SP142 Antibody Research Articles

PD-L1 in breast cancer: comparative analysis of 3 different antibodies

The interaction of programmed cell death-1 and its ligand-1 (PD-L1) serves as a regulatory check against excessive immune response to antigen and autoimmunity. We compared the performance of 3 different PD-L1 antibodies (Ventana SP263, Dako 22C3, and BioCare RbMCAL10 antibodies) in 136 invasive ductal carcinoma specimens including 43 primary, 48 locally metastatic, and 46 distantly metastatic diseases. PD-L1 expression was correlated with clinicopathologic parameters including tumor size, grade, lymphovascular invasion, estrogen receptor, progesterone receptor, HER2, Ki67, molecular type, and triple-negative status. There was excellent agreement between the 3 antibodies, with highly significant κ values (P≤.001). PD-L1 expression was more likely to be associated with higher tumor grade and estrogen receptor-negative, progesterone receptor-negative, triple-negative, and highly proliferative tumors (P<.001). When we studied PD-L1 expression at 0, 1%-9%, 10%-49%, and ≥50% cutoff points by the 3 antibodies, there were 20 discordant cases between the antibodies. Sixteen were of inconsequential impact as far as low and high PD-L1 expression. The 4 differences between antibodies did exhibit an interesting pattern of expression, where there was a general agreement between the BioCare and Ventana antibodies with consistently higher PD-L1 expression compared with the Dako antibody. Given the high concordance, it is not surprising that all 3 antibodies demonstrated the same associations with all pathologic and clinical parameters studied. Standardization studies to identify reliable biomarkers that help in patient selection for immune therapy to improve the risk-benefit ratio for these drugs are still needed.

Immunohistochemical assays incorporating SP142 and 22C3 monoclonal antibodies for detection of PD-L1 expression in NSCLC patients with known status of EGFR and ALK genes.

Different immunohistochemical (IHC) assays were approved for PD-L1 expression examination on tumor cells in qualification to immune-checkpoint inhibitors therapy in NSCLC patients. These assays have some similarities, but also very serious differences. We assessed 2 IHC tests for PD-L1 expression evaluation in NSCLC tumors with different pathological diagnoses and genetic abnormalities.We enrolled 48 NSCLC patients (median age: 65 years) with known status of EGFR and ALK genes. We compared the effectiveness of PD-L1 expression examination of two IHC assays with 22C3 (Dako) and SP142 antibodies (Ventana). IHC tests were performed in resected tissue samples and in cellblocks from bronchoscopy biopsies (formalin-fixed paraffin-embedded). IHC staining was carried out on Dako Autostainer Link 48 and Ventana Benchmark GX.The percentage of tumors with PD-L1 expression of ≥5% and ≥50% on tumor cells was significantly (p<0.05) higher in assay with 22C3 (66.7% and 45.8%) than with SP142 antibody (39.6% and 22.9%). The median percentage of tumor cells with PD-L1 expression was significantly (p<0.0001) higher in test with 22C3 than with SP142 antibody. Percentage of squamous cell carcinoma (SCC) patients with PD-L1 expression was significantly higher than of non-SCC patients. Large group of patients without PD-L1 expression on tumor cells was identified among patients with common EGFR mutations and ALK rearrangement.Our results support that the highest PD-L1 expression on tumor cells occurs in SCC patients and in adenocarcinoma patients without common, druggable genetic abnormalities. The above mentioned results were clearly visible in IHC assay with 22C3 (strong cell staining).

Programmed death-ligand 1 expression and T790M status in EGFR-mutant non-small cell lung cancer

BackgroundDifferential biology and prognosis between T790M+ and T790M- populations imply immunological differences also. MethodsWe retrospectively analyzed programmed death-ligand 1 (PD-L1) expression and T790M status in rebiopsied samples of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). PD-L1 immunohistochemistry was performed using the SP142 antibody for tumour cell (TC) and tumour-infiltrating immune cell (IC) and the 28-8 antibody for TC. PD-L1+ was defined as TC or IC ≥1%. ResultsWe investigated 67 available rebiopsied histologic samples in 47 patients. Using the SP142, prevalence of PD-L1 any+, moderate+, and strong+ in T790M+ vs. T790M- samples were 31% vs. 61%, 8% vs. 15%, and 0% vs. 2%, respectively, representing PD-L1+ prevalence of T790M+ samples was significantly lower than that of T790M- (p=0.0149). Prevalence of any TC+/IC+ in T790M+ vs. T790M- samples were TC: 31% vs. 51% (p=0.0997) and IC: 8% vs. 27% (p=0.0536), respectively. Using the 28-8, median percentage of PD-L1+ in T790M+ samples was 1.9 (range, 0–27.2), whereas T790M- was 4.1 (range, 0–89.8) (p=0.0801). Prevalence of PD-L1+ ≥1%, ≥5%, and ≥10% in T790M+ vs. T790M- samples were 77% vs. 83% (p=0.5476), 31% vs. 49% (p=0.1419), and 12% vs. 27% (p=0.1213), respectively. In 9 of 11 patients receiving multiple rebiopsies, T790M and/or PD-L1 expression revealed temporal dynamism. Survival curves according to PD-L1 expression/T790M status suggested better prognosis in PD-L1-/T790M+ population. ConclusionsT790M+ status was correlated to lower PD-L1 expression. PD-L1 expression might have a prognostic value and interaction with T790M mutation in EGFR-mutant NSCLC.

