Percentage Of Positive Tumor Cells Research Articles

Metabolic reprogramming and angiogenesis in primary cutaneous Merkel cell carcinoma: expression of hypoxia-inducible factor-1α and its central downstream factors.

Metabolic reprogramming and altered gene expression mediated by hypoxia-inducible factors play crucial roles during tumour growth and progression. Nevertheless, studies analysing the expression of hypoxia-inducible factor-1α and its downstream targets in Merkel cell carcinoma (MCC) are lacking but are warranted to shed more light on MCC pathogenesis and to potentially provide new therapeutic options. To analyse the immunohistochemical expression of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor-A (referred to as VEGF throughout the manuscript), VEGF receptor-2 (VEGFR-2), VEGF receptor-3 (VEGFR-3), glucose transporter-1 (Glut-1), monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CAIX) in primary cutaneous MCC. The 16 paraffin-embedded primary cutaneous MCCs (Merkel cell polyomavirus (McPyV) positive/negative: 11/5) were analysed by immunohistochemistry, namely HIF-1α, VEGF, VEGFR-2 (KDR), VEGFR-3 (FLT4), Glut-1, MCT4 and CAIX. An established quantification score (QS) was applied to quantitate the protein expression by considering the percentage of positive tumour cells (0: 0%; 1: up to 1%; 2: 2-10%; 3: 11-50%; 4: >50%) in relation to the staining intensity (0: negative; 1: low; 2: medium; 3: strong). HIF-1α was expressed in all MCCs and predominantly found at the invading edges of tumour margins. The HIF-1α downstream factors Glut-1, MCT4 and CAIX were expressed in 13 of 16 MCC (81%), 14 of 16 MCC (88%) and 16 of 16 MCC (100%), respectively. Interestingly, VEGF and VEGFR-2 were not expressed in tumour cells, whereas VEGFR-3 was expressed in all MCCs. HIF-1α was expressed significantly stronger in McPyV+ tumours (QS: 10.36±2.41) than in McPyV- tumours (QS: 5.40±1.34; P=0.002). Similarly, VEGFR-3 was also expressed significantly stronger in McPyV+ tumours (QS: 10.00±2.52) than in McPyV- tumours (QS: 5.40±3.43, P=0.019). Our data provide first evidence for a role of HIF-1α in induced metabolic reprogramming contributing to MCC pathogenesis. The metabolic signatures of McPyV+ and McPyV- tumours seem to show relevant differences.

Distribution of PD-L1 expression level across major tumor types.

e15176 Background: Programmed death-ligand (PD-L1) expression is an efficacy predictive biomarker in cancer immunotherapy. PD-1/PD-L1 axis can restore the host immunity against malignancies causing durable tumor remissions, and thus the level of PD-L1 protein expression is of critical clinical significance. However, the knowledge of PD-L1 expression level of patients with different malignancies is still inadequate understanding. Methods: Based on the percentage of positive tumor cells (TC value) identified by immunohistochemistry (IHC) method, the protein expression of PD-L1 was divided into three groups as g1 (TC < 1%), g2 (1% ≤ TC < 50%) and g3 (TC ≥ 50%). The expressions of PD-L1 from 1772 patients with malignancies across eight types including 133 patients with liver cancer, 35 patients with bile duct cancer, 94 patients with gastric cancer, 146 patients with colorectal cancer, 48 patients with pancreatic cancer, 76 patients with melanoma cancer, 33 patients with glioma and 1207 patients with lung cancer cases were made. The relationships between cancer types and PD-L1 expression were calculated via χ2 test using SPSS v22.0. Results: The ratios of PD-L1 expression in three groups (g1, g2 and g3, respectively) across eight cancer types were 67.7, 30.8 and 1.5% in liver cancer, 65.7, 28.6 and 5.7% in bile duct cancer, 77.7, 20.2 and 2.1% in gastric cancer, 81.5, 18.5 and 0% in colorectal cancer, 64.6, 31.2 and 4.2% in pancreatic cancer, 50.5, 35.7 and 13.8% in melanoma, 30.3, 63.1 and 6.6% in glioma and 55.9, 34.0, 10.1% in lung cancer, respectively. Significant correlation between the PD-L1 expression and cancer types were found (χ2 = 140.543, Cramer’s V coefficient = 0.199, p = 5.53×10−23). The expression of PD-L1 in patients with lung cancer tended to be distributed in g3, in patients with melanoma tended to be distributed in g2, and in patients with gastric cancer, glioma, and colorectal cancer tended to be distributed in g1. Conclusions: Our study suggested the protein expression of PD-L1 was significantly heterogeneous among various cancer types and offered new references for cancer-specific characters of PD-L1 expression. As a retrospective study with a relatively small population, the conclusions of this study needed to be verified with a larger sample.

The frequency of HPD gene mutation profile in solid tumors with PD-L1 expression positive.

e15175 Background: Program death-ligand (PD-L1) expression and tumor mutational burden (TMB) are the promising predictive biomarkers in immunotherapy for solid tumors. Immunotherapy could be harmful in patients harboring hyper-progressive disease (HPD) gene mutations. Nevertheless, the distributions of HPD gene mutation profile in patients with PD-L1 positive solid tumors especially in East Asian populations have not been well characterized yet. Methods: In this study, tumor tissues from 354 patients using 539 genes panel based on next generation sequencing were performed. Single nucleotide variants (SNVs) of DNMT3A gene and copy number variants (CNVs) of M DM2, MDM4, C CND1, F GF3, F GF4 and F GF19 genes were evaluated as HPD mutations. The protein expression of PD-L1 was divided into three groups as g1 (TC < 1%), g2 (1% ≤ TC < 50%) and g3 (TC ≥ 50%) via the percentage of positive tumor cells (TC value) identified by immunohistochemistry (IHC) method. The ratios of patients with or without HPD mutations in different PD-L1 expression groups were calculated by χ2 test using program SPSS v22.0. Results: In 354 patients with solid tumors, 33 of 354 (9.3%) were found harboring HPD mutations. The ratios of patients with HPD mutations in three PD-L1 expression groups were 8.6% in g1, 10.4 % in g2 and 8.7% in g3, respectively. Meanwhile, the ratios of patients without HPD related mutations in three PD-L1 expression groups were 91.4%, 89.6 % and 91.3%, respectively. There were no significant differences of HPD frequency between different PD-L1 expression groups via χ2 test (χ2 = 0.323, p = 0.851). Conclusions: The frequency of HPD gene mutations was not significantly heterogeneous between different PD-L1 expression groups and indicated that immunotherapy for patients with PD-L1 expression positive needed to test HPD related gene mutations. Our study provided meaningful information to the precise administration of immunotherapy in cancer clinical practice. As a retrospective study with a relatively small population, the conclusions of this study needed to be verified with a larger sample.

