Weak Staining Research Articles

Tacrolimus action pathways in an ointment base for hypertrophic scar prevention in a rabbit ear model

BackgroundTacrolimus is used to prevent unaesthetic scars due to its action on fibroblast activity and collagen production modulation. ObjectivesTo evaluate the action pathways, from the histopathological point of view and in cytokine control, of tacrolimus ointment in the prevention of hypertrophic scars. MethodsTwenty-two rabbits were submitted to the excision of two 1-cm fragments in each ear, including the perichondrium. The right ear received 0.1% and 0.03% tacrolimus in ointment base twice a day in the upper wound and in the lower wound respectively. The left ear, used as the control, was treated with petrolatum. After 30 days, collagen fibers were evaluated using special staining, and immunohistochemistry analyses for smooth muscle actin, TGF-β and VEGF were performed. ResultsThe wounds treated with 0.1% tacrolimus showed weak labeling and a lower percentage of labeling for smooth muscle actin, a higher proportion of mucin absence, weak staining, fine and organized fibers for Gomori's Trichrome, strong staining and organized fibers for Verhoeff when compared to controls. The wounds treated with 0.03% tacrolimus showed weak labeling for smooth muscle actin, a higher proportion of mucin absence, strong staining for Verhoeff when compared to the controls. There was absence of TGF-β and low VEGF expression. Study limitationsThe analysis was performed by a single pathologist. Second-harmonic imaging microscopy was performed in 2 sample areas of the scar. ConclusionsBoth drug concentrations were effective in suppressing TGF-β and smooth muscle actin, reducing mucin, improving the quality of collagen fibers, and the density of elastic fibers, but only the higher concentration influenced elastic fiber organization.

Immunohistochemical expression and prognostic significance of MAGE-A in canine oral malignant melanoma

Canine oral malignant melanoma (COMM) is considered a chemo-resistant cancer with a poor long-term prognosis. The melanoma-associated antigen A (MAGE-A) genes, which belong to the cancer-testis antigen family, are expressed in several different canine cancers but not in normal somatic tissue. This study evaluates the expression of MAGE-A proteins and their prognostic role in COMM. The study was conducted in 2 parts. During the first part, biopsies from oral malignant melanomas from 43 dogs were examined and immunohistochemically assessed for expression of MAGE-A proteins. For the second part, the association between MAGE-A expression and outcome was assessed using follow-up data which was available for 20 dogs whose primary tumour had been controlled with surgery +/- radiation therapy. MAGE-A proteins were expressed in 88.4% (38/43) of oral malignant melanomas and had a predominantly cytoplasmic expression pattern. Immunopositivity was observed in more than 50% of the cells in 21 dogs (48.8%). Immunostaining intensity was classified as weak, moderate and intense in 16 (37%), 16 (37%) and 6 (14%) cases, respectively. No staining for MAGE-A was seen in 5 dogs (11%). Dogs whose COMM had weak MAGE-A staining intensity had a median survival time (MST) of 320days while this was 129days for dogs with moderate and intense immunostaining (p=0.161). Dogs whose COMM had >50% of positive staining neoplastic cells had an MST of 141days and dogs with a staining <50% had an MST of 320days (p=0.164). MAGE-A expression did not influence survival in our cohort.

Clinicopathological features and prognosis of nodal nevi

Objective: To investigate the clinicopathological characteristics, differential diagnosis and prognosis of nodal nevi (NN). Methods: Eighteen cases of NN diagnosed at Fudan University Shanghai Cancer Center, Shanghai, China from 2009 to 2019 were collected. The clinicopathological characteristics and follow-up data were retrospectively analyzed. Histopathologic evaluation and immunohistochemical studies were carried out. The Vysis Melanoma FISH Probe Kit, combined with 9p21(CDKN2A) and 8q24(MYC) assays were performed in 2 cases. Results: There were 2 males and 16 females in the case series. The age of the patients ranged from 36 to 70 years (average 48.2 years). Fifteen cases located in axillary lymph nodes, 1 in inguinal lymph node, 1 in cervical lymph node, and 1 in external iliac lymph node. NN was found in only one lymph node in each case. Histologically, the nevus cell aggregates were found in capsule of lymph nodes in all cases. Nevus cells grew along the capsule into trabeculae in 8 cases, with 3 of them scattered in parenchyma. In one of these 8 cases, nevus cell aggregates massively occupied the parenchyma of the lymph node. The largest lesions in the 18 NN cases measured from 0.2 to 6.5 mm. All of the NN cases were classified as conventional nevi. The majority of the cases were composed of uniform nevus-like cells and identical to cutaneous pigmented nevi without atypia, necrosis, or mitosis. In the NN case that massively occupied parenchyma, some areas had abundant nevus cells and displayed atypical cytologic features, including increased nucleo-cytoplasmic ratio, small nucleoli, and occasional mitotic figures. Immunohistochemistry was performed in 13 cases. All of them were positive for S-100, SOX10, Melan A, and p16. HMB45 showed weak staining in rare cells of only one case out of 13 cases. Ki-67 labeling index <1% was found in all 13 cases. Additionally, the results of FISH assay were both negative. All patients were followed up for 13 to 129 months (median 31.5 months). Except that one patient died of the salivary gland carcinoma, the other patients all survived without tumor during the follow-up period. Conclusions: NN is a benign melanocytic lesion in lymph node. It is important to distinguish NN from metastatic melanoma when nevus cells occur in parenchyma and subcapsular sinus of lymph nodes, or show some atypical cytologic features. The morphology of bland nevus cells in capsule and trabeculae is a valuable clue. Besides, immunohistochemical profiling and FISH assay are helpful in the differential diagnosis.