Abstract 2625: Clinicopathological features of programmed cell death ligand 1 (PD-L1) expression in resected non-small cell lung cancers

Abstract Background: Antibodies against programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) have recently demonstrated a survival benefit in several types of cancer, including melanoma and non-small cell lung cancer (NSCLC). PD-L1 expression has provided a predictive biomarker for anti-PD-1/PD-L1 therapy. However, the relationship between PD-L1 expression and clinical and clinicopathological characteristics remains unclear in NSCLCs. The aim of this study was to investigate the relevance of PD-L1 expression to clinical and clinicopathological factors in resected NSCLCs. Materials and Methods: PD-L1 expression was evaluated in 154 surgically resected NSCLC specimens by immunohistochemistry (IHC) using the SP142 antibody. Before the IHC of clinical samples, we performed a concordance study of Western blot analysis and IHC in 17 lung cancer cell lines. We evaluated the relationship between PD-L1 expression and clinical and clinicopathological factors by univariate and multivariate analyses. Survival curves were estimated by the Kaplan-Meier methods, and differences in survival distributions were evaluated by the log-rank test. Results: PD-L1 expression was well concordantly observed in Western blot analysis and IHC in the lung cancer cell lines analyzed. PD-L1 expression was observed in 51 of 154 (33%) surgically resected NSCLCs. PD-L1 expression was significantly correlated with male, heavy smoking history, squamous cell carcinoma, and moderate/poor differentiation of tumors by univariate analysis. In multivariate analysis, tumor differentiation had a strong relationship with PD-L1 expression (odds ratio: 6.32, 95% CI: 1.70-23.49, P = 0.006). The survival was not statistically different between patients with high and low PD-L1 expression (5-year survival rate: 44.1% versus 58.1%, P = 0.22). PD-L1 expression was not an independent prognostic factor in univariate and multivariate analyses. Conclusion: PD-L1 expression was strongly associated with tumor differentiation in resected NSCLCs. PD-L1 expression was not a prognostic factor in this cohort of resected NSCLCs. Citation Format: Yoshihito Ohhara, Ichiro Kinoshita, Utano Tomaru, Kanako C. Hatanaka, Yutaka Hatanaka, Rio Honma, Satoshi Takeuchi, Yasushi Shimizu, Kichizo Kaga, Yoshihiro Matsuno, Hirotoshi Dosaka-Akita. Clinicopathological features of programmed cell death ligand 1 (PD-L1) expression in resected non-small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2625. doi:10.1158/1538-7445.AM2017-2625

Abstract 2616: Upregulation of program death ligand 1 expression after a single cycle of chemotherapy is associated with inferior survival in patients with local advanced esophageal squamous cell carcinoma treated with preoperative chemoradiotherapy

Abstract Introduction Previous studies have demonstrated that the expression of program death-ligand 1 (PD-L1), a ligand of an immune checkpoint, on cancer cells or on immune cells may have prognostic significance in multiple cancer types including esophageal squamous cell carcinoma (ESCC). We evaluated the expression of PD-L1 and its dynamic changes following one cycle of chemotherapy before standard preoperative chemoradiotherapy (CRT) in a cohort of locally advanced ESCC patients. Patients and Methods A cohort of 66 patients of locally advanced ESCC have been enrolled to a phase II clinical trial of a single-cycle chemotherapy (a combination of weekly paclitaxel, cisplatin and 24 hour infusional 5-fluorouracil, 2 week on plus one week off, followed by paclitaxel/cisplatin (TP) -based preoperative CRT and radical surgery. Paired tumor tissues from endoscopic biopsy before and after the single-cycle chemotherapy were available in 21 patients. Immunohistochemical stain for PD-L1 expression was performed using the Clone SP142 antibody (Spring Bioscience, Pleasanton, CA, USA). The expression of PD-L1 was scored as previously reported. Tumor cells were scored according to percentage of positively-stained cells: TC3≥50%, TC2≥5% and &amp;lt;50%, TC1≥1% and &amp;lt;5%, and TC0&amp;lt;1%; Immune cells: IC3≥10%, IC2≥5% and &amp;lt;10%, IC1≥1% and &amp;lt;5%, and IC0&amp;lt;1%. Results In 21 baseline ESCC tissues, 7 of 19 (37%) exhibited positive PD-L1 expression on tumor cells, and 10 of 21 (48%) exhibited positive expression on immune cells. In 21 ESCC tissues obtained a median of 18 days after initiating chemotherapy, 9 of 18 (50%) and 15 of 21 (71%) demonstrated positive PD-L1 expression on tumor cells and immune cells, respectively. The expression of PD-L1 was significantly increased in post-chemotherapy ESCC tissues compared with baseline tissues (p=0.010); and the increased PD-L1 expression between paired tissues was significant on immune cells (p=0.0056) but did not reach statistical significance on tumor cells (p=0.11). The positive PD-L1 expression in baseline ESCC tissues was correlated with better survival (HR: 0.37, p=0.023). Besides, patients with more increase of PD-L1 expression after chemotherapy had significantly worse survival than those with less (median survival: 19.4 vs 71.20 months, HR: 4.07, p=0.0089). Conclusion PD-L1 expression of ESCC can be up-regulated by chemotherapy. The up-regulation of PD-L1 is associated with poor outcome for locally advanced ESCC patients. (The study was supported by the grant NTUH 104-M2891 and the grant MOST 105-2314-B-002-186-MY3) Citation Format: Ta-Chen Huang, Cher-Wei Liang, Chia-Chi Lin, Ya-Jhen Chen, Kuan-Ling Lin, Ann-Lii Cheng, Chin-Hung Hsu. Upregulation of program death ligand 1 expression after a single cycle of chemotherapy is associated with inferior survival in patients with local advanced esophageal squamous cell carcinoma treated with preoperative chemoradiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2616. doi:10.1158/1538-7445.AM2017-2616

