PD-L1 Staining Research Articles

Biomarkers of immune checkpoint inhibitor response and toxicity: Challenges and opportunities.

Immune checkpoint inhibitors have transformed cancer therapy, but their optimal use is still constrained by lack of response and toxicity. Biomarkers of response may facilitate drug development by allowing appropriate therapy selection and focusing clinical trial enrollment. However, aside from PD-L1 staining in a subset of tumors and rarely mismatch repair deficiency, no biomarkers are routinely used in the clinic. In addition, severe toxicities may cause severe morbidity, therapy discontinuation, and even death. Here, we review the state of the field with a focus on our research in therapeutic biomarkers and toxicities from immune checkpoint inhibitors.

Expression and role of the immune checkpoint regulator PD-L1 in the tumor-stroma interplay of pancreatic ductal adenocarcinoma.

Immune checkpoint inhibitors (ICI), e.g., targeting programmed cell death protein 1-ligand 1 (PD-L1) or its receptor PD-1, have markedly improved the therapy of many cancers but so far failed in pancreatic ductal adenocarcinoma (PDAC). Macrophages represent one of the most abundant immune cell populations within the tumor microenvironment (TME) of PDAC being able to either support or restrain tumor progression depending on their phenotype. To better understand treatment failure of PD-L1/PD-1 inhibitors in PDAC, this study examined PD-L1 expression in the context of a dynamic TME in PDAC with a particular focus on the impact of macrophages. Formalin-fixed and paraffin embedded tissue samples of primary PDAC tissues and corresponding liver metastases were used for immunohistochemical analyses. Serial sections were stained with antibodies detecting Pan-Cytokeratin, CD68, CD163, CD8, and PD-L1.To investigate whether the PD-1/PD-L1 axis and macrophages contribute to immune escape of PDAC cells, a stroma enriched 3D spheroid coculture model was established in vitro, using different PDAC cell lines and macrophages subtypes as well as CD8+ T cells. Functional and flow cytometry analyses were conducted to characterize cell populations. Immunohistochemical analyses revealed that PD-L1 is mainly expressed by stroma cells, including macrophages and not PDAC cells in primary PDAC tissues and corresponding liver metastases. Notably, high local abundance of macrophages and strong PD-L1 staining were commonly found at invasion fronts of tumoral lesions between CD8+ T cells and tumor cells. In order to investigate whether PD-L1 expressing macrophages impact the response of PDAC cells to treatment with PD-L1/PD-1 inhibitors, we developed a spheroid model comprising two different PDAC cell lines and different ratios of in vitro differentiated primary M1- or M2-like polarized macrophages. In line with our in situ findings, high PD-L1 expression was observed in macrophages rather than PDAC cells, which was further increased by the presence of PDAC cells. The effector phenotype of co-cultured CD8+ T cells exemplified by expression of activation markers and release of effector molecules was rather enhanced by PDAC macrophage spheroids, particularly with M1-like macrophages compared to mono-culture spheroids. However, this was not associated with enhanced PDAC cell death. ICI treatment with either Durvalumab or Pembrolizumab alone or in combination with Gemcitabine hardly affected the effector phenotype of CD8+ T cells along with PDAC cell death. Thus, despite strong PD-L1 expression in macrophages, ICI treatment did not result in an enhanced activation and cytotoxic phenotype of CD8+ T cells. Overall, our study revealed novel insights into the interplay of PDAC cells and macrophages in the presence of ICI.

#3789 SERUM AND URINARY SOLUBLE PD-1, PD-L1 AND PD-L2 AS BIOMARKERS FOR CHECKPOINT INHIBITOR RELATED ACUTE INTERSTITIAL NEPHRITIS

Abstract Background and Aims Acute interstitial nephritis (AIN) related to Immune Checkpoint Inhibitors (ICI) is a frequently described adverse effect, although the mechanisms are not fully understood. The gold standard for the diagnosis is kidney biopsy. Although it is invasive, it is necessary as AIN can be confounded with other causes of acute kidney injury (AKI) in oncologic patients. The diagnosis of AIN has therapeutical consequences such as the need to withdraw the drug if confirmed, possibly diminishing patients’ life expectancy. Few biomarkers have been proposed for non-invasive diagnosis or the follow-up of these patients, which could be useful when the biopsy is contraindicated or not available. Our aim was to study the Immune Checkpoint pathway during the event, as well as analyse potential biomarkers directly related to the pathway. Method We performed an observational study. We recruited patients with incident diagnosis of AIN related to ICI in our institution from 2018 to 2022. We obtained serum and urine at the time of diagnosis after signing informed consent. We recruited n = 18 patients with the diagnosis of AIN related to ICI. For comparison, we recruited patients with non ICI-related AIN (n = 18), patients with active ANCA-vasculitis (n = 35), patients with acute tubular necrosis (ATN) (n = 22), and controls (n = 36) (patients with diagnosis of malignancy without AKI (n = 22), and healthy controls (n = 14)). We determined soluble PD-1 (sPD-1), sPD-L1 and sPD-L2 in serum and urine using a multiplex bead-based Luminex assay. Besides, we performed PD-L1 immunohistochemistry and PD-L2 immunofluorescence staining of kidney biopsies from patients with ICI-related AIN (n = 14) and compared to patients with non ICI-related AIN (n = 10). Results Serum sPD-1 was higher in patients with AIN compared to controls (p = 0.0004) and patients with ATN (p = 0.021). There were no differences between controls and ATN (p = 0.76). Urinary sPD-1 was lower in subjects with AKI compared to controls (control vs AIN (p = 0.019), control vs active vasculitis (p<0.0001) and control vs ATN (p = 0.0304)). Patients with ATN exhibited the lowest urinary sPD-1 levels, and there were statistically significant differences when compared to AIN (p = 0.0304). Focusing on oncologic patients, urinary sPD-1 concentration <129.3 pg/ml had a 71.43% sensitivity and 94.12% specificity to differentiate ATN from AIN, with a likelihood ratio of 12.14. Serum and urinary sPD-L1 and sPD-L2 did not show statistically significant differences between AIN and ATN. In kidney biopsies, patients with ICI related AIN showed higher density of PD-L1 positive tubules than patients with non-ICI related AIN (p = 0.037). Proportion of patients having more than 2.64/mm2 PD-L2 positive tubules was higher among patients with ICI related AIN compared to non ICI-related AIN (p = 0.034). There was a positive correlation (p = 0.009, r = 0.72) between urinary sPD-1 and the density of PD-L1 positive tubules (tubules/mm2). As for PD-L2, serum PD-L2 was higher in patients with positive tubular PD-L2 expression (p = 0.009). Conclusion Serum sPD-1 increases in AIN, contrary to patients with ATN and controls. Urinary sPD-1 decreases in patients with AKI. This decrease is more intense in the case of patients with ATN, thus being useful in the differentiation between ATN and AIN, especially in patients with neoplasms. Patients with ICI-related AIN exhibit higher positivity for PD-L1 and PD-L2 staining compared to other causes of AIN. Altogether, our findings suggest a role for these soluble molecules in the ICI-related AIN pathophysiology.

