Longer Metastasis-free Survival Research Articles

Post-surgery statin use contributes to favorable outcomes in patients with early breast cancer

BackgroundStatins are a group of lipid-lowering drugs with pleiotropic effects that include, but are not limited to the inhibition of cholesterol synthesis resulting in a wide range of anti-inflammatory, anti-tumor, immunomodulatory, and anti-thrombotic properties. This study aimed to determine the impact of the prior to- or after- breast surgery usage of statins on the tumor prognosis in breast cancer (BC) patients. MethodsA cohort of patients diagnosed with early invasive ductal BC (n=301) at the Hospital Italiano de Buenos Aires, Argentina, with a minimum follow-up period of 10 years after the surgical procedure were included and stratified according to the time of use of statins and type of statin used. Then, local relapse-free survival (RFS), metastasis-free survival (MFS), bone metastasis-free survival (BMFS), visceral metastasis-free (VMFS), mixed metastasis (bone and visceral)-free survival (mix-MFS) and overall survival (OS) were analyzed. ResultsStatins usage after breast surgery was related with lesser metastatic occurrence (p=0.017), lower number of metastatic foci (p=0.034) and fewer dead events (p=0.041), as well as longer MFS (p=0.013) and OS (p=0.027). When stratified by the nature of statins (hydrophilic or lipophilic), only the relatively hydrophilic statin rosuvastatin (ROSU) had an impact on the increase of MFS and OS (p=0.018 and p=0.030, respectively). ConclusionPost-surgery statins usage was associated with increased MFS and OS, with increased benefits of ROSU over simvastatin (SIM) or atorvastatin (ATOR). These results set the rationale for additional studies addressing the use of statins, and particularly, rosuvastatin, to improve the outcome of BC patients.

Abstract 6270: Anti-metastatic effects of estrogen receptor β in inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) is a rare but highly aggressive form of breast cancer with a 5-year survival rate of less than 50%. Although 20-30% of women with IBC have distant metastasis at the time of diagnosis, the biology underlying the aggressive behavior of the disease is poorly understood and molecular markers that distinguish IBC from non-IBC have not yet been identified. In response to the urgent need of discovering new targeted therapies in IBC, we have studied the second estrogen receptor (ERβ) since prior work from our lab correlated higher tumor expression of ERβ in samples from IBC patients with longer metastasis free survival. We showed that ERβ exerted anti-metastatic activity in IBC by inhibiting actin-based cell migration through the suppression of RhoC GTPase signaling. However, since estrogen receptors elicit their effects by inducing global cellular alterations, we have now adopted an integrated genomic approach to fully understand the anti-metastatic effects of ERβ in IBC and delineate the underlying transcriptional regulatory circuits. We found ERβ binding sites in IBC cells and combined this information with gene expression data to identify downstream direct target genes and associated pathways. Our findings suggest the regulation of tumor micro-environment and metabolism as the primary targets of ERβ and we are currently working to pinpoint the crucial regulatory components in these functions. Our findings thus offer an opportunity to better understand the mechanism of ERβ action in aggressive breast cancer, serving as a critical step in validating its tumor repressive role. Citation Format: Harika Nagandla, Spyros Tastsoglou, Kim Cuong Cap, Aireana Phillips, Wei Qian, Jianying Zhou, Jenny Chang, Savitri Krishnamurthy, Naoto Ueno, Artemis Hatzigeorgiou, Christoforos Thomas. Anti-metastatic effects of estrogen receptor β in inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6270.

Prognostic Role of Prolactin-Induced Protein (PIP) in Breast Cancer.

