PD-L1 Positivity Research Articles

The Predictive Value of CD3+/CD8+ Lymphocyte Infiltration and PD-L1 Expression in Colorectal Cancer.

The immune system plays an important role in tumor development and treatment. In this study, we aimed to determine the relationships among the expressions of PD-L1, CD3, CD8, MMR proteins, clinicopathological features, and prognosis of CRC. Immunohistochemistry was used to determine the expression of PD-L1, CD3, and CD8 in 771 patients with CRC. The expression of PD-L1 in TC was related to the right colon, adenocarcinoma, and dMMR, and in IC, it was related to younger CRC patients and the TNM stage. The expression of CD3 and CD8 in tumor-infiltrating lymphocytes was related to lymph node metastasis and the TNM stage. The expression of PD-L1 in TC and IC was correlated with the infiltration of CD3+ and CD8+ lymphocytes. Univariate survival analysis showed that the expression of PD-L1 in TC, IC, and dMMR was related to a better prognosis. Multivariate survival analysis showed that age, TNM stage, and dMMR were independent prognostic factors for CRC. The OS of the chemotherapy was significantly higher than that of the non-chemotherapy in III-IV TNM stage patients; CRC patients with positive PD-L1 expression in TC or IC and dMMR did not benefit from chemotherapy. PD-L1 expression in TC and IC was closely related to the density of CD3 and CD8 infiltration in tumor-infiltrating lymphocytes. The expression of CD3 and CD8 in tumor-infiltrating lymphocytes and the expression of PD-L1 in IC were linked to the TNM stage of CRC patients. PD-L1 expression in TC and IC and MMR status may act as an important biomarker for guiding the postoperative treatment of III-IV TNM stage CRC patients.

PD-L1 and HIF-2α Upregulation in Head and Neck Paragangliomas after Embolization

Hypoxia activates pathways associated with tumor progression, metastatic spread, and alterations in the immune microenvironment leading to an immunosuppressive phenotype. In particular, the upregulation of PD-L1, a target for therapy with checkpoint inhibitors, is well-studied in several tumors. However, the relationship between hypoxia and PD-L1 regulation in pheochromocytomas and paragangliomas (PPGL), and especially in paragangliomas treated with embolization, is still largely unexplored. We investigated the expression of the hypoxia-marker HIF-2α and of PD-L1 in a PPGL-cohort with and without embolization as potential biomarkers that may predict the response to treatment with HIF-2α and checkpoint inhibitors. A total of 29 tumor samples from 25 patients who were operated at a single center were included and analyzed utilizing immunohistochemistry (IHC) for PD-L1 and HIF-2α. Embolization prior to surgery was performed in seven (24%) tumors. PD-L1 expression in tumor cells of head and neck paragangliomas (HNPGLs) receiving prior embolization (median PD-L1 positivity: 15%) was significantly higher as compared to PD-L1 expression in HNPGLs without prior embolization (median PD-L1 positivity: 0%) (p = 0.008). Consistently, significantly more HNPGLs with prior embolization were positive for HIF-2α (median nuclear HIF-2α positivity: 40%) as compared to HNPGLs without prior embolization (median nuclear HIF-2α positivity: 0%) (p = 0.016). Our results support the hypothesis that embolization with subsequent hypoxia leads to the upregulation of both PD-L1 and HIF-2α in HNPGLs, and could thus facilitate targeted treatment with HIF-2α and checkpoint inhibitors in the case of inoperable, locally advanced, or metastatic disease.

Real-world cohort study of PD-1 blockade plus lenvatinib for advanced intrahepatic cholangiocarcinoma: effectiveness, safety, and biomarker analysis.

In clinical practice, some patients with advanced intrahepatic cholangiocarcinoma (ICC) cannot tolerate or refuse chemotherapy due to the toxicity, necessitating alternative treatments. PD-1 blockade combined with lenvatinib showed promising results in phase II studies with small sample size, but there is a lack of data on the routine use with this regimen. This study aimed to evaluate the effectiveness and safety of the regimen in patients with advanced ICC, and to identify predictors for treatment response and prognosis. We conducted a retrospective cohort study of patients treated with PD-1 inhibitors plus lenvatinib for advanced ICC between July 2017 and August 2022. The study endpoints were progression-free survival (PFS), overall survival (OS), objective response rate (ORR), disease control rate (DCR), and safety. Biomarker analysis for CA19-9 and PD-L1 expression was performed. Exploratory analysis for genetic alternation was conducted. The study included 103 patients. It demonstrated a median PFS of 5.9months and a median OS of 11.4months. ORR was 18.4% and DCR was 80.6%. The incidence of grade 3 or 4 adverse events was 50.5%. Positive PD-L1 expression (TPS ≥ 1%) was associated with higher ORR (P = 0.013) and prolonged PFS (P = 0.023). Elevated CA19-9 (> 37U/ml) was associated with decreased ORR (P = 0.019), poorer PFS (P = 0.005) and OS (P = 0.034). Patients with IDH1 mutations exhibited a favorable response to the treatment (P = 0.011), and patients with TP53 mutations tended to have worse OS (P = 0.031). PD-1 blockade plus lenvatinib is effective and safe in routine practice. PD-L1 expression and CA19-9 level appear to predict the treatment efficacy. IDH1 mutations might indicate a better treatment response. NCT03892577.