The effect of codeine administration on oxidative stress biomarkers and the expression of the neuron-specific enolase in the brain of Wistar rats.

The study aimed to assess the effects of codeine medication on some oxidative stress parameters and how it affects the expression of enolase in neuronal cells. The codeine medication used for the study was Archilin™ with codeine syrup and dihydrocodeine 30mg. The study used 30 male Wistar rats which were grouped in five: A, B, C, D, and E (n = 6), while treatments were administered for 21days. Based on the LD50s of 6.09ml/kg body weight (b.wt.) Archilin™ with codeine syrup and 3.145mg/kg b.wt. dihydrocodeine, group A served as control and were given normal saline; groups B and C were treated with 1mg/kg and 2mg/kg b.wt. dihydrocodeine, respectively; while groups D and E were treated with 2ml/kg and 4ml/kg b.wt. Archilin™ with codeine syrup, respectively. After treatments, animals were sacrificed via cervical dislocation and the brains were harvested and prepared for determination of oxidative stress biomarkers as well as immunohistochemical studies of neuron-specific enolase (NSE) to assess for neuronal cell integrity. Significantly decreased mean values (p < 0.05) of superoxide dismutase (SOD) and catalase (CAT) activities were observed while malondialdehyde (MDA) is significantly increased (p < 0.05) among treated groups. The expression of enolase was downregulated in treatment groups when compared to control. Animals in group A which are control showed strong staining intensity of the prefrontal cortex compared to groups C, D, and E which showed mild staining. The scoring of group A for cerebellum showed strong staining intensity, groups B and C showed mild staining, while groups D and E showed weak staining intensity. From the findings of this study, prolonged codeine syrup administration causes oxidative stress and this affects the expression of enolase in neuronal cells resulting in glucose hypometabolism which eventually results in functional brain failure.

C1Q‐TNF‐related peptide 8 (CTRP8) in human prostate cancer

Introduction C1Q Tumor Necrosis Factor related Peptide 8 (CTRP8) is the one of the least characterized members of the C1Q/TNF family of proteins which is engaged in diverse functions ranging from metabolism to immunity and is one of only two CTRP family members absent in the mouse genome. CTRP8 was identified in our lab as a novel ligand of the RXFP1 receptor and was found to promote invasiveness and chemoresistance to temozolomide in glioblastoma (GB)(Glogowska et al., 2013; Thanasupawat et al., 2018). We have generated high-affinity rabbit antisera to human CTRP8 to identify a selective mast cell subpopulation in human prostate cancer tissues (Hempel Sullivan et al., 2020). Mast cells are innate immune cells which are primarily known for their role in the mediation of allergic responses. They have been detected within prostate cancer tissues and serve as potential prognostic marker in prostate cancer (Johansson et al., 2010; Hempel Sullivan et al., 2020). Methods Affinity-purified rabbit polyclonal antisera directed against an epitope unique to human CTRP8 were characterized by Western blot, immunofluorescence, and immunohistochemistry in prostate cancer tissue sections and human prostate cancer tissue microarrays (TMAs). Results Of several Flag-tagged human and mouse CTRP family members transiently expressed in HEK293T cells, the CTRP8 antisera exclusively detected human CTRP8 as assessed by Western blot, demonstrating the specificity of the antisera. We studied the expression of CTRP8 in human prostate tissues. CTRP8 was expressed in a subpopulation of human mast cells distributed in prostate cancer tissues, as demonstrated by co-localization of CTRP8 immunoreactivity and toluidine blue positive mast cells in prostate cancer tissues. This was further validated by dual immunofluorescence of CTRP8 with Tryptase, a marker for mature mast cells. Initial analysis of prostate cancer tissue microarrays (TMAs) (n= 360) revealed a comparative increase in the proportion of Tryptase+/ CTRP8+ cells versus total number of mast cells (Tryptase+/ CTRP8+ and Tryptase+/ CTRP8- cells) in the extra-tumoral compartment as compared to intra-tumoral regions for Gleason grades 6 and 7 prostate cancer samples. Weak staining of CTRP8 was detected in prostate epithelial cells. The RXFP1+ human prostate cancer cell line PC3 responded to CTRP8 treatment with increased protein kinase C delta signaling and enhanced motility as determined by real-time migration assays. Conclusions Here we have identified the expression of adipokine family member CTRP8 in prostate epithelial cells and in a subpopulation of mature mast cells in patient prostate cancer tissues, thus, identifying CTRP8 as a novel player in prostate cancer and associated mast cell compartment. References Glogowska, A., et al. J. Pathol., 231, 466–479, 2013 Thanasupawat, T., et al. Mol. Oncol. 12, 1464–1479, 2018 Johansson, A., Am. J. Pathol, 177, 1031–1041, 2010 Hempel Sullivan H, et al. J. Pathol., 2020 Hempel Sullivan H, et al. Cancer Epidemiol Biomarkers Prev., 2020

Expression of kisspeptin and KISS1 receptor in pituitary neuroendocrine tumours - an immunohistochemical study.