Abstract 1636: Increased expression of programmed death-ligand 1 (PD-L1) on infiltrating immune cells of hepatocellular carcinoma (HCC) tissues after sorafenib treatment

Abstract Background: PD-L1 expression in tumor microenvironment of HCC was reported to associate with tumor aggressiveness and recurrence. The impact of sorafenib treatment on PD-L1 expression on tumor cells (TC) or tumor-infiltrating immune cells (IC) of HCC has been unclear. Patients and Methods: We reviewed patients with HCC who had received sorafenib for advanced diseases at National Taiwan University Hospital, Taipei, Taiwan. Patients with paired HCC tissues, obtained before and after sorafenib treatment, were included. Immunohistochemistry (IHC) assay with clone SP142 antibody (Spring Bioscience, Pleasanton, CA, USA) was performed to analyze PD-L1 expression on TC and IC in paired specimens obtained before and after sorafenib. PD-L1 expression was scored as IHC 0, 1, 2, or 3 if &amp;lt;1%, ≥1% but &amp;lt;5%, ≥5% but &amp;lt;10%, or ≥10% of cells were PD-L1 positive, respectively. Further IHC assay was employed to characterize the PD-L1-positive IC. Associations between the PD-L1 expression and overall survival (OS) or duration of sorafenib treatment of the patients were analyzed. Results: Twenty-three advanced HCC patients (Male: Female= 20: 3, median age of 64 years) with paired HCC tissues were included. All of the post-sorafenib HCC tissues were obtained after disease progression. The median duration of sorafenib treatment was 4.3 months (range: 1.3 to 18.7). The PD-L1 expression on IC was significantly increased in post-sorafenib HCC tissues, compared with tissues obtained before sorafenib (pre-sorafenib vs post-sorafenib IHC 0/1/2/3: 10/5/5/3 vs 5/5/2/11, p=0.046). However, the PD-L1 expression on TC was not significantly different between pre- and post-sorafenib tissues (IHC 0/1/2/3: 19/2/0/2 vs 14/5/0/4, p=0.065). By using IHC staining of CD68, CD66b, CD11b, and CD3 on the consecutive slides, we found the PD-L1-expressing IC were mainly CD68-positive macrophages, but not neutrophils or T lymphocytes. Neither the PD-L1 expression levels on IC of pre-sorafenib or those of post-sorafenib tissues were associated with OS or duration of sorafenib treatment. Conclusions: The PD-L1 expression on IC, especially on macrophages, in tumor microenvironment was significantly increased in post-sorafenib progression HCC tissues. Whether this increased PD-L1 expression contributes to treatment failure of sorafenib warrants further investigation (This work was supported by the grants of NTUH 105-M3232 and MOST 105-2314-B-002-180). Citation Format: Li-Chun Lu, Yi-Hsuan Lee, Chun-Jung Chang, Yu-Yun Shao, Tsung-hao Liu, Ann-Lii Cheng, Chih-Hung Hsu. Increased expression of programmed death-ligand 1 (PD-L1) on infiltrating immune cells of hepatocellular carcinoma (HCC) tissues after sorafenib treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1636. doi:10.1158/1538-7445.AM2017-1636

PD-L1 protein expression in tumour cells and immune cells in mismatch repair protein-deficient and -proficient colorectal cancer: the foundation study using the SP142 antibody and whole section immunohistochemistry

AimsRoutine application of PD-L1 immunohistochemistry (IHC) in colorectal cancer (CRC) is limited due to lack of standardized scoring criteria, antibody clones, and intratumoral staining heterogeneity. We assessed PD-L1 protein expression...