Prolactin-inducible protein (PIP), also referred to as gross cystic disease fluid protein 15 (GCDFP-15), has been a trending topic in recent years due to its potential role as a specific marker in breast cancer. PIP binds to aquaporin-5 (AQP5), CD4, actin, fibrinogen, β-tubulin, serum albumin, hydroxyapatite, zinc α2-glycoprotein, and the Fc fragment of IgGs, and the expression of PIP has been demonstrated to be modulated by various cytokines, including IL4/13, IL1, and IL6. PIP gene expression has been extensively studied due to its captivating nature. It is influenced by various factors, with androgens, progesterone, glucocorticosteroids, prolactin, and growth hormone enhancing its expression while estrogens suppress it. The regulatory mechanisms involve important proteins such as STAT5A, STAT5B, Runx2, and androgen receptor, which collaborate to enhance PIP gene transcription and protein production. The expression level of PIP in breast cancer is dependent on the tumor stage and subtype. Higher expression is observed in early-stage tumors of the luminal A subtype, while lower expression is associated with luminal B, basal-like, and triple-negative subtypes, which have a poorer prognosis. PIP expression is also correlated with apocrine differentiation, hormone receptor positivity, and longer metastasis-free survival. PIP plays a role in supporting the immune system's antitumor response during the early stages of breast cancer development. However, as cancer progresses, the protective role of PIP may become less effective or diminished. In this work, we summarized the clinical significance of the PIP molecule in breast cancer and its potential role as a new candidate for cell-based therapies.

Bone-matrix mineralization dampens integrin-mediated mechanosignalling and metastatic progression in breast cancer.

In patients with breast cancer, lower bone mineral density increases the risk of bone metastasis. Although the relationship between bone-matrix mineralization and tumour-cell phenotype in breast cancer is not well understood, mineralization-induced rigidity is thought to drive metastatic progression via increased cell-adhesion forces. Here, by using collagen-based matrices with adjustable intrafibrillar mineralization, we show that, unexpectedly, matrix mineralization dampens integrin-mediated mechanosignalling and induces a less proliferative stem-cell-like phenotype in breast cancer cells. In mice with xenografted decellularized physiological bone matrices seeded with human breast tumour cells, the presence of bone mineral reduced tumour growth and upregulated a gene-expression signature that is associated with longer metastasis-free survival in patients with breast cancer. Our findings suggest that bone-matrix changes in osteogenic niches regulate metastatic progression in breast cancer and that in vitro models of bone metastasis should integrate organic and inorganic matrix components to mimic physiological and pathologic mineralization.

HORMAD1 overexpression predicts response to anthracycline-cyclophosphamide and survival in triple-negative breast cancers.

Triple negative breast cancers (TNBCs) represent 15-20% of all breast cancers and are associated with higher recurrence and distant metastasis rate. Standard of care for early stage TNBC is anthracyclines combined with cyclophosphamide (AC) followed by taxanes, in the neo-adjuvant or adjuvant setting. This work aimed to identify predictive biomarkers of AC response in patient-derived xenograft (PDX) models of TNBC and to validate them in the clinical setting. By gene and protein expression analysis of 39 PDX with different responses to AC, we found that high expression of HORMAD1 was associated with better response to AC. Both gene and protein expression were associated with promoter hypomethylation. In a cohort of 526 breast cancer patients, HORMAD1 was overexpressed in 71% of TNBC. In a second cohort of 186 TNBC patients treated with AC, HORMAD1 expression was associated with longer metastasis-free survival (MFS). In summary, HORMAD1 overexpression was predictive of an improved response to AC in PDX and is an independent prognostic factor in TNBC patients treated with AC.

Screening and Identification of Key Biomarkers in Metastatic Uveal Melanoma: Evidence from a Bioinformatic Analysis.

Purpose: To identify key biomarkers in the metastasis of uveal melanoma (UM). Methods: The microarray datasets GSE27831 and GSE22138 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified, and functional enrichment analyses were performed. A protein−protein interaction network was constructed, and four algorithms were performed to increase the reliability of hub genes. Biomarker analysis and metastasis-free survival analysis were performed to screen and verify prognostic hub genes. Results: A total of 138 DEGs were identified, consisting of 71 downregulated genes and 67 upregulated genes. Four genes (ROBO1, FMN1, FYN and FXR1) were selected as hub genes. Biomarker analysis and metastasis-free survival analysis showed that ROBO1, FMN1, FYN and FXR1 were factors affecting the metastasis and metastasis-free survival of UM (all p < 0.05). High expression of ROBO1 and low expression of FMN1 were associated with longer metastasis-free survival. Multivariable logistic regression and Cox analyses in GSE 27831 indicated that ROBO1 was an independent factor affecting metastasis and metastasis-free survival of UM (p = 0.010 and p = 0.009), while ROBO1 and FMN1 were independent factors affecting metastasis and metastasis-free survival of UM in GSE22138 (all p < 0.05). Conclusions: ROBO1, FMN1, FYN and FXR1 should be regarded as diagnostic biomarkers for the metastasis of UM, especially ROBO1 and FMN1. High expression of ROBO1 and low expression of FMN1 were associated with longer metastasis-free survival. This study may facilitate the understanding of the molecular mechanisms underlying the metastasis of UM.

