p21 Protein Outperforms Clinico-pathological Criteria in Predicting Liver Metastases in Pancreatic Endocrine Tumors.

BACKGROUND/AIM: P21 is a cyclin-dependent kinase inhibitor regulating the cell cycle as a tumor suppressor. Using a p21 immunohistochemistry (IHC) assay, we compared tumor p21 levels with conventional clinico-pathological criteria in primary pancreatic endocrine tumor subsets with and without liver metastases. MATERIALS AND METHODS: Sections from tissue microarray (TMA) including 13 archival metastatic primary and 18 non-metastatic primary pancreatic endocrine carcinomas/tumors (MP-PECAs/NMP-PETs) were stained with a monoclonal anti-p21WAFI,CIP primary antibody. Tumor p21 IHCs were scored as the sum of intensity (0-3) and proportion scores (0-5) (Total Allred score: 0-8), and as p21% labelling index in the tumor. ROC curve analysis was used for most optimal p21 score cut-off (4 or >) and Fisher's exact test was used to compare the association among tumor p21 scores, conventional prognostic criteria, and liver metastases. RESULTS: For PET/PECA patients, mean ages were 55.6 years (27-73) and 49.3 years (28-71), M/F ratios were 7/11 and 7/6. Mean p21 labelling index (%) for MP- PECAs was 24% (range=3-63%) vs. 9% for NMP-PETs (range=1-25%) (p=0.022). The mean p21 index in MP-PECAs was significantly higher (24%) as compared to PIs (7%) (p=0.0047). Using a p21 Allred score of ≥4, high p21 IHC score had strong association with the presence of liver metastases (p-value <0.001). High tumor p21 IHC score had a 93% sensitivity, 68% specificity, 78% predictive accuracy, 66% positive, and 94% negative predictive values. CONCLUSION: In patients with primary PETs, p21 IHC is superior to conventional criteria in predicting presence or absence of liver metastases.

An Analysis of the Impact of COVID-19 Pandemic-related Lockdown Measures on a Large Gastrointestinal Pathology Service in the United States.

BACKGROUND/AIM: The coronavirus disease 2019 (COVID-19) pandemic prompted global recommendations to delay non-urgent endoscopic procedures to limit the spread of SARS-COV-2, but such delays had unprecedented impact on the delivery of healthcare. Being a large specialty GI Pathology service, we sought to analyze the effect of the pandemic on the frequency of GI malignancies in our department. PATIENTS AND METHODS: Based on the electronic search of departmental pathology records, we compared the total numbers of cancer diagnoses (primary and metastatic) from various GI biopsy sites during the 12-month pre- and post-pandemic periods. We summarized patient demographics and analyzed pertinent histopathologic data. RESULTS: For all GI biopsy sites, the number of intramucosal/invasive malignancies reported during the one-year pre-COVID-19 pandemic (pre-COVID) and post-COVID-19 pandemic national lockdown (post-COVID) observation periods were 146 and 218, respectively. Among these, 32 and 70 malignancies were reported for the first quarter (representing the earliest post-lockdown period), 29 and 53 for the second, 41 and 54 for the third, and 44 and 41 for the fourth quarter. During the first two quarters of the post-COVID observation period, the increase in malignant diagnoses was most profound, showing 119% post-COVID increase compared to the pre-COVID levels. Of the two main primary histologic types of large intestinal carcinomas [adenocarcinoma (ADC) and squamous cell carcinoma (SCC)], the most profound post-COVID increase was noted in SCCs (136% vs. 58% for ADCs). CONCLUSION: Compared to the pre-pandemic baseline, the COVID-19 pandemic caused a major increase in biopsy diagnoses of GI cancers in our department. The most plausible explanations for this trend include inevitable lockdowns to minimize the spread of SAR-COV2, which affected GI endoscopy procedure schedules/re-schedules as well as patient response and adaptation to emerging post-COVID GI healthcare patterns. The COVID-19 pandemic's long-term impact on the health of GI cancer patients will need to be determined through systematic analyses by multi-disciplinary teams.

Expression of DNA Mismatch Repair Proteins, PD1 and PDL1 in Barrett's Neoplasia.

