Intense Membrane Staining Research Articles

Abstract PD7-02: Value-based medicine: Are the 2013 guidelines for HER2 testing clinically significant and cost effective?

Abstract Background: In the U.S., around 252,710 new breast cancer cases will be diagnosed in the year 2017. About 15-20% of these patients will be candidates for treatment with Her-2 directed therapy. The College of American Pathologists (CAP) published initial guidelines for immunohistochemistry (IHC) in 2003 with revisions in 2007 and 2013. 2013 guidelines define 3+(positive) as >10% intense complete membrane staining similar to 2003; >30% complete in 2007. Equivocal 2+ category in 2013 includes incomplete staining > 10% for the first time; this was negative in 2003 and 2007.Hypothesis: The 2013 guidelines do not result in more true positives but increased equivocal cases resulting in clinical uncertainty and increased cost. Methods : A retrospective analysis was performed of all IHC and FISH testing done at a single institution with a single pathology reader from 2003-2016. Criteria for IHC and FISH positivity were followed for each time period and compared to one another for positive and negative HER2 expression. The equivocal categories than compared for outcome by FISH. Ultimate numbers for percentage in each category compared for statistical significance. Results: IHC NegativeEquivocalPositiveTotal(1) 2003279, 70.6%71, 17.9%45, 11.3%395(2) 2007187, 71.6%49, 18.7%25, 9.5%261(3) 2013181, 61.7%79, 26.9%33, 11.2%293Total647199103949Chi-SquareDFValueProbOverall410.40.03 Period 1 vs 220.50.75Period 1 vs 328.20.01Period 2 vs 326.40.04 IHC Equivocal Reflex to FISH NegativeEquivocalPositiveTotal(1) 200358, 82.8%1, 1.4%11, 15.7%70(2) 200734, 80.9%0, 0.0%8, 19.0%42(3) 201362, 82.6%6, 8.0%7, 9.3%75Total154726187DF4Chi-Square8.3Asymptotic Pr> ChiSq0.08Exact Pr ≥ ChiSq0.07 When further analysis was carried out, period 1 and 2 were added together and compared to period 3. When all positives and negatives were compared, there was no statistical difference between the periods. However in the equivocal category, Period 1 and 2 were statistically different than period 3. In fact the only change in period 3 was the increase in the equivocal cases (same case-equivocal IHC and FISH). Conclusion: In the 2013 CAP guidelines, 2+ IHC now includes incomplete staining in >10% of cells. This does not result in more positive cases as was the intention but an increase in the equivocal category by 8.19%. This adds to clinical uncertainty as to how to treat this group of patients. A send out for FISH is labor intensive, slow and costs on average of $650/case. With over 250,000 new cases expected this year in the U.S. this cost exceeds $13million. From all our data, the best parameters for IHC testing would be; positive-strong, complete staining in >10% cells and equivocal to eliminate the incomplete staining category. This would yield the highest number of true positives by FISH and almost eliminate ultimate equivocal cases. Citation Format: Sharma A, Vadehra D, Talal K, Hegde P, Wu R, Tannenbaum S. Value-based medicine: Are the 2013 guidelines for HER2 testing clinically significant and cost effective? [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD7-02.

A decision tree-based prediction model for fluorescence in situ hybridization HER2 gene status in HER2 immunohistochemistry-2+ breast cancers: a 2538-case multicenter study on consecutive surgical specimens.

Objective: To investigate the proportion of HER2 gene amplifications and the association between the HER2-IHC-staining pattern and gene status in IHC-2+ breast cancers according to 2013 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines.Methods: We retrospectively analyzed and re-evaluated the IHC-staining pattern of 2538 IHC-2+ surgical specimens of breast cancer from November 2014 to October 2015 in 12 institutions. All cases used for building a prediction model of HER2 gene amplification according to the IHC-staining pattern and were randomly divided into a training set (n = 1914) or validation set (n = 624).Results: The overall HER2 fluorescence in situ hybridization (FISH) amplification, non-amplification and equivocation rates in HER2 IHC-2+ cases were 17.8%, 76.2% and 6.0%, respectively. In the training set, cases that had ≤ 10% of cells with intense, complete and circumferential membrane staining or had > 85% of cells with complete membrane staining of any staining intensity tended to be HER2 gene amplified (77.0% and 60.5%, respectively). And cases with weak and incomplete membrane staining had the lowest amplification rate of 6.1%. The prediction model was constructed based on IHC-staining pattern in the training set and validated using a validation set. The positive and negative prediction values were 51.6% and 79.2%, respectively, in the validation set. Moreover, the HER2 copy number per cell was much higher in cases with amplification-associated staining patterns (7.84 and 8.75) than in cases with non-amplification-associated staining patterns (2.97 to 4.41, P < 0.05).Conclusions: In HER2 IHC-2+ breast cancers, the staining pattern is associated with the HER2 gene status. This finding is compatible with recommendations of 2013 ASCO/CAP guidelines.