A Prospective, Multi-institutional, Pathologist-Based Assessment of 4 Immunohistochemistry Assays for PD-L1 Expression in Non–Small Cell Lung Cancer

ImportanceFour assays registered with the US Food and Drug Administration (FDA) detect programmed cell death ligand 1 (PD-L1) to enrich for patient response to anti–programmed cell death 1 and anti–PD-L1 therapies. The tests use 4 separate PD-L1 antibodies on 2 separate staining platforms and have their own scoring systems, which raises questions about their similarity and the potential interchangeability of the tests.ObjectiveTo compare the performance of 4 PD-L1 platforms, including 2 FDA-cleared assays, 1 test for investigational use only, and 1 laboratory-developed test.Design, Setting, and ParticipantsFour serial histologic sections from 90 archival non–small cell lung cancers from January 1, 2008, to December 31, 2010, were distributed to 3 sites that performed the following immunohistochemical assays: 28-8 antibody on the Dako Link 48 platform, 22c3 antibody on the Dako Link 48 platform, SP142 antibody on the Ventana Benchmark platform, and E1L3N antibody on the Leica Bond platform. The slides were scanned and scored by 13 pathologists who estimated the percentage of malignant and immune cells expressing PD-L1. Statistical analyses were performed from December 1, 2015, to August 30, 2016, to compare antibodies and pathologists’ scoring of tumor and immune cells. Main Outcomes and MeasuresPercentages of malignant and immune cells expressing PD-L1.ResultsAmong the 90 samples, the SP142 assay was an outlier, with a significantly lower mean score of PD-L1 expression in both tumor and immune cells (tumor cells: 22c3, 2.96; 28-8, 3.26; SP142, 1.99; E1L3N, 3.20; overall mean, 2.85; and immune cells: 22c3, 2.15; 28-8, 2.28; SP142, 1.62; E1L3N, 2.28; overall mean, 2.08). Pairwise comparisons showed that the scores from the 28-8 and E1L3N tests were not significantly different but that the 22c3 test showed a slight (mean difference, 0.24-0.30) but statistically significant reduction in labeling of PD-L1 expression in tumor cells. Evaluation of intraclass correlation coefficients (ICCs) between antibodies to quantify interassay variability for PD-L1 expression in tumor cells showed high concordance between antibodies for tumor cell scoring (0.813; 95% CI, 0.815-0.839) and lower levels of concordance for immune cell scoring (0.277; 95% CI, 0.222-0.334). When examining variability between pathologists for any single assay, the concordance between pathologists’ scoring for PD-L1 expression in tumor cells ranged from ICCs of 0.832 (95% CI, 0.820-0.844) to 0.882 (95% CI, 0.873-0.891) for each assay, while the ICCs from immune cells for each assay ranged from 0.172 (95% CI, 0.156-0.189) to 0.229 (95% CI, 0.211-0.248).Conclusions and RelevanceThe assay using the SP142 antibody is an outlier that detected significantly less PD-L1 expression in tumor cells and immune cells. The assay for antibody 22c3 showed slight yet statistically significantly lower staining than either 28-8 or E1L3N, but this significance was detected only when using the mean of 13 pathologists’ scores. The pathologists showed excellent concordance when scoring tumor cells stained with any antibody but poor concordance for scoring immune cells stained with any antibody. Thus, for tumor cell assessment of PD-L1, 3 of the 4 tests are concordant and reproducible as read by pathologists.

A Comprehensive Analysis of Programmed Cell Death Ligand-1 Expression With the Clone SP142 Antibody in Non–Small-Cell Lung Cancer Patients

BackgroundProgrammed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1) have been identified as novel targets for immunotherapy, with anti–PD-1 therapy currently the standard treatment for non–small-cell lung cancer (NSCLC) patients after the failure of first-line chemotherapy treatment. The recent phase II POPLAR and phase III OAK studies showed that atezolizumab, a representative PD-L1 inhibitor, exhibited a survival benefit compared with standard therapy in patients with NSCLC. Patients and MethodsWe examined PD-L1 expression in NSCLC using the clone SP142 of POPLAR and OAK studies. PD-L1 expression in 499 surgically resected NSCLC patients was evaluated using immunohistochemistry using SP142. We set cutoff values as 1%, 5%, 10%, and 50%. ResultsThe samples from 189 (37.9%), 119 (23.8%), 71 (14.2%), and 39 (7.8%) patients were positive for PD-L1 expression at cutoff values of 1%, 5%, 10%, and 50%, respectively. Fisher exact tests showed that PD-L1 positivity was significantly associated with male sex, smoking, advanced stage, the presence of vascular invasion, squamous cell carcinoma, and wild type epidermal growth factor receptor gene mutation status at all cutoff values. Univariate and multivariate survival analyses revealed that PD–L1-positive patients had a worse prognosis than PD–L1-negative patients only at the 1% cutoff value. Forest plot analyses showed that the 1% cutoff provided a more sensitive value for the prediction of postoperative prognosis. ConclusionPD-L1 expression varied greatly according to different cutoff values. This study might be a useful reference to understand the results of POPLAR and OAK studies and to select patients likely to benefit from atezolizumab.

PD-L1 Status in Refractory Lymphomas.

