Lys-27-Met mutations in histone 3 genes (H3K27M) characterize a subgroup of deadly gliomas and decrease genome-wide H3K27 trimethylation. Here we use primary H3K27M tumor lines and isogenic ...CRISPR-edited controls to assess H3K27M effects in vitro and in vivo. We find that whereas H3K27me3 and H3K27me2 are normally deposited by PRC2 across broad regions, their deposition is severely reduced in H3.3K27M cells. H3K27me3 is unable to spread from large unmethylated CpG islands, while H3K27me2 can be deposited outside these PRC2 high-affinity sites but to levels corresponding to H3K27me3 deposition in wild-type cells. Our findings indicate that PRC2 recruitment and propagation on chromatin are seemingly unaffected by K27M, which mostly impairs spread of the repressive marks it catalyzes, especially H3K27me3. Genome-wide loss of H3K27me3 and me2 deposition has limited transcriptomic consequences, preferentially affecting lowly-expressed genes regulating neurogenesis. Removal of H3K27M restores H3K27me2/me3 spread, impairs cell proliferation, and completely abolishes their capacity to form tumors in mice.
The ability to effectively monitor key indicators is important for continuous quality improvement in laboratory immunohistochemistry. This article deals specifically with laboratory turnaround time ...(TAT) as a key delivery indicator and the impact of laboratory workflow on laboratory TATs. While our laboratory has traditionally relied on the manual calculation of slide-TAT (S-TAT) to monitor delivery, we have determined that automated calculation of case-TAT (C-TAT) would be superior as a delivery indicator. AABACUS (Automatable Activity-Based Approach to Complexity Unit Scoring) is an activity-based workload model designed to function primarily as a decision support tool to monitor pathologist staffing levels. We devised a high-level proof-of-principle approach to determine whether it is possible to apply AABACUS as a decision support tool for quality improvement through analysis of alternative laboratory workflows that have potential to impact C-TAT. Our use of AABACUS in this proof-of-principle quality improvement endeavour was two-fold: (1) we leveraged the ability of AABACUS to link data at the slide level to data at the case level, which enabled the automated calculation of C-TAT; and (2) we adapted AABACUS to evaluate the impact of laboratory workflow activities (specifically workflow bifurcation activities) on the calculated C-TATs. We have coined the term 'L-AABACUS' to describe the adaptation of AABACUS to the analysis of laboratory workflow.
Diverse functions have been reported for lipocalin 2. To investigate these functions in vivo, we generated gene-targeted lipocalin 2-deficient mice ($Lcn2^{-/-}$mice). In vitro studies have suggested ...that lipocalin 2 is important for cellular apoptosis induced by IL-3 withdrawal, and for the induction of kidney differentiation during embryogenesis. Analysis of$Lcn2^{-/-}$mice showed normal cell death upon IL-3 withdrawal and normal kidney development. However, we found that$Lcn2^{-/-}$mice exhibited an increased susceptibility to bacterial infections, in keeping with the proposed function of lipocalin 2 in iron sequestration. Neutrophils isolated from$Lcn2^{-/-}$mice showed significantly less bacteriostatic activity compared with WT controls. The bacteriostatic property of the WT neutrophils was abolished by the addition of exogenous iron, indicating that the main function of lipocalin 2 in the antibacterial innate immune response is to limit this essential element. Another important function ascribed to lipocalin 2 has been its protective role against kidney ischemia-reperfusion injury. We analyzed$Lcn2^{-/-}$mice using a mouse model for severe renal failure and could not detect any significant differences compared with their WT littermates.
•High quality ROS1 IHC assays have high clinical utility.•Adjusting readout can increase diagnostic accuracy in ROS1 IHC.•ROS1 IHC readout must be balanced with the analytical sensitivity of the ...assay.•A multi-institutional approach abets the validation of rare disease biomarker assays.
Patients with non-small cell lung cancer (NSCLC) harboring ROS proto-oncogene 1 (ROS1) gene rearrangements show dramatic response to the tyrosine kinase inhibitor (TKI) crizotinib. Current best practice guidelines recommend that all advanced stage non-squamous NSCLC patients be also tested for ROS1 gene rearrangements. Several studies have suggested that ROS1 immunohistochemistry (IHC) using the D4D6 antibody may be used to screen for ROS1 fusion positive lung cancers, with assays showing high sensitivity but moderate to high specificity. A break apart fluorescence in situ hybridization (FISH) test is then used to confirm the presence of ROS1 gene rearrangement. The goal of Canadian ROS1 (CROS) study was to harmonize ROS1 laboratory developed testing (LDT) by using IHC and FISH assays to detect ROS1 rearranged lung cancers across Canadian pathology laboratories.