Role of E-cadherin in Progression of Oral Squamous Cell Carcinoma: A Retrospective Immunohistochemical Study

The aim of present study was to assess the role of E-cadherin in oral carcinogenesis by comparing their expressions in normal oral mucosa, oral epithelial dysplasia (OED), and oral squamous cell carcinoma (OSCC). To elucidate the pattern of E-cadherin expression in oral carcinogenesis, 21 archival cases of formalin-fixed paraffin-embedded sections of OSCC, 21 OED, and 7 normal oral mucosa samples as control were used for the study. We observed reduction/loss of E-cadherin in membranous expression pattern and staining intensity with progression from dysplasia to oral cancer. A decrease in staining intensity and loss of E-cadherin membranous expression were noted from dysplasia to carcinoma, suggesting its role as a tumor suppressor gene. E-cadherin can be used as a biomarker to assess and evaluate the progression and prognosis of oral dysplastic lesions and OSCC.

PD-L1 Expression of Tumor Cells, Macrophages, and Immune Cells in Non-Small Cell Lung Cancer Patients with Malignant Pleural Effusion.

Whether immunohistochemical staining of programmed death ligand 1 (PD-L1) on cells of pleural effusion could be used to predict response to immunotherapy treatment has not been reported. We retrospectively enrolled patients who had undergone malignant pleural effusion drainage and had effusion cell block specimens from 2014 to 2016. Immunohistochemical staining for PD-L1 was performed with tumor cells, immune cells, and macrophages of all cell block specimens. Immunoactivity was scored as 0 for absence of staining and 1+ for faint, 2+ for moderate, and 3+ for intense membranous staining. Patients' clinicopathological characteristics were also collected. PD-L1 expression of pleural effusion tumor cells was associated with the PD-L1 expression of macrophages (p= 0.003) and immune cells (p < 0.001). However, the PD-L1 expression of immune cells was not associated with that of macrophages. The PD-L1 expression of tumor cells was correlated with sex (p= 0.012), smoking status (p= 0.032), and Eastern Cooperative Oncology Group performance status (p= 0.017). The PD-L1 expression of immune cells was associated with the overall survival of patients (p= 0.004). These results suggest that there might be an immune interaction between pleural effusion tumor cells and macrophages. The low intensity of PD-L1 expression in immune cells is associated with the poor survival of patients with lung cancer with malignant pleural effusion.

Photophysical, Cellular‐Uptake, and Bioimaging Studies of Luminescent Ruthenium(II)–Polypyridine Complexes Containing a d‐Fructose Pendant

Two luminescent ruthenium(II)–polypyridine complexes, appended with a d‐fructose unit, [Ru(N^N)2(bpy‐fructose)](PF6)2 [bpy‐fructose = 4‐(1‐deoxy‐d‐fructos‐1‐yl)aminocarbonyl‐4′‐methyl‐2,2′‐bipyridine; N^N = 4,7‐diphenyl‐1,10‐phenanthroline (Ph2‐phen) (1a) and 2,2′‐bipyridine (bpy) (2a)] and their d‐fructose‐free counterparts [Ru(N^N)2(bpy‐Et)](PF6)2 [bpy‐Et = 4‐(ethylaminocarbonyl)‐4′‐methyl‐2,2′‐bipyridine; N^N = Ph2‐phen (1b) and bpy (2b)] are synthesized and characterized. The photophysical properties, lipophilicity, cytotoxicity, and cellular uptake of the complexes are studied. Laser‐scanning confocal microscopy reveals that both complexes 1a and 1b display intense membrane staining toward human breast adenocarcinoma (MCF‐7) cells. However, while complex 1a retains its membrane‐staining characteristic when incubated with human cervical epithelioid carcinoma (HeLa) cells, complex 1b is localized in the mitochondria. Additionally, fructose‐dependent cellular‐uptake experiments suggest that complex 1a is transported into MCF‐7 cells through a glucose transporter (GLUT) mediated pathway.

EHD4 is a novel regulator of urinary water homeostasis.

The Eps15-homology domain-containing (EHD) protein family comprises 4 members that regulate endocytic recycling. Although the kidney expresses all 4 EHD proteins, their physiologic roles are largely unknown. This study focused on EHD4, which we found to be expressed differentially across nephron segments with the highest expression in the inner medullary collecting duct. Under baseline conditions, Ehd4-/- [EHD4-knockout (KO)] mice on a C57Bl/6 background excreted a higher volume of more dilute urine than control C57Bl/6 wild-type (WT) mice while maintaining a similar plasma osmolality. Urine excretion after an acute intraperitoneal water load was significantly increased in EHD4-KO mice compared to WT mice, and although EHD4-KO mice concentrated their urine during 24-h water restriction, urinary osmolality remained significantly lower than in WT mice, suggesting that EHD4 plays a role in renal water handling. Total aquaporin 2 (AQP2) and phospho-S256-AQP2 (pAQP2) protein expression in the inner medulla was similar in the two groups in baseline conditions. However, localization of both AQP2 and pAQP2 in the renal inner medullary principal cells appeared more dispersed, and the intensity of apical membrane staining for AQP2 was reduced significantly (by ∼20%) in EHD4-KO mice compared to WT mice in baseline conditions, suggesting an important role of EHD4 in trafficking of AQP2. Together, these data indicate that EHD4 play important roles in the regulation of water homeostasis.-Rahman, S. S., Moffitt, A. E. J., Trease, A. J., Foster, K. W., Storck, M. D., Band, H., Boesen, E. I. EHD4 is a novel regulator of urinary water homeostasis.