BACKGROUND/AIM: Cancers with a microsatellite instability-high (MSI-H) or deficient mismatch repair (dMMR) status respond to immune checkpoint inhibition (ICI). Regardless of the tumor type, MSI-H/dMMR status is a reliable biomarker for ICI responsiveness. This study aimed at determining the MSI-H status in precursor lesions to esophageal adenocarcinoma (EAC) such as Barrett's esophagus (BE) and BE with either low-grade dysplasia (LGD) or high-grade dysplasia (HGD). PATIENTS AND METHODS: We performed immunohistochemical staining (IHC) for PMS2, MSH6, PD1, and PD-L1. RESULTS: All cases of BE (50), LGD (48), and HGD (50) had intact PMS2 and MSH6 nuclear expression; were negative for PD1; and had a PD-L1 combined positive score (CPS) score <1. One EAC case (2%) was negative for PMS2 nuclear expression. One HGD case (2%) and two EAC cases (4%) were PD1 positive (CPS score <1 applied to PD1). One EAC case (2%) had a CPS score >1, and one EAC case (2%) was MSI-H. MSI-H tumors usually show PD-L1 expression, although the MSI-H EAC in this study had a PD-L1 CPS score of <1. CONCLUSION: Further studies investigating EAC and its precursor lesions for PD1, PD-L1, and dMMR status may be informative regarding the immunogenicity of the evolution of EAC.

Clinicopathologic analysis of primary gastroenteropancreatic poorly differentiated neuroendocrine carcinoma; A ten year retrospective study of 68 cases at Moffit Cancer Center.

OBJECTIVE: To assess clinicopathological characteristics of primary gastro-entero-pancreatic poorly differentiated neuroendocrine carcinomas (GEP-PDNECAs) and evaluate overall survival in patients treated with systemic platinum and etoposide therapy. METHODS: A detailed retrospective review of clinico-pathologic data (1999-2009) on 68 consecutive adult patients with primary GEP-PDNECAs was carried out, from H Lee Moffit Cancer Center and Research Institute, Tampa, Florida; USA, based on electronic patient records, specialty consultation files, tumor registry, social security index and pathology archives. All available tumor slides were reviewed and subtyped by neuro-endocrine pathologists. Clinicopathologic data and patient survival were analyzed. RESULTS: Of 68 patients 41 were males and 27 females with a mean age of 42 years (range: 25-76 years). Regarding the site of origin, 39 patients were of the colorectal location, 19 from the pancreas, 04 from small intestines, 03 from stomach and 03 were multi-focal from colon, small intestine and pancreas. Sixty three of 68 (93%) patients presented with lymph node/distant metastases. Of 68 tumors 37 (54%) were classified as small cell carcinoma (SCCA), 16 (24%) large cell carcinoma (LCCA), 5 (7%) mixed small and large cell (MSLCCA) and 10 (15%) poorly differentiated carcinoma with neuroendocrine features (PDCA-NEF). Neuroendocrine differentiation was confirmed by positivity for chromogranin in 38/65 (55%), synaptophysin in 62/67 (92%) and CD56 in 17/21 (81%) cases. One neuroendocrine marker was positive in 22/68 (32%), 2 in 40/68 (59%) and all 3 were positive in 9/68 (13%) cases. Fifty-eight of 68 (85%) patients were treated with platinum and etoposide. Overall patient survival at 1, 3 5 and 10 years was 85%, 40%, 16% and 1.5% respectively. Patient survival was independent of age (r= 0.1022), sex (r= -0.909) and histologic tumor subtype (r=0.1028) (p= 0.128) but was related to distant metastases (r=0.306; p=0.0383). CONCLUSIONS: GEP-PDNECA occurred in many part of the GI tract, most commonly in the colorectal region. Positivity of neuroendocrine markers was variable, which helped to confirm neuro-endocrine differentiation and to avoid under-diagnosis of GEP-PDNECA, especially in metastatic setting. Overall prognosis of GEP-PDNECA patients following platinum and etoposide therapy in our series was relatively favorable but remained poor in the presence of distant metastases.

Cross-Platform Comparison of Computer-assisted Image Analysis Quantification of In Situ mRNA Hybridization in Investigative Pathology.

