Objective and Impact Statement: We present a panel of virtual staining neural networks for lung and heart transplant biopsies, providing rapid and high-quality histological staining results while bypassing the traditional histochemical staining process. Introduction: Allograft rejection is a common complication of organ transplantation, which can lead to life-threatening outcomes if not promptly managed. Histological examination is the gold standard method for evaluating organ transplant rejection status, as it provides detailed insights into rejection signatures at the cellular level. Nevertheless, the traditional histochemical staining process is time-consuming, costly, and labor-intensive since transplant biopsy evaluations typically necessitate multiple stains. Furthermore, once these tissue slides are stained, they cannot be reused for other ancillary tests. More importantly, suboptimal handling of very small tissue fragments from transplant biopsies may impede their effective histochemical staining, and color variations across different laboratories or batches can hinder efficient histological analysis by pathologists. Methods: To mitigate these challenges, we developed a panel of virtual staining neural networks for lung and heart transplant biopsies, which digitally convert autofluorescence microscopic images of label-free tissue sections into their bright-field histologically stained counterparts-bypassing the traditional histochemical staining process. Specifically, we virtually generated hematoxylin and eosin (H&E), Masson's Trichrome (MT), and elastic Verhoeff-Van Gieson stains for label-free transplant lung tissue, along with H&E and MT stains for label-free transplant heart tissue. Results: Blind evaluations conducted by 3 board-certified pathologists confirmed that the virtual staining networks consistently produce high-quality histology images with high color uniformity, closely resembling their well-stained histochemical counterparts across various tissue features. The use of virtually stained images for the evaluation of transplant biopsies achieved comparable diagnostic outcomes to those obtained via traditional histochemical staining, with a concordance rate of 82.4% for lung samples and 91.7% for heart samples. Moreover, virtual staining models create multiple stains from the same autofluorescence input, eliminating structural mismatches observed between adjacent sections stained in the traditional workflow, while also saving tissue, expert time, and staining costs. Conclusion: The presented virtual staining panels provide an effective alternative to conventional histochemical staining for transplant biopsy evaluation. These virtual staining panels have the potential to enhance the clinical diagnostic workflow for organ transplant rejection and improve the performance of downstream automated models for the analysis of transplant biopsies.

Histopathological staining of human tissue is essential for disease diagnosis. Recent advances in virtual tissue staining technologies using artificial intelligence alleviate some of the costly and tedious steps involved in traditional histochemical staining processes, permitting multiplexed staining and tissue preservation. However, potential hallucinations and artefacts in these virtually stained tissue images pose concerns, especially for the clinical uses of these approaches. Quality assessment of histology images by experts can be subjective. Here we present an autonomous quality and hallucination assessment method, AQuA, for virtual tissue staining and digital pathology. AQuA autonomously achieves 99.8% accuracy when detecting acceptable and unacceptable virtually stained tissue images without access to histochemically stained ground truth and presents an agreement of 98.5% with the manual assessments made by board-certified pathologists, including identifying realistic-looking images that could mislead diagnosticians. We demonstrate the wide adaptability of AQuA across various virtually and histochemically stained human tissue images. This framework enhances the reliability of virtual tissue staining and provides autonomous quality assurance for image generation and transformation tasks in digital pathology and computational imaging.

