Pathomic Features Reveal Immune and Molecular Evolution From Lung Preneoplasia to Invasive Adenocarcinoma.

Recent statistics on lung cancer, including the steady decline of advanced diseases and the dramatically increasing detection of early-stage diseases and indeterminate pulmonary nodules, mark the significance of a comprehensive understanding of early lung carcinogenesis. Lung adenocarcinoma (ADC) is the most common histologic subtype of lung cancer, and atypical adenomatous hyperplasia is the only recognized preneoplasia to ADC, which may progress to adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) and eventually to invasive ADC. Although molecular evolution during early lung carcinogenesis has been explored in recent years, the progress has been significantly hindered, largely due to insufficient materials from ADC precursors. Here, we employed state-of-the-art deep learning and artificial intelligence techniques to robustly segment and recognize cells on routinely used hematoxylin and eosin histopathology images and extracted 9 biology-relevant pathomic features to decode lung preneoplasia evolution. We analyzed 3 distinct cohorts (Japan, China, and United States) covering 98 patients, 162 slides, and 669 regions of interest, including 143 normal, 129 atypical adenomatous hyperplasia, 94 AIS, 98 MIA, and 205 ADC. Extracted pathomic features revealed progressive increase of atypical epithelial cells and progressive decrease of lymphocytic cells from normal to AAH, AIS, MIA, and ADC, consistent with the results from tissue-consuming and expensive molecular/immune profiling. Furthermore, pathomics analysis manifested progressively increasing cellular intratumor heterogeneity along with the evolution from normal lung to invasive ADC. These findings demonstrated the feasibility and substantial potential of pathomics in studying lung cancer carcinogenesis directly from the low-cost routine hematoxylin and eosin staining.

Multi-omics Analysis Reveals Immune Features Associated with Immunotherapy Benefit in Patients with Squamous Cell Lung Cancer from Phase III Lung-MAP S1400I Trial.

PURPOSE: Identifying molecular and immune features to guide immune checkpoint inhibitor (ICI)-based regimens remains an unmet clinical need. EXPERIMENTAL DESIGN: Tissue and longitudinal blood specimens from phase III trial S1400I in patients with metastatic squamous non-small cell carcinoma (SqNSCLC) treated with nivolumab monotherapy (nivo) or nivolumab plus ipilimumab (nivo+ipi) were subjected to multi-omics analyses including multiplex immunofluorescence (mIF), nCounter PanCancer Immune Profiling Panel, whole-exome sequencing, and Olink. RESULTS: Higher immune scores from immune gene expression profiling or immune cell infiltration by mIF were associated with response to ICIs and improved survival, except regulatory T cells, which were associated with worse overall survival (OS) for patients receiving nivo+ipi. Immune cell density and closer proximity of CD8+GZB+ T cells to malignant cells were associated with superior progression-free survival and OS. The cold immune landscape of NSCLC was associated with a higher level of chromosomal copy-number variation (CNV) burden. Patients with LRP1B-mutant tumors had a shorter survival than patients with LRP1B-wild-type tumors. Olink assays revealed soluble proteins such as LAMP3 increased in responders while IL6 and CXCL13 increased in nonresponders. Upregulation of serum CXCL13, MMP12, CSF-1, and IL8 were associated with worse survival before radiologic progression. CONCLUSIONS: The frequency, distribution, and clustering of immune cells relative to malignant ones can impact ICI efficacy in patients with SqNSCLC. High CNV burden may contribute to the cold immune microenvironment. Soluble inflammation/immune-related proteins in the blood have the potential to monitor therapeutic benefit from ICI treatment in patients with SqNSCLC.

Automated Cellular-Level Dual Global Fusion of Whole-Slide Imaging for Lung Adenocarcinoma Prognosis.

Histopathologic whole-slide images (WSI) are generally considered the gold standard for cancer diagnosis and prognosis. Survival prediction based on WSI has recently attracted substantial attention. Nevertheless, it remains a central challenge owing to the inherent difficulties of predicting patient prognosis and effectively extracting informative survival-specific representations from WSI with highly compounded gigapixels. In this study, we present a fully automated cellular-level dual global fusion pipeline for survival prediction. Specifically, the proposed method first describes the composition of different cell populations on WSI. Then, it generates dimension-reduced WSI-embedded maps, allowing for efficient investigation of the tumor microenvironment. In addition, we introduce a novel dual global fusion network to incorporate global and inter-patch features of cell distribution, which enables the sufficient fusion of different types and locations of cells. We further validate the proposed pipeline using The Cancer Genome Atlas lung adenocarcinoma dataset. Our model achieves a C-index of 0.675 (±0.05) in the five-fold cross-validation setting and surpasses comparable methods. Further, we extensively analyze embedded map features and survival probabilities. These experimental results manifest the potential of our proposed pipeline for applications using WSI in lung adenocarcinoma and other malignancies.

