Expression In Brain Metastases Research Articles

Abstract PO4-14-07: Genomic and immune profiling of breast cancer brain metastases

Abstract BACKGROUND: Brain metastases (BrM) arising from breast cancer (BC) are an increasing consequence of advanced disease, with up to half of patients (pts) with metastatic HER2+ or triple negative breast cancer experiencing central nervous system (CNS) recurrence. The genomic alterations driving CNS recurrence, along with contribution of the immune microenvironment, particularly by BC subtype remains unclear. METHODS: We characterized BrM from a cohort of n=42 BC pts by sequencing whole-exome DNA and total RNA libraries from frozen (n=31) and FFPE (n=34) BCBrM, FFPE extracranial (ECT, n=12) and blood DNA (n=26) tissues from the Duke Brain Tumor Biorepository. Analyses conducted and planned include inference of PAM50 intrinsic subtypes, somatic mutations, copy number alterations, immune cell type decompositions by CIBERSORTx, differential RNA expression, driver analysis, and associations with clinical outcomes. RESULTS: PAM50 subtypes across 31 frozen BrM were 23% Luminal (Lum) A, 13% LumB, 33% HER2-enriched, 27% Basal-like, 3% Normal-like. For 34 FFPE BrM, 15% were LumA, 24% LumB, 33% HER2-enriched, 24% Basal-like, 3% Normal-like. Across 26 paired FFPE and frozen BrM, subtype discrepancy was seen in 23% (6) cases of which frozen to FFPE classification differed: 3 LumA to LumB, 1 LumB to LumA, 1 Normal to LumB, and 1 Basal to LumB. Among 10 paired FFPE BrM and ECT, subtype concordance was observed in 70%. Discordance was seen in 1 case each for ECT to BrM: LumB to HER2, LumB to LumA, Basal to Normal-like. WES demonstrated frequent copy number alterations (CNA) in clinically-relevant genes including TGFB1, NOTCH1, CDK4, and ERBB3, in both BrM and ECT. For pts with matched blood, ERBB3 CNA (gain and loss) were more commonly found in FFPE BrM (7/19, 37%) compared to ECT (1/8, 13%). Among BrM, the most frequently altered clinically-relevant genes included TP53 (~75%), PTEN, EGFR, RB1, PIK3CA, NF1 and ESR1 (all ≤15%); in ECT, TP53 (67%), BRCA2, FBXW7, and ATM (all ≤33%). PI3K pathway genes (e.g. PTEN, PIK3CA) alterations were exclusive to BrM. One pt’s ECT showed an ATM mutation that was not observed in the paired BrM. Conversely, BRAF and CCND2 mutations were observed in BrM, but not ECT for 1 pt each. Inferring the relative abundance of immune populations in frozen BrM illustrated that 23% were CD4+ resting memory T cells, 25% M2 tumor-permissive macrophages, 13% M0 macrophages; M1 tumor-inhibiting macrophages were only 2%. When comparing immune cell populations between FFPE BrM and ECT, ECT had more M1 macrophages (Chi-sq 4.23, P = 0.04), while other immune cell populations were of similar relative abundance. Immune cell fractions did not vary by subtype in BrM or ECT with one exception: M2 macrophages were lower in Basal compared to LumB tissues (frozen BrM: Chi-sq = 9.28, P = 0.05; FFPE BrM: Chi-sq = 4.61, P = 0.33; FFPE EC: Chi-sq = 9.78, P = 0.04). In n=8 pt-matched FFPE BrM/ECT, hallmark pathways upregulated in BrM included MYC and E2F targets and oxidative phosphorylation, while those with lower expression in BrM included epithelial-mesenchymal transition, interferon gamma response, and JAK-STAT signaling. CONCLUSION: This analysis showed moderate discrepancy in subtype call of BrM by tissue preparation (frozen vs. FFPE), with LumB classification showing the highest discrepancy, and more commonly called in FFPE tissues. Subtype concordance between ECT and BrM was relatively high. Analysis of CNA illustrated deletions and amplifications in targetable genes, notably ERBB3 preferentially in BrM compared to ECT. Mutational analysis identified targetable alterations exclusive to BrM; this knowledge could lead to BrM-targeted treatments. Inferred immune cell populations illustrated a tumor permissive microenvironment in BrM. Therapeutic strategies repolarizing macrophages toward a tumor-inhibiting phenotype in BrM are warranted. Analyses of associations between genomic data and clinical outcomes, as well as driver analyses, are ongoing. Citation Format: Amanda Van Swearingen, Marissa Lee, Layne Rogers, Alexander Sibley, Pixu Shi, Xiaodi Qin, Michael Goodin, Kouros Owzar, Carey Anders. Genomic and immune profiling of breast cancer brain metastases [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-07.

