age 50 are left sided. Our aim was to analyze the location and histology of benign colorectal lesions found in a cohort of patients younger than 50, documenting the presence of advanced histology. METHODS: We used the database in the Department of Pathology to retrospectively review the location and histology of all benign colorectal neoplasms in patients under age 50 submitted to pathology examination during 2006-2016. RESULTS: A total of 8364 lesions were examined from 4773 patients, and 3534 (65.5%) of the patients had only one polyp and the rest had multiple. Mean age was 41.9 years (range 16-49) while 3843 (72.8%) of the patients were between the ages of 40 and 49. In total, 4570/8364 lesions (54.6%) were distal to the splenic flexure. The most common pathology was tubular adenoma (63.7%), then hyperplastic polyps (16.6%), sessile serrated lesions (SSLs) (13.1%), and tubulovillous adenomas (6.3%). Tubulovillous adenomas, villous lesions, advanced adenomas, and adenomas with high-grade dysplasia were all predominantly left sided (left colon and rectum = 77.6%, 85%, 78.3%, and 87.6% respectively). Of the SSLs, 71.5% were in the right colon while 16.6% of hyperplastic lesions were right sided. CONCLUSIONS: High-risk advanced adenomas are predominantly left sided. This focuses attention on the rectum and left colon where carcinogenesis is strong in the young.

Specific Histopathologic Features Aid in Distinguishing Diffuse-type Gastric Adenocarcinoma From Metastatic Lobular Breast Carcinoma.

Metastatic invasive lobular carcinoma (mILC) may masquerade as primary diffuse gastric adenocarcinoma (PDGA) by demonstrating significant clinical and pathologic overlap. Accurate distinction is of therapeutic and prognostic significance. On the basis of anecdotal cases of mILC that lacked estrogen receptor and/or GATA3 expression, we analyzed the cytoarchitectural features of 28 mILC and 44 PDGA specimens obtained from women to assess features that would help in this distinction and prompt ancillary work-up. In addition to performing an interobserver agreement analysis among 3 pathologists, we also evaluated SATB2 expression in this setting. Eighteen of 20 (90%) patients had a history of ILC. The mean interval between initial diagnosis of breast cancer and metastasis was 7.3 years (range: 1 to 36 y). Compared with mILC, PDGA was significantly associated with full-thickness mucosal involvement (47% vs. 80%; P=0.015), a nested/sheet-like growth pattern (32% vs. 68%; P=0.004), anastomosing cords (0% vs. 100%; P=0.001), multivacuolated cells (0% vs. 61%; P<0.0001), pleomorphic nuclei (4% vs. 70%; P<0.0001) and enlarged nuclei (4% vs. 70%; P<0.0001). Single file growth pattern (P<0.0001) and superficial lamina propria involvement (P=0.009) were more common in mILC. Estrogen receptor and GATA3 were expressed in all but 5 mILC cases; SATB2 was only seen in 30% of PDGA cases. Our results demonstrate that in a biopsy specimen, careful morphologic assessment can be extremely helpful in distinguishing mILC from PDGA and guiding ancillary work-up, especially when a history of breast cancer may not be readily available or when the neoplasm lacks expression of conventional breast markers.

Fibrous Extracellular Spheroids in an Endoscopic Ultrasound-Guided Pancreatic Fine Needle Aspiration Correlating to a Gyriform Pancreatic Endocrine Tumor with a Unique Cobblestone Pavement Growth Pattern.

Pancreatic neuroendocrine neoplasms (PanNENs) are uncommon tumors. Fine needle aspiration (FNA) samples from PanNENs are typically of high cellularity and lack necrosis. In cytology slides from these tumors, dyscohesive cells are usually reported with variably round to oval to plasmacytoid forms exhibiting coarsely granular chromatin and showing immunoreactivity for synaptophysin. We present an unusual, and to our knowledge not previously described, example of an FNA of a PanNEN with large extracellular fibrous spheroids containing intrinsic fibroblasts and rimmed by small to intermediate sized neoplastic epithelial cells with high nuclear cytoplasmic ratios. The cytomorphology of the PanNEN in this case was in some ways reminiscent of that expected in adenoid cystic carcinomas of the salivary glands that most often contain large extracellular globules of basement membrane material and a somewhat biphasic population of lesional cells. The cytomorphology in this case was found to correlate well with the resection specimen histomorphology of an exaggerated gyriform pattern of growth resulting in a unique cobblestone-pavement like microscopic appearance. Knowledge of this potential cytomorphology will aid the cytology community through recognition and reporting of this previously undescribed pattern in an uncommon disease.