Cell lines expressing different levels of ROS1 (high, low, none) were used to calibrate IHC protocols after which participating laboratories ran the calibrated protocols on a reference set of 24 NSCLC cases (9 ROS1 rearranged tumors and 15 ROS1 non-rearranged tumors as determined by FISH). Results were compared using a centralized readout. The stained slides were evaluated for the cellular localization of staining, intensity of staining, the presence of staining in non-tumor cells, the presence of non-specific staining (e.g. necrosis, extracellular mater, other) and the percent positive cells. H-score was also determined for each tumor.
Analytical sensitivity and specificity harmonization was achieved by using low limit of detection (LOD) as either any positivity in the U118 cell line or H-score of 200 with the HCC78 cell line. An overall diagnostic sensitivity and specificity of up to 100% and 99% respectively was achieved for ROS1 IHC testing (relative to FISH) using an adjusted H-score readout on the reference cases.
This study confirms that LDT ROS1 IHC assays can be highly sensitive and specific for detection of ROS1 rearrangements in NSCLC. As NSCLC can demonstrate ROS1 IHC positivity in FISH-negative cases, the degree of the specificity of the IHC assay, especially in highly sensitive protocols, is mostly dependent on the readout cut-off threshold. As ROS1 IHC is a screening assay for a rare rearrangements in NSCLC, we recommend adjustment of the readout threshold in order to balance specificity, rather than decreasing the overall analytical and diagnostic sensitivity of the protocols.
Different clones, protocol conditions, instruments, and scoring/readout methods may pose challenges in introducing different PD-L1 assays for immunotherapy. The diagnostic accuracy of using different ...PD-L1 assays interchangeably for various purposes is unknown. The primary objective of this meta-analysis was to address PD-L1 assay interchangeability based on assay diagnostic accuracy for established clinical uses/purposes. A systematic search of the MEDLINE database using PubMed platform was conducted using "PD-L1" as a search term for 01/01/2015 to 31/08/2018, with limitations "English" and "human". 2,515 abstracts were reviewed to select for original contributions only. 57 studies on comparison of two or more PD-L1 assays were fully reviewed. 22 publications were selected for meta-analysis. Additional data were requested from authors of 20/22 studies in order to enable the meta-analysis. Modified GRADE and QUADAS-2 criteria were used for grading published evidence and designing data abstraction templates for extraction by reviewers. PRISMA was used to guide reporting of systematic review and meta-analysis and STARD 2015 for reporting diagnostic accuracy study. CLSI EP12-A2 was used to guide test comparisons. Data were pooled using random-effects model. The main outcome measure was diagnostic accuracy of various PD-L1 assays. The 22 included studies provided 376 2×2 contingency tables for analyses. Results of our study suggest that, when the testing laboratory is not able to use an Food and Drug Administration-approved companion diagnostic(s) for PD-L1 assessment for its specific clinical purpose(s), it is better to develop a properly validated laboratory developed test for the same purpose(s) as the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic, than to replace the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic with a another PD-L1 Food and Drug Administration-approved companion diagnostic that was developed for a different purpose.
PD-L1 immunoexpression in head and neck squamous-cell carcinoma (HNSCC) determines immunotherapy eligibility. Patients are often diagnosed using fine-needle aspiration (FNA) of metastatic lymph ...nodes, however, the cytohistologic correlation of the combined positive score (CPS) is largely unknown.
This study retrospectively identified 96 paired histologic (HS) and cytologic specimens (CyS), between 2016 and 2020, diagnosed with HNSCC. Cases with <100 tumor cells (n = 54) or missing block(s) (n = 8) were excluded. All 34 case pairs were scored with CPS using the PD-L1 22C3 pharmDx assay at clinically relevant cut-offs of <1%, 1%-19%, and ≥20% independently by three observers blinded to the case pairs (CyS with corresponding HS).
The CPS (<1/1-19/≥20) for CyS and HS were as follows: 10(29.4%)/10(29.4%)/14(41.2%) and 2(5.9%)/13(38.2%)/19(55.9%), respectively. There was fair overall cytohistologic agreement (OA) of 76.5% (k = 0.261) at the CPS cut-off of 1. The OA did not differ significantly between site-matched (n = 13) and -unmatched (n = 21) case pairs (p = .4653). CyS has a specificity and positive predictive value (PPV) of 100% but a negative predictive value (NPV) of only 20% as compared to its paired HS.