TPS2697 Background: The prognosis for patients with unresectable and metastatic solid tumors that progress on standard of care therapy remains poor due to limited treatment options. Compelling evidence suggests that the induction and activation of tumor-residing conventional type-1 dendritic cells (cDC1) is critical to elicit anti-tumor immunity. Recently, we have demonstrated that a combinatorial regimen comprised of in situ delivery of Fms-like tyrosine kinase 3 ligand (Flt3L), radiation therapy (9Gy), and dual TLR3/CD40 stimulation 1) mobilizes cDC1 to the tumor microenvironment; 2) induces maturation of cDC1; 3) facilitates trafficking of cDC1 carrying tumor antigens to tumor-draining lymph nodes; 4) elicits de novo adaptive T cell immunity; 5) triggers regression of primary tumors, as well as non-irradiated distant tumors; and 6) develops tumor-specific systemic immunological memory using multiple syngeneic orthotopic murine models (1-4). Based on results from preclinical studies, we have initiated a phase 1 study as below. Methods: This is a phase 1 study of in situ immunomodulation with CDX-301 (Flt3L), radiation therapy, CDX-1140 and Poly-ICLC (dual CD40/TLR3 stimulation) in patients with unresectable and metastatic solid tumors. Eligibility for this study includes patients ≥ 18 yrs who have clinically or pathologically confirmed diagnosis of unresectable and metastatic melanoma, cutaneous squamous cell carcinoma, Merkel cell carcinoma, high-grade bone and soft tissue sarcoma or HER2/neu (-) breast cancer with no curative treatment options, and progressed on at least one line of standard systemic therapy. The unresectable disease to be irradiated and injected with medications must be located in breast, dermal, subcutaneous, or soft tissue, or lymph nodes with the longest axis of the tumor 2-7 cm, and should be considered safe for injection by the investigator. The metastatic disease must be measurable per irRECIST criteria. Patients will be treated with daily intratumoral injection of CDX-301 for 5 days (Day 1-5), radiation therapy (9Gy, Day 8) followed by administration of CDX-1140 and Poly-ICLC (Day 9). Up to 4 cycles of this combination therapy can be given every 3 weeks. The primary endpoints are safety/tolerability and to evaluate the MTD of for injection (intratumoral in the cohort A and intratumoral and intravenous in the cohort B) of CDX-1140. A standard 3+3 design, allowing for dose de-escalation, will be used within each cohort. Secondary endpoints are to evaluate immune signatures in blood and the tumor. This phase 1 trial opened to enrollment at the University of Southern California Norris Comprehensive Cancer Center in January 2023 (recruitment is currently 4 on February 6, 2024). 1. Nature Communications 2020. 2. J. Immunology 2021. 3. Cancer Research 2021. 4. Scientific Reports 2022. Clinical trial information: NCT04616248 .

Systemic amyloidosis is a group of diseases characterized by the deposition of misfolded proteins in various organs and tissues, leading to progressive organ dysfunction and failure. Congo red stain is the gold standard chemical stain for the visualization of amyloid deposits in tissue sections, as it forms complexes with the misfolded proteins and shows a birefringence pattern under polarized light microscopy. However, Congo red staining is tedious and costly to perform, and prone to false diagnoses due to variations in the amount of amyloid, staining quality and expert interpretation through manual examination of tissue under a polarization microscope. Here, we report the first demonstration of virtual birefringence imaging and virtual Congo red staining of label-free human tissue to show that a single trained neural network can rapidly transform autofluorescence images of label-free tissue sections into brightfield and polarized light microscopy equivalent images, matching the histochemically stained versions of the same samples. We demonstrate the efficacy of our method with blind testing and pathologist evaluations on cardiac tissue where the virtually stained images agreed well with the histochemically stained ground truth images. Our virtually stained polarization and brightfield images highlight amyloid birefringence patterns in a consistent, reproducible manner while mitigating diagnostic challenges due to variations in the quality of chemical staining and manual imaging processes as part of the clinical workflow.