Molecular profiling of primary uveal melanomas with tumor-infiltrating lymphocytes.

In contrast to other cancers, the presence of tumor-infiltrating lymphocytes (TILs) in uveal melanoma is associated with a poor prognosis. However, how TILs may promote disease progression and what regulates their infiltration has not yet been established. To address these clinically relevant outstanding questions, T cell, immune regulatory, and chemokine gene expression profiles of 57 enucleated uveal melanoma tumors were compared, encompassing 27 with TILs and 30 without,. Tumors with infiltrating lymphocytes expressed more CD8A mRNA, as well as IFNG, TGFB1, and FOXP3 transcripts. Other T helper associated cytokines and T helper transcription factors were not differentially expressed, nor were mediators of lymphocyte cytotoxicity. The immune inhibitors INDO, PDCA1, CTLA4, and LAG3, and the non-classical MHC Class I target of CD8+ T regulatory cells, HLA­E, were significantly higher in tumors with TILs. FAS was also significantly higher. The C-C chemokine ligands CCL4, CCL5, and CCL20 were higher in tumors with TILs. Levels of CCL5 were most strongly correlated with levels of CD8A. Chemokine receptors were not differentially expressed. Molecular profiling of uveal melanoma tumors with TILs supports the existence of an immunosuppressive tumor microenvironment and suggests roles for CD8+ regulatory T cells, as well as specific chemokines, in fostering uveal melanoma disease progression.

Multifocal Primary Uveal Melanoma: Clinical and Molecular Characteristics.

We report two patients who developed a second distinct choroidal melanoma in the same eye following successful regression of their first choroidal melanoma after iodine-125 plaque brachytherapy. Neither patient demonstrated ocular melanocytosis, local tumor recurrence, or vitreous seeding. One patient had the second tumor arising from a previously documented choroidal nevus, and after undergoing enucleation, there was no detectable connection between the tumors on histopathologic examination. Germline BAP1 mutation was absent in both cases. Multifocal primary uveal melanoma is a rare entity in which the second tumor may occur either de novo or from a malignant transformation of a choroidal nevus. Known risk factors include ocular melanocytosis or germline BAP1 mutation. Additional underlying mechanisms have yet to be elucidated.

Case Report: Primary Orbital Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) of the orbit is almost uniformly the result of local invasion from a cutaneous primary, extension by perineural invasion, or the result of metastasis. This is owed to the lack of native squamous epithelium in the orbit. After review of the literature, to date, only 6 reports of 8 patients with primary orbital SCC exist. Of those cases, only 2 reported non-apical orbital SCC. There are 2 reports of orbital SCC after retina surgery with proposed transplanted conjunctival epithelium and subsequent malignant transformation of a conjunctival cyst. The initial signs and symptoms can be vague and lead to delay in diagnosis. We present a case of primary orbital SCC and discuss the workup, imaging, and multidisciplinary management of this rare condition.

A rare case of paraganglioma of the cystic duct.

INTRODUCTION: Biliary system paragangliomas are rare neuroendocrine tumors of embryonic neural crest origin. The majority is asymptomatic and incidentally found due to gallbladder functional disorders. Herein, we present a non-functional, 2.25 mm focus in the cystic duct, which to our knowledge, is the first reported paraganglioma of the cystic duct. PRESENTATION OF CASE: The patient presented to the Emergency Department complaining of a sudden-onset, right upper abdominal and epigastric pain. Ultrasound and Computed Tomography were both consistent with signs of early cholecystitis. Laparoscopic cholecystectomy was performed without major complications. In addition to cholelithiasis and chronic cholecystitis, pathological examination reported a neuroendocrine proliferation in the cystic duct measuring 2.25 mm favoring paraganglioma. Incidentally, the patient is unique in that they were also found to have an adrenal nodule and a normocalcemic primary hyperparathyroidism that raised suspicion for an underlying endocrinopathy. Nevertheless, genetic testing was negative. DISCUSSION: Extensive literature review demonstrates only nine cases of gallbladder paraganglioma, and three cases of hepatic ducts paraganglioma, but no cases of paraganglioma occurring at the cystic duct. Although a gene mutation and syndrome was not identified in the patient, the fact that an adrenal nodule and normocalcemic primary hyperparathyroidism were present, suggests that a complete hormonal workup should be obtained in these patients. CONCLUSION: It is important to realize that biliary system paragangliomas, although rare, may occur. As they have an association with multiple endocrine neoplasia syndrome, a thorough endocrine investigation should be made.

