Low PD-L1 Expression Research Articles

Machine learning modeling of real-world primary resistance and hyperprogression in immune checkpoint inhibitor (ICI) treated advanced non-small cell lung cancer (aNSCLC) patients.

e21127 Background: Many ICI-eligible patients display primary resistance (10-res), with some experiencing hyperprogressive disease (HPD). Machine learning (ML) on real-world data (RWD) can improve our clinical and mechanistic understanding related to ICI response. Methods: XGBoost models were built on ConcertAI’s oncology clinicogenomic database, comprising structured EMR, curated records from unstructured documents, and NGS reports, to predict 10-res and HPD in aNSCLC patients treated with ICI or chemotherapy (CT) within their 1st three lines between January 2015 and June 2021. HPD was defined as either tumor progression, treatment (Tx) discontinuation, or death within 49 days from Tx start (index date). 10-res was defined as any of these events after 49 days but within 180 days from Tx start. Models were trained on a clinical (N = 7647, ICI = 3265, 10-res = 3909, HPD = 2076), and a clinicogenomic (CG) (2304, 1271, 1084, 596) cohort. A 60:20:20 split was used for training, validation, and testing. Baseline period was defined as 180 days prior to the index date. Prognostic and predictive factors were identified from Shapley Additive Explanation (SHAP) values. Results: The clinical model was superior at predicting HPD; the CG model was better at predicting 10-res (AUROC = 0.67 for both). Total metastases (≥ 2) and lower levels of hemoglobin (HGB), and lymphocytes, higher ALP were risk factors for both HPD and 10-res. Elevated WBC and bilirubin, higher heart rate, lower body temperature, and history of anti-inflammatory medications were risk factors for HPD. Non-ICI-based regimens, low TMB, lower PD-L1 expression, and mutations in EGFR or KEAP1 were risk factors for 10-res. Mutation in KRAS was protective of 10-res. In addition to PD-L1 and TMB, the models identified prior lung radiation, smokers, higher baseline comorbidities, and positive MMR status as predictive of lower risk of 10-res from ICI but not in CT. STK11 mutation was predictive of higher risk of 10-res from ICI vs. CT. Non-adenocarcinoma histology was predictive of higher risk of HPD from ICI vs. CT. Conclusions: ML on RWD generated evidence to support both established and emerging prognostic and predictive markers for ICI response.[Table: see text]

Impact of STK11 copy loss on clinical outcomes to PD-(L)1 blockade in non–small cell lung cancer.

9059 Background: STK11 is among the most commonly altered genes in non-squamous lung cancers. While STK11 mutation is associated with diminished efficacy of immune checkpoint inhibition (ICI), particularly in KRAS mutated tumors, it is not known whether STK11 copy deletion influences outcomes to ICI. Methods: Patients with advanced non-squamous non-small cell lung cancer (NSCLC) treated with ICI whose tumors underwent genomic profiling were included. Clinical outcomes to ICI were analyzed according to KRAS mutation and STK11 deletions. STK11 copy number variations (CNVs) were determined using an internal informatic pipeline. Kaplan-Meier methodology was used to estimate event-time distributions. Results: Of 559 patients with non-squamous NSCLCs (Nsq-NSCLC), 40.4% (N = 226) had a KRAS mutation ( KRASmut), 18.4% (N = 103) had an oncogenic STK11 mutation ( STK11mut), and 22.5% had either single (N = 123), or bi-allelic (N = 3) deletion ( STK11del). Given that 32.5% of STK11del cases had a concurrent oncogenic STK11 mutation in our cohort, to isolate the impact of STK11del on ICI outcomes we excluded samples with STK11mut from this analysis. In all comers with NSCLC, STK11del had no impact on objective response rate (22.2% versus 23.8%, P = 0.8), progression-free (PFS, HR 0.90, P = 0.30), and overall survival (OS, HR 0.96, P = 0.79) to ICI. When we examined the impact of STK11del on clinical outcomes to PD-(L)1 blockade among KRASmut cases we found that STK11del was associated with a numerically lower ORR (13.3% versus 30.0%, P = 0.12), and a significantly shorter PFS (HR 0.57, P = 0.018) compared to cases without STK11del. No difference in OS were observed between these groups (HR 0.77, P = 0.39). Among KRASwt NSCLCs, STK11del cases had a similar ORR (22.6% versus 22.9%, P = 0.99), PFS (HR 0.92, P = 0.63), and OS (HR 1.18, P = 0.32) to PD-(L)1 inhibition compared to cases without STK11del. Among KRASmut but not KRASwt NSCLCs, cases with STK11del had significantly lower PD-L1 expression compared to those without STK11 deletions (27.5% versus 70%, P = 0.01). Conclusions: STK11 deletion is associated with low response rate and short progression-free survival among KRAS mutant NSCLCs. Future analyses will incorporate additional cases to increase sample size and immunopathologic findings to assess impact of mono and bi-allelic deletion on protein expression.

