RESUMEN
Tissue sections offer the opportunity to understand a patient's condition, to make better prognostic evaluations and to select optimum treatments, as evidenced by the place pathology holds today in clinical practice. Yet, there is a wealth of information locked up in a tissue section that is only partially accessed, due mainly to the limitations of tools and methods. Often tissues are assessed primarily based on visual analysis of one or two proteins, or 2-3 DNA or RNA molecules. Even while analysis is still based on visual perception, image analysis is starting to address the variability of human perception. This is in contrast to measuring characteristics that are substantially out of reach of human perception, such as parameters revealed through co-expression, spatial relationships, heterogeneity, and low abundance molecules. What is not routinely accessed is the information revealed through simultaneous detection of multiple markers, the spatial relationships among cells and tissue in disease, and the heterogeneity now understood to be critical to developing effective therapeutic strategies. Our purpose here is to review and assess methods for multiplexed, quantitative, image analysis based approaches, using new multicolor immunohistochemistry methods, automated multispectral slide imaging, and advanced trainable pattern recognition software. A key aspect of our approach is presenting imagery in a workflow that engages the pathologist to utilize the strengths of human perception and judgment, while significantly expanding the range of metrics collectable from tissue sections and also provide a level of consistency and precision needed to support the complexities of personalized medicine.
Asunto(s)
Inmunohistoquímica/métodos , Tiramina/química , Animales , Automatización , Biomarcadores de Tumor , Neoplasias de la Mama/metabolismo , ADN/química , Femenino , Colorantes Fluorescentes/química , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Queratinas/química , Neoplasias/metabolismo , Reconocimiento de Normas Patrones Automatizadas , Percepción , Medicina de Precisión , ARN/química , Programas InformáticosRESUMEN
BACKGROUND: Emerging data suggest predictive biomarkers based on the spatial arrangement of cells or coexpression patterns in tissue sections will play an important role in precision immuno-oncology. Multiplexed immunofluorescence (mIF) is ideally suited to such assessments. Standardization and validation of an end-to-end workflow that supports multisite trials and clinical laboratory processes are vital. Six institutions collaborated to: (1) optimize an automated six-plex assay focused on the PD-1/PD-L1 axis, (2) assess intersite and intrasite reproducibility of staining using a locked down image analysis algorithm to measure tumor cell and immune cell (IC) subset densities, %PD-L1 expression on tumor cells (TCs) and ICs, and PD-1/PD-L1 proximity assessments. METHODS: A six-plex mIF panel (PD-L1, PD-1, CD8, CD68, FOXP3, and CK) was rigorously optimized as determined by quantitative equivalence to immunohistochemistry (IHC) chromogenic assays. Serial sections from tonsil and breast carcinoma and non-small cell lung cancer (NSCLC) tissue microarrays (TMAs), TSA-Opal fluorescent detection reagents, and antibodies were distributed to the six sites equipped with a Leica Bond Rx autostainer and a Vectra Polaris multispectral imaging platform. Tissue sections were stained and imaged at each site and delivered to a single site for analysis. Intersite and intrasite reproducibility were assessed by linear fits to plots of cell densities, including %PDL1 expression by TCs and ICs in the breast and NSCLC TMAs. RESULTS: Comparison of the percent positive cells for each marker between mIF and IHC revealed that enhanced amplification in the mIF assay was required to detect low-level expression of PD-1, PD-L1, FoxP3 and CD68. Following optimization, an average equivalence of 90% was achieved between mIF and IHC across all six assay markers. Intersite and intrasite cell density assessments showed an average concordance of R2=0.75 (slope=0.92) and R2=0.88 (slope=0.93) for breast carcinoma, respectively, and an average concordance of R2=0.72 (slope=0.86) and R2=0.81 (slope=0.68) for NSCLC. Intersite concordance for %PD-L1+ICs had an average R2 value of 0.88 and slope of 0.92. Assessments of PD-1/PD-L1 proximity also showed strong concordance (R2=0.82; slope=0.75). CONCLUSIONS: Assay optimization yielded highly sensitive, reproducible mIF characterization of the PD-1/PD-L1 axis across multiple sites. High concordance was observed across sites for measures of density of specific IC subsets, measures of coexpression and proximity with single-cell resolution.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Técnica del Anticuerpo Fluorescente/métodos , Inmunohistoquímica/métodos , Laboratorios Clínicos/normas , Análisis de Matrices Tisulares/métodos , Femenino , Humanos , MasculinoRESUMEN
Importance: The true incidence of sudden unexplained death in childhood (SUDC), already the fifth leading category of death among toddlers by current US Centers for Disease Control and Prevention estimates, is potentially veiled by the varied certification processes by medicolegal investigative offices across the United States. Objective: To evaluate the frequency of SUDC incidence, understand its epidemiology, and assess the consistency of death certification among medical examiner and coroner offices in the US death investigation system. Design, Setting, and Participants: In this case series, 2 of 13 forensic pathologists (FPs) conducted masked reviews of 100 cases enrolled in the SUDC Registry and Research Collaborative (SUDCRRC). Children who died aged 11 months to 18 years from 36 US states, Canada, and the United Kingdom had been posthumously enrolled in the SUDCRRC by family members from 2014 to 2017. Comprehensive data from medicolegal investigative offices, clinical offices, and family members were reviewed. Data analysis was conducted from December 2014 to June 2020. Main Outcomes and Measures: Certified cause of death (COD) characterized as explained (accidental or natural) or unexplained, as determined by SUDCRRC masked review process. Results: In this study of 100 cases of SUDC (mean [SD] age, 32.1 [31.8] months; 58 [58.0%] boys; 82 [82.0%] White children; 92 [92.0%] from the United States), the original pathologist certified 43 cases (43.0%) as explained COD and 57 (57.0%) as unexplained COD. The SUDCRRC review process led to the following certifications: 16 (16.0%) were explained, 7 (7.0%) were undetermined because of insufficient data, and 77 (77.0%) were unexplained. Experts disagreed with the original COD in 40 cases (40.0%). These data suggest that SUDC incidence is higher than the current Centers for Disease Control and Prevention estimate (ie, 392 deaths in 2018). Conclusions and Relevance: To our knowledge, this is the first comprehensive masked forensic pathology review process of sudden unexpected pediatric deaths, and it suggests that SUDC may often go unrecognized in US death investigations. Some unexpected pediatric deaths may be erroneously attributed to a natural or accidental COD, negatively affecting surveillance, research, public health funding, and medical care of surviving family members. To further address the challenges of accurate and consistent death certification in SUDC, future studies are warranted.