Abstract 607: PDL1 expression associated with triple negative breast ductal carcinomas in African American women

Abstract Background: Tumor cells avoid host immune response through expression of inhibitory T cell regulator, Programmed cell death ligand (PDL1).Our objective was to evaluate PDL1 expression by immunohistochemistry in the four major subtypes of breast carcinoma (Luminal A, Luminal B, HER2, and Triple Negative) in a population of 202 African-American (AA) women with other clinicopathological factors. Design: Tissue microarrays (TMAs) were constructed from FFPE tumor blocks from primary ductal breast carcinomas in 202 African-American females. Two separate 1 mm cores represented each case. Five micrometer sections were stained with rabbit monoclonal antibody against PDL1. The sections were evaluated for the percentage and intensity of membrane staining in both tumor and immune cells. Cut off was &amp;gt; 1%. Bivariate analysis was done via χ2 analysis and survivability data was calculated via the generation of Kaplan-Meier curves (SPSS v19). Statistical significance was assumed if p &amp;lt; 0.05. Results: PDL1 expression in only tumor cells, only immune cells and in combination was associated with ER negative (p&amp;lt;0.0001), PR negative (p&amp;lt;0.0001), Triple negative subtype (p&amp;lt;0.0001) and high grade (p&amp;lt;0.0001) breast ductal cancers. Conclusion: Our study finding of selective expression of PDL1 in triple negative breast ductal cancers (TNBC) in AA women suggests that inhibition of adaptive immune response is involved in the progression of these cancers. PDL1/PD1 is the inhibitory check point of immune response resulting in decreased T cell activation and cytokine production. Some studies have shown that PDL1 expression is associated with loss of PTEN and phosphatide inositol 3 kinase pathway (PI3K) activation. Our previous study showed loss of PTEN in TNBC. Combining PDL1 inhibitors with PI3K inhibitors may be useful therapy for aggressive TNBC. Citation Format: Farhan Khan, Yasmine Kanaan, Luisel J. Ricks-Santi, Rabia Zafar, Hagos Aymut, Tammey Naab. PDL1 expression associated with triple negative breast ductal carcinomas in African American women [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 607. doi:10.1158/1538-7445.AM2017-607

High Prevalence of Claudin 18.2 Expression in Japanese Patients with Gastric Cancer.

e15584 Background: Expression of the gastric mucosal tight junction protein, Claudin-18.2 (CLDN18.2), is altered in gastric cancer (GC). In a phase 2 clinical trial (NCT01630083; FAST), a monoclonal antibody against CLDN18.2 (IMAB362) significantly increased overall survival in European patients with CLDN18.2-positive (CLDN18.2+) gastric and gastroesophageal junction adenocarcinomas when added to an EOX chemotherapy regimen. As the GC occurrence is high in Japan, this study evaluated the prevalence of CLDN18.2 expression in Japanese patients with GC. Methods: CLDN18.2 expression was assessed in primary GC tumors and corresponding lymph node metastases (LNM) by cell membrane staining intensity using a validated, semi-quantitative, immunohistochemical assay for CLDN18.2 expression. Samples showing any positivity for specific staining with at least 1+ intensity were defined as CLDN18.2+. Correlation between percentage of CLDN18.2+cells in primary tumors and corresponding LNM was determined with Spearman’s rank correlation. Results: A total of 263 samples, including tissues from 134 primary GC tumor/LNM matched pairs, 128 primary GC tumors only, and 1 LNM only were collected at Kurume University Medical Center, Japan, between 2000 and 2012. Overall, CLDN18.2 was detected in 87% (n = 228/262) of all primary tumors and 80% (n = 108/135) of LNM. Strong CLDN18.2 expression (≥2+ membrane staining intensity in ≥40% of tumor cells; eligibility criterion in FAST) was observed in 52% (n = 135/262) of primary tumors and 45% (n = 61/135) of LNM. Individual tumor samples consisted of a mixture of tumor cells expressing CLDN18.2 at different staining intensities; samples with a high fraction of CLDN18.2 reactivity had a large proportion of tumor cells staining with 3+ intensity (Spearman r = .8413, P&lt; .0001). In matched pairs, 72% of samples were CLDN18.2+ (n = 96/134) and 9% were CLDN18.2– (n = 12/134). The fraction of CLDN18.2+ tumor cells at any staining intensity strongly correlated in matched paired samples (Spearman r = .6942; P&lt; .0001). Conclusions: CLDN18.2 expression is highly prevalent in Japanese GC patients, making it a targetable alteration and supporting development of IMAB362 as a therapeutic agent for this patient population.

Relationship Between Circulating Tumor Cells and Annexin A2 in Early Breast Cancer Patients.

