Development of an Immunohistochemical Assay to Detect the Ataxia-Telangiectasia Mutated (ATM) Protein in Gastric Carcinoma.

Ataxia-telangiectasia mutated (ATM), a key activator of DNA damage response mechanisms, represents a potential biomarker for targeted gastric carcinoma therapies. A phase II study (Study 39; NCT01063517) designed to investigate the combination olaparib plus paclitaxel in patients with recurrent or metastatic gastric cancer did not meet its primary endpoint of progression-free survival; however, an improvement in the secondary endpoint of overall survival was recorded with a greater overall survival benefit noted in patients with ATM-negative tumors. An ATM immunohistochemical (IHC) diagnostic assay was developed to identify patients who may respond favorably to targeted therapies and deployed in the confirmatory phase III GOLD trial (NCT01924533). The VENTANA ATM (Y170) assay was developed for investigational use in formalin-fixed, paraffin-embedded gastric carcinoma samples using an anti-ATM rabbit monoclonal antibody (clone Y170) and was optimized with OptiView DAB IHC Detection Kit on a BenchMark ULTRA instrument. The assay was deployed in studies assessing sensitivity, specificity, robustness, precision, and determining optimal ATM staining cutoff to define ATM-deficiency (ATM-low). The ATM (Y170) assay met all predefined product development acceptance criteria. Multiple parameters were characterized, including repeatability, reproducibility, analytical sensitivity, specificity, robustness, and product stability. The scoring algorithm was defined; gastric carcinoma samples were considered ATM-negative or ATM-positive when <25% or ≥25%, respectively, of tumor cell nuclei expressed ATM at any IHC stain intensity and nuclei of immune and/or endothelial cells expressed ATM at a moderate stain intensity (internal positive control). Results highlight reproducibility of the assay, supporting suitability for investigational use for evaluation of gastric carcinoma samples using tumor cell staining cutoff of <25% to define ATM-deficiency. Using this ATM assay, phase III GOLD trial (NCT01924533) clinical trial did not meet its primary endpoint, only suggesting, but not demonstrating, that assessment of ATM levels by IHC could possibly be useful in assessing the degree of benefit that may be achieved by adding olaparib to paxitaxel when treating gastric carcinoma. The utility of ATM (Y170) assay as a companion diagnostic requires further clinical validation.

Impact of Pre-Analytical Conditions on the Antigenicity of Lung Markers: ALK and MET.

Diagnostic assays for molecular alterations highly correlated with prognosis, predictive efficacy or safety of therapeutics are valuable clinical tools and in some cases approved as companion diagnostics (CDx) by the Federal Food and Drug Administration. For example, assays that determine echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) translocation status have been approved as CDx assay for therapies that target this molecular alteration. Characterizing the parameters that may compromise diagnostic accuracy for molecular biomarkers is critical for optimal patient care. To investigate the impact of pre-analytical handling and processing of tumor tissue on commonly used diagnostic immunohistochemistry-based assays for ALK and mesenchymal epithelial transition protein [c-mesenchymal epithelial transition (c-MET)], we investigated the effects of cold ischemia, fixative type, fixation time, and cut-slide age on staining consistency and intensity using human lung xenograft tumor tissue. Cold ischemia times for up to 5 to 6 hours for c-MET or ALK, respectively had minimal impact on staining. The optimal fixation conditions for both assays were found to be at least 6 hours and up to 48 hours for c-MET or 72 hours for ALK, in 10% neutral buffered formalin and Zinc formalin. The ALK antigen demonstrated marked staining intensity differences across non-neutral buffered formalin fixative types and times. Finally, cut-slide age influenced assay performance for both ALK and c-MET, with maximum stability observed when cut slides were stored at ambient temperatures (30°C) for no longer than 3, and 5 months, respectively. This study highlights the potential for pre-analytical factors to confound diagnostic test result interpretation.

Intertumoral Heterogeneity of CD3+ and CD8+ T-Cell Densities in the Microenvironment of DNA Mismatch-Repair-Deficient Colon Cancers: Implications for Prognosis.

PURPOSE: Colorectal cancers with deficient DNA mismatch repair (dMMR) are presumed to uniformly have dense lymphocytic infiltration that underlies their favorable prognosis and is critical to their responsiveness to immunotherapy, as compared with MMR-proficient (pMMR) tumors. We examined T-cell densities and their potential heterogeneity in a large cohort of dMMR tumors. EXPERIMENTAL DESIGN: CD3+ and CD8+ T-cell densities were quantified at the invasive margin (IM) and tumor core (CT) in 561 stage III colon cancers (dMMR, n = 278; pMMR, n = 283) from a phase III adjuvant trial (N0147). Their association with overall survival (OS) was determined using multivariable Cox analysis. RESULTS: Although CD3+ and CD8+ T-cell densities in the tumor microenvironment were higher in dMMR versus pMMR tumors overall, intertumoral heterogeneity in densities between tumors was significantly higher by 30% to 88% among dMMR versus pMMR cancers (P < 0.0001 for all four T-cell subtypes [CD3+IM, CD3+CT, CD8+IM, CD8+CT]). A substantial proportion of dMMR tumors (26% to 35% depending on the T-cell subtype) exhibited T-cell densities as low as that in the bottom half of pMMR tumors. All four T-cell subtypes were prognostic in dMMR with CD3+IM being the most strongly prognostic. Low (vs. high) CD3+IM was independently associated with poorer OS among dMMR (HR, 4.76; 95% confidence interval, 1.43-15.87; P = 0.0019) and pMMR tumors (P = 0.0103). CONCLUSIONS: Tumor-infiltrating T-cell densities exhibited greater intertumoral heterogeneity among dMMR than pMMR colon cancers, with CD3+IM providing robust stratification of both dMMR and pMMR tumors for prognosis. Potentially, lower T-cell densities among dMMR tumors may contribute to immunotherapy resistance.