Demonstrating the interference of tissue processing in the evaluation of tissue biomarkers: The case of PD-L1

The immunohistochemical (IHC) expression of PD-L1 in cancer models is used as a predictive biomarker of response to immunotherapy. We aimed to evaluate the impact of the usage of 3 different tissue processors in the IHC expression of PD-L1 antibody clones: 22C3 and SP142. Three different topographies of samples (n = 73) were selected at the macroscopy room: 39 uterine leiomyomas, 17 placentas and 17 palatine tonsils. Three fragments were collected from each sample and were inked with a specific color that represented their separate processing in a different tissue processor (A, B or C). During embedding, the 3 fragments with distinct processing were ensemble in the same cassette for sectioning of 3 slides/each: hematoxylin-eosin, 22C3 PDL1 IHC staining and SP142 PD-L1 IHC staining, that were blindly observed by 2 pathologists under digital environment.All but one set of 3 fragments were considered adequate for observation even in the presence of artifacts associated with processing issues that were recorded as high as 50.7 % for processor C.The occurrence of background non-specific staining and the presence of false positive results appear to be unrelated with the PD-L1 clone or the type of tissue processing.22C3 PD-L1 was more frequently considered adequate for evaluation than SP142 PD-L1 that, in 29.2 % of WSIs (after tissue processor C) were considered not adequate for observation due to lack of the typical pattern of expression. Similarly, the intensity of PD-L1 staining was significantly decreased in fragments processed by C (both PD-L1 clones) in tonsil and placenta specimens, and by A (both clones) in comparison with those processed by B.This study demonstrates the need to standardize the tissue processing in pathology to cope with the growing needs of precision medicine quantifications and the production of high-quality material necessary for computational pathology usage.

KRAS G12C-mutated pancreatic cancer: Clinical outcomes based on chemotherapeutic regimen.

4150 Background: Frontline treatment for advanced pancreatic ductal adenocarcinoma (PDAC) has been either 5-fluorouracil, oxaliplatin and irinotecan (FOLFIRINOX) or gemcitabine and nab-paclitaxel (GP) for the past decade. While the NAPOLI-3 trial, utilizing liposomal irinotecan, highlighted the superiority of a triplet regimen over GP, the question remains whether certain subgroups may derive particular benefit from GP. Pre-clinical data in lung cancer suggest that KRAS G12C may facilitate enhanced DNA adduct removal after platinum chemotherapy and confer resistance to this drug class. While multiple KRAS G12C inhibitors have shown early promise in PDAC, multi-agent chemotherapy remains the frontline standard and will likely remain an important therapeutic tool. This study aimed to investigate clinical outcomes after platinum and non-platinum-based chemotherapy in patients with advanced KRAS G12C-mutated PDAC. Methods: PDAC samples were tested using whole transcriptome sequencing (WTS; Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) at Caris Life Sciences (Phoenix, AZ). Significance was determined by X2 and Fisher-Exact and p adjusted for multiple comparisons (q). Real-world overall survival (rwOS) was obtained from insurance claims data and calculated from first of treatment to last contact with comparison done by Kaplan-Meier test. Results: A total of 5,555 PDAC patients harboring KRAS pathogenic variants were identified, including 109 with a G12C mutation. For KRAS G12C mutants, median overall survival was 470 days for those who received GP (n=22) compared to 240 days for patients who received FOLFIRINOX (n=9) with a median difference of 230 days (HR 0.32, CI 0.12-0.82, p=0.013). In contrast, median overall survival was higher for patients harboring G12D (392 vs 297 days, p=0.003), G12V (500 vs 356 days, p=0.012) or G12R (469 vs 391 days, p=0.095) mutations who received FOLFIRINOX compared to GP. KRAS G12C mutated PDAC showed the highest rate of PDL1+ staining (27%) compared to other variants (G12R 13% and G12D 19%, q<0.01). TMB-H and MSI-H prevalence were highest in G12C compared to other variants (no statistical significance observed). Conclusions: In patients with advanced PDAC and a G12C mutation, median overall survival appears significantly longer in those treated with GP compared to FOLFIRINOX. The opposite trend was seen in patients with other KRAS variants including G12D, G12V, and G12R, consistent with the recently presented NAPOLI-3 trial. PDL1 staining was also highest in the KRAS G12C cohort. While this is the largest reported analysis of outcomes to frontline chemotherapy in KRAS G12C-mutated PDAC, the sample size is small and needs validation in additional datasets. Next steps include evaluating DNA mutational status and RNA expression of genes involved in DNA repair in G12C vs other KRAS variants to better understand the outcome data presented here.