Annexin A2 (ANXA2) is a phospholipid-binding protein involved in fibrinolysis, cell proliferation, migration and metastatic dissemination. Circulating tumor cells (CTCs) are cells responsible for tumor dissemination and have a prognostic value in several types of cancers including breast cancer. Previously, we found correlation between CTCs and activation of coagulation. This study aimed to correlate CTCs with ANXA2 expression on CTCs, tumor cells and tumor associated stroma in primary breast cancer (PBC) patients. This prospective study included 101 PBC patients treated by primary surgery. CTCs were detected by quantitative real-time polymerase chain reaction (qRT-PCR) assay for the expression of epithelial (CK19) or epithelial-mesenchymal transition (EMT) genes [TWIST1, SNAI1, SNAI2, zinc finger E-box-binding homeobox 1 (ZEB1)]. ANXA2 expression on CTCs was detected by qRT-PCR, while expression of ANXA2 in tumor specimen was evaluated by immunohistochemistry and expressed by a weighted histoscore, evaluating both the percentage of positive cells and the intensity of membrane and cytoplasmic staining. Results of hormone receptors, HER2 status, B-cell lymphoma 2 (bcl-2) protein expression and protein p53 were reported as either positive or negative on histopathology report without further quantification. CTCs were detected in 24.8% patients. Patients with epithelial CTCs had a significantly higher ANXA2 expression on CTCs than those of patients without CTCs (p=0.01). There was no association between CTCs and ANXA2 protein expression in tumor cells. However, patients, whom CTCs with EMT phenotype were detected in, had higher ANXA2 expression in tumor stroma when compared to those with absent EMT CTCs (p=0.04). Hormone-negative tumors had significantly higher ANXA2 expression in tumor cells compared to hormone-positive tumors (p=0.03). Similarly, tumors without bcl-2 protein expression had higher tumor levels of ANXA2 compared to tumor cells that were bcl-2 positive (p=0.05). ANXA2 stromal expression might play a key role in aggressive tumor phenotype associated with increased EMT CTCs release, however, other factors beyond ANXA2 are responsible for coagulation activation mediated by CTCs in breast cancer patients.

Expression of claudin 1, claudin 4, and claudin 7 in colorectal cancer and its relation with CLDN DNA methylation patterns.

Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p ≤ 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.

SC10.03 Anti-EGFR Monoclonal Antibodies in Squamous Cell NSCLC

SC10.03 Anti-EGFR Monoclonal Antibodies in Squamous Cell NSCLC

Expression of epidermal growth factor receptor in patients with insulin resistance and seborrheic keratomas

to study the expression of epidermal growth factor receptor (EGFR) in patients with seborrheic keratomas (SK) and insulin resistance (type 2 diabetes mellitus (DM2)) and in those without concomitant carbohydrate metabolic disturbances. 80 patients with SK were examined. According to the presence or absence of DM2, the patients were divided into 2 groups: 1) 40 patients with concomitant DM2; 2) 40 people without carbohydrate metabolic disturbances. DM2 was diagnosed on the basis of laboratory studies and an endocrinologist's consultation. Histological and immunohistochemical (IHC) examinations using anti-EGFR antibodies were performed; two intact skin sections from the patients with DM2 and two intact skin sections from those without carbohydrate metabolic disturbances were used as a control. The ICH examination using anti-EGFR monoclonal antibodies revealed that Group 1 showed intense diffuse membrane staining of more than 50% of the cells in 32 (80%) patients, moderate (30-50% cells) and weak (10-30% cells) staining in 6 (15%) and 2 (5%) patients, respectively. Marked EGFR expression was also noted in two intact skin biopsy specimens taken from patients with DM2. In Group 2, the staining intensity was weak in more than 10% but less than 30% of the SK cells in 28 (70%) patients; moderate EGFR expression was observed in 9 (22.5%) and diffuse, pronounced, staining in more than 50% of the SK cells was in 3 (7.5%) patients. The intact skin biopsy specimens taken from 2 patients without carbohydrate metabolic disturbances displayed a weak EGFR expression in the basal cell layer of the epidermis. EGFR overexpression in SK may be a result of metabolic disorders rather than a diagnostic sign of malignant neoplasms of the internal organs. The increased EGFR expression revealed in patients with SK and concomitant DM2 is caused by insulin resistance and hyperinsulinemia, in which the dysregulation of insulin signal transmission into the cell leads to changes in EGF synthesis and signaling pathway that regulates cell proliferation and growth.

Abstract A084: High OX-40 expression on the tumor immune infiltrate is a prognostic factor of better survival in non-small cell lung cancer

