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Metastatic tumors (MTs) may show different characteristics of the immune microenvironment from primary tumors (PTs) in non-small cell lung cancer (NSCLC). The heterogeneity of immune markers in metastatic NSCLC and its associated factors has not been well demonstrated. In this study, 64 surgically resected specimens of paired PTs and MTs were obtained from 28 patients with NSCLC. Multiplex immunofluorescence (mIF; panel including programmed death-ligand 1 (PD-L1), Cytokeratin, CD8, and CD68) was performed on whole sections. The heterogeneity of the immune contexture of PD-L1 expression, infiltrating lymphocytes, and immune-to-tumor cell distances was quantified via digital image analysis. In a quantitative comparison of MTs and corresponding PTs, MTs showed higher PD-L1 expression levels, lower density of CD8+ cytotoxic T lymphocytes (CTLs), and longer spatial distance between CTLs and tumor cells. Subgroup analysis, which associated clinical factors, revealed that the heterogeneity of immune markers was more obvious in extrapulmonary, metachronous, and treated MTs, while fewer differences were observed in intrapulmonary, synchronous, and untreated MTs. In particular, MTs showed significantly higher PD-L1 expression and lower lymphocyte infiltration in metastatic NSCLC with EGFR mutations. Prognosis analysis showed that an increased density of CD8+ CTLs in MTs was associated with better overall survival (OS). Therefore, significant discrepancies in PD-L1 expression and lymphocyte infiltration in metastatic NSCLC are most likely associated with temporal heterogeneity with a history of anti-treatment and correlated with EGFR mutations. The detection of immune markers in re-obtained metastatic specimens may be required for immunotherapy prediction in these patients with metastatic NSCLC.

The heterogeneity of programmed death-ligand 1 (PD-L1) status between core needle biopsies (CNBs) from one tumor has not been well studied before. The current study attempts to find out the best index using multiple core biopsies from one tumor which can better reflect the actual PD-L1 status. Random CNB was performed in surgical specimens from 170 consecutive non-small cell lung cancer samples. Fifty-one cases (41 cases with PD-L1 positive and 10 cases with PD-L1 negative) and 216 matched CNBs were analyzed by DAKO 22C3 PharmDx Link 48 Autostainer. The PD-L1 status was compared between the surgical specimens and matched CNBs. Heterogeneity of PD-L1 status between CNBs from one tumor was observed in 56% of PD-L1 positive cases. Different tumor proportion score (TPS) statistical forms with regard to the highest, mean, median, weighted average TPS, as well as TPS showed by the longest biopsy specimen and the biopsy with most tumor volume were compared. At a cut-off of 1%, the concordance rates were 94.1%, 88.2%, 90.2%, 86.3%, 86.3%, and 86.3%; At a cut-off of 50%, the concordance rates were 92.2%, 86.3%, 84.3%, 82.4%, 82.4%, and 86.3%, respectively. The CNB with the highest TPS can best represent PD-L1 status estimated by whole surgical specimen. The highest TPS among the multiple biopsies is a robust evaluation of the PD-L1 status, but not mean TPS, at the 1% and 50% cut-offs.

BACKGROUND: Aquaporin2 (AQP2) is water channel protein that is widely distributed among mammalian tissues and plays a major role in water homeostasis. However, little is known about the expression and significance of AQP2 in human adrenal tumors. Thus, we performed an immunohistochemical investigation of AQP2 in normal and neoplastic adrenal tissues. METHODS: AQP2 protein expression was investigated in 190 adrenal tumor patients using immunohistochemistry. Correlation between protein expression and clinicopathological features was statistically analyzed. RESULTS: We demonstrated immunopositivity for AQP2 in all adrenal medulla-originating tumors, including 96 pheochromocytomas (PCC), 7 metastatic pheochromocytomas (MPCC), and 10 neuroblastic tumors (NT) and 13 extra-adrenal paragangliomas (EAPGL). Whereas, APQ2 was absent from the 52 adrenal cortical adenomas and 2 adrenal cortical carcinomas examined. The 10 metastatic carcinomas examined in adrenal tissue were also negative for AQP2. In 82 (85%) of the 96 samples from patients with PCC, we studied the relationship between clinicopathologic factors and AQP2 expression and our findings suggested that the tumors that exhibited diffuse expression pattern of AQP2 were larger in diameter than those exhibiting focal (P=0.007) or mediate expression pattern (P=0.001). CONCLUSIONS: AQP2 protein is significantly expressed in normal adrenal medullary tissues and medullary tumors (including PCC, MPCC and NT) as well as EAPGL. AQP2 expression may indicate the origin of normal adrenal tissues, and its expression in cancer tissue may reflect the maintenance of water metabolism via AQP2 during tumorigenesis. AQP2 may serve as a valuable marker for the differential diagnosis of adrenal tumors.