A primary luminal/HER2 negative breast cancer patient with mismatch repair deficiency

Here, we present the case of a 47-year-old woman diagnosed with luminal B breast cancer subtype and provide an in-depth analysis of her gene mutations, chromosomal alterations, mRNA and protein expression changes. We found a point mutation in the FGFR2 gene, which is potentially hyper-activating the receptor function, along with over-expression of its ligand FGF20 due to genomic amplification. The patient also harbors somatic and germline mutations in some mismatch repair (MMR) genes, with a strong MMR mutational signature. The patient displays high microsatellite instability (MSI) and tumor mutational burden (TMB) status and increased levels of CTLA-4 and PD-1 expression. Altogether, these data strongly implicate that aberrant FGFR signaling, and defective MMR system might be involved in the development of this breast tumor. In addition, high MSI and TMB in the context of CTLA-4 and PD-L1 positivity, suggest the potential benefit of immune checkpoint inhibitors. Accurate characterization of molecular subtypes, based on gene mutational and expression profiling analyses, will be certainly helpful for individualized treatment and targeted therapy of breast cancer patients, especially for those subtypes with adverse outcome.

An Analysis of Patients Treated with Stereotactic Body Radiotherapy for Metastatic Urinary Tract Tumors to Identify Predictors of Response.

To identify selection criteria linked to outcomes in patients treated with stereotactic body radiotherapy (SBRT) for metastatic tumors of the urinary tract (UT). Single institution retrospective analysis of SBRT treated patients for oligometastatic/progressive UT tumors from 2006-2022. Charts were queried for M1 status at diagnosis or during disease course, treatment details (surgery, SBRT, systemic therapy), metabolic status (diabetes [DM], BMI) and outcomes. A linear quadratic formula was used to calculate the biologically effective dose (BED) using an α/β of 10 for tumor. Descriptive statistics portrayed the cohort, and analyses were done at patient and site level. Time-to-event analyses, including overall survival (OS) and progression-free survival (PFS) from SBRT, were assessed by the Kaplan-Meier method. Cox regression was used for univariable (UVA) and multivariable analyses (MVA) to identify predictors of outcomes. A total of 35 patients were treated at 44 metastatic sites, including: bone (25%), node (36.4%), lung (20.5%), soft tissue (13.6%) and liver (4.5%). Most were male (74.3%) with a median age of 70 (range: 51-89), without DM (60%) having a median BMI of 29.8, and ECOG <2 (97.1%) at time of SBRT. Six (17.1%) patients were M1 at diagnosis. Of the 29 non-M1 patients, 86.2% received definitive local therapy (LT), 58.6% had at least T3/N+ disease, 75.8% received systemic therapy with a median of 2 agents (range: 1-6) prior to SBRT. Sixteen (45.7%) received immunotherapy (IO) with most receiving this before (75%) and after (56.2%) SBRT. Six patients had positive PD-L1 status (n = 10). The median RT dose, fractionation and BED was 40 Gy (range: 14-46), 5 fractions, and 72 (range: 28-132), respectively. At a median follow-up of 34.8, the median OS was 18.4 m (range: 9.3-27.4) with a 2-year OS of 35.9%. At patient level, 62.8% recurred after SBRT. The median PFS after SBRT was 5.3 m (range: 1.8-8.7) with a 2-yr PFS of 29.3%. Patient-level PFS was improved with LT (6.7 vs 1.4 m; p = 0.001) and DM (NR vs 2.9 m; p = 0.015), whereas improved OS was related with LT (18.9 vs 6.6 m; p = 0.03), DM (p = 0.04), ECOG (p = 0.004), and no relapse after SBRT (NR vs 9.8 m; p <0.001). Exposure to < 3 systemic agents prior to SBRT portended better PFS (6.7 vs 2.6 m; p = 0.04) without any impact by IO. At site level, 20.4% of sites had local relapse with 4 being the first event. Site was related with PFS (p = 0.009) with order of increased relapse risk being liver > bone > soft tissue > node > lung. No dosimetric feature was related with recurrence risk. On MVA, both DM (p = 0.02) and LT (p = 0.002) were predictive for PFS. Only recurrence after SBRT predicted for OS on MVA (HR: 6.7, 95% CI: 1.4-31; p = 0.014). In the IO subset, median PFS was 5.3 m and OS was 9.4 m, with no difference seen with IO-SBRT sequence or PDL1 status. Optimized selection criteria for metastasis-directed therapy in patients with UT tumors is unclear, notably with IO. Future studies may benefit by assessing circulating tumor markers prior to SBRT.

Is Programmed Death Ligand 1(PD-L1) Expression in Vulvar Cancer Prognostic for Locoregional Control?