Although availability of automated platforms has proliferated, there is no standard practice for computer-assisted generation of scores for mRNA in situ hybridization (ISH) visualized by brightfield microscopic imaging on tissue sections. To address this systematically, an ISH for peptidylprolyl isomerase B (PPIB) (cyclophilin B) mRNA was optimized and applied to a tissue microarray of archival non-small cell lung carcinoma cases, and then automated image analysis for PPIB was refined across 4 commercially available software platforms. Operator experience and scoring results from ImageScope, HALO, CellMap, and Developer XD were systematically compared with each other and to manual pathologist scoring. Markup images were compared and contrasted for accuracy, the ability of the platform to identify cells, and the ease of visual assessment to determine appropriate interpretation. Comparing weighted scoring approaches using H-scores (Developer XD, ImageScope, and manual scoring) a correlation was observed (R value=0.7955), and association between the remaining 2 approaches (HALO and CellMap) was of similar value. ImageScope showed the highest R value in comparison with manual scoring (0.7377). Mean-difference plots showed that HALO produced the highest relative normalized values, suggesting higher relative sensitivity. ImageScope overestimated PPIB ISH signal at the high end of the range scores; however, this tendency was not observed in other platforms. HALO emerged with the highest number of favorable observations, no apparent systematic bias in score generation compared with the other methods, and potentially higher sensitivity to detect ISH. HALO may serve as a tool to empower teams of investigative pathology laboratory scientists to assist pathologists readily with quantitative scoring of ISH.

Efficacy and safety of ramucirumab-containing chemotherapy in patients with pretreated metastatic gastric neuroendocrine carcinoma.

BACKGROUND: Ramucirumab (RAM), a monoclonal antibody for vascular endothelial growth factor 2 (VEGFR2), has been effective for advanced gastric adenocarcinoma (AC). However, little is known about the efficacy of RAM-containing chemotherapy (RAM-CTx) in gastric neuroendocrine carcinoma (G-NEC). METHODS: We retrospectively analysed and compared the clinical outcomes of patients (pts) with G-NEC receiving RAM-CTx, G-NEC receiving CTx without RAM and AC receiving RAM-CTx in our hospital. G-NEC was defined by neuroendocrine carcinoma features, regardless of the proportion, based on histology and neuroendocrine markers (synaptophysin, chromogranin A or CD56). VEGFR2 expression in tumour vessels was evaluated in archival primary G-NEC tissues by immunohistochemistry using the same anti-VEGFR2 primary antibody and scoring scheme (vascular VEGFR2 H-score) as in the REGARD trial. RESULTS: Seventeen G-NEC receiving RAM-CTx, 13 G-NEC receiving CTx without RAM and 173 AC pts receiving RAM-CTx were analysed. The overall response rate (59% vs 8 % vs 28%), progression-free survival (median 7.7 vs 1.8 vs 3.3 months) and overall survival (median 16.1 vs 8.6 vs 9.6 months) were significantly better in pts with G-NEC receiving RAM-CTx than G-NEC receiving CTx without RAM or AC receiving RAM-CTx. No severe or unexpected adverse events occurred. The median vascular VEGFR2 H-score, based on available G-NEC tissues from 12 pts receiving RAM-CTx, was 220 (range 150-260), which was markedly higher than that reported on AC tissues from the REGARD trial as historical control (median 35, range 0-240). CONCLUSIONS: RAM-CTx showed a promising activity without severe or unexpected safety profile in pts with G-NEC. This may in part be explained by higher vascular VEGFR2 expression in G-NEC tissues.

Differential expression of VEGFR2 protein in HER2 positive primary human breast cancer: potential relevance to anti-angiogenic therapies.