Cellular engagement and interaction in the tumor microenvironment predict non-response to PD-1/PD-L1 inhibitors in metastatic non-small cell lung cancer

Immune checkpoint inhibitors (ICI) with anti-PD-1/PD-L1 agents have improved the survival of patients with metastatic non-small cell lung cancer (mNSCLC). Tumor PD-L1 expression is an imperfect biomarker as it does not capture the complex interactions between constituents of the tumor microenvironment (TME). Using multiplex fluorescent immunohistochemistry (mfIHC), we modeled the TME to study the influence of cellular distribution and engagement on response to ICI in mNSCLC. We performed mfIHC on pretreatment tissue from patients with mNSCLC who received ICI. We used primary antibodies against CD3, CD8, CD163, PD-L1, pancytokeratin, and FOXP3; simple and complex phenotyping as well as spatial analyses was performed. We analyzed 68 distinct samples from 52 patients with mNSCLC. Patients were 39–79 years old (median 67); 44% were male and 75% had adenocarcinoma histology. The most used ICI was atezolizumab (48%). The percentage of PD-L1 positive epithelial tumor cells (EC), degree of cytotoxic T lymphocyte (CTL) engagement with EC, and degree of CTL engagement with helper T lymphocytes (HTL) were significantly lower in non-responders versus responders (p = 0.0163, p = 0.0026 and p = 0.0006, respectively). The combination of these 3 characteristics generated the best sensitivity and specificity to predict non-response to ICI and was also associated with shortened overall survival (p = 0.0271). The combination of low CTL engagement with EC and HTL along with low expression of EC PD-L1 represents a state of impaired endogenous immune reactivity. Together, they more precisely identified non-responders to ICI compared to PD-L1 alone and illustrate the importance of cellular interactions in the TME.

The regulation of CD73 in non-small cell lung cancer

BackgroundLung cancer is the leading cause of global cancer-related mortality. Although immune checkpoint therapy has achieved remarkable results in lung cancer, EGFR-mutant or ALK-positive non-smallcell lung cancer patients show limited benefit. Besides the low tumor mutational burden, PD-L1 expression and CD8+ tumor-infiltrating T cells, upregulation of CD73/adenosine pathway also contributes to the immune-inert microenvironment of EGFR-mutant NSCLC. However, the detailed mechanism underlying the regulation of CD73 is unclear. MethodsTCGA data was used to analyze the CD73 expression in cancer patients. Western blotting, qPCR, and ChIP-PCR were performed in multiple NSCLC cancer cell lines and patient derived organoids were used to explore the regulation of CD73 expression using western blotting. ResultsCD73 expression was highly expressed in multiple cancer types. Pharmacological or genetic inhibition of EGFR, MEK, KRAS, or ALK dramatically reduced the CD73 mRNA and protein expression in NSCLC cancer cells and patient-derived organoids with EGFR mutation, KRAS mutation or ALK-rearrangement. C-Jun overexpression-induced CD73 mRNA and protein expression. ChIP assay showed that c-Jun bind to CD73 genomic regions. ConclusionsHigher CD73 expression in NSCLC cancer cells and patient-derived organoids with EGFR mutation, KRAS mutation or ALK-rearrangement. Mechanistically, CD73 is regulated by ERK-Jun pathway, wherein c-Jun regulates CD73 expression via binding to CD73 genomic regions.

The Role of PD-L1 Expression in Prediction and Stratification of Recurrent or Refractory Extranodal Natural Killer/T-Cell Lymphoma.