Objective: To investigate the correlation and overlaps between PD-L1 expression and classical genomic aberrations in Chineselung adenocarcinoma (LADC) patients. Methods: We reviewed 428 consecutive, surgically resected cases of LADC from October 2015 to December 2016 from our center.PD-L1 expression was evaluated based on tumor proportion score (TPS). Correlation and co-occurrence of PD-L1 expression levelwith those of classical driver genes, such as EGFR, ALK, ROS-1, and KRAS and with clinical variables and disease-free survival(DFS) were analyzed. Results: Seventy of the 428 cases (16.4%) showed TPS ≥ 1%, and 21 cases (4.9%) showed TPS ≥ 50%. PD-L1 positive expressionwas significantly associated with male gender, smoking, advanced TNM stage, and solid histologic subtype. Both TPS ≥ 1% and ≥50% were correlated with the absence of an EGFR mutation (P < 0.001) and the presence of ALK rearrangement (P = 0.024).KRAS mutation was associated with TPS ≥ 50% (P = 0.035). PD-L1 positivity commonly overlapped with the alterations ofclassical driver oncogenes (58.5% with TPS ≥ 1% and 42.9% with TPS ≥ 50%). Approximately three-quarters of PD-L1 positivecases co-occurred with classical therapeutic-gene aberrations in cases with stage III/IV cancer or cancer progression. LADC couldbe divided into four subgroups based on the expression profile of current routine biomarkers for potential therapeutic strategies. Conclusions: PD-L1 expression is not only closely correlated with classic gene alterations but also commonly overlaps with theaberrations of classical driver oncogenes in Chinese LADC patients. These findings provide a useful overview of clinical strategiesthat rely on the profile of routinely used molecular biomarkers. Cite this article as: Wu J, Sun W, Wang H, Huang X, Wang X, Jiang W, et al.The correlation and overlaps between PD-L1 expression and classicalgenomic aberrations in Chinese lung adenocarcinoma patients: a single centercase series. Cancer Biol Med. 2019; 16: 811-21. doi: 10.20892/j.issn.2095-3941.2019.0209

AimsThis study aimed to validate the application of combined multiplex immunofluorescence (mIF) and digital image analysis (DIA) in formalin-fixed and paraffin-embedded tissues for the quantitative assessment of programmed death-ligand 1(PD-L1) and immune cells (ICs) in non-small cell lung cancer (NSCLC).MethodsFifty resected samples of NSCLC were sequentially stained with a DNA-tagged mIF (panel including PD-L1, CKpan, CD8, CD68 and 4′,6-diamidino-2-phenylindole (DAPI)) and conventional immunohistochemistry (cIHC). The assessment of cell density and consistency of tumour proportion score (TPS) via DIA were compared with those by pathologists.ResultsA strong correlation in the cell population of immune markers was obtained between mIF and cIHC (for PD-L1: R=0.9304, CKpan: R=0.8231, CD8: R=0.9314 and CD68: R=0.8366) within 95% limits of agreement. The continuous TPS calculated using mIF was highly consistent with the IHC staining results which were evaluated by pathologists (R=0.9362). However, in the comparison of TPS using interval variables, a poor agreement was obtained at a cut-off of 1% (κ=0.197), whereas excellent agreement was achieved at cut-offs of 50% (κ=0.908) and 5% (κ=0.823). DIA on mIF showed that PD-L1 commonly colocalised with CD68+ macrophages and CD8+ cytotoxic cells were closer to PD-L1-/CK+ tumour cells (TCs) than to PD-L1+/CK+ TCs in spatial distribution.ConclusionsA combination of mIF and DIA is useful for the quantification of PD-L1 expression and IC populations in NSCLC. Further validation of TPS at a cut-off of 1% and assay harmonisation is essential for translating this method in a diagnostic setting.