Analysis of cell-free DNA (cfDNA) fragmentomes and prediction scores across distinct molecular subtypes.

e15029 Background: Liquid biopsies have the advantage of being convenient in collecting tumor-derived DNA for clinical use. Genome-wide analysis of cell-free DNA (cfDNA) fragmentomes has emerged as a promising approach to detect lung cancer based on the examination of sequencing features other than actionable mutations. Here we report initial analyses of the DELFI (DNA evaluation of fragments for early interception) technology in patients enrolled in the Dutch Lung Cancer Early Molecular Assessment (LEMA) trial (NCT02894853), a prospective, observational, and multicenter study, to evaluate fragmentation patterns in genotyped blood samples with actionable alterations irrespective of disease stage and histology subtype. Methods: Plasma samples from 576 patients with an established diagnosis of lung cancer but awaiting the start of definitive treatment were processed and examined using low-coverage whole-genome sequencing. A lasso model that incorporated genome-wide fragmentation profiles was trained on an internal Delfi Diagnostics cohort and used to generate patient-specific DELFI scores for participants in LEMA. Results: Enrollees comprised 531 individuals with non-small cell lung cancer, 38 with small-cell lung cancer, and 7 with unknown lung cancer type. Most patients were diagnosed with late-stage disease (28% stage I, 10% stage II, 26% stage III, and 36% stage IV). Current smokers (N = 238; 41%) and former smokers (N = 295, 51%) outnumbered never smokers (N = 43; 8%). Analysis showed a stepwise increase of the DELFI scores from T1 to T4 and N0 to N1-3 disease, associating aberrant fragmentation profiles with the overall tumor burden. Small-cell carcinomas and squamous cell carcinomas presented with higher DELFI scores than adenocarcinomas ( p< 1e-7, generalized linear model). Patients without a smoking history had similar scores to those who had a previous history of smoking. When adjusted for stage, mutation status in ALK, BRAF, EGFR, ERBB2, KRAS, MET, RET, and ROS were not associated with DELFI scores ( p= 0.5, likelihood ratio test). Similarly, PD-L1 staining did not significantly affect DELFI scores when a 1% cutoff was applied to cases with PD-L1 staining data ( p= 0.07, generalized linear model). Conclusions: Our results reveal that DELFI scores increase stepwise with stage and are higher in small-cell and squamous cell carcinomas. The LEMA study reinforces the potential usefulness of genome-wide cfDNA fragmentation profiling as a liquid biopsy approach for identifying patients with lung cancer regardless of smoking status, somatic driver mutation, and PD-L1 expression scores.

Immunoscore immune checkpoint using spatial quantitative analysis of CD8 and PD-L1 markers is predictive of the efficacy of anti- PD1/PD-L1 immunotherapy in non-small cell lung cancer.

Anti-PD-1 and PD-L1 antibodies (mAbs) are approved immunotherapy agents to treat metastatic non-small cell lung cancer (NSCLC) patients. Only a minority of patients responds to these treatments and biomarkers predicting response are currently lacking. Immunoscore-Immune-Checkpoint (Immunoscore-IC), an invitro diagnostic test, was used on 471 routine single FFPE-slides, and the duplex-immunohistochemistry CD8 and PD-L1 staining was quantified using digital-pathology. Analytical validation was performed on two independent cohorts of 206 NSCLC patients. Quantitative parameters related to cell location, number, proximity and clustering were analysed. The Immunoscore-IC was applied on a first cohort of metastatic NSCLC patients (n=133), treated with anti-PD1 or anti-PD-L1 mAbs. Another independent cohort (n=132) served as validation. Anti-PDL1 clone (HDX3) has similar characteristics as anti-PD-L1 clones (22C3, SP263). Densities of PD-L1+ cells, CD8+ cells and distances between CD8+ and PD-L1+ cells were quantified and the Immunoscore-IC classification was computed. Using univariate Cox model, 5 histological dichotomised variables (CD8 free of PD-L1+ cells, CD8 clusters, CD8 cells in proximity of PD-L1 cells, CD8 density and PD-L1 cells in proximity of CD8 cells) were significantly associated with Progression-Free Survival (PFS) (all P<0.0001). Immunoscore-IC classification improved the discriminating power of prognostic model, which included clinical variables and pathologist PD-L1 assessment. In two categories, the Immunoscore-IC risk-score was significantly associated with patients' PFS (HR=0.39, 95% CI (0.26-0.59), P<0.0001) and Overall Survival (OS) (HR=0.42, 95% CI (0.27-0.65), P<0.0001) in the training-set. Further increased hazard ratios (HR) were found when stratifying patients into three-category Immunoscore-IC (IS-IC). All patients with Low-IS-IC progressed in less than 18 months, whereas PFS at 36 months were 34% and 33% of High-IS-IC patients in the training and validation sets, respectively. Immunoscore-IC is a powerful tool to predict the efficacy of immune-checkpoint inhibitors (ICIs) in patients with NSCLC. Veracyte, INSERM, Labex Immuno-Oncology, Transcan ERAnet European project, ARC, SIRIC, CARPEM, Ligue Contre le Cancer, ANR, QNRF, INCa France, Louis Jeantet Prize Foundation.