Abstract Background: OX-40 expression on tumor-infiltrating lymphocytes correlates with better survival in several human cancers, suggesting that OX-40 signals may play a critical role in establishing an antitumor immune response. To better understand the prognostic role of OX-40 expression on the tumor immune infiltrate in non small cell lung cancer (NSCLC), we have profiled 100 surgically resected tumor specimens from patients with stage I-III NSCLC with known clinico-pathological characteristics using a panel of several markers of immune suppression and activation by immunohistochemistry (IHC) and correlated them with survival outcomes and gene expression profiles. Methods: 100 formalin fixed paraffin embedded NSCLC specimens (61 adenocarcinoma, 31 squamous cell carcinoma) were stained by IHC using an automated stainer with a panel of antibodies targeting the following immune markers: OX-40, PD-L1, PD-1, CD3, CD4, CD8, CD45RO, CD57, CD68, FOXP3, Granzyme B, ICOS. PD-L1 expression was evaluated in the tumor cells using H-score (range 0-300) taking into consideration the percentage of positive cells showing membrane staining pattern (0 to 100) and intensity of the staining (0 to 3+). The remaining markers were evaluated as density of cells, defined as the number of positive cells per area regardless of the intensity. The final score for each marker was expressed as the average score of the five areas analyzed within the tumor region. mRNA expression was determined in the tumors by Illumina microarray and correlated with the IHC score by Spearman correlation test. Results: OX-40 IHC expression was localized in the membrane of the tumor immune infiltrate (median value 271, range 56 to 1246). No statistical correlation was observed in tumor immune infiltrate with high versus low OX-40 IHC expression (by median value) and gender, age, race, smoking status, stage, EGFR and KRAS-mutation status. In contrast, high OX-40 expression levels in the tumor immune infiltrate correlated with better overall survival (OS) by IHC median score (p = 0.002) and by quartile distribution analysis (p = 0.001). This statistical association was maintained in the squamous cell carcinoma subgroup by median value (p = 0.04) and quartile distribution (p = 0.001). To understand the prognostic role of the OX-40 expression in relationship with the CD3+/CD8+ immune infiltrate, we analyzed the prognostic significance of CD3+/CD8+ with or without OX-40 positivity. The patients whose tumors showed high levels of CD3, CD8 and OX-40 IHC staining in the tumor immune infiltrate had a statistically significant better OS than the counterpart with high CD3+/CD8+ but low OX-40 expression (p = 0.007). High OX-40 IHC score was associated with increased expression of immune-related genes such as CD3, INFgamma, ICOS, CD8, CCL5, EOMES, granzyme B, BTK (p&amp;lt;0.001). Conclusion: High OX-40 IHC expression in the tumor immune infiltrate has a strong prognostic association with better OS in patients with surgically resected stage I-III NSCLC. Our study suggests the potential for OX-40 agonistic antibodies, currently in clinical development for NSCLC, to enhance the efficacy of existing checkpoint inhibition therapies. (This study was supported by the Immunotherapy Platform at M.D. Anderson Cancer Center). Citation Format: Erminia Massarelli, Edwin R. Parra, Jaime Rogriguez-Canales, Carmen Behrens, Lixia Diao, Jorge Blando, Jing Wang, Lauren A. Byers, Ignacio I. Wistuba, Padmanee Sharma, James P. Allison, John V. Heymach. High OX-40 expression on the tumor immune infiltrate is a prognostic factor of better survival in non-small cell lung cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A084.

Abstract 2225: Image analysis-based PD-L1 companion and complementary diagnostics

Abstract The PD-1 pathway, comprised of the immune cell co-receptor Programmed Death 1 (PD-1) and its ligands PD-L1 and PD-L2, mediates local immunosuppression in the tumor microenvironment. Immune checkpoint modulators are designed to block the local immunosuppression caused by this pathway. The FDA approved anti-PD-1 antibody therapies Opdivo® (nivolumab; Bristol-Myers Squibb) and Keytruda® (prembrolizumab; Merck &amp; Co) rely on PD-L1 immunohistochemistry (IHC) in vitro diagnostic (IVD) tests to determine the PD-L1 status in patients in non-small cell lung cancer (NSCLC), in order to predict response to these drugs. The current complementary diagnostic for Opdivo® (Dako 28-8 PharmDx®) relies on a pathologist scoring paradigm which considers any patient with ≥1% positive tumor cells an optimal candidate for Opdivo® treatment. However, overall survival (OS) is further increased when patients have ≥5% or ≥10% PD-L1 positive tumor cells. This scoring approach is vastly different than the PD-L1 scoring approach used in the Keytruda® companion diagnostic (Dako 22C3 PharmDx®), which utilizes a ≥50% positive tumor cells value to predict a positive Overall Response Rate (ORR; OS not yet determined). Thus, the 28-8 test for Opdivo® utilizes a more precise approach than the 22C3 test for Keytruda®, and requires a more calibrated scoring approach. This calibrated approach for Opdivo® requires the difficult challenge of pathologists reliably distinguishing membrane staining to define the fine gradations of 1%, 5% and 10% PD-L1 positive neoplastic cells. To best meet this challenge, we developed a digital Tissue Image Analysis (TIA) solution which enabled accurate, unbiased quantification of PD-L1 on a cell-by-cell basis to classify the percentage positive tumor cells in patients with high granularity. Using Flagship's proprietary CellMapTM algorithm, we evaluated 40 formalin-fixed paraffin-embedded (FFPE) NS-NSCLC samples which were stained using the Dako 28-8 PharmDx® PD-L1 IHC test. The TIA strategy digitally separated tumor cells from other cell types, and quantified membrane staining intensity according to a consistent threshold. The performance of the resulting IHC-TIA assay was evaluated in the context of a CLIA validation study performed by Flagship. The results demonstrated equivalency to the manually scored IVD reference standard; however, the TIA scoring of this assay provided consistent, unbiased, and more detailed scoring of PD-L1 stained tissues for determining the patients with ≥1, ≥5, and ≥10% PD-L1 positive tumor cells with greater confidence than a manual scoring approach. Moving forward, these TIA tools can be utilized to assess PD-L1 positive cell frequencies with greater reliability and granularity to identify optimal treatment cutpoints for these and other PD-L1 IHC tests used to predict response to PD-L1/PD-1 inhibitors. Citation Format: Joseph S. Krueger, Roberto Gianani, Brooke Hirsch, Stefan Pieterse, Famke Aeffner, David Young. Image analysis-based PD-L1 companion and complementary diagnostics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2225.