Targeted immunotherapy based on PD-1/PD-L1 suppression has revolutionized the treatment of various solid tumors. A remarkable improvement has also been observed in the treatment of patients with refractory/relapsing classical Hodgkin lymphoma (cHL). We investigated PD-L1 status in a variety of treatment resistant lymphomas. Tumor samples from 78 patients with therapy resistant lymphomas were immunohistochemically (IHC) investigated for the expression of PD-L1 using two antibody clones (SP142 and SP263, Ventana). Thirteen PD-L1+ cases were further analyzed for gene copy number variations (CNV) by NGS and for PD-L1/JAK2/PD-L2 co-amplification using fluorescent in-situ hybridization assay (FISH). PD-L1 positivity (≥5% positive cancer cells, IHC) was present in 32/77 (42%) and 33/71 cases (46%) using SP142 and SP263 antibodies, respectively. Concordance between the two anti-PD-L1 clones was high with only three (4%) discrepant cases. The strongest and consistent (10/11 cases) expression was observed in cHL and primary mediastinal B-cell lymphomas (3/3). Diffuse large B-cell lymphomas (DLBCL) were frequently positive (13/26) irrespective of subtype. Follicular (1/8), peripheral T-cell (3/11) and mantle cell (1/8) lymphomas were rarely positive, while small lymphocytic lymphoma/CLL and marginal zone lymphomas were consistently negative (3/3). Co-amplification/CNVs of PD-L1/JAK2/PD-L2 were observed in 3 cases of DLBCL and cHL, respectively. Of note, all three cHL-amplified cases were positive by FISH, but not by NGS. Since only a fraction of the IHC positive lymphoma cases were positive by FISH and NGS assays, other mechanisms are involved in PD-L1 upregulation, especially in DLBCL. FISH assay may be more suitable than NGS assay for determination of PD-L1 alterations in cHL.

O.04: Profiling of MET-Amplified Non-Small Cell Lung Cancer (NSCLC), Correlation to cMET Protein Expression/MET Exon 14 Skipping

Recent genomic studies of molecular subtypes of NSCLC revealed a plethora of data surrounding its underlying biology. Knowledge of EGFR-driven NSCLC has changed the way one molecular subtype is being managed. While MET-driven NSCLC has been documented, a lot remains unknown about this molecular subtype of NSCLC, in contrast to EGFR- and KRAS-mutated molecular subtypes. The purpose of this study is to investigate the genomics of MET-amplified NSCLC to further understand its tumor biology, and to indicate potential for clinical trials. In total, 3,540 NSCLC specimens underwent multiplatform testing at a CAP/ISO/CLIA-certified laboratory (Caris Life Sciences). Analysis included protein expression by immunohistochemistry (IHC), gene amplification by in situ hybridization (ISH), fusions by ArcherDx FusionPlex assay, and copy number variance (CNV)/mutation by next generation sequencing (NGS). The laboratory validated CISH assay, utilized a gene copy number > 5 to assess gene amplification. In this cohort, 3.1% (109/3,540) patients were MET-amplified. In MET-amplified tumors (n=109), protein expression by IHC showed no ALK expression (0/36), positive EGFR at 47.5% (38/80), and positive PD-L1 at 56.0% (36/64, using 5% cut off value with SP142 antibody), with a sub-group showing a PD-L1 positive rate (50% cut off) of 68.8% (11/16) using the recently FDA approved companion diagnostics kit (22C3 antibody). For gene translocation by FISH, ALK was 2.5% (2/79) and ROS1 was 0.0% (0/97). For mutational analysis, BRAF mutations were 6.5% (6/92), EGFR was 23.1% (21/91), ERBB2 was 1.1% (1/92), and KRAS was 13.0% (12/92). ForMET-amplified NSCLC specimens, cMET protein expression was detected in 92.5% (74/80), MET amplification by CNV was 66.7% (4/6), MET mutation rates were 5.4% (5/92), and MET fusion was found in 20.0% (1/5). Evaluation of the 3,540 specimens revealed 17 MET exon 14 skipping mutations with only two showing amplification (11.8%, 2/17). Our analysis reveals potentially targetable aberrations in MET-driven NSCLC. High PD-L1 rates in this group indicate potential for targeting with PD-1blockade therapy and potential for combined immune blockade/MET inhibition therapy. Differences in cMET alterations indicate the need to analyze patient specimens for MET using different technologies and to find the optimal threshold for MET. Further studies of this molecular subtype of NSCLC are urgently needed to elucidate the optimal therapy for these patients.

PD-1/PD-L1 expression and regorafenib clinical efficacy on refractory pancreatic cancer patient.

e15684 Background: Regorafenib, a potent multikinase inhibitor, targets a broad range of receptor tyrosine kinases (RTKs), including but not limited to VEGFR, PDGFR, and FGFR, which play an integral role in angiogenesis and in making a immunosuppressive tumor microenvironment. The results of the correlation between PD-1/PD-L1 expression and RTKs inhibition remains controversial in lung cancer and limited data exist in pancreatic cancer. Methods: We analysed a cohort of 69 chemorefractory metastatic pancreatic cancer patients, including 56 KRAS mutated, treated with Regorafenib as third line of treatment. PD-L1 (MRQ-22 antibody) and PD-1 (SP142 antibody) expression were assessed by immunohistochemistry. All cases with moderate or strong staining (2+/3+) in > 5% of tumour cells and tumor infiltrating lymphocytes (TILs) were considered as positive. Results: Pancreatic tumors expressed PD-L1 at a frequency of 20% (14/69), and PD-1 at a frequency of 46% (32/69). PD-1 positive (+) was significantly associated with previous alcohol abuse status (p = 0.04) and with the presence of KRAS mutations (p = 0.008). Patients with tumors overespressing PD-1/PD-L1 positivity, (Group A: 46/69) had a median progression free survival (PFS) was 4.58 months, whereas it was 1.45 months in the group without the combined expression (Group B: 23/69) (p = 0.0012). Median overall survival (OS) was 7,16 months and 2.18 months in group A and B respectively (p = 0.031). At multivariate analysis a baseline high platelet count was related to a worse OS (p = 0.057). Conclusions: Our analysis suggests that PD-L1/PD-1 positivity is a predictive factor for clinical outcome in pancreatic cancer patients treated with regorafenib. Further confirmations in larger trials are needed.