Little is known about the efficacy of COVID‐19 vaccines during acute lymphoblastic leukemia therapy (ALL); data for COVID‐19 vaccine immune responses in pediatric leukemia remain sparse. We conducted a single center study of patients aged 5–25 years undergoing ALL chemotherapy who received COVID‐19 vaccination. Twenty‐one patients were enrolled; efficacy was evaluable in 20. Twenty were vaccinated while receiving chemotherapy. Twenty received the BNT162b2 mRNA vaccine. Spike reactive antibodies (S‐IgG) and/or T‐cells (SRT) were detected in 16 of 20 (80%) vaccinated patients; 13 (65%) and 9 (45%) were positive for S‐IgG and SRT, respectively. Six (30%) showed both spike reactive B and T‐cell responses. Eleven of the 13 with S‐IgG positivity were negative for anti‐Nucleocapsid IgG, an antibody profile consistent with a vaccine induced immune response. All 13S‐IgG+ patients showed neutralizing antibodies. SRT included CD4+ (7) and CD8+ (6) T‐cells; both CD4+ and CD8+ SRT were seen in 4. SRT were multifunctional (producing multiple cytokines) in most patients (8 of 9); 4 showed SRT with triple cytokine and B‐cell co‐stimulatory responses, indicating a multimodal adaptive immune response. Immune responses were seen among patients vaccinated in the settings of lymphopenia (6 of 12) intensive chemotherapy (3 of 4), and Peg allergy (6 of 8). Sequencing revealed public CD4+ and CD8+ TCR sequences reactive to epitopes across the spike protein. In conclusion, COVID‐19 vaccination induced B and/or T‐cell responses in a majority of children and young adults undergoing ALL chemotherapy.

Histological examination is a crucial step in an autopsy; however, the traditional histochemical staining of post-mortem samples faces multiple challenges, including the inferior staining quality due to autolysis caused by delayed fixation of cadaver tissue, as well as the resource-intensive nature of chemical staining procedures covering large tissue areas, which demand substantial labor, cost, and time. These challenges can become more pronounced during global health crises when the availability of histopathology services is limited, resulting in further delays in tissue fixation and more severe staining artifacts. Here, we report the first demonstration of virtual staining of autopsy tissue and show that a trained neural network can rapidly transform autofluorescence images of label-free autopsy tissue sections into brightfield equivalent images that match hematoxylin and eosin (H E) stained versions of the same samples, eliminating autolysis-induced severe staining artifacts inherent in traditional histochemical staining of autopsied tissue. Our virtual H E model was trained using >0.7 TB of image data and a data-efficient collaboration scheme that integrates the virtual staining network with an image registration network. The trained model effectively accentuated nuclear, cytoplasmic and extracellular features in new autopsy tissue samples that experienced severe autolysis, such as COVID-19 samples never seen before, where the traditional histochemical staining failed to provide consistent staining quality. This virtual autopsy staining technique can also be extended to necrotic tissue, and can rapidly and cost-effectively generate artifact-free H E stains despite severe autolysis and cell death, also reducing labor, cost and infrastructure requirements associated with the standard histochemical staining.

Abstract Demonstrating a combination of high prevalence, late detection, and low survival, Lung cancer is the leading cause of cancer related deaths worldwide (Sainz de Aja J, 2021) and Lung Adenocarcinoma (LUAD) is its most prevalent pathologic subtype (Zhou, 2021). Emerging from within the distal alveolar epithelium, LUAD presents with vast heterogeneity in histology, mutational spectra, and epigenomic dysregulation (Sainz de Aja J, 2021) (Zhou, 2021). Additionally, LUAD’s considerable intratumoral heterogeneity manifests vast genomic and phenotypic diversity across tumor cells and cellular components of the tumor microenvironment (TME) within the same tumor tissue. LUAD also reveals significant racial disparities in incidence, survival and mortality rates between White and Black men in the United States. The occurrence of LUAD is approximately 68.3 per capita for Black men and 61.5 per capita for White men, reflecting an 11% higher rate in the former. Further, survival outcomes indicate that Black men generally exhibit lower 5-year relative survival rates compared to White men, highlighting greater mortality risk and conspicuously poorer prognosis (Siegel RL et al., 2023). While addressing the social determinants of health is vital for the mitigation of health disparities, understanding the genetic and molecular foundations that underly racial health inequities is essential for developing comprehensive interventions and advancing more precise therapeutic modalities for more diverse patient populations. Single Nucleotide Polymorphisms (SNPs) can influence oncogenic transformation and may be associated with distinct molecular landscapes in tumors and adjacent TME. Furthermore, ancestry may play a pivotal role in shaping individual susceptibility and risk for mitochondrial dysfunction and ensuing oncogenic transformation. To elucidate the biological underpinnings of racial health disparities in LUAD, 10 Black and 10 White male LUAD patients’ tumor and normal corresponding adjacent tissues underwent molecular profiling for KRAS mutation status, Whole Exome Sequencing (WES) to determine ancestry, and analysis of associated LUAD induced Mitochondrial Somatic Nucleotide Variants (mtSNVs). We also employed the 10X Visium HD spatial transcriptomic platform to evaluate spatially resolved molecular and cellular differences in KRAS mediated LUAD between Black and White men. Hypothesis: Thus, we propose that global ancestry is associated with variations in intratumoral heterogeneity, TME composition, and mitochondrial dynamics, indicating a potential role in the observed disparities in susceptibility, malignancy and mortality in LUAD across diverse patient populations. Citation Format: Eun Kyu Sung, Yuxin Jin, Yonatan Amzaleg, John Carpten, William Dean Wallace, Dan Raz, Aaron M. Neely. Investigating the effects of ancestry in KRAS mediated lung adenocarcinoma: Exploring intratumoral heterogeneity and mitochondrial dynamics [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C169.

