Scoring PD-L1 Expression in Urothelial Carcinoma: An International Multi-Institutional Study on Comparison of Manual and Artificial Intelligence Measurement Model (AIM-PD-L1) Pathology Assessments.

Assessing programmed death ligand 1 (PD-L1) expression on tumor cells (TCs) using Food and Drug Administration-approved, validated immunoassays can guide the use of immune checkpoint inhibitor (ICI) therapy in cancer treatment. However, substantial interobserver variability has been reported using these immunoassays. Artificial intelligence (AI) has the potential to accurately measure biomarker expression in tissue samples, but its reliability and comparability to standard manual scoring remain to be evaluated. This multinational study sought to compare the %TC scoring of PD-L1 expression in advanced urothelial carcinoma, assessed by either an AI Measurement Model (AIM-PD-L1) or expert pathologists. The concordance among pathologists and between pathologists and AIM-PD-L1 was determined. The positivity rate of ≥ 1%TC PD-L1 was between 20-30% for 8/10 pathologists, and the degree of agreement and scoring distribution for among pathologists and between pathologists and AIM-PD-L1 was similar both scored as a continuous variable or using the pre-defined cutoff. Numerically higher score variation was observed with the 22C3 assay than with the 28-8 assay. A 2-h training module on the 28-8 assay did not significantly impact manual assessment. Cases exhibiting significantly higher variability in the assessment of PD-L1 expression (mean absolute deviation > 10) were found to have patterns of PD-L1 staining that were more challenging to interpret. An improved understanding of sources of manual scoring variability can be applied to PD-L1 expression analysis in the clinical setting. In the future, the application of AI algorithms could serve as a valuable reference guide for pathologists while scoring PD-L1.

Comparing anti-tumor and anti-self immunity in a patient with melanoma receiving immune checkpoint blockade.

BACKGROUND: Tumor regression following immune checkpoint blockade (ICB) is often associated with immune-related adverse events (irAEs), marked by inflammation in non-cancerous tissues. This study was undertaken to investigate the functional relationship between anti-tumor and anti-self immunity, to facilitate irAE management while promoting anti-tumor immunity. METHODS: Multiple biopsies from tumor and inflamed tissues were collected from a patient with melanoma experiencing both tumor regression and irAEs on ICB, who underwent rapid autopsy. Immune cells infiltrating melanoma lesions and inflamed normal tissues were subjected to gene expression profiling with multiplex qRT-PCR for 122 candidate genes. Subsequently, immunohistochemistry was conducted to assess the expression of 14 candidate markers of immune cell subsets and checkpoints. TCR-beta sequencing was used to explore T cell clonal repertoires across specimens. RESULTS: While genes involved in MHC I/II antigen presentation, IFN signaling, innate immunity and immunosuppression were abundantly expressed across specimens, irAE tissues over-expressed certain genes associated with immunosuppression (CSF1R, IL10RA, IL27/EBI3, FOXP3, KLRG1, SOCS1, TGFB1), including those in the COX-2/PGE2 pathway (IL1B, PTGER1/EP1 and PTGER4/EP4). Immunohistochemistry revealed similar proportions of immunosuppressive cell subsets and checkpoint molecules across samples. TCRseq did not indicate common TCR repertoires across tumor and inflammation sites, arguing against shared antigen recognition between anti-tumor and anti-self immunity in this patient. CONCLUSIONS: This comprehensive study of a single patient with melanoma experiencing both tumor regression and irAEs on ICB explores the immune landscape across these tissues, revealing similarities between anti-tumor and anti-self immunity. Further, it highlights expression of the COX-2/PGE2 pathway, which is known to be immunosuppressive and potentially mediates ICB resistance. Ongoing clinical trials of COX-2/PGE2 pathway inhibitors targeting the major COX-2 inducer IL-1B, COX-2 itself, or the PGE2 receptors EP2 and EP4 present new opportunities to promote anti-tumor activity, but may also have the potential to enhance the severity of ICB-induced irAEs.