PD-1/PD-L1 expression and molecular associations in HPB malignancies.

289 Background: Cholangiocarcinoma (CC), hepatocellular carcinoma (HCC), and pancreatic ductal adenocarcinoma (PDAC) are all devastating malignancies. Limited data exists about PD-1 and PDL1 expression in these cancers. We assessed expression of PD1/PDL1 in a large cohort of patients with hepato-pancreatico-biliary (HPB) cancers and explored the existence of accompanying genomic mutations associated with expression of PD-1/PD-L1. Methods: 524 patients with HPB cancers (354 PDAC, 58 HCC, 54 intrahepatic (ICC), 18 extrahepatic (ECC), and 40 gallbladder (GBC)) were included in the study and tumors tested centrally at a CLIA lab (Caris Life Sciences, Phoenix, AZ). Tests included one or more of the following: gene sequencing (next generation sequencing, Illumina TruSeq), protein expression (immunohistochemistry [IHC]) and gene amplification). PD-1 (SP142 antibody) and PD-L1 (MRQ-22 antibody) status was tested in all samples. Two-tailed Fisher's exact test was performed to test where proportions of positive results were different by subgroup (p²0.05). Results: Among those with PDAC, HCC, ICC, ECC, and GBC, rates of PD1 expression on tumor infiltrating lymphocytes (TILs) and PD-L1 expression on tumor cells is detailed in the Table. In PD-1+, compared with PD-1- HPB cancers, mutations in the following were more prevalent, though not statistically significant: BRCA2, ATM, CTNNB1, and PIK3CA. Among all theranostic biomarkers tested, TOP2A expression (IHC) was significantly increased in PD-L1+ versus PD-L1- tumors (82% vs 60%; p=0.0083). Conclusions: HPB tumors express PD-L1 at a frequency of 4-18%, and PD-1 at a frequency of 29-45%. A statistically significant association of mutations with PD-1+ or PD-L1+ tumors was not identified in this group of tumors. PD-L1 expression associates with TOP2a expression, a marker of proliferation and also anthracycline sensitivity. Further evaluation of this correlation and PDL1/PD-1 + anthracycline combination therapy may be warranted. [Table: see text]

English

Background: Breast cancer markers are becoming increasingly important in breast cancer research due to their impact on prognosis, treatment and survival. The present retrospective study was carried out to quantify the proportion of estrogen (ER), progesterone (PR), and human epithelial receptor 2 (HER2) expressions and their association with tumour grade, age, and tumour size in breast cancer patients in Nigeria. Materials and methods: The paraffin embedded tissue sections were analysed for breast cancer markers using monoclonal antibody SP1 for ER and SP2 for PR and polyclonal antibody ErbB2 for HER2. Results: A total of 286 breast cancer paraffin wax tissue sections were analysed for ER, PR and HER2 expression. Of all the tissue samples examined, 20 (7%) were ERpositive, 6 (2.1%) were PR-positive, 11 (3.8%) were HER2-positive whereas 248 (87%) were triple-negative breast carcinoma. ER- and PR-positivity was associated with early grade I and II tumours (P  0.010-0.009) and tumour sizes of  50mm (p  0.001). HER2positivity was positively (P  0.009) associated with grade II tumours and negatively (P  0.0001) associated with grade III tumours. Triple-negative breast cancer was associated with grade III tumours (P  0.0001) and larger tumour sizes of  50mm (P  0.0001). Conclusion: A small proportion of Nigerian women with breast cancer are ER/PR-positive which are associated with less aggressive, better prognosis and benefit from endocrine therapy. An even smaller proportion of patients with aggressive tumors were HER2posivite but responsive to Herceptin treatment. Unfortunately, a very high proportion of cases were triple-negative which is associated with very aggressive tumours and no targeted treatment, which may explain the high mortality rates from breast cancer in Nigeria.