Retrospective analysis of NUT carcinoma: The Northwestern experience.

e18096 Background: NUT carcinoma (NC) is a rare, highly aggressive carcinoma that originates from a wide variety of organs, typically presenting as poorly differentiated squamous cell carcinoma. Cases typically are aggressive, with a median overall survival (OS) of only 6.5 months. Given only 40-50 cases are diagnosed in the United States annually, NC is an orphan disease with no standard treatment options and the outlook is generally poor. A previously published case of thyroid NUT carcinoma demonstrated a prolonged response to pembrolizumab for &gt; 3 years. Methods: Patients (pts) harboring NUTM1 rearrangements were retrospectively identified at our institution. We reviewed characteristics related to tumor immune microenvironments by tumor immunohistochemistry and next generation sequencing as well as clinical outcomes. Results: 10 NMC pts were identified (5 female, 5 male): lung (6), thyroid (2), primary mediastinal (1), maxillary sinus (1). The median age at diagnosis was 49 years (range of 27-75). The thyroid cases were diagnosed at ages 51 and 75. 8/10 had metastatic or unresectable disease at diagnosis, 3 of which with central nervous system metastases. In 8 evaluable patients, NUTM1 rearrangements included BRD2 (1), BRD3 (1), BRD4 (2), NSD3 (3), SCL12A6 (1). Median OS was 10.8 months (range 1.1-75.8m); 3/10 patients were deceased. 3 patients received IO (2 ipilimumab/nivolumab, 1 pembrolizumab) after either surgery, RT, or chemotherapy combined with RT. Patients who received IO had a median survival of 18 months (range 10.9-76.0 months) compared with 8.5 months in the 8 patients not receiving IO (p = 0.07). All patients with tissue available in this cohort all had PD-L1 staining &lt; 5%, were microsatellite stable (MSS) and had a low median tumor mutation burden (TMB) of 3.1 mut/Mb. Conclusions: In our series, NC originating from the thyroid gland accounted for more cases than expected, thyroid NC patients were older than NC cases arising in other locations and with the more typical squamous histology. Despite relatively low PD-L1 staining, TMB, and MS status, our data suggests NMC tumors may be amenable to treatment with immune checkpoint inhibitors, warranting further investigation.

RNA sequencing as a confirmatory assay and its impact on patient care in multiple cancer types.

e15058 Background: Increasing evidence shows the ability of RNA sequencing (RNA-seq) to detect expression of multiple biomarkers with improved sensitivity and scalability, offering a more objective and reproducible interpretation of data compared to pathological assessment methods by immunohistochemistry (IHC). Further, ongoing research demonstrating strong correlation between RNA-seq gene expression values and IHC values suggests the utility of RNA-seq as an objective diagnostic approach. Here, we highlight 4 cases where discordance in expression levels measured by IHC and RNA-seq resulted in re-evaluation of IHC values and a subsequent change in the clinical course. Methods: All patients underwent RNA and DNA sequencing of tumor tissue as part of a standard-of-care workflow. Sequencing was performed and reported with the BostonGene Tumor Portrait test, utilizing whole-exome sequencing (WES) and transcriptome sequencing data for tumor analysis. Gene expression levels were evaluated by comparison to a reference cohort of patients with a similar diagnosis. Results: In one case, a 53-year-old female was diagnosed with metastatic cholangiocarcinoma. While IHC of her biopsy reported positive HER2 (+3) expression, RNA-seq revealed low expression. A repeated IHC assay confirmed negative HER2 expression, leading to a change in therapy options. Another 61-year-old female was diagnosed with triple negative breast cancer (TNBC) (IHC: ER 0%, PR 0%, HER2 1+). Integrated WES and RNA-seq analysis detected HER2 amplification (+8 copies), ERBB2-activating mutation, high ERBB2 (HER2) expression and a HER2-enriched PAM50 subtype. IHC was repeated, now showing positive HER2 (+3) expression. In order to target HER2, systemic treatment was changed to a combination containing trastuzumab and pertuzumab, resulting in pathological complete response. In the third case of an 85-year-old female with relapsed TNBC, IHC demonstrated 1% PDL-1 staining. In contrast, RNA-seq detected high PD-L1 expression leading the treating physician to repeat IHC. Staining showed high PDL-1 (CPS50), prompting a change in therapy to an anti-PDL-1 (pembrolizumab) therapy. Lastly, while IHC of a 21-year-old female diagnosed with diffuse large B-cell lymphoma (DLBCL) showed negative CD30, subsequent RNA-seq detected relatively high CD30 expression that prompted a second IHC evaluation confirming positive CD30 expression. Eventually, the diagnosis was changed to primary mediastinal B-cell lymphoma and DA-EPOCH was selected as the therapy. Conclusions: RNA-seq is emerging as an objective tool to evaluate key diagnostic and targetable events in multiple cancer types. The use of RNA-seq can also add value as an objective measurement to confirm critical IHC findings, potentially changing clinical care.

Models including pathological and radiomic features vs clinical models in predicting outcome of patients with metastatic non-small cell lung cancer treated with immunotherapy.