Our study demonstrates fair CPS cytohistologic correlation in HNSCC specimens using the PD-L1 IHC 22C3 pharmDx assay with high PPV but low NPV. This suggest that determining PD-L1 status in FNA specimens can play an important role in the clinical management of HNSCC patients.
Based largely on studies in xenograft models, lipocalin-2 (Lcn2) has been implicated in the progression of multiple types of human tumors, including breast cancer. Here we examine the role of Lcn2 in ...mammary tumorigenesis and lung metastasis using an in vivo molecular genetics approach. We crossed a well-characterized transgenic mouse model of breast cancer, the MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen) mouse, with two independent gene-targeted Lcn2⁻/⁻ mouse strains of the 129/Ola or C57BL/6 genetic background. The onset and progression of mammary tumor development and lung metastasis in the female progeny of these crosses were monitored over a 20-week period. Female Lcn2⁻/⁻MMTV-PyMT mice of the 129/Ola background (Lcn2⁻/⁻PyMT¹²⁹) showed delayed onset of mammary tumors, and both Lcn2⁻/⁻PyMT¹²⁹ mice and Lcn2⁻/⁻MMTV-PyMT mice of the C57BL/6 background (Lcn2⁻/⁻PyMTB⁶) exhibited significant decreases in multiplicity and tumor burden (~2- to 3-fold), as measured by total tumor weight and volume. At the molecular level, mammary tumors derived from Lcn2⁻/⁻PyMTB⁶ females showed reduced matrix metalloproteinase-9 (MMP-9) activity and a lack of high molecular weight MMP activity. However, although increased MMP-9 activity has been linked to tumor progression, neither Lcn2⁻/⁻PyMTB⁶ nor Lcn2⁻/⁻PyMT¹²⁹ female mice showed a reduction in lung metastases compared to Lcn2⁺/⁺PyMT controls. Our results demonstrate, using an in vivo animal model approach, that Lcn2 is a potent inducer of mammary tumor growth but not a significant promoter of lung metastasis.
The Trp53 gene family member Trp73 encodes two major groups of protein isoforms, TAp73 and DeltaNp73, with opposing pro- and anti-apoptotic functions; consequently, their relative ratio regulates ...cell fate. However, the precise roles of p73 isoforms in cellular events such as tumor initiation, embryonic development, and cell death remain unclear. To determine which aspects of p73 function are attributable to the TAp73 isoforms, we generated and characterized mice in which exons encoding the TAp73 isoforms were specifically deleted to create a TAp73-deficient (TAp73(-/-)) mouse. Here we show that mice specifically lacking in TAp73 isoforms develop a phenotype intermediate between the phenotypes of Trp73(-/-) and Trp53(-/-) mice with respect to incidence of spontaneous and carcinogen-induced tumors, infertility, and aging, as well as hippocampal dysgenesis. In addition, cells from TAp73(-/-) mice exhibit genomic instability associated with enhanced aneuploidy, which may account for the increased incidence of spontaneous tumors observed in these mutants. Hence, TAp73 isoforms exert tumor-suppressive functions and indicate an emerging role for Trp73 in the maintenance of genomic stability.
PD-L1 testing by immunohistochemistry (IHC) has presented significant challenges not only for clinical laboratories, but also for external quality assurance (EQA) entities that provide proficiency ...testing (PT) for clinical laboratories. Canadian Immunohistochemistry Quality Control (CIQC) has used educational runs to explore approaches to sample design and analysis of results that would enhance patient safety. As PT for predictive biomarkers requires modeling at every level (design of the run, assessment of the run, and reporting of "pass" or "fail") based on "fit-for-purpose" principles, CIQC has applied those principles to PD-L1 PT runs. Each laboratory received unstained slides with TMA tissue cores from 104 randomly selected primary NSCLC and tonsil tissues to test with their current PD-L1 assay. Diagnostic sensitivity and specificity were calculated against designated gold standards based on the "3D" approach (drug-disease-diagnostic assay). Depending on the selection of fit-for-purpose gold standards and also on the selection of what was considered fit-for-purpose cut-off points, great variation in the performance (accuracy) of both companion/complementary diagnostic assays and laboratory developed tests was seen. "Fit-for-purpose" in PT for PD-L1 testing entails that the purpose(s) of each PT run is declared a priori, that the PT program has selected/designated purpose-specific gold standard results for the PT challenge, and that the PT materials for the PT run are designed and constructed to enable calculations of diagnostic accuracy.
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