Matched Lymph Node Metastases Research Articles

Abstract PO4-05-14: Gene expression in metastatic breast cancer – patterns and prognostic potential

Abstract Introduction Metastatic breast cancer (mBC) is the leading cause of breast cancer mortality. However, mBC has a considerable variance in prognosis and course of disease, reflecting a substantial biological heterogeneity. Despite this, there is a lack of prognostic biomarkers validated for mBC. New prognostic markers and improved understanding of the molecular variants of mBC could aid in prognostication and personalizing treatments, and lead to the discovery of novel drug targets. The primary objective of this study was to explore the prognostic value of gene expression (GEX) in the distant metastasis (DM) of mBC. Secondly, we aimed to identify molecular subtypes of mBC, and describe their biological niches. Methods We used a cohort of mBC patients enrolled in a prospective trial (NCT01322893) that includes tissue from primary tumor (PT) and matched lymph node metastasis (LNM) and/or DM (1). The inclusion criteria were diagnosed mBC with a life expectancy of >2 months, ECOG score of 0-2, and an age of >18 years. RNA was extracted from formalin-fixed, paraffin-embedded tumor tissue. GEX data were acquired for n=123 PT, n=71 LNM, and n=74 DM using the NanoString Breast Cancer 360 assay, which comprises 758 genes and >30 GEX signatures. The relationships between GEX and the endpoints overall survival (OS) and progression-free survival (PFS) were assessed using cox proportional hazards regression with GEX as a continuous score. Multivariable cox regressions were adjusted for PAM50 of the DM, number of metastatic sites, visceral metastasis, ECOG, and age at mBC diagnosis. Visualization by heatmap and k-means clustering were used to identify GEX patterns. When clustering single genes, we included only the 100 genes with the highest standard deviation. Results In relation to PFS, we found prognostically unfavorable roles of DM GEX of the signatures for Treg cells (HR=1.3, p=0.02), cytotoxicity (HR=1.3, p=0.03), and p53 (HR=1.3, p=0.02). Further, our data indicate prognostically favorable value of DM GEX of ESR1 (HR=0.77, p=0.02), PGR (HR=0.65, p=0.002), FOXA1 (HR=0.76, p=0.02), and AR (HR=0.68, p=0.0006). The prognostic value of AR remained in multivariable analysis. In relation to OS the first 2 years after mBC prognosis, we found prognostically favorable value of DM GEX of B7H3 (HR=0.65, p=0.04), TGFB (HR=0.53, p=0.02) and the signatures for stroma (HR=0.7, p=0.05) and claudin-low (HR=0.61, p=0.02). Finally, our data suggests that the previously published PAM50MET panel performs well as a prognostic indicator, especially when considered on a continuous scale, for PFS (HR=1.3, p=0.0023) and OS (HR=1.3, p=0.003) (2). Based on the 100 most highly variable genes, we identified five stable tumor clusters with distinct GEX profiles, where some strongly associated with metastatic site. Using the same approach on the GEX signatures, we established four GEX profiles able to outperform PAM50 in identifying tumors with poor OS in this material. Conclusion In this study, we demonstrate prognostic value for GEX-based panels in DM samples. First, we find GEX signatures related to hormone responsiveness to be favorable, and genetic instability to be unfavorable for mBC prognosis. We also demonstrate AR to be an independent marker for better PFS. Further, we confirm the prognostic value of PAM50MET, a previously published panel with the advantage of including only PAM50 genes and clinical parameters. Finally, we identify new GEX profiles related to metastatic site and outcome. These results illuminate the biological differences between mBC in relation to outcome and metastatic site. Better understanding of the mBC GEX subtypes may open new venues for tailored treatment.

Assessment of programmed death-ligand 1 expression in primary tumors and paired lymph node metastases of gastric adenocarcinoma.