Entinostat, nivolumab and ipilimumab for women with advanced HER2-negative breast cancer: a phase Ib trial.

We report the results of 24 women, 50% (N = 12) with hormone receptor-positive breast cancer and 50% (N = 12) with advanced triple-negative breast cancer, treated with entinostat + nivolumab + ipilimumab from the dose escalation (N = 6) and expansion cohort (N = 18) of ETCTN-9844 ( NCT02453620 ). The primary endpoint was safety. Secondary endpoints were overall response rate, clinical benefit rate, progression-free survival and change in tumor CD8:FoxP3 ratio. There were no dose-limiting toxicities. Among evaluable participants (N = 20), the overall response rate was 25% (N = 5), with 40% (N = 4) in triple-negative breast cancer and 10% (N = 1) in hormone receptor-positive breast cancer. The clinical benefit rate was 40% (N = 8), and progression-free survival at 6 months was 50%. Exploratory analyses revealed that changes in myeloid cells may contribute to responses; however, no correlation was noted between changes in CD8:FoxP3 ratio, PD-L1 status and tumor mutational burden and response. These findings support further investigation of this treatment in a phase II trial.

Neoadjuvant radioimmunotherapy in pancreatic cancer enhances effector T cell infiltration and shortens their distances to tumor cells.

Radiotherapy is hypothesized to have an immune-modulating effect on the tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) to sensitize it to anti-PD-1 antibody (a-PD-1) treatment. We collected paired pre- and posttreatment specimens from a clinical trial evaluating combination treatment with GVAX vaccine, a-PD-1, and stereotactic body radiation (SBRT) following chemotherapy for locally advanced PDACs (LAPC). With resected PDACs following different neoadjuvant therapies as comparisons, effector cells in PDACs were found to skew toward a more exhausted status in LAPCs following chemotherapy. The combination of GVAX/a-PD-1/SBRT drives TME to favor antitumor immune response including increased densities of GZMB+CD8+ T cells, TH1, and TH17, which are associated with longer survival, however increases immunosuppressive M2-like tumor-associated macrophages (TAMs). Adding SBRT to GVAX/a-PD-1 shortens the distances from PD-1+CD8+ T cells to tumor cells and to PD-L1+ myeloid cells, which portends prolonged survival. These findings have guided the design of next radioimmunotherapy studies by targeting M2-like TAM in PDACs.

SREBP-dependent regulation of lipid homeostasis is required for progression and growth of pancreatic ductal adenocarcinoma.

Metabolic reprogramming is a necessary component of oncogenesis and cancer progression that solid tumors undergo when their growth outstrips local nutrient supply. The supply of lipids such as cholesterol and fatty acids is required for continued tumor cell proliferation, and oncogenic mutations stimulate de novo lipogenesis to support tumor growth. Sterol regulatory element-binding protein (SREBP) transcription factors control cellular lipid homeostasis by activating genes required for lipid synthesis and uptake. SREBPs have been implicated in the progression of multiple cancers, including brain, breast, colon, liver, and prostate. However, the role the SREBP pathway and its central regulator SREBP cleavage activating protein (SCAP) in pancreatic ductal adenocarcinoma (PDAC) has not been studied in detail. Here, we demonstrated that pancreas-specific knockout of Scap has no effect on mouse pancreas development or function, allowing for examination of the role for Scap in the murine KPC model of PDAC. Notably, heterozygous loss of Scap prolonged survival in KPC mice, and homozygous loss of Scap impaired PDAC tumor progression. Using subcutaneous and orthotopic xenograft models, we showed that S CAP is required for human PDAC tumor growth. Mechanistically, chemical or genetic inhibition of the SREBP pathway prevented PDAC cell growth under low serum conditions due to a lack of lipid supply. Highlighting the clinical importance of this pathway, the SREBP pathway is broadly required for cancer cell growth, SREBP target genes are upregulated in human PDAC tumors, and increased expression of SREBP targets genes is associated with poor survival in PDAC patients. Collectively, these results demonstrate that SCAP and the SREBP pathway activity are essential for PDAC cell and tumor growth in vitro and in vivo , identifying SCAP as a potential therapeutic target for PDAC. SIGNIFICANCE: Our findings demonstrate that SREBP pathway activation is a critical part of the metabolic reprogramming that occurs in PDAC development and progression. Therefore, targeting the SREBP pathway has significant therapeutic potential.