BACKGROUND: Clinically relevant predictive biomarkers to tailor anti-angiogenic therapies to breast cancer (BRC) patient subpopulations are an unmet need. METHODS: We analyzed tumor vascular density and VEGFR2 protein expression in various subsets of primary human BRCs (186 females; Mean age: 59 years; range 33-88 years), using a tissue microarray. Discrete VEGFR2+ and CD34+ tumor vessels were manually scored in invasive ductal, lobular, mixed ductal-lobular and colloid (N = 139, 22, 18, 7) BRC cores. RESULTS: The observed CD34+ and VEGFR2+ tumor vascular counts in individual cases were heterogeneous. Mean CD34+ and VEGFR2+ tumor vessel counts were 11 and 3.4 per tumor TMA core respectively. Eighty-nine of 186 (48%) cases had >10 CD34+ tumor vessels, while 97/186 (52%) had fewer CD34+ vessels in each TMA core. Of 169 analyzable cores in the VEGFR2 stained TMA, 90 (53%) showed 1-5 VEGFR2+ tumor vessels/TMA core, while 42/169 (25%) cores had no detectable VEGFR2+ tumor vessels. Thirteen of 169 (8%) cases also showed tumor cell (cytoplasmic/membrane) expression of VEGFR2. Triple-negative breast cancers (TNBCs) appeared to be less vascular (Mean VD = 9.8, range 0-34) than other breast cancer subtypes. Overall, VEGFR2+ tumor vessel counts were significantly higher in HER2+ as compared to HR+ (p = 0.04) and TNBC (p = 0.02) tissues. Compared to HER2- cases, HER2+ breast cancers had higher VEGFR2+ tumor vessel counts (p = 0.007). CONCLUSION: Characterization of pathologic angiogenesis in HER2+ breast cancer provides scientific rationale for future investigation of clinical activity of agents targeting the VEGF/VEGFR2 axis in this clinically aggressive breast cancer subtype.

Phase 1 study of narnatumab, an anti-RON receptor monoclonal antibody, in patients with advanced solid tumors.

Purpose Macrophage-stimulating 1-receptor (RON) is expressed on macrophages, epithelial cells, and a variety of tumors. Narnatumab (IMC-RON8; LY3012219) is a neutralizing monoclonal antibody that blocks RON binding to its ligand, macrophage-stimulating protein (MSP). This study assessed safety, maximum tolerated dose (MTD), pharmacokinetics, pharmacodynamics, and efficacy of narnatumab in patients with advanced solid tumors. Methods Narnatumab was administered intravenously weekly at 5, 10, 15, or 20 mg/kg or every 2 weeks at 15, 20, 30, or 40 mg/kg in 4-week cycles. Results Thirty-nine patients were treated, and 1 dose-limiting toxicity (DLT) (grade 3 hyponatremia, 5 mg/kg) was reported. The most common narnatumab-related adverse events (AEs) were fatigue (20.5%) and decreased appetite, diarrhea, nausea, and vomiting (10.3% each). Except for 2 treatment-related grade 3 AEs (hyponatremia, hypokalemia), all treatment-related AEs were grade 1 or 2. Narnatumab had a short half-life (<7 days). After Cycle 2, no patients had concentrations above 140 µg/mL (concentration that demonstrated antitumor activity in animal models), except for 1 patient receiving 30 mg/kg biweekly. Eleven patients had a best response of stable disease, ranging from 6 weeks to 11 months. Despite only 1 DLT, due to suboptimal drug exposure, the dose was not escalated beyond 40 mg/kg biweekly. This decision was based on published data reporting that mRNA splice variants of RON are highly prevalent in tumors, accumulate in cytoplasm, and are not accessible by large-molecule monoclonal antibodies. Conclusions Narnatumab was well tolerated and showed limited antitumor activity with this dosing regimen.

Profiling of Vascular Endothelial Growth Factor Receptor Heterogeneity Identifies Protein Expression-defined Subclasses of Human Non-small Cell Lung Carcinoma.

BACKGROUND: the vascular endothelial growth factor (VEGF) pathway plays a prominent role in the growth and progression of human cancer, including non-small cell lung carcinoma (NSCLC). The key mediators of VEGF signaling are a family of related receptor tyrosine kinases that include VEGFR1, VEGFR2, and VEGFR3. The relative expression levels, activity, and cross-talk among these receptors may contribute to response of NSCLC to anti-angiogenic therapies, and a better systematic, translatable approach to categorizing tumors is needed. MATERIALS AND METHODS: We comparatively evaluated immunohistochemical expression of the three VEGFRs in archival primary NSCLC tissues (n=96). RESULTS: VEGFR1 and VEGFR2 were localized both in vessels and tumor cells, while VEGFR3 was only localized in tumor vessels. A set of eight VEGFR staining subclasses were identified: Triple VEGFR positive (n=11, 11.5%), VEGFR1 predominant (n=22, 22.9%), VEGFR2 predominant (n=9, 9.4%), VEGFR3 predominant (n=3, 3.1%), VEGFR1/2 predominant (13, 13.5%), VEGFR1/3 predominant (2, 2.1%), VEGFR2/3 predominant (n=8, 8.3%), and triple VEGFR negative (n=28, 29.2%). An objective categorization based on K-means clustering revealed four clusters, three of which showed high VEGFR2 compared to VEGFR3 (30.7% of cases), cases high in both VEGFR2 and VEGFR3 (18.2%), and cases that were negative/low for both VEGFR2 and VEGFR3 (45.4%). A positive association between VEGFR2 and VEGFR3 was found, however no associations were observed between VEGFR1 and VEGFR2, nor VEGFR1 and VEGFR3. CONCLUSION: The proposed subclasses of NSCLC are an approach for complementing lines of investigation of anti-angiogenic therapies beginning with systematic characterization of the disease.