Abstract P5-01-15: Anti-ER monoclonal antibody SP1 seems more sensitive in the identification of ER alpha positive breast cancer cases than anti-ER monoclonal antibody 1D5

Abstract Background: Accurate pathological analysis of biopsies is crucial for conclusive and precise disease diagnosis, prognosis and therapeutic guidelines. Evaluation of estrogen receptor (ER) expression in breast cancer (BC) is imperative in determining the disease prognosis, and patients' eligibility for hormonotherapy. Herein, ER alpha status was evaluated in 61 BC cases using two different anti-ER alpha monoclonal antibodies, SP1 (rabbit) and 1D5 (mouse). Methods: Primary BC cases registered in two reference hospitals, with correspondent written informed consent, were used to generate in house tissue microarray platforms. ER alpha expression was accessed by immunohistochemistry using the monoclonal antibodies anti-ER alpha, SP1 and 1D5. Staining scoring followed the Allred method (PS, proportion score or percentage of stained cells; IS, intensity score). Concordance and correlation parameters were calculated using Kappa factor; Pearson, Spearman's, or intraclass correlation coefficient (CF). Staining patterns were compared by paired T-Test and Wilcoxon test. Findings: SP1 and 1D5 provided equivalent results (Concordance rate, 96.7%; Kappa factor, 0.921), whereas SP1 was more prone for positive results than 1D5 (2 samples diverged). Total concordance of PS was obtained (Pearson and intraclass CF, 0.7351 and 0.6193, respectively); however, concordance between the antibodies seems more accurate in higher PS values. Excellent SI correlation between antibodies was observed throughout the population (Spearman's CF, ρ=0.9150). Following the Allred score, 17 out of 42 positive BC samples diverged; always pointing 1D5 to weaker staining than SP1. When calculating Spearman's CF of Total Score (TS) within the population, excellent correlation between both the antibodies (ρ=0.9238) was noted; nonetheless, results were less concordant result among the BC positive cases (ρ=0.7743). Indeed, 20 samples were differentially classified using the antibodies (only 3 had higher TS with 1D5). Considering the mean TS of all samples, or of invasive ductal carcinoma, SP1 provided higher scores than 1D5 (p &amp;lt; 0.05). Interpretation: Despite the equivalence between the anti-ER alpha monoclonal antibodies, SP1 and 1D5, the former has proven superior to the latter with respect to the IS and TS. Therefore, we suggest the clinical use of SP1 while diagnosing BC, due to its higher accuracy in examining ER status; thus, enabling more accurate therapeutic decisions to treat the disease. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-01-15.

Comparison of Anti–Estrogen Receptor Antibodies SP1, 6F11, and 1D5 in Breast Cancer

We compared the anti-estrogen receptors (ER) SP1, 6F11, and 1D5 antibodies in breast carcinoma cases with different ranges of positive cells to evaluate whether this could generate different therapies for patients. We selected 66 cases of breast cancer, each of which was immunostained with the 3 antibodies. 1D5 was less sensitive than SP1 and 6F11, as seen in 26, 20, and 21 negative cases, respectively. Nine cases showed differences in endocrine-therapy indications, of which 8 1D5-negative cases showed low positivity for SP1 and/or 6F11. However, these cases were prevalently G3, progesterone receptor-negative or low-positive, with high Ki-67 and positive HER-2 findings, all biological features associated with endocrine resistance. Finally ER values obtained with these 3 antibodies had no implications for chemotherapy.

Abstract 3600: Comparison of estrogen receptor expression in breast cancer cells by immunohistochemistry using the monoclonal antibodies SP1 and 1D5

Abstract Background: Accurate pathological analysis of biopsies is crucial for conclusive and precise disease diagnosis, prognosis and therapeutic guidelines. Evaluation of estrogen receptor (ER) expression in breast cancer (BC) is imperative in determining the disease prognosis, and patients' eligibility for hormonotherapy. Herein, ERα status was evaluated in 61 BC cases using two different anti-ERα monoclonal antibodies, Sp1 (rabbit) and 1D5 (mouse). Methods: Primary BC cases registered in two reference hospitals, with correspondent written informed consent, were used to generate in house tissue microarray platforms. ERα expression was accessed by immunohistochemistry using the monoclonal antibodies anti-ERα, SP1 and 1D5. Staining scoring followed the Allred method (PS, proportion score or percentage of stained cells; IS, intensity score). Concordance and correlation parameters were calculated using Kappa factor; Pearson, Spearman's, or intraclass correlation coefficient (CF). Staining patterns were compared by paired T-Test and Wilcoxon test. Findings: SP1 and 1D5 provided equivalent results (Concordance rate, 96.7%; Kappa factor, 0.921), whereas SP1 was more prone for positive results than 1D5 (2 samples diverged). Total concordance of PS was obtained (Pearson and intraclass CF, 0.7351 and 0.6193, respectively); however, concordance between the antibodies seems more accurate in higher PS values. Excellent SI correlation between antibodies was observed throughout the population (Spearman's CF, α=0.9150). Following the Allred score, 17 out of 42 positive BC samples diverged; always pointing 1D5 to weaker staining than SP1. When calculating Spearman's CF of Total Score (TS) within the population, excellent correlation between both the antibodies (α=0.9238) was noted; nonetheless, results were less concordant result among the BC positive cases (α=0.7743). Indeed, 20 samples were differentially classified using the antibodies (only 3 had higher TS with 1D5). Considering the mean TS of all samples, or of invasive ductal carcinoma, SP1 provided higher scores than 1D5 (p&amp;lt;0.05). Interpretation: Despite the equivalence between the anti-ERα monoclonal antibodies, SP1 and 1D5, the former has proven superior to the latter with respect to the IS and TS. Therefore, we suggest the clinical use of SP1 while diagnosing BC, due to its higher accuracy in examining ER status; thus, enabling more accurate therapeutic decisions to treat the disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3600. doi:1538-7445.AM2012-3600