Abstract Originating from within the distal lung, lung adenocarcinoma (LUAD) is the most common pathologic subtype of non-small cell lung carcinoma (NSCLC) and encompasses an assortment of histologic presentations. Colocalized within the distal alveolar epithelium are two alveolar epithelial cell (AEC) subpopulations: alveolar epithelial type 1 (AT1) and alveolar epithelial type 2 (AT2) cells. We previously demonstrated that signatures unique to each AEC subpopulation were detected in LUAD tumors formed by oncogenic KRASG12D activation in mice and were associated with disparate survival outcomes, transcriptomic patterning, and histologic presentation. Epithelial growth factor receptor (EGFR) mutations are among the most frequent oncogenic drivers in thoracic oncology. Therefore, we set out to determine if EGFR oncogenic activation resulted in differential LUAD presentation and outcomes based on the cell of origin in which it was activated. To activate EGFR oncogenic mutations in AT1 or AT2 cells, we employed triple transgenic mouse models (AT1: Gramd2-CreERT2:EF1-LSL-tTA:TetO-EGFRdel20/T790 and AT2: Sftpc-CreERT2:EF1-LSL-tTA:TetO-EGFRdel20/T790), which allowed for EGFR activation in either Gramd2+ AT1 or Sftpc+ AT2 cells. Longitudinal analysis of hematoxylin and eosin-stained lungs over the course of 20 weeks was performed. Blinded pathology review demonstrated that EGFR oncogenic activation in Gramd2+ AT1 cells resulted in histologically defined LUAD with lepidic properties at early time points and solid adenocarcinoma properties at later time points. This differed from EGFR activation in Sftpc+ AT2 cells, which resulted in pre-carcinogenic atypical adenomatous hyperplasia (AAH) formation that did not progress to adenocarcinoma. While preliminary, our results suggest that cell of origin strongly influences EGFR-mediated lung oncogenesis and concomitant pathology and histologic presentation. This work may advance our understanding while informing patient stratification and treatment strategies for LUAD patients. Citation Format: Aaron M. Neely, Andrea Lanz, William Dean Wallace, Anthony W. Kim, Zea Borok, Crystal N. Marconett. Elucidating the tumorigenic properties of alveolar epithelial type 1 cells in EGFR-mediated LUAD tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6997.

We present deep learning-based virtual staining of unlabeled lung and heart tissue sections to diagnose organ transplant rejection, achieving comparable diagnostic accuracy to histochemical staining methods, while significantly reducing staining-related time, cost and labor.

