Biphenotypic Branchioma: A Better Name Than Ectopic Hamartomatous Thymoma for a Neoplasm with HRAS Mutation.

Ectopic hamartomatous thymoma is a rare neck lesion originally thought to represent a non-neoplastic hamartoma, even though thymic origin has been questioned, and there is uncertainty about whether the lesion is a neoplasm. We investigated the genetics by performing targeted next generation sequencing (NGS). Three cases were identified from the authors' consultation files. A custom, targeted NGS panel including 1385 pan-cancer-related genes was performed on all cases. Three patients included 2 males and 1 female, aged 50, 58 and 70 years, respectively (mean 59.3 years), with tumors arising in the low anterior neck. All cases showed classical histologic features of EHT, with one case showing intraductal carcinoma in association with the EHT. By targeted NGS, one case harbored a hotspot HRAS mutation (p.Gln61Lys), while the other two cases only showed non oncogenic variants. Dual mesoderm and endoderm derivation/differentiation (biphenotypic) has been previously recognized, with epithelial and myoepithelial components, and arising from the apparatus contributing to neck development (branchial apparatus). Thus, EHT has been shown to have genetic alterations in HRAS. These findings, without evidence of thymic derivation or an ectopic tissue location, strongly support that EHT is a true neoplasm. The name biphenotyic branchioma more correctly reflects the true nature of this dual mesoderm and endoderm derived tumor occurring in the lower neck.

Avoiding pitfalls in diagnostic immunohistochemistry-important technical aspects that every pathologist should know.

This review focuses on technical aspects of diagnostic immunohistochemistry (IHC), with an emphasis on aspects of methodology and interpretation that may be problematic for practicing pathologists. Pitfalls in IHC are reviewed, and the importance of good controls and the the use of multi-tissue controls is discussed. Also covered is the optimal use of IHC in cytologic specimens and specimens where no paraffin block is available. Artifacts encountered in IHC are discussed and illustrated, and a number of useful techniques are described in detail, including preparation of multi-tissue control material, tissue and cell transfer techniques, and tissue protection techniques.

Differential expression of BAFF and its receptors in discoid lupus erythematosus patients.

BACKGROUND: B-cell activating factor of the TNF family (BAFF) promotes the maturation and survival of B cells. Because BAFF levels are elevated in systemic lupus erythematosus (SLE) patients, BAFF has been the target of emerging therapies for SLE, such as belimumab. Levels of BAFF and its receptors in discoid lupus erythematosus (DLE) patients are unknown. OBJECTIVE: To compare skin and blood mRNA and protein levels of BAFF and its receptors BAFF-R, TACI, and BCMA in DLE subjects with (DLE+/SLE+ (N=28)) and without SLE (DLE+/SLE- (N=35)), psoriasis subjects (N=11), and normal subjects (N=42). METHODS: We used quantitative real-time PCR to measure blood and skin BAFF, BAFF-R, TACI, and BCMA mRNA, sandwich ELISAs to measure sera BAFF, and immunohistochemistry to evaluate BAFF and BAFF-R skin protein expression. RESULTS: BAFF mRNA and protein levels were highest in DLE+/SLE+blood, followed by DLE+/SLE-, psoriasis, and normal blood. BAFF protein also correlated with anti-nuclear antibodies, and autoantibodies against double-stranded DNA, single-stranded DNA, and ribonucleoprotein, and Systemic Lupus Erythematosus Disease Activity Index scores in DLE patients. While showing no difference between DLE+/SLE+ and DLE+/SLE- skin, BAFF and its receptors mRNA were up-regulated in DLE skin vs. normal and psoriasis skin. DLE skin had higher percentages of BAFF-R⁺ inflammatory cells, likely T cells and macrophages, than psoriasis and normal skin. CONCLUSIONS: BAFF may be a serologic marker of systemic disease in DLE patients. BAFF and its receptors are elevated in DLE skin, suggesting that targeted therapies against these proteins could treat refractory DLE patients.

