Maria Vassilakopoulou

Quantitative Assessment of Effect of Preanalytic Cold Ischemic Time on Protein Expression in Breast Cancer Tissues

BACKGROUND Companion diagnostic tests can depend on accurate measurement of protein expression in tissues. Preanalytic variables, especially cold ischemic time (time from tissue removal to fixation in formalin) can affect the measurement and may cause false-negative results. We examined 23 proteins, including four commonly used breast cancer biomarker proteins, to quantify their sensitivity to cold ischemia in breast cancer tissues. METHODS A series of 93 breast cancer specimens with known time-to-fixation represented in a tissue microarray and a second series of 25 matched pairs of core needle biopsies and breast cancer resections were used to evaluate changes in antigenicity as a function of cold ischemic time. Estrogen receptor (ER), progesterone receptor (PgR), HER2 or Ki67, and 19 other antigens were tested. Each antigen was measured using the AQUA method of quantitative immunofluorescence on at least one series. All statistical tests were two-sided. RESULTS We found no evidence for loss of antigenicity with time-to-fixation for ER, PgR, HER2, or Ki67 in a 4-hour time window. However, with a bootstrapping analysis, we observed a trend toward loss for ER and PgR, a statistically significant loss of antigenicity for phosphorylated tyrosine (P = .0048), and trends toward loss for other proteins. There was evidence of increased antigenicity in acetylated lysine, AKAP13 (P = .009), and HIF1A (P = .046), which are proteins known to be expressed in conditions of hypoxia. The loss of antigenicity for phosphorylated tyrosine and increase in expression of AKAP13, and HIF1A were confirmed in the biopsy/resection series. CONCLUSIONS Key breast cancer biomarkers show no evidence of loss of antigenicity, although this dataset assesses the relatively short time beyond the 1-hour limit in recent guidelines. Other proteins show changes in antigenicity in both directions. Future studies that extend the time range and normalize for heterogeneity will provide more comprehensive information on preanalytic variation due to cold ischemic time.

Evaluation of PD-L1 Expression and Associated Tumor-Infiltrating Lymphocytes in Laryngeal Squamous Cell Carcinoma

Purpose: Programmed death-ligand 1 (PD-L1; also known as CD274 or B7-H1) expression represents a mechanism of immune escape for cancer. Our purpose was to characterize tumor PD-L1 expression and associated T-cell infiltration in primary laryngeal squamous cell carcinomas (SCC). Experimental Design: A well-annotated cohort of 260 operable primary laryngeal SCCs [formalin-fixed paraffin-embedded (FFPE) specimens] was morphologically characterized for stromal tumor-infiltrating lymphocytes (TIL), on hematoxylin/eosin-stained whole sections and for PD-L1 mRNA expression by qRT-PCR in FFPE specimens. For PD-L1 protein expression, automated quantitative protein analysis (AQUA) was applied on tissue microarrays consisting of two cores from these tumors. In addition, PD-L1 mRNA expression in fresh-frozen tumors and normal adjacent tissue specimens was assessed in a second independent cohort of 89 patients with primary laryngeal SCC. Results: PD-L1 mRNA levels were upregulated in tumors compared with surrounding normal tissue (P = 0.009). TILs density correlated with tumor PD-L1 AQUA levels (P = 0.021). Both high TILs density and high PD-L1 AQUA levels were significantly associated with superior disease-free survival (DFS; TILs: P = 0.009 and PD-L1: P = 0.044) and overall survival (OS; TILs: P = 0.015 and PD-L1: P = 0.059) of the patients and retained significance in multivariate analysis. Conclusions: Increased TILs density and PD-L1 levels are associated with better outcome in laryngeal squamous cell cancer. Assessment of TILs and PD-L1 expression could be useful to predict response to immune checkpoint inhibitors. Clin Cancer Res; 22(3); 704–13. ©2015 AACR.

Pathways and targets in hepatocellular carcinoma

The incidence of hepatocellular carcinoma (HCC) has been rising in several western low-incidence areas over the past decade. The purpose of this review was to summarize the current knowledge on the ‘state of the art’ management of HCC focusing on targeted systemic therapies. The information for this review was compiled by searching the PubMed and MEDLINE databases for articles published until 1 June 2012. Cytotoxic chemotherapy has failed to affect outcome of HCC. Treatment with sorafenib is associated with survival gain in HCC but the responses are not durable. In addition, sorafenib is associated with substantial dermatologic and gastrointestinal toxicity. In this review, the authors summarize molecular targets and signal transduction pathways in HCC and provide an update of published and ongoing studies. Many targeted agents against angiogenesis, Ras/Raf/MAPK, EGF receptor, PI3K/AKT/mTOR, HGF/Met and IGF/IGF receptor are being tested in clinical trials.