This study aimed to identify cellular and molecular pathways mediating severe maternal immune activation (MIA) effects on placental permeability and vasculature in a mouse model. Pharmacological and immunohistological methods identified the role of cyclooxygenase-2 (COX2) pathway and cellular localization of COX2 molecular pathway components to identify key players and potential therapeutic targets. Pregnant mice were injected with saline or a viral mimetic, polyinosinic-polycytidylic acid (polyI:C; 10 mg/kg), to induce inflammation on gestational day 13. After 24 hours, mice either received celecoxib (20 mg/kg) or vehicle only. On gestational day 15, live MRI was performed and specimens collected. Immunohistochemistry was used to evaluate placental pericyte coverage of endothelial cells and cellular localization of key elements of the COX 2 pathway. Live MRI revealed that severe MIA increases placental permeability to a gadolinium contrast agent. In vivo pharmacological inhibition experiments demonstrated that this effect is from a COX2-dependent signal which increases macrophage activity and decreases pericyte coverage of endothelial cells in the placental villi. Similar responses were observed in human placental histological samples from patients with infections. Immunohistological studies revealed COX2 expression in decidual cells, microsomal Prostaglandin E synthase-1 (mPGES1) in syncytiotrophoblasts, and Prostaglandin E₂ receptor 3 (EP3) in adjacent pericytes. Our results suggest that severe gestational MIA activates a decidual COX2 pathway leading to immune activation in placental villi and disruption of the blood-placental barrier integrity via EP3 receptor signaling in pericytes. Disruption of placental vascular structure and permeability may increase the risk of abnormal fetal development and negatively impact long-term health due to exposure to pathogens and inflammatory molecules. These results have implications extending beyond infection as COX2-mediated disruption of pericyte coverage of endothelial cells is observed in in vivo models of preeclampsia.

Clear cell renal cell carcinoma (ccRCC) is a common cancer and could result in poor prognosis. Understanding individual tumor immune microenvironment (TIME) in ccRCC patients may predict prognosis and response to therapy. In this work, we explore the concept of using radiomic features extracted from computer tomography (CT) imaging to correlate the TIME measurements from multiplex immunohistochemistry (mIHC) analysis. Since CT imaging has long been the standard for evaluation of RCCs, it has the potential to provide non-invasive approximations of the tissue-based mIHC biomarkers. We selected two biomarkers that were grounded by clinical research: PD-L1 expression and CD8(+)PD-1(+) T cell to CD8(+) T cell ratio of the tumor epithelium. Then we extracted these two markers from a preliminary set of 52 patients using automated mIHC analysis. We used Random Forest, AdaBoost and ElasticNet to classify each sample as either expressing high or low levels of these markers. We found the radiomic features can correlate tumor epithelium PD-L1 >5%, PD-L1 >10%, and CD8(+)PD1(+)/CD8(+) >37% with AUROC 0.75, 0.85 and 0.71, respectively.