Mechanistic and Pharmacodynamic Studies of Adct-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies

ADCT-301, currently in Phase I clinical trial, is an ADC composed of a recombinant human IgG1, HuMax®-TAC against human IL-2R-α (CD25) conjugated through a cleavable linker to a PBD dimer warhead with a drug-antibody ratio of 2.3. In vitro and ex vivo, ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and targeted cytotoxicity against a panel of human lymphoma cell lines. On release, PBD dimers bind in the DNA minor groove and exert their cytotoxic action via the formation of DNA interstrand cross-links. In vivo, ADCT-301 demonstrates dose-dependent antitumor activity against subcutaneous and disseminated lymphoma models. For example, in the Karpas 299 xenograft model, 10/10 tumor-free survivors are observed following a single dose of 0.5 mg/kg, whereas Adcetris® gives only a modest delay in mean tumor growth at 0.5 mg/kg, despite this tumor expressing three-fold higher target antigen levels for this drug. The current study aimed to further define the mechanism of action of ADCT-301 and validate pharmacodynamic assays for clinical development.In Karpas 299 cells, evidence for internalization of ADCT-301 was shown by a reduction of CD25 molecules on the cell surface over the first three hours post-treatment followed by a return to pre-treatment levels by 16 hours. This is consistent with the documented rapid recycling of CD25 to the membrane after exposure to IL-2 (Hemar et al Journal of Cell Biology 1995). Furthermore, ADCT-301 on the cell surface declined by >70% over four hours. Following a two-hour exposure to ADCT-301, DNA interstrand cross-linking, measured using a modification of the single cell gel electrophoresis (comet) assay, reached a peak between 4 and 8 hours after which cross-links persisted up to 36 hours. In contrast, the peak of cross-link formation for an equimolar concentration of warhead was immediately following drug exposure and a non-targeted PBD-containing ADC did not produce crosslinks in these cells. A strong correlation (r = 0.97) between loss of viability and DNA cross-link formation provides support for this DNA damage being the critical initiating mechanism of cytotoxicity of ADCT-301.We have previously shown that PBD-induced DNA interstrand cross-links elicit a robust, but delayed γ-H2AX response (Wu et al Clinical Cancer Research 2013). In Karpas 299 cells phosphorylation of H2AX was observed 24 hours after a two-hour exposure to sub-GI50 concentrations of ADCT-301. In these cells continuous exposure to ADCT-301 resulted in a dose-dependent G2/M arrest, peaking at 48 hours, later than for the naked warhead. The peak of the early apoptosis marker annexin-V on the cell surface of Karpas 299 cells was observed between 60 and 72 hours and maximal loss of viability was at 96 hours.Significant bystander killing of CD25-negative human Burkitt's lymphoma-derived Ramos cells was demonstrated for ADCT-301 both by co-culture experiments with CD25-positive Karpas 299 cells, and by media transfer from Karpas 299 cells treated with ADCT-301. This is important as many lymphomas are heterogeneous in their CD25 expression profile (Strauchen et al American Journal of Pathology 1987).In SCID mice with Karpas 299 subcutaneous tumors a single dose of ADCT-301 was administered at 0.2 or 0.6 mg/kg. 24 hours after treatment, excised tumors showed a dose proportional increase in intensity of membrane and cytoplasmic staining by an anti-PBD payload antibody. Cross-linking was determined as 23% (0.2 mg/kg) vs 49% (0.6 mg/kg) (p ≤ 0.01) reduction in Tail Moment using the comet assay and dose-dependent γ-H2AX formation measured by immunohistochemistry was observed. No cross-linking was observed in matched lymphocyte samples.These data confirm the mechanism of cell killing of ADCT-301 and provide relevant pharmacodynamic assays for use in the clinical development of PBD-based ADCs. DisclosuresFlynn:Spirogen/Medimmune: Employment. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zammarchi:ADC Therapeutics: Employment. Tyrer:Spirogen/Medimmune: Employment. Williams:Spirogen/Medimmune: Employment. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Abstract 1686: CD70 as a target in non-Hodgkin lymphoma (NHL) and renal cell carcinoma (RCC)