e21164 Background: Overall survival of patients with metastatic non-small cell lung cancer (NSCLC) has increased with the use of anti-PD-1 immune checkpoint inhibitors. However, the duration of response remains highly variable between patients, and only 20-30% of patients are alive at 2 years. Thus, new biomarkers for predicting response to treatment and patient outcomes are still needed to guide therapeutic decision. In this study, we retrospectively investigated multimodal approaches that might improve the limited predictive power of clinical data. Methods: We studied a cohort of 317 patients with metastatic NSCLC treated with first-line immune checkpoint inhibitors alone or combined with platinum-based chemotherapy. Clinical data were collected for all patients, pathological slides (HES and PD-L1 staining) and baseline 18-FDG PET/CT scans were available in 237 and 130 patients respectively. An automatic cell type detection algorithm was applied to each pathological slide and pathomic features were extracted from the resulting annotations. After semi-automated segmentation of all tumor foci in the PET/CT scans, radiomic features were calculated for each tumor lesion and aggregated across all the lesions of each patient. Prognostic models were built using random forest and XGboost classifiers to predict patient survival at 12 months based on 1) features from single modalities (clinical, pathomic, or radiomic), 2) features from multiple modalities, where early fusion and late fusion strategies were investigated. The models were trained and tested with cross-validation and their performances were established using the area under the ROC curve (AUC) computed on the same 88 test patients for whom all the modalities were available. Results: Unimodal strategies yielded AUC of 0.62 ± 0.08 (1 std), 0.64 ± 0.07, 0.59 ± 0.08 for clinical, radiomic and pathomic features respectively. With late fusion, bimodal models consistently outperformed the clinical model, with the combination of radiomic and clinical features giving the best performance (AUC = 0.67 ± 0.07). The trimodal model outperformed all other modality combinations with an AUC of 0.69 ± 0.07; in particular, it was significantly superior to the clinical model (p-value &lt; 0.001, paired t-test). The early fusion experiments confirmed the superiority of every bimodal approach over the clinical model. However, the trimodal model did not outperform the best bimodal model with early fusion. Validation will be performed on independent cohorts from external centers. Conclusions: Our study highlighted the potential of multimodal approaches for predicting the outcome of metastatic NSCLC patients treated with immunotherapy. Models integrating medical images and pathological slides usually collected from routine care outperformed a model trained on clinical data alone.

Characterization of immune checkpoints expression and lymphocyte densities of iranian breast cancer patients; the co-expression status and clinicopathological associates

BackgroundBreast malignancies are now the most common and deadliest type of neoplasms among women worldwide. Novel therapeutic approaches are needed to combat advanced stages of breast cancer. In this study, we aimed to investigate the expression and co-expression status of three immune checkpoints (PD-1, PD-L1, and LAG-3), as well as tumor-infiltrating lymphocytes (TIL) scores, and to further establish their potential correlations with clinicopathologic features.MethodsWe performed a retrospective study on 361 pathologic samples of breast cancer. Immunohistochemistry was performed to assess the status of the immune checkpoint markers, and H&E staining was used to score TILs. The correlations of the immune checkpoint markers of tumor cells and tumor-associated immune cells and TIL scores with clinicopathological characteristics were analyzed.ResultsOut of 361 assessed samples, LAG-3 was positive in 51%, while IC PD-L1 and TC PD-L1 were detectable in 36% and 8.9%, respectively. Moreover, both IC PD-L1 and LAG-3 stained positively in 24.4% of samples. IC PD-L1 expression was significantly higher in tumors with higher nuclear, mitotic, and overall grades and tubule formation. In addition, TC PD-L1 and LAG-3 exhibited a similar trend for higher overall grading. Tumors with positive estrogen- and progesterone-receptor (ER and PR) expression had significantly lower IC PD-L1 and TC PD-L1 staining, while LAG-3 positivity was more prevalent in HER2 positive samples. Tumors that were positive for these biomarkers had significantly higher Ki-67 scores. LAG-3 expression showed significant correlations with PD-1 and IC PD-L1 expression. Besides, the co-expression of LAG-3 and IC PD-L1 was significantly more encountered in luminal B and triple-negative subtypes, compared to the luminal A subtype. Regarding TILs, their scoring was significantly higher in ER and PR negative and HER2 positive samples. Intriguingly, samples with positive staining for LAG-3, IC PD-L1, and TC PD-L1 had significantly higher TIL scorings.ConclusionsImmune checkpoints show differentially different levels of expression in certain molecular subtypes of breast cancer. Moreover, they reveal a meaningful correlation with each other, proliferation indices, and histologic grades. Finally, a sizable proportion of breast cancers co-express PD-L1 and LAG-3, which will make them appropriate targets for future combined ICIs.

Using cell-free DNA to predict response to immunotherapy in non-small cell lung cancer pre- and post-treatment.

e21198 Background: Despite revolutionizing cancer therapy, immune checkpoint inhibitors (ICI) still do not benefit a significant proportion of patients. The risks associated with ICI-related adverse events, mixed performance of PD-L1 staining in predicting treatment response and its high cost, present a clinical need for more precise methods to define disease states in the context of ICI treatment. ICI response is thought to depend on the phenotype of the tumor and the associated immune response, especially functional state of CD8+ T cells in the tumor microenvironment (TME). Methods: Here we show that the CD8+ T cell signatures from plasma cell-free DNA (cfDNA) could be used to predict response of patients to anti-PD-1 therapy, using samples collected before and shortly after start of treatment. Blood samples were drawn just before the first dose, 1 day to 1 week before the start of treatment and 45-60 days since start of treatment. The treatment duration varied depending on response. Response was evaluated by CT scans every 8-12 weeks. 11 of the samples are from patients with no or minor response ( &lt; 6 months of treatment), and 12 are from patients with prolonged benefit of the medication ( &gt; 1 year of treatment). Exhausted CD8+ T cells, one of the targets of immunotherapy treatment are epigenetically regulated. So, we utilized naïve and PD-1high CD8+ T cell ATAC-seq data to define the repertoire of accessible chromatin specific to these cell types. We found an enrichment of short cfDNA fragments at a significant fraction of these sites, enabling us to define responder-specific and non-responder-specific accessible PD-1high regions from cfDNA. Results: We used the enrichment of short- versus long-cfDNA fragments (reflecting transcription factor-nucleosome dynamics in tissue of origin of cfDNA) as a scoring function, and repeated-cross-validation as a classifier model to identify differentially enriched features between responders and non-responders. Our model accurately predicts the two classes of response both pre-treatment (mean test AUC = 0.86; SD = 0.14). Conclusions: Notably, in addition to generating an accurate classifier, our analysis enabled us to identify predominant transcription factor motifs from predictive ATAC-seq peaks that characterize both response and lack of response, paving the way for further understanding the mechanistic basis of patient-specific response to ICI. Our results suggest the possibility of personalized prediction of treatment response that is independent of specific tumor genotype.