Background and AimsThe clinical outcome of relapsed and refractory (RR) extranodal natural killer/T-cell lymphoma (ENKTL) is poor. It is necessary to identify RR patients in ENKTL and find novel therapeutic targets to improve the prognosis of patients with RR ENKTL.MethodsA total of 189 ENKTL patients with effective clinical characteristics were enrolled. Paraffin specimens were collected for PD-L1 expression identification. Kaplan-Meier curve analysis was performed for survival analysis. Whole exome sequencing (WES) was performed for identifying the mutational characterization of RR and effective treatment (ET) patients.ResultsUnivariate and multivariate Cox proportional hazards regression analysis showed that negative PD-L1 expression (HR = 1.132, 95% CI = 0.739-1.734, P = 0.036) was an independent predictor of poor prognosis in patients with ENKTL. The overall survival (OS) of PD-L1 positive patients was significantly higher than that of PD-L1 negative patients (P = 0.009). Then, we added PD-L1 expression as a risk factor to the model of Prognostic Index of Natural Killer Lymphoma (PINK), and named as PINK+PD-L1. The PINK+PD-L1 model can significantly distinguish RR patients, ET patients, and the whole cohort. Moreover, our data showed that PD-L1 expression was lower than 25% in most RR patients, suggesting that RR subtypes may be associated with low expression of PD-L1 (P = 0.019). According to the whole exome sequencing (WES), we found that the mutation frequencies of JAK-STAT (P = 0.001), PI3K-AKT (P = 0.02) and NF-kappa B (P < 0.001) pathways in RR patients were significantly higher than those in ET patients.ConclusionPatients tend to show RR when PD-L1 expression is lower than 25%. The model of PINK+PD-L1 can stratify the risk of different groups and predict OS in ENKTL patients. The mutational profile of ENKTL patients with RR is different from that of patients with ET.

Restored CD8+PD-1+ T Cells Facilitate the Response to Anti-PD-1 for Patients With Pancreatic Ductal Adenocarcinoma.

PurposeWe aimed to investigate the restoration of CD8+PD-1+ T cells through adoptive T-cell therapy (ACT) in relation to the prognosis and the therapeutic response to anti-PD-1 in patients with advanced pancreatic cancer (APC).MethodsA total of 177 adult patients who underwent tumor resection as initial treatment for pancreatic ductal adenocarcinoma (PDAC) from February 2013 to July 2019 at Zhongnan Hospital of Wuhan University were enrolled in this study. Another cohort of 32 patients with APC was prospectively enrolled from Capital Medical University Cancer Center between June 1, 2013, and May 30, 2019.ResultsOf the 177 patients who received tumor resection, 67 tumor samples showed overexpression of PD-L1 and 110 patients with low expression of PD-L1. We found that overexpressed PD-L1 was a significant prognostic factor related to overall survival (OS). Furthermore, we tested the percentage of peripheral CD8+PD-1+ T cells in all patients and found that it was significantly correlated with the PD-L1 expression and the prognosis of patients with PDAC. The peripheral blood T lymphocyte subtypes were tracked for 30 months, and CD8+PD-1+ cells were shown to decrease. After that, we performed ACT for patients with APC in another cancer center. We found that the ratios of posttreatment of ACT/pre-ACT CD8+PD-1+ T cells were significantly related to the prognosis of patients with APC. Moreover, patients with combined treatment of ACT with anti-PD-1 had significantly favorable OS.ConclusionsThis study showed that the CD8+PD-1+ T-cell level was related to the expression of PD-L1. Restoring CD8+PD-1+ T cells in patients with APC by treatment of ACT significantly benefits the prognosis and facilitates the response to anti-PD-1.

The “digital biopsy” in non-small cell lung cancer (NSCLC): a pilot study to predict the PD-L1 status from radiomics features of [18F]FDG PET/CT

The present pilot study investigates the putative role of radiomics from [18F]FDGPET/CT scans to predict PD-L1 expression status in non-small cell lung cancer (NSCLC) patients. In a retrospective cohort of 265 patients with biopsy-proven NSCLC, 86 with available PD-L1 immunohistochemical (IHC) assessment and [18F]FDGPET/CT scans have been selected to find putative metabolic markers that predict PD-L1 status (< 1%, 1-49%, and ≥ 50% as per tumor proportion score, clone 22C3). Metabolic parameters have been extracted from three different PET/CT scanners (Discovery 600, Discovery IQ, and Discovery MI) and radiomics features were computed with IBSI compliant algorithms on the original image and on images filtered with LLL and HHH coif1 wavelet, obtaining 527 features per tumor. Univariate and multivariate analysis have been performed to compare PD-L1 expression status and selected radiomic features. Of the 86 analyzed cases, 46 (53%) were negative for PD-L1 IHC, 13 (15%) showed low PD-L1 expression (1-49%), and 27 (31%) were strong expressors (≥ 50%). Maximum standardized uptake value (SUVmax) demonstrated a significant ability to discriminate strong expressor cases at univariate analysis (p = 0.032), but failed to discriminate PD-L1 positive patients (PD-L1 ≥ 1%). Three radiomics features appeared the ablest to discriminate strong expressors: (1) a feature representing the average high frequency lesion content in a spherical VOI (p = 0.009); (2) a feature assessing the correlation between adjacent voxels on the high frequency lesion content (p = 0.004); (3) a feature that emphasizes the presence of small zones with similar grey levels inside the lesion (p = 0.003). The tri-variate linear discriminant model combining the three features achieved a sensitivity of 81% and a specificity of 82% in the test. The ability of radiomics to predict PD-L1 positive patients was instead scarce. Our data indicate a possible role of the [18F]FDG PET radiomics in predicting strong PD-L1 expression; these preliminary data need to be confirmed on larger or single-scanner series.