Vulvar cancer is a rare female genital neoplasm in which surgery and radiotherapy play an integral role in the treatment paradigm; however, locoregional recurrence remains the predominant pattern of failure. Little is known about the impact of PD-L1 status in vulvar cancer and its value for clinical outcomes and response prediction to immunotherapy. We sought to explore clinical outcomes of patients with positive PD-L1 expression in vulvar cancer. Single-institution retrospective analysis of patients with surgically resectable invasive vulvar carcinoma from 2001-2021 was performed. Patients with locally advanced disease not amendable to upfront surgery or de novo metastatic disease were excluded. Immunohistochemical PD-L1 expression was assessed using the Combined Positive Score (CPS) with positive expression defined as ≥1, and Tumor Proportion Score (TPS) with positive expression defined as ≥1%. Survival and disease control outcomes were calculated using the Kaplan-Meier Method with log-rank t-test. Multivariable analysis was conducted using a parsimonious cox regression analysis using forward conditional selection. A total of 85 patients were identified with a median age of 69 years old (IQR: 59-78), 54% (n = 46) FIGO stage I-II, 97% (n = 82) squamous cell carcinoma histology, 41% (n = 35) p16 positive status, 74% (n = 63) without a history of lichen sclerosis, 40% (n = 34) without co-existing vulvar intraepithelial neoplasm (VIN), and 49% (n = 42) treated with surgery alone. There were 72% (n = 61) with positive PD-L1 TPS (≥1%), and 81% (n = 69) with positive PD-L1 CPS (≥1) expression. The median follow up was 49 months (IQR: 21-75 months). The 5-year OS was 79% (95% CI, 70%-89%), DFS 55% (95% CI, 43%-67%), local control (LC) 59% (95% CI, 47%-72%), regional control (RC) 86% (95% CI, 78%-94%), and distant metastasis (DM) 96% (95% CI, 92%-100%). PD-L1 expression was associated with lower LC and DFS by TPS ≥1%. The 5-year LC of 82% (95% CI, 65%-98%) for PD-L1 negative versus 50% (95% CI, 34%-65%) positive disease (p = 0.03). The 5-year DFS was 77% (95% CI, 59%-95%) for PD-L1 negative versus 46% (95% CI, 31%-61%) positive disease (p = 0.03). No significant DFS or LC difference was noted by CPS levels ≥1. No significant difference was observed for RC, DM, or OS. On multivariable analysis, PD-L1 TPS remained a significant predictor for LC (HR = 3.01, 95% CI, 1.07-8.95, p = 0.04). No significant difference in DFS was observed for PD-L1 TPS on multivariable analysis. PD-L1 expression is associated with higher rates of local recurrence and may represents a potentially important actionable target independent of p16 status to improve the predominant pattern of relapse in this uncommon malignancy.

PD-L1 mRNA Detection in Immunohistochemically Negative Patients: A Complementary Method for a Better Treatment Selection?

PD-L1 inhibitors have been approved for cisplatin-ineligible urothelial cancer patients relapsing after radical cystectomy. A prerequisite for therapy is a positive PD-L1 expression in the tumor tissue, whereas no options are available for patients with negative PD-L1 status. However, studies revealed that many PD-L1-negative patients also responded to PD-L1 therapy. This study investigated the feasibility of PD-L1 mRNA complementary RNA in situ hybridization (RNAish) analysis to detect PD-L1-responders independent of PD-L1 protein status. Immunohistochemistry and RNA in situ hybridization were used to assess PD-L1 protein and mRNA in radical cystectomy tissue from patients with advanced and metastasized urothelial cancer. In this study, PD-L1 protein and mRNA were detected in ≥90% of the examined tissue. Positive PD-L1 mRNA expression (≥1%) on TC and IC could be evaluated in 77% and 31% of the cases, respectively. Moreover, scatterplot analysis revealed a PD-L1 mRNA positive and PD-L1 protein negative subpopulation. According to the CPS score, positive PD-L1 protein expression could be evaluated in 88% and positive PD-L1 mRNA expression in 71% of the cases. Scatterplot analysis of the CPS scores revealed a CPS protein negative but CPS mRNA positive small subpopulation. The feasibility of RNAish on formalin-fixed tissue could be proven. Moreover, complementary PD-L1 RNAish identified a sub-population of PD-L1 protein-negative and PD-L1 mRNA-positive patients, which may benefit from PD-L1 therapy.

HPV Infection and Immunohistochemical Analysis of P16, P53, and PD-L1 Expression as Prognostic Biomarkers in Squamous Cell Anal Cancer Patients Receiving Definitive Radiotherapy/Chemoradiotherapy

The treatment of anal squamous cell carcinoma (SCC) with definitive radiotherapy (RT)/chemoradiotherapy (CRT) has a high likelihood of success; nevertheless, treatment resistance and recurrence rates cannot be ignored. Therefore, we aimed to identify the relationship between immunohistochemical (IHC) evaluation, treatment response, and prognosis. This retrospective study included 42 patients with anal SCC treated with definitive RT/CRT at a single institution between 2006 and 2020. Detection of high-risk HPV-DNA and IHC analysis of p16, p53, and PD-L1 expression was performed from diagnostic formalin-fixed, paraffin-embedded (FFPE) biopsies. Positive staining was accepted as >5% in tumor cells for p16 and p53 expression in addition to ≥1% combined positive score (CPS) [(PD-L1 positive tumor, lymphocyte, macrophage count/total viable cell count) x100] for PD-L1 expression. Thirty patients (71.4%) had a complete response to definitive RT/CRT. Recurrence was observed in 16 (38.1%) patients, with 3 (7.1%) having locoregional recurrence (LRR), 6 (14.3%) having distant metastases (DM), and 7 (16.7%) having both LRR and DM. Twenty-four (57.1%) patients were alive. Thirty (71.4%) patients were HPV+, while 12 (28.6%) were HPV-. There was a significant correlation between HPV+ and p16+ status (p<0.001). HPV- status was associated with the male gender (p = 0.001). HPV- and p16- status were significantly associated with a lack of complete response to definitive RT/CRT (p<0.001, p<0.001, respectively). Furthermore, there was a significant relationship between lack of complete response and increased recurrence (p = 0.016) and distant metastases (p = 0.015). Ten of the 16 patients with recurrence were p53+; a significant correlation was found between recurrence and p53+ status (p = 0.006). Similarly, p53+ status was associated with increased LRR (p = 0.014). PD-L1 CPS ≥ 1% was found in 31 (73.8%) patients. PD-L1 positivity was significantly correlated with HPV+ (p = 0.026) and p16+ (p = 0.013) status. All 10 (23.8%) patients with LRR were PD-L1+; PD-L1 CPS ≥ 1% was associated with poor local control (p = 0.031). In univariate analysis, age [<65] (p = 0.049), complete response (p = 0.015), and HPV+ status (p = 0.010) were related to increased 5-year (y) overall survival (OS); complete response (p = 0.001), HPV+ status (p = 0.025) and p53- status (p = 0.010) were associated with increased 5-y disease-free survival (DFS). In multivariate analysis, age [<65] (p = 0.010) and HPV+ status (p = 0.002) were significant prognostic factors for 5-y OS, whereas complete response (p = 0.007) and p53- status (p = 0.038) were significant prognostic factors for 5-y DFS. Patients with HPV- status and/or poor prognostic biomarkers should be identified at diagnosis. Thus, better outcomes can be achieved with different treatment options, such as combining immunotherapy and standard CRT.