The Reliability of Rabbit Monoclonal Antibodies in the Immunohistochemical Assessment of Estrogen Receptors, Progesterone Receptors, and HER2 in Human Breast Carcinomas

The reliability of the rabbit monoclonal antibodies SP1, SP2, SP3, and 4B5 was immunohistochemically assessed on a range of 96 invasive breast carcinomas and the results compared with those achieved with established antibody markers for estrogen receptors (6F11), progesterone receptors (PgR636), and HER2 (polyclonal A0485 and clone CB11), with HER2 status validated by fluorescence in situ hybridization (FISH) and silver in situ hybridization. Optimal results depended on the duration of microwave antigen-retrieval time and the use of a high pH buffer for rabbit and mouse estrogen receptor antibodies (SP1 and 6F11), although only on antigen-retrieval duration for the progesterone receptors SP2 and PgR636. The highest rate of concordance between HER2 overexpression and HER2 gene amplification was with the rabbit monoclonal antibodies (SP3 and 4B5) and FISH. Rabbit monoclonal antibodies are reliable alternatives to established antibody markers for the immunohistochemical testing of estrogen receptors, progesterone receptors, and HER2 in breast cancer.

Comparison of immunostaining for estrogen receptor in breast cancer using the rabbit monoclonal antibody SP1 or the mouse monoclonal antibody 6F-11

11562 Background: A high-quality assay to detect estrogen receptor (ER) expression is crucial as ER status guides many therapeutic decisions in breast cancer. Previously the rabbit monoclonal antibody (Ab) SP1 was shown to be more sensitive than the mouse monoclonal Ab 1D5. Results were similar in a comparison of mouse Abs 6F11 and 1D5. This study compares the ER status in breast cancer using SP1 on cases previously interpreted as negative with 6F11. Methods: All low grade (1 or 2), ER negative, invasive breast carcinomas, reviewed at the British Columbia Cancer Agency between 2002 and 2006 were reviewed. Cases that had been stained at the Vancouver site (VCC) with 6F11 were selected and those with available tissue blocks were re-stained with SP1. The patient’s oncologist was notified of any changes in ER status. Results: 123 cases of low grade, ER negative invasive breast tumors were initially selected. 49 of these cases had been stained at VCC with 6F11. Tissue blocks were available for 45/49 (92%) of cases. 5/45 cancers (11%) were grade 1 and 40/45 (89%) were grade 2. With SP1 the ER status changed from negative to positive in 3/45 (6.7%) of the cases. For 2 cases the ER status changed because current scoring criteria regard a test as positive even if only rare cells stain. Review of original 6F11 stained sections revealed these 2 cases would be scored as ER positive using the new criteria. The third case was a core biopsy with scant tumor and only focal staining. Review revealed the change in ER status was likely due to sampling difference in the deeper section. Conclusions: Of 45 previously 6F11 ER negative cases restained with SP1, the ER status changed in 2 because of revised more sensitive scoring criteria and in 1 because of a sampling difference between the original and subsequent sections. The rabbit monoclonal Ab SP1 appears to be as sensitive as the mouse monoclonal Ab 6F11 in detecting ER expression in breast cancer. This highlights the need for good quality ER testing with highly sensitive antibodies. Additional data for grade 3 cases will be presented. No significant financial relationships to disclose.

Immunohistochemical Detection Using the New Rabbit Monoclonal Antibody SP1 of Estrogen Receptor in Breast Cancer Is Superior to Mouse Monoclonal Antibody 1D5 in Predicting Survival

Estrogen receptor (ER) expression predicts improved breast cancer-specific survival and reduced risk of recurrence and is targeted in breast cancer therapy. A high-quality antibody to identify ER-positive patients plays an important role in clinical decision making for women with breast cancer. This study evaluates immunohistochemistry using two anti-ER antibodies, a new rabbit monoclonal antibody (SP1) and the mouse monoclonal antibody (1D5), in relation to biochemical ER assay results and clinical data on survival and adjuvant systemic therapy. A population-based tissue microarray series of 4,150 invasive breast cancers was constructed. All patients had staging, pathology, treatment, and follow-up information. The median follow-up was 12.4 years and the median age at diagnosis 60 years. Survival analysis and log-rank tests were used to evaluate the prognostic value of ER status and correlations with clinical data. Among the 4,105 samples interpretable for both antibodies, SP1 detected ER positivity in 69.5% and 1D5 in 63.1% of cases. Both monoclonal antibodies are demonstrated to be good prognostic indictors for breast cancer-specific and relapse-free survival. In multivariate analysis, including age, tumor size, grade, and lymphovascular and nodal status, SP1 was a better independent prognostic factor than 1D5. Among patients with discrepant ER results, the 8% of patients who were SP1 positive/1D5 negative showed good outcomes, and the 2% SP1-negative/1D5 positive had poor outcomes. Maintaining the same 92% specificity and 98% positive predictive value, SP1 is 8% more sensitive than 1D5 using biochemical assay as gold standard. SP1 represents an improved standard for ER immunohistochemistry assessment in breast cancer.