Incidence of HER2-expressing brain metastases in patients with HER2-null breast cancer: a matched case analysis

The HER2-directed antibody-drug conjugate trastuzumab deruxtecan is active against lower levels of HER2 expression than prior-generation therapies. The rate of HER2 expression in brain metastases among patients with initially HER2-null breast cancer is undefined, and receptor discordance in advanced breast cancer with brain metastases may underestimate CNS response potential in the absence of brain metastasis sampling. In this cohort study including 136 patients with 401 samples scored according to ASCO/CAP guidelines, 15/28 patients (54%) with HER2-null primary breast cancer have detectable HER2 expression in subsequently resected brain metastases, a significant discordant population.

Fibroblast activation protein as a potential theranostic target in brain metastases of diverse solid tumours

Brain metastases are a very common and serious complication of oncological diseases. Despite the vast progress in multimodality treatment, brain metastases significantly decrease the quality of life and prognosis of patients. Therefore, identifying new targets in the microenvironment of brain metastases is desirable. Fibroblast activation protein (FAP) is a transmembrane serine protease typically expressed in tumour-associated stromal cells. Due to its characteristic presence in the tumour microenvironment, FAP represents an attractive theranostic target in oncology. However, there is little information on FAP expression in brain metastases. In this study, we quantified FAP expression in samples of brain metastases of various primary origin and characterised FAP-expressing cells. We have shown that FAP expression is significantly higher in brain metastases in comparison to non-tumorous brain tissues, both at the protein and enzymatic activity levels. FAP immunopositivity was localised in regions rich in collagen and containing blood vessels. We have further shown that FAP is predominantly confined to stromal cells expressing markers typical of cancer-associated fibroblasts (CAFs). We have also observed FAP immunopositivity on tumour cells in a portion of brain metastases, mainly originating from melanoma, lung, breast, and renal cancer, and sarcoma. There were no significant differences in the quantity of FAP protein, enzymatic activity, and FAP+ stromal cells among brain metastasis samples of various origins, suggesting that there is no association of FAP expression and/or presence of FAP+ stromal cells with the histological type of brain metastases. In summary, we are the first to establish the expression of FAP and characterise FAP-expressing cells in the microenvironment of brain metastases. The frequent upregulation of FAP and its presence on both stromal and tumour cells support the use of FAP as a promising theranostic target in brain metastases.

P11.26.A STAT3 expression in brain metastases from breast cancer: correlations with different molecular subtypes and clinical outcome

Abstract Background STAT3 expression in peritumoral reactive astrocytes (RA) of brain metastases (BM) may favor a pro-metastatic environment. The aim of the study was to evaluate in a retrospective cohort of surgically resected BM from breast cancer (BC) the expression of pSTAT3 in RA of peritumoral tissue of BM, identify different patterns of expression according to molecular subtypes, and correlate with intracranial progression-free survival (i-PFS). Material and Methods Patients with histologically proven BM diagnosis from BC were identified from the biobank of Pathology Unit of University of Turin and Spanish national BrM network (RENACER). pSTAT3 expression was evaluated and scored in RA of peritumoral tissue using GFAP and STAT3 immunohistochemistry, according to Priego et al. (Nat Med 2018). Data on histological diagnosis, molecular subtypes, and i-PFS were retrieved by chart review. Intracranial progression was defined based on MRI reports. Results Eighty-five BM specimens from BC of 85 female patients with a median age of 54 years (range 30-81 years) were available for analysis. Immunohistochemistry for GFAP and pSTAT3 was feasible in 68/85 (80%). Fifteen out of 68 patients (21.1%) had BM from luminal BC, 27/68 (39.7%) from HER2-positive BC, and 26/68 (39.2%) from TNBC. Fifty-six out of 68 (82.4%) showed positive staining of pSTAT3 in peritumoral RA, of which 9/68 (13.3%) scored with 3, 26/68 (38.2%) with 2, and 21/68 (30.9%%) with 1, while pSTAT3 expression was negative (score 0) in 12/68 (17.6%). High pSTAT3 expression (score 2-3) was observed in 17/27 (62.9%) BM from HER2-positive BC and in 15/26 (57.7%) BM from TNBC, while most of BM from luminal BC (12/15 - 80%) had low or absent pSTAT3 (score 0-1) (p=0.021). Overall i-PFS was 16 months (range 7-41): low pSTAT3 BM (score 0-1) had a median i-PFS of 21 months versus 12 months for high pSTAT3 BM (score 2-3). A shorter median i-PFS was observed in high pSTAT3 BM from TNBC (4 months) as compared with low pSTAT3 BM (11 months). Conversely, i-PFS of high pSTAT3 BM (7 months) was similar to low pSTAT3 BM (6 months) in HER2-positive BC. Conclusion pSTAT3 expression in RA of peritumoral tissue of BM from TNBC and HER2-positive BC is higher than in BM from luminal BC. Of note, patients with high pSTAT3 BM from TNBC progressed earlier in comparison with those with low pSTAT3, suggesting that pSTAT3 expression has an influence on the outcome.