Preanalytical variables and phosphoepitope expression in FFPE tissue: quantitative epitope assessment after variable cold ischemic time

Individualized targeted therapies for cancer patients require accurate and reproducible assessment of biomarkers to be able to plan treatment accordingly. Recent studies have shown highly variable effects of preanalytical variables on gene expression profiling and protein levels of different tissue types. Several publications have described protein degradation of tissue samples as a direct result of delay of formalin fixation of the tissue. Phosphorylated proteins are more labile and epitope degradation can happen within 30 min of cold ischemic time. To address this issue, we evaluated the change in antigenicity of a series of phosphoproteins in paraffin-embedded samples from breast tumors as a function of time to formalin fixation. A tissue microarray consisting of 93 breast cancer specimens with documented time-to-fixation was used to evaluate changes in antigenicity of 12 phosphoepitopes frequently used in research settings as a function of cold ischemic time. Analysis was performed in a quantitative manner using the AQUA technology for quantitative immunofluorescence. For each marker, least squares univariate linear regression was performed and confidence intervals were computed using bootstrapping. The majority of the epitopes tested revealed changes in expression levels with increasing time to formalin fixation. Some phosphorylated proteins, such as phospho-HSP27 and phospho-S6 RP, involved in post-translational modification and stress response pathways increased in expression or phosphorylation levels. Others (like phospho-AKT, phosphor-ERK1/2, phospho-Tyrosine, phospho-MET, and others) are quite labile and loss of antigenicity can be reported within 1–2 h of cold ischemic time. Therefore specimen collection should be closely monitored and subjected to quality control measures to ensure accurate measurement of these epitopes. However, a few phosphoepitopes (like phospho-JAK2 and phospho-ER) are sufficiently robust for routine usage in companion diagnostic testing.

Prognostic biomarkers in phase II trial of cetuximab-containing induction and chemoradiation in resectable HNSCC: Eastern cooperative oncology group E2303.

Purpose: We sought to evaluate the correlation between tissue biomarker expression (using standardized, quantitative immunofluorescence) and clinical outcome in the E2303 trial. Experimental Design: Sixty-three eligible patients with operable stage III/IV head and neck squamous cell cancer (HNSCC) participated in the Eastern Cooperative Oncology Group (ECOG) 2303 phase II trial of induction chemotherapy with weekly cetuximab, paclitaxel, and carboplatin followed by chemoradiation with the same regimen. A tissue microarray (TMA) was constructed and EGF receptor (EGFR), ERK1/2, Met, Akt, STAT3, β-catenin, E-cadherin, EGFR Variant III, insulin-like growth factor-1 receptor, NF-κB, p53, PI3Kp85, PI3Kp110a, PTEN, NRAS, and pRb protein expression levels were assessed using automated quantitative protein analysis (AQUA). For each dichotomized biomarker, overall survival (OS), progression-free survival (PFS), and event-free survival (EFS) were estimated by the Kaplan–Meier method and compared using log-rank tests. Multivariable Cox proportional hazards models were used to estimate HRs and test for significance. Results: Forty-two of 63 patients with TMA data on at least one biomarker were included in the biomarker analysis. Tumor extracellular signal–regulated kinase (ERK)1/2 levels were significantly associated with PFS [HR (low/high), 3.29; P = 0.026] and OS [HR (low/high), 4.34; P = 0.008]. On multivariable Cox regression analysis, ERK1/2 remained significantly associated with OS (P = 0.024) and PFS (P = 0.022) after controlling for primary site (oropharynx vs. non-oropharynx) and disease stage (III vs. IV), respectively. Clustering analysis revealed that clusters indicative of activated RAS/MAPK/ERK and/or PI3K/Akt pathways were associated with inferior OS and/or PFS and maintained significance in multivariable analysis. Conclusions: These results implicate PI3K/Akt and RAS/MAPK/ERK pathways in resistance to cetuximab-containing chemoradiation in HNSCC. Large prospective studies are required to validate these results. Clin Cancer Res; 20(11); 3023–32. ©2014 AACR.

Anticancer treatment and fertility: Effect of therapeutic modalities on reproductive system and functions

The significant improvement of cancer treatments entailed a longer life in cancer survivors and raised expectations for higher quality of life with minimized long-term toxicity. Infertility and gonadal dysfunction are adverse effects of anticancer therapy or may be related to specific tumors. In female cancer survivors, premature ovarian failure is common after antineoplastic treatments resulting in infertility and other morbidities related to oestrogen deficiency such as osteoporosis. In male cancer survivors, infertility and persistent a zoospermia is a more common long-term adverse effect than hypogonadism because germ cells are more sensitive to chemotherapy and radiotherapy than leydig cells. Gonadal toxicity and compromise of reproductive functions will be more efficiently prevented and treated if addressed before treatment initiation. This review focuses on these issues in young cancer survivors of childbearing age, where methods of protecting or restoring endocrine function and fertility need to be considered.