MARCKS as a Potential Therapeutic Target in Inflammatory Breast Cancer.

Inflammatory breast cancer (IBC) is the most pro-metastatic form of breast cancer (BC). We previously demonstrated that protein overexpression of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein was associated with shorter survival in IBC patients. MARCKS has been associated with the PI3K/AKT pathway. MARCKS inhibitors are in development. Our objective was to investigate MARCKS, expressed preferentially in IBC that non-IBC (nIBC), as a novel potential therapeutic target for IBC. The biologic activity of MPS, a MARCKS peptide inhibitor, on cell proliferation, migration, invasion, and mammosphere formation was evaluated in IBC (SUM149 and SUM190) and nIBC (MDA-MB-231 and MCF7) cell lines, as well as its effects on protein expression in the PTEN/AKT and MAPK pathways. The prognostic relevance of MARCKS and phosphatase and tensin homolog (PTEN) protein expression as a surrogate marker of metastasis-free survival (MFS) was evaluated by immunohistochemistry (IHC) in a retrospective series of archival tumor samples derived from 180 IBC patients and 355 nIBC patients. In vitro MPS impaired cell proliferation, migration and invasion, and mammosphere formation in IBC cells. MARCKS inhibition upregulated PTEN and downregulated pAKT and pMAPK expression in IBC cells, but not in nIBC cells. By IHC, MARCKS expression and PTEN expression were negatively correlated in IBC samples and were associated with shorter MFS and longer MFS, respectively, in multivariate analysis. The combination of MARCKS-/PTEN+ protein status was associated with longer MFS in IBC patient only (p = 8.7 × 10−3), and mirrored the molecular profile (MARCKS-downregulated/PTEN-upregulated) of MPS-treated IBC cell lines. In conclusion, our results uncover a functional role of MARCKS implicated in IBC aggressiveness. Associated with the good-prognosis value of the MARCKS-/PTEN+ protein status that mirrors the molecular profile of MPS-treated IBC cell lines, our results suggest that MARCKS could be a potential therapeutic target in patients with MARCKS-positive IBC. Future preclinical studies using a larger panel of IBC cell lines, animal models and analysis of a larger series of clinical samples are warranted in order to validate our results.

High C-Reactive Protein Levels Are Relatedto Better Survival in Patients with Uveal Melanoma.

PurposeTo determine whether peripheral blood leukocyte numbers and serum markers of inflammation can be used to predict which patients with primary uveal melanoma will develop metastasis.DesignRetrospective study.ParticipantsMedical records of patients with uveal melanoma (UM) who received treatment for primary UM between February 1992 and December 2020 at the Erasmus University Medical Center (Rotterdam, The Netherlands) and the Rotterdam Eye Hospital (Rotterdam, The Netherlands) were reviewed.MethodsInclusion criteria were the presence of a melanoma of the choroid or ciliary body and the availability of data from peripheral blood samples taken before treatment of the melanoma. Data including patient demographics, C-reactive protein (CRP) levels; erythrocyte sedimentation rate (ESR); number of leukocytes, neutrophils, monocytes, and lymphocytes; and histopathologic findings were obtained from medical records. Neutrophil-to-lymphocyte ratio (NLR) and lymphocyte-to-monocyte ratio (LMR) were calculated.Main Outcome MeasuresMetastasis-free survival.ResultsOf the 807 patients with UM, serum and leukocyte data were available for 183 of them at the time of primary tumor treatment. In the total group, no correlation was found between ESR before treatment; the number of leukocytes; percentages of neutrophils, monocytes, and lymphocytes; or NLR or LMR values and any of the clinical characteristics or metastasis-free survival. Among patients who underwent enucleation, those with negative BAP1 findings showed significantly lower numbers of leukocytes (P < 0.05). In the entire cohort, a significant association was found between high CRP levels and longer metastasis-free survival (MFS; P = 0.049).ConclusionsThe total blood leukocyte number was related to loss of BAP1 staining in patients who underwent enucleation, with lower leukocyte counts correlating with absent BAP1 staining. Higher CRP levels were associated with a longer MFS in the entire cohort. Neither the NLR nor the LMR is a good predictor for metastasis developing in patients with UM.