264P Increased membrane HER3 expression in brain metastases compared to primary tumors in breast cancer

Breast cancer (BC) is a common cause of brain metastases occurring in at least 10–16 % of patients. Tucatinib is FDA-approved for patients with metastatic HER2-positive BC, including patients with brain metastases, whereas no approved targeted therapies are available for HER2-negative brain metastasis. This study aims to elucidate the expression patterns of HER2 and HER3 in primary tumors and brain metastases.

Study on the Predictive Value of P53 Protein Expression in Brain Metastasis in NSCLC and the Mechanism of miR-424 Reversing Platinum Resistance in NSCLC.

In order to analyze the predictive value of P53 protein expression in brain metastases in NSCLC and the mechanism of miR-424 reversing platinum resistance in NSCLC, a retrospective analysis is conducted in this study. Eighty-two NSCLC patients who received relevant diagnosis and treatment in our hospital from September 2020 to September 2021 are chosen. The prognosis of the patients is observed, and the patients were divided into two groups according to the occurrence of BMS. The comparison of clinical baseline data and the expression of P53 protein and miR-424 after surgery are performed. Furthermore, the predictive value of the P53 protein gene on the occurrence of BMS in NSCLC is analyzed by the ROC curve, and the expression of miR-424 in serum of the patients before and after drug resistance is compared. The results demonstrate that the expression of P53 protein has a high predictive value for predicting the occurrence of BRAIN metastases in NSCLC patients. Also, the high expression of miR-424 suggests that it is closely related to the occurrence of platinum resistance in NSCLC patients.

BSCI-21 COX4I1 EXPRESSION IN BRAIN METASTASES

BACKGROUNDCOX4I1 (Cytochrome C oxidase, subunit 4, isoform 1) is a mitochondrial enzyme involved in the process of switching from glycolysis to oxidative phosphorylation. A previously published prospective biomarker study in glioblastoma cells found that Cytochrome C oxidase (CcO) activity was associated with resistance to treatment with both radiation and temozolomide (TMZ). The current study was designed to retrospectively assess COX4I1 expression in brain metastases from various primary cancers. METHODSThis single-institution, blinded, retrospective biomarker study evaluated 24 patients with paired brain metastases and primary cancers including lung cancer, malignant melanoma, breast cancer, colorectal cancer, renal cell carcinoma, and urothelial carcinoma. COX4I1 immunohistochemical expression in primary and metastatic samples was assessed using the H-score method. A paired t-test was used to assess the difference in total H-score between primary and brain metastasis tissue samples. Cox regression was used to assess the association between COX4I1 expression and overall survival (OS). For OS, time was calculated from metastatic tissue sample retrieval to death due to any cause. RESULTSBrain metastasis tissue samples were found to have a significantly lower total H-score, on average, when compared to primary cancer tissue samples (p=0.01, mean difference of -33.3). 83.3% of tissue samples from metastases were not radiated. 58.3% of patients were on systemic treatment 6 months prior to tumor resection of brain metastases. COX4I1 expression was not associated with overall survival. CONCLUSIONSThere is significantly increased COX4I1 expression in primary cancers as compared to brain metastases. Anti-cytochrome C oxidase therapies may be beneficial for treatment of primary tumors. No patient or treatment variables were significantly associated with overall survival in paired patients.KEYWORDSCOX4I1, cytochrome c oxidase, brain metastases

Musashi1 expression is negatively correlated with numb expression in brain metastases.

The expression of tumor stem cell markers musashi1 (msi1) and numb in brain metastases were detected to explore their roles in the development of brain metastases.A total of 51 cases of brain metastasis, 29 cases of primary tumor and 15 cases of normal brain tissue were selected. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to detect msi1 and numb expression at the protein and mRNA levels. Correlation between msi1 and numb in brain metastases were evaluated.Immunohistochemistry and RT-PCR showed that no significant difference in the expression of msi1 and numb between brain metastases and primary tumors was observed (P > .05); the expression of msi1 and numb in brain metastases was significantly higher than that in normal brain tissues (P < .05); and the expression of msi1 and numb in primary tumors was significantly higher than that in normal brain tissues (P < .05). In general, the expression of msi1 gene was negatively correlated with the expression of numb at mRNA level by Pearson correlation analysis (r = −0.345, P < .05). Additionally, the expression of msi1 and numb in brain metastases was not related to gender, age, and tissue origin (P > .05).Msi1 is highly expressed in brain metastases and primary tumors, while numb is lowly expressed in brain metastases and primary tumors; msi1 and numb are negatively correlated in brain metastases, suggesting that msi1 and numb may have regulatory mechanisms in the development of brain metastases.