Association between pathologic response and survival after neoadjuvant therapy in lung cancer.

Neoadjuvant immunotherapy plus chemotherapy improves event-free survival (EFS) and pathologic complete response (0% residual viable tumor (RVT) in primary tumor (PT) and lymph nodes (LNs)), and is approved for treatment of resectable lung cancer. Pathologic response assessment after neoadjuvant therapy is the potential analog to radiographic response for advanced disease. However, %RVT thresholds beyond pathologic complete response and major pathologic response (≤10% RVT) have not been explored. Pathologic response was prospectively assessed in the randomized, phase 3 CheckMate 816 trial (NCT02998528), which evaluated neoadjuvant nivolumab (anti-programmed death protein 1) plus chemotherapy in patients with resectable lung cancer. RVT, regression and necrosis were quantified (0-100%) in PT and LNs using a pan-tumor scoring system and tested for association with EFS in a prespecified exploratory analysis. Regardless of LN involvement, EFS improved with 0% versus >0% RVT-PT (hazard ratio = 0.18). RVT-PT predicted EFS for nivolumab plus chemotherapy (area under the curve = 0.74); 2-year EFS rates were 90%, 60%, 57% and 39% for patients with 0-5%, >5-30%, >30-80% and >80% RVT, respectively. Each 1% RVT associated with a 0.017 hazard ratio increase for EFS. Combining pathologic response from PT and LNs helped differentiate outcomes. When compared with radiographic response and circulating tumor DNA clearance, %RVT best approximated EFS. These findings support pathologic response as an emerging survival surrogate. Further assessment of the full spectrum of %RVT in lung cancer and other tumor types is warranted. ClinicalTrials.gov registration: NCT02998528 .

Discovery and characterization of cross-reactive intrahepatic antibodies in severe alcoholic hepatitis.

The pathogenesis of antibodies in severe alcoholic hepatitis (SAH) remains unknown. We analyzed immunoglobulins (Ig) in explanted livers from SAH patients (n=45) undergoing liver transplantation and tissues from corresponding healthy donors (HD, n=10) and found massive deposition of IgG and IgA isotype antibodies associated with complement fragment C3d and C4d staining in ballooned hepatocytes in SAH livers. Ig extracted from SAH livers, but not patient serum exhibited hepatocyte killing efficacy. Employing human and Escherichia coli K12 proteome arrays, we profiled the antibodies extracted from explanted SAH, livers with other diseases, and HD livers. Compared with their counterparts extracted from livers with other diseases and HD, antibodies of IgG and IgA isotypes were highly accumulated in SAH and recognized a unique set of human proteins and E. coli antigens. Further, both Ig- and E. coli-captured Ig from SAH livers recognized common autoantigens enriched in several cellular components including cytosol and cytoplasm (IgG and IgA), nucleus, mitochondrion, and focal adhesion (IgG). Except IgM from primary biliary cholangitis livers, no common autoantigen was recognized by Ig- and E. coli-captured Ig from livers with other diseases. These findings demonstrate the presence of cross-reacting anti-bacterial IgG and IgA autoantibodies in SAH livers.

PSMA-Targeted PET Radiotracer [18F]DCFPyL as an Imaging Biomarker in Inflammatory Bowel Disease.