Investigation on the survival implications of PD-L1 expression status in ALK- rearranged advanced non-small cell lung cancer treated with first-line crizotinib

BackgroundProgrammed cell death–ligand 1 (PD-L1) expression has been associated with shorter progression-free survival (PFS) of crizotinib-treated patients with anaplastic lymphoma kinase (ALK) rearranged non-small cell lung cancer (NSCLC). However, the association between PD-L1 expression and overall survival (OS) in ALK-rearranged NSCLC remains unclear. In this study, we investigated the survival implication of baseline PD-L1 expression status in crizotinib-treated patients with ALK-rearranged advanced NSCLC. MethodsBetween October 1, 2015, and October 31, 2021, we retrospectively analyzed the baseline PD-L1 expression levels using immunohistochemistry 22C3 assay of tissue samples from 128 patients with ALK-rearranged advanced lung adenocarcinoma who were treated with first-line crizotinib. ResultsOf the 128 baseline tumor specimens analyzed, a majority (76.6%, n = 98) had low PD-L1 expression (tumor proportion score (TPS) < 50%), wherein 58.6% (n = 75) had < 1% and 18.0% (n = 23) had 1%–49%, and the remaining 23.4% (n = 30) had high PD-L1 expression level (TPS ≥ 50%). High baseline PD-L1 expression was not associated with any clinical characteristic examined. Patients with high baseline PD-L1 (n = 30) expression level had significantly shorter median PFS (6 vs 11 months, p = 0.011) and OS (17 vs 53 months, p = 0.023) on crizotinib treatment than those with low PD-L1 level (n = 98). ConclusionsA subset of patients with ALK-rearranged NSCLC having high baseline PD-L1 expression level (TPS of ≥ 50%) had poorer survival outcomes despite crizotinib therapy. Our study raises the need to investigate alternative treatment strategies to improve survival outcomes of this patient subset.

Multiple eruptive squamoproliferative lesions during anti-PD1 immunotherapy for metastatic melanoma: Pathogenesis, immunohistochemical analysis and treatment.

Treatment with anti-PD1 inhibitors may enhance the risk for developing low grade squamoproliferative skin tumors. Immunohistochemical (IHC) analysis of the immune tumor microenvironment (TME) allows exploration of the pathogenesis and relationship with the PD1/PDL1 axis. Patients with eruptive keratoacanthoma (KA)-like lesions were recruited from the Melanoma Institute Australia, a tertiary referral specialist melanoma treatment center from January 2015 to August 2017. Clinicopathologic evaluation and IHC features of tumor cells (PDL1 expression) and peritumoral microenvironment (PD1, FOXP3, PDL1, CD4:CD8 expressing cells) in 12 eruptive KA-like lesions, were compared with solitary KAs in age and sex matched non-anti-PD1 treated controls. Four patients with repeated episodes of eruptive KA-like and lichenoid lesions developing 2-7 months after commencing pembrolizumab for AJCC stage IV melanoma, were recruited. Eruptive KA-like squamoproliferative lesions occurred in sun exposed sites and in areas of resolving, concomitant or delayed lichenoid reactions. Histologically, the lesions were well-differentiated squamoproliferative lesions resembling infundibulocystic squamous cell carcinoma or KA. IHC of cases and controls revealed low PDL1 expression of both squamous tumor cells and the TME immune cells. The numbers of immunosuppressive FOXP3 positive Tregs and PD1-expressing T-cells were higher in the cases than the controls but the CD4:CD8 ratio (2:1) was similar. The patients best responded to acitretin and were managed surgically if they demonstrated neoplastic features. Accelerated squamoproliferative growth in actinically damaged keratinocytes associated with lichenoid eruptions may be unmasked in patients treated with anti-PD1 immunotherapy potentially contributed to by a local cutaneous immunosuppressed TME.

Correlation Between Immune Cell Infiltration and PD-L1 Expression and Immune-Related lncRNA Determination in Triple-Negative Breast Cancer.