Pituitary neuroendocrine tumours (PitNETs), traditionally designated as pituitary adenomas, show elatively frequent invasive growth with exceptional metastatic potential, the causes of which are not entirely elucidated. Kisspeptins, which perform their activity through KISS1 receptor (KISS1R), are recognised as metastatic suppressors in many malignant tumours. This study aimed to investigate the immunohistochemical expression of kisspeptin and KISS1R in different types of PitNETs and to compare it with the expression in the normal anterior pituitary, using tissue microarray. The experimental group consisted of 101 patients with PitNETs, with 45 (37.3%) being of gonadotroph, 40 (33.9%) somatotroph, 4 (3.4%) corticotroph, 4 (3.4%) thyrotroph, 3 (2.5%) lactotroph, and 6 (5.1%) null-cell type. The control group consisted of anterior pituitary tissue accidentally removed during the surgery for PitNETs in 17 patients. Kisspeptin expression was observed in both experimental and control groups, without statistically significant differences in the staining intensity. Negative kisspeptin staining was detected in 10 (9.9%), weak in 79 (78.2%), and moderate in 12 tumours (11.9%); none of the tumours had strong staining intensity. The weak staining intensity was predominant in all PitNET types except thyrotroph tumours. Significant statistical difference in terms of kisspeptin expression between types of PitNET and the control group was not observed. Immunohistochemical expression of KISS1R was not observed in the control group or in the experimental group. We conclude that immunohistochemistry, as a method, cannot confirm the involvement of kisspeptin in tumourigenesis and aggressiveness of PitNETs, but potentially supports its antimetastatic role. The absence of KISS1R immunohistochemical expression in all anterior pituitaries and PitNETs in our cohort needs verification through the use of different procedures designed for the detection of the presence and localisation of proteins in the cell.

Expression of uroplakin II and GATA-3 in bladder cancer mimickers: caveats in the use of a limited panel to determine cell of origin in bladder lesions

Antibodies targeting uroplakin II (UPII) are highly specific for urothelial cells and are frequently used to determine if a primary bladder lesion or a metastatic lesion originates from the urothelium. However, to date, no studies have tested the expression of UPII in histological mimickers of bladder cancer that are nonurothelial in origin. Given the potential risk of misdiagnosis, immunohistochemical markers are often used to better characterize these lesions. In the present study, we analyzed the immunohistochemical expression of UPII in a set of urothelial carcinoma mimickers that included conventional nephrogenic adenoma (n=8), papillary nephrogenic adenoma (n=6), endometriosis/endosalpingiosis (n=5), inflammatory myofibroblastic tumor (n=4), ectopic prostate tissue (n=2), and malakoplakia (n=2). We also examined the expression of GATA-3, another commonly used immunohistochemical marker in bladder cancer diagnosis, in the same lesions. Weak immunoreactivity for UPII was identified in 6 of 27 mimickers (22%), and GATA-3 was expressed in 16 of 27 mimickers (59%). Strong immunoreactivity for UPII appeared to be a specific marker for urothelial cell of origin, although weak staining was seen in a significant proportion of mimickers. GATA-3 immunostaining was present in a greater number and broader spectrum of mimickers; however, only one case of papillary nephrogenic adenoma showed dual positivity for UPII and GATA-3. These findings support the immunohistochemical panel-based approach in the diagnosis of bladder lesions, especially if nonurothelial bladder cancer mimickers are in the differential diagnosis. Additional larger studies would be of value to expand on these findings.

Immunohistochemical localization and pharmacokinetics of the anti-MRSA drug teicoplanin in rat kidney using a newly developed specific antibody.

We prepared a polyclonal antibody against a teicoplanin (TEIC)-bovine serum albumin conjugate that was specific to both conjugated and free forms of TEIC. We demonstrated that this antibody could be used to detect the time-dependent localization of TEIC in rat kidneys. Immunohistochemistry revealed immunoreactivity specifically in the microvilli and apical cytoplasm of epithelial cells in proximal tubule segments S1 and S2, 1h after intravenous TEIC injection, with higher staining intensity in the S2 segments. The epithelial cells of S3 segments showed moderate immunostaining with a few cells exhibiting nuclear staining. Furthermore, we found that the distal tubules and collecting ducts contained both TEIC-positive and -negative cells. TEIC immunoreactivity decreased rapidly over time; only weak staining remained in the S3 segments, distal tubules, and collecting ducts 24h after administration. No staining was detected 7days after injection. These results were significantly different from those of our previous study obtained using vancomycin, which showed moderate staining in the proximal tubule segments S1 and S2, distal tubules, and the collecting ducts 8days after administration. The lower TEIC accumulation in tissues may account for a lower risk of adverse events compared to that using vancomycin.

Outcome, prognostic factors and histological characterization of canine gastrointestinal sarcomas.