Macrophages/Microglia Represent the Major Source of Indolamine 2,3-Dioxygenase Expression in Melanoma Metastases of the Brain.

The manifestation of brain metastases in patients with advanced melanoma is a common event that limits patient's survival and quality of life. The immunosuppressive properties of the brain parenchyma are very different compared to the rest of the body, making it plausible that the current success of cancer immunotherapies is specifically limited here. In melanoma brain metastases, the reciprocal interplay between immunosuppressive mediators such as indoleamine 2, 3-dioxygenase (IDO) or programmed cell death-ligand 1 (PD-L1) in the context of neoplastic transformation are far from being understood. Therefore, we analyzed the immunoreactive infiltrate (CD45, CD3, CD8, Forkhead box P3 [FoxP3], CD11c, CD23, CD123, CD68, Allograft Inflammatory factor 1[AIF-1]) and PD-L1 with respect to IDO expression and localization in melanoma brain metastases but also in matched metastases at extracranial sites to correlate intra- and interpatient data with therapy response and survival. Comparative tissue analysis identified macrophages/microglia as the major source of IDO expression in melanoma brain metastases. In contrast to the tumor infiltrating lymphocytes, melanoma cells per se exhibited low IDO expression levels paralleled by cell surface presentation of PD-L1 in intracranial metastases. Absolute numbers and pattern of IDO-expressing cells in metastases of the brain correlated with recruitment and localization of CD8+ T cells, implicating dynamic impact on the regulation of T cell function in the brain parenchyma. However, paired analysis of matched intra- and extracranial metastases identified significantly lower fractions of cytotoxic CD8+ T cells in intracranial metastases while all other immune cell populations remain unchanged. In line with the already established clinical benefit for PD-L1 expression in extracranial melanoma metastases, Kaplan-Meier analyses correlated PD-L1 expression in brain metastases with favorable outcome in advanced melanoma patients undergoing immune checkpoint therapy. In summary, our data provide new insights into the landscape of immunosuppressive factors in melanoma brain metastases that may be useful in the implication of novel therapeutic strategies for patients undergoing cancer immunotherapy.

Clinicopathologic significance of nuclear HER4 and phospho-YAP(S127) in human breast cancers and matching brain metastases.

Background:Human epidermal growth factor receptor-4 (HER4) and yes-associated protein-1 (YAP) are candidate therapeutic targets in oncology. YAP’s transcriptional coactivation function is modulated by the HER4 intracellular domain (HER4-ICD) in vitro, but the clinical relevance of this has not been established. This study investigated the potential for targeting the HER4-YAP pathway in brain metastatic breast cancer.Methods:We performed immuno-phenotypic profiling of pathway markers in a consecutive breast cancer series with 25 years of clinical follow up (n = 371), and patient-matched breast and metastatic brain tumours (n = 91; 30 pairs).Results:Membrane localisation of phospho-HER4 [pHER4(Y1162)] was infrequent in primary breast cancer, but very frequent in brain metastases (5.9% versus 75% positive), where it was usually co-expressed with pHER3(Y1289) (p < 0.05). The presence of YAP in tumour cell nuclei was associated directly with nuclear pERK5(T218/Y210) (p = 0.003). However, relationships with disease-specific survival depended on oestrogen receptor (ER) status. Nuclear pYAP(S127) was associated with smaller, good prognostic ER+ breast tumours (log-rank hazard-ratio 0.53; p = 9.6E−03), but larger, poor prognostic triple-negative cancers (log-rank hazard-ratio 2.78; p = 1.7E−02), particularly when co-expressed with nuclear HER4-ICD (p = 0.02). This phenotype was associated with stemness and mitotic instability markers (vimentin, SOX9, ID1, SPAG5, TTK, geminin; p < 0.05). YAP expression in brain metastases was higher than matched primary tumours; specifically, nuclear pYAP(S127) in ER-negative cases (p < 0.05). Nuclear YAP was detected in ~70% of ER-negative, HER4-activated brain metastases.Discussion:Our findings suggest that the canonical-mechanism where Hippo pathway-mediated phosphorylation of YAP ostensibly excludes it from the nucleus is dysfunctional in breast cancer. The data are consistent with pYAP(S127) having independent transcriptional functions, which may include transducing neuregulin signals in brain metastases. Consistent with mechanistic studies implicating it as an ER co-factor, nuclear pYAP(S127) associations with breast cancer clinical outcomes were dependent on ER status.Conclusion:Preclinical studies investigating HER4 and nuclear YAP combination therapy strategies are warranted.