Concordance among the three commercially available PD-L1 assays for esophageal squamous cell carcinoma.

4058 Background: Immune checkpoint inhibitors are now standard-of-care for patients with esophageal squamous cell carcinoma (ESCC). The predictive significance of PD-L1 expression in ESCC has been investigated by different antibodies, scoring algorithms and cutoff values in several phase III trials. However, it remains controversial whether PD-L1 is a predictive biomarker for response to PD-1/PD-L1 blockade. Evaluating the concordance of the commercially available PD-L1 assays is essential to understand the discrepancy in the ESCC clinical trials, and helpful to further investigate the predictive value of PD-L1 expression. Methods: 145 archival tumor samples were obtained from 131 patients with ESCC (stage I-IV) at National Taiwan University Hospital. Formalin-fixed, paraffin-embedded archival tumor samples were assessed by three commercially available PD-L1 assays: VENTANA SP263, Dako 22C3 and Dako 28-8 assays. Assays were performed in a College of American Pathologists accredited central laboratory and scored for PD-L1 staining by using multiple metrics including tumor cell score (TC), immune cell score (IC) and combined positive score (CPS) or tumor area percentage (TAP). Analytical concordance was calculated pairwise between assays using the Spearman (ρ) rank correlation coefficient. Classification concordance, including agreement between clinically relevant scoring algorithms, was investigated using positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) at multiple cutoff values to assess the overlap between populations by using different assays. Results: SP263, 22C3 and 28-8 assays showed good analytical correlation for TC staining (Spearman’s rank correlation coefficient 0.8–0.9). Correlation was lower for IC (Spearman’s rank correlation coefficient 0.59-0.61). There was moderate overlap between populations identified by SP263 and 28-8 or 22C3 based on CPS or TAP algorithm at multiple cutoff values. OPAs for these 3 assays ranged from 68%–88% at various matched algorithms. The SP263 and 22C3 PD-L1 assays appeared relatively more sensitive, assigning a higher proportion of patients as PD-L1 positive or high, compared to 28-8 assay. When using assay-specific clinically relevant algorithm, moderate classification agreement was seen for SP263 versus 22C3 or 28-8. Differences were observed between patient populations with tumor classified as PD-L1 high versus PD-L1 low/negative using CPS≥10, TAP≥10% and TC≥1%. The PPA between 22C3 CPS≥10 and SP263 TAP≥10 or 28-8 CPS≥10 or 28-8 TC≥1% were 75%, 57% and 68% respectively. Conclusions: This study is the first dataset to compare various PD-L1 assays in ESCC. Differences in classification of patients with PD-L1 high versus low/negative using clinically relevant algorithms suggest that caution should be taken when comparing data across the trials.

A single-arm, phase II study of durvalumab (D) and tremelimumab (T) for relapsed/refractory germ cell tumors (GCT).

5040 Background: Patients (pts) with GCT and progressive disease (PD) after salvage high-dose chemotherapy (HDCT) have dismal outcomes with median progression free survival (PFS) and overall survival (OS) of 1.0 and 4.7 months (mo), respectively (Feldman et al. Cancer 2012). Immune checkpoint blockade (ICB) has resulted in durable benefit in multiple tumor types. We tested D (anti-PD-L1) + T (anti-CTLA-4) for pts with relapsed/refractory GCTs in a phase II trial (NCT03158064). Methods: Eligible pts were ≥ 18 years old with GCT of any primary site who had PD after first-line chemotherapy and HDCT or were not candidates for HDCT or other curative options. Pts received D (1500 mg) and T (75 mg) every 4 weeks (wks) for 4 cycles followed by D (1500 mg) every 4 wks for up to 1 year (schedule 1). The study was later amended to one cycle of D (1500 mg) and T (300 mg) followed by D (1500 mg) every 4 wks for up to 1 year (schedule 2). The primary endpoint was initially overall response rate (ORR) in a Simon’s two-stage design with null and alternate rates of 5% and 20%, respectively. The study was then amended to use a primary endpoint of 16-wk PFS in a new Simon’s two-stage design with null and alternate rates of 5% and 25%. The regimen would be considered promising if 16-wk PFS was achieved in ≥ 3 of 20 pts. Secondary endpoints included PFS, OS, safety, and association of PD-L1 staining with outcome. Results: Between July 2017 and Jan 2022, 29 pts were treated, the last 7 (24%) on schedule 2. Median age was 37 (range, 21-70). Histology was non-seminoma in 25 pts (86%), and primary sites were testis in 20 (69%), mediastinum in 7 (24%), ovary in 1 (3%), and unknown in 1 (3%). Six (21%) pts had late relapse and 21 (72%) received prior HDCT. Median AFP was 310 ng/mL (range, 1–18,087) and HCG 471 mIU/mL (range, 3.3-62,302). Ten pts were treated with the ORR primary endpoint with no responses in stage 1, but 2 pts had tumor reductions of 20% and 22% with PFS of 9.9 and 10.4 mo, respectively, and 1 patient with elevated AFP but no RECIST measurable disease had a PFS of 5.7 mo. Of 19 pts treated with the 16-wk PFS primary endpoint, one had an ongoing partial response (59% tumor reduction) with 33 months follow-up; all others had PD in &lt;16 wks (17 with PD and 1 with stable disease). Thus, the study did not meet its primary endpoint. Among 29 treated pts, the 16-wk PFS was 13.8% (range, 4.3-28.6), and median PFS and OS were 1.4 (95% CI: 4.1, 7.1) and 7.3 mo (95% CI: 3.2, 10.9), respectively. PD-L1 expression data was available for 20/29 pts - 3/13 (23%) with low/negative and 1/7 (14%) with high expression achieved PFS &gt;16 wks. Six (21%) pts had grade 3-4 treatment-related adverse events, 3 (10%) of whom required high-dose steroids. Conclusions: D+T had limited antitumor activity in pts with relapsed/refractory GCT, failing to meet protocol-defined success. However, some pts did appear to benefit, including one exceptional responder. Additional correlative studies are ongoing. Clinical trial information: NCT03158064 .