Anti-programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) immunotherapy has demonstrated promising results on gastric cancer (GC). However, PD-L1 can express differently between metastatic sites and primary tumors (PT). To compare PD-L1 status in PT and matched lymph node metastases (LNM) of GC patients and to determine the correlation between the PD-L1 status and clinicopathological characteristics. We retrospectively reviewed 284 GC patients who underwent D2-gastrectomy. PD-L1 was evaluated by immunohistochemistry (clone SP142) using the combined positive score. All PD-L1+ PT staged as pN+ were also tested for PD-L1 expression in their LNM. PD-L1(-) GC with pN+ served as the comparison group. Among 284 GC patients included, 45 had PD-L1+ PT and 24 of them had pN+. For comparison, 44 PD-L1(-) cases with pN+ were included (sample loss of 4 cases). Of the PD-L1+ PT, 54.2% (13/24 cases) were also PD-L1+ in the LNM. Regarding PD-L1(-) PT, 9.1% (4/44) had PD-L1+ in the LNM. The agreement between PT and LNM had a kappa value of 0.483. Larger tumor size and moderate/severe peritumoral inflammatory response were associated with PD-L1 positivity in both sites. There was no statistical difference in overall survival for PT and LNM according to the PD-L1 status (P = 0.166 and P = 0.837, respectively). Intra-patient heterogeneity in PD-L1 expression was observed between the PT and matched LNM. This disagreement in PD-L1 status may emphasize the importance of considering different tumor sites for analyses to select patients for immunotherapy.

Targeting TGF beta receptor 1 in head and neck squamous cell carcinoma.

The transforming growth factor-Beta (TGF-ß) pathway may be involved in the radioresistance of head and neck squamous cell carcinoma (HNSCC). This study analyzed TGF-ß receptor 1 (TGFBR1) expression in HNSCC patients and evaluated the antineoplastic and radiosensitizing effects of vactosertib, a novel TGFBR1 inhibitor, in vitro. TGFBR1 expression was examined in HNSCC patients at the mRNA level in silico and the protein level by immunohistochemistry, including surgical specimens of primary tumors, matched lymph node metastasis, and recurrent disease. Furthermore, a novel small molecule TGFBR1 inhibitor was evaluated in HNSCC cell lines. Finally, an indirect coculture model using patient-derived cancer-associated fibroblasts was applied to mimic the tumor microenvironment. Patients with high TGFBR1 mRNA levels showed significantly worse overall survival in silico (OS, p = 0.024). At the protein level, an association between TGFBR1+ tumor and OS was observed for the subgroup with TGFBR1-stroma (p = 0.001). Those results prevailed in multivariable analysis. Inhibition of TGFBR1 showed antineoplastic effects in vitro. In combination with radiation, vactosertib showed synergistic effects. Our results indicate a high risk of death in tumorTGFBR1+|stromaTGFBR1- expressing patients. In vitro data suggest a potential radiosensitizing effect of TGFBR1 inhibition by vactosertib.

Detection of Cellular Senescence Reveals the Existence of Senescent Tumor Cells within Invasive Breast Carcinomas and Related Metastases

Oncogene-induced senescence is thought to constitute a barrier to carcinogenesis by arresting cells at risk of malignant transformation. However, numerous findings suggest that senescent cells may conversely promote tumor growth and metastatic progression, for example, through the senescence-associated secretory phenotype (SASP) they produce. Here, we investigated the degree to which senescent tumor cells exist within untreated human primary breast carcinomas and whether the presence of senescent cancer cells in primary tumors is recapitulated in their matched lymph node metastases. For the detection of senescence, we used SA-β-galactosidase (SA-β-gal) staining and other senescence markers such as Ki67, p21, p53, and p16. In patients with invasive luminal A and B breast carcinomas, we found broad similarities in the appearance of cancer cells between primary tumors and their corresponding metastases. Analysis of lymph nodes from patients with other breast cancer subtypes also revealed senescent tumor cells within metastatic lesions. Collectively, our findings show that senescent tumor cells exist within primary breast carcinomas and metastatic lesions. These results suggest a potential role for senescent breast tumor cells during metastatic progression and raise the question as to whether the targeting of senescent tumor cells with anti-senescent drugs might represent a novel avenue for improved treatment of breast and other cancers.

Multiplex imaging of breast cancer lymph node metastases identifies prognostic single-cell populations independent of clinical classifiers

Although breast cancer mortality is largely caused by metastasis, clinical decisions are based on analysis of the primary tumor and on lymph node involvement but not on the phenotype of disseminated cells. Here, we use multiplex imaging mass cytometry to compare single-cell phenotypes of primary breast tumors and matched lymph node metastases in 205 patients. We observe extensive phenotypic variability between primary and metastatic sites and that disseminated cell phenotypes frequently deviate from the clinical disease subtype. We identify single-cell phenotypes and spatial organizations of disseminated tumor cells that are associated with patient survival and a weaker survival association for high-risk phenotypes in the primary tumor. We show that p53 and GATA3 in lymph node metastases provide prognostic information beyond clinical classifiers and can be measured with standard methods. Molecular characterization of disseminated tumor cells is an untapped source of clinically applicable prognostic information for breast cancer.