PD-L1, PD-1, LAG-3, and TIM-3 in Melanoma: Expression in Brain Metastases Compared to Corresponding Extracranial Tumors.

BackgroundMetastatic melanoma to the brain carries a particularly poor prognosis that may be associated with an attenuated antitumor response in the presence of central nervous system malignancies. Thus, the development of brain metastases could theoretically accelerate cancer progression both locally and systemically. Although dysregulation of checkpoint markers, such as programmed death-ligand 1 (PD-L1), programmed cell death receptor 1 (PD-1), lymphocyte activation gene 3 (LAG-3), and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), have been implicated in immune dysfunction, the exact relationship between these markers and brain tumor-mediated immune suppression remains unclear. Thus, the objective of this study was to explore whether there exists a differential expression of the above checkpoint markers in the intracranial milieu as compared to tumors in the periphery, which may shed light on the mechanism behind the diminished antitumor response.MethodsWe identified nine patients with extracranial melanomas and matched intracranial metastases. Formalin-fixed, paraffin-embedded slides were stained for PD-L1, PD-1, LAG-3, and TIM-3 via immunohistochemistry. Qualitative analysis was performed to assess the staining of the markers in the neoplastic and lymphocytic cells, which were the two cell lineages in each biopsy. ResultsExpression of PD-1 and TIM-3 between extracranial and intracranial tumoral sites was conserved. Specifically, in lymphocytes, PD-1 expression was observed in 100% of extracranial and 100% of intracranial slides, whereas TIM-3 expression was seen in 33.33% of extracranial and 33.33% of intracranial slides. Neither marker stained tumor cells, as expected. PD-L1 showed a slight variation in staining between sites, with lymphocyte staining in 100% of extracranial and 88.89% of intracranial slides, and the same percentages per site for tumor cells. The greatest variability was observed in LAG-3 lymphocyte staining, with staining in 77.78% of extracranial and 33.33% of intracranial slides. No LAG-3 staining of tumor cells was noted, as expected.ConclusionPreliminary analysis revealed the conservation of PD-L1, PD-1, LAG-3, and TIM-3 expression intra- and extracranially. This could suggest that these markers are important in maintaining an immunosuppressive phenotype at both sites. Another possibility is that this pattern of expression is associated with patients who develop brain metastasis, as this was the only subset of patients included in this study. Interestingly, LAG-3 staining of lymphocytes appeared more prominent in extracranial over intracranial tumors. Future studies should include more samples to draw out potential patterns masked by the small sample size, as well as to compare checkpoint expression in other patient groups, such as those with non-brain metastasis or those with no metastasis at all.

Distinct gene expression profiles between primary breast cancers and brain metastases from pair-matched samples

Our objectives were to determine whether clinic-pathological markers and immune-related gene signatures in breast cancer exhibit any change upon brain metastasis and whether previously reported genes significantly associated with brain metastases and the epithelial-mesenchymal transition (EMT) were reproducible and consistent in our dataset. Sixteen pair-matched samples from primary breast cancers and brain metastases diagnosed were collected from the Japan Clinical Oncology Group Breast Cancer Study Group. Gene expression profiles for immune-, brain metastases-, and EMT-related genes were compared between primary breast cancers and brain metastases. Potential therapeutic target genes of 41 FDA-approved or under-investigation agents for brain metastases were explored. Immune-related signatures exhibited significantly lower gene expression in brain metastases than in primary breast cancers. No significant differences were detected for the majority of genes associated with brain metastases and EMT in the two groups. Among 41 therapeutic target candidates, VEGFA and DNMT3A demonstrated significantly higher gene expression in brain metastases. We found that distinct patterns of gene expression exist between primary breast cancers and brain metastases. Further studies are needed to explore whether these distinct expression profiles derive from or underlie disease status and compare these features between metastases to the brain and other sites.