A Sensitive ALK Immunohistochemistry Companion Diagnostic Test Identifies Patients Eligible for Treatment with Crizotinib.

INTRODUCTION: The availability of high-quality, rigorously validated diagnostic tests that can be broadly implemented is necessary to efficiently identify patients with anaplastic lymphoma kinase (ALK)-positive NSCLC who can potentially benefit from treatment with crizotinib. Here we present data on the recently approved Ventana ALK (D5F3) CDx Assay (Ventana Medical Systems, Tucson, AZ), the only immunohistochemistry (IHC)-based assay linked to treatment outcome. METHODS: NSCLC specimens prospectively tested for anaplastic lymphoma receptor tyrosine kinase gene (ALK) status by flourescent in situ hybridization (FISH) in the PROFILE 1014 clinical trial of crizotinib versus chemotherapy (N = 1018, including 179 ALK-positive and 754 ALK-negative specimens) were evaluated using the ALK (D5F3) CDx assay. Hazard ratios for progression-free survival comparing crizotinib and chemotherapy for ALK IHC-positive patients and ALK FISH-positive patients, as well as for concordance with the enrollment ALK FISH assay, were determined. RESULTS: Results from both assays were obtained for 933 cases. Percent positive, negative, and overall agreement rates were 86.0% , 96.3%, and 94.3%, respectively. There were 53 discrepant cases, of which 25 were ALK FISH-positive/ALK IHC-negative and 28 were ALK FISH-negative/ALK IHC-positive. The hazard ratios using observed outcomes were 0.401 for ALK FISH-positive/ALK IHC-positive cases and 0.407 for all ALK FISH-positive cases tested with ALK IHC versus 0.454 for all ALK FISH-positive cases enrolled in the trial. Outcome data for ALK FISH-negative/ALK IHC-positive cases were not available for analysis. Between-reader agreement rates for ALK IHC involving three independent laboratories exceeded 98%. CONCLUSIONS: The ALK (D5F3) CDx assay is a stand-alone companion diagnostic test for identification of patients for treatment with crizotinib. This automated assay provides an effective option to accurately and rapidly identify patients with ALK-positive NSCLC. The simple binary scoring algorithm results in high reader-to-reader precision.

Validation of an electronic program for pathologist training in the interpretation of a complex companion diagnostic immunohistochemical assay.

Companion diagnostics assay interpretation can select patients with the greatest targeted therapy benefits. We present the results from a prospective study demonstrating that pathologists can effectively learn immunohistochemical assay-interpretation skills from digital image-based electronic training (e-training). In this study, e-training was used to train board-certified pathologists to evaluate non-small cell lung carcinoma for eligibility for treatment with onartuzumab, a MET-inhibiting agent. The training program mimicked the live training that was previously validated in clinical trials for onartuzumab. A digital interface was developed for pathologists to review high-resolution, static images of stained slides. Sixty-four pathologists practicing in the United States enrolled while blinded to the type of training. After training, both groups completed a mandatory final test using glass slides. The results indicated both training modalities to be effective. Overall, 80.6% of e-trainees and 72.7% of live trainees achieved passing scores (at least 85%) on the final test. All study participants reported that their training experience was "good" and that they had received sufficient information to determine the adequacy of case slide staining to score each case. This study established that an e-training program conducted under highly controlled conditions can provide pathologists with the skills necessary to interpret a complex assay and that these skills can be equivalent to those achieved with face-to-face training using conventional microscopy. Programs of this type are scalable for global distribution and offer pathologists the potential for readily accessible and robust training in new companion diagnostic assays linked to novel, targeted, adjuvant therapies for cancer patients.

Oligonucleotide PIK3CA/Chromosome 3 Dual in Situ Hybridization Automated Assay with Improved Signals, One-Hour Hybridization, and No Use of Blocking DNA.

The PIK3CA gene at chromosome 3q26.32 was found to be amplified in up to 45% of patients with squamous cell carcinoma of the lung. The strong correlation between PIK3CA amplification and increased phosphatidylinositol 3-kinase (PI3K) pathway activities suggested that PIK3CA gene copy number is a potential predictive biomarker for PI3K inhibitors. Currently, all microscopic assessments of PIK3CA and chromosome 3 (CHR3) copy numbers use fluorescence in situ hybridization. PIK3CA probes are derived from bacterial artificial chromosomes whereas CHR3 probes are derived mainly from the plasmid pHS05. These manual fluorescence in situ hybridization assays mandate 12- to 18-hour hybridization and use of blocking DNA from human sources. Moreover, fluorescence in situ hybridization studies provide limited morphologic assessment and suffer from signal decay. We developed an oligonucleotide-based bright-field in situ hybridization assay that overcomes these shortcomings. This assay requires only a 1-hour hybridization with no need for blocking DNA followed by indirect chromogenic detection. Oligonucleotide probes produced discrete and uniform CHR3 stains superior to those from the pHS05 plasmid. This assay achieved successful staining in 100% of the 195 lung squamous cell carcinoma resections and in 94% of the 33 fine-needle aspirates. This robust automated bright-field dual in situ hybridization assay for the simultaneous detection of PIK3CA and CHR3 centromere provides a potential clinical diagnostic method to assess PIK3CA gene abnormality in lung tumors.