As a key element of the tumor microenvironment (TME), immune cell infiltration (ICI) is a frequently observed histologic finding in people with triple-negative breast cancer (TNBC), and it is linked to immunotherapy sensitivity. Nonetheless, the ICI in TNBC, to the best of our knowledge, has not been comprehensively characterized. In our current work, computational algorithms based on biological data from next-generation sequencing were employed to characterize ICI in a large cohort of TNBC patients. We defined various ICI patterns by unsupervised clustering and constructed the ICI scores using the principal component analysis (PCA). We observed patients with different clustering patterns had distinct ICI profiles and different signatures of differentially expressed genes. Patients with a high ICI score tended to have an increased PD-L1 expression and improved outcomes, and these patients were associated with decreased tumor mutational burden (TMB). Interestingly, it was showed that patients with high TMB exhibited an ameliorated overall survival (OS) than patients with low TMB. Furthermore, TMB scores only affected the prognosis of TNBC patients in the low-ICI score group but not in the high group. Finally, we identified a new immune-related lncRNA (irlncRNA) signature and established a risk model for the TNBC prognosis prediction. In addition, the high-risk group was related to poor prognosis, a high infiltration level of plasma B cells, monocytes, M2 macrophages, and neutrophils and a low PD-L1 expression. Therefore, the characterization and systematic evaluation of ICI patterns might potentially predict the prognosis and immunotherapy response in TNBC patients.

Lung Neuroendocrine Tumors: How Does Molecular Profiling Help?

Lung neuroendocrine tumors (NETs)-typical carcinoids and atypical carcinoids-have unique molecular alterations that are distinct from neuroendocrine carcinomas of the lung and non-small cell lung cancers. Here, we review the role of molecular profiling in the prognosis and treatment of lung NETs. There have been no recently identified molecular prognostic factors for lung NETs and none that have been routinely used to guide management of patients with lung NETs. Previous findings suggest that patients with loss of chromosome 11q may have a worse prognosis along with upregulation of anti-apoptotic pathways (e.g., loss of CD44 and OTP protein expression). Lung NETs rarely harbor driver mutations commonly found in non-small cell lung cancer (NSCLC) or TP53/RB1 mutations found universally in small cell lung cancer. Lung NETs also have low tumor mutation burden and low PD-L1 expression. Everolimus, an mTOR inhibitor and the only FDA approved therapy for unresectable lung NETs, is an effective treatment but the presence of a molecular alteration in the PI3K/AKT/mTOR pathway is not known to predict treatment response. The predominant mutations in lung NETs occur in genes regulating chromatin remodeling and histone modification, with potential targeted therapies emerging in clinical trials. Lung NETs have recurring alterations in genes that regulate the epigenome. Future targeted therapy interfering with epigenetic pathways may hold promise.

Component with abundant immune-related cells in combined hepatocellular cholangiocarcinoma identified by cluster analysis.

Combined hepatocellular cholangiocarcinoma (cHCC‐CCA) is a heterogeneous tumor sharing histological features with hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). The tumor immune microenvironment (TIME) of cHCC‐CCA is unclear. We compared the TIME of cHCC‐CCA with that of HCC and iCCA. Twenty‐three patients with cHCC‐CCA after hepatectomy were evaluated in this study. Twenty‐three patients with iCCA and HCC were also included. iCCA was matched for size, and HCC was matched for size and hepatitis virus infection with cHCC‐CCA. Immune‐related cells among the iCCA‐component of cHCC‐CCA (C‐com), HCC‐component of cHCC‐CCA (H‐com), iCCA, and HCC were assessed using multiplex fluorescence immunohistochemistry. Among C‐com, H‐com, iCCA, and HCC, multiple comparisons and cluster analysis with k‐nearest neighbor algorithms were performed using immunological variables. Although HCC had more T lymphocytes and lower PD‐L1 expression than iCCA (P < 0.05), there were no significant differences in immunological variables between C‐com and H‐com. C‐com tended to have more T lymphocytes than iCCA (P = 0.09), and C‐com and H‐com had fewer macrophages than HCC (P < 0.05). In cluster analysis, all samples were classified into two clusters: one cluster had more immune‐related cells than the other, and 12 of 23 H‐com and eight of 23 C‐com were identified in this cluster. The TIME of C‐com and H‐com may be similar, and some immunological features in these components were different from those in HCC and some iCCA. Cluster analysis identified components with abundant immune‐related cells in cHCC‐iCCA.

Withdrawal.