Recovery of the injured external anal sphincter after injection of local or intravenous mesenchymal stem cells.

OBJECTIVES: To understand the endogenous process of wound healing after anal sphincter injury and to determine possible mechanisms by which mesenchymal stem cells (MSCs) exert their regenerative potential. METHODS: Virginal female rats (n=204) underwent anal sphincter laceration and repair. Thereafter, animals were randomly assigned to control injection, injection with intravenous MSCs, or direct injection of MSCs into the injured sphincter. Twenty uninjured animals served as baseline controls. Sphincters were analyzed for contractile function and parameters of wound healing 24 hours, 48 hours, 7 days, and 21 days after injury. RESULTS: Direct injection of MSCs into the injured anal sphincter resulted in improved contractile function 21 days after injury compared with controls. Although expression of both proinflammatory (cyclooxygenase-2 and interleukin-6) and anti-inflammatory (interleukin-10 and tumor necrosis factor-α-stimulated gene-6) genes were increased dramatically and transiently after injury, MSCs did not alter this response. In contrast, transforming growth factor (TFG)-ß1 (an important mediator of matrix deposition by mesenchymal cells) and lysyl oxidase (an enzyme important for synthesis of collagen and elastin) expression increased dramatically at earlier time points in the direct MSC injection group compared with controls. Increased expression of TFG-ß1 and lysyl oxidase in directly injected sphincters was associated with increased collagen deposition and engraftment of MSCs in the sphincter. CONCLUSION: In this preclinical animal model, direct, but not intravenous, injection of MSCs into the injured anal sphincter at the time of repair resulted in improved contractile function of the sphincter after injury, increased matrix deposition in the external anal sphincter, and increased expression of TFG-ß1 and lysyl oxidase in the acute phase after injury.

Effects of pregnancy, parturition, and anal sphincter transection on function of the external anal sphincter in an animal model.

OBJECTIVE: To estimate the effects of pregnancy, parturition, and anal sphincter laceration (with repair) on external anal sphincter morphology and neurophysiology and to define the time course of these effects after injury. METHODS: Within 4 hours of vaginal delivery, 80 rats underwent either sham or anal sphincter laceration with repair. After 3 days, 3 weeks, and 3 and 6 months (n=20 for each time point), animals were killed, and the anal sphincter complexes dissected and removed for neurophysiologic studies. Twitch tension, peak tetanic force, fatigue, and maximal electrical field-stimulated force generation were determined. Sphincters were then fixed and serially sectioned (5-micrometer thickness) at 100-micrometer intervals for histologic analysis. RESULTS: Maximal electrical field-stimulated force generation, maximal tetanic contraction, and twitch tension were decreased in the external anal sphincter 3 days after anal sphincter laceration with repair compared with sham-operated parturient rats (3.3 g compared with 11.6 g, 4.5 g compared with 14.5 g, and 0.6 g compared with 2.0 g, respectively, all P<.02). Increased fatigability of the sphincter muscle was observed in all newly parturient rats-sham and anal sphincter laceration with repair; recovery occurred in the shams by 3 months. A gradual recovery occurred in all these neurophysiologic measures, with no significant differences between anal sphincter laceration with repair and shams by 6 months postpartum. CONCLUSION: Repaired anal sphincter transection in periparturient animals results in short-term severe compromise of neurophysiologic function of the external anal sphincter. Over time, however, force generation recovers and approximates that of postpartum rats with intact anal sphincters.

Reactivity with TdT in Merkel cell carcinoma: a potential diagnostic pitfall.