A survey of cancer testis antigen (CTA) expression across T-cell lymphoma subtypes.

e15246 Background: T-cell lymphomas (TCL) are a rare and heterogeneous group of non-Hodgkin lymphomas (NHL) that originate from T- and NK-cells and portends a poor prognosis. One promising therapeutic strategy involves generation of tumor associated antigen directed T-cells (TAA-T) against specific CTA. RNAseq have established that combinations of histone deacetylase inhibitors and hypomethylating agents uniquely induce genes characterized as cancer testes antigens (CTA), which may increase immunogenicity. There is a lack of data on the baseline expression of CTA across the TCL, limiting application of TAA-T. We sought to establish the baseline expression of CTA in PTCL using immunohistochemistry to better understand treatment effects. Methods: A tissue microarray was created using formalin-fixed paraffin embedded tissue from 40 patients with diverse T-cell neoplasms. Expression of WT1, SCP1, survivin, PLAC1, SSX2, and PRAME was evaluated by immunohistochemistry. The percentage of positive tumor cells (divided into quartiles), staining intensity (weak, moderate, strong), and subcellular localization (nuclear, cytoplasmic) was assessed. Results: Forty patients with T-cell lymphomas were included in the analysis. The median age of diagnosis and median number of treatments was 60.2 years and 2 (0-9), respectively. Thirty-nine (98%) patients expressed at least one CTA. A high frequency of survivin, which is associated with cell proliferation and is expressed on normal lymphoid tissue, was expressed in 98% of TCL, 5% expressed WT1, and 12.5% expressed SCP1, and expression of other CTA was not observed. Of five patients with anaplastic large cell lymphoma, 3 samples were positive for SCP1 and one was also positive for WT1. One of 9 angioimmunoblastic T-cell lymphoma samples was positive for SCP1. Of 4 T-lymphoblastic lymphoma patients, 1 was positive for WT1, and of 3 transformed mycosis fungoides specimens, 1 demonstrated SCP1 expression. In this sample set, we did not find a correlation with expression and response to chemotherapy. There were no patients with CTA expression who were treated with epigenetic treatments. Conclusions: We described low expression of CTA on T-cell lymphomas, which is consistent with the literature on expression by RT-PCR. By establishing this baseline expression of CTA, we have the foundation on which to measure changes in CTA expression pre- and post-treatment by immunohistochemistry.

Delta-Like Protein 3 Expression and Targeting in Merkel Cell Carcinoma.

Delta-like protein 3 (DLL3) is being developed as a predictive biomarker for DLL3-targeting antibody-drug conjugate and other therapies. Given the neuroendocrine features of Merkel cell carcinoma (MCC), we sought to evaluate DLL3 expression and its role in MCC. Formalin-fixed and paraffin-embedded MCC cases were consecutively selected. Immunohistochemistry was performed for DLL3 (SC16.65 antibody) and polyomavirus large T-antigen (sc-136172 antibody). Slides were read out for percentage of positive tumor cells. Cox proportional hazards model was applied to assess the association between DLL3 expression and overall survival (OS). A patient with a DLL3-expressing MCC was treated with rovalpituzumab tesirine (Rova-T) in the "other tumor" cohort of NCT02709889 and assessed for response. The median H-score of DLL3 expression of 65 patients included was 60 (interquartile range, 30-100). Fifty-eight cases (89%) had ≥1% tumor cells positive for DLL3 expression with any intensity, of which the median DLL3 expression was 50% (interquartile range, 25%-70%). Thirty-four cases (52%) had ≥50% tumor cells positive for DLL3 expression with any intensity. Higher H-score of DLL3 expression was associated with higher polyomavirus nuclear expression (p = .003) when it was dichotomized to negative versus positive. H-score of DLL3 expression did not predict OS of patients with MCC (p = .4) after being adjusted for common clinicopathological factors. A patient treated with Rova-T for refractory metastatic MCC achieved partial response. DLL3 overexpression is very common in MCC by immunohistochemistry. The response to treatment suggests that DLL3 expression may have predictive relevance for DLL3-targeting therapies in MCC. Delta-like protein 3 (DLL3) is being developed as a predictive biomarker to identify patients for treatment with DLL3-targeting agents. Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin. It was found that DLL3 overexpression is very common in MCC by immunohistochemistry and significantly associated with Merkel cell polyomavirus expression. Despite the lack of prognostic significance in this cohort, DLL3 expression may have predictive relevance for DLL3-targeting therapies in MCC. The high levels of DLL3 expression in a subset of MCC may potentially be used to select patients to receive DLL3-targeting therapies.

Clinicopathologic Correlation of Kaposi Sarcoma Involving the Ocular Adnexa: Immunophenotyping of Diagnostic and Therapeutic Targets.

