BRCA1 Protein Expression Predicts Survival in Glioblastoma Patients from an NRG Oncology RTOG Cohort.

PURPOSE: Glioblastoma, the most common malignant brain tumor, was associated with a median survival of <1 year in the pre-temozolomide (TMZ) era. Despite advances in molecular and genetic profiling studies identifying several predictive biomarkers, none has been translated into routine clinical use. Our aim was to investigate the prognostic significance of a panel of diverse cellular molecular markers of tumor formation and growth in an annotated glioblastoma tissue microarray (TMA). METHODS AND MATERIALS: A TMA composed of archived glioblastoma tumors from patients treated with surgery, radiation, and non-TMZ chemother-apy, was provided by RTOG. RAD51, BRCA-1, phosphatase and tensin homolog tumor suppressor gene (PTEN), and miRNA-210 expression levels were assessed using quantitative in situ hybridization and automated quantitative protein analysis. The objectives of this analysis were to determine the association of each biomarker with overall survival (OS), using the Cox proportional hazard model. Event-time distributions were estimated using the Kaplan-Meier method and compared by the log-rank test. RESULTS: A cohort of 66 patients was included in this study. Among the 4 biomarkers assessed, only BRCA1 expression had a statistically significant correlation with survival. From univariate analysis, patients with low BRCA1 protein expression showed a favorable outcome for OS (p = 0.04; hazard ratio = 0.56) in comparison with high expressors, with a median survival time of 18.9 versus 4.8 months. CONCLUSIONS: BRCA1 protein expression was an important survival predictor in our cohort of glioblastoma patients. This result may imply that low BRCA1 in the tumor and the consequent low level of DNA repair cause vulnerability of the cancer cells to treatment.

Molecular and Clinical Activity of CDX-3379, an Anti-ErbB3 Monoclonal Antibody, in Head and Neck Squamous Cell Carcinoma Patients.

PURPOSE: ErbB3 and its ligand neuregulin-1 (NRG1) are widely expressed in head and neck squamous cell carcinoma (HNSCC) and associated with tumor progression. A "window-of-opportunity" study (NCT02473731) was conducted to evaluate the pharmacodynamic effects of CDX-3379, an anti-ErbB3 mAb, in patients with HNSCC. PATIENTS AND METHODS: Twelve patients with newly diagnosed, operable HNSCC received two infusions of CDX-3379 (1,000 mg) at a 2-week interval prior to tumor resection. The primary study objective was to achieve ≥50% reduction in tumor ErbB3 signaling (phosphorylation of ErbB3; pErbB3) in ≥30% of patients. Other potential tumor biomarkers, pharmacokinetics, safety, and tumor measurements were also assessed. RESULTS: pErbB3 was detectable in all tumors prior to treatment and decreased for 10 of 12 (83%) patients following CDX-3379 dosing, with ≥50% reduction in 7 of 12 (58%; P = 0.04; 95% confidence interval, 27.7%-84.8%). Target trough CDX-3379 serum levels were achieved in all patients. CDX-3379 treatment-related toxicity was grade 1-2 and included diarrhea, fatigue, and acneiform dermatitis. Five of 12 (42%) patients had shrinkage in tumor burden, including a marked clinical response in a patient with human papillomavirus-negative oral cavity HNSCC. All patients with tumor shrinkage had tumors that expressed both NRG1 and ErbB3 and demonstrated reduced pErbB3 with CDX-3379 treatment. CONCLUSIONS: This study demonstrates that CDX-3379 can inhibit tumor ErbB3 phosphorylation in HNSCC. CDX-3379 was well tolerated and associated with measurable tumor regression. A phase II study (NCT03254927) has been initiated to evaluate CDX-3379 in combination with cetuximab for patients with advanced HNSCC.

Tumor Pre-Analytics in Molecular Pathology: Impact on Protein Expression and Analysis.