Significance of Programmed Death Receptor Ligand One Expression in Brain Metastases of Non-Small Cell Lung Carcinoma

Survival in patients with metastatic non-small cell lung carcinoma (NSCLC) has considerably increased in recent years, in part due to the advent of programmed death/programmed death ligand one (PD-1/PD-L1) inhibitors. There is increasing evidence that patients with NSCLC brain metastases (BMs) also benefit from these novel therapies. PD-L1 expression as assessed by immunohistochemistry is known to predict response to PD-L1/PD-1 checkpoint inhibitors. However, it is not known whether this biomarker has prognostic significance in patients with BMs. Therefore, the aim of this study was to assess whether PD-L1 expression predicts survival in patients with NSCLC BMs treated with radiotherapy or neurosurgery as well as PD-1/PD-L1 inhibitors, after adjusting for the lung-Graded Prognostic Assessment (lung-GPA). This was a multi-institutional retrospective study. Patient records from three hospitals were reviewed to identify NSCLC BM patients who were treated with PD-1/PD-L1 inhibitors after intracranial treatment (resection, SRS (stereotactic radiosurgery/radiotherapy (SRS/SRT)), or whole-brain radiotherapy) but before intracranial progression. PD-L1 expression was considered positive in case of >1% histopathological staining. Cox proportional hazards model was used to assess prognostic significance of PD-L1 expression. Forty-eight patients with available PD-L1 expression were identified; median follow-up in this cohort was 24.7 months. Intracranial treatment consisted of SRS (n = 37), SRT (n = 3), resection (n = 7), or WBRT (n = 1). Intracranial treatment for newly diagnosed BMs was given to 30 patients; while 18 were treated for a recurrent BM. PD-L1 expression was positive in 33 patients (69%). In this cohort, median survival was 21.7 months. When split by lung-GPA, median survival in months was as follows: GPA 0-1: 11.4 (n = 6), GPA 1.5 – 2: 16.3 (n = 22), GPA 2.5 – 3: median not reached (n = 17); GPA 3.5 – 4: 14.6 (n = 3). In univariate analysis, PD-L1 expression was a borderline significant predictor for overall survival (HR = 0.44; 95% CI 0.19 – 1.00; p = 0.05). In multivariate analysis, PD-L1 was prognostic for overall survival (HR = 0.36, 95% CI = 0.14 – 0.90; p = 0.03) after correcting for lung-GPA, type of intracranial treatment, and newly diagnosed vs recurrent BMs. PD-L1 expression may be a prognostic factor independent of lung-GPA in patients with NSCLC BMs who underwent treatment with PD-1/PD-L1 checkpoint inhibitors. Larger studies are required to validate this finding.

Abstract 1114: Upregulation of ALCAM is a marker for non-small-cell lung cancer brain metastases

Abstract Non-small-cell lung cancer (NSCLC) is the most common cause of cancer-related death with still a 5-year survival rate of &amp;lt;10%. Every second patient with advanced NSCLC will develop brain metastases and in 50% of those the brain is the only site of tumor relapse (oligo-metastasis). Cancer cells need to have specific properties to access and colonize the brain microenvironment leading to the question which metastatic factors foster brain metastasis formation. Different cell adhesion molecules (CAMs) have been shown to be involved in the process of metastasis in different tumor entities. The activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein of the immunoglobulin superfamily and involved in the maintenance of cell-cell contacts and has a possible role in the “homing” of metastatic cells in distant organs. ALCAM is also involved in the leukocyte transmigration across the blood-brain barrier. Here we therefore investigated the role of ALCAM in NSCLC brain metastases formation. Our studies on patient material revealed that the ALCAM protein expression in brain metastases (n=71) is significantly increased (50.7% positive, p=0.023) compared to the primary tumor (21.7% positive, n=47). Analysis of matched pairs of primary tumors and brain metastases confirmed this observation, as in 33% a de novo ALCAM expression in the matching brain metastases could be observed. Additionally, patients with a strong ALCAM expression in either their primary tumors or brain metastases showed a significantly shortened overall survival (p=0.017 and p=0.035 respectively). In vitro analysis of the effect of ALCAM knock out in the H460 lung cancer cell line showed no effect on proliferation, migration or colony-forming behavior. However, cell adhesion was severely hampered in the knock out cells. Currently in vivo mice experiments are running. Together these findings indicate an important role for ALCAM in brain metastasis formation. Citation Format: Desiree Loreth, Justine Münsterberg, Cecile Maire, Stefan Werner, Monja Gandrass, Christian Bernreuther, Jolanthe Kropidlowski, Manfred Westphal, Stefan Steurer, Katrin Lamszus, Markus Glatzel, Klaus Pantel, Harriet Wikman. Upregulation of ALCAM is a marker for non-small-cell lung cancer brain metastases [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 1114.