Abstract Background CD70 is a member of the TNF superfamily expressed primarily on activated lymphocytes. CD70 interacts with CD27 to regulate B and T cell functions. Among normal, nonlymphoid tissues, CD70 is only expressed on stromal cells of the thymic medulla and mature dendritic cells. CD70 expression in RCC and NHL highlights its potential as a target for cancer therapy. Methods An indirect immunohistochemistry (IHC) assay was developed using an anti-CD70 mouse monoclonal antibody (Ryan 2010), and validated in a CLIA-compliant laboratory. Control cell lines had receptor numbers and IHC staining intensities of ∼190,000 (3+), ∼21000 (3+), ∼4000 (1+) and 0 (0). Archived tumor tissue from consenting patients with relapsed/refractory (RR) NHL or metastatic RCC were stained and read by a board-certified pathologist at Quest Diagnostics, reporting the percentage of cells staining for CD70, intensity, and subcellular localization of the stain. Results The rates and homogeneity of combined membrane and non-membrane CD70 staining intensity ≥1+ and 3+ for each tumor type are shown in the table below. The majority of NHL (88%) and RCC (80%) samples exhibited membranous CD70 staining ≥1+; &amp;gt;75% exhibited 3+ staining. Among the NHL and RCC subtypes sampled, high levels of 3+ intensity were observed in diffuse large B cell lymphoma (DLBCL; N = 44, 84%), follicular lymphoma (FL; N = 26, 77%), mantle cell lymphoma (MCL; N = 6, 67%), clear cell RCC (ccRCC; N = 138, 93%), and papillary RCC (pRCC; N = 15, 67%). No chromophobe RCC samples showed membranous staining (N = 5). CD70 expression in NHL and RCC tumor samples% cells in sample expressing CD70Samples positive for membrane and non-membrane CD70 expression, n (%)NHLRCCDLBCL, N = 44Other NHL, N = 41Clear Cell, N = 138Other RCC, N = 32&amp;gt;/ = 1+ intensity3+ intensity&amp;gt;/ = 1+ intensity3+ intensity&amp;gt;/ = 1+ intensity3+ intensity&amp;gt;/ = 1+ intensity3+ intensity02 (5)7 (16)6 (15)12 (29)4 (3)9 (7)11 (34)14 (44)1-209 (20)10 (23)11 (27)17 (41)12 (9)26 (19)7 (22)7 (22)21-404 (9)6 (14)8 (20)3 (7)14 (10)19 (14)2 (6)3 (9)41-604 (9)3 (7)3 (7)4 (10)10 (7)18 (13)2 (6)5 (16)61-807 (16)7 (16)5 (12)1 (2)12 (9)26 (19)4 (13)2 (6)81-10018 (41)11 (25)8 (20)4 (10)86 (62)40 (29)6 (19)1 (3) Conclusions CD70 is expressed in the majority of DLBCL, FL, MCL, ccRCC, and pRCC samples tested. Based on the unmet medical needs of these diseases, limited expression of CD70 on normal tissues, predominant membrane expression of CD70, and observed preclinical antitumor activity (Jeffrey 2013), a CD70-targeted antibody-drug conjugate, SGN-CD70A, has been advanced into clinical development in CD70-positive RR NHL and metastatic RCC (NCT# NCT02216890). Citation Format: Kenneth W. Wood, Elaina M. Gartner, Mechthild Jonas, Maureen Ryan, Dana A. Kennedy. CD70 as a target in non-Hodgkin lymphoma (NHL) and renal cell carcinoma (RCC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1686. doi:10.1158/1538-7445.AM2015-1686

Sodium Iodide Symporter Expression in Adenoid Cystic Carcinoma of the Head and Neck.

Sodium iodide symporter (NIS) expression in adenoid cystic carcinoma (ACC) has not been fully elucidated in the literature, and it is unclear whether radioactive iodine may be a potential therapeutic modality. To our knowledge, the present study includes the largest ACC tumor sample size to evaluate for NIS expression. To assess whether ACC of the head and neck expresses NIS by using immunohistochemical staining techniques, as well as assess whether the presence or intensity of staining correlates with tumor or patient variables. Immunohistochemical analysis of NIS expression was performed on 20 ACC specimens from various head and neck subsites obtained from January 1, 1988, to May 6, 2013, at a single academic tertiary care medical center. Staining intensity was graded on a scale of 0 to 3+ (higher numbers indicate greater staining intensity) and was analyzed according to multiple patient and tumor variables. Tumors were eliminated from the study if the patient had undergone prior surgical resection, chemotherapy, or radiotherapy, or was receiving thyroid hormone supplementation at the time of the operation. Presence or absence of staining was the primary outcome measured; secondary outcomes were the intensity and localization of staining. Sodium iodide symporter staining was positive in 15 of the 20 tumor specimens (75%). Staining was largely localized to the cytoplasm and was of low intensity. There was no significant association between the presence or intensity of staining and the tumor subtype, tumor location, or any of the patient variables assessed (P > .05). No association between staining intensity and tumor growth pattern was shown on χ² analysis: 1+ (P = .53), 2+ (P = .14), or a combination of 1+ and 2+ staining (P = .64). Parotid control tissue demonstrated intense membranous staining of the striated parotid gland ducts. Sodium iodide symporter was expressed in the cytoplasm with low intensity in most of the tumor specimens examined in this study. These staining characteristics are also commonly found in thyroid cancer cells. Further investigation is required to determine the significance of this finding. We are optimistic that future studies using endogenous NIS stimulation and identification of genes associated with NIS plasma membrane localization could be applied to the treatment of ACC with radioactive iodine techniques.