Background: Prostate-specific membrane antigen (PSMA) is highly and specifically upregulated in active-inflamed mucosa of patients with inflammatory bowel disease (IBD). We hypothesized that this upregulation would be detectable using a PSMA-targeted positron emission tomography/computed tomography (PET/CT) imaging agent, [18F]DCFPyL, enabling non-invasive visualization of inflammation. A noninvasive means of detecting active inflammation would have high clinical value in localization and management of IBD. Study: We performed [18F]DCFPyL imaging in three IBD patients with active disease. Abnormally increased gastrointestinal [18F]DCFPyL uptake was observed in areas with endoscopic, histologic, and immunohistochemical inflammation, demonstrating partial overlap of segments of bowel with abnormal [18F]DCFPyL uptake and active inflammation. Conclusion: This study demonstrates that PSMA-targeted [18F]DCFPyL PET can effectively detect regions of inflamed mucosa in patients with IBD, suggesting its utility as a non-invasive imaging agent to assess location, extent, and disease activity in IBD.

Immune landscape of tertiary lymphoid structures in hepatocellular carcinoma (HCC) treated with neoadjuvant immune checkpoint blockade.

Neoadjuvant immunotherapy is thought to produce long-term remissions through induction of antitumor immune responses before removal of the primary tumor. Tertiary lymphoid structures (TLS), germinal center-like structures that can arise within tumors, may contribute to the establishment of immunological memory in this setting, but understanding of their role remains limited. Here, we investigated the contribution of TLS to antitumor immunity in hepatocellular carcinoma (HCC) treated with neoadjuvant immunotherapy. We found that neoadjuvant immunotherapy induced the formation of TLS, which were associated with superior pathologic response, improved relapse free survival, and expansion of the intratumoral T and B cell repertoire. While TLS in viable tumor displayed a highly active mature morphology, in areas of tumor regression we identified an involuted TLS morphology, which was characterized by dispersion of the B cell follicle and persistence of a T cell zone enriched for ongoing antigen presentation and T cell-mature dendritic cell interactions. Involuted TLS showed increased expression of T cell memory markers and expansion of CD8+ cytotoxic and tissue resident memory clonotypes. Collectively, these data reveal the circumstances of TLS dissolution and suggest a functional role for late-stage TLS as sites of T cell memory formation after elimination of viable tumor.

Balancing Safety and Efficacy to Determine the Most Suitable Size of Imaging-Visible Embolic Microspheres for Bariatric Arterial Embolization in a Preclinical Model.

OBJECTIVES: To identify the most suitable size of imaging-visible embolic agents with balanced safety and efficacy for bariatric arterial embolization (BAE) in a preclinical model. MATERIALS AND METHODS: Twenty-seven pigs were divided into 3 cohorts. In Cohort I, 16 pigs were randomized to receive (n = 4 each) 40-100-µm microspheres in 1 or 2 fundal arteries, 70-340-µm radiopaque microspheres in 2 fundal arteries, or saline. In Cohort II, 3 pigs underwent renal arterial embolization with either custom-made 100-200-µm, 200-250-µm, 200-300-µm, or 300-400-µm radiopaque microspheres or Bead Block 300-500 µm with microsphere distribution assessed histologically. In Cohort III, 8 pigs underwent BAE in 2 fundal arteries with tailored 100-200-µm radiopaque microspheres (n = 5) or saline (n = 3). RESULTS: In Cohort I, no significant differences in weight or ghrelin expression were observed between BAE and control animals. Moderate-to-severe gastric ulcerations were noted in all BAE animals. In Cohort II, renal embolization with 100-200-µm microspheres occluded vessels with a mean diameter of 139 µm ± 31, which is within the lower range of actual diameters of Bead Block 300-500 µm. In Cohort III, BAE with 100-200-µm microspheres resulted in significantly lower weight gain (42.3% ± 5.7% vs 51.6% ± 2.9% at 8 weeks; P = .04), fundal ghrelin cell density (16.1 ± 6.7 vs 23.6 ± 12.6; P = .045), and plasma ghrelin levels (1,709 pg/mL ± 172 vs 4,343 pg/mL ± 1,555; P < .01) compared with controls and superficial gastric ulcers (5/5). CONCLUSIONS: In this preclinical model, tailored 100-200-µm microspheres were shown to be most suitable for BAE in terms of safety and efficacy.

Spatial transcriptomics analysis of neoadjuvant cabozantinib and nivolumab in advanced hepatocellular carcinoma identifies independent mechanisms of resistance and recurrence.