Canine gastrointestinal sarcomas, a group of tumours that includes leiomyosarcomas (LMSAs), gastrointestinal stromal tumours (GISTs) and other rarer sarcomas, comprise about 10-30% of all gastrointestinal tumours. This study aims to characterize the histologic characteristics and clinical behaviour in order to identify prognostic factors predictive of outcome. A single institution database search for surgically treated gastrointestinal sarcomas yielded 47 cases with adequate tissue remaining for histologic analysis and 42 cases available for analysis of clinical outcome. Tumours were then prospectively evaluated for mitotic count, necrosis, haemorrhage and inflammation, as well as categorized via immunohistochemical (IHC) staining for smooth muscle actin, c-KIT and DOG-1. IHC analysis defined 32 tumours as GISTs, 14 as LMSAs and one as a sarcoma not otherwise specified. For both GISTs and LMSAs, the overall median survival time (MST) is 1024 days (range 31-1456), which did not differ statistically between tumour types (p=.92). The overall metastatic rate of GISTs in this study was 32.1% (n=9) which was not significantly different to that of LMSAs at 15.3% (n=2, p=.45). In multivariate analysis, mitotic count under 9 in GIST patients and complete surgical excision in all tumour types correlated with improved MST. For patients with GISTs, the intensity of c-KIT staining also correlated positively with survival, with an MST of 250 days in cases with weak staining and an MST of 1418 days in cases with moderate or strong c-KIT staining (p=.005).

Immunolocalization of Adrenoceptors in the Reproductive Tract of Male Domestic Cats in Comparison to Rats.

Simple SummaryIn cats, semen is collected by pharmacological stimulation. The administration of a drug that stimulates α2-adrenoceptors causes the expulsion of spermatozoa into the urethra. However, as the results are not always satisfactory, this method needs to be improved. There are nine subtypes of adrenoceptors that are involved in the contraction of smooth muscle, including those in the reproductive tract, so adrenoceptors other than the α2-subtype are potential targets in any new, optimized protocol. The aim of this study was to analyze the immunolocalization of the adrenergic receptors in the reproductive tract of the male cat for the first time in this species. The expression of all adrenoceptor subtypes was noted in the peritubular smooth muscle in cats, indicating a potential clinical application for agonists of these receptors for the optimization of the pharmacological semen collection in felids. In a broader context, the development of a new procedure for semen collection in the male cat, using active substances from groups other than those currently used, will support the wider application of reproductive biotechnologies in felids.Adrenoceptors mediate the action of the sympathetic nervous system, including the contraction of the epididymis and vas deferens. The aim of this study was to immunolocalize the adrenergic receptors in the reproductive tract of the male cat, as this information is not yet available. The epididymis and vas deferens of domestic cats and rats (the biological controls) were analyzed by immunohistochemistry to determine the localization of the α1A-, α1B-, α1D-, α2A-, α2B-, α2C-, β1-, β2-, and β3-adrenoceptors. All the receptors were expressed in the peritubular smooth muscles of the cat, but the α1D-, α2C-, and β1-adrenoceptors were not detected in this tissue in the rat. For the α2A-adrenoceptor, the intensity of immunostaining differed significantly between the caput epididymis (weakest staining) and the vas deferens (strongest staining). The presence of all the types of the receptors was also detected in the cytoplasm of the epithelial cells in all the regions of the reproductive tract. The strong expression of the α2A-adrenoreceptor suggests it has a leading role in the contraction of the reproductive tract in the cat. The presence of other adrenergic receptors in the smooth muscle of the epididymis and vas deferens indicates a potential clinical application for α1-mimetics in the optimization of pharmacological semen collection in felids.

Mesothelin Expression in Human Tumors: A Tissue Microarray Study on 12,679 Tumors.

Mesothelin (MSLN) represents an attractive molecule for targeted cancer therapies. To identify tumors that might benefit from such therapies, tissue microarrays including 15,050 tumors from 122 different tumor types and 76 healthy organs were analyzed for MSLN expression by immunohistochemistry. Sixty-six (54%) tumor types showed at least occasional weak staining, including 50 (41%) tumor types with at least one strongly positive sample. Highest prevalence of MSLN positivity had ovarian carcinomas (serous 97%, clear cell 83%, endometrioid 77%, mucinous 71%, carcinosarcoma 65%), pancreatic adenocarcinoma (ductal 75%, ampullary 81%), endometrial carcinomas (clear cell 71%, serous 57%, carcinosarcoma 50%, endometrioid 45%), malignant mesothelioma (69%), and adenocarcinoma of the lung (55%). MSLN was rare in cancers of the breast (7% of 1138), kidney (7% of 807), thyroid gland (1% of 638), soft tissues (0.3% of 931), and prostate (0 of 481). High expression was linked to advanced pathological tumor (pT) stage (p < 0.0001) and metastasis (p < 0.0001) in 1619 colorectal adenocarcinomas, but unrelated to parameters of malignancy in 1072 breast-, 386 ovarian-, 174 lung-, 757 kidney-, 171 endometrial-, 373 gastric-, and 925 bladder carcinomas. In summary, numerous important cancer types with high-level MSLN expression might benefit from future anti-MSLN therapies, but MSLN’s prognostic relevance appears to be limited.