To describe the clinicopathologic characteristics and the expression of diagnostic/treatment targets in ocular adnexal Kaposi Sarcoma. We conducted a clinical-pathologic retrospective case series. Immunohistochemical staining for cluster of differentiation 31 (CD31), human herpesvirus-8 (HHV8), platelet-derived growth factor receptor alpha (PDGFR-A), vascular endothelial growth factor receptor-1 (VEGF), tyrosine-protein kinase Kit (c-Kit), and programmed cell death protein 1 (PD-1) were performed. Percentage of positive tumor cells was recorded for PD-1; staining intensity and distribution (H-score) were determined for the remaining stains. A Friedman non-parametric ANOVA analysis evaluated the staining. The study cohort included 13 patients (age 25 to 95 years; mean 46): 7 lesions were in the eyelid, 5 in the conjunctiva, and 1 in the cornea. Nine of 11 lesions (82%) were in human immunodeficiency syndrome-positive patients (human immunodeficiency syndrome status was unknown in 2 cases). Staging included 6 plaques and 7 nodules. The mean H-scores of CD31, HHV8, c-Kit, VEGF, and PDGF-A were 8.00, 8.23, 2.77, 11.54, and 10.31, respectively. Mean PD-1 staining was 6.46%. The Friedman non-parametric ANOVA analysis showed VEGF, PDGF-A, CD31, and HHV8 differed significantly, and all differed significantly from c-Kit. Programmed cell death protein 1 staining was not significant with any clinical variable. Cluster of differentiation 31 and HHV8 are helpful diagnostic adjuncts for ocular adnexal Kaposi Sarcoma. Platelet-derived growth factor receptor alpha and VEGF are promising treatment targets. Programmed cell death protein 1/PD-L1 and c-Kit are targets that are useful in several tumors; their roles in ocular adnexal Kaposi Sarcoma warrant further studies.

Quantitative assessment and clinical relevance of VEGFRs-positive tumor cells in refractory brain tumors

Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)1 and 2 signaling is a potent activator of tumor angiogenesis. Although the expressions of VEGFR1 and VEGFR2 were initially thought to be limited to the endothelial cells, it is now known that both the receptors are expressed in tumor cells. This is the first study wherein VEGFRs-positive tumor cells are quantitatively evaluated for brain tumors with upregulated VEGF/VEGFR signaling. The percentage of VEGFRs-positive tumor cells was quantitatively evaluated in various brain tumors (10 glioblastomas, 22 neurofibromatosis type 2 [NF2]-related schwannomas, 21 sporadic schwannomas, 27 chordomas, 36 meningiomas, 29 hemangioblastomas, 11 hemangiopericytoma, and 13 ependymomas) using immunohistochemistry. VEGF-A expression was also analyzed using quantitative real-time polymerase chain reaction. Double immunofluorescence staining using anti-PDGFR-β and anti-CD34 antibody, microvessel density, and vessel diameter were analyzed to evaluate the vascular characteristics. Chordomas demonstrated an extremely higher percentage of VEGFR1 and VEGFR2-positive tumor cells than other tumors. In contrast, meningiomas and hemangiopericytomas showed few VEGFRs-positive tumor cells. The percentage of positive tumor cells in chordomas, hemangioblastomas, and NF2 schwannomas was associated with clinical courses, such as shorter progression free survival, and growth speed. Glioblastomas and NF2 schwannomas showed larger tumor vessels without pericyte coverage. The present study is the first to quantitatively analyze VEGFR1- and VEGFR2- positive tumor cells in various types of refractory brain tumors. This novel parameter significantly correlated with the progressive clinical courses.

Abstract P3-08-13: Prognostic significance of intraepithelial CD103-positive tumor-infiltrating lymphocytes in breast cancer

Abstract Cluster of differentiation 103 (CD103), also known as αEβ7 integrin, is expressed on subsets of CD8+ T lymphocytes and a biomarker for tissue resident memory CD8+ T cells (TRM). CD103 expression is upregulated upon antigen recognition concurrent with transforming growth factor β (TGFβ) stimulation. CD103 is primarily expressed in the intra-epithelial tumor-infiltrating lymphocytes (iTILs) compared with that in stromal TILs (sTILs). Here, we investigated the prognostic significance of CD103+ iTILs in invasive breast cancer (IBC). Immunohistochemistry was performed for CD103, TGFβ isoforms (1, 2, and 3), and TGFβ receptors on tissue microarrays of 1173 IBC samples. CD103 expression was analyzed using Aperio ScanScope slide scanner. An area with the highest density of CD103+ iTILs was captured at a magnification of × 200, and CD103+ iTILs per unit area (mm2) were enumerated. The threshold for CD103+ iTILs (high vs low) was determined based on the mean number. Immunohistochemical staining for TGFβ isoforms (1, 2, and 3) and their receptors was quantitated using H-score and calculated as: staining intensity (0-3) × the percentage of positive tumor cells (0-100%). For statistical analyses, we categorized cases as positive or negative based on the mean value of H-score. The number of CD103+ iTILs were compared with clinicopathological characteristics, and tumor cell expression of TGFβ and its receptors. Further, the relationship between CD103+ iTILs density and prognosis was investigated according to the molecular subtypes. CD103+ iTILs were observed in 713 (60.8%) cases with a mean ± standard deviation of 34.2 ± 94.6. Increased abundance of CD103+ iTILs was found to be significantly associated with histological grade 3, high Ki-67 labelling index, non-luminal subtypes, high sTIL density, and positive expression of TGFβ3 and its receptor (all, P< 0.001). Furthermore, high CD103+ iTIL density was significantly associated with prolonged overall survival (OS) (P = 0.02) and disease-free survival (DFS) (P = 0.001). Subgroup analysis according to molecular subtypes showed that CD103+ iTILs was prognostic only for triple-negative breast cancer (OS, P = 0.003; DFS, P = 0.005). Multivariate analysis of CD103+ iTILs with clinicopathological prognostic factors revealed that CD103+ iTILs was an independent prognostic factor for OS (P = 0.005) and DFS (P = 0.006) for the triple-negative subtype. Our study revealed that breast cancer cell expression of TGFβ3 may be involved in recruitment of CD103+ iTILs. In breast cancer, CD103+ iTILs indicates good prognosis which is an independent favorable prognostic factor in patients with triple negative subtype and more reliable than sTIL density. Citation Format: Young Kyung Bae, Min Hui Park, Jung Eun Choi, Su Hwan Kang, Gyungyub Gong. Prognostic significance of intraepithelial CD103-positive tumor-infiltrating lymphocytes in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-13.