Thyroglobulin expression, Ki-67 index, and lymph node ratio in the prognostic assessment of papillary thyroid cancer

The clinical significance of thyroglobulin (Tg) expression in papillary thyroid cancer (PTC) has not been systematically explored in relation to the Ki-67 index, lymph node ratio (LNR), or other conventional prognostic predictors. In this retrospective study of 327 patients with PTC, we investigated the immunohistochemical expression of Tg in both primary tumors and their matching lymph node metastases in relation to the Ki-67 index, LNR, and clinical data. Tumoral Tg immunoreactivity was inversely correlated to the Ki-67 index and tumor recurrence. The Ki-67 index was higher in lymph node metastases (mean 4%) than in the primary tumors (mean 3%). Reduced Tg expression, estimated as 0–25% Tg positive tumor cells, was more common in lymph node metastases compared to primary tumors. In addition to advanced metastatic burden (defined as N1b stage and LNR ≥ 21%), low Tg expression (0–25% positive tumor cells) in lymph node metastases had a significant prognostic impact with shorter recurrence-free survival. These findings support the potential value of histopathological assessment of Tg expression and Ki-67 index in lymph node metastases as complementary predictors to anticipate the prognosis of PTC patients better.

Genomic Landscape Alterations in Primary Tumor and Matched Lymph Node Metastasis in Hormone-Naïve Prostate Cancer Patients.

Simple SummaryThe aim of the present study is to investigate genetic changes associated with lymph node metastasis in a cohort of hormone-naïve prostate cancer patients. We found mostly concordance between both primary PCa samples and matched lymph node metastasis in terms of genomic alteration. Indeed, the analysis of hotspot regions in genes such as ERBB4, AKT1, FGFR2 and MLH1 underline that specific alterations in the primary tumor are extremely important for cancer prognosis prediction.Background: Prostate cancer (PCa) is a disease with a wide range of clinical manifestations. Up to the present date, the genetic understanding of patients with favorable or unfavorable prognosis is gaining interest for giving the appropriate tailored treatment. We aimed to investigate genetic changes associated with lymph node metastasis in a cohort of hormone-naïve Pca patients. Methods: We retrospectively analyzed data from 470 patients who underwent surgery for PCa between 2010 and 2020 at the Department of Urology, University of Catania. Inclusion criteria were patients with lymph node metastasis and patients with PCa with extra capsular extension (pT3) and negative lymph node metastasis. The final cohort consisted of 17 different patients (11 PCa with lymph node metastasis and 6 PCa without lymph node metastasis). Through the cBioPortal online tool, we analyzed gene alterations and their correlations with clinical factors. Results: A total of 688 intronic, synonym and nonsynonym mutations were sequenced. The gene with the most sequenced mutations was ERBB4 (83 mutations, 12% of 688 total), while the ones with the lower percentage of mutations were AKT1, FGFR2 and MLH1 (1 mutation alone, 0.14%). Conclusion: In the present study we found mostly concordance concerning the ERBB4 mutation between both primary PCa samples and matched lymph node metastasis, underlining that the identification of alterations in the primary tumor is extremely important for cancer prognosis prediction.

Tumor expression of Nectin-1–4 and its clinical implication in muscle invasive bladder cancer: An intra-patient variability of Nectin-4 expression

There is limited evidence regarding the tumor expression of nectins and their clinical implications in muscle invasive bladder cancer. Herein, we evaluated expression of Nectins 1–4 in 64 patients with muscle invasive bladder cancer who underwent radical cystectomy using a histochemical scoring method (H-score; immunohistochemical staining intensity multiplied by the percentage of positive-staining cells). The cutoff values were defined based on the median H-scores. Of the 64 patients, 45 (70%) had residual tumors in radical cystectomy specimens, while 13 (20%) had lymph node metastasis. The median (interquartile range) H-scores of Nectin-1, − 2, − 3, and − 4 expression were 0 (0−10), 80 (30−180), 5 (0−30), and 100 (33−160), respectively. The Nectin-4 H-score of the neuroendocrine variant was significantly lower than that of pure urothelial carcinoma (P = 0.015). Post-neoadjuvant chemotherapy pathological response (<ypT2N0 residual disease and pN-negative) was achieved in 18 (49%) of the 37 patients who received neoadjuvant chemotherapy. Clinical stage II, not Nectin expression, was an independent factor associated with pathological response (P = 0.019, adjusted odds ratio 6.9, vs stage III/IV). There was no correlation in Nectin-4 tumor expression between transurethral resection and matched radical cystectomy specimens and between radical cystectomy specimens and matched lymph node metastatic lesions. However, there was a significant decrease in Nectin-4 expression in post-neoadjuvant chemotherapy radical cystectomy specimens compared to pre-neoadjuvant chemotherapy transurethral resection specimens (P = 0.008). Given the downregulation of Nectin-4 by chemotherapy and the significant discrepancy between radical cystectomy and matched lymph node metastasis specimens, baseline primary tumors may not be a suitable material for evaluating Nectin-4 expression and its potential as a predictive biomarker for enfortumab vedotin treatment.