Merkel cell carcinoma (MCC) is a high-grade neuroendocrine carcinoma of skin characterized by cells with a "blastic" appearance, scant cytoplasm, and fine, evenly distributed chromatin. Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase present in thymic T cells, lymphoblastic lymphoma/leukemia, and some cases of acute myeloid leukemia. After observing TdT immunoreactivity in a case of MCC, we analyzed 26 tumors by immunohistochemical analysis to determine their spectrum of reactivity with TdT and identified TdT in 19 (73%) of 26 MCCs. Staining intensity was variable but was often moderate to strong and present in a significant percentage of cells. Because MCC has cytomorphologic features similar to those of lymphoblastic lymphoma and may manifest as metastatic disease, reactivity with TdT in MCC could represent a diagnostic pitfall in the differential diagnosis with lymphoblastic lymphoma, particularly because the latter may lack CD45 and/or CD20, yet both neoplasms may express PAX-5, a B-cell-associated marker.

Effect of prolonged vaginal distention and sphincter transection on physiologic function of the external anal sphincter in an animal model.

OBJECTIVE: To estimate the effect of prolonged vaginal distention and anal sphincter transection on contractile properties of the external anal sphincter. METHODS: Young female virgin rats were randomly assigned to four treatment groups (sham, vaginal distention, transection of anal sphincter plus repair, or combined distention and transection plus repair). After 3 weeks, the anal sphincter complex was analyzed for twitch tension, maximal tetanic force, fatigue, and maximal responses to electrical field stimulation. Each sphincter was serially cross-sectioned and stained with hematoxylin and eosin to evaluate sphincter integrity. Statistical evaluation was performed using analysis of variance (Student-Newman-Keuls) and chi2 analysis. RESULTS: The function of a transected and repaired anal sphincter is compromised significantly after 3 weeks. Vaginal distention alone did not alter function of the sphincter. Further, prolonged vaginal distention together with sphincter transection did not result in statistically significant differences in sphincter function compared with transection alone. CONCLUSION: In an animal model, there is a major effect on external anal sphincter function in vitro 3 weeks after laceration with repair. Although prolonged vaginal distention had no significant effect alone and minimal adverse effects on the lacerated anal sphincter, laceration of the sphincter has major adverse effects on its morphology and function. LEVEL OF EVIDENCE: II.

Use of p63/P504S monoclonal antibody cocktail in immunohistochemical staining of prostate tissue.

Diagnosis of prostate needle biopsies can be challenging, particularly when the atypical areas of interest are very small. The utility of immunostains for high-molecular-weight cytokeratin (34betaE12) to highlight prostatic basal cells in these cases is well established, and recent reports also document the utility of immunostains for p63 (a marker that stains the nuclei of prostate basal cells) for this purpose. Several investigators have demonstrated that immunostaining for P504S, a cytoplasmic protein that is overexpressed in a high percentage of prostate cancers and in many cases of high-grade prostatic intraepithelial neoplasia (PIN), can also be of use in the diagnosis of prostate biopsies. Because of the cytoplasmic localization of P504S and nuclear localization of p63, the authors hypothesized that a cocktail of these two antibodies might allow simultaneous demonstration of P504S and p63 using a single immunostain. In this report the authors describe the successful use of a cocktail of p63/P504S for immunohistochemical staining of prostate tissue. Two different staining approaches were investigated, with essentially identical results. This cocktail localizes P504S in the cytoplasm of prostate carcinoma cells and high-grade PIN and demonstrates the nuclei of prostatic basal cells, providing information on both the status of P504S and the presence or absence of basal cells with a single immunostain. This cocktail can be of great utility in the examination of diagnostically challenging prostate specimens.

Immunohistochemical analysis of forensic evidence from a double homicide.

We report the use of immunohistochemical staining for analysis of forensic evidence from a double homicide. A 38-year-old woman and her 7-year-old daughter were murdered by multiple blows to the head and face with a tomahawk, resulting in multiple fragments of brain tissue scattered about the murder scene. The victims' husband and father was the main suspect, who maintained that he was out of town on business during the evening of the murders. However, a shirt taken from the suspect's car on the morning after the murders (secured by the police before the suspect visited the murder scene) was found to have two small stains. DNA analysis on the stains showed the presence of the deceased wife's DNA, and immunohistochemical stains on shirt fragments conclusively documented the presence of deep central nervous system tissue, providing the critical piece of evidence needed to arrest and prosecute the suspect. This report demonstrates that shirt or similar cloth fragments can be processed into paraffin blocks and subsequently immunostained to search for and classify types of tissue fragments that may be present on the fabric.