Evaluation of VEGF expression correlates with COX-2 expression in pleomorphic adenoma, mucoepidermoid carcinoma and adenoid cystic carcinoma

Pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (AdCC) are the most common benign and malignant salivary gland tumors. Cyclooxygenase-2 (COX-2) is a key regulatory enzyme that its overexpression in various tumors is correlated with progression, metastasis, and apoptosis inhibition. Vascular endothelial growth factor (VEGF) is a potent angiogenic mediator that has an important role in neoplastic angiogenesis. The aim of this study was to immunohistochemically analyze the expression of COX-2 and VEGF and to compare the expression of benign and two malignant salivary gland tumors with varied structures. In this cross-sectional study, 90 specimens including 30 cases of each tumor were retrieved. Immunohistochemical staining of COX-2 and VEGF was performed for all the samples. The percentage of positive tumor cells and staining intensity was evaluated by two pathologists blindly. Data were analyzed by Chi-square and Gamma test and P < 0.05. A statistically significant difference was noted between the expression and intensity of COX-2 and VEGF in PA, MEC, and AdCC (P < 0.05). A significant correlation was observed between COX-2 and VEGF expression in MEC and AdCC (P < 0.05). However, no significant correlation was found between the expression and intensity of COX-2 and VEGF with histologic grade and lymph node metastasis in MEC and AdCC (P < 0.05). High expression of VEGF and COX-2 in malignant tumors compared to PA suggested the role of both markers in malignant transformation. The significant correlation of VEGF expression with COX-2 may represent the role of COX-2 in tumor angiogenesis by modulating VEGF production.

Programmed Death Ligand 1 Expression in Laryngeal Squamous Cell Carcinomas and Prognosis.

Aim:We aimed to show the immunohistochemical expression of programmed death ligand 1 (PD-L1) in laryngeal squamous cell carcinomas (SCCs).Materials and methods:The study includes 52 laryngeal SCC cases that underwent surgical resection. Immunohistochemical staining of PD-L1 (Clone 22C3) was applied to the sections obtained from paraffin blocks. Combined Positive Score (CPS) was evaluated as described in manuals. Tumor Proportion Score (TPS) was assessed by the percentage of positive tumor cells which were designated as positive if ⩾1% of the tumor cells showed membranous staining.Results:There were 35 cases (67.3%) having CPS < 1 and 17 cases (32.7%) having CPS ⩾ 1. There was no relationship between CPS, TPS, and the clinicopathological data.Conclusion:Further studies with a large number of advanced-stage cases are needed.

Immunoexpression of Ki-67 Labelling Proliferation Index in Phyllodes Tumour of Breast by Polymer-based Detection Method

Phylloides tumours (PTs) are rare breast neoplasms with a variable clinical course depending on the tumour category. The classification of PTs proposed by the World Health Organization (WHO) into benign, borderline and malignant is based on a combination of several histologic features, including Stromal cellularity, nuclear atypia, mitotic activity, stromal overgrowth and tumour margin appearance. However, there are no defined criteria or clear cut-offs for individual histologic parameters. Thus, the diagnosis of PTs based on the integration of morphology remains challenging. Along with the grade, additional study of proliferative markers such as Ki-67 are essential to identify those with potential for aggressive behaviour. This study was undertaken to assess the histopathological characters and correlate Ki-67 expression in different subtypes of PTs. In this study, 30 cases of PTs were studied. Regarding histologic features, routine H&amp;E stains were taken into consideration for diagnosis and classification of tumours. Immunostaining for Ki-67 was also performed by polymer-based detection method. Ki-67 labelling index (LI) was categorized as 0-10, 11-30, 31-50, 51 and above depending on the percentage of positive tumor cells and was correlated with histologic grade and clinical features in each case. Twenty cases (66.7%) of benign phyllodes tumour (BPT), 3 cases (10%) of borderline PT, and 7 cases (23.3%) cases of malignant phylloides tumour (MPT) were examined in this study. Among 20 cases of BPT, 3 cases (15%) were recurrent tumours. Average Ki-67 LI in BPT was 5% (range 1-10%) and borderline PT was 17.5% (range 15-20%). MPT exhibited Ki-67 LI range of 15-35% with average LI of 25%. A significant association was seen between expression of Ki-67 in different grades of PT(p=0.01). So, Ki-67 LI should be performed in routine histopathology reporting of phylloides for sub-categorisation of phylloides.

Diagnostic and prognostic value of glucose transporters in melanocytic lesions.

We have previously reported increased glucose transporter 1 (GLUT1) expression in melanoma compared to benign nevi, associated with a significantly lower survival rate. GLUT1 upregulation was highly specific for distinguishing melanoma from benign nevi, yet poorly sensitive, likely because of expression of other GLUT isoforms. The purpose of this study was to evaluate GLUT2 and GLUT3, as melanoma biomarkers. A tissue microarray, consisting of 91 primary melanomas, 18 melanoma metastases, and 56 nevi, was examined using GLUT2 and GLUT3 immunohistochemistry. A semiquantitative scoring method was used to determine the percentage of positive tumor cells and staining intensity. GLUT2 was negative in all melanomas and benign nevi examined. Increased GLUT3 expression was more frequent in melanoma than in nevi (P < 0.0001), and in metastatic melanoma than in primary melanomas (P < 0.001). Of melanoma cases, 85.3% expressed either GLUT1 or GLUT3 or both, 39.4% of melanoma cases coexpressed GLUT1 and GLUT3, 17.4% of melanoma cases only expressed GLUT1, 28.4% of melanoma cases only expressed GLUT3, and 14.7% of melanoma cases were negative for both markers. Patients whose melanoma exhibited a high level of GLUT3 had significantly lower survival rates than those with low GLUT3 expression (P = 0.002). Evaluating both GLUT1 and GLUT3 increased the diagnostic value by increasing the sensitivity while the specificity remained high. In conclusion, GLUT2 was not expressed in melanocytes. GLUT3 expression was upregulated in melanoma compared with nevi, especially in those with worse prognosis. Similar to GLUT1, GLUT3 may serve as a useful diagnostic and prognostic marker.