PD-L1 Expression of Lung Cancer Cells, Unlike Infiltrating Immune Cells, Is Stable and Unaffected by Therapy During Brain Metastasis

BackgroundApproximately 50% of brain metastases originate from non–small-cell lung cancer. The median survival of patients with brain metastases is 1 month without treatment. Novel immunotherapeutic strategies, such as those targeting the programmed death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) axis, are promising in patients with advanced systemic disease but are often preferentially administered to patients with tumors showing PD-L1 positivity. Patients and MethodsSurgically resected paired primary lung adenocarcinoma and brain metastasis samples of 61 patients were analyzed. We compared the paired samples regarding the amount of peritumoral and stromal mononuclear infiltration, PD-L1 expression of tumor and immune cells, and PD-1 expression of immune cells. We investigated the effect of radiotherapy, chemotherapy, and steroid therapy on PD-L1 expression in brain metastases. ResultsThere was significant positive correlation regarding the PD-L1 expression of tumor cells between the paired primary lung adenocarcinoma and brain metastatic samples with the use of different cutoff levels (1%, 5%, 50%). We found no impact of chemotherapy or steroid therapy on the changes of PD-L1 expression of tumor cells between the 2 sites. There is no or only limited concordance of the proportion of PD-1– or PD-L1–positive tumor-associated immune cells between the paired tumor samples, which suggests that brain metastases develop their own immune environment. ConclusionWe observed a strong correlation of PD-L1 positive tumor cells between primary lung adenocarcinoma cases and their corresponding brain metastases, which is not significantly influenced by chemotherapy or steroid therapy.

Clinical Significance of PD-L1 Expression in Brain Metastases from Non-small Cell Lung Cancer.

To investigate the association between positivity for programmed cell death-ligand 1 (PD-L1) in brain metastases (BM) and the prognosis or clinical factors in patients with non-small cell lung cancer (NSCLC). Thirty-two patients with surgically resected brain-metastatic NSCLC were enrolled. The PD-L1 expression in BM was analyzed using the antibody against human PD-L1 (clone SP142). The PD-L1 positivity was defined as PD-L1 expression on brain-metastatic tumor cells of ≥5%. Seven (21.9%) out of 32 patients showed PD-L1 positivity in BM. The PD-L1-positive BM group had a significantly shorter brain-specific disease-free survival than the PD-L1-negative BM group (p<0.05). PD-L1 positivity in BM was significantly associated with a heavy smoking history and the administration of radiotherapy for BM before surgery (p<0.05 and p<0.05, respectively). The PD-L1 expression in BM from NSCLC may be associated with local recurrence following surgery, and the smoking- or radiotherapy-derived effects.

Abstract A39: Expression of DNA methylation-related proteins in metastatic breast cancer

Abstract Purpose: To investigate the expressions of methylation-related proteins (5-methylcytosine [5-meC] and DNA [cytosine-5-]-methyltransferase 1 [DNMT1]) in metastatic breast cancers of variable sites and their associations with clinicopathologic factors. Methods: A total of 126 metastatic breast cancers (31, 36, 11, and 48 bone, brain, liver, and lung metastases, respectively) were made into tissue microarrays, and immunohistochemical staining was performed for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER-2), Ki-67, 5-meC, and DNMT1. Molecular classification was made on the basis of the immunohistochemical staining results of ER, PR, HER-2, and Ki-67, as luminal A, luminal B, HER-2-positive, and triple-negative breast cancers. Results: Methylation-related proteins were differentially expressed based on the metastatic sites. Tumoral and stromal 5-meC showed the lowest expression in bone metastases (P &amp;lt; 0.001), while tumoral DNMT1 showed the lowest expression in bone metastases and the highest expression in brain metastases (P &amp;lt; 0.001). Expression of DNMT1 correlated with ER-negativity (P = 0.004), PR-negativity (P = 0.011), HER-2-positivity (P = 0.016), higher Ki-67 labeling indices (P = 0.016), and non-luminal A type (P = 0.017). Univariate analysis revealed that DNMT1-positivity was associated with shorter overall survival in patients with bone (P = 0.017) and lung metastases (P = 0.028). Conclusion: In metastatic breast cancer, methylation-related proteins are differentially expressed according to the metastatic sites. Tumoral and stromal 5-meC showed the lowest expression in bone metastases, while tumoral DNMT1 expression was low in bone metastases and the highest in brain metastases. Citation Format: Lee Yukyung, Kim Eun-Sol, Koo Ja Seung. Expression of DNA methylation-related proteins in metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A39.

Role of HYAL1 expression in primary breast cancer in the formation of brain metastases.