Stereotactic Radiosurgery (SRS) for Large Brain Metastases: Dosimetric and Clinical Predictors of Local Progression and Radionecrosis

Stereotactic radiosurgery (SRS) provides high rates of local control for small brain metastases with low rates of radionecrosis (RN). Larger targets are associated with increased risk of both local progression (LP) and RN. In this analysis, we hypothesized that dosimetric and clinical parameters predict for risk of LP and RN in SRS targets larger than two centimeters. We retrospectively reviewed patients with one or more targets with either an intact versus post-operative cavity larger than 2.0 cm treated with LINAC-based SRS between 2017 and 2022 at one institution. We assessed for association between patient, treatment, and disease variables with LP and RN. Variables assessed included tumor resection status, PDL1 positivity, target volume, maximum and minimum target dose, EQD2 and BED (a/b = 2 for necrosis and a/b = 10 for tumor control), as well as receipt of steroids, bevacizumab, or systemic therapy before or after SRS. Radionecrosis was determined by characteristic radiographic changes. Analyses were performed for the entire cohort and within subsets including by resection status and dose fractionation. A total of 178 lesions in 143 patients were included. Targets with volume diameters measuring at least 2 cm were used. Median follow-up was 2.3 years. Overall survival at 1 and 2 years was 56% and 32%, respectively. Most lesions (n = 119) were resected and treated with SRS post-operatively. The most common dose and fractionation schemes used were 30 Gy in 5 fractions (n = 89) and 27 Gy in 3 fractions (n = 63). For the entire cohort, the cumulative incidence of LP 1 and 2 years was 26% and 34%, respectively. The cumulative incidence of radiographic radionecrosis at 1 and 2 years was 12% and 17%, respectively. There was no difference in LP or RN between 27 Gy in 3 fractions versus 30 Gy in 5 fractions (p>0.5 for both). Median planning target volume (PTV) size was 18.5 cc for the 27 Gy in 3 fraction group compared to 21.9 cc in the 30 Gy in 5 fraction group. Minimum or maximum dose within the target was not associated with increased risk of LP or RN. Among patients receiving 27 Gy in 3 fractions, patients treated with resection followed by SRS had lower risk of LP compared to those treated with SRS alone (HR: 0.15, 95% CI: 0.03-0.64, p = 0.011). Among patients receiving 30 Gy in 5 fractions, patients who received corticosteroids prior to SRS had a lower risk of RN (HR: 0.14, 95% CI: 0.03-0.66, p = 0.013). For the entire cohort as well as within all subgroups, PD-L1≥1% was associated with increased risk of RN (p<0.001 for all). Selecting the optimal SRS dose fractionation and planning parameters to minimize both LP and RN remains a challenge for large targets. In this analysis, 27 Gy in 3 fractions appeared to provide equivalent LP and RN compared to 30 Gy in 5 fractions, and may be more convenient for patients. Patients with PD-L1≥1% with large brain targets treated with SRS may be at increased risk of RN; corticosteroid prophylaxis may be considered in this population.

Cost-effectiveness analysis of atezolizumab in patients with non-small-cell lung cancer ineligible for treatment with a platinum-containing regimen: a United Kingdom health care perspective.

Cost-effectiveness of atezolizumab, as a treatment for advanced non-small-cell lung cancer (NSCLC) patients who cannot receive a platinum-containing regimen,was still unknown. Our objective was to evaluate the cost-effectiveness of atezolizumab vs. chemotherapy in this indication from the perspective of UK healthcare system. From the global, randomised, open-label, phase III IPSOS trial, clinical inputs and patient characteristics were obtained. A partitioned survival model with three health states was built: Progression-free survival, progressed disease and death. A lifetime time horizon was applied, with an annual discount rate of 3.5%. Additionally, the willingness-to-pay threshold of £50,000/QALY was utilized. Primary outcomes were quality-adjusted life-year (QALY), costs, and incremental cost-effectiveness ratio (ICER). Sensitivity, scenario, and subgroup analyses were used to assess the reliability of base-case results. Price simulations were carried out in order to provide information for the pricing strategy at specific willingness-to-pay threshold. In the base-case analysis, atezolizumab resulted in a gain of 0.28 QALYs and an ICER of £94,873/QALY compared to chemotherapy, demonstrating no cost-effectiveness. Price simulation results revealed that atezolizumab would be preferred at a price lower than £2,215 (a reduction of 41.8%) at the willingness-to-pay threshold of £50,000. Sensitivity, scenario and subgroup analyses revealed these conclusions were generally robust, the model was most sensitive to the price of atezolizumab and subsequent medication. Furthermore, atezolizumab was found to be more cost-effective for patients displaying a positive PD-L1 expression, with an ICER of £72,098/QALY as compared to chemotherapy. Atezolizumab is not cost-effective for patients with advanced NSCLC ineligible for platinum-containing regimen, potential price reduction is necessary.