Aquaporin 3 (AQP3) is the membrane channel of water and involved in fluid homeostasis. The aim of this study was to reveal the expression and significance of AQP3 in cutaneous lesions. We analyzed AQP3 mRNA levels using RT-PCR in 311 cutaneous lesions and confirmed AQP3 expression in these lesions by immunohistochemistry. AQP3 mRNA was detected in normal epidermis, seborrheic keratosis, solar keratosis, Bowen’s disease, squamous cell carcinoma, eccrine poroma, apocrine carcinoma, and sebaceoma; however, AQP3 mRNA was absent in basal cell carcinoma, nevocellular nevus, or malignant melanoma. By immunohistochemistry, diffuse AQP3 expression was seen in all keratotic lesions including seborrheic keratosis, verruca vulgaris, molluscum contagiosum, solar keratosis, Bowen’s disease, and squamous cell carcinoma. Diffuse AQP3 expression was also present in all extramammary Paget’s disease. No AQP3 staining was obtained in basal cell carcinoma. Positive AQP3 staining was seen in sweat gland tumors including hidradenoma, eccrine poroma, and apocrine carcinoma. Among sebaceous tumors, AQP3 expressed diffusely in all sebaceous hyperplasia and sebaceous adenoma, but not in sebaceous carcinomas. Only focal AQP3 staining was seen in nevocellular nevus and no AQP3 staining in melanoma. Our findings indicate the function of AQP3 maintained in most skin tumors. AQP3 may be used for differential diagnosis in skin tumors.

BackgroundThis study proposed a computational method to detect the cancer areas and calculate the tumor proportion score (TPS) of PD-L1 immunohistochemistry (IHC) expression for lung adenocarcinoma based on deep learning and transfer learning.Patients and methodsPD-L1 22C3 and SP142 IHC slides of lung adenocarcinoma samples on digitized whole-slide images (WSI) database were employed. We build a deep convolutional neural network (DCNN) to automatically segment cancer regions. TPS was calculated based on segmented areas and then compared with the interpretations of pathologists.ResultsWe trained a DCNN model based on 22C3 dataset and fine-tuned it with SP142 dataset. We obtain a robust performance on cancer region detection on both datasets, with a sensitivity of 93.36% (22C3) and 92.80% (SP142) and a specificity of 93.97% (22C3) and 89.25% (SP142). With all the coefficient of determinations larger than 0.9 and Fleiss’ and Cohen’s Kappa larger than 0.8 (between mean or median of pathologists and TPS calculated by our method), we also found out the strong correlation between the TPS estimated by our computational method and estimation from multiple pathologists’ interpretations of 22C3 and SP142 respectively.ConclusionWe provide an AI method to efficiently predict cancer region and calculate TPS in PD-L1 IHC slide of lung adenocarcinoma on two different antibodies. It demonstrates the potential of using deep learning methods to conveniently access PD-L1 IHC status. In the future, we will further validate the AI tool for automated scoring PD-L1 in large volume samples.