Background Hepatoblastoma (HB) is the most common malignant embryonic liver tumor in children. In this study, we used the NGS method to detect the genotype characteristics of HB, summarized the correlation between common mutation genotypes of HB and clinical treatment and prognosis. Methods Firstly, DNA was extracted from tumor tissue specimens and peripheral blood of 19 children patients with HB. Then, DNA panel and NGS methods were used to detect tumor diagnosis and treatment-related genes, followed by summary of genotype characteristics. Thirdly, In order to further study the application of immunotherapy in HB, we performed programmed cell PDL1 immunohistochemical detection. Finally, the clinical treatment effect and prognosis of the children patients were statistically analyzed according to the characteristics of the genotype. Results In this study, some mutated genes including NFE2L2, CTNNB1, MYCN, TP53, AXIN1 and APC were related to the pathogenesis of HB. The immunohistochemical results showed lower expression of PDL1. The complete remission (CR) rate of children patients in the gene abnormality group was lower than that of the non-reported disease-related gene abnormality group. Conclusion The CR rate of the children patients with altered genotype was lower than those children patients without altered genotype. In addition, low expression of PDL1 limited the immunotherapy of children patients with HB.

Comprehensive genomic profiling (CGP) of chromophobe renal cell carcinoma (chrRCC) compared with clear cell RCC (ccRCC): Impact of FLCN genomic alteration (GA) status.

292 Background: FLCN is a tumor suppressor gene associated with cutaneous hair follicle development. FLCN germline mutations are linked to inherited chrRCC in the Birt-Hogg-Dube (BHD) syndrome. We queried whether clinically sporadic chrRCC featured FLCN mutations by comparing the genomic profiles of chrRCC with ccRCC. Methods: 108 chrRCC and 2110 ccRCC underwent hybrid-capture based comprehensive genomic profiling (CGP) to evaluate all classes of genomic alterations (GA). Tumor mutational burden (TMB) was determined on up to 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. PD-L1 expression was determined by IHC (Dako 22C3). Results: Patients (pts) with chrRCC were more frequently female and younger than pts with ccRCC p &lt;.0001). None of the submitted clinical records in the chrRCC cases listed signs of BHD syndrome. FLCN GA were identified in only 2.3% of the ccRCC cases with 37% of the GA predicted to be germline. chrRCC did not reveal somatic or germline FLCN GA. GA/tumor were slightly higher in ccRCC vs chrRCC (3.6 vs 2.4; NS). GA more frequent in chrRCC included TP53, RB1 and PTEN. GA more frequent in the ccRCC included VHL, BAP1, PBRM1, SETD2, CDKN2A/B, ARID1A, NF2, PIK3CA and TERT. Putative biomarkers of immune checkpoint inhibitor (ICPI) response were infrequent in both groups with only a slightly higher, but still low, mean TMB in ccRCC vs chrRCC cases. IHC revealed moderate PD-L1 expression at low and minimal PD-L1 expression at high staining level, which was slightly increased in the chrRCC group. Conclusions: FLCN mutations that are associated with the familial incidence of chrRCC were not associated with sporadic chrRCC. Sporadic chrRCC has substantially different genomic profile from ccRCC and may harbor a few ‘targetable’ GA. The prediction of response to ICPI in RCC remains challenging with chrRCC featuring slightly higher PD-L1 expression and ccRCC featuring higher PBRM1 GA and higher TMB.[Table: see text]

Association of RB1 mutational status with overall genomic landscape in neuroendocrine prostate cancer (NEPC).

156 Background: NEPC is a high-grade aggressive form of prostate cancer. We queried whether RB1 mutation status would impact the genomic features of NEPC. We hypothesized that there would be differences in GA frequencies in RB1 mutated vs non mutated cases. Methods: From a series of 13,496 cases of clinically advanced PC, 415 (3.1%) histologically defined NEPC were sequenced using a hybrid capture-based FDA-approved clinical genomic profiling (CGP) assay to detect all classes of genomic alterations (GA). Tumor mutational burden (TMB) was determined on 0.8 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 95 loci. PD-L1 expression was determined by IHC (Dako 22C3) with low tumor cell positive staining 1-49% and high staining ≥50% expression. Results: 253 (61%) of NEPC feature GA in RB1 (RB1 mut+). This contrasts with a 5.8% frequency of RB1 GA in the non-NEPC (P &lt;.0001). The RB1 mut+ NEPC featured a slightly greater number of driver GA/tumor than the RB1 mut- NEPC (5.1 vs 4.2). Age and TMPRSS2-ERG fusion frequency were similar between the groups. RB1 Mut- NEPC was associated with significantly higher amplifications (amp) and total GA in AR compared to RB1 mut+ NEPC. RB1 mut+ NEPC featured significantly greater frequency of PTEN GA. GA frequencies in targetable kinases and DNA repair GA including BRCA1/2 and ATM linked to PARP inhibitor (PARPi) response were similar in both groups. For potential immune-oncology (IO) biomarkers, RB1 Mut+ NEPC featured significantly greater frequency of low positive PD-L1 expression and lower frequencies of MDM2 and CDK12 GA. CD274 ( PD-L1) amplification, MSI-high status and cases with TMB ≥10 mut/Mb were very uncommon in both groups. Conclusions: In NEPC, the presence of RB1 mutation is associated with various GA that may have clinical relevance. Limitations include possible selection and confounding biases, and lack of clinical annotation. Further study of RB1 status as a guide of trial designs on therapy selection for men with NEPC is warranted.[Table: see text]

Immune characterization of urothelial bladder carcinoma integrating transurethral resection of bladder tumor (TURBT) samples and serum: A feasibility study.