Targeted amplicon sequencing for primary tumors and matched lymph node metastases in patients with extrahepatic cholangiocarcinoma.

Lymph node metastasis (LNM) is one of the most adverse prognostic factors in extrahepatic cholangiocarcinoma (EHCC) cases. As next-generation sequencing technology has become more widely available, the genomic profile of biliary tract carcinoma has been clarified. However, whether LNMs have additional genomic alterations in patients with EHCC has not been investigated. Here, we aimed to compare the genomic alterations between primary tumors and matched LNMs in patients with EHCC. Sixteen patients with node-positive EHCCs were included. Genomic DNA was extracted from tissue samples of primary tumors and matched LNMs. Targeted amplicon sequencing of 160 cancer-related genes was performed. Among the 32 tumor samples from 16 patients, 91 genomic mutations were identified. Genomic mutations were noted in 31 genes, including TP53, MAP3K1, SMAD4, APC, and ARID1A. TP53 mutations were most frequently observed (12/32; 37.5%). Genomic mutation profiles were highly concordant between primary tumors and matched LNMs (13/16; 81.3%), and an additional genomic mutation of CDK12 was observed in only one patient. Genomic mutations were highly concordant between primary tumors and matched LNMs, suggesting that genotyping of archived primary tumor samples may help predict genomic mutations of metastatic tumors in patients with EHCC.

PD-L1 expression in bladder cancer: Which scoring algorithm in what tissue?

IntroductionFor cisplatin-ineligible patients, approval of first-line immune-checkpoint inhibitor therapy relies on the programmed death ligand 1 (PD-L1) expression assay employed, namely, the combined positive score (CPS) or immune cell (IC) score. This study compares PD-L1 diagnostic scores and positivity in primary and matched metastatic bladder cancer tissue. MethodsA total of 108 patients undergoing radical cystectomy for urothelial bladder cancer and lymphatic spread (pN+) were included. PD-L1 expression was compared by immunohistochemistry (IHC) between the primary bladder tumor and associated lymph node metastases using Ventana SP263 anti-PD-L1 antibody. In a subset of cases further IHC was performed with Ventana SP142 and Dako 22C3 antibodies. Second, the PD-L1 scoring algorithms for the CPS and IC score were compared. Correlation of PD-L1 positivity with clinical parameters and tumor stage was assessed. Intra- and intertissue analyses were performed with Pearson's chi square test, McNemar test and Spearman correlation employing IBM SPSS 25. ResultsPD-L1 expression did not correlate with clinicopathological parameters. The CPS (43.5% vs. 35.6%; P=0.006) and the IC score (28.7% vs. 21.2%; P=0.002) yielded PD-L1 positivity significantly more often in primary BC than in matched lymph node metastasis. Both the CPS (r=0.775; P<0.001) and the IC score (r=0.711; P<0.001) correlated between primary and metastatic bladder cancer tissue. Employing CPS vs. IC led to significantly more PD-L1-positive classified cases in primary BC (CPS vs. IC; 43.5% vs. 28.7%; P<0.001) and lymph node metastasis (CPS vs. IC; 35.6% vs. 21.2%, P<0.001). ConclusionCompared to lymph node analysis, bladder tissue yields more PD-L1 positivity assessed with the CPS and IC scores. This is particularly evident when employing the CPS. The findings highlight that employing both PD-L1 assays may maximize eligibility for first-line checkpoint-inhibitors to treat bladder cancer patients unfit for cisplatin-based chemotherapy.

Tumor Immune Microenvironment Characterization of Primary Lung Adenocarcinoma and Lymph Node Metastases.