The incidence of brain metastases in breast cancer patients has increased in the last years. However, the knowledge about tumor cell invasion in the brain is still very limited. Based on our recent study on cDNA microarray data of breast cancer patients, we hypothesized that two enzymes involved in the hyaluronan metabolism, namely, hyaluronan synthase 2 (HAS2) and hyaluronidase 1 (HYAL1) are associated with brain metastases formation. Protein expression levels of hyaluronan, HAS2, and HYAL1 were analyzed in primary breast cancer, and metastatic tissue samples from different localizations (brain, bone, skin, liver, and lung) were included in four different cohorts by immunohistochemistry. Correlations of expression levels with clinical and pathological parameters were performed within the individual cohorts. Higher HYAL1 expression was detected among primary tumors from patients with subsequent brain metastases compared with those without brain metastases (p=0.011). Interestingly, brain metastatic tissue showed a significantly reduced HYAL1 expression compared with the corresponding primary tumor (p=0.003). HYAL1 expression in brain metastases was also significantly lower than in skin, liver, and lung metastases. Further, hyaluronan staining in brain metastases was mainly located on the surface of the tumor cells, whereas in all other metastatic sites hyaluronan was only detected in the extracellular matrix. We could not show an association of HAS2 with the formation of brain metastases. In conclusion, our results suggest that the enzyme HYAL1 plays a role in tumor dissemination and brain-specific colonization, rather than in subsequent metastatic out-growth.

Differential Site-Based Expression of Pentose Phosphate Pathway-Related Proteins among Breast Cancer Metastases.

Purpose. We aimed to investigate the expression of pentose phosphate pathway- (PPP-) related proteins in metastatic breast cancer and its relationship with clinicopathologic factors. Methods. Tissue samples from 126 metastatic breast cancers were included in a tissue microarray. Expression of PPP-related proteins [glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), 6-phosphogluconate dehydrogenase (6PGDH), and nuclear factor erythroid 2-related factor (NRF2)] was determined by immunohistochemistry. Results. G6PDH (p = 0.011) and cytoplasmic NRF2 (p = 0.001) showed the highest expression in brain metastases. Human epidermal growth factor receptor (HER-2) positivity was associated with G6PDH (p < 0.001) and cytoplasmic NRF2 (p = 0.015) positivity. A high Ki-67 labeling index (LI) was correlated with nuclear NRF2 positivity (p = 0.037), and HER-2-positive luminal B type was associated with G6PDH positivity (p = 0.001). On multivariate Cox analysis, independent risk factors of short overall survival were 6PGL positivity in bone metastasis (HR 4.180, 95% CI 1.160–15.06, p = 0.029) and low Ki-67 LI in lung metastasis (HR 11.853, 95% CI 1.841–76.30, p = 0.009). Conclusion. Differential expression of PPP-related proteins correlated with different prognoses and metastatic sites, with the highest expression in brain metastases, and could be a potential therapeutic target.

Tumor infiltrating lymphocytes and PD-L1 expression in brain metastases of small cell lung cancer (SCLC).

Brain metastases (BM) are frequent in small cell lung cancer (SCLC). Novel insights into their pathobiology are needed for development of better therapies. We investigated tumor-infiltrating lymphocyte (TIL) subsets (CD3+, CD8+, CD45RO+, FOXP3+ and PD-1+) and expression of PD-L1 in a series of 32 SCLC BM specimens and four matched primary tumor specimens using immunohistochemistry. 30/32 (93.8 %) BM specimens showed TIL infiltration. CD3+ TILs were observed in 30/32 (93.8 %) BM specimens, CD8+ TILs in 25/32 (78.1 %), CD45RO+ TILs in 15/32 (46.9 %), FOXP3+ TILs in 15/32 (46.9 %) and PD-1+ TILs in 1/32 (3.1 %) BM specimens. Patients with infiltration of CD45RO+ TILS had a significantly longer median survival time (11months; 95 % CI 0.000-26.148) as compared to patients without the presence of CD45RO+ TILs (5months; 95 % CI 0.966-9.034; p = 0.007; log rank test). Membranous PD-L1 on tumor cells was observed in 24/32 (75.0 %) BM specimens, with 11/32 (34.4 %) cases showing PD-L1 expression in over 5 % of viable BM tumor cells. PD-L1 expression on TILs was seen in 8/32 (25.0 %) and on tumor infiltrating macrophages in 9/32 (28.1 %) cases. Patients with PD-L1 expression on TILs presented with improved survival prognosis (6 versus 29months; p = 0.002; log rank test). Among matched primary tumors, all (4/4; 100 %) specimens showed TIL infiltration, while PD-L1 expression found in only 1/4 (25.0 %) specimen. TIL infiltration and PD-L1 expression are commonly found in SCLC BM and presence of CD45RO+ memory T-cells and PD-L1+ TILs in SCLC BM seem to associate with favorable survival times. Our data suggest an active immune microenvironment in SCLC BM that may be targetable by immune-modulating drugs.