Cutaneous melanoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†.

Cutaneous melanoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†.

Comparative Assessment of Surface CD19 and CD20 Expression on B-Cell Lymphomas from Clinical Biopsies: Implications for Targeted Therapies

Background: CD19 and CD20 are B-cell lineage-specific antigens expressed on the cell surface of most B-cell lymphomas. While CD20 is acquired during late stages of B-cell lymphogenesis and is then lost upon differentiation into plasma cells, CD19 expression covers the entire spectrum of early B-cell genesis and maturation. CD20-targeting agents have been broadly integrated into the therapeutic armamentarium for B-cell lymphomas. More recently, CD19-targeting agents have emerged as promising alternatives with demonstrated therapeutic value. Given the imminent availability of both CD19 and CD20 targeted therapies, and potential for combinational approaches, we studied the surface expression of these antigens at the single-cell level on lymphoma cells and benign background lymphoid subsets from biopsy specimens. Methods: Flow cytometric analysis (seven-color) was performed on biopsy specimens from 47 patients with newly diagnosed B-cell lymphomas, including diffuse large B-cell lymphoma (n=15), follicular lymphoma (n=15), marginal zone lymphoma (n=9), mantle cell lymphoma (n=9), Burkitt lymphoma (n=2), and unclassifiable low-grade B-cell lymphoma (n=2). Small lymphocytic lymphoma was intentionally excluded, given its well-described loss of CD20 expression. Biopsies from eight additional patients with persistent/recurrent B-cell lymphomas after anti-CD20 therapy (greater than 6 months after last dose) were also evaluated. Thresholds for CD19 or CD20 antigen positivity were defined for each case, based on the 95th percentile fluorescence intensity of the respective marker on tumor-infiltrating T-cells (internal negative control). In addition, CD19 or CD20 fluorescence intensities of tumor cells were normalized to background benign B-cells (internal positive control) using the median fluorescence ratio (MFR). Results: Both CD19 and CD20 were highly expressed on CD20 treatment naïve tumor cells, with a slightly higher median percentage of positive tumor cells for CD19 (98%) compared with CD20 (93%) (p=0.003), and one case lacking CD20 expression. When compared with background benign B-cells, CD20 was frequently overexpressed on tumor cells (mean MFR=1.8), while CD19 expression was overall similar to background benign B-cells (mean MFR=0.9) (p=0.001). As the surface density of CD20 on benign B-cells is reportedly higher than CD19 (~100,000 vs ~20,000 molecules per cell, respectively), these findings are consistent with a higher density of surface CD20 than CD19 on most B-cell lymphomas. However, CD20 expression was more heterogeneous (within individual patient samples and across patients), with a higher median percentage of CD20-negative tumor events (median=0.5%, range=0-98%) compared with CD19-negative events (median=0%, range=0-28%) (p=0.003). Interestingly, expression of CD19 within the CD20-negative tumor subsets was largely preserved (mean % CD19-positive events=97.86%, min=40%). In addition, percentages of CD19/CD20 double-negative tumor events were very small (median=0%, range=0-4.9%), and only detectable in 15 cases (32%). Of eight additional cases studied post-anti-CD20 immunotherapy (6-84 months after last dose), the percentage of antigen-positive events by tumor cells was largely preserved for both CD19 (median=99.6%, range=93.5-100%) and CD20 (median=95.7%, range=55.6-100%), similar to the pre-therapy cohort. Conclusions: CD19 and CD20 are both highly and consistently expressed in B-cell lymphomas. While CD20 has a higher average density of surface molecules per tumor cell, CD19 expression is more homogenous and is preserved in small CD20-negative tumor subsets and after anti-CD20 targeted therapy. These findings support the clinical evaluation of anti-CD19 immunotherapies and combinational therapies targeting both surface antigens. Disclosures Horna: MorphoSys AG: Research Funding. Nowakowski:Selvita: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Curis: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Genentech, Inc.: Research Funding; MorphoSys: Consultancy, Research Funding; NanoString: Research Funding. Endell:MorphoSys AG: Employment, Patents &amp; Royalties. Boxhammer:MorphoSys AG: Employment, Patents &amp; Royalties.

EP1.09-03 Interobserver Variability Study of PD-L1 Immunostaining in Non-Small Cell Lung Cancer

Pathologic assessment of programmed cell death ligand 1 (PD-L1) immunohistochemical staining now have been used for treatment of non-small cerll lung cancer in advanced stage. But, due to misinterpretation or unspecified staining pattern, interpretation of PD-L1 staining may be difficult. This study was to investigate interobserver variability between three different pathologists to see if the staining can be safely assessed in practice. PD-L1 immunostaining (SP263 and SP142) was performed in representative sections of 70 adenocarcinoma cases and 58 squamous cell carcinoma cases which surgically resected at the chonnam National University Hwasun Hospital, Republic of Korea between 2013 to 2015. Three pathologists individually evaluated the percentage of positive tumor cells, scoring each sample applying cutoff levels used in clinical practice: <1% positive tumor cells (score 0), 1–4% (score 1), 5–9% (score 2), 10–49% (score 3), and >50% positive tumor cells (score 4). Three pathologists were in agreement in 121 of 128 (94.5%) of scored cases in the SP263 test and 109 of 128 (85.2%) in the SP142 test. In 26 remaining cases, at least two pathologists were in aggrement of scored cases. Weighted kappa value for interobserver variability between pathologists was respectively 0.892 in the SP263 test and 0.837 in the SP142 test. The number of differently classified cases was significantly higher for the ≥1% cutoff value than every other cutoff value. Also, a significantly better agreement between pathologists was seen using ≥50% as cutoff. No statistically significant differences were seen between adenocarcinoma and squamous cell carcinoma. Interobserver agreement remains an important challenge, and the ≥1% cutoff value seems to be problematic. But, this may be overcome by training, longer experience and optimization of cutoff value.