Heterogeneity of Vascular Endothelial Growth Factor Receptors 1, 2, 3 in Primary Human Colorectal Carcinoma.

BACKGROUND: The vascular endothelial growth factor (VEGF) pathway plays an important role in growth and progression of human cancer, including colorectal carcinomas (CRC). The key mediators of VEGF signaling are VEGFR1, VEGFR2, and VEGFR3, part of a family of related receptor tyrosine kinases. The relative expression, activity, or interplay among these receptors may determine the response of CRC patients to anti-angiogenic therapies. MATERIALS AND METHODS: We developed technically sound immunohistochemical (IHC) assays to quantify VEGFR1, 2 and 3, and using a well-annotated CRC tissue microarray (TMA), we carried out comprehensive comparative evaluation of the three VEGFRs in archival primary CRC tissues (n=84). For each TMA core, tumor cell VEGFR1 expression was reported as H-score (range=0-300); vascular VEGFR2/VEGFR3 expression was manually scored as the number of receptor-positive tumor stromal vessels. Each case was defined as VEGFR1/ VEGFR2/VEGFR3-negative, low, medium or high. RESULTS: Based on the differential expression of the three VEGFRs, eight VEGFR staining profiles were observed: Triple VEGFR positive (n=12, 14%), VEGFR1 predominant (n=17, 20%), VEGFR2 predominant (n=7, 8%), VEGFR3 predominant (n=1, 1%), VEGFR1/2 predominant (n=39, 46%), VEGFR1/3 predominant (n=2, 2%), VEGFR2/3 predominant (n=3, 4%), and triple-VEGFR-negative (n=3, 4%). CONCLUSION: Herein we demonstrated heterogeneity of expression of VEGFRs in human CRC stromal vessels and tumor cells. The observed VEGFR expression-based subsets of human CRCs may reflect differences in biology of pathologic angiogenesis in primary CRC tissues. Furthermore, the heterogeneity of expression of VEGFRs unraveled in this analysis merits independent validation in larger cohorts of primary and metastatic human CRC tissues and in pertinent experimental models treated with various anti-angiogenic therapies.

Efficacy and Safety of Abemaciclib, an Inhibitor of CDK4 and CDK6, for Patients with Breast Cancer, Non-Small Cell Lung Cancer, and Other Solid Tumors.

UNLABELLED: We evaluated the safety, pharmacokinetic profile, pharmacodynamic effects, and antitumor activity of abemaciclib, an orally bioavailable inhibitor of cyclin-dependent kinases (CDK) 4 and 6, in a multicenter study including phase I dose escalation followed by tumor-specific cohorts for breast cancer, non-small cell lung cancer (NSCLC), glioblastoma, melanoma, and colorectal cancer. A total of 225 patients were enrolled: 33 in dose escalation and 192 in tumor-specific cohorts. Dose-limiting toxicity was grade 3 fatigue. The maximum tolerated dose was 200 mg every 12 hours. The most common possibly related treatment-emergent adverse events involved fatigue and the gastrointestinal, renal, or hematopoietic systems. Plasma concentrations increased with dose, and pharmacodynamic effects were observed in proliferating keratinocytes and tumors. Radiographic responses were achieved in previously treated patients with breast cancer, NSCLC, and melanoma. For hormone receptor-positive breast cancer, the overall response rate was 31%; moreover, 61% of patients achieved either response or stable disease lasting ≥6 months. SIGNIFICANCE: Abemaciclib represents the first selective inhibitor of CDK4 and CDK6 with a safety profile allowing continuous dosing to achieve sustained target inhibition. This first-in-human experience demonstrates single-agent activity for patients with advanced breast cancer, NSCLC, and other solid tumors. Cancer Discov; 6(7); 740-53. ©2016 AACR.See related commentary by Lim et al., p. 697This article is highlighted in the In This Issue feature, p. 681.