Stereotactic Radiosurgery (SRS) for Brain Metastasis in the Era of Evolving Systemic Treatment (Immunotherapy or Targeted Therapy) in Patients with Non-Small Cell Lung Cancer (NSCLC)

With evolving role for targeted and immunomodulatory agents, the survival rate of NSCLC has been improved even with brain metastasis. This study analyzed the outcome of brain metastasis treated with SRS in conjunction with immunotherapy and target therapy as well as chemotherapy. After obtaining institutional review board (IRB) approval, 229 patients with NSCLC with brain metastasis treated with SRS at our institution were retrospectively reviewed from 2008-2021. Demographic information (e.g., age, sex, race), performance status (PS), extracranial metastasis (ECM), sequence of systemic therapy (SST) and surgery for BM (SBM) were reviewed and collected. Overall survival (OS) and intracranial recurrence free survival (ICRFS) were analyzed using the Kaplan-Meier method/Cox regression, and time to each event were measured from the date of the first SRS treatment. All statistical analyses were performed using SAS version 9.4 (Carey, NC). Of the 229 pts, the median age of pts at first SRS was 64.1 (range, 34.8- 83.0 years). 38.6% were biomarker (EGFR/ALK/ROS/PDL1) positive, and 30% were PDL1 positive. 28.8 % had surgery before SRS. The median number of SRS was 1 (range, 1 - 6), and 10 patients received salvage whole brain radiation therapy (WBRT) after SRS. With a median follow-up of 9.6 months (mo), median OS and ICRFS were 14.0 mo and 8.1 mo respectively. On univariate outcome analysis, PS, ECM, SBM and PDL1 were associated with OS while biomarker, SBM and SST were closely related to ICRFS. Pts with poor PS had worse median OS of 8.3 mo compared to 29.1 mo with good PS (p = 0.001). Median OS for no ECM vs. ECM were 20.7 mo vs. 9.6 mo respectively (p = 0.002). Patients who underwent SBM initially had better median OS of 28.7 mo compared to 9.9 mo without SBM (p<0.001). There was a trend of significant impact of positive PDL1 on median OS (21.1 mo vs. 11.7 mo, p = 0.06). Similarly, SBM was found closely related to ICRFS with median ICRFS of 8.1 mo vs. 4.9 mo for those with and without SBM respectively. Both positive biomarker (13.1 mo vs. 6.4 mo, P = 0.17) and SRS first SST (8.1 mo vs. 4.9 mo, p = 0.11) demonstrated a trend of increased median ICRFS. On multivariable analyses, no SBM was linked with worse OS (HR: 1.85; 95% CI:1.15 - 2.97; p = 0.01) and decreased ICRFS (HR: 1.65; 95% CI: 1.09 - 2.51; p = 0.02). Of 31 pts with information on cause of death, 3.2% died from brain metastasis related cause. This study demonstrated effective treatment of brain metastasis in NSCLC with multiple SRSs, especially for patients with favorable prognosis. Of noticed there was only a small number of patients received salvage WBRT and most patients didn't die from brain metastasis related progression.

Differential outcomes and immune checkpoint inhibitor response among endometrial cancer patients with MLH1 hypermethylation versus MLH1 “Lynch-like” mismatch repair gene mutation

ObjectivesTo identify differential survival outcomes and immune checkpoint inhibitor (ICI) response in MLH1 hypermethylated versus MLH1 mutated (“Lynch-like”) endometrial tumors and determine whether their molecular profiles can elucidate the differential outcomes. Methods1673 mismatch repair deficient endometrial tumors were analyzed by next-generation sequencing and whole transcriptome sequencing (Caris Life Sciences, Phoenix, AZ). PD-L1, ER, and PR were tested by immunohistochemistry and immune cell infiltrates were calculated using MCP-counter. Significance was determined using Chi-square and Mann-Whitney U tests and adjusted for multiple comparisons. Overall survival (OS) was depicted using Kaplan-Meier survival curves. ResultsThe endometrial cancer cohort comprised 89.2% patients with MLH1 hypermethylated tumors and 10.8% with MLH1 mutated tumors, with median ages of 67 and 60 years, respectively (p < 0.01). Patients with MLH1 hypermethylated tumors had significantly worse OS and trended toward worse OS following ICI treatment than patients with MLH1 mutated tumors. The immune microenvironment of MLH1 hypermethylated relative to MLH1 mutated was characterized by decreased PD-L1 positivity, immune checkpoint gene expression, immune cell infiltration, T cell inflamed scores, and interferon gamma (IFNγ) scores. MLH1 hypermethylation was also associated with decreased mutation rates in TP53 and DNA damage repair genes, but increased rates of JAK1, FGFR2, CCND1, and PTEN mutations, as well as increased ER and PR positivity. ConclusionsEndometrial cancer patients with MLH1 hypermethylation display significantly decreased survival and discrepant immunotherapy responses compared to patients with MLH1 mutated tumors, which was associated with differential mutational profiles, a more immune cold phenotype, and increased ER/PR expression in MLH1 hypermethylated tumors. Providers may consider early transition from single agent ICI to a multi-agent regimen or hormonal therapy for patients with MLH1 hypermethylated tumors.

Tumor Infiltrating Lymphocytes (TILS) and PD-L1 Expression in Breast Cancer: A Review of Current Evidence and Prognostic Implications from Pathologist's Perspective.