SATB2 is a sensitive marker for colorectal adenocarcinomas. No study has investigated its diagnostic utility in metastatic Krukenberg tumors (MKTs) of the ovary. Here we performed immunohistochemical staining SATB2 in 70 MKTs of various origins (stomach 27, colorectum 13, appendix 20 including 19 metastatic adenocarcinomas ex goblet cell carcinoids [AdexGCC] and 1 conventional poorly differentiated carcinoma with signet ring cells, breast 5, bladder 3, lung 2) to assess its diagnostic utility. We also compared SATB2 with CDX2, CK7, CK20, chromogranin, and synaptophysin in MKTs of gastric origin (MKTs-stomach), those of colorectal origin (MKTs-colorectum) and those due to appendiceal AdexGCCs (MKT-AdexGCCs) for their sensitivity and specificity to distinguish these tumors. SATB2 staining was seen in 1/27 (4%) MKTs-stomach (40% cells), 7/13 (54%) MKTs-colorectum (mean: 17% cells, median: 7%, range: 2% to 60%), and 19/19 (100%) of MKT-AdexGCCs (mean: 97% cells, median: 100%, range: 80% to 100%) (P0.01 between any two). SATB2 staining was seen in 1/1 metastatic appendiceal poorly differentiated carcinoma with signet ring cells (5% cells), 1/3 MKTs of bladder origin (60% cells), 0/2 MKTs of pulmonary origin, and 1/5 MKTs of breast origin (10% cells). SATB2 staining was diffuse strong in MKT-AdexGCCs whereas in other MKTs it was focal and weak in the signet ring and nonsignet ring nonglandular cells and from focal weak to diffuse strong in well-formed glands. MKTs-stomach, MKTs-colorectum, and MKT-AdexGCCs showed no significant staining difference in CDX2 (100%, 100%, 100% cases, respectively; P=1.0), CK20 (96%, 100%, 100%, respectively; P=1.0), chromogranin (59%, 31%, 63%, respectively; P0.05) or synaptophysin (59%, 63%, 84%, respectively; P0.05) but they had significant difference in CK7 staining (93%, 8%, 42%, respectively; P0.05). Among these 6 markers, SATB2 is the best one to distinguish MKT-AdexGCCs from MKTs-stomach (100% sensitivity, 96% specificity) and MKTs-colorectum (100% sensitivity and 100% specificity if staining more than 75% tumor cells as the cutoff). In distinguishing MKTs-stomach from MKTs-colorectum, SATB2 is not as good as CK7 which is the best marker. Our results indicate that SATB2 is a highly sensitive marker (100% sensitivity) for metastatic MKT-AdexGCCs with high specificity (100% specificity when showing strong staining in at least 75% cells) among MKTs. SATB2 is a useful marker for determining the primary sites of MKTs of the ovary.

Background Special AT-rich sequence-binding protein 2 (SATB2) is a novel marker for colorectal adenocarcinomas but little is known about its expression in appendiceal adenocarcinomas. We aim to investigate SATB2 in these tumors and colorectal adenocarcinomas with comparison to CDX2. Methods Immunohistochemical stains for SATB2 and CDX2 were performed in 49 appendiceal adenocarcinomas (23 conventional, 26 adenocarcinoma ex goblet cell carcinoids (AdexGCCs)) and 57 colorectal adenocarcinomas. Their expression was correlated with tumor differentiation and growth patterns. Results SATB2 staining was positive in 26/26 (100%) appendiceal AdexGCCs and 15/23 (65%) appendiceal conventional adenocarcinomas (P = 0.001). Their mean percentage of SATB2-positive cells was 93% and 34%, respectively (P < 0.0001). CDX2 staining was seen in 26/26 (100%) AdexGCCs and 22/23 (96%) appendiceal conventional adenocarcinomas (P = 0.4694). SATB2 and CDX2 showed similar staining in AdexGCCs but CDX2 labeled more tumor cells than SATB2 in conventional adenocarcinomas (mean 84% vs. 34%, P < 0.0001). SATB2 and CDX2 staining was seen in 82% (47/57) and 96% (55/57) colorectal adenocarcinomas, respectively (P = 0.01). The mean percentage of cells positive for SATB2 and CDX2 was 48% and 91%, respectively (P < 0.00001). Decreased SATB2 immunoreactivity was associated with non-glandular differentiation particularly signet ring cells in colorectal (P = 0.001) and appendiceal conventional adenocarcinomas (P = 0.04) but not in appendiceal AdexGCCs. Conclusions SATB2 is a highly sensitive marker for appendiceal AdexGCCs with similar sensitivity as CDX2. In colorectal and appendiceal conventional adenocarcinomas, SATB2 is not as sensitive as CDX2 and its immunoreactivity is dependent on tumor differentiation.

Background Immune checkpoint blockade targeting PD-1/PD-L1 has shown efficacy in several types of cancers. However, the correlation between PD-L1/PD-1 expression and the specific clinicopathological features in papillary thyroid carcinoma (PTC) has not been investigated. Methods We examined the immunohistochemical expression of PD-L1, PD-1, and BRAF V600E on whole-tissue sections from 126 cases of primary PTC more than 1 cm in size. The correlation between the PD-L1/PD-1 expression and the clinicopathological features was evaluated. Results PD-L1 was positively expressed in 53.2% PTCs, and its expression was positively correlated with rich tumor-infiltrating lymphocytes (TILs), background chronic lymphocytic thyroiditis (CLT), female gender, absence of psammoma bodies, and PD-1 expression. Among these parameters, rich TILs, female gender, and absence of psammoma bodies were independent factors affecting PD-L1 expression on the multivariate logistic regression analysis. PD-1 expression was detected in the TILs and was positively correlated with rich TILs, background CLT, and absence of stromal calcification. Lack of stromal calcification was an independent factor affecting PD-1 expression. Neither PD-L1 nor PD-1 expression showed significant correlation with BRAF V600E expression. Conclusions Our results show that the distinctive pathological features of PTCs, including TILs, background CLT, female gender, psammoma bodies, and stromal calcification, are useful parameters for predicting PD-L1 or PD-1 expression.