PURPOSE OF REVIEW: Precision medicine promises patient tailored, individualized diagnosis and treatment of diseases and relies on clinical specimen integrity and accuracy of companion diagnostic testing. Therefore, pre-analytics, which are defined as the collection, processing, and storage of clinical specimens, are critically important to enable optimal diagnostics, molecular profiling, and clinical decision-making around harvested specimens. This review article discusses the impact of tumor pre-analytics on molecular pathology focusing on biospecimen protein expression and analysis. RECENT FINDINGS: Due to busy clinical schedules and workflows that have been established for many years and to lack of standardization and limited assessment tools to quantify variability in pre-analytical processing, the effects of pre-analytics on biospecimen integrity are often overlooked. Several studies have recently emphasized an emerging crisis in science and reproducibility of results. SUMMARY: Biomarker instability due to pre-analytical variables affects comprehensive analysis and molecular phenotyping of patients' tissue. This problematic emphasizes the critical need for standardized protocols and technologies to be applied in the clinical and research setting.

ErbB activation signatures as potential biomarkers for anti-ErbB3 treatment in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) accounts for 3-5% of all tumor types and remains an unmet medical need with only two targeted therapies approved to date. ErbB3 (HER3), the kinase-impaired member of the EGFR/ErbB family, has been implicated as a disease driver in a number of solid tumors, including a subset of HNSCC. Here we show that the molecular components required for ErbB3 activation, including its ligand neuregulin-1 (NRG1), are highly prevalent in HNSCC and that HER2, but not EGFR, is the major activating ErbB3 kinase partner. We demonstrate that cetuximab treatment primarily inhibits the ERK signaling pathway and KTN3379, an anti-ErbB3 monoclonal antibody, inhibits the AKT signaling pathway, and that dual ErbB receptor inhibition results in enhanced anti-tumor activity in HNSCC models. Surprisingly, we found that while NRG1 is required for ErbB3 activation, it was not sufficient to fully predict for KTN3379 activity. An evaluation of HNSCC patient samples demonstrated that NRG1 expression was significantly associated with expression of the EGFR ligands amphiregulin (AREG) and transforming growth factor α (TGFα). Furthermore, NRG1-positive HNSCC cell lines that secreted high levels of AREG and TGFα or contained high levels of EGFR homodimers (H11D) demonstrated a better response to KTN3379. Although ErbB3 and EGFR activation are uncoupled at the receptor level, their respective signaling pathways are linked through co-expression of their respective ligands. We propose that NRG1 expression and EGFR activation signatures may enrich for improved efficacy of anti-ErbB3 therapeutic mAb approaches when combined with EGFR-targeting therapies in HNSCC.

Nuclear IRF-1 expression as a mechanism to assess "Capability" to express PD-L1 and response to PD-1 therapy in metastatic melanoma.

BACKGROUND: Predictive biomarkers for antibodies against programmed death 1 (PD-1) remain a major unmet need in metastatic melanoma. Specifically, response is seen in tumors that do not express programmed death ligand 1 (PD-L1), highlighting the need for a more sensitive biomarker. We hypothesize that capacity to express PD-L1, as assessed by an assay for a PD-L1 transcription factor, interferon regulatory factor 1 (IRF-1), may better distinguish patients likely to benefit from anti-PD-1 immunotherapy. METHODS: Samples from 47 melanoma patients that received nivolumab, pembrolizumab, or combination ipilimumab/nivolumab at Yale New Haven Hospital from May 2013 to March 2016 were collected. Expression of IRF-1 and PD-L1 in archival pre-treatment formalin-fixed, paraffin-embedded tumor samples were assessed by the AQUA method of quantitative immunofluorescence. Objective radiographic response (ORR) and progression-free survival (PFS) were assessed using modified RECIST v1.1 criteria. RESULTS: Nuclear IRF-1 expression was higher in patients with partial or complete response (PR/CR) than in patients with stable or progressive disease (SD/PD) (p = 0.044). There was an insignificant trend toward higher PD-L1 expression in patients with PR/CR (p = 0.085). PFS was higher in the IRF-1-high group than the IRF-1-low group (p = 0.017), while PD-L1 expression had no effect on PFS (p = 0.83). In a subset analysis, a strong association with PFS is seen in patients treated with combination ipilimumab and nivolumab (p = 0.0051). CONCLUSIONS: As a measure of PD-L1 expression capability, IRF-1 expression may be a more valuable predictive biomarker for anti-PD-1 therapy than PD-L1 itself.