Tenascin-C immobilizes infiltrating T lymphocytes through CXCL12 promoting breast cancer progression.

Immune checkpoint therapy, where CD8 tumor infiltrating T lymphocytes (TIL) are reactivated, is a promising anti‐cancer treatment approach, yet with low response rates. The extracellular matrix, in particular tenascin‐C, may generate barriers for TIL. To investigate this possibility, we used a MMTV‐NeuNT and syngeneic mammary gland grafting model derived thereof with engineered tenascin‐C levels and observed accumulation of CD8 TIL in tenascin‐C‐rich stroma. Inhibition studies revealed that tenascin‐C induced CXCL12 through TLR4. By binding CXCL12, tenascin‐C retained CD8 TIL in the stroma. Blockade of CXCR4, the receptor of CXCL12, enhanced macrophage and CD8 TIL infiltration and reduced tumor growth and subsequent metastasis. Retention of CD8 TIL by tenascin‐C/CXCL12 was also observed in human breast cancer by tissue staining. Moreover, whereas high CD8 TIL numbers correlated with longer metastasis‐free survival, this was not the case when also tenascin‐C and CXCL12 levels were high. Altogether, these results may be useful for improving tumor immunity as diagnostic tool and to formulate a future “TIL‐matrix‐release‐and‐reactivate” strategy.

POLG Gene Variants in Cervical Cancer Patients and Their Associations with Clinical and Pathomorphological Tumor Characteristics.

Cervical cancer is one of the most common cancers in women worldwide. Human papillomaviruses are known to be the main, but not the only risk factor, of this cancer type. Despite all the knowledge on this cancer type, it is still a challenge to predict the course of the disease, and therefore, minimally invasive biomarkers are needed. This study aimed to analyze single-nucleotide variants in the POLG gene and assess the associations with tumor phenotype and patient outcome. A total of 172 cervical cancer patients were included in this study. Clinical and tumor data were gathered from medical records retrospectively. Single nucleotide variations were determined using TaqMan probes with Real-Time PCR. Significant associations between POLG rs3087374 and cervical cancer patients’ tumor histological type, stage, and tumor size were determined. The CA genotype and A allele of rs3087374 increased the probability of adenocarcinoma histological tumor type, IIIA stage, and T3 tumor size compared to CC genotype and C allele, respectively. Furthermore, patients with AA genotype in rs2072267 had longer metastasis-free survival than those with the GG genotype. Our data suggest that mitochondrial polymerase gamma encoded by nuclear POLG gene is important for specific tumor phenotype formation and patient outcome in cervical cancer.

633P Tolerability and treatment response to darolutamide (DARO) in patients with non-metastatic castration-resistant prostate cancer (nmCRPC) in the phase III ARAMIS trial