BACKGROUND: Novel immunotherapy combination therapies have improved outcomes for patients with hepatocellular carcinoma (HCC), but responses are limited to a subset of patients. Little is known about the inter- and intra-tumor heterogeneity in cellular signaling networks within the HCC tumor microenvironment (TME) that underlie responses to modern systemic therapy. METHODS: We applied spatial transcriptomics (ST) profiling to characterize the tumor microenvironment in HCC resection specimens from a prospective clinical trial of neoadjuvant cabozantinib, a multi-tyrosine kinase inhibitor that primarily blocks VEGF, and nivolumab, a PD-1 inhibitor in which 5 out of 15 patients were found to have a pathologic response at the time of resection. RESULTS: ST profiling demonstrated that the TME of responding tumors was enriched for immune cells and cancer-associated fibroblasts (CAF) with pro-inflammatory signaling relative to the non-responders. The enriched cancer-immune interactions in responding tumors are characterized by activation of the PAX5 module, a known regulator of B cell maturation, which colocalized with spots with increased B cell marker expression suggesting strong activity of these cells. HCC-CAF interactions were also enriched in the responding tumors and were associated with extracellular matrix (ECM) remodeling as there was high activation of FOS and JUN in CAFs adjacent to the tumor. The ECM remodeling is consistent with proliferative fibrosis in association with immune-mediated tumor regression. Among the patients with major pathologic responses, a single patient experienced early HCC recurrence. ST analysis of this clinical outlier demonstrated marked tumor heterogeneity, with a distinctive immune-poor tumor region that resembles the non-responding TME across patients and was characterized by HCC-CAF interactions and expression of cancer stem cell markers, potentially mediating early tumor immune escape and recurrence in this patient. CONCLUSIONS: These data show that responses to modern systemic therapy in HCC are associated with distinctive molecular and cellular landscapes and provide new targets to enhance and prolong responses to systemic therapy in HCC.

Relationship of Hepatocellular Carcinoma Stage and Hepatic Function to Health-Related Quality of Life: A Single Center Analysis.

Health-related quality of life (HRQoL) is known to be an important prognostic indicator and clinical endpoint for patients with hepatocellular carcinoma (HCC). However, the correlation of the Barcelona Clinic Liver Cancer (BCLC) stage with HRQoL in HCC has not been previously studied. We examined the relationship between BCLC stage, Child-Pugh (CP) score, and Eastern Cooperative Oncology Group (ECOG) performance status on HRQoL for patients who presented at a multidisciplinary liver cancer clinic. HRQoL was assessed using the Functional Assessment of Cancer Therapy-Hepatobiliary (FACT-Hep) questionnaire. Fifty-one patients met our inclusion criteria. The FACT-Hep total and subscales showed no significant association with BCLC stages (p = 0.224). Patients with CP B had significantly more impairment in FACT-Hep than patients with CP A. These data indicate that in patients with HCC, impaired liver function is associated with reduced quality of life, whereas the BCLC stage poorly correlates with quality of life metrics. Impairment of quality of life is common in HCC patients and further studies are warranted to determine the impact of early supportive interventions on HRQoL and survival outcomes.

Multi-Institutional Evaluation of Pathologists' Assessment Compared to Immunoscore.

BACKGROUND: The Immunoscore (IS) is a quantitative digital pathology assay that evaluates the immune response in cancer patients. This study reports on the reproducibility of pathologists' visual assessment of CD3+- and CD8+-stained colon tumors, compared to IS quantification. METHODS: An international group of expert pathologists evaluated 540 images from 270 randomly selected colon cancer (CC) cases. Concordance between pathologists' T-score, corresponding hematoxylin-eosin (H&E) slides, and the digital IS was evaluated for two- and three-category IS. RESULTS: Non-concordant T-scores were reported in more than 92% of cases. Disagreement between semi-quantitative visual assessment of T-score and the reference IS was observed in 91% and 96% of cases before and after training, respectively. Statistical analyses showed that the concordance index between pathologists and the digital IS was weak in two- and three-category IS, respectively. After training, 42% of cases had a change in T-score, but no improvement was observed with a Kappa of 0.465 and 0.374. For the 20% of patients around the cut points, no concordance was observed between pathologists and digital pathology analysis in both two- and three-category IS, before or after training (all Kappa < 0.12). CONCLUSIONS: The standardized IS assay outperformed expert pathologists' T-score evaluation in the clinical setting. This study demonstrates that digital pathology, in particular digital IS, represents a novel generation of immune pathology tools for reproducible and quantitative assessment of tumor-infiltrated immune cell subtypes.