Neuroprotective effects of vitamin C and garlic on glycoconjugates changes of cerebellar cortex in lead-exposed rat offspring

The deteriorating effects of Lead (Pb) on central nervous system (CNS) such as cerebellum has been demonstrated in previous studies. Glycoconjugates with the important role in CNS development may be affected by Pb-exposure. Utilization of antioxidant agents and herbal plants has attracted a great deal of attention on attenuating neurotoxicants-induced damage. Thus, in this study the neuroprotective effects of vitamin C and garlic on content of glycoconjugates of cerebellar cortex in Pb-exposed animals were investigated. Wistar pregnant rats were divided into: control (C), Pb-exposed (Pb) (1500 ppm lead acetate in drinking water), Pb plus vitamin C (Pb + Vit C) (500 mg/kg) intraperitoneally, Pb plus garlic (Pb + G) (1 mL /100 g body weight fresh garlic juice via gavage), Pb plus vitamin C and garlic (Pb + Vit C + G), and sham groups (Sh). Finally, levels of Pb in blood were measured in both rats and offspring on postnatal day 50 (PND50). Also, the cerebellums were removed for measuring Pb-levels and performing lectin histochemistry. Blood and cerebellar Pb-levels were increased in Pb-exposed group compared to control group (P < 0.001), whereas they were decreased significantly in Pb + Vit C, Pb + G, and Pb + Vit C + G groups (P < 0.01). By using MPA, UEA-1, and WGA lectin histochemistry, Pb-exposed group showed weak staining intensity compared to other groups. Besides, significant decrease was observed in the density of lectin-positive neurons of Pb-exposed group compared to the control group (P < 0.001). Moreover, strong staining intensity and high lectin-positive neurons were found in Pb + Vit C, Pb + G and Pb + Vit C + G groups than Pb-exposed group (P < 0.001). The present study revealed that Pb-exposure can result in alteration in the cerebellar glycoconjugates contents and co-administration of vitamin C and garlic could attenuate the adverse effects of Pb. The findings of this study revealed the ameliorating effects of vitamin C and garlic against Pb, suggesting the potential use of vitamin C and garlic as preventive agents in Pb poisoning.

Application of Neuron-Selective Fluorescent Probe, NeuA, To Identify Mouse Retinal Degeneration.

The retina is part of the central nerve system (CNS) and has various interneurons and sensory neurons such as photoreceptor cells. Retinitis pigmentosa (RP) is an inherited condition that is characterized by photoreceptor degeneration. Herein, we developed a fluorescent probe-NeuA-for detecting retinal neuronal cells and applied NeuA to discriminate between healthy and RP retinas. The staining pattern of NeuA in the retinas of healthy and RP mouse models was examined in vitro, ex vivo and in vivo using confocal microscopy, the fluorescent fundus microscopy and optical coherent tomography (OCT). NeuA strongly stained the outer segment layer of photoreceptor cells and some bipolar cells in the healthy retina, but there was only weak staining in the photoreceptor degenerated retinas. Therefore, NeuA probe can be used as the detecting RP tools in the preclinical conditions.

Role of EGFR and HER-2/NEU Expression in Gall Bladder Carcinoma (GBC)

Background Gall bladder carcinoma (GBC) is the most common malignancy of the biliary tract. Being known for its geographical and racial variations, and compared with the global statistics, its incidence is higher in the Indian subcontinent, mainly in the northern and eastern regions, accounting for 80 to 95% of cases. Aims and Objectives This study was conducted to evaluate the clinic-pathological spectrum and expression of EGFR and HER-2/NEU in GBCs and to understand their relation to prognosis, paving the way for targeted therapies for better treatment outcomes and patient survival. Materials and Methods This is a prospective study performed in a tertiary care hospital in 30 resected specimens of GBC cases recorded in our Department of Pathology from November 2017 to November 2019. Clinical history including the radiological reports and demographic parameters were included in the study pro forma. Immunohistochemical (IHC) staining for EGFR and HER-2/NEU was performed on all the selected cases. Clinicopathologic parameters like age, sex, histologic type, perineural, and lymphovascular invasion were compared and correlated with EGFR and HER-2/NEU status. Results Expression of EGFR was found in 93.33% of cases, which showed a highly significant correlation with histological tumor type ( p = 0.000). HER-2/NEU expression was found in 56.66% of cases, which also showed a significant correlation with histological tumour type ( p = 0.021). We found most of the cases with strong EGFR immunoreactivity (3+) were poorly differentiated tumors and most of the cases showing weak immunoreactivity for EGFR (1+) were well-differentiated. Conversely, in case of HER-2/NEU immunoreactivity, strong staining (3+) was seen in well-differentiated tumors and weak staining (1+) in poorly differentiated tumors. A significant correlation was also found between EGFR and HER-2/NEU expression ( p = 0.000) and between cholelithiasis and EGFR expression ( p = 0 .033). Conclusion EGFR is expressed in most cases of GBC. Its expression is more in poorly differentiated carcinomas as compared to the well-differentiated carcinomas, whereas HER-2/NEU expression is more in well-differentiated carcinomas. Therefore, they may serve as independent prognostic factors and also as targets for molecular therapy in GBCs.