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples.

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system's image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

Pathological Response and Immune Biomarker Assessment in Non-Small-Cell Lung Carcinoma Receiving Neoadjuvant Immune Checkpoint Inhibitors.

Lung cancer is the leading cause of cancer incidence and mortality worldwide. Adjuvant and neoadjuvant chemotherapy have been used in the perioperative setting of non-small-cell carcinoma (NSCLC); however, the five-year survival rate only improves by about 5%. Neoadjuvant treatment with immune checkpoint inhibitors (ICIs) has become significant due to improved survival in advanced NSCLC patients treated with immunotherapy agents. The assessment of pathology response has been proposed as a surrogate indicator of the benefits of neaodjuvant therapy. An outline of recommendations has been published by the International Association for the Study of Lung Cancer (IASLC) for the evaluation of pathologic response (PR). However, recent studies indicate that evaluations of immune-related changes are distinct in surgical resected samples from patients treated with immunotherapy. Several clinical trials of neoadjuvant immunotherapy in resectable NSCLC have included the study of biomarkers that can predict the response of therapy and monitor the response to treatment. In this review, we provide relevant information on the current recommendations of the assessment of pathological responses in surgical resected NSCLC tumors treated with neoadjuvant immunotherapy, and we describe current and potential biomarkers to predict the benefits of neoadjuvant immunotherapy in patients with resectable NSCLC.

Multiplex Immunofluorescence and the Digital Image Analysis Workflow for Evaluation of the Tumor Immune Environment in Translational Research.

A robust understanding of the tumor immune environment has important implications for cancer diagnosis, prognosis, research, and immunotherapy. Traditionally, immunohistochemistry (IHC) has been regarded as the standard method for detecting proteins in situ, but this technique allows for the evaluation of only one cell marker per tissue sample at a time. However, multiplexed imaging technologies enable the multiparametric analysis of a tissue section at the same time. Also, through the curation of specific antibody panels, these technologies enable researchers to study the cell subpopulations within a single immunological cell group. Thus, multiplexed imaging gives investigators the opportunity to better understand tumor cells, immune cells, and the interactions between them. In the multiplexed imaging technology workflow, once the protocol for a tumor immune micro environment study has been defined, histological slides are digitized to produce high-resolution images in which regions of interest are selected for the interrogation of simultaneously expressed immunomarkers (including those co-expressed by the same cell) by using an image analysis software and algorithm. Most currently available image analysis software packages use similar machine learning approaches in which tissue segmentation first defines the different components that make up the regions of interest and cell segmentation, then defines the different parameters, such as the nucleus and cytoplasm, that the software must utilize to segment single cells. Image analysis tools have driven dramatic evolution in the field of digital pathology over the past several decades and provided the data necessary for translational research and the discovery of new therapeutic targets. The next step in the growth of digital pathology is optimization and standardization of the different tasks in cancer research, including image analysis algorithm creation, to increase the amount of data generated and their accuracy in a short time as described herein. The aim of this review is to describe this process, including an image analysis algorithm creation for multiplex immunofluorescence analysis, as an essential part of the optimization and standardization of the different processes in cancer research, to increase the amount of data generated and their accuracy in a short time.

Pathology Quality Control for Multiplex Immunofluorescence and Image Analysis Assessment in Longitudinal Studies.

Immune profiling of formalin-fixed, paraffin-embedded tissues using multiplex immunofluorescence (mIF) staining and image analysis methodology allows for the study of several biomarkers on a single slide. The pathology quality control (PQC) for tumor tissue immune profiling using digital image analysis of core needle biopsies is an important step in any laboratory to avoid wasting time and materials. Although there are currently no established inclusion and exclusion criteria for samples used in this type of assay, a PQC is necessary to achieve accurate and reproducible data. We retrospectively reviewed PQC data from hematoxylin and eosin (H&E) slides and from mIF image analysis samples obtained during 2019. We reviewed a total of 931 reports from core needle biopsy samples; 123 (13.21%) were excluded during the mIF PQC. The most common causes of exclusion were the absence of malignant cells or fewer than 100 malignant cells in the entire section (n = 42, 34.15%), tissue size smaller than 4 × 1 mm (n = 16, 13.01%), fibrotic tissue without inflammatory cells (n = 12, 9.76%), and necrotic tissue (n = 11, 8.94%). Baseline excluded samples had more fibrosis (90 vs 10%) and less necrosis (5 vs 90%) compared with post-treatment excluded samples. The most common excluded organ site of the biopsy was the liver (n = 19, 15.45%), followed by soft tissue (n = 17, 13.82%) and the abdominal region (n = 15, 12.20%). We showed that the PQC is an important step for image analysis and that the absence of malignant cells is the most limiting sample characteristic for mIF image analysis. We also discuss other challenges that pathologists need to consider to report reliable and reproducible image analysis data.