Deoxycholic Acid and the Marginal Mandibular Nerve: A Cadaver Study.

BACKGROUND: One of the rare but serious complications observed with deoxycholic acid administration is damage to the marginal mandibular nerve. In this study, we evaluated if deoxycholic acid directly induces histologic damage to fresh cadaveric marginal mandibular nerve. METHODS: A segment of marginal mandibular nerve was harvested from 12 hemifaces of 6 fresh cadavers. The nerve specimen was exposed to either 0.9% sterile saline for 24 h, deoxycholic acid (10 mg/ml) for 20 min, or deoxycholic acid (10 mg/ml) for 24 h. The nerve specimens were then fixed in glutaraldehyde for a minimum of 24 h. Toluidine blue stained sections were evaluated for stain intensity using light microscopy and color deconvolution image analysis. Supraplatysmal fat was harvested as a positive control and exposed to the same treatments as the marginal mandibular nerve specimens, then evaluated using transmission electron microscopy. RESULTS: Toluidine blue staining was less in the marginal mandibular nerve exposed to deoxycholic acid when compared to saline. The specimen exposed to deoxycholic acid for 24 h showed less toluidine blue staining than that of the nerve exposed to deoxycholic acid for 20 min. Transmission electron microscopy of submental fat exposed to deoxycholic acid revealed disruption of adipocyte cell membrane integrity and loss of cellular organelles when compared to specimens only exposed to saline. CONCLUSIONS: Deoxycholic acid (10 mg/ml) damages the marginal mandibular nerve myelin sheath in fresh human cadaver specimens. Direct deoxycholic acid neurotoxicity may cause marginal mandibular nerve injury clinically. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Isolated Conjunctival Inflammation as a Manifestation of IgG4-Related Disease.

PURPOSE: To report a unique case of isolated conjunctival inflammation from IgG4-related disease (IgG4-RD) confirmed by pathology. METHODS: A single interventional case of conjunctival IgG4-RD. RESULTS: A 63-year-old woman presented with a chronic, solitary, vascularized, tan-colored, and raised conjunctival lesion measuring 7.5 × 8.0 × 1.2 mm located at the temporal bulbar conjunctiva. An excisional biopsy was diagnostic of IgG4-RD based on the classic fibrosis pattern, 120 IgG4-positive plasma cells per high-power field, and an overwhelming majority of IgG4-positive cells among IgG plasma cells. No orbital or systemic involvement was found on clinical examination, imaging, and laboratory workup. The serum IgG4 level was normal (87.1 mg/dL). The patient was free of recurrence at 6-month follow-up. CONCLUSIONS: Isolated conjunctival inflammation without orbital involvement can be a presentation of IgG4-RD.

Multidisciplinary Conference and Clinical Management of Rectal Cancer.

BACKGROUND: Presentation of rectal cancer cases at a colorectal cancer multidisciplinary conference (CRC-MDC) is a required standard for the newly formed National Accreditation Program for Rectal Cancer administered by the Commission on Cancer. The aim of this study was to determine the frequency and manner in which CRC-MDC changed the management of rectal cancer patients at a tertiary academic center. STUDY DESIGN: All rectal cancer cases presented at a weekly CRC-MDC between July 2015 and June 2016 were prospectively included. Patient demographics and clinical information were recorded. The presenting physician completed a uniform written questionnaire outlining any changes in management as a result of the discussion. RESULTS: There were 408 rectal cancer cases included, and survey responses were obtained for 371 (91%). Thirty-nine patients (11%) had stage IV disease and 20 (5%) had locally recurrent cancer. There was a documented change in plan as a result of the CRC-MDC discussion in 97 of 371 (26%) cases surveyed. Changes in management included a change in therapy or change in therapy sequence in 76 cases, and recommendation of additional evaluation in 36 cases. Rates of management change were similar regardless of surgeon experience. Changes occurred in 23%, 28%, and 26% of cases presented by surgeons with <10, 10 to 20, and >20 years of experience, respectively (chi-square p = 0.63). CONCLUSIONS: The CRC-MDC changes clinical management for a significant portion of rectal cancer patients at a tertiary center, independent of the presenting surgeon's years of clinical experience. Our results support the CRC-MDC standard for the National Accreditation Program for Rectal Cancer.