P38.16 Expression of ROS1 Gene Rearrangement in Pulmonary Inflammatory Myofibroblastic Tumor With Cerebral Metastasis: Case Report

Primary pulmonary inflammatory myofibroblastic tumor (IMT) is a rare benign neoplasm; the tumor has been characterized by the application of immunohistochemical techniques, molecular biology and cytogenetics, which are very precious for the diagnosis. The purpose of this case was to determine whether immunohistochemistry for ROS1 could predict ROS1 rearrangement in inflammatory myofibroblastic tumor. A 50-year-old Moroccan man with history of mitral stenosis and mechanical valve replacement; presented to our hospital with a complaint of fatigue; headaches and lower back pain for one month. Contrast-enhanced computed tomography of his chest revealed a heterogenous well-defined mass lesion with central irregular calcification with no mediastinal lymphadenopathy or satellite nodules; this mass involving the left seventh rib and vertebral body and left atrial cavity. CT brain showed four solid enhancing irregular shaped mass lesions with hypodense areas consistent with metastasis CT-guided biopsy of the chest lesion showed fibrosis with dystrophic calcification; and non-specific chronic inflammation. He underwent radical excision of the right frontal lesion, and histopathology was distinctive fascicular growth of spindle cells with with a prominent inflammatory infiltrate, showed weak, diffuse and dot-like cytoplasmic staining with anaplastic lymphoma kinase (ALK)-negative immunohistochemistry and Fluorescence in situ hybridization (FISH) analysis was performed on interphase nuclei on 5 micron thin sections of formalin-fixed paraffin-embedded tissue that was found to be positive for TFG-ROS1 fusion. Because surgical excision was not possible crizotinib therapy (250 mg, bid) was chosen for the existence of ROS1 fusion gene with brain radiotherapy boost to metastasis. Treatment with glucocorticoids (methylprednisolone 1 mg/kg) and radiotherapy (5 days/week for 3 weeks at 3 Gy/fraction, 45 Gy/15 days) was started for cerebral metastasis . After taking crizotinib for 3 months, the strong changes in the size of the pulmonary mass was showed at chest chest computed tomography with decreased infiltration around the adjacent structures. Inflammatory pseudotumour of the lung with brain metastasis is very uncommon; we must continued therapy with crizotinib and long-term follow-up is necessary, for detection of recurrence, metastasis and to understand the natural course, especially in patients with myofibroblastic differentiation ROS1 positive Crizotinib is effective for pulmonary IMT, and should be taken into consideration for the treatment of inoperable pulmonary IMT patients who lack ALK rearrangement.

KM55 Monoclonal Antibody Staining in IgA-Dominant Infection-Related Glomerulonephritis

Introduction: IgA-dominant infection-related glomerulonephritis (IgA-IRGN) is a unique form of IRGN, which needs to be distinguished from IgA nephropathy (IgAN), due to overlapping clinical and pathological features. The key factor in the pathogenesis of IgAN is galactose-deficient IgA1 (Gd-IgA1). However, the mechanism of glomerular IgA deposition in patients with IgA-IRGN is unclear. Therefore, we evaluated whether Gd-IgA1 could be a useful biomarker to distinguish between these 2 diseases. Methods: A case-control study was conducted to analyze the clinical and pathological characteristics of 12 patients with IgA-IRGN. The intensity and distribution of glomerular Gd-IgA1 (KM55) staining in renal biopsies were assessed. The control group consisted of 15 patients diagnosed with IgAN and an additional 17 patients with glomerulopathy involving IgA deposition. Results: The main clinical manifestations of patients with IgA-IRGN were nephrotic-range proteinuria, hematuria, acute renal injury, and hypocomplementemia. Active lesions were the leading pathological feature, while focal segmental sclerosis was rare. Half of the patients exhibited hump-shaped subepithelial deposits. Glomerular KM55 staining was negative in 7 patients, trace in 4 patients, and 2+ in 1 patient. The median intensity of KM55 staining in IgA-IRGN patients was 0 (range 0∼2+), which was significantly lower than that of primary IgAN patients (median 2+, range 1+∼3+). The receiver operating characteristic analysis demonstrated that the optimal cutoff level to identify these 2 diseases was 0.5+. Conclusions: Glomerular KM55 staining intensity might be helpful to distinguish IgA-IRGN from primary IgAN. Weak or negative staining may favor IgA-IRGN. In addition, integrated analysis including clinical data, pathological findings, and prognostic information would further improve the differential diagnosis.

Abstract PS18-37: Loss of Trop2 protein in metaplastic carcinoma of breast by IHC