With the rise of novel immunotherapies able to stimulate the antitumor immune response, increasing literature concerning the immunogenicity of breast cancer has been published in recent years. Numerous clinical studies have been conducted in order to identify novel biomarkers that could reflect the immunogenicity of BC and predict response to immunotherapy. In this regard, TILs have emerged as an important immunological biomarker related to the antitumor immune response in BC. TILs are more frequently observed in triple-negative breast cancer and HER2+ subtypes, where increased TIL levels have been linked to a better response to neoadjuvant chemotherapy and improved survival. PD-L1 is a type 1 transmembrane protein ligand expressed on T lymphocytes, B lymphocytes, and antigen-presenting cells and is considered a key inhibitory checkpoint involved in cancer immune regulation. PD-L1 immunohistochemical expression in breast cancer is observed in about 10-30% of cases and is extremely variable based on tumor stage and molecular subtypes. Briefly, TNBC shows the highest percentage of PD-L1 positivity, followed by HER2+ tumors. On the other hand, PD-L1 is rarely expressed (0-10% of cases) in hormone-receptor-positive BC. The prognostic role of PD-L1 expression in BC is still controversial since different immunohistochemistry (IHC) clones, cut-off points, and scoring systems have been utilized across published studies. In the present paper, an extensive review of the current knowledge of the immune landscape of BC is provided. TILS and PD-L1 expression across different BC subtypes are discussed, providing a guide for their pathological assessment and reporting.

Discordance in PD-L1 expression using 22C3 and SP142 assays between primary and metastatic triple-negative breast cancer.

SP142 and 22C3 assays are approved companion diagnostic assays for anti-PD-1/PD-L1 therapy selection in metastatic triple-negative breast cancer (TNBC). The discordance in PD-L1 status between primary and metastatic tumors in the same patient has been poorly characterized. Here, we examined the concordance of PD-L1 status between the two assays and between primary tumors and metastases for each assay. We retrospectively evaluated tumor samples from 160 patients with TNBC, including 45 patients with paired primary and metastatic tumors. PD-L1 status was assessed using SP142 and 22C3 assays, to determine the immune cell (IC) score, tumor cell (TC) score (SP142 and 22C3), and combined proportion score (CPS: 22C3). The concordance of PD-L1 positivity at diagnostic cutoffs for SP142 (IC ≥ 1) and 22C3 (CPS ≥ 10) was substantial (κ = 0.80) in primary tumors and moderate (κ = 0.60) in metastatic tumors. In comparison, between primary and metastatic tumors, the concordance with 22C3 was moderate (κ = 0.50), whereas that with SP142 was poor (κ = -0.03). Among patients who were PD-L1 negative for both assays in primary tumors, 7/30 (23.3%) were PD-L1 positive for both or either 22C3 or SP142 in the metastatic tumors. The inter-assay concordance of PD-L1 positivity at diagnostic cutoffs was substantial in primary tumors and moderate in metastatic tumors. Discordance between PD-L1 status in primary and metastatic tumors was frequently observed, especially with SP142. Some patients with a PD-L1-negative status in primary tumors may still be candidates for immunotherapy, depending on the PD-L1 status in their metastatic tumors.

Correlation of tumor PD-L1 expression in different tissue types and outcome of PD-1-based immunotherapy in metastatic melanoma – analysis of the DeCOG prospective multicenter cohort study ADOREG/TRIM

PD-1-based immune checkpoint inhibition (ICI) is the major backbone of current melanoma therapy. Tumor PD-L1 expression represents one of few biomarkers predicting ICI therapy outcome. The objective of the present study was to systematically investigate whether the type of tumor tissue examined for PD-L1 expression has an impact on the correlation with ICI therapy outcome. Pre-treatment tumor tissue was collected within the prospective DeCOG cohort study ADOREG/TRIM (CA209-578; NCT05750511) between February 2014 and May 2020 from 448 consecutive patients who received PD-1-based ICI for non-resectable metastatic melanoma. The primary study endpoint was best overall response (BOR), secondary endpoints were progression-free (PFS) and overall survival (OS). All endpoints were correlated with tumor PD-L1 expression (quantified with clone 28-8; cutoff ≥5%) and stratified by tissue type. Tumor PD-L1 was determined in 95 primary tumors (PT; 36.8% positivity), 153 skin/subcutaneous (34.0% positivity), 115 lymph node (LN; 50.4% positivity), and 85 organ (40.8% positivity) metastases. Tumor PD-L1 correlated with BOR if determined in LN (OR=0.319; 95% CI=0.138-0.762; P=0.010), but not in skin/subcutaneous metastases (OR=0.656; 95% CI=0.311-1.341; P=0.26). PD-L1 positivity determined on LN metastases was associated with favorable survival (PFS, HR=0.490; 95% CI=0.310-0.775; P=0.002; OS, HR=0.519; 95% CI=0.307-0.880; P=0.014). PD-L1 positivity determined in PT (PFS, HR=0.757; 95% CI=0.467-1.226; P=0.27; OS; HR=0.528; 95% CI=0.305-0.913; P=0.032) was correlated with survival to a lesser extent. No relevant survival differences were detected by PD-L1 determined in skin/subcutaneous metastases (PFS, HR=0.825; 95% CI=0.555-1.226; P=0.35; OS, HR=1.083; 95% CI=0.698-1.681; P=0.72). For PD-1-based immunotherapy in melanoma, tumor PD-L1 determined in LN metastases was stronger correlated with therapy outcome than that assessed in PT or organ metastases. PD-L1 determined in skin/subcutaneous metastases showed no outcome correlation and therefore should be used with caution for clinical decision making. Bristol-Myers Squibb (ADOREG/TRIM, NCT05750511); German Research Foundation (DFG; Clinician Scientist Program UMEA); Else Kröner-Fresenius-Stiftung (EKFS; Medical Scientist Academy UMESciA).