Background: Triple-negative breast cancer (TNBC) is a molecularly heterogeneous group of clinically aggressive malignancies. There are well-recognized health disparities in TNBC outcomes, and the risk of TNBC is higher among African-Americans (AA). It is unclear whether immunological features of the tumor microenvironment (TME) associated with disease stage, socioeconomic factors, or comorbidities such as obesity may affect tumor immunity. The incidence of TNBC and obesity in Louisiana is among the highest in the nation, and we have documented disparities in incidence linked to race and disparities in mortality linked to social determinants of health. Recent studies described immunologic characteristics of the TNBC TME. However, the possible association of immunogenomic portraits of TNBCs with race, comorbidities or socioeconomic factors remains understudied. Methods: We studied the expression of immunity-associated genes in clinically annotated TNBCs from Louisiana AA and European-American (EA) patients with or without obesity. Primary invasive breast cancer cases with confirmed TNBC diagnosis were identified by the Louisiana Tumor Registry (LTR). Sections of FFPE tissue containing ≥ 50% tumor were identified and processed for RNA-Sequencing [(n = 256; White women:125 (Lean: 50 and Obese:75) and Black women:131 (Lean:28 and Obese: 103)] at Translational Genomic Core, LSUHSC. Categorical outcomes were compared via Chi-squared tests, and survival was compared via log-rank tests. Spearman correlation analysis was used to determine associations between CIBERSORT cell populations and stage of disease at diagnosis. Results: We found that race was associated with the stage of TNBC, and AA patients were more often diagnosed with a later stage of TNBC (p=0.0447). However, race was not associated with survival (p=0.4673). Obesity was not associated with stage at diagnosis (p=0.7256). Stage at diagnosis was the strongest determinant of survival (p<0.0001). We utilized CIBERSORT analysis to identify and quantify immune cell populations within the TME. Later stage at diagnosis was associated with increased T follicular helper cells (p=0.0038), M1 macrophages (p= 0.0032), and activated mast cells (p=0.0487). Conversely, later stage of disease was associated with decreased resting mast cells (p=0.0004) and monocytes (p=0.0455). Immunosuppressive Treg cells were positively associated with stage at diagnosis in AA patients (p=0.0273) but not in EA patients (p=0.9141). Conclusions: Stage at diagnosis was the strongest determinant of survival and was associated with significant differences in TME immune cell populations. Stage, race and obesity were associated with the presence of immunosuppressive Treg cells. If confirmed, these findings may help understand the variability in immunotherapy responses in TNBC. Citation Format: Fokhrul Hossain, Denise Danos, Jovanny Zabaleta, Jiande Wu, Mary Anne Lynch, Luis Del Valle, Xiao-Cheng Wu, Augusto Ochoa, Chindo Hicks, Lucio Miele. Understanding triple-negative breast cancer immune microenvironment by disease stages, obesity, and race [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2525.

Introduction: KK-LC-1 is a cancer testis antigen expressed notably in lung, breast, and gastric cancer making it an attractive target for T-cell receptor therapy. Here, we evaluate cell densities of NSCLC tissue specimens with known KK-LC-1 gene expression to better characterize the tumor immune microenvironment in such patients. Methods: A seven-plex mIF panel (FoxP3, PDL1, PanCK, PD1, CD8, KK-LC-1, and CD68) was developed based on a previously established mIF Lung-cancer panel and addition of KK-LC-1 following rigorous optimization of KK-LC-1 antibody titer and panel order with qualitative assessment of monoplex immunofluorescent assays. Serial sections from 23 NSCLC cases (14 biopsy samples and 9 excision specimens) with known KK-LC-1 gene expression values were collected. Tissue sections were stained and imaged on an Akoya Biosciences Vectra Polaris multispectral imaging platform. The regions of interest were identified and stamped with the Phenochart mIF image management software. Spectral unmixing, tissue segmentation and cell phenotyping were performed with Akoya Biosciences InForm image analysis software using the multispectral fields workflow. The 4-tier positive staining thresholds for PD-L1 and KK-LC-1 were determined by a pathologist (WDW). Cell densities in the tumor and adjacent stroma as well as the KK-LC-1 H-score were assessed by linear regression analysis. Student’s t-test was used to compare between the top quartile of tumors based on KK-LC-1 gene expression (Q4) and the remaining 3 quartiles (Q1-Q3). Results: The study consisted of 17/23 adenocarcinoma (73.9%), 4/23 squamous cell carcinoma (SCC) (17.3%), and 2/23 (8.6%) unclear or mixed histology tumors. Adenocarcinoma trended to have higher KK-LC-1 gene expression compared to SCC tumors (mean 17.34 vs. 0.65, p-value = 0.204). 13/23 (56.5%) were pan-wild type, 6/23 (26.0%) were EGFR mutated, 2/23 (8.7%) were KRAS mutated, and 1/23 (4.4%) were MET Exon 14 skip and multiple alterations. We saw significantly higher cell density of FoxP3 in tumor cells in Q4 versus Q1-Q3 (mean 7.17 vs. 0.8 cells/mm2, p-value = 0.004), and there was a trend towards a higher FoxP3/CD8 cell density ratio (mean 3.28 vs. 0.05, p = .110) and higher PD-1 cell density (mean 186.7 vs. 32.8 cells/mm2, p=0.1548) in tumor cells in Q4 versus Q1-Q3. There was significant correlation towards increased CD8+ T cell density ratio between tumor and stroma cells in cases with higher KK-LC-1 gene expression (r2= 0.307, p = 0.011). Conclusion: KK-LC-1 expression in lung cancer is seen in multiple molecular subsets and is associated with increase in both regulatory T cells in tumor and in CD8+ T-cells in tumor relative to stroma. Strategies to deplete regulatory T cells and to further amplify CD8+ T cells should be pursued in the development of T-cell receptor therapy targeting KK-LC-1. Citation Format: Ainaz Dory Barkhordarzadeh, Robert C. Hsu, Rongfu Wang, Sue E. Martin, Jorge J. Nieva, William Dean Wallace. Immune infiltrates in Kita-Kyushu lung cancer antigen 1 (KK-LC-1) expressing non-small cell lung cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2526.