Automated measurement of estrogen receptor in breast cancer: a comparison of fluorescent and chromogenic methods of measurement.

Whereas FDA-approved methods of assessment of estrogen receptor (ER) are 'fit for purpose', they represent a 30-year-old technology. New quantitative methods, both chromogenic and fluorescent, have been developed and studies have shown that these methods increase the accuracy of assessment of ER. Here, we compare three methods of ER detection and assessment on two retrospective tissue microarray (TMA) cohorts of breast cancer patients: estimates of percent nuclei positive by pathologists and by Aperio's nuclear algorithm (standard chromogenic immunostaining), and immunofluorescence as quantified with the automated quantitative analysis (AQUA) method of quantitative immunofluorescence (QIF). Reproducibility was excellent (R(2)>0.95) between users for both automated analysis methods, and the Aperio and QIF scoring results were also highly correlated, despite the different detection systems. The subjective readings show lower levels of reproducibility and a discontinuous, bimodal distribution of scores not seen by either mechanized method. Kaplan-Meier analysis of 10-year disease-free survival was significant for each method (Pathologist, P=0.0019; Aperio, P=0.0053, AQUA, P=0.0026); however, there were discrepancies in patient classification in 19 out of 233 cases analyzed. Out of these, 11 were visually positive by both chromogenic and fluorescent detection. In 10 cases, the Aperio nuclear algorithm labeled the nuclei as negative; in 1 case, the AQUA score was just under the cutoff for positivity (determined by an Index TMA). In contrast, 8 out of 19 discrepant cases had clear nuclear positivity by fluorescence that was unable to be visualized by chromogenic detection, perhaps because of low positivity masked by the hematoxylin counterstain. These results demonstrate that automated systems enable objective, precise quantification of ER. Furthermore, immunofluorescence detection offers the additional advantage of a signal that cannot be masked by a counterstaining agent. These data support the usage of automated methods for measurement of this and other biomarkers that may be used in companion diagnostic tests.

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.

Quantitative measurement of cancer tissue biomarkers in the lab and in the clinic.

Detection of biomolecules in tissues provides contextual information and the possibility to assess the interaction of different cell types and markers. Routine qualitative assessment of immune- and oligonucleotide-based methods in research and the clinic has been associated with assay variability because of lack of stringent validation and subjective interpretation of results. As a result, the vast majority of in situ assays in clinical usage are nonquantitative and, although useful, often of questionable scientific validity. Here, we revisit the reporters and methods used for single- and multiplexed in situ visualization of protein and RNA. Then we examine methods for the use of quantitative platforms for in situ measurement of protein and mRNA levels. Finally, we discuss the challenges of the transition of these methods to the clinic and their potential role as tools for development of companion diagnostic tests.

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.

Tools to assess tissue quality.

Biospecimen science has recognized the importance of tissue quality for accurate molecular and biomarker analysis and efforts are made to standardize tissue procurement, processing and storage conditions of tissue samples. At the same time the field has emphasized the lack of standardization of processes between different laboratories, the variability inherent in the analytical phase and the lack of control over the pre-analytical phase of tissue processing. The problem extends back into tissue samples in biorepositories, which are often decades old and where documentation about tissue processing might not be available. This review highlights pre-analytical variations in tissue handling, processing, fixation and storage and emphasizes the effects of these variables on nucleic acids and proteins in harvested tissue. Finally current tools for quality control regarding molecular or biomarker analysis are summarized and discussed.

A tissue quality index: an intrinsic control for measurement of effects of preanalytical variables on FFPE tissue.