Stromal-Based Signatures for the Classification of Gastric Cancer.

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573-86. ©2016 AACR.

Overexpression of Vascular Endothelial Growth Factor A in Invasive Micropapillary Colorectal Carcinoma.

BACKGROUND: Invasive micropapillary carcinoma (IMPC) is a rare variant of colorectal cancer with an adverse prognosis. "Retraction artifact" around tumor cells is a feature of IMPC. The aim of this study was to assess the nature of the retractions around the tumor cells and to describe the histopathological features of a group of 18 cases of IMPC. METHODS: A pathology review of 128 consecutive colorectal cancers identified 18 cases of histologically proven IMPC using 5% of the total tumor volume comprised of a micropapillary component as the diagnostic criterion. Immunostains for D2-40, CD31, CD34, vascular endothelial growth factor A (VEGF-A), and mucin 1 (MUC-1) were performed using the avidin-biotin complex method. RESULTS: Cases of IMPC were characterized by pseudomicropapillae surrounded by lacunar-like clear spaces. These structures exhibited the inside-out growth pattern as highlighted by MUC-1 staining. The lining of the lacunar spaces was immunoreactive to CD31 but not CD34 or D2-40, indicating that they are neovascular structures. Furthermore, the tumor cells strongly and diffusely expressed VEGF-A. CONCLUSIONS: The strong coexpression of VEGF-A and CD31 suggests a prominent role of neoangiogenesis in these tumors.

Tumor cell expression of vascular endothelial growth factor receptor 2 is an adverse prognostic factor in patients with squamous cell carcinoma of the lung.

A robust immunohistochemical (IHC) assay for VEGFR2 was developed to investigate its utility for patient tailoring in clinical trials. The sensitivity, specificity, and selectivity of the IHC assay were established by siRNA knockdown, immunoblotting, mass spectrometry, and pre-absorption experiments. Characterization of the assay included screening a panel of multiple human cancer tissues and an independent cohort of non-small cell lung carcinoma (NSCLC, n = 118) characterized by TTF-1, p63, CK5/6, and CK7 IHC. VEGFR2 immunoreactivity was interpreted qualitatively (VEGFR2 positive/negative) in blood vessels and by semi-quantitative evaluation using H-scores in tumor cells (0-300). Associations were determined among combinations of VEGFR2 expression in blood vessels and tumor cells, and clinico-pathologic characteristics (age, sex, race, histologic subtype, disease stage) and overall survival using Kaplan-Meier analyses and appropriate statistical models. VEGFR2 expression both in blood vessels and in tumor cells in carcinomas of the lung, cervix, larynx, breast, and others was demonstrated. In the validation cohort, 99/118 (83.9%) NSCLC tissues expressed VEGFR2 in the blood vessels and 46/118 (39.0%) showed high tumor cell positivity (H-score ≥10). Vascular and tumor cell expression were inversely correlated (p = 0.0175). High tumor cell expression of VEGFR2 was associated with a 3.7-fold reduction in median overall survival in lung squamous-cell carcinoma (SCC, n = 25, p = 0.0134). The inverse correlation between vascular and tumor cell expression of VEGFR2 and the adverse prognosis associated with high VEGFR2 expression in immunohistochemically characterized pulmonary SCC are new findings with potential therapeutic implications. The robustness of this novel IHC assay will support further evaluation of its utility for patient tailoring in clinical trials of antiangiogenic agents.

Cytoplasmic Clusterin expression correlates with pancreatic neuroendocrine tumor size and pathological stage.

