Analytical validation of automated multiplex chromogenic immunohistochemistry for diagnostic and predictive purpose in non-small cell lung cancer.

OBJECTIVES: The evaluation of an increasing number of diagnostic and predictive markers is playing a central role in precision thoracic oncology. Multiplex immunohistochemistry (mIHC), alongside next-generation sequencing, is ideally situated for this purpose and maximizes tumor tissue preservation for molecular analyses that use increasingly large panels. However, the standardization and validation of mIHC that supports routine clinical laboratory processes are mandatory. After a previous proof-of-concept study, we now (i) optimized two automated four-plex assays on a commercially available IHC autostainer for use in daily practices worldwide and (ii) evaluated the repeatability and concordance of the assessment of the cell density. PATIENTS AND METHODS: Two four-plex mIHC assays [i) TTF1, p40, PD-L1, CD8; and, ii) ALK, ROS1, BRAFV600E, NTRK] were optimized on the BenchMark ULTRA autostainer (Ventana Medical Systems, Inc.), as determined in comparison to conventional IHC chromogenic assays. Intra-site repeatability was evaluated on serial tumor sections from non-small cell lung carcinomas (NSCLC). The concordance was assessed by linear fit to plots of the percentage staining evaluated on tumor sections from 89 NSCLC patients. RESULTS: Following optimization, an average concordance for a staining rate of 95.4% was achieved between conventional IHC and mIHC across all selected markers. Assessment of intra-site repeatability showed strong concordance for all these markers (average, R2 = 0.96; P-value < 0.001). CONCLUSIONS: Our optimized mIHC assay gave a sensitive and repeatable assessment of two panels of eight diagnostic and predictive biomarkers for NSCLC. The availability of standardized protocols to determine these biomarkers on a widely available IHC platform will expand the number of pathology laboratories able to determine the eligibility of patients with NSCLC for targeted treatment or immunotherapy in a reliable and concordant manner, thus providing a unique sample-sparing tool to characterize limited tissue samples in thoracic oncology.

Automated chromogenic multiplexed immunohistochemistry assay for diagnosis and predictive biomarker testing in non-small cell lung cancer.

OBJECTIVES: The current challenge in the management of non-small cell lung cancer (NSCLC) in pathology laboratories is to combine immunohistochemistry (IHC) and molecular approaches on increasingly smaller biopsies and the need to reserve a fair amount of tumor material for molecular analyses with increasingly larger panels. The latest lung cancer classification, especially in the setting of poorly differentiated tumors, requires an IHC workup to allow for accurate diagnosis and also to preserve as much tissue as possible for molecular testing. Thus, it is recommended to reduce use of the term NSCLC not otherwise specified as much as possible and classify tumors according to their specific histologic subtype. This implies limiting the number of tissue slides despite the existence of specific and sensitive biomarkers (ALK, ROS1, BRAF V600E, PD-L1) and the obligation to distinguish lung adenocarcinoma (TTF-1 positive) from squamous cell carcinoma (p40 positive). MATERIALS AND METHODS: Samples from 18 patients with NSCLC, previously characterized for histologic and genomic/immune features, were included. Two multiplexed IHC assays were developed, for diagnosis and immunophenotyping including TTF1, p40, PD-L1, and pan-Keratin antibodies, and for molecular profiling panel including ALK, ROS1 and BRAF V600E antibodies. RESULTS: We developed two sensitive multiplexed IHC assays to comprehensively characterize major NSCLC histotypes and FDA-cleared predictive biomarkers, without antigenicity loss, steric interference or increased cross-reactivity. The assays rely on standard antigen retrieval and automated staining protocols, limiting the need for validation strategies. CONCLUSION: Our multiplexed IHC approach provides a unique sample-sparing tool to characterize limited tissue samples in lung oncology and making it an alternative method in the clinical setting for therapeutic decision making of advanced NSCLC, provided that validation in a larger population is performed.

Chromogenic Multiplex Immunohistochemistry Reveals Modulation of the Immune Microenvironment Associated with Survival in Elderly Patients with Lung Adenocarcinoma.

With underrepresentation of elderly patients with lung adenocarcinoma (LADC) in anti-PD-1/PD-L1 clinical trials, better understanding of the interplay of PD-L1 and tumor-associated immune cells (TAICs) could assist clinicians in stratifying these patients for immunotherapy. One hundred and one patients with LADCs, stratified by age, were included for analysis of PD-L1 expression and density of TAICs expressing CD4, CD8, and CD33, by using multiplex chromogenic immunohistochemistry (IHC) assays and automated digital quantification. The CD4⁺/CD8⁺ ratio was significantly higher in elderly patients. In patients <75 years, the density of CD4⁺, CD8⁺, and PD-L1 in TAICs showed a positive significant correlation with PD-L1 expression in tumor cells (TCs), while a lower correlation was observed in the elderly population. In the latter, a high CD4⁺/CD8⁺ ratio, and combined PD-L1 expression ≥1% TCs with a low CD8⁺ density, low CD33⁺ density, and a high CD4⁺ density correlated to worse overall survival. We identified differences according to age in the CD4⁺/CD8⁺ ratio and in correlation between PD-L1 expression and the density of TAICs in LADC patients. Distinct groups of tumor microenvironments had an impact on the OS of elderly patients with LADC.

Structural study of the catalytic domain of PKCzeta using infrared spectroscopy and two-dimensional infrared correlation spectroscopy.

The secondary structure of the catalytic domain from protein kinase C zeta was studied using IR spectroscopy. In the presence of the substrate MgATP, there was a significant change in the secondary structure. After heating to 80 degrees C, a 14% decrease in the alpha-helix component was observed, accompanied by a 6% decrease in the beta-pleated sheet; no change was observed in the large loops or in 3(10)-helix plus associated loops. The maximum increase with heating was observed in the aggregated beta-sheet component, with an increase of 14%. In the presence of MgATP, and compared with the sample heated in its absence, there was a substantial decrease in the 3(10)-helix plus associated loops and an increase in alpha-helix. Synchronous 2D-IR correlation showed that the main changes occurred at 1617 cm(-1), which was assigned to changes in the intermolecular aggregated beta-sheet of the denaturated protein. This increase was mainly correlated with the change in alpha-helix. In the presence of MgATP, the main correlation was between aggregated beta-sheet and the large loops component. The asynchronous 2D-correlation spectrum indicated that a number of components are transformed in intermolecularly aggregated beta-sheet, especially the alpha-helix and beta-sheet components. It is interesting that changes in 3(10)-helix plus associated loops and in alpha-helix preceded changes in large loops, which suggests that the open loops structure exists as an intermediate state during denaturation. In summary, IR spectroscopy revealed an important effect of MgATP on the secondary structure and on the thermal unfolding process when this was induced, whereas 2D-IR correlation spectroscopy allowed us to show the establishment of the denaturation pathway of this protein.

The RNA binding protein FMRP: new connections and missing links.

The loss of the fragile X mental retardation protein (FMRP) is responsible for the most common cause of inherited mental retardation called the fragile X syndrome. FMRP is suspected to participate in the synaptic plasticity of neurons by acting on posttranscriptional control of gene expression. FMRP is an RNA binding protein that associates with mRNAs together with other proteins to form large ribonucleoprotein complexes. These complexes are proposed to participate in the transport, localization and translation of target mRNAs. Progress has been made recently in the identification of the mRNAs and the proteins present in these complexes and a possible connection with the micro-RNA dependent regulatory pathway has been established.