HER2 is an orphan receptor tyrosine kinase of the EGFR families and is considered to be a key tumor driver gene [1]. Breast cancer and gastric cancer with HER2 amplification can be effectively treated by its neutralizing antibody, Herceptin. In clinic, Immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification [2]. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted with another family member HER4 [3]. IHC staining with 4B5 also indicated that there was strong non-specific cytoplasmic and nuclear signals in normal gastric mucosal cells and some gastric cancer samples. Using a protein lysate array which covers 85% of the human proteome, we have confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and nuclear staining of 4B5 of normal gastric epithelium. Finally, we have developed a novel mouse HER2 monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. Therefore, UMAB36 could provide as an alternative highly specific IHC reagent for testing HER2 amplification in gastric cancer populations.

Gelatins are important and frequently-used food additives, but the quality control of gelatins is always an intractable problem in routine analysis. In this work, a new strategy for simultaneously rapid identification and quantification of gelatins from various species using ultrasound assisted digestion-UPLC-MS/MS was described. Fourteen peptide markers were used for authenticity of gelatins by the presence or absence of these species-specific peptides. In the meantime, adulteration ratios could be calculated based on the peak area of different species peptide markers. With 10 g/L trypsin and ultrasound application, digestion time could be decreased from >12 h (traditional method) to 5 min, and the determination of animal gelatins could be achieved in 20 min with a single analysis run. Twenty-five commercial gelatin samples were screened, among which two deer-hide gelatin samples were adulterated with 900 g/kg and 265 g/kg cattle-hide gelatin (CHG); two donkey-hide gelatin (ACC) samples were adulterated with 66 g/kg and 381 g/kg horse-hide gelatin; and one ACC sample was adulterated with 786 g/kg CHG. This strategy provides an efficient and sensitive authentication and traceability of gelatin-containing products, and could also be applied to processed meat or protein food.

The mechanism of carcinogenesis of gastric cancer (GC) is still unclear now. This study aimed to explore the correlations among PD-L1, Epstein-Barr virus (EBV) infection, lymphocyte infiltration, HER2 expression, HER2 gene status, histology, and other clinicopathological factors in GC. A total of 44 GC patients with massive lymphocyte infiltration (GC-MLI) and 93 GC patients without massive lymphocyte infiltration were involved in this study. Immunohistochemical analysis was used to test the expression levels of PD-L1 and HER2. Fluorescence in situ hybridization was used on HER2-positive cases with a score of 2+ to test the HER2 gene status. EBV-encoded RNA was used to test for EBV infection. In univariate analysis, PD-L1 expression was significantly associated with GC-MLI (P

Background Recent research supports a significant role of immune checkpoint inhibitors in the treatment of solid tumors. However, relevant reports for programmed death-ligand 1 (PD-L1) and CD8+ tumor-infiltrating lymphocytes (TILs) in pulmonary neuroendocrine tumors (PNETs) have not been fully studied. Therefore, we investigated PNETs for the expression of PD-L1 and infiltration by CD8+ TILs as well as the prognostic value of both factors. Methods In total, 159 specimens of PNETs (35 TC, 2 AC, 28 LCNEC, 94 SCLC) were included in this study. Immunohistochemistry (IHC) was used to detect the expression of PD-L1 in these cases. Cases demonstrating ≥5% tumor cell expression or any expression (> 1%) of PD-L1 on immune cells were considered positive. CD8+ TILs both within stroma and tumor areas of invasive carcinoma were analyzed using whole-slide digital imaging. Manual regional annotation and machine cell counts were performed for each case. Results Positive expression of PD-L1 was observed in 72 cases (45.3%), including 9 cases (5.7%) with expression exclusively on tumor cells, 46 cases (28.9%) with expression exclusively on immune cells, and 17 cases (10.7%) with the expression on tumor cells and immune cells. PD-L1 expression was associated with necrosis (p