OBJECTIVES: Cytoplasmic clusterin (Clusterin), a ubiquitous multifunctional secretory sulfated glycoprotein, plays a role in apoptosis and is reportedly overexpressed in a variety of tumors. The role of Clusterin in pancreatic neuroendocrine tumors (PNETs) has not been investigated. In this study, Clusterin expression was evaluated in a subset of PNETs, and the results were correlated with the clinical-pathological features of the tumors. METHODS: Fifty-nine surgical cases were used to evaluate the immunohistochemical expression of Clusterin in PNETs. Using the avidin-biotin complex method, tissue sections from each case were stained with a rabbit anticlusterin antibody (Abcam, Cambridge, Mass). The immunohistochemical reactions were qualitatively and semiquantitatively evaluated by 2 pathologists. RESULTS: Strong Clusterin reactivity was identified in 36 (61%) of 59 PNETs. In 23 (39%) of 59 cases, the Clusterin score was 3 or less. Clusterin expression scores significantly associated with tumor size (P = 0.03) and with tumor stage (P = 0.02). The immunohistochemical score index did not correlate with tumor grade (P = 0.15). CONCLUSIONS: We report the expression of Clusterin in PNETs. The correlation of Clusterin with tumor size and stage suggests involvement of this molecule in pancreatic neuroendocrine tumor progression. Clusterin may represent a new target of therapy for PNETs.

A phase 1 dose escalation, pharmacokinetic, and pharmacodynamic evaluation of eIF-4E antisense oligonucleotide LY2275796 in patients with advanced cancer.

PURPOSE: The antisense oligonucleotide LY2275796 blocks expression of cap-binding protein eukaryotic initiation factor 4E (eIF-4E), an mRNA translation regulator upregulated in tumors. This phase I study sought an appropriate LY2275796 dose in patients with advanced tumors. EXPERIMENTAL DESIGN: A 3-day loading dose, then weekly maintenance doses, were given to 1 to 3 patient cohorts, beginning with 100 mg and escalating. Plasma samples were collected to determine LY2275796 concentrations and tumor biopsies to quantify eIF-4E mRNA/protein. RESULTS: Thirty patients with stage 4 disease received 1 or more LY2275796 dose. A dose-limiting toxicity was observed at 1,200 mg, with 1,000 mg the maximum-tolerated dose. Across all dose levels, most patients (87%) had only grade 1 to 2 toxicities. LY2275796 pharmacokinetics supported the dosing regimen. Comparison of pre- and postdose biopsies showed eIF-4E decreased in most patients. Fifteen patients had progressive disease, and 7 patients achieved stable disease (minimum of 6 weeks) as best response, with 2 patients on therapy for more than 3 months (one with melanoma, one with cystadenocarcinoma of the head/neck). CONCLUSIONS: LY2275796 was well tolerated up to 1,000 mg. Because tumor eIF-4E expression was decreased, but no tumor response observed, LY2275796 should be studied combined with other treatment modalities.

Palladin is a marker of liver metastasis in primary pancreatic endocrine carcinomas.

BACKGROUND: Palladin is a metastasis-associated gene regulating cell motility. The expression of palladin protein in pancreatic neuroendocrine tumors (PET) and carcinomas (PECA) is not known. MATERIALS AND METHODS: A tissue microarray (TMA) of well-differentiated (WD) PETs/PECAs (AJCC 2010) and non-neoplastic, histologically normal pancreatic tissue/islets (HNPIs) was immunostained with palladin antibody and quantified using the Allred score. The results were correlated with the presence or absence of liver metastases. RESULTS: The retrospective study included 19 males and 19 females of age 27-79 years (mean 54). Tumor size was 0.9-11.5 cm (mean 3.8). Palladin expression was cytoplasmic and/or membranous. The tumors with high palladin expression were associated with liver metastasis (p<0.0001). All 14 primary PECA with hepatic metastases (MP-PECAs) exhibited palladin expression whereas 14 out of 24 (58%) clinically-localized primary PET (CLP-PETs) expressed palladin (p<0.01) with median Allred scores of 5 (range 3-7) and 2 (range 0-6) respectively (p<0.0001). The mean Allred score for the HNPIs in the MP-PECAs (N=6) was higher (4.2) as compared to that in the CLP-PETs (2.5,N=11) (p=0.23). CONCLUSION: Palladin may identify primary pancreatic endocrine neoplasms with a propensity to metastasize to the liver.

Ischemic time impacts biological integrity of phospho-proteins in PI3K/Akt, Erk/MAPK, and p38 MAPK signaling networks.