Best Practices for Technical Reproducibility Assessment of Multiplex Immunofluorescence.

Multiplex immunofluorescence (mIF) tyramide signal amplification is a new and useful tool for the study of cancer that combines the staining of multiple markers in a single slide. Several technical requirements are important to performing high-quality staining and analysis and to obtaining high internal and external reproducibility of the results. This review manuscript aimed to describe the mIF panel workflow and discuss the challenges and solutions for ensuring that mIF panels have the highest reproducibility possible. Although this platform has shown high flexibility in cancer studies, it presents several challenges in pre-analytic, analytic, and post-analytic evaluation, as well as with external comparisons. Adequate antibody selection, antibody optimization and validation, panel design, staining optimization and validation, analysis strategies, and correct data generation are important for reproducibility and to minimize or identify possible issues during the mIF staining process that sometimes are not completely under our control, such as the tissue fixation process, storage, and cutting procedures.

Multiplex Immunofluorescence Tyramide Signal Amplification for Immune Cell Profiling of Paraffin-Embedded Tumor Tissues.

Every day, more evidence is revealed regarding the importance of the relationship between the response to cancer immunotherapy and the cancer immune microenvironment. It is well established that a profound characterization of the immune microenvironment is needed to identify prognostic and predictive immune biomarkers. To this end, we find phenotyping cells by multiplex immunofluorescence (mIF) a powerful and useful tool to identify cell types in biopsy specimens. Here, we describe the use of mIF tyramide signal amplification for labeling up to eight markers on a single slide of formalin-fixed, paraffin-embedded tumor tissue to phenotype immune cells in tumor tissues. Different panels show different markers, and the different panels can be used to characterize immune cells and relevant checkpoint proteins. The panel design depends on the research hypothesis, the cell population of interest, or the treatment under investigation. To phenotype the cells, image analysis software is used to identify individual marker expression or specific co-expression markers, which can differentiate already selected phenotypes. The individual-markers approach identifies a broad number of cell phenotypes, including rare cells, which may be helpful in a tumor microenvironment study. To accurately interpret results, it is important to recognize which receptors are expressed on different cell types and their typical location (i.e., nuclear, membrane, and/or cytoplasm). Furthermore, the amplification system of mIF may allow us to see weak marker signals, such as programmed cell death ligand 1, more easily than they are seen with single-marker immunohistochemistry (IHC) labeling. Finally, mIF technologies are promising resources for discovery of novel cancer immunotherapies and related biomarkers. In contrast with conventional IHC, which permits only the labeling of one single marker per tissue sample, mIF can detect multiple markers from a single tissue sample, and at the same time, deliver extensive information about the cell phenotypes composition and their spatial localization. In this matter, the phenotyping process is critical and must be done accurately by a highly trained personal with knowledge of immune cell protein expression and tumor pathology.

Gastric hepatoid adenocarcinoma with multiple liver metastasis: a case report.

Hepatoid adenocarcinoma is a poorly differentiated alpha-fetoprotein-producing (AFP) tumor frequently located in the stomach, ovary, and pancreas. Presentation in the stomach has a high mortality rate due to late diagnosis, which offers the patient few therapeutic alternatives. On February 22, 2019, a 44-year-old woman from Lima entered the emergency department for pain in the right hypochondrium for 4 months, weight loss, nausea, and asthenia. On physical examination, hepatomegaly presented with a liver spam of 17 cm. Serology showed severe anemia and AFP of 49,800. The tomography showed multiple hypodense lesions in the liver and the presence of nodes. Endoscopy showed Bormann III gastric malignancy. Gastric biopsy determined undifferentiated epithelial malignancy; the immunohistochemical mark (+) for AFP and PAS Diastase confirmed a hepatoid gastric adenocarcinoma. A rare variant of gastric adenocarcinoma was evident, which often mimics an HCC. In this case, multiple liver metastases were observed that differed from the diagnosis of HCC, so this variant must always be taken into account when a primary gastric tumor presents with hepatic metastases.