Background The essential roles of the tumor microenvironment (TME) have been recognized during the initiation and progression of primary lung adenocarcinoma (LUAD). The aim of the present study was to delineate the immune landscape in both primary cancer and matched lymph node metastasis from a cohort of locally advanced stage LUAD patients with distinct outcomes. Methods Formalin-fixed, paraffin-embedded samples were collected from 36 locally advanced LUAD patients. Transcriptome data of the tumor immune microenvironment were resolved using an immune oncology panel RNA sequencing platform. Bioinformatics approaches were used to determine the differentially expressed genes (DEGs), dysregulated pathways, and immune cell fraction between patients with early recurrence (ER) and late recurrence (LR). Results Here, we showed that in primary cancer tissues, 23 DEGs were obtained between patients with ER and LR. Functional analysis revealed that the LR in LUAD patients may be associated with enriched gene sets belonging to the antigen presentation and MHC protein complex, innate immune response, and IFN-γ signaling pathways. Next, the transcriptome data were adopted to quantify immune cell fractions, indicating that high infiltration of mast cells and neutrophils was correlated with ER. Interestingly, similar findings were observed in metastatic lymph nodes from patients suffering from ER or LR. By analyzing the shared immune features of primary cancers and lymphatic metastases, we unraveled the prognostic value and joint utility of two DEGs, CORO1A and S100A8. Conclusions In LUAD, the enrichment in antigen presentation, MHC protein complex, and IFN-γ signaling, and low infiltration of neutrophils in primary or metastatic nodules may be indications for a favorable prognosis. Integrated with bioinformatics approaches, transcriptome data of immune-related genes from formalin-fixed, paraffin-embedded (FFPE) samples can effectively profile the landscape of the tumor immune microenvironment and help predict clinical outcomes.

PD-L1 expression in non-small cell lung cancer: heterogeneity by pathologic types, tissue sampling and metastasis.

BackgroundProgrammed cell death ligand-1 (PD-L1) is a predictive marker of anti-PD-1/PD-L1 therapy response. Intra-tumour heterogeneity of PD-L1 expression has been reported in non-small cell lung cancer (NSCLC), but comprehensive studies regarding the determination of PD-L1 expression in different materials are still lacking. Therefore, we aimed to compare PD-L1 expression in paired tumour samples and in different specimen types.MethodsA total of 1,002 resected NSCLC specimens, 35 biopsy specimens and 54 endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) samples were performed PD-L1 immunohistochemistry (IHC) testing using the 22C3 assay. PD-L1 expression was evaluated using the tumour proportion score (TPS) and categorized into three levels: negative (TPS <1%), low expression (TPS 1–49%) and high expression (TPS ≥50%).ResultsA total of 1,002 resected NSCLC specimens, including 852 adenocarcinomas (ADCs) and 150 squamous cell carcinomas (SCCs); 35 paired biopsy and resected samples; 54 paired cell block and biopsy samples; 53 paired blocks from the same resected tissue and 49 paired primary and metastatic lesion samples were included in this study. Interestingly, high PD-L1 expression was significantly more frequent in poorly differentiated subtypes than in well-differentiated subtypes in the ADC subgroup (P<0.001). In the SCC subgroup, high PD-L1 expression was significantly more associated with the nonkeratinizing type than the keratinizing type (P=0.001). PD-L1 expression differed between cell blocks and matched biopsy specimens (discordance rate =11.1%, 6/54) and between biopsy and matched resected specimens (discordance rate =31.4%, 11/35). PD-L1 expression differed between different paraffin blocks from the same resected specimen (discordance rate =35.8%, 19/53), and the discordance rate of PD-L1 expression between primary tumours and matched lymph node metastases was 28.6% (14/49).ConclusionsDiscordant PD-L1 expression is not uncommon in NSCLC and warrants additional studies and serious consideration when interpreting PD-L1 test results. Initial negative test results may lead to repeat PD-L1 testing in additional samples or the use of a different clone if necessary.

Combined use of immunohistochemical markers of basal and luminal subtypes in urothelial carcinoma of the bladder: Association with clinicopathological features and outcomes