Post-translational modifications of proteins, such as phosphorylation, are labile events dynamically regulated by opposing kinase and phosphatase activities. Preanalytical factors, such as ischemic time before fixation, affect these activities and can have a significant impact on the ability to elucidate signaling pathways in tissue. Immunohistochemical analysis of phosphorylated proteins involved in PI3K/Akt, Erk/MAPK, and p38 MAPK signaling networks was performed in human cell line xenografts from lung, brain, ovary, and prostate tumors. In order to replicate real-world practices, the tissues were subjected to ischemic times of 0 (baseline), 1, 4, and 24 hours before fixation in formalin. Two key concepts emerge from this analysis: (1) the stability of different phospho-epitopes within a given tumor type is variable (e.g. phospho-PRAS40 is more labile than phospho-S6 ribosomal protein) and (2) the stability of a given phospho-epitope (e.g. phospho-MAPKAPK2) varies significantly across different tumor types. These results highlight the importance of proper tissue acquisition and rapid fixation to preserve the biological integrity of signal transduction pathways that may guide therapeutic decision making.

Novel molecular markers of malignancy in histologically normal and benign breast.

To detect the molecular changes of malignancy in histologically normal breast (HNB) tissues, we recently developed a novel 117-gene-malignancy-signature. Here we report validation of our leading malignancy-risk-genes, topoisomerase-2-alpha (TOP2A), minichromosome-maintenance-protein-2 (MCM2) and "budding-uninhibited-by-benzimidazoles-1-homolog-beta" (BUB1B) at the protein level. Using our 117-gene malignancy-signature, we classified 18 fresh-frozen HNB tissues from 18 adult female breast cancer patients into HNB-tissues with low-grade (HNB-LGMA; N = 9) and high-grade molecular abnormality (HNB-HGMA; N = 9). Archival sections of additional HNB tissues from these patients, and invasive ductal carcinoma (IDC) tissues from six other patients were immunostained for these biomarkers. TOP2A/MCM2 expression was assessed as staining index (%) and BUB1B expression as H-scores (0-300). Increasing TOP2A, MCM2, and BUB1B protein expression from HNB-LGMA to HNB-HGMA tissues to IDCs validated our microarray-based molecular classification of HNB tissues by immunohistochemistry. We also demonstrated an increasing expression of TOP2A protein on an independent test set of HNB/benign/reductionmammoplasties, atypical-ductal-hyperplasia with and without synchronous breast cancer, DCIS and IDC tissues using a custom tissue microarray (TMA). In conclusion, TOP2A, MCM2, and BUB1B proteins are potential molecular biomarkers of malignancy in histologically normal and benign breast tissues. Larger-scale clinical validation studies are needed to further evaluate the clinical utility of these molecular biomarkers.

RUNX1T1: a novel predictor of liver metastasis in primary pancreatic endocrine neoplasms.

OBJECTIVES: Using gene expression profiling on frozen primary pancreatic endocrine tumors (PETs), we discovered RUNX1T1 as a leading candidate progression gene. This study was designed (1) to validate the differential expression of RUNX1T1 protein on independent test sets of metastatic and nonmetastatic PETs and (2) to determine if RUNX1T1 underexpression in primary tumors was predictive of liver metastases. METHODS: Immunohistochemical expression of RUNX1T1 protein was quantified using Allred scores on archival metastatic (n = 13) and nonmetastatic (n = 24) primary adult PET tissues using custom-designed tissue microarrays. Wilcoxon rank sum/Fisher exact tests and receiver operating characteristic curves were used in the data analysis. RESULTS: Median RUNX1T1 scores were 2 (2-7) and 6 (3-8) in metastatic versus nonmetastatic primaries (P < 0.0001). Eleven of 13 metastatic and 1 of 24 nonmetastatic primaries exhibited RUNX1T1-scores of 4 or less (P < 0.0001). Low RUNX1T1 expression was highly associated with hepatic metastases (P < 0.0001), whereas conventional histological criteria (Ki-67 index, mitotic rate, necrosis) were weakly associated with metastases (P = 0.08-0.15). Considering RUNX1T1 expression (Allred) score of 4 or less to be predictive, the sensitivity to predict hepatic metastases was 85%, with a specificity of 96%. CONCLUSIONS: RUNX1T1 protein is underexpressed in well-differentiated metastatic primary PETs relative to nonmetastatic primaries and emerges as a promising novel biomarker for prediction of liver metastases.