Androgen receptor inhibitors (ARIs) approved for treating nmCRPC are associated in varying degrees with certain adverse events (AEs), e.g., fatigue, risk of falls or rash. In phase III studies, dose modification and discontinuation due to AEs was higher with ARIs vs placebo. Reduced dose, compliance patterns and treatment fidelity may impact drug efficacy. Tolerability of DARO and association of prostate-specific antigen (PSA) decline in response to DARO treatment with metastasis-free survival (MFS) in the ARAMIS trial are reviewed. 1509 patients with nmCRPC and PSA doubling time (PSADT) ≤10 months were randomised 2:1 to DARO 600 mg twice daily (n=955) or placebo (PBO; n=554) while continuing androgen deprivation therapy. AEs and dose modifications were assessed every 16 weeks. The association between PSA decline from baseline in response to DARO treatment and MFS was evaluated using the Cox proportional hazards model. An association with overall survival will be investigated. DARO was well tolerated; 97.2% of patients on DARO received the full planned dose. Patients treated with DARO did not have an increase in treatment discontinuation due to AEs vs PBO (8.9% vs 8.7%). Few patients had dose modifications for any reason (15.2% vs 9.7% for DARO vs PBO). Almost all of the patients on DARO with discontinuation due to AEs or dose modifications were able to resume and re-establish the indicated dose (91.7% vs 88.9% for DARO vs PBO). In the ARAMIS trial, 50.9% of patients on DARO had a maximal PSA response (≥90% decrease from baseline) vs 1.8% of patients on PBO. Pharmacodynamic modelling showed that longer MFS was positively associated with maximum decrease in PSA from baseline. Favourable tolerability of DARO at the recommended dose of 600 mg twice daily was associated with low rates of dose reduction and treatment discontinuation, which in turn may lead to extended survival with longer duration of treatment with DARO. It is important to further assess the real-world tolerance of different ARIs in men with nmCRPC.

Abstract 5425: Novel molecular subtypes identified in prostate cancer: Results from the SPARTAN study

Abstract Background: Results from SPARTAN, a phase III placebo (PBO)-controlled study in patients (pts) with nonmetastatic castration-resistant prostate cancer (nmCRPC), show that apalutamide (APA) plus ongoing androgen deprivation therapy (ADT) significantly improves metastasis-free survival (MFS) compared with PBO + ADT. This analysis investigated the effects of APA in biologically distinct molecular subclasses of prostate cancer defined by gene expression profiles. Methods: Gene expression profiles (DECIPHER® prostate test, San Diego, CA) were generated from 233 archival primary prostate tumors; data were summarized based on 160 predefined gene signatures indicative of clinical prognosis and prostate cancer-related biology. Unsupervised consensus clustering identified sets of co-regulated expression signatures, and associations between signature expression, treatment, and signature-treatment interaction with MFS were evaluated using Kaplan-Meier analysis and Cox proportional hazards models. Results: Four co-regulated expression signature classes, each with distinct biological pathway functions, were identified. Class C1 included prognosis-related (risk) signatures; C2 included steroid homeostasis-related signatures; C3 included hormonal therapy nonresponsive basal and neuroendocrine-like signatures; C4 included immune and stromal signatures. Increased C1 expression was associated with shorter MFS in the PBO group (HR [95% CI], 2.18 [1.11-4.28], p = 0.02), while it was associated with longer MFS in the APA group (interaction HR [95% CI], APA vs PBO, 0.36 [0.14-0.95], p = 0.04). Similarly, increased C2 expression was associated with shorter MFS in the PBO group (HR [95% CI], 1.42 [1.02-1.98], p = 0.04), while it was associated with longer MFS in the APA group (interaction HR [95% CI], APA vs PBO, 0.57 [0.35-0.93], p = 0.02). Although there is no significant interaction effect between signature and treatment in C3, pts with low expression of C3 (adeno-like) showed longer MFS on APA vs PBO (HR [95% CI], 0.23 [0.13-0.40], p &amp;lt; 0.0001) compared with high-C3-expressing neuroendocrine-like tumors. Increased C4 expression was associated with decreased risk of metastasis in the APA group (HR [95% CI], 0.55 [0.35-0.86], p = 0.008) compared with PBO (interaction HR [95% CI], APA vs PBO, 0.53 [0.28-0.98], p = 0.04). Conclusion: Expression signatures were clustered into 4 correlated unique biological subclasses. Clinical benefit of APA + ADT was observed in pts with expression profiles with high risk (C1), C2, C3, or C4 compared with PBO + ADT. Citation Format: Clemente Aguilar, Michael Gormley, Shibu Thomas, Paul N. Mainwaring, David Olmos, Fred Saad, Simon Chowdhury, Elai Davicioni, Yang Liu, Deborah S. Ricci, Angela Lopez-Gitlitz, Margaret K. Yu, Matthew R. Smith, Eric J. Small, Felix Feng. Novel molecular subtypes identified in prostate cancer: Results from the SPARTAN study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5425.

