IntroductionPatient suitability to anti–programmed death ligand 1 (PD-L1) immune checkpoint inhibition is key to the treatment of NSCLC. We present, applied to PD-L1 testing: a comprehensive cross-validation of two immunohistochemistry (IHC) clones; our descriptive experience in diagnostic reflex testing; the concordance of IHC to in situ RNA (RNA-ISH); and application of digital pathology.MethodsEight hundred thirteen NSCLC tumor samples collected from 564 diagnostic samples were analyzed prospectively, and 249 diagnostic samples analyzed retrospectively in tissue microarray format. Validated methods for IHC and RNA-ISH were tested in tissue microarrays and full sections and the QuPath system were used for digital pathology analysis.ResultsAntibody concordance of clones SP263 and 22C3 validation was 97% to 98% in squamous cell carcinoma and adenocarcinomas, respectively. Clinical NSCLC cases were reported as PD-L1–negative (48%), 1% to 49% (23%), and more than 50% (29%), with differences associated to tissue-type and EGFR status. Comparison of IHC and RNA-ISH was highly concordant in both subgroups. Comparison of digital assessment versus manual assessment was highly concordant. Discrepancies were mostly around the 1% clinical threshold. Challenging IHC interpretation included 1) calculating the total tumor cell denominator and the nature of PD-L1 expressing cell aggregates in cytology samples; 2) peritumoral expression of positive immune cells; 3) calculation of positive tumor percentages around clinical thresholds; and 4) relevance of the 100 malignant cell rule.ConclusionsSample type and EGFR status dictate differences in the expected percentage of PD-L1 expression. Analysis of PD-L1 is challenging, and interpretative guidelines are discussed. PD-L1 evaluations by RNA-ISH and digital pathology appear reliable, particularly in adenocarcinomas.

Understanding the connections between scientific inquiry and digital literacy in informal learning environments is essential to furthering students' critical thinking and technology skills. The Habitat Tracker project combines a standards-based curriculum focused on the nature of science with an integrated system of online and mobile computing technologies designed to help students learn about and participate in scientific inquiry in formal classroom settings and informal learning environments such as science museums or wildlife centers. This research documents the digital literacy skills elementary students used while participating in the Habitat Tracker project, exploring the connections between the scientific inquiry practices they developed and the digital literacy skills they employed as they engaged with the Habitat Tracker curriculum. The results of this research have implications for researchers and practitioners interested in fostering both the scientific inquiry practices and digital literacy skills of elementary students in formal and informal learning environments.

IHC staining can be used to screen for ROS1 gene rearrangements, with patients herein showing a response to crizotinib. Patients with tumors that test positive according to IHC staining but negative according to FISH were also identified, which may have implications for treatment selection.

Introduction:Detection of the ALK rearrangement in a solid tumor gives these patients the option of crizotinib as an oral form of anticancer treatment. The current test of choice is fluorescence in situ hybridization (FISH), but various cheaper and more convenient immunohistochemical (IHC) assays have been proposed as alternatives.Methods:Fifteen FISH-positive cases from patients, seven with data on crizotinib therapy and clinical response, were evaluated for the presence of ALK protein using three different commercially available antibodies: D5F3, using the proprietary automated system (Ventana), ALK1 (Dako), and 5A4 (Abcam). A further 14 FISH-negative and three uncertain (<15% rearrangement detected) cases were also retrieved. Of the total 32 specimens, 17 were excisions and 15 were computed tomography-guided biopsies or cytological specimens. All three antibodies were applied to all cases. Antibodies were semiquantitatively scored on intensity, and the proportion of malignant cells stained was documented. Cutoffs were set by receiver operating curve analysis for positivity to optimize correct classification.Results:All three IHC assays were 100% specific but sensitivity did vary: D5F3 86%, ALK 79%, 5A4 71%. Intensity was the most discriminating measure overall, with a combination of proportion and intensity not improving the test. No FISH-negative IHC-positive cases were seen. Two FISH-positive cases were negative with all three IHC assays. One of these had been treated with crizotinib and had failed to show clinical response. The other harbored a second driving mutation in the EGFR gene.Conclusions:IHC with all three antibodies is especially highly specific (100%) although variably sensitive (71%-86%), specifically in cases with scanty material. D5F3 assay was most sensitive in these latter cases. Occasional cases are IHC-positive but FISH-negative, suggesting either inaccuracy of one assay or occasional tumors with ALK rearrangement that do not express high levels of ALK protein.