Informing virtual clinical trials of hepatocellular carcinoma with spatial multi-omics analysis of a human neoadjuvant immunotherapy clinical trial.

Human clinical trials are important tools to advance novel systemic therapies improve treatment outcomes for cancer patients. The few durable treatment options have led to a critical need to advance new therapeutics in hepatocellular carcinoma (HCC). Recent human clinical trials have shown that new combination immunotherapeutic regimens provide unprecedented clinical response in a subset of patients. Computational methods that can simulate tumors from mathematical equations describing cellular and molecular interactions are emerging as promising tools to simulate the impact of therapy entirely in silico. To facilitate designing dosing regimen and identifying potential biomarkers, we developed a new computational model to track tumor progression at organ scale while reflecting the spatial heterogeneity in the tumor at tissue scale in HCC. This computational model is called a spatial quantitative systems pharmacology (spQSP) platform and it is also designed to simulate the effects of combination immunotherapy. We then validate the results from the spQSP system by leveraging real-world spatial multi-omics data from a neoadjuvant HCC clinical trial combining anti-PD-1 immunotherapy and a multitargeted tyrosine kinase inhibitor (TKI) cabozantinib. The model output is compared with spatial data from Imaging Mass Cytometry (IMC). Both IMC data and simulation results suggest closer proximity between CD8 T cell and macrophages among non-responders while the reverse trend was observed for responders. The analyses also imply wider dispersion of immune cells and less scattered cancer cells in responders' samples. We also compared the model output with Visium spatial transcriptomics analyses of samples from post-treatment tumor resections in the original clinical trial. Both spatial transcriptomic data and simulation results identify the role of spatial patterns of tumor vasculature and TGFß in tumor and immune cell interactions. To our knowledge, this is the first spatial tumor model for virtual clinical trials at a molecular scale that is grounded in high-throughput spatial multi-omics data from a human clinical trial.

NCCN Guidelines® Insights: Biliary Tract Cancers, Version 2.2023.

In 2023, the NCCN Guidelines for Hepatobiliary Cancers were divided into 2 separate guidelines: Hepatocellular Carcinoma and Biliary Tract Cancers. The NCCN Guidelines for Biliary Tract Cancers provide recommendations for the evaluation and comprehensive care of patients with gallbladder cancer, intrahepatic cholangiocarcinoma, and extrahepatic cholangiocarcinoma. The multidisciplinary panel of experts meets at least on an annual basis to review requests from internal and external entities as well as to evaluate new data on current and emerging therapies. These Guidelines Insights focus on some of the recent updates to the NCCN Guidelines for Biliary Tract Cancers as well as the newly published section on principles of molecular testing.

A platform trial of neoadjuvant and adjuvant antitumor vaccination alone or in combination with PD-1 antagonist and CD137 agonist antibodies in patients with resectable pancreatic adenocarcinoma.