Abstract Background: Trop2 (trophoblast cell surface antigen) is a transmembrane calcium transducer which has been associated with tumor growth, aggressiveness and metastasis. Compared to normal tissue, Trop2 is expressed at much higher levels in many epithelial tumors, which makes it an excellent target for ADCs (antibody-drug conjugates). Sacituzumab govitecan-hziy, an ADC that combines Trop-2 antibody with a SN-38 payload has been recently approved for treatment of refractory metastatic triple-negative breast cancer, and several other TROP2 targeted therapies are in development. To better understand Trop 2 expression in breast tumor that may help to identify patients who could benefit most from Trop2-targeted therapy, we aimed to screen Trop2 protein in multiple types of breast tumor. Design: Using chromogenic immunohistochemistry (IHC), Trop2 protein was examined in 42 formalin fixed paraffin embedded (FFPE) specimens from surgically resected breast tumors, including 21 TNBC, 16 HR+, 4 HER2+ (3 HR+/HER2+ and 1 HR-/HER2+), and 1 sarcoma. Sections were stained on an automated staining system (BOND-MAX; Leica Microsystems) using anti-TROP2 antibody (clone ERP20043, Abcam, cat# 214488). Membrane staining was assessed. The percentage of positivity (0% to 100%) and the staining intensity (0 = no staining, 1+ = weak staining, 2+ = moderate staining, and 3+ = strong staining) were evaluated and multiplied to generate H-score (0-300). Results: Among of 41 breast carcinoma cases, 38 (92.6%) cases expressed Trop2 protein in tumor cells, with a median H-score of 162.5, including 18/21 TNBC, 16/16 HR+, 4/4 HER2+ cases. Trop2 was expressed in several breast cancer histopathology subtypes, including 32 invasive ductal carcinomas (H score ranged from 29 - 290), 2 invasive lobular carcinomas (H score 140 and 145), 1 invasive papillary carcinoma (H score 70), 1 invasive micropapillary carcinoma (H score 210), 1 invasive squamous cell carcinoma (H score 245) and 1 metaplastic carcinoma with mesenchymal differentiation (cartilaginous sarcomatoid type) (H score 17). Three breast carcinomas were negative for Trop2 (H score 0). All three cases are metaplastic carcinomas, including two spindle cell carcinomas and one with mesenchymal differentiation, which are TNBC. Only two of 5 (40%) metaplastic carcinomas tested expressed Trop2 (p = 0.0034). One breast sarcoma was tested and had no TROP2 expression. Conclusion: Trop2 is commonly expressed in breast cancer. However, metaplastic carcinoma of breast has shown a trend of Trop2 protein loss. Further validation on Trop2 loss in metaplastic carcinoma might help with identifying breast cancer patients less likely to benefit from Trop2-targeted therapy. Citation Format: Fei Yang, Yasmeen Rizvi, Erkan Yuca, Ming Zhao, Kurt Evans, Xiaofeng Zheng, Aysegul Sahin, Funda Meric-Bernstam. Loss of Trop2 protein in metaplastic carcinoma of breast by IHC [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-37.

Abstract PS2-02: Prognostic relevance of the HER2 status of circulating tumor cells in metastatic breast cancer patients screened for participation in the DETECT study program

Abstract Background Circulating tumor cells (CTCs) have been reported to predict clinical outcome in metastatic breast cancer (MBC). Biology of CTCs may differ from the primary tumor and HER2-positive CTCs are found in some patients with HER2-negative disease. In this analysis, we evaluated the clinical relevance of the HER2 status of CTCs in patients screened for participation in the DETECT trials, the largest study program for CTC-based therapy interventions in MBC. Methods Patients with HER2-negative MBC were screened for CTCs using CellSearch. CTCs were labeled with an anti-HER2 antibody and classified according to staining intensity (negative, weak, moderate, or strong staining). Patients with HER2-positive CTCs were invited to participate in the randomized phase III DETECT III trial, which evaluated treatment with physicians' choice therapy with and without lapatinib. Patients with HER2-negative CTCs could participate in the DETECT IVa/b trials which investigated serial blood measurements during in-label systemic therapy. Patients who participated in the screening but were not enrolled in the DETECT III or DETECT IV trials were treated at discretion of their physician and followed up for progression and death. Results Screening blood samples were analyzed in 1933 patients with HER2-negative MBC. Out of these, 102 were enrolled in the DETECT III and 213 in the DETECT IVa/b trials, respectively. 1217 out of the 1933 screened patients (63.0%) had ≥ 1 CTC per 7.5 ml blood. ≥ 5 CTCs were detected in 735 patients (38.0%; range 1 – 35,078 CTCs, median 8 CTCs). Patients with ER-positive tumors were more likely to be CTC-positive than patients with ER-negative disease (≥ 1 CTC in 64.7% vs. 57.1% patients, respectively, p = 0.011; ≥ 5 CTCs in 40.8% vs. 29.5%, p &amp;lt; 0.001). CTC status was also associated with ECOG performance status (≥ 1 CTC in 63.2% of patients with ECOG 0 vs. 69.0% with ECOG 1-3, p = 0.020; ≥ 5 CTCs in 36.3% vs. 47.2%, p &amp;lt; 0.001). HER2 status of CTCs was assessed in 1159 CTC-positive patients. At least one CTC with strong HER2 staining was found in 174 (15.0%) patients. The proportion of CTCs with strong HER2 staining among all CTCs of an individual patient ranged between 0.06% to 100% (mean: 15.8%). Patients with ER- and PR-positive tumors were more likely to harbor ≥ 1 CTC with strong HER2 staining. For survival analysis, 52 patients receiving anti-HER2-therapy with lapatinib in the interventional arm of the DETECT III trial were excluded. CTC status was significantly associated with OS (median OS in patients with ≥ 1 CTC: 15.5 [95%-CI: 14.2-16.8] months vs. 37.2 [32.7-41.7] months in CTC-negative patients, p &amp;lt; 0.001, HR 2.359; median OS in ≥ 5 CTCs vs. &amp;lt; 5 CTCs: 12.0 [10.0-14.0] vs. 28.6 [25.5-31.6] months, p &amp;lt; 0.001, HR 2.204). Detection of ≥ 1 CTC with strong HER2 staining was associated with shorter OS (9.7 [7.1-12.3] vs. 16.5 [14.9-18.1] months in patients with CTCs with negative-to-moderate HER2 staining only, p = 0.013). In the multivariate analysis, only age, ER status, PR status, ECOG performance status, therapy line, and CTC status independently predicted OS. Conclusion To our knowledge, this is the largest analysis to date regarding the clinical relevance of HER2 status of CTCs in MBC. We confirm the high prognostic value of CTC detection in patients with HER2-negative disease. Presence of ≥ 1 CTC with strong HER2 staining was significantly associated with shorter OS in univariate analysis. However, the CTC-HER2 status did not correlate with survival in multivariate analysis. * VM and MBP contributed equally to this work Citation Format: Volkmar Müller, Maggie Banys-Paluchowski, Thomas WP Friedl, Peter A Fasching, Andreas Schneeweiss, Andreas Hartkopf, Diethelm Wallwiener, Brigitte Rack, Franziska Meier-Stiegen, Jens Huober, Matthias Rübner, Oliver Hoffmann, Lothar Müller, Wolfgang Janni, Pauline Wimberger, Bernadette Jäger, Klaus Pantel, Nadia Harbeck, Tanja Fehm. Prognostic relevance of the HER2 status of circulating tumor cells in metastatic breast cancer patients screened for participation in the DETECT study program [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-02.