Background GATA-binding protein 3 (GATA3) has been identified as a sensitive marker for breast carcinoma but its sensitivity in primary genital extramammary Paget diseases (EMPDs) has not been well studied. Methods Here we investigated immunohistochemical expression of GATA3 in 72 primary genital EMPDs (35 from female, 37 from male; 45 with intraepithelial disease only, 26 with both intraepithelial disease and invasive adenocarcinoma including 14 also metastasis, 1 with metastatic adenocarcinoma only for study). We also compared GATA3 to gross cystic disease fluid protein 15 (GCDFP15) for their sensitivity. Results Positive GATA3 staining was seen in all 71 (100%) intraepithelial diseases, 25/26 (96%; female 10/10, male 15/16) invasive adenocarcinomas and 14/15 (93%; female 3/3, male 11/12) metastatic adenocarcinomas, respectively. Positive GCDFP15 staining was seen in 46/71 (65%; female 28/34 or 82%, male 18/37 or 49%) intraepithelial diseases, 20/26 (77%; female 9/10, male 11/16) invasive adenocarcinomas, and 12/15 (80%; female 2/3, male 10/12) metastatic adenocarcinomas, respectively (GATA3 versus GCDFP15: p

Immune checkpoint inhibitor therapies targeting PD-L1/PD-1 have been shown to be effective in treating several types of human cancer. In papillary thyroid carcinoma (PTC), little is known about the expression of PD-L1/PD-1 in the tumor microenvironment or its potential correlation with BRAF V600E mutation status. In this study, we examined the expression of PD-L1, PD-1, and BRAF V600E in PTC by immunohistochemistry and investigated the clinical significance of expression status. We studied the expression of PD-L1, PD-1, and BRAF V600E by immunohistochemical staining in 110 cases of PTC with a diameter > 1 cm. Cases with a background of chronic lymphocytic thyroiditis (CLT) were excluded, as differentiating lymphocytes in the context of CLT from tumor-infiltrating lymphocytes (TILs) is difficult. We classified PD-L1+/PD-1+ expression as type 1 (41%), PD-L1−/PD-1− as type 2 (17%), PD-L1+/PD-1− as type 3 (5%), and PD-L1−/PD-1+ as type 4 (37%). Significant correlations were found between expression of BRAF V600E and that of PD-L1 and PD-1. The positive correlation observed between expression of BRAF V600E and PD-L1/PD-1 suggests that immunotherapies targeting PD-L1/PD-1 might be effective for PTC patients with the BRAF V600E mutation, which are refractory to radioiodine therapy.

Nasal-type extranodal NK/T cell lymphoma (NK/TCL) is a rare malignancy that usually presents as a destructive midline lesion in the nasal region. The incidence of nasal-type extranodal NK/TCL has been reported to be much higher in Asia and South America than in North America and Europe. Its diagnosis mainly relies on histopathological and immunohistochemical examinations. Most patients demonstrate rapid disease progression and short survival. This report concerns a 37-year-old male with nasal-type extranodal NK/TCL showing severe facial disfigurement who was initially misdiagnosed as having Aspergillus infection. The report emphasizes the clinical manifestations and the importance of pathological diagnosis.

Human epidermal growth factor receptor 2 (HER2) is an orphan receptor tyrosine kinase member of the EGFR families and is found to be a key tumor driver gene. In breast cancer and gastric cancer, HER2 amplification can be effectively treated by its neutralizing antibody, Herceptin. In clinic, the HER2 immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted to another family member HER4. IHC staining also indicated that it has strong non-specific cytoplasmic and nucleus staining in normal gastric mucosal cells and some gastric cancer samples. Using a protein lysate array which covers 85% of the human proteome, we have successfully identified and confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and unclear staining of 4B5 on normal gastric epithelium. Finally, we have deve...