909PD - NIVOREN GETUG-AFU 26 translational study: CD8 infiltration and PD-L1 expression are associated with outcome in patients (pts) with metastatic clear cell renal cell carcinoma (mccRCC) treated with nivolumab (N)

BackgroundThe NIVOREN GETUG-AFU 26 study reported safety and efficacy of N in mccRCC pts in a “real world setting”. A translationnal research program was launched to characterize immune cell populations in and around the tumor by immunohistochemistry (IHC) and correlate them with outcome on N. MethodsAll pts treated with N in the GETUG AFU 26 NIVOREN trial who consented for translational program and with available archived paraffin-embedded (FFPE) tumor tissue samples were eligible. Tumor were centrally reviewed. Densities of CD3-, CD8- and CD20-cells and tertiary lymphoid structures (TLS) were estimated by IHC. PD-L1 expression was quantified as percentage of positive tumor cells (TC) and immune cells (IC). ResultsOverall 324 pts were included. Pts had similar baseline characteristics (IMDC Good, Intermediate, Poor in 18%, 60% and 22%, respectively) than overall trial population. Median PFS was 4.5 (2.9-5.2) months and median OS 25.4 (23.6-NE) months. Highest density of CD8 T cells at the invasive margin (IM) was associated with worse PFS (2.3 vs 4.6 months, HR=3.96 (1.84-8.51), p=0.0001) and OS (8.6 vs 25.4 months, HR=2.43 (0.99-5.95), p=0.0451). PD-L1 expression on TC (³1%) was associated with worse OS (22.7 vs 29.1 months, HR=1.51 (1.06-2.15), p=0.0232) but not PFS (3.5 vs 4.6 months, p=0.5121). PD-L1 expression on IC (³1%) was associated with numerically shorter median OS (24.7 months vs NR, HR=1.34 (0.95-1.88), p=0.0955). CD8 densities, either in the T or at the IM were associated with high PD-L1 expression (>1%) by TC or by IC (all p<0.0001). Densities of CD3, CD20 and TLS were not significantly associated with OS or PFS. More data such as response rates and combinations of markers will be presented at the meeting. ConclusionsWe report the largest translational analysis supporting that highest CD8 density at the IM is associated with worse OS and PFS while PD-L1 expression by ³1% TC or IC is associated with worse OS in pts with mccRCC receiving N. Clinical trial identificationNCT03013335. Legal entity responsible for the studyUNICANCER. FundingInstitut National du Cancer, Direction Générale de l’Offre de Soins, Bristol-Myers-Squibb. DisclosureY. Vano: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Roche; Honoraria (self), Advisory / Consultancy: Ipsen; Honoraria (self), Advisory / Consultancy: Pfizer; Travel / Accommodation / Expenses: MSD; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: BMS; Honoraria (self), Advisory / Consultancy: Merck; Travel / Accommodation / Expenses: Janssen-Cilag; Honoraria (self), Advisory / Consultancy: Sanofi; Honoraria (self), Advisory / Consultancy: Astellas. N. Rioux-Leclercq: Honoraria (self), Advisory / Consultancy: Pfizer; Honoraria (self), Advisory / Consultancy: BMS; Honoraria (self), Advisory / Consultancy: Janssen-Cilag; Honoraria (self), Advisory / Consultancy: Ipsen. B. Beuselinck: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): BMS. M. Gross-Goupil: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: BMS; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Ipsen; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Roche; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Pfizer; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: GSK. S. Negrier: Honoraria (self), Advisory / Consultancy: Pfizer; Honoraria (self), Advisory / Consultancy: BMS; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy: Ipsen; Honoraria (self), Advisory / Consultancy: Eusa Pharma. D. Borchiellini: Honoraria (self), Advisory / Consultancy: BMS; Honoraria (self), Advisory / Consultancy: Pfizer; Honoraria (self), Advisory / Consultancy: Ipsen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Roche; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): MSD; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Janssen-Cilag; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Astellas; Honoraria (self), Advisory / Consultancy: Sanofi. B. Escudier: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: BMS; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Pfizer; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Novartis; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Ipsen; Honoraria (self), Advisory / Consultancy: Eusa Pharma; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Aveo. L. Albiges: Honoraria (institution), Advisory / Consultancy: Pfizer; Honoraria (institution), Advisory / Consultancy: BMS; Honoraria (institution), Advisory / Consultancy: Ipsen; Honoraria (institution), Advisory / Consultancy: Roche; Honoraria (institution), Advisory / Consultancy: MSD; Honoraria (institution), Advisory / Consultancy: AstraZeneca; Honoraria (institution), Advisory / Consultancy: Novartis. All other authors have declared no conflicts of interest.