Impact of a pathologist-driven molecular testing protocol on veterans with non-small cell lung cancer.

e21209 Background: Precision medicine has shifted the paradigm of cancer treatment in the last two decades. Currently, with over ten targetable mutations in non-small cell lung cancer (NSCLC) and increasingly emerging biomarkers for immunotherapy, genomic testing is necessary for every NSCLC patient. However, this multiple biomarker-defined treatment approach is often compromised by many challenges including slow turnaround time (TAT), high rate of quantity not sufficient (QNS) resulting in inaccurate molecular testing or needs for second biopsy. These barriers lead to increased risk of developing medical complications from re-biopsy or treatment delay as well as additional social and financial support, which is not always available for veterans. A recent study reported that up a one third of veterans with NSCLC containing highly actionable mutations were not prescribed targeted treatment, among which, 25% of patients do not have a documented molecular testing results. Herein, we reported a pathologist-driven protocol to enhance the implement of molecular testing on guiding NSCLC treatment in veteran population. Methods: A pathologist-driven molecular testing protocol is applied West Los Angeles VA hospital since 2018. First, in preparation for diagnosis and molecular testing, tissue block will be cut in serial sections in the following priority: 2 slides for H&amp;E, 4 slides for IHC, 2 slides for PD-L1 staining. An additional slide is made for quality control (QC) purpose before 10 additional slides are cut for molecular testing and 6 for FISH studies. The reminder of the tissue block will be cut in serial sections and preserved in a vial along with tissue shaved at initial block effacement. Every tissue block will be exhausted. Upon initial evaluation of H&amp;E slides, the reviewing pathologist, independent of the treating oncologist, will order molecular testing including EGFR, ALK, ROS and BRAF by NGS if NSCLC is identified. PD-L1 IHC staining is also ordered automatically regardless of the histology. If a clinically actionable mutation is detected, a mutation notification is immediately sent via email by the molecular pathologist to alert the treating oncologist. Results: After implementing this protocol, we observed a decreased rate of QNS (~30% reduction) and subsequently the need for 2ndbiopsy dropped substantially. We also observed a faster TAT between initial cancer diagnosis (within 7 days) and the resulting of molecular testing to guide subsequent treatment. Conclusions: Refined tissue processing and pathologist driven ordering system can significantly impact initial management of patients with NSCLC as it reduces the rate of QNS and repeat biopsy/TAT. This protocol can be easily implemented in VA system or other community based healthcare system.

Reproducibility in PD-L1 Immunohistochemistry Quantification through the Tumor Proportion Score and the Combined Positive Score: Could Dual Immunostaining Help Pathologists?

We studied the pathologists’ agreements in quantifying PD-L1 expression through the tumor proportion score (TPS) and the combined positive score (CPS) using single PD-L1 immunohistochemistry (S-IHC) and double immunohistochemistry (D-IHC) combining PD-L1 staining and tumor cell markers. S-IHC and D-IHC were applied to 15 cancer samples to generate 60 digital IHC slides (30 whole slides images and 30 regions of interest of 1 mm2) for PD-L1 expression quantification using both TPS and CPS, twice by four pathologists. Agreements were estimated calculating intraclass correlation coefficients (ICC). Both S-IHC and D-IHC slides analyses resulted in excellent (for TPS, ICC &gt; 0.9) to good (for CPS, ICC &gt; 0.75) inter- and intra-pathologist agreements with slightly higher ICC with D-IHC than with S-IHC. S-IHC resulted in higher TPS and CPS than D-IHC (+5.6 and +6.1 mean differences, respectively). High reproducibility in the quantification of PD-L1 expression is attainable using S-IHC and D-IHC.

Development of a Pathomics-Based Model for the Prediction of Malignant Transformation in Oral Leukoplakia

Accurate prognostic stratification of oral leukoplakia (OLK) with risk of malignant transformation into oral squamous cell carcinoma is crucial. We developed an objective and powerful pathomics-based model for the prediction of malignant transformation in OLK using hematoxylin and eosin (H&E)-stained images. In total, 759 H&E-stained images from multicenter cohorts were included. A training set (n = 489), validation set (n = 196), and testing set (n = 74) were used for model development. Four deep learning methods were used to train and validate the model constructed using H&E-stained images. Pathomics features generated through deep learning combined with machine learning algorithms were used to develop a pathomics-based model. Immunohistochemical staining of Ki67, p53, and PD-L1 was used to interpret the black box of the model. Pathomics-based models predicted the malignant transformation of OLK (validation set area under curve [AUC], 0.899; testing set AUC, 0.813) and significantly identified high-risk and low-risk populations. The prediction performance of malignant transformation from dysplasia grading (validation set AUC, 0.743) was lower than that of the pathomics-based model. The expressions of Ki67, p53, and PD-L1 were correlated with various pathomics features. The pathomics-based model accurately predicted the malignant transformation of OLK and may be useful for the objective and rapid assessment of the prognosis of patients with OLK.