OBJECTIVES:Whole genome expression profiles allow the stratification of bladder urothelial carcinoma into basal and luminal subtypes which differ in histological patterns and clinical behavior. Morpho-molecular studies have resulted in the discovery of immunohistochemical markers that might enable discrimination between these two major phenotypes of urothelial carcinoma.METHODS:We used two combinations of immunohistochemical markers, i.e., cytokeratin (CK) 5 with CK20 and CK5 with GATA3, to distinguish subtypes, and investigated their association with clinicopathological features, presence of histological variants, and outcomes. Upon searching for tumor heterogeneity, we compared the findings of primary tumors with their matched lymph node metastases. We collected data from 183 patients who underwent cystectomy for high-grade muscle-invasive urothelial carcinoma, and representative areas from the tumors and from 76 lymph node metastasis were organized in tissue microarrays.RESULTS:Basal immunohistochemical subtype (CK5 positive and CK20 negative, or CK5 positive and GATA3 negative) was associated with the squamous variant. The luminal immunohistochemical subtype (CK5 negative and CK20 positive, or CK5 negative and GATA3 positive) was associated with micropapillary and plasmacytoid variants. Remarkably, only moderate agreement was found between the immunohistochemical subtypes identified in bladder tumors and their lymph node metastasis. No significant difference in survival was observed when using either combination of the markers.CONCLUSION:This study demonstrates that these three routinely used immunohistochemical markers could be used to stratify urothelial carcinomas of the bladder into basal and luminal subtypes, which are associated with several differences in clinicopathological features.

Spatial profiling of gastric cancer patient-matched primary and locoregional metastases reveals principles of tumour dissemination

ObjectiveEndoscopic mucosal biopsies of primary gastric cancers (GCs) are used to guide diagnosis, biomarker testing and treatment. Spatial intratumoural heterogeneity (ITH) may influence biopsy-derived information. We aimed to study ITH...

A four-antibody immunohistochemical panel can distinguish clinico-pathological clusters of urothelial carcinoma and reveals high concordance between primary tumor and lymph node metastases.

Urothelial carcinoma of the bladder (UC) has a poor prognosis, partly because of chemotherapy resistance. Molecular classifications have shown their interest and can help to offer personalized treatment. In this study, we evaluated the feasibility of an immunohistochemical study to divide advanced UC into clinico-pathological-molecular subgroups and evaluate phenotypic correspondence between primary UC and matched lymph node metastases (LMN). An eight-antibody immunohistochemical panel was performed on UC and matched LMN from patients treated with radical cystectomy. One hundred eighty-seven UCs (100 pN0 tumor and 87 pN+ tumor) were tested. Multiple correspondence analysis showed that UC expressing GATA3 also expressed FOXA1 (p = 0.010) and did not stain for CK5/6 (p = 0.031) nor CK14 (p = 0.003). UC expressing CK14 coexpressed CK5/6 (p < 0.0001), had high Ki67 (p = 0.010) and no GATA3 (p = 0.003) nor FOXA1 (p = 0.011) expression. Loss of expression of STAG2 was associated with high Ki67 (p = 0.001). Sixty-seven percent of [CK5/6 CK14]+ [GATA3 FOAXA1]- patients had high Ki67 expression vs 37% of [GATA3 FOXA1]+ [CK5/6 CK14]- patients (p = 0.024). The majority of [CK5/6 CK14]+ [GATA3 FOAXA1]- patients (92%) had advanced disease (pT3-pT4) whilst 86% of pT1-T2 cases were [GATA3 FOXA1]+ [CK5/6 CK14]- (p = 0.041). Differential antigen expression between 63 pN+ primary tumors and their corresponding LNM showed the following concordance percentages: p53 (76%), p63 (75%), CK5/6 (65%), CK14 (89%), GATA3 (75%), FOXA1 (68%), STAG2 (65%), and Ki-67 (71%). These results support the interest of immunohistochemistry for subtype profiling in metastatic UC, using CK5/6, CK14, GATA3, and FOXA1, highlighting also few phenotypical modifications when tumor spreads to lymph nodes.

TCF21 Promotes Luminal-Like Differentiation and Suppresses Metastasis in Bladder Cancer.

Little is known regarding the subclone evolution process in advanced bladder cancer, particularly with respect to the genomic alterations that lead to the development of metastatic lesions. In this project, we identify gene expression signatures associated with metastatic bladder cancer through mRNA expression profiling of RNA isolated from 33 primary bladder cancer and corresponding lymph node (LN) metastasis samples. Gene expression profiling (GEP) was performed on RNA isolated using the Illumina DASL platform. We identified the developmental transcription factor TCF21 as being significantly higher in primary bladder cancer compared with LN metastasis samples. To elucidate its function in bladder cancer, loss- and gain-of-function experiments were conducted in bladder cancer cell lines with high and low expression of TCF21, respectively. We also performed GEP in bladder cancer cell lines following TCF21 overexpression. We identified 2,390 genes differentially expressed in primary bladder cancer and corresponding LN metastasis pairs at an FDR cutoff of 0.1 and a fold change of 1. Among those significantly altered, expression of TCF21 was higher in the primary tumor compared with LN metastasis. We validated this finding with qPCR and IHC on patient samples. Moreover, TCF21 expression was higher in luminal cell lines and knockdown of TCF21 increased invasion, tumor cell dissemination, and metastasis. In contrast, overexpression of TCF21 in highly metastatic basal bladder cancer cell lines decreased their invasive and metastatic potential. IMPLICATIONS: TCF21 is differentially overexpressed in primary bladder cancer compared with matched LN metastasis, with in vitro and in vivo studies demonstrating a metastasis suppressor function of this transcription factor.