538 Background: Urothelial bladder cancer (UBC) is characterized by high immunogenicity. Multiple immunotherapies both in the perioperative and advanced setting are being developed. However, we lack good predictive biomarkers of response to immunotherapy in UBC. We aimed to characterize the immune profile of UBC patients using TURBT and serum samples. Methods: 13 UBC biopsies from TURBT samples were analyzed along with sera of these patients. Biopsy samples were divided in two equal parts. One part was formalin-fixed paraffin embedded and stained for immune histochemistry [IHC] for the following markers (CD3, CD4, CD8, CD20, CD68 and PD-L1 using SP142 antibody) and results expressed in percentages. The other part was homogenized and processed for immune cell isolation (human tumor isolation Miltenyi Kit). Tumor cells were stained with PHK and double positive CD8 and PHK were isolated using a MACSTM procedure. Different immune cell populations were analyzed by flow cytometry for the following markers (CD3, CD4, CD8, CD20 and CD68) and results expressed in percentages. Cytokines were examined in sera using the Luminex multiparametric procedure and results were expressed as pg/mL. Results: IHC and flow cytometry data were comparable and identified three groups according to the immune cells associated with the tumors (Table). Groups 1 and 2 showed high and moderate levels of tumor activated CD8+ T cells (1-4%) along with a higher expression of PD-L1 (40-20%). Group 3 lacked tumor activated CD8+ T cells and show lower expression of PD-L1 (15%). All groups showed significant levels of Th2 cytokines (IL-4, IL-6 or IL-10) while very low or lack of Th1 cytokines (IFN or IL-2) was observed except in group 2. Conclusions: TURBT biopsies and serum analysis of cytokines could represent a valid approach to further evaluate potential predictive biomarkers of response to immunotherapy in UBC. Different immune populations subgroups were identified in our series and could potentially predict for diverse responses to treatment. Further prospective validation in larger series is ongoing to confirm these results.[Table: see text]

Genetic mutation profiles and immune microenvironment analysis of pulmonary enteric adenocarcinoma

BackgroundPulmonary enteric adenocarcinoma (PEAC) has distinctive clinical outcomes, radiographic, pathological and molecular characteristics. The prognosis of patients with PEAC was poor. However, molecular profiles and therapeutic biomarkers of PEAC remain elusive.MethodsIn the present study, the hospitalized patients with PEAC admitted to Tongji Hospital in Wuhan from January 1, 2014 to November 20, 2020 were retrospectively enrolled and followed until December 10, 2020. Comprehensive genomic profiling of tumor tissue from the PEAC patients were performed and compared with lung adenocarcinoma, colorectal cancer and metastatic colorectal carcinoma. Tumor immune microenvironment analysis were evaluated.ResultsThere were 10 patients with PEAC enrolled. 70% of patients were male and the median age of onset was 63 years (interquartile range, 55–72). There were six early-stage patients (Stage IA to IIB) and four stage IV patients. Molecular analysis revealed the most common gene mutations included TP53 (57%, 4/7) and KRAS (57%, 4/7) mutations. There were 40% mutations occurred in genes encoding receptor tyrosine kinases (RTKs). 100% of patients (8/8) were microsatellite stability (MSS). The median level of TMB was 6.0 (interquartile range, 4.5–7.0) mutations/Mb. Three of 10 patients showed low PD-L1 expression (tumor proportion score < 10%) and the others were PD-L1 negative. A small subset of CD8+, CD3+, CD68+ T cells were observed and were mainly distributed in the cancer stroma.ConclusionThis study demonstrated that PEAC was characterized by low-frequency RTK gene mutation, high KRAS mutation, low PD-L1 expression, low TMB, and low CD8+ T cells infiltration.