CONTEXT.—:The adoption of digital capture of pathology slides as whole slide images (WSI) for educational and research applications has proven utility.OBJECTIVE.—:To compare pathologists' primary diagnoses derived from WSI versus the standard microscope. Because WSIs differ in format and method of observation compared with the current standard glass slide microscopy, this study is critical to potential clinical adoption of digital pathology.DESIGN.—:The study enrolled a total of 2045 cases enriched for more difficult diagnostic categories and represented as 5849 slides were curated and provided for diagnosis by a team of 19 reading pathologists separately as WSI or as glass slides viewed by light microscope. Cases were reviewed by each pathologist in both modalities in randomized order with a minimum 31-day washout between modality reads for each case. Each diagnosis was compared with the original clinical reference diagnosis by an independent central adjudication review.RESULTS.—:The overall major discrepancy rates were 3.64% for WSI review and 3.20% for manual slide review diagnosis methods, a difference of 0.44% (95% CI, -0.15 to 1.03). The time to review a case averaged 5.20 minutes for WSI and 4.95 minutes for glass slides. There was no specific subset of diagnostic category that showed higher rates of modality-specific discrepancy, though some categories showed greater discrepancy than others in both modalities.CONCLUSIONS.—:WSIs are noninferior to traditional glass slides for primary diagnosis in anatomic pathology.

Background: The majority of NSCLC patients do not respond to single agent PD-1/PD-L1 inhibitors, in part due to the lack of cytolytic T cell infiltration at the tumor site. To improve the efficacy of checkpoint blockade, in situ vaccination with functional antigen presenting cells (APCs) is designed to take advantage of the full repertoire of tumor antigens. This converts the tumor into a lymph node-like environment to promote T cell activation both locally and systemically. The chemokine CCL21 promotes co-localization of naive T cells and DCs to facilitate T cell activation. Our preclinical studies and recently completed phase I trial demonstrated that intratumoral (IT) administration of DC genetically modified to overexpress CCL21 (CCL21-DC) augments tumor antigen presentation in situ, resulting in more effective T cell responses and systemic antitumor immunity. We also observed increased PD-L1 expression in the tumor microenvironment (TME) in a subset of patients, suggesting that tumor-mediated impairment of T cell function may be forestalling a more robust antitumor response. Similarly, the lack of PD-1/PD-L1 inhibitor efficacy could be combated by enhanced T cell infiltration and augmented APC function following IT CCL21-DC delivery. Therefore, in a phase I trial initiated in November 2018, we are combining IT CCL21-DC with intravenous (IV) pembrolizumab to amplify host antitumor immunity in NSCLC with low or absent baseline PD-L1 expression, who often do not respond to PD-1 inhibition alone.Methods: This is a single institution, non-randomized, dose-escalating, multi-cohort trial followed by an expansion cohort at the dose established during dose escalation. Patients will be included with pathologically confirmed and radiographically measurable stage IV NSCLC expressing PD-L1 in less than 50% of cells without EGFR or ALK mutations, who have tumor accessible by either CT-guided intervention or bronchoscopy, and who are naïve to systemic treatment for NSCLC. Up to 12 patients will be evaluated in a dose escalation cohort. An additional 12 patients will be enrolled in an expansion cohort. Patients will receive three IT injections of autologous CCL21-DC (days 0, 21, 42) together with IV pembrolizumab. The IV pembrolizumab will continue every 3 weeks for up to 1 year. All patients will be monitored for clinical efficacy as well as local and systemic immune responses to define potential determinants of the response. The primary objective in the dose escalation will be to determine the safety and maximum tolerated dose (MTD) of IT CCL21-DC (5x106, 1x107, 3x107) when combined with IV pembrolizumab. The primary objective in the dose expansion will be to evaluate the objective response rate (ORR) in patients treated with MTD of CCL21-DC combined with IV pembrolizumab. This trial is registered at ClinicalTrial.gov (NCT03546361), and is currently open for enrollment.Citation Format: Bin Liu, Edward B. Garon, Aaron E. Lisberg, Ramin Salehi-Rad, Jay M. Lee, Linh M. Tran, Kostyantyn Krysan, Rui Li, Raymond J. Lim, Manash Paul, Ying Lin, Zhe Jing, Fereidoun Abtin, Robert D. Suh, Scott Oh, Denise R. Aberle, Lauren E. Winter, William Dean Wallace, David Elashoff, Sherven Sharma, Steven M. Dubinett. A Phase I trial of in situ vaccination with autologous CCL21-modified dendritic cells (CCL21-DC) combined with pembrolizumab for advanced NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT226.