Values of alkaline phosphatase at the diagnosis of castration resistance and response to primary androgen deprivation therapy as predictors of subsequent metastasis in non-metastatic castration-resistant prostate cancer.

Among various therapeutic options available for metastatic castration-resistant prostate cancer (mCRPC), only apalutamide and enzalutamide have shown evidences of improved metastasis-free survival (MFS) for non-metastatic castration-resistant prostate cancer (nmCRPC). However, there is a paucity of evidence to indicate who may be targeted for aggressive therapy among patients with nmCRPC. The objectives of this retrospective study were to explore predictors of metastasis in patients with nmCRPC and to identify a subpopulation of patients with nmCRPC who may benefit from aggressive therapy. A total of 115 patients with CRPC who had no metastasis detected at the time of diagnosis of CRPC were included in this retrospective study. All patients were treated at Jikei University and its affiliated hospitals. The primary outcome measure was MFS from the time of diagnosis of CRPC. Predictors of MFS were also explored with a multivariate Cox hazard model. The median observation period after diagnosis of CRPC was 30months (range 2-143months). Kaplan-Meier analysis revealed a median MFS of 76. Multivariate analysis demonstrated that low alkaline phosphatase (ALP) values at diagnosis of CRPC and favorable response to primary androgen deprivation therapy (ADT) were significant predictors of longer MFS (P = 0.011, and 0.031, respectively). Results of this study suggest that high ALP values at diagnosis of CRPC and poor response to primary ADT may predict the propensity of metastasis in patients with nmCRPC. Further prospective studies will be required enrolling more patients to confirm our findings.

Alkaline phosphatase (ALP) value at diagnosis of CRPC and response to primary androgen deprivation therapy may predict metastasis in patients with nonmetastatic castration-resistant prostate cancer (nmCRPC).

294 Background: Numerous therapeutic options are available for metastatic castration-resistant prostate cancer (mCRPC). However, current therapeutic options remain inadequate for non-metastatic CRPC (nmCRPC); furthermore, there is a paucity of clear evidence to show who may be targeted for aggressive therapy among nmCRPC patients. Therefore, the objective of this retrospective study was to explore predictors of metastasis in patients with nmCRPC and to identify a subpopulation of nmCRPC patients who may benefit from aggressive therapy. Methods: A total of 115 patients with CRPC who had no metastasis at the time of diagnosis of CRPC were included in this retrospective study. All patients were treated at Jikei University Hospital. The primary outcome measure was metastasis-free survival (MFS) from the time of diagnosis of CRPC. Predictors of MFS were also explored with a multivariate Cox model. Results: The median observation period after diagnosis of CRPC in these patients was 30 months. Kaplan-Meier analysis revealed a median MFS of 76 months in these patients. Multivariate analysis demonstrated that low ALP values at diagnosis of CRPC and favorable response to primary androgen deprivation therapy (ADT) were significant predictors of longer MFS ( P = 0.011, and 0.031, respectively). Conclusions: Study results suggest that high ALP values at diagnosis of CRPC and poor response to primary ADT may predict metastasis in patients with nmCRPC. Further prospective studies will be required in more patients to confirm our findings. Univariate/multivariate analysis of factors contributing to MFS in nmCRPC patients. [Table: see text]

An 11-gene-based prognostic signature for uveal melanoma metastasis based on gene expression and DNA methylation profile.

Uveal melanoma (UM) is the most common intraocular tumor worldwide. We proposed to identifya vital gene signature that has prognostic value for UM metastasis. For this purpose, we obtained a published DNA methylation and gene expression data set associated with UM from the Gene Expression Omnibus. The genes whose aberrant expression significantly associated with UM patients' metastasis-free survival (MFS) were identified by applying a univariate Cox proportional hazards model to the gene expression data set followed by a robust likelihood-based survival analysis to screen the optimal prognostic gene signatures (PGS). A formula for calculating the risk score that represents UM metastasis risk was constructed by including the PGSs' expression values weighted by their regression coefficients, which were obtained by a multivariate Cox regression analysis. As a result, aberrant expression of 2884 genes were found to be significantly associated with UM patients' MFS, which were referred to as MFSGs, and 11 out of those MFSGs, GJC1, TCEA