While efforts are made to improve tissue quality and control preanalytical variables, pathologists are often confronted with the challenge of molecular analysis of patient samples of unknown quality. Here we describe a first attempt to construct a tissue quality index (TQI) or an intrinsic control that would allow a global assessment of protein status based on quantitative measurement of a small number of selected, informative epitopes. Quantitative immunofluorescence (QIF) of a number of proteins was performed on a series of 93 breast cancer cases where levels of expression were assessed as a function of delayed time to formalin fixation. A TQI was constructed based on the combination of proteins that most accurately reflect increased and decreased levels of expression in proportion to delay time. The TQI, defined by combinations of measurements of cytokeratin, ERK1/2 and pHSP-27 and their relationship to cold ischemic time were validated on a second build of the training series and on two independent breast tissue cohorts with recorded time to formalin fixation. We show an association of negative TQI values (an indicator for loss of tissue quality) with increasing cold ischemic time on both validation cohorts and an association with loss of ER expression levels on all three breast cohorts. Using expression levels of three epitopes, we can begin to assess the likelihood of delayed time to fixation or decreased tissue quality. This TQI represents a proof of concept for the use of epitope expression to provide a mechanism for monitoring tissue quality.

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.

Hypoxia-induced protein CAIX is associated with somatic loss of BRCA1 protein and pathway activity in triple negative breast cancer.

The purpose of this study is to explore the relationship between tumor hypoxia assessed by CA IX protein expression and loss of BRCA1 function in triple negative breast cancer (TNBC). Protein expression of CA IX and BRCA1 was evaluated by AQUA™ technology on two breast cancer cohorts: an unselected cohort of 637 breast cancer patients and a TNBC cohort of 120 patients. Transcriptional profiling was performed on FFPE samples from the TNBC cohort to evaluate a gene expression signature associated with BRCA1 mutation (van't Veer et al., Nature 415(6871):530-536, 2002). CA IX is expressed in 7 % of the unselected breast cancer cohort and in 25 % of the TNBCs and is significantly associated with the triple negative phenotype. CA IX protein expression and BRCA1 protein expression are inversely correlated in both cohorts. Patients expressing high levels of CA IX show significantly worse overall survival (p = 0.02). Importantly, high CA IX protein expression occurs in patients who show the BRCA1 mutant signature and low levels of BRCA1 protein. These data suggest that elevated CA IX protein in TNBC is associated with a BRCA1 mutant signature and loss of BRCA1 function. CA IX may be a useful biomarker to identify triple negative patients with defective homologous recombination, who might benefit from PARP inhibitor therapy.

Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features.

Some breast cancers have been shown to contain a small fraction of cells characterized by CD44(+)/CD24(-/low) cell-surface antigen profile that have high tumor-initiating potential. In addition, breast cancer cells propagated in vitro as mammospheres (MSs) have also been shown to be enriched for cells capable of self-renewal. In this study, we have defined a gene expression signature common to both CD44(+)/CD24(-/low) and MS-forming cells. To examine its clinical significance, we determined whether tumor cells surviving after conventional treatments were enriched for cells bearing this CD44(+)/CD24(-/low)-MS signature. The CD44(+)/CD24(-/low)-MS signature was found mainly in human breast tumors of the recently identified "claudin-low" molecular subtype, which is characterized by expression of many epithelial-mesenchymal-transition (EMT)-associated genes. Both CD44(+)/CD24(-/low)-MS and claudin-low signatures were more pronounced in tumor tissue remaining after either endocrine therapy (letrozole) or chemotherapy (docetaxel), consistent with the selective survival of tumor-initiating cells posttreatment. We confirmed an increased expression of mesenchymal markers, including vimentin (VIM) in cytokeratin-positive epithelial cells metalloproteinase 2 (MMP2), in two separate sets of postletrozole vs. pretreatment specimens. Taken together, these data provide supporting evidence that the residual breast tumor cell populations surviving after conventional treatment may be enriched for subpopulations of cells with both tumor-initiating and mesenchymal features. Targeting proteins involved in EMT may provide a therapeutic strategy for eliminating surviving cells to prevent recurrence and improve long-term survival in breast cancer patients.