EGFR expression is associated with decreased benefit from trastuzumab in the NCCTG N9831 (Alliance) trial

Background:Epidermal growth factor receptor (EGFR) has been hypothesised to modulate the effectiveness of anti-HER2 therapy. We used a standardised, quantitative immunofluorescence assay and a novel EGFR antibody to evaluate the correlation between EGFR expression and clinical outcome in the North Central Cancer Treatment Group (NCCTG) N9831 trial.Methods:Tissue microarrays were constructed that allowed analysis of 1365 patients randomly assigned to receive chemotherapy alone (Arm A), sequential trastuzumab after chemotherapy (Arm B) and chemotherapy with concurrent trastuzumab (Arm C). Measurement of EGFR was performed using the EGFR antibody, D38B1, on the fluorescence-based AQUA platform. The result was validated using an independent retrospective metastatic breast cancer cohort (n=130).Results:Epidermal growth factor receptor assessed as a continuous (logarithmic transformed) variable shows an association with disease-free survival in Arm C (P=0.009) but not in Arm A or B. High EGFR expression was associated with worse outcome (Hazard ratio (HR)=2.15; 95% CI 1.28–3.60, P=0.004). Validation in a Greek metastatic breast cancer cohort showed an HR associated with high EGFR expression of 1.92 (P=0.0073).Conclusions:High expression of EGFR appears to be associated with decreased benefit from adjuvant concurrent trastuzumab. Since other treatment options exist for HER2-driven tumours, further validation of these data may select patients for alternative or additive therapy.

In Situ Quantitative Measurement of HER2mRNA Predicts Benefit from Trastuzumab-Containing Chemotherapy in a Cohort of Metastatic Breast Cancer Patients

Background We sought to determine the predictive value of in situ mRNA measurement compared to traditional methods on a cohort of trastuzumab-treated metastatic breast cancer patients. Methods A tissue microarray composed of 149, classified as HER2-positive, metastatic breast cancers treated with various trastuzumab-containing chemotherapy regimens was constructed. HER2 intracellular domain(ICD), HER2 extracellular domain(ECD) and HER2 mRNA were assessed using AQUA. For HER2 protein evaluation, CB11 was used to measure ICD and SP3 to measure ECD of the HER2 receptor. In addition, HER2 mRNA status was assessed using RNAscope assay ERRB2 probe. Kaplan – Meier estimates were used for depicting time-to-event endpoints. Multivariate Cox regression models with backward elimination were used to assess the performance of markers as predictors of TTP and OS, after adjusting for important covariates. Results HER2 mRNA was correlated with ICD HER2, as measured by CB11 HER2, with ECD HER2 as measured by SP3 (Pearson’s Correlation Coefficient, r = 0.66 and 0.51 respectively) and with FISH HER2 (Spearman’s Correlation Coefficient, r = 0.75). All markers, HER2 mRNA, ICD HER2 and ECD HER2, along with FISH HER2, were found prognostic for OS (Log-rank p = 0.007, 0.005, 0.009 and 0.043 respectively), and except for FISH HER2, they were also prognostic for TTP Log-rank p = 0.036, 0.068 and 0.066 respectively) in this trastuzumab- treated cohort. Multivariate analysis showed that in the presence of pre-specified set of prognostic factors, among all biomarkers only ECD HER2, as measured by SP3, is strong prognostic factor for both TTP (HR = 0.54, 95% CI: 0.31–0.93, p = 0.027) and OS (HR = 0.39, 95%CI: 0.22–0.70, p = 0.002). Conclusions The expression of HER2 ICD and ECD as well as HER2 mRNA levels was significantly associated with TTP and OS in this trastuzumab-treated metastatic cohort. In situ assessment of HER2 mRNA has the potential to identify breast cancer patients who derive benefit from Trastuzumab treatment.

Inside the USCAP Journals

Neuroendocrine tumors (NETs) are often readily diagnosed, but the site of origin may be challenging to determine. Knowing the site of origin is important for grading and staging, clinical management, and application of targeted therapies. Kerr et al addressed this conundrum by analyzing both metastatic and primary tumors of gastrointestinal, pulmonary, Merkel cell, pancreatic, pheochromocytoma, and medullary thyroid types using quantitative reverse transcription–PCR with a previously validated 92-gene panel. The specimens were enriched to 80–90% tumor nuclei content via manual or laser-capture microdissection. Overall, 99% of tumors were correctly classified, and 95% were correctly assigned to the known site of origin previously established by expert clinicopathologic diagnosis. Not surprisingly, identification was more accurate in well-differentiated NETs than in poorly differentiated cases. The tumors consisted of a mixture of primary and metastatic foci, and the test performed well in the latter setting.