P2.09-35 Proposal to Revise VENTANA ALK Scoring Interpretation Guide for Non-Small Cell Lung Carcinoma: Interpretation of ALK Heterogeneity

A small number of non-small cell lung cancer (NSCLC) cases showed heterogeneity of anaplastic lymphoma kinase (ALK) by VENTANA immunohistochemistry (IHC) in clinical practice. According to the ALK Scoring Interpretation Guide for VENTANA anti-ALK (D5F3), the presence of strong granular cytoplasmic staining in tumor cells (any percentage of positive tumor cells) is called positive for ALK. However, we know little about ALK heterogeneous cases. Multiple detection platforms are used to analyze molecular variability and pathological features in anticipation of clinical treatment decisions for such cases. A total of 2228 NSCLC cases with successful ALK IHC (VENTANA, D5F3, Roche) detection in Guangdong Provincial People`s Hospital were recruited between January 2012 and April 2018. Positive and negative system control and a negative agent control were established for each case. ALK IHC positivity was defined as the presence of strong granular cytoplasmic staining in 100% tumor cells; ALK IHC heterogeneity as the presence of strong granular cytoplasmic staining in 1-99% tumor cells; ALK IHC negativity as no tumor cells show strong granular cytoplasmic staining. Fluorescence in-situ hybridization (FISH) (Vysis ALK Break Apart FISH Probe Kit, Abbott) and next-generation sequencing (NGS) (OseqTMLung Cancer Gene Detection, BGI, China) was performed for cases showed ALK (D5F3) IHC heterogeneity. ALK (D5F3) double-blind review analysis showed 201 (9.0%) ALK-positive cases, 10 (0.4%) ALK-heterogeneous cases, and 2017 (90.5%) ALK-negative cases. The heterogeneity cases included 2 large cell neuroendocrine carcinomas, 1 lymphoid epithelioid carcinoma, and 7 squamous cell carcinomas. The percentages of tumor cells with strong granular cytoplasmic staining were 1% to 30%. The ALK FISH break apart signal of these ten cases were 0% to 12%, indicating ALK FISH negativity. Nine ALK-heterogeneous cases were successfully detected by NGS, and no ALKgene variations (including gene fusion, copy number variation, insertion/deletion or single nucleotide variation) were found. Immunohistochemical staining showed that some ALK-heterogeneous cases showed neuroendocrine differentiation.TableComparison of IHC, FISH and NGS results of the ALK-heterogeneous cases.Case No.GenderAgeBiopsy/surgeryDiagnosisTumor cell contentALK IHCALK FISHALK FISHALK NGSALK NGS8thTNMStrong positive staining tumor cellsBreak apart signalInterpretation resultALK fusionALK INDEL/SNV/CNV1M69Wedge resectionLarge cell neuroendocrine carcinoma70%2%0%NegativeNegativeNegativepT1bN0M02M55LobectomyLarge cell neuroendocrine carcinoma85%20%4%NegativeNegativeNegativepT2bN0M03F33LobectomyLymphoid epithelioid carcinoma60%5%0%NegativeNegativeNegativepT3N0M04M68LobectomySquamous cell carcinoma70%5%6%NegativeNegativeNegativepT3N0M05M69LobectomySquamous cell carcinoma70%5%12%NegativeNegativeNegativepT2aN0M06M52LobectomySquamous cell carcinoma80%1%0%NegativeNegativeNegativepT2aN1M07M73LobectomySquamous cell carcinoma90%5%2%NegativeNegativeNegativepT3N0M08M70LobectomySquamous cell carcinoma80%5%4%NegativeNegativeNegativepT2bN1M09M69BiopsySquamous cell carcinoma85%15%0%NegativeNegativeNegativesT3N1M010M60BiopsySquamous cell carcinoma85%30%0%NegativeNANAcT2bN3M0Abbreviations: INDEL, insertion and deletion; SNV, single nucleotide variation; CNV, copy number variation; NA, NGS was not performed due to insufficient tumor tissues. Open table in a new tab Abbreviations: INDEL, insertion and deletion; SNV, single nucleotide variation; CNV, copy number variation; NA, NGS was not performed due to insufficient tumor tissues. Multi-platform detection of ALK-heterogeneous cases did not show evidence of ALKgene variation, and the effect of ALK-TKI treatment was unknown. therefore, the VENTANA ALK scoring interpretation guide for non-small cell lung carcinoma should be revised. It is recommended that ALK-heterogeneous cases be defined as ALK (D5F3) uncertain equivocal cases. The molecular pathology report should clearly state that the clinical significance is unclear, and it is recommended to conduct further testing by FISH or NGS.

Hormone receptor expression patterns define clinically meaningful subgroups of endometrioid ovarian carcinoma

BackgroundNumerous studies have investigated the association between hormone receptor expression and clinical outcome in ovarian carcinoma (OC); however, these have largely focussed on serous OCs, with few studies reporting specifically on endometrioid OCs (EnOC). Where analyses have been stratified by histotype, expression has been assessed using the percentage of positive tumor cells, without accounting for nuclear expression intensity. MethodsHere we assess the expression levels of progesterone receptor (PR), estrogen receptor alpha (ER) and androgen receptor (AR) using histoscore – a nuclear scoring method incorporating both proportion of positive cells and the intensity of nuclear staining – across a cohort of 107 WT1 negative EnOCs. ResultsHierarchical clustering by PR, ER and AR histoscores identified four EnOC subgroups (PR+/ER+, PR+/ER−, PR−/ER+ and PR−/ER−). EnOC patients in the PR+/ER+ and PR+/ER− groups displayed favorable outcome (multivariable HR for disease-specific survival 0.05 [0.01–0.35] and 0.05 [0.00–0.51]) compared to the PR−/ER+ group. Ten-year survival for stage II PRhigh and PRlow cases was 94.1% and 42.4%. ERhigh EnOC patients (PR+/ER+, PR−/ER+) had higher body mass index compared to ERlow cases (P = 0.015) and high grade serous OC patients (P < 0.001). ConclusionThese data demonstrate that endometrioid OC cases with high PR expression display markedly favorable outcome. Stage II EnOCs with high PR expression represent potential candidates for de-escalation of first-line therapy. Future work should seek to characterise the sensitivity of PR and ER positive EnOCs to endocrine therapy.