Heterogeneity in PD-L1 expression on infiltrating immune cells between the primary and metastatic breast cancers

Objective: To investigate the expression difference in PD-L1 on mesenchymal infiltrating immune cells between the primary and metastatic breast cancers, and to explore its relationship with clinicopathological parameters. Methods: All cases of primary breast cancer and their matched metastases diagnosed at the Fourth Hospital of Hebei Medical University between January 2010 and December 2018 were included. Immunohistochemistry (IHC) was used to detect the expression of PD-L1 (SP142) in interstitial infiltrating immune cells, and the expression of ER, PR, HER2 and Ki-67 in primary and matched metastases was detected. Statistical software SPSS 24.00 was used for statistical analysis. Kappa test was used for concordance/agreement analysis and McNemar test for difference analysis. Results: Among the 140 identified primary breast cancers, there were 52 cases with matched lymph node metastasis, 88 cases with distant metastasis, including 35 cases with liver metastasis, 21 cases with lung metastasis, 13 cases with chest wall metastasis, 11 cases with bone metastasis, 6 cases with brain metastasis, 1 case with small intestine metastasis, and 1 case with eyeball metastasis. The overall concordance rate of the PD-L1 expression on mesenchymal immune cells between primary breast cancer and paired metastatic breast cancer was 72.9% (κ=0.441). The concordance rate of PD-L1 expression between primary breast cancers and paired lymph node metastases, and that between primary breast cancers and distant metastases were 75.0% (κ=0.472) and 71.6% (κ=0.472), respectively. The inconcordance rate of interstitial immune cell PD-L1 expression between primary breast cancer and matched lung metastasis was 28.6%(6/21), and the difference was statistically significant (P=0.031). The expression of PD-L1 in mesenchymal immune cells of primary breast cancer was significantly correlated with tumor size, histological grade, vascular invasion, lymph node metastasis, and Ki-67 index (P<0.05). The PD-L1 expression was independently associated with lymph node metastasis (P<0.05), while the expression of PD-L1 in metastatic breast cancer interstitial immune cells was significantly related to the expression of ER (P<0.05). Conclusions: The expression of PD-L1 in the primary breast cancer is moderately concordant with that in paired metastases, but different from that in paired lung metastases. Therefore, the expression of PD-L1 in distant metastasis needs to be re-evaluated to optimize the treatment outcomes of PD-L1 based therapy.

Plasticity and Polarization of Human NK Cells in the Tumor Microenvironment

Abstract The presence and function of innate lymphoid cells (ILC), including natural killer (NK) cells, within human tumors has been poorly characterized. Here, we have assessed the heterogeneity of NK and ILC populations by single-cell RNA sequencing (scRNAseq) of hundreds of individual NK cells and ILCs within human head and neck squamous cell carcinoma (HNSCC), matched lymph node metastases, matched peripheral blood, and blood from healthy donors. Fresh tumor specimens and blood were obtained from 8 patients undergoing surgical resection of HNSCC. Unsupervised clustering revealed 8 different clusters of NK cells and ILC subsets. We observed significant heterogeneity, with distinct subsets showing profiles consistent with that of conventional NK cells, ILC1-like cells and ILC3-like cell. Importantly, peripheral NK cells were distinct from intratumoral NK cells. The presence of these different cell subsets within primary HNSCC tumors was confirmed by flow cytometry. Further, plasticity between the NK cell subsets was supported by in vitro and in vivo experimental data. Given the ability of NK and ILCs to polarize the immune responses through the secretion of cytokines and the ability of certain subsets to kill target cells, we hypothesize that the differences observed in NK and ILC populations and their plasticity may result in different immune responses, and may influence clinical outcomes following therapy.