We have identified 25 lesions involving alveolar lung parenchyma characterized by nodular proliferation of bland bilayered bronchiolar-type epithelium containing a continuous layer of basal cells. These lesions shared some histologic features with the recently described entity of ciliated muconodular papillary tumor (CMPT); however, the majority did not fit all diagnostic criteria in that they exhibited only focal or absent papillary architecture, and they had variable number of ciliated and mucinous cells, with some lesions entirely lacking 1 or both of these components. The morphologic and immunohistochemical features ranged from those resembling proximal bronchioles (proximal-type: moderate to abundant mucinous and ciliated cells; negative or weak TTF1 in luminal cells; n=8) to those resembling respiratory bronchioles (distal-type: scant or absent mucinous and ciliated cells; positive TTF1 in luminal cells; n=17). The hallmark of all lesions was a continuous layer of basal cells (p40 and CK5/6-positive). We provisionally designated these lesions as bronchiolar adenomas (BAs) and analyzed their clinicopathologic and molecular features. All BAs were discrete, sharply circumscribed lesions with a median size of 0.5 cm (range, 0.2 to 2.0 cm). Most lesions were either entirely flat (n=14) or contained focal papillary architecture (n=7); only 4 lesions, all proximal-type, were predominantly papillary, fitting the classic description of CMPT. Notably, of 9 lesions submitted for frozen section evaluation, 7 were diagnosed as adenocarcinoma. No postsurgical recurrences were observed for any lesions (median follow-up, 11 mo). Twenty-one BAs underwent next-generation sequencing and/or immunohistochemistry for BRAF V600E, revealing mutation profiles similar to those previously described for CMPTs, including BRAF V600E mutations (n=8, 38%), unusual EGFR exon 19 deletions (n=2, 10%), EGFR exon 20 insertions (n=2, 10%), KRAS mutations (n=5, 24%), and HRAS mutations (n=1, 5%). The mutation profiles were similar in proximal-type and distal-type lesions. In conclusion, we describe a family of putatively benign clonal proliferations with a spectrum of morphology recapitulating various levels of the bronchiolar tree, of which only a minor subset fits the classic description of CMPT. Comparable mutation profiles and partially overlapping morphologic features across the spectrum of these lesions support their nosological relationship. We propose designating this entire family of lesions as BAs, and that lesions currently designated CMPTs represent a subgroup of this family.