Morphometric properties of the posterior vaginal wall in women with pelvic organ prolapse.

OBJECTIVE: Our purpose was to analyze the morphometric properties of the posterior vaginal wall and compare the smooth muscle distribution in the posterior vaginal muscularis in women with and without pelvic organ prolapse. STUDY DESIGN: Specimens were taken from the apex of the posterior vaginal wall after hysterectomy from 15 women with pelvic organ prolapse and from 8 healthy control subjects. Smooth muscle cells of the posterior vaginal wall were identified by immunohistochemistry with antibodies to smooth muscle alpha-actin. Morphometric analysis was performed on histologic cross-sections of the posterior vaginal wall to determine the fractional area of nonvascular smooth muscle in the muscularis. The innervation pattern of the vaginal wall was determined by use of S100 immunostaining. Statistical comparisons between two groups were conducted by a Student t test. Comparisons between multiple groups were conducted with a one-way analysis of variance followed by a post-hoc Student-Neuman-Keuls test. RESULTS: The fractional area of nonvascular vaginal smooth muscle in the muscularis of women with posterior wall prolapse was significantly decreased compared with that of healthy control subjects. Nerve bundles were located in the deep vaginal muscularis and adventitia of the posterior vaginal wall. In women with posterior wall prolapse, nerve bundles were smaller and fewer in number. CONCLUSION: Morphologic features of the posterior vaginal wall are significantly altered in women with posterior wall prolapse compared with asymptomatic control subjects.

Morphometric analysis of smooth muscle in the anterior vaginal wall of women with pelvic organ prolapse.

OBJECTIVE: The purpose of this study was to compare the smooth muscle content of the anterior vaginal wall in normal women and women with pelvic organ prolapse. STUDY DESIGN: Specimens were taken from the apex of the anterior vaginal cuff after abdominal hysterectomy from 28 women with pelvic organ prolapse and 12 control subjects. Smooth muscle cells of the anterior vaginal wall were identified by immunohistochemistry with antibodies to smooth muscle alpha-actin. Morphometric analysis was used to determine the fractional area of nonvascular smooth muscle in the muscularis in histologic cross-sections of the anterior vaginal wall. RESULTS: The fractional area of nonvascular vaginal smooth muscle in the muscularis of women with prolapse was significantly decreased compared with that of control subjects. This decreased fraction of smooth muscle in the anterior vaginal wall was not related to age, race, or stage of prolapse. In women with prolapse, vaginal smooth muscle content was most diminished in specimens from postmenopausal women with no estrogen replacement. The fractional area of muscularis smooth muscle was also decreased significantly in premenopausal women with prolapse. CONCLUSION: The fraction of smooth muscle in the muscularis of the anterior vaginal wall is significantly decreased in women with pelvic organ prolapse compared with normal control subjects.

Tissue protection immunohistochemistry: a useful adjunct in the interpretation of prostate biopsy specimens and other selected cases in which immunostains are needed on minute lesions.

Performing immunohistochemical analysis on minute lesions is a challenging task, primarily because they frequently disappear when the paraffin block is recutfor immunostaining purposes. This is a particularly common occurrence with prostate biopsy specimens, in which immunostains for high-molecular-weight cytokeratin commonly are used as an adjunct to H&E examination for aiding in the interpretation of minute "suspicious" lesions. We describe an original method designated tissue protection immunohistochemistry, that allows the performance of high-molecular-weight cytokeratin immunostains (or other immunostains) on previously stained H&E slides. The method described does not require destaining of H&E-stained sections, and it allows the preservation of the H&E stain on adjacent levels that may be present on the same slide. The method described requires that the original H&E-stained sections be placed on adhesive slides, but it has the advantages of eliminating the requirement of a paraffin block for immunostaining and eliminating the need for saving intervening unstained sections for possible immunohistochemical analysis.