Mucinous Histology Signifies Poor Oncologic Outcome in Young Patients With Colorectal Cancer.

BACKGROUND: The incidence of colorectal cancer in the young (under age 40) is increasing, and this population has worse oncologic outcomes. Mucinous histology is a potential prognostic factor in colorectal cancer, but has not been evaluated specifically in young patients. OBJECTIVE: The objective of the study was to determine factors associated with poor outcome in young patients with colorectal cancer (≤40 years) and to determine relationships between mucinous histology and oncologic outcomes in this population. DESIGN: This is a retrospective study. SETTING: Patients from a single-institution tertiary care center were studied. PATIENTS: A total of 224 patients with colorectal cancer under 40 years of age diagnosed between 1990 and 2010 were included (mean age, 34.7 years; 51.3% female). 34 patients (15.2%) had mucinous histology. INTERVENTIONS(S): There were no interventions. MAIN OUTCOME MEASURES: Oncologic outcomes were analyzed according to the presence of mucinous histology. RESULTS: The mucinous and nonmucin colorectal cancer study populations were statistically similar in age, sex, tumor location, pathological stage, differentiation, and adjuvant chemotherapy use. Five-year disease-free survival was 29.1% versus 71.3% (p < 0.0001) and 5-year overall survival was 54.7% versus 80.3% (p < 0.0001) for mucinous and nonmucinous patients, respectively. Mucinous colorectal cancers recurred earlier at a median time of 36.4 months versus 94.2 months for nonmucin colorectal cancers (p < 0.001). On multivariate analysis, pathological stage (stage II HR, 3.61; 95% CI, 1.37-9.50; stage III HR, 5.27; 95% CI, 2.12-12.33), positive margins (HR, 1.95; 95% CI, 1.12-3.23), angiolymphatic invasion (HR, 2.15; 95% CI, 1.26-3.97), and mucinous histology (HR, 2.36; 95% CI, 1.44-3.96) were independently associated with worse disease-free and overall survival. LIMITATIONS: This is a retrospective study without genetic information. CONCLUSIONS: Mucinous histology is a negative prognostic factor in young patients with colorectal cancer. This is associated with early and high recurrence rates, despite use of standard neoadjuvant and adjuvant regimens. Physicians need to be aware of this association and potentially explore novel treatment options. See Video Abstract at http://links.lww.com/DCR/A575.

Microanatomical Location of Hyaluronic Acid Gel Following Injection of the Upper Lip Vermillion Border: Comparison of Needle and Microcannula Injection Technique.

PURPOSE: To compare needle and microcannula injection techniques in regards to the microanatomical location of hyaluronic acid (HA) gel injected in the upper lip vermillion border of cadaver specimens. METHODS: The upper lip vermillion border was injected transcutaneously with HA gel in 8 fresh hemifaces of 4 female human cadavers. Each hemiface was injected by a single experienced injector, the right side using a 27-gauge microcannula and the left side using a 30-gauge needle. A 2-cm region of each lip was excised lateral to a point 1-cm lateral to the philtrum. Specimens were fixed in 95% alcohol, embedded in paraffin, and stained with hematoxylin-eosin for histologic examination. RESULTS: Most HA injected with either a needle or a microcannula was located within the orbicularis oris muscle, and the remaining HA resided within the subcutaneous fat. In 3/4 right (microcannula) hemifaces, 100% of the HA was located within the muscle. Only 2/4 left (needle) hemifaces had at least 95% of the HA located within the muscle. Overall, in right (microcannula) hemifaces, 93% of the filler was located within the muscle, and in left (needle) hemifaces, 79% of the filler was located within the muscle (p =0.14). CONCLUSIONS: Most HA filler injected into the vermillion border after either microcannula or needle injection resides within the orbicularis oris muscle rather than in a subcutaneous/submucosal location. Injection with a microcannula shows a trend for more uniform intramuscular location compared with needle injection.