Clinicopathological characteristics of gastric SMARCA4-deficient undifferentiated/rhabdoid carcinoma

Objective: To investigate the clinicopathological features and immunohistochemical phenotypes of gastric SMARCA4-deficient undifferentiated carcinoma, and to discuss the daily diagnostics of this entity and analyze its prognosis. Methods: The cases of gastric SMARCA4-deficient undifferentiated carcinoma diagnosed at the Department of Pathology, Peking University Cancer Hospital, China from January 2010 to August 2022 were collected. The histological sections were reviewed, the immunohistochemical results and clinicopathological features were analyzed, and relevant literature was reviewed. Results: Pure foci of undifferentiated carcinoma were seen in 7 cases, and 1 case was accompanied by a moderately differentiated tubular adenocarcinoma component. Undifferentiated carcinoma foci showed similar sheet-like or solid diffuse growth pattern, medium-sized tumor cells characterized by 1-2 nucleoli, and abundant cytoplasm and rhabdoid appearance. The average patient age was 65±8 years. Six patients were male and 2 were female. Immunohistochemical staining showed that undifferentiated carcinoma of all 8 tumors were negative for SMARCA4 (BRG1). Among 7 patients who underwent SMARCA2 (BRM) and SMARCB1 (INI1) staining, 4 cases showed loss of BRM expression, 2 cases showed weakly positive staining, and 1 case was diffusely positive, but all 7 cases were diffusely strong positive for INI1. The neuroendocrine marker, synaptophysin, was weakly positive in 5 cases, while CgA and CD56 were negative in 8 cases. Ki-67 index was more than 70%. Two cases were mismatch repair deficient and showed the loss of MLH1/PMS2 expression, while 1 case showed only MSH2 loss. PD-L1 staining showed that combined positive score (CPS)≥1 in 4 cases (CPS ranging from 1 to 55) and CPS<1 in the other 3 cases. Four patients had clinical stage Ⅳ disease. Two of them died within 3 months after diagnosis. Conclusions: Gastric SMARCA4-deficient undifferentiated carcinoma/rhabdoid carcinoma is a rare group of highly malignant tumors with a poor prognosis. Loss of the core subunit of SWI/SNF complex may be associated with the development of dedifferentiated histological pattern and aggressive tumor progression, which may be more frequently accompanied with mismatch repair deficiency.

Expression of Tryptophan Metabolism Enzymes in Patients with Diffuse Large B-cell Lymphoma and NK/T-cell Lymphoma.

Metabolites of tryptophan (Trp) metabolism in the tumor microenvironment play crucial immunosuppressive roles in various cancers. However, the role of Trp metabolism in diffuse large B-cell lymphoma (DLBCL) or natural killer/T-cell lymphoma (NK/TCL) remains unelucidated. We investigated the potential role of Trp metabolism in a cohort of 43 patients with DLBCL and 23 with NK/TCL. We constructed tissue microarrays and performed in situ staining of Trp-catabolizing enzymes and PD-L1 using immunohistochemistry (IHC). We observed 14.0% positive staining of IDO1 in DCBCL and 60.9% in NK/TCL; 55.8% of IDO2 in DCBCL and 95.7% in NK/TCL; 79.1% of TDO2 in DCBCL and 43.5% in NK/TCL; 29.7% of IL4I1 in DCBCL and 39.1% in NK/TCL. However, IDO1, IDO2, TDO2, and IL4I1 positivity did not significantly differ between PD-L1+ and PD-L1- biopsy tissue samples of NK/TCL; nonetheless, a positive correlation of IDO1 (r = 0.87, p < 0.001), IDO2 (r = 0.70, p < 0.001), TDO2 (r = 0.63, p < 0.001), and IL4I1 (r = 0.53, p < 0.05) with PD-L1 expression was observed in the TCGA-DLBCL dataset. Finally, immunohistochemical (IHC) analysis revealed the lack of superior prognostic effect with higher expression of Trp enzymes in DLBCL and NK/TCL. Furthermore, IDO1, IDO2, TDO2, and IL4I1 expression, as well as survival rates, did not significantly differ across all groups in the TCGA-DLBCL cohort. Collectively, our findings provide novel insights into the enzymes involved in Trp metabolism in DLBCL and NK/TCL and their association with PD-L1 expression, which offers potential strategies to combine Trp-metabolism enzyme inhibitors with anti-PD-L1 or other immunotherapeutic strategies in clinical DLBCL or NK/TCL treatment.

Investigation of the PD-1/PD-L1 Expression in the Lesional Skins of Patients With Psoriasis.

Psoriasis is an immune-mediated, chronic and inflammatory disease whose pathogenesis is affected by the interactions of several immune cells and cytokines. PD-1 is an inhibitor receptor that is expressed to a large extent in T lymphocytes and responsible for regulating autoimmunity and self-tolerance. In this study, we aimed to investigate the expression of PD-1/PD-L molecules in the lesioned skins of psoriasis patients. The study included 30 psoriasis patients, and 15 healthy volunteers as the control group. Anti PD-1 and PD-L1 antibodies were applied to the skin biopsy samples that were collected from the patient and control groups. Cytoplasmic and membranous staining of PD-1 and PD-L1 were considered positive. The number of stained immune cells that was examined for each case. The percentage of the tissues with high PD-1 (+) and PDL-1 (+) immune cell counts were significantly higher in the psoriasis patients compared to healthy controls (P values = 0.004 and 0.002, respectively). A negative and statistically significant correlation was detected between PDL-1(+) immune cell numbers and PASI scores (P = 0.033, r=-0.57). In the lesioned skin samples of psoriasis patients, the PD-1 and PD-L1 expressions were significantly higher in immune cells than that in the skin samples of the healthy controls. This study was the first investigation of the expression of PD-1/PD-L molecules in the immune cells in found the lesioned skins of psoriasis patients.