PD-L1 protein expression by Combined Positive Score (CPS) in patients with muscle invasive or advanced urothelial carcinoma: a single institution experience

IntroductionImmune checkpoint inhibitors have revolutionized the treatment of patients with advanced urothelial carcinoma (UC) in the frontline and relapsed settings. Lebanon has one of the highest incidence of UC worldwide, yet no data exists regarding the expression of PD-L1 by Combined Positive Score (CPS) in advanced disease.MethodsWe reviewed all patients treated at our institution for high grade UC, stage pT2 and above, between January 2017 and March 2021. We assessed the expression of PD-L1 by immunohistochemistry using 22C3 clone, and analyzed the association between PD-L1 expression and clinicopathological characteristics. PD-L1 positivity was defined as CPS score ≥ 10.ResultsA total of 101 patients with advanced UC were included, with a median age of 71 years (range, 38 to 96 years); 78% were ever-smokers. Ninety-three of 101 patients (92%) had conventional UC and 43 patients (43%) had positive PD-L1 expression, with 12 patients having CPS of 100. The analysis by molecular subtype showed that patients with maximal CPS of 100 were enriched in “basal” molecular subtype. However, no association was found between PD-L1 expression (positive versus negative) and clinicopathological characteristics.ConclusionThe positivity of PD-L1 expression as assessed by CPS using the 22C3 clone in our population was almost comparable to the results reported in the occidental literature. Therefore, PD-L1 expression, as a potential predictor of response to immunotherapy, concerns the same percentage of the Lebanese UC patients.

Circulating and intratumoral immune determinants of response to atezolizumab plus bevacizumab in patients with variant histology or sarcomatoid renal cell carcinoma

Abstract Background Renal cell carcinoma of variant histology (vRCC) encompasses approximately 20% of RCC diagnoses. Due to a poor understanding of the different biologies of vRCCs, there is currently no standard of care for this type of tumor and guidelines are extrapolated from clear-cell RCC trials. A phase II trial of atezolizumab plus bevacizumab in patients with vRCC or sarcomatoid differentiation had an acceptable safety profile and evidence of clinical activity, particularly in tumors with positive PD-L1 expression or sarcomatoid differentiation. To better understand the determinants of immunotherapy response in this population, we characterized blood- and tissue-based immune markers for patients with vRCC, or any RCC histology with sarcomatoid differentiation, enrolled in this phase II trial of atezolizumab and bevacizumab. Methods We used patient data and biospecimens (blood and tumor) from a prospective phase-II clinical trial of the anti-PD-L1 antibody atezolizumab and the anti-VEGF-A antibody bevacizumab in patients with metastatic RCC with variant histology and/or sarcomatoid differentiation (NCT02724878). Blood samples were collected at baseline, prior to therapy (C1D1: cycle one day one), and on-treatment, following two cycles of therapy (C3D1: cycle three day one). Plasma was assayed for the levels of soluble factors (e.g., cytokines and growth factors), and peripheral blood mononuclear cells (PBMCs) were immunophenotyped using flow cytometry. In addition, formalin-fixed, paraffin-embedded (FFPE) archival or pre-treatment study tumor biopsies were analyzed by immunohistochemistry and immunofluorescence for enrolled subjects with available biospecimen. Baseline demographics, clinical characteristics, and treatment outcomes, including progression-free survival (PFS), overall survival (OS), and clinical benefit, were collected, and analyzed from the trial database. This study was approved by each participating institution's institutional review board, and all patients provided written informed consent. We used R (version 4.1.1) to carry out our analyses. We assessed the relationships between biomarker variables were analyzed using Pearson correlations and hierarchical clustering using Euclidean distance. All biomarker variables were Z-score normalized. We calculated an inflammatory module, the systemic inflammation score (SIS), which corresponds to the average score of the five highly correlated inflammatory cytokines: MIP-1b, IL-1, MCP-1, IL-6, and IL-13. We applied pair-wise Wilcoxon tests to evaluate the association of biomarkers with clinical benefit groups and log-rank-tests and univariate Cox regression models to evaluate the relationship of biomarkers with PFS and OS. Results With the exception of two angiogenic cytokines (VEGF-A and Angiopoietin 2) that were more abundant in patients with non-sarcomatoid tumors, baseline circulating cytokines (plasma) had similar distributions between sarcomatoid and non-sarcomatoid subgroups. Baseline circulating inflammatory cytokines were highly correlated with one another, forming an “inflammatory module” that was increased in IMDC-poor risk patients and was associated with worse PFS (p=0.028) and with resistance to therapy (p=0.033). At baseline, an elevated circulating VEGF-A level was associated with a lack of response (p=0.03) and worse PFS (p=0.021). However, a larger increase in on-treatment levels of circulating VEGF-A was associated with clinical benefit (p=0.01) and improved overall survival (p=0.0058). Among peripheral immune cell populations, an on-treatment decrease in circulating PD-L1+ T cells was associated with improved outcomes, with a reduction in CD4+ PD-L1+ (HR:0.62[0.49-0.91], p=0.016) and CD8+ PD-L1+ T cells (HR:0.59[0.39-0.87], p=0.009) correlated with improved PFS. Within the tumor itself, a higher percentage of terminally exhausted (PD-1+ and either TIM-3+ or LAG-3+) CD8+ T cells was associated with worse PFS (p=0.028). We observed similar trends for all of these results in different subgroups according to sarcomatoid status and histology. Conclusions Overall, these findings support the value of tumor and blood-based immune assessments in determining therapeutic benefit for patients with RCC receiving atezolizumab plus bevacizumab. Furthermore, they provide a foundation for future biomarker studies for patients with variant histology RCC receiving immunotherapy-based combinations.

Retracted: Risk Analysis of Positive PD-L1 Expression and Clinicopathological Features and Survival Prognosis in Patients with Colorectal Cancer: Systematic Review and Meta-Analysis.