[Trends in drugs usage for COVID-19 during the first wave of the pandemic in a hospital in Lima, Peru]. / Tendencias en el uso de fármacos para la COVID-19 durante la primera ola de la pandemia en un hospital de Lima, Perú.

This study aimed to evaluate the pharmacological trends in patients with COVID-19 from a hospital in Peru during the first wave of the pandemic. Retrospective study conducted between April and September 2020. The medical records of patients hospitalized for COVID-19 pneumonia at the Dos de Mayo National Hospital (Lima, Peru) were reviewed. Of a total of 3103 patients, 381 medical records were included (77.4% male, median age: 44 years). The use of four prehospital drugs increased (azithromycin, ivermectin, corticosteroids, and ceftriaxone), while the in-hospital use of six drugs (ceftriaxone, azithromycin, hydroxychloroquine, ivermectin, corticosteroid pulse, and prophylactic anticoagulation) decreased and in-hospital anticoagulation use decreased. These findings suggest that the management of COVID-19 has varied during the first wave of the pandemic, typically increasing prehospital drug use and decreasing inpatient use.

Con el objetivo de evaluar las tendencias en el uso de fármacos en pacientes con la COVID-19 de un hospital del Perú durante la primera ola de la pandemia se realizó un estudio retrospectivo entre abril y septiembre del 2020. Se revisaron las historias clínicas de pacientes hospitalizados por neumonía por COVID-19 en el Hospital Nacional Dos de Mayo (Lima, Perú). De un total de 3103 pacientes, se incluyeron 381 historias clínica (77,4% varones y mediana de edad de 44 años). Se observó un incremento del uso de cuatro fármacos prehospitalarios (azitromicina, ivermectina, corticoides y ceftriaxona), y una disminución del uso de seis fármacos intrahospitalarios (ceftriaxona, azitromicina, hidroxicloroquina, ivermectina, pulso de corticoides y anticoagulación profiláctica); además, el uso de anticoagulación intrahospitalaria aumentó. Estos hallazgos sugieren que el manejo de la COVID-19 varió durante la primera ola de la pandemia, aumentando el uso de fármacos prehospitalarios y disminuyendo el uso de fármacos intrahospitalarios.

Tendencias en el uso de fármacos para la COVID-19 durante la primera ola de la pandemia en un hospital de Lima, Perú / Trends in drugs usage for COVID-19 during the first wave of the pandemic in a hospital in Lima, Peru

Resumen Con el objetivo de evaluar las tendencias en el uso de fármacos en pacientes con la COVID-19 de un hospital del Perú durante la primera ola de la pandemia se realizó un estudio retrospectivo entre abril y septiembre del 2020. Se revisaron las historias clínicas de pacientes hospitalizados por neumonía por COVID-19 en el Hospital Nacional Dos de Mayo (Lima, Perú). De un total de 3103 pacientes, se incluyeron 381 historias clínica (77,4% varones y mediana de edad de 44 años). Se observó un incremento del uso de cuatro fármacos prehospitalarios (azitromicina, ivermectina, corticoides y ceftriaxona), y una disminución del uso de seis fármacos intrahospitalarios (ceftriaxona, azitromicina, hidroxicloroquina, ivermectina, pulso de corticoides y anticoagulación profiláctica); además, el uso de anticoagulación intrahospitalaria aumentó. Estos hallazgos sugieren que el manejo de la COVID-19 varió durante la primera ola de la pandemia, aumentando el uso de fármacos prehospitalarios y disminuyendo el uso de fármacos intrahospitalarios.

Abstract This study aimed to evaluate the pharmacological trends in patients with COVID-19 from a hospital in Peru during the first wave of the pandemic. Retrospective study conducted between April and September 2020. The medical records of patients hospitalized for COVID-19 pneumonia at the Dos de Mayo National Hospital (Lima, Peru) were reviewed. Of a total of 3103 patients, 381 medical records were included (77.4% male, median age: 44 years). The use of four prehospital drugs increased (azithromycin, ivermectin, corticosteroids, and ceftriaxone), while the in-hospital use of six drugs (ceftriaxone, azithromycin, hydroxychloroquine, ivermectin, corticosteroid pulse, and prophylactic anticoagulation) decreased and in-hospital anticoagulation use decreased. These findings suggest that the management of COVID-19 has varied during the first wave of the pandemic, typically increasing prehospital drug use and decreasing inpatient use.