Abstract 1536: Tumor heterogeneity and primary versus metastatic evaluation of PD-L1

Abstract Immunotherapy with checkpoint inhibitors allowing recovery of effector cell function, has been demonstrated to be highly effective in many tumor types and represents a true revolution in oncology. However, current assays for the prognostic and/or predictive role of tumor PD-L1 expression are not fully standardized with respect to either quantity or distribution of expression, or regarding the concordance of expression in primary tumor versus metastasis. To assess tumor heterogeneity, we evaluated PD-L1 expression using the Ventana SP-263 assay in paired formalin fixed paraffin embedded (FFPE) blocks from the same primary tumor for each of 22 lung squamous cell carcinoma (SCC), 6 lung adenocarcinoma (ADC), 53 gastric ADC, 55 colorectal ADC, 60 urothelial bladder carcinoma, 3 pancreatic ductal ADC and 4 head and neck SCC. In addition, PD-L1 expression in primary tumor and synchronous metastasis was evaluated in 32 cases of lung neoplasm (10 ADC +22 SCC), 30 cases of gastric adenocarcinoma, 55 cases of colorectal adenocarcinoma, and 16 head and neck SCC, each with corresponding synchronous metastases. PD-L1 expression was determined by pathologist evaluation of the percentage of positive tumor cells and cases were grouped into categories representing &amp;lt;1%, 2-9%, 10-25%, 26-49%, 50-75% and 76-100%, with those samples expressing more than 25% tumor positivity considered as expressing “high” PD-L1 levels. In lung carcinoma, just 1 case of SCC exhibited a change in PD-L1 classification when two blocks from the same tumor were evaluated (0% vs 5%), and also for 1 case of ADC (2% vs 0%) . In gastric ADC we recorded a change in classification in 1 out of 53 cases (5% vs 0%), and for CRC ADC we found a difference between the two blocks evaluated in just 1 of the 55 cases (0% vs 50%). For the remaining tumor cohorts (60 bladder carcinoma, 4 head and neck SCC and 3 pancreatic ADC) 100% concordance was observed between two blocks from the same tumor. Evaluation of primary tumors and synchronous metastasis revealed no differences in PD-L1 scores for the 32 cases of lung carcinoma, or for the 16 head and neck SCC cases. In the 30 gastric ADC cases, there was a discordant PD-L1 expression score for just one pair of tumors (primary tumor 3% and metastasis 0%). Similarly, 1 of 55 CRC ADC examined revealed a primary tumor PD-L1 score of 50% while the metastatic tissue was negative. Taken together, these data demonstrate that PD-L1 expression between two sites in the same tumour, and between primary versus synchronous metastases is remarkably consistent. The conclusions drawn are that a representative PD-L1 score may be achieved from a single FFPE tumour block, and that for cases where obtaining tissue from a primary tumor may be challenging, PD-L1 could be evaluated in metastatic tissue. Citation Format: Lorenzo Colarossi, Eleonora Aiello, Marie Cumberbatch, Cristina Colarossi, Chris Womack, Lorenzo Memeo. Tumor heterogeneity and primary versus metastatic evaluation of PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1536.

Abstract 3171: Delta-like protein 3 expression in Merkel cell carcinoma

Abstract Purpose: Delta-like protein 3 (DLL3) is being developed as a predictive biomarker to identify small cell lung cancer (SCLC) patients for treatment with the DLL3-targeting antibody-drug conjugate rovalpituzumab tesirine (Rova-T). Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin, associated with Merkel cell polyomavirus. Given the neuroendocrine features of SCLC and MCC, we sought to evaluate DLL3 expression, its association with common clinicopathological factors, and its prognostic role in MCC. Methods: A total of 65 formalin-fixed and paraffin-embedded MCC cases were consecutively identified from an institutional biospecimen repository and were cut at 5 µm. Each case was stained for DLL3 protein with SC16.65 mouse monoclonal antibody at 3 µg/mL with the FLEX+ system (DAKO). Slides were then scanned at 20X with AT2 (Leica/Aperio) and read out for percentage of positive tumor cells with 1+, 2+, and 3+ intensity side-by-side with isotype control (IgG2a). Any subcellular staining pattern (membranous, cytoplasmic or punctate) was counted as overall positive. Polyomavirus status was determined by immunohistochemistry (IHC) using an antibody that binds to MCPyV large T-antigen (sc-136172, Santa Cruz Biotechnology, Inc.). A generalized additive model was used to visualize the trend between common clinical factors and overall survival (OS). A multivariable Cox proportional hazards model was applied to assess the association between DLL3 expression and OS. The association between DLL3 expression and other clinicopathological factors was evaluated with median and t tests. Results: A total of 67 patients were included with a median age of 74 years. Thirty-six (55%) had stage I/II disease; 29 (45%) had stage III/IV disease. Thirty-five (52%) patients died from MCC. Among the 65 cases with successful DLL3 stain, the median H-score of DLL3 expression was 60 (interquartile range: 30-100). Fifty-eight cases (89%) had ≥1% tumor cells positive for DLL3 expression with any intensity, of which the median DLL3 expression was 50% (interquartile range: 25-70%). Thirty-four cases (52%) had ≥50% tumor cells positive for DLL3 expression with any intensity. Older age significantly predicted shorter overall survival (p = 0.03). However, higher AJCC stage did not predict shorter OS (p = 0.3). H-score of DLL3 expression was not associated with AJCC stages (p = 0.4). However, higher H-score of DLL3 expression was associated with higher polyomavirus nuclear expression (p = 0.02) when it was dichotomized to 0/1+ and 2+/3+. In a multivariable Cox regression analysis, H-score of DLL3 expression did not predict OS of patients with MCC (p = 0.8). Conclusions: DLL3 overexpression is very common in MCC by IHC. DLL3 expression was significantly associated with Merkel cell polyomavirus expression but was not prognostic for OS in this cohort. The high levels of DLL3 expression in a subset of MCC may potentially be used to select patients to receive Rova-T. Citation Format: Hao Xie, Kumiko Isse, Yan Sun, Johanna Ramoth, Dorothy M. French, Laura R. Saunders, Aaron S. Mansfield. Delta-like protein 3 expression in Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3171.