A neoadjuvant immunotherapy platform clinical trial allows for rapid evaluation of treatment-related changes in tumors and identifying targets to optimize treatment responses. We enrolled patients with resectable pancreatic adenocarcinoma into such a platform trial (NCT02451982) to receive pancreatic cancer GVAX vaccine with low-dose cyclophosphamide alone (Arm A; n = 16), with anti-PD-1 antibody nivolumab (Arm B; n = 14), and with both nivolumab and anti-CD137 agonist antibody urelumab (Arm C; n = 10), respectively. The primary endpoint for Arms A/B - treatment-related change in IL17A expression in vaccine-induced lymphoid aggregates - was previously published. Here, we report the primary endpoint for Arms B/C: treatment-related change in intratumoral CD8+ CD137+ cells and the secondary outcomes including safety, disease-free and overall survivals for all Arms. Treatment with GVAX+nivolumab+urelumab meets the primary endpoint by significantly increasing intratumoral CD8+ CD137+ cells (p = 0.003) compared to GVAX+Nivolumab. All treatments are well-tolerated. Median disease-free and overall survivals, respectively, are 13.90/14.98/33.51 and 23.59/27.01/35.55 months for Arms A/B/C. GVAX+nivolumab+urelumab demonstrates numerically-improved disease-free survival (HR = 0.55, p = 0.242; HR = 0.51, p = 0.173) and overall survival (HR = 0.59, p = 0.377; HR = 0.53, p = 0.279) compared to GVAX and GVAX+nivolumab, respectively, although not statistically significant due to small sample size. Therefore, neoadjuvant and adjuvant GVAX with PD-1 blockade and CD137 agonist antibody therapy is safe, increases intratumoral activated, cytotoxic T cells, and demonstrates a potentially promising efficacy signal in resectable pancreatic adenocarcinoma that warrants further study.

Uncovering the spatial landscape of molecular interactions within the tumor microenvironment through latent spaces.

Recent advances in spatial transcriptomics (STs) enable gene expression measurements from a tissue sample while retaining its spatial context. This technology enables unprecedented in situ resolution of the regulatory pathways that underlie the heterogeneity in the tumor as well as the tumor microenvironment (TME). The direct characterization of cellular co-localization with spatial technologies facilities quantification of the molecular changes resulting from direct cell-cell interaction, as it occurs in tumor-immune interactions. We present SpaceMarkers, a bioinformatics algorithm to infer molecular changes from cell-cell interactions from latent space analysis of ST data. We apply this approach to infer the molecular changes from tumor-immune interactions in Visium spatial transcriptomics data of metastasis, invasive and precursor lesions, and immunotherapy treatment. Further transfer learning in matched scRNA-seq data enabled further quantification of the specific cell types in which SpaceMarkers are enriched. Altogether, SpaceMarkers can identify the location and context-specific molecular interactions within the TME from ST data.

Multi-Institutional Study of Pathologist Reading of the Programmed Cell Death Ligand-1 Combined Positive Score Immunohistochemistry Assay for Gastric or Gastroesophageal Junction Cancer.

The assessment of the expression of programmed cell death ligand-1 (PD-L1) using immunohistochemistry (IHC) has been controversial since its introduction. The methods of assessment and the range of assays and platforms contribute to confusion. Perhaps the most challenging aspect of PD-L1 IHC is the combined positive score (CPS) method of interpretation of IHC results. Although the CPS method is prescribed for more indications than any other PD-L1 scoring system, its reproducibility has never been rigorously assessed. In this study, we collected a series of 108 gastric or gastroesophageal junction cancer cases, stained them using the Food and Drug Administration-approved 22C3 assay, scanned them, and then circulated them to 14 pathologists at 13 institutions for the assessment of interpretative concordance for the CPS system. We found that higher cut points (10 or 20) performed better than a CPS of <1 or >1. We used the Observers Needed to Evaluate Subjective Tests algorithm to assess how the CPS system might perform in the real-world setting and found that the cut points of <1 or >1 showed an overall percent agreement of only 30% among the pathologist raters, with a plateau occurring at 8 raters. The raters performed better at higher cut points. However, the best cut point of <20 versus that of >20 was still disappointing, with a plateau at an overall percent agreement of 70% (at 7 raters). Although there is no ground truth for CPS, we compared the score with quantitative messenger RNA measurement and showed no relationship between the score (at any cut point) and messenger RNA amount. In summary, we showed that CPS shows high subjective variability among pathologist readers and is likely to perform poorly in the real-world setting. This system may be the root cause of the poor specificity and relatively low predictive value of IHC companion diagnostic tests for PD-1 axis therapies that use the CPS system.