P8.02 - Overexpression of PD-1 and PD-L1 in renal cell carcinoma is associated with poor prognosis in metastatic patients treated with sunitinib

Anti vascular growth epithelial factor (VEGF) therapies are currently used in first line of metastatic clear cell renal cell carcinoma (ccRCC), but some patients are inherently resistant to these treatments. Immunotherapy based on disruption of immune checkpoints, such as Programmed Death 1 (PD-1) and Programmed Death Ligand 1 (PD-L1), has been showed promising results. PD-1 and PD-L1 are mainly expressed by T-Cells and tumor cells respectively. Interaction between PD-1 and PD-L1 down-regulates antitumor activity and could be involved in resistance to anti-VEGF therapies. This study assessed PD-1 and PD-L1 expressions in primary ccRCC of metastatic patients with sunitinib first-line treatment and its correlation with clinical outcomes. Formalin-fixed paraffin samples were obtained from primary ccRCC before any systemic treatment. Prognostic pathological criteria and clinical data were collected with a median follow up of 27 months after introduction of sunitinib. PD-1 and PD-L1 expressions were evaluated by immunohistochemistry on the most inflammatory and the highest Fuhrman nuclear grade areas respectively, considering tumor heterogeneity. Kaplan Meier survival curves were compared by log rank test. PD-1 and PD-L1 were considered overexpressed when at least a moderate to marked density of positive immune cells and at least 30% of tumor cells were positive with moderate to strong membranous staining intensity, respectively. Overexpression of both PD-1 and PD-L1 was observed in a subgroup of 17/47 primary ccRCC (36%). Patients in this subgroup compared to others had a worse progression free survival (p = 0.001) with medians of 9 months and 14 months respectively. Even if statistical significance was not reached, there was also a trend toward a worse median overall survival (18 months versus 32). This is the first study to assess the PD-1 and PD-L1 expressions in primary ccRCC of metastatic patients with sunitinib first-line treatment. The subgroup with both PD-1 and PD-L1 overexpression should be individualized as having a peculiar immune phenotype. These patients experienced poor prognosis when treated by sunitinib and may be good candidates from anti-PD-1 or anti-PD-L1 immunotherapies.

Evaluation of immunohistochemistry HER2 results interpretation in invasive micropapillary carcinoma of the breast

To evaluate the standards of HER2 immunohistochemistry (IHC) interpretation in invasive micropapillary carcinoma of the breast (IMPC). HER2 expression in 60 cases of IMPC was evaluated by IHC and fluorescence in situ hybridization (FISH) using TMA-based techniques. The characteristics between cases with HER2 IHC and HER2 gene amplification results were compared. Using 2007 American Society of Clinical Oncology/College of American Pathologist (ASCO/CAP) criteria, among the 52 cases that were successfully stained by IHC, 40 were HER2 IHC negative and 12 were equivocal (2+). Fifteen cases of HER2 IHC 0 were negative for amplification by FISH. Twenty-five cases with IHC 1+ were tested by FISH; and of these, one showed HER2 amplification, 2 were equivocal, and the others were not amplified. All cases of IHC 2+ showed HER2 amplification by FISH. IHC staining of HER2 was located at cell-cell membrane or basolateral membrane of micropapillary structure, but not in the cytoplasmic membrane facing the stroma in all 13 cases which were HER2 amplified, including 12 showing very strong staining and one showing moderate staining. Among the 37 non amplified HER2 cases, 22 showed IHC staining at cell-cell membrane or basolateral membrane (including 15 weak staining and 7 moderate staining). HER2 IHC detection in IMPC is characterized by staining at cell-cell membrane or basolateral membrane of the micropapillary structure, and absence of staining in the cytoplasmic membrane. It is suggested that interpretation of HER2 IHC staining should be based on membrane staining intensity, but not the completeness of the membrane staining in IMPC. It is suggested to determine the HER2 gene amplification status by using FISH when IHC staining shows moderate or strong intensity.

HER2 immunohistochemical assessment with A0485 polyclonal antibody: is it time to refine the scoring criteria for the "2+" category?

An accurate determination of human epidermal growth factor receptor 2 (Her2) status in women with breast cancer is mandatory to identify patients who will benefit from trastuzumab-based therapy. Her2 immunohistochemical analysis (IHC) (performed with A0485 polyclonal antibody) on 943 invasive breast cancer cases was evaluated independently and blindly twice by 3 of us (V.A., I.P., and A.C.) according to DAKO scoring criteria. A total of 230 cases of invasive breast cancer scored 2+ at IHC and consequently evaluated by FISH were reviewed first independently, and then simultaneously by 3 of us (V.A., I.P., and A.C.) at a multiheaded microscope assessing the following parameters: overall signal intensity, granularity and continuity of membrane staining, and the presence of band-like membrane pattern in >25% of tumor cells. The frequencies of HER2 gene amplification for all the immunohistologic parameters (individually considered or in combination) were compared by Pearson χ analysis. Combinations of staining patterns did not give any statistically significant results, except when combining strong staining intensity and continuity of membrane signal. In fact, only 9 of the 86 cases with a weak-to-moderate staining intensity, which showed a fragmented membrane signal, resulted in being amplified by FISH, whereas 19 of the 51 cases presenting an overall strong IHC reaction and some extent of continuous membrane signal were FISH amplified (P=0.002). Combined intensity and linearity of membrane signal, although limited, resulted in the best aid (P=0.0002) in making the final score decision in borderline IHC Her2 tests similar to what is envisaged in the Her2 scoring system for gastric cancer.