Abstract 4902: Positive Ki-67 expression in post neoadjuvant chemotherapy (NAC) radical cystectomy samples are associated with lack of tumor downstaging and shorter overall survival (OS) in patients with muscle invasive bladder cancer (MIBC)

Abstract Background: We recently reported single institution data on tumor downstaging and survival outcomes of 332 patients who underwent platinum based NAC followed by radical cystectomy for MIBC [Peyton et al. JAMA Oncol. 2018]. High expression levels of cell proliferation marker Ki-67 are associated with poor outcomes in chemotherapy naïve bladder cancer. Androgen receptor (AR) expression is associated with resistance to cisplatin in bladder cancer cell line models. We therefore studied Ki-67 and AR expression in our cohort of post NAC radical cystectomy samples and correlated them with tumor downstaging and OS. Methods: Tissue microarrays (TMAs) were constructed from 130 post-NAC cystectomy samples and up to 5 cores were taken from each radical cystectomy sample. Matched lymph node metastases if present were included in TMAs. The expression of Ki-67 and AR were evaluated with immunohistochemistry and the average of the H score was used to represent each radical cystectomy sample and its matched lymph node metastasis. Results: The median survival of this cohort of 130 patients was 33.4 months (range: 1.13 -127 months). 40 patients (31%) had tumor downstaging and 21 patients (16%) achieved pathological complete response. Using a Cox regression model for OS, positive Ki-67 expression in post NAC radical cystectomy samples is associated with poorer overall survival (hazard ratio=2.412, 95% CI:1.076-5.408, p=0.033), independent of the pathological N stage. Positive Ki-67 was significantly associated with lack of tumor downstage in a multivariable logistic regression model (odds ratio=0.081, 95% CI:0.014-0.464, p=0.004) while adjusting for adjuvant chemotherapy and pathological complete response. No statistically significant associations between AR and OS or AR and tumor downstaging were observed in multivariable analysis. When AR expression is evaluated as a binary variable (AR+, AR-), post NAC AR- expression is significantly associated with tumor downstaging compared to post NAC AR+ samples (Fisher’s exact test, p=0.002). When stratified by sex, a trend towards better survival was observed in females (36 patients) compared to males (94 patients) (p=0.089). Post NAC AR- female patients also demonstrated a trend toward better survival (Log rank test, p=0.497) when compared to post NAC AR+ females. Conclusions: Positive Ki-67 expression in post NAC radical cystectomy sample is associated with poorer OS and lack of tumor downstaging. If confirmed by a larger data set, Ki-67 can serve as a biomarker to select MIBC patients for post cystectomy adjuvant therapy. Note: This abstract was not presented at the meeting. Citation Format: Selene Rubino, Youngchul Kim, Junmin Zhou, Charles Peyton, Scott Gilbert, Wade Sexton, Jingsong Zhang. Positive Ki-67 expression in post neoadjuvant chemotherapy (NAC) radical cystectomy samples are associated with lack of tumor downstaging and shorter overall survival (OS) in patients with muscle invasive bladder cancer (MIBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4902.

Analysis of Mucosal Melanoma Whole-Genome Landscapes Reveals Clinically Relevant Genomic Aberrations.

Unlike advances in the genomics-driven precision treatment of cutaneous melanomas, the current poor understanding of the molecular basis of mucosal melanomas (MM) has hindered such progress for MM patients. Thus, we sought to characterize the genomic landscape of MM to identify genomic alterations with prognostic and/or therapeutic implications. Whole-genome sequencing (WGS) was performed on 65 MM samples, including 63 paired tumor blood samples and 2 matched lymph node metastases, with a further droplet digital PCR-based validation study of an independent MM cohort (n = 80). Guided by these molecular insights, the FDA-approved CDK4/6 inhibitor palbociclib was tested in an MM patient-derived xenograft (PDX) trial. Besides the identification of well-recognized driver mutations of BRAF (3.1%), RAS family (6.2%), NF1 (7.8%), and KIT (23.1%) in MMs, our study also found that (i) mutations and amplifications in the transmembrane nucleoporin gene POM121 (30.8%) defined a patient subgroup with higher tumor proliferation rates; (ii) enrichment of structural variations between chromosomes 5 and 12 defined a patient subgroup with significantly worse clinical outcomes; (iii) over 50% of the MM patients harbored recurrent focal amplification of several oncogenes (CDK4, MDM2, and AGAP2) at 12q13-15, and this co-occurred significantly with amplification of TERT at 5p15, which was verified in the validation cohort; (iv) the PDX trial demonstrated robust antitumor effects of palbociclib in MMs harboring CDK4 amplification. Our largest-to-date cohort WGS analysis of MMs defines the genomic landscape of this deadly cancer at unprecedented resolution and identifies genomic aberrations that could facilitate the delivery of precision cancer treatments.See related commentary by Shoushtari, p. 3473.