Biology subjects

Dillon, L. M.

Publications and source records attributed to Dillon, L. M..

3 recordsLinked to original sources

bioRxiv2022

Development of a LAG-3 Immunohistochemistry Assay for Melanoma

AimsA robust immunohistochemistry (IHC) assay was developed to detect lymphocyte-activation gene 3 (LAG-3) expression by immune cells (ICs) in tumor tissues. LAG-3 is an immuno-oncology target with demonstrable clinical benefit, and there is a need for a standardized, well-characterized assay to measure its expression. This study aims to describe LAG-3 scoring criteria and present the specificity, sensitivity, analytical precision, and reproducibility of this assay. MethodsThe specificity of the assay was investigated by antigen competition and with LAG3 knockout cell lines. A melanin pigment removal procedure was implemented to prevent melanin interference in IHC interpretation. Formalin-fixed, paraffin-embedded (FFPE) human melanoma samples with a range of LAG-3 expression levels were used to assess the sensitivity and analytical precision of the assay with a [≥]1% cutoff to determine LAG-3-positivity. Interobserver and intraobserver reproducibility were evaluated with 60 samples in intralaboratory studies and 70 samples in interlaboratory studies. ResultsThe LAG-3 IHC method demonstrated performance suitable for analysis of LAG-3 IC expression in clinical melanoma samples. The pretreatment step effectively removed melanin pigment that could interfere with interpretation. LAG-3 antigen competition and analysis of LAG3 knockout cell lines indicated that the 17B4 antibody clone binds specifically to LAG-3. The intrarun repeatability, interday, interinstrument, interoperator, and interreagent lot reproducibility demonstrated a high scoring concordance (>95%). The interobserver and intraobserver reproducibility and overall interlaboratory and intralaboratory reproducibility also showed high scoring concordance (>90%). ConclusionsWe have demonstrated that the assay reliably assesses LAG-3 expression in FFPE human melanoma samples by IHC. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSLymphocyte-activation gene 3 (LAG-3) is an immune checkpoint receptor expressed on immune cells that limits T-cell activity and is being actively explored as a target for immunotherapy. What this study addsAn immunohistochemistry assay was developed to detect the LAG-3 protein in formalin-fixed paraffin-embedded human tumor tissue specimens. This study describes scoring criteria and shows the specificity, sensitivity, analytical precision, and reproducibility of this assay as an aid to determine LAG-3 expression in melanoma patients using a [≥]1% expression on immune cells threshold. How this study might affect research, practice or policyThe study describes a key immuno-oncology checkpoint immunohistochemistry assay that is robust and suitable for clinical trials. The assay was used in RELATIVITY-047 (NCT03470922), a phase 2/3 clinical trial that compared combined nivolumab and relatlimab treatment with nivolumab monotherapy, to stratify patients based on the percentage of LAG-3-positive immune cells within the tumor region. This assay is also being used in several ongoing clinical trials evaluating clinical response to relatlimab.

pathology↗

bioRxiv2022

Consistent measurement of LAG-3 expression across multiple staining platforms with the 17B4 antibody clone

ContextAn immunohistochemistry (IHC) assay developed to detect lymphocyte-activation gene 3 (LAG-3), a novel immune checkpoint inhibitor target, has demonstrated high analytical precision and interlaboratory reproducibility using a Leica staining platform, but has not been investigated on other IHC staining platforms. ObjectiveTo evaluate the performance of LAG-3 IHC assays using the 17B4 antibody clone across widely used IHC staining platforms: Agilent/Dako Autostainer Link 48 (ASL-48) and VENTANA BenchMark ULTRA (VBU) compared with Leica BOND-RX (BOND-RX). DesignEighty formalin-fixed paraffin-embedded melanoma tissue blocks were cut into consecutive sections and evaluated using staining platform-specific IHC assays with the 17B4 antibody clone. Duplicate testing was performed on the BOND-RX platform to assess intraplatform agreement. LAG-3 expression using a numerical score was evaluated by a pathologist and with a digital scoring algorithm. LAG-3 positivity was determined from manual scores using a [≥] 1% cutoff. ResultsLAG-3 IHC staining patterns and intensities were visually similar across all 3 staining platforms. Pearson correlation was [≥] 0.88 for interplatform and BOND-RX intraplatform concordance when LAG-3 expression was evaluated with a numerical score determined by a pathologist. Correlation increased with a numerical score determined with a digital scoring algorithm (Pearson correlation [≥] 0.93 for all comparisons). Overall percentage agreement was [≥] 77.5% for interplatform and BOND-RX intraplatform comparisons when a [≥] 1% cutoff was used to determine LAG-3 positivity. ConclusionsData from this study demonstrate that LAG-3 expression can be robustly and reproducibly assessed across 3 major commercial IHC staining platforms using the 17B4 antibody clone.

pathology↗

bioRxiv2020

AMPK activation by metformin promotes survival of dormant ER+ breast cancer cells

PurposeDespite adjuvant anti-estrogen therapy for patients with estrogen receptor alpha (ER)-positive breast cancer, dormant residual disease can persist for years and eventually cause tumor recurrence. We sought to deduce mechanisms underlying the persistence of dormant cancer cells to identify therapeutic strategies. Experimental DesignMimicking the aromatase inhibitor-induced depletion of estrogen levels used to treat patients, we developed preclinical models of dormancy in ER+ breast cancer induced by estrogen withdrawal in mice. We analyzed tumor xenografts and cultured cancer cells for molecular and cellular responses to estrogen withdrawal and drug treatments. Publicly available clinical breast tumor gene expression datasets were analyzed for responses to neoadjuvant anti-estrogen therapy. ResultsDormant breast cancer cells exhibited upregulated 5 adenosine monophosphate-activated protein kinase (AMPK) levels and activity, and upregulated fatty acid oxidation. While the anti-diabetes AMPK-activating drug metformin slowed the estrogen-driven growth of cells and tumors, metformin promoted the persistence of estrogen-deprived cells and tumors through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival. ConclusionsAMPK has context-dependent effects in cancer, cautioning against the widespread use of an AMPK activator across disease settings. The development of therapeutics targeting fat metabolism is warranted in ER+ breast cancer. Statement of Translational RelevanceDormant cancer cells that survive adjuvant therapy can ultimately give rise to recurrent/advanced tumors that frequently develop resistance to all approved therapies. Patients with early-stage estrogen receptor alpha (ER)-positive breast cancer are typically treated with surgical resection followed by [≥]5 years of adjuvant anti-estrogen therapy that neutralizes ER and suppresses, but often does not eliminate, tumor-initiating cells. Estrogen withdrawal, which mimics aromatase inhibitor therapy, induced activation of the metabolic sensor 5 adenosine monophosphate-activated protein kinase (AMPK) and upregulated fatty acid oxidation (FAO) in preclinical models. Treatment with the anti-diabetes AMPK-activating drug metformin or high dietary fat intake promoted survival of dormant ER+ breast cancer cells, while anti-anginal drugs that inhibit FAO induced clearance of dormant tumor cells. These findings caution against using AMPK modulators with anti-estrogens in patients with ER+ breast cancer, and warrant testing of FAO inhibitors as anti-cancer agents in combination with anti-estrogens.

cancer biology↗