Asunto(s)
Síndrome de Brugada/etiología , Causas de Muerte/tendencias , Certificado de Defunción , Síndrome de Brugada/epidemiología , Canadá/epidemiología , Preescolar , Testimonio de Experto , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Oportunidad Relativa , Factores de Riesgo , Estados Unidos/epidemiologíaRESUMEN
PURPOSE: Tumor-infiltrating lymphocytes (TIL) are associated with benefit to trastuzumab and chemotherapy in patients with early-stage HER2+ breast cancer. The predictive value of TILs, TIL subsets, and other immune cells in patients receiving chemotherapy-sparing lapatinib plus trastuzumab treatment is unclear.Experimental Design: Hematoxylin and eosin-stained slides (n = 59) were used to score stromal (s-)TILs from pretreatment biopsies of patients enrolled in the neoadjuvant TBCRC006 trial of 12-week lapatinib plus trastuzumab therapy (plus endocrine therapy for ER+ tumors). A 60% threshold was used to define lymphocyte-predominant breast cancer (LPBC). Multiplexed immunofluorescence (m-IF) staining (CD4, CD8, CD20, CD68, and FoxP3) and multispectral imaging were performed to characterize immune infiltrates in single formalin-fixed paraffin-embedded slides (n = 33). RESULTS: The pathologic complete response (pCR) rate was numerically higher in patients with LPBC compared with patients with non-LPBC (50% vs. 19%, P = 0.057). Unsupervised hierarchical clustering of the five immune markers identified two patient clusters with different responses to lapatinib plus trastuzumab treatment (pCR = 7% vs. 50%, for cluster 1 vs. 2 respectively; P = 0.01). In multivariable analysis, cluster 2, characterized by high CD4+, CD8+, CD20+ s-TILs, and high CD20+ intratumoral TILs, was independently associated with a higher pCR rate (P = 0.03). Analysis of single immune subpopulations revealed a significant association of pCR with higher baseline infiltration by s-CD4, intratumoral (i-) CD4, and i-CD20+ TILs. CONCLUSIONS: LPBC was marginally associated with higher pCR rate than non-LPBC in patients with lapatinib plus trastuzumab treated HER2+ breast cancer. Quantitative assessment of the immune infiltrate by m-IF is feasible and may help correlate individual immune cell subpopulations and immune cell profiles with treatment response.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos/inmunología , Terapia Neoadyuvante/métodos , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Estudios de Seguimiento , Humanos , Lapatinib/administración & dosificación , Linfocitos/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Persona de Mediana Edad , Pronóstico , Trastuzumab/administración & dosificaciónRESUMEN
The diversity and heterogeneity within high-grade serous ovarian cancer (HGSC), which is the most lethal gynecologic malignancy, is not well understood. Here, we perform comprehensive multi-platform omics analyses, including integrated analysis, and immune monitoring on primary and metastatic sites from highly clinically annotated HGSC samples based on a laparoscopic triage algorithm from patients who underwent complete gross resection (R0) or received neoadjuvant chemotherapy (NACT) with excellent or poor response. We identify significant distinct molecular abnormalities and cellular changes and immune cell repertoire alterations between the groups, including a higher rate of NF1 copy number loss, and reduced chromothripsis-like patterns, higher levels of strong-binding neoantigens, and a higher number of infiltrated T cells in the R0 versus the NACT groups.
Asunto(s)
Cistadenocarcinoma Seroso/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Adulto , Femenino , Perfilación de la Expresión Génica/métodos , Genómica/métodos , Humanos , Metabolómica/métodos , Persona de Mediana Edad , Neoplasias Ováricas/genéticaRESUMEN
Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4',6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.
Asunto(s)
Carcinoma/patología , Técnica del Anticuerpo Fluorescente/métodos , Formaldehído/uso terapéutico , Inmunohistoquímica/métodos , Humanos , Microambiente TumoralRESUMEN
The International Conference on Harmonization (ICH) E5 guidelines were developed to provide a general framework for evaluating the potential impact of ethnic factors on the acceptability of foreign clinical data, with the underlying objective to facilitate global drug development and registration. It is well recognized that all drugs exhibit significant inter-subject variability in pharmacokinetics and pharmacologic response and that such differences vary considerably among individual drugs and depend on a variety of factors. One such potential factor involves ethnicity. The objective of the present work was to perform an extensive review of the world literature on ethnic differences in drug disposition and responsiveness to determine their general significance in relation to drug development and registration. A few examples of suspected ethnic differences in pharmacokinetics or pharmacodynamics were identified. The available literature, however, was found to be heterologous, including a variety of study designs and research methodologies, and most of the publications were on drugs that were approved a long time ago.