Seronegative Villous Atrophy in Children: Clinical and Immunohistochemical Features.

Villous atrophy (VA) is not pathognomonic of celiac disease (CD). We aimed at reporting distribution, clinical, and immunohistochemical features of seronegative VA (SNVA) in a pediatric population. We retrospectively collected data from patients who underwent intestinal biopsies between 2010 and 2017 and showed VA without serum CD-associated autoantibodies. Marsh-Oberhuber grading was used. Density of intraepithelial lymphocytes (IELs) expressing CD3 or TCRγδ+ receptor and of lamina propria CD25+ cells was assessed by immunohistochemistry. Intestinal deposits of anti-tissue tranglutaminase2 (anti-TG2) were also investigated by double immunofluorescence. Over a 7-year period, 64 out of 1282 patients with VA had negative serum CD serology. Diagnoses were: inflammatory bowel diseases (IBD) (21/64), Gastro-Esophageal Reflux Disease (GERD) (12/64), food allergy (8/64), infections (7/64, of which 3 HIV infections), immune deficiency (3/64), short bowel syndrome (3/64), congenital diarrhea (2/64), other/inconclusive diagnosis (8/64). Forty-four, 15, and 5 showed Marsh 3a, 3b, and 3c lesion, respectively. The latter category included 2 patients with Crohn disease, 2 with immunodeficiencies, 1 with lymphohistiocytosis. In 41/46 (89%) patients, mononuclear CD25+ cells were above the cut-off, indicating mucosal inflammation but only 18/46 (39%) had IELs and TCRγδ + IELs above limits of normality. In 10 of 46 (22%) patients, a positive immunofluorescence indicated the presence of anti-TG2 mucosal antibodies. SNVA is not rare representing up to 5% of the cases of VA. Most patients have a Marsh 3a lesion. Immunohistochemical analysis may be helpful in excluding CD, whereas the finding of mucosal anti-TG2, particularly with a weak staining, shows no absolute specificity for CD.

Assessment of HER2 using the 2018 ASCO/CAP guideline update for invasive breast cancer: a critical look at cases classified as HER2 2+ by immunohistochemistry.

In 2018, the American Society of Clinical Oncology/College of American Pathologists revised the criteria for HER2 immunohistochemistry (IHC) equivocal (2+) classification in their updated guideline. We reviewed invasive breast cancer specimens originally classified as equivocal (2+) under the 2018 guideline that underwent HER2 fluorescence in situ hybridization (FISH) testing from August 2018 to August 2019 at our Canadian reference hospital to investigate cases with ambiguous staining patterns between the 1+ and 2+ definitions. Demographics, pathologic features, and pre-analytic conditions were recorded. The H&E and corresponding HER2 IHC slides were reviewed to confirm tumor type and grade, and classify as HER2 indeterminate, 0, 1+, 2+, or "Intermediate" (staining features between the 1+ and 2+ classifications). FISH testing was performed on 289 cases and 273 met inclusion criteria. The FISH-amplified rate was 12.1%. Upon IHC review, 44.7% (122/273) of cases were reclassified as Intermediate. These cases had incomplete staining with moderate intensity (43/122, 35.3%) and/or <10% complete weak or moderate staining (102/122, 83.6%). Intermediate cases had a significantly lower frequency of amplified FISH results than 2+ cases (p < 0.0001), with only four (3.3%) FISH positive and two (1.6%) FISH heterogeneous. Our study highlights the ambiguity in the current guideline for classifying some HER2 IHC patterns. As the rate of gene amplification in these cases was low (4.9%), we recommend adhering to the 2018 HER2 2+ criteria for reflex FISH testing. However, cases with <10% moderate complete staining and certain heterogeneous patterns warrant special consideration. Further descriptive clarification of 1+ criteria is needed.