[This retracts the article DOI: 10.1155/2022/8212486.].

Effect of spatial transcriptomics on tumor immune microenvironment resistant to immune checkpoint inhibitors in metastatic urothelial carcinoma.

68 Background: Immune checkpoint inhibitors (ICBs) for metastatic urothelial carcinoma (mUC) are widely used, but their response rates are inadequate, around 20%. Currently, analyzing samples prior to ICB administration has accumulated so far; thereby predicting response to ICBs by high tumor mutation burden levels and PD-L1 positivity. However, little is known about what kind of tumor microenvironment is formed in recurrent or residual tumors post ICBs, and how it contributes to ICB resistance. Therefore, we applied large datasets of spatial transcriptome analysis to mUC samples post ICBs in order to reveal the diversity of the tumor microenvironment after ICB administration. Methods: The Visium spatial transcriptome analysis was performed on FFPE samples from 12 mUC patients with viable tumor resection after ICB administration. Further, 4 FFPE samples of non-metastatic UC treated-surgically were subjected to spatial transcriptome analysis and were used as ICB-naïve tumor controls. First, the tumor and stromal regions were pathologically annotated. We then identified CD4-positive, CD8A-positive, and FOXP3-positive spots in both tumor and stromal regions, respectively, and analyzed changes in differentially expressed genes (DEGs) and pathways in the tumor space where cancer cells survive after ICB treatment. Results: For DEGs and pathway analysis, 8082 spots in the tumor region and 10964 spots in the stromal region were used. In the tumor and stromal areas, 15.4% and 15.1% were CD4 positive, 6.5% and 6.3% were CD8A positive, and 2.9% and 2.0% were FOXP3 positive, respectively. In tumor regions of ICB-treated specimens, the expression of SULF1, an extracellular matrix-related gene, was increased in all CD4-, CD8A-, and FOXP3-positive spots compared to negative spots. In addition, the expression of complement-related genes such as C1QB, C1QC, and C1R was upregulated in CD8A-positive spots in the tumor regions after ICB. Pathway analysis revealed that oxidative phosphorylation was enriched in CD8A-positive spots in tumor regions prior to ICB, while angiogenesis was enriched in CD8A-positive spots after ICB. Conclusions: Spatial transcriptome analysis of a large dataset of UC samples showed distinct features of immune infiltration involved in therapeutic resistance in the tumor microenvironment under immunotherapy. Notably, T-cells may be exhausted by metabolic changes in CD8A-positive spots in ICB-refractory tumor environment spaces.

Skip metastasis at the esophageal resection margin in radical gastrectomy: clinical characteristics of 30 cases

Objective: To summarize the clinical characteristics of patients with skip metastasis at esophageal resection margin during radical gastrectomy. Methods: This is a descriptive study of case series. Relevant data from 2006 to 2022 were collected from two major gastric cancer consultation and treatment centers: Nanjing Drum Tower Hospital and Jinling Hospital.Characteristics, surgical approach, number of dissected lymph nodes, immunohistochemical staining, and pathological staging were summarized and analyzed. The distribution of residual tumor cells at the esophageal margins was further analyzed at the cellular and tissue levels. Skip metastasis at the esophageal resection margin was defined as a negative esophageal margin with a positive margin in the cephalad donut. Results: Thirty (0.33%, 30/8972) eligible patients, 24 (80.0%) of whom were male, were identified in the two centers. The mean age was 63.9±11.0 years. Seventeen (56.7%) of these patients had papillary or tubular adenocarcinomas, including 13 (43.3%) poorly- and four (13.3%) moderately-differentiated tumors; four (13.3%) had signet-ring cell carcinomas; four (13.3%) mucinous adenocarcinomas; three (10.0%) mixed adenocarcinomas, including two with poorly-differentiated tubular adenocarcinomas mixed with signet-ring cell carcinoma and mucinous adenocarcinoma; and one had a poorly differentiated tubular adenocarcinoma mixed with signet-ring cell carcinoma. Two patients (6.7%) had other types of cancer, namely adenosquamous carcinoma in one patient and undifferentiated carcinoma in the other one. The predominant tumor sites were the lesser curvature (n=26, 86.7%) and the cardia (n=24, 80.0%). The mean tumor diameter was 6.6 cm, mean distance between tumor and esophageal resection margin was 1.5 cm, and proportions of tumor invasion into the dentate line, nerves, and vessels were 80.0% (24/30), 86.7%(26/30), and 93.3% (28/30), respectively. The mean number of lymph nodes resected was 20.4±8.9. The pathological stage was mainly T4 (n=18, 60.0%) and N3 (n=21, 70.0%), the median Ki67 was 52.7%, and the rates of positivity for HER2, EGFR, VEGFR, E-cadherin and PD-L1 were 40.0% (12/30), 46.7% (14/30), 80.0% (24/30), 86.7% (26/30) and 16.7% (5/30), respectively. At the cellular level, cancer cells were mainly distributed in small focal areas, as cell masses, or as tumor thrombi; large numbers of widely distributed atypic cells were seldom observed. At the tissue level, cancer cells were located in the mucosal layer in seven patients (23.3%), in the submucosal layer in 18 (60.0%), and in the muscular layer in five (16.7%); no cancer cells were identified in the outer membrane. Five of the seven tumors were located in the lamina propria, two in the muscularis mucosae, and none in the mucosal epithelium. Conclusion: Patients with skip metastasis at the esophageal resection margin at radical gastrectomy have unfavorable tumor biology and a high proliferation index, are at a late pathological stage, and the residual cancer is mostly located in the submucosa.