Abstract PD3-04: Multi-omics characterization of triple-negative breast cancer identifies therapeutic vulnerabilities and epigenetic immune suppression in the mesenchymal subtype

Abstract Triple-negative breast cancer (TNBC) is a collection of biologically diverse cancers characterized by distinct transcriptional patterns, biology and immune composition. TNBCs display transcriptional diversity with at least four tumor-intrinsic subtypes that include two basal-like (BL1, BL2), a mesenchymal (M) and a luminal androgen receptor (LAR) subtype. Using integrative analyses of transcriptomic, epigenetic, proteomic and phospho-proteomic patterns we have identified subtype-specific vulnerabilities, which advanced our understanding of the cellular origins of TNBC subtypes. In mesenchymal subtype tumors we observed high mutation load and genomic instability, absence of immune cells, low PD-L1 expression, loss of global DNA methylation and transcriptional repression of antigen presentation genes through the polycomb repressor complex 2 (PRC2). Using cell line models, we demonstrate that MHC-I expression is epigenetically silenced by the PRC2 in mesenchymal TNBC cell lines and inhibition of EZH2 restores expression. Pharmacological inhibition of EZH2 enhances chemotherapy efficacy in syngeneic murine tumor models providing a rationale for using PRC2 inhibitors in PD-L1 negative mesenchymal tumors. Citation Format: Brian D Lehmann, Antonio Colaprico, Tiago C Silva, Jianjiao Chen, Hanbing An, Yuguang Ban, Lily Wang, Jamaal L James, Justin Balko, Paula I Gonzalez-Ericsson, Melinda E Sanders, Bing Zhang, Jennifer A Pietenpol, Xi S Chen. Multi-omics characterization of triple-negative breast cancer identifies therapeutic vulnerabilities and epigenetic immune suppression in the mesenchymal subtype [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-04.

Comprehensive analysis of m6A regulator-based methylation modification patterns characterized by distinct immune profiles in colon adenocarcinomas

Mounting evidences have indicated that RNA N6-methyladenosine (m6A) modification played important roles in tumor formation and growth. However, it is rarely reported that m6A modifications are involved in the immune regulation and tumor microenvironment (TME) formation. In this study, we aimed to investigate the correlation between m6A modifications and TME regulation of colon adenocarcinoma (COAD) by bioinformatic analysis. NMF algorithm was applied to carry out consensus molecular subtype analysis on 36 selected m6A regulators regarding methylation modification, to identify m6A modification patterns and characteristics of m6A related genes in colon adenocarcinoma (COAD). Further, the relative infiltration levels of different immune cell subsets were quantified by ssGSEA and CIBERSORT algorithms, and a m6Sig scoring scheme was constructed to predict the prognosis and evaluate the response to immunotherapy in the patients with COAD. Among 579 COAD samples, we identified three different m6A modification patterns which were related to different biological pathways and clinical outcomes. Then, a scoring scheme termed “m6Sig score” was developed based on m6A-related characteristic genes, and was utilized to score patients with COAD into groups. We found that COAD patients with lower m6Sig scores exhibited prolonged survival and potentiated immune infiltration, which were associated with higher tumor mutation load, lower PD-L1 expression, and higher mutation rates of SMG (such as TTN and KRAS). Moreover, analysis regarding evaluation of immune response revealed that the patients with lower m6Sig scores had higher Immunophenoscore. Collectively, our study provided in depth insight into the interactions between m6A modification and regulation of TME. In addition, the quantitative evaluation of m6A modification patterns in our results may have implications in further immunotherapy for individual COAD patients.

ARID1A Downregulation Predicts High PD-L1 Expression and Worse Clinical Outcome in Patients With Gallbladder Cancer.

BackgroundRecent studies have confirmed that AT-rich interactive domain-containing protein 1A (ARID1A) plays a critical role in tumorigenesis, but its role in gallbladder cancer (GBC) remains unclear.MethodsIn total, 224 patients from Zhongshan Hospital were recruited for this retrospective study. The clinicopathological and baseline characteristics of the patients were collected. Bioinformatics analysis was performed to reveal variations in genes and signaling pathways, and ARID1A and PD-L1 expression and the number of PD1+ tumor-infiltrating lymphocytes (TILs) were measured by immunohistochemical staining.ResultsARID1A expression was negatively correlated with overall survival in patients with GBC, and multivariate analysis identified ARID1A as an independent prognostic factor for overall survival. A heatmap and gene set enrichment analysis suggested that cytotoxic T lymphocyte signatures and immune-related signaling pathways were downregulated in ARID1A low tumors. Subsequent immunohistochemical staining confirmed that ARID1A expression was negatively correlated with PD-L1 expression and PD1+ TILs in the tumor microenvironment. The Kaplan–Meier analysis suggested that high ARID1A expression combined with low PD-L1 expression or low PD1+ TIL counts is associated with the best prognosis in patients with GBC.ConclusionARID1A inactivation can lead to a worse prognosis in patients with GBC, potentially by mediating immune evasion through the PD1/PD-L1 pathway.