Xenotransplantation of human cancers into immunodeficient mice is a very useful approach for studying human tumor biology. However, the occasional occurrence of lymphomagenesis in some mice can spoil the model and must be investigated in detail. We found that a high percentage (32.5%, 26/80) of cancer patient-derived xenografts (PDXs) resembled lymphoma in NOD/SCID mice. Of the 26 xenografts, 23 were human-derived expressing human CD45 (hCD45+) and proved to be of the B-cell subtype (CD3-/CD20+) and they were all positive for Epstein - Barr virus (EBV). The remaining 3 xenografts proved to be mouse-derived for both hCD45- and negative amplification of a human gene. The most interesting finding is that gastric cancer had much higher rates (24/126, 19.0%) of lymphoma formation in the PDX model than did colorectal cancer (1/43, 2.3%). Statistical analysis revealed that cancer type and inflammation in the parent tumor are significantly associated with lymphomagenesis. Further validation discovered lymphomagenesis by inoculating only gastritis mucosa. Therefore, our findings suggest that it is necessary to take precautions when directly xenografting cancer tissues with remarkable baseline inflammation, such as gastric cancer into immunodeficient NOD/SCID strains. Further, the established xenograft models should be validated by both leukocyte markers and human gene signatures.

Increased sensitivity and specificity of immunohistochemical (IHC) detection of HER2 expression is crucial as the role of Trastuzumab have expanded in the treatment of HER2 positive non-breast cancer patients such as gastric cancer patients. Non-specific nuclear and cytoplasmic staining of the HER2 antibody clone 4B5 has been reported by other labs. In this study, we evaluated the specificity of HER2 clone 4B5 and a new HER2 antibody clone UMAB36 using a suite of methods including protein lysate arrays, western blots, FISH and IHC correlation screens on 129 breast and 158 colon cancer cases for HER2 expression. The protein lysate array and western results revealed that clone 4B5 recognizes three proteins: HER2, HER4, and ZSCAN18, a nuclear transcription protein. In comparison, clone UMAB36 recognized only HER2 protein in the same protein lysate array and western screen as clone 4B5. False negative results, based on correlation of IHC with FISH HER2 positives, were generated using clone 4B5 in 1 breast and 5 gastric of the total 287 cancer cases screened. Comparatively, no false negative results were observed using clone UMAB36 in which there was a 100% correlation between IHC and FISH screen. In this study, areas of normal gastric tissue often stained positive by HER2 clone 4B5, so we performed further analysis of 470 normal gastric cases using Ventana's BenchMark instrument. The results show that 278 normal gastric cases had positive stain with clone 4B5 compared to 3 cases with clone UMAB36. The high background by clone 4B5 may be due HER4 being upregulated in adjacent normal gastric tissue. Our results indicate clone UMAB36 has higher specificity and sensitivity than clone 4B5 in screening gastric tumors. Citation Format: Lixin Zhou, Kehu Yuan, Fangfang Ren, Lili Ki, Min Zhou, Wei Fu, Xiaozheng Huang, Rachel M. Gonzalez, Youmin Shu, Yi Shen, Guangli Wang, Donghui Ma, Wei-Wu He, Jian Chen. A screen of breast and colon cancers with HER2 antibody clone UMAB36 does not exhibit the cross-reactivity of clone 4B5 with HER4 protein. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3921.

Human epidermal growth factor receptor 2 (HER2) is an orphan receptor tyrosine kinase member of the EGFR families and is found to be a key tumor driver gene. In breast cancer and gastric cancer, HER2 amplification can be effectively treated by its neutralizing antibody, trastuzumab. In clinic, the HER2 immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted to another family member HER4. IHC staining also indicated that it has strong non-specific cytoplasmic and nucleus staining in normal gastric mucosal cells and some gastric cancer samples. Using a protein lysate array which covers 85% of the human proteome, we have successfully identified and confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and unclear staining of 4B5 on normal gastric epithelium. Finally, we have developed a novel HER2 mouse monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. UMAB36 could provide an alternative high specific IHC reagent for HER2 amplification testing in gastric cancer population. Citation Format: Lixin Zhou, Kehu Yuan, Fangfang Ren, Lili Qi, Zhongwu Li, Guiyin Wu, Xiaozheng Huang, Yi Shen, Min Zhao, Wei Fu, Huibo Liu, Boyang Chu, Guangli Wang, Youmin Shu, Donghui Ma, Wei-Wu He, Jian Chen. Using protein chips to develop a highly specific HER2 antibody for HER2 amplification testing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3399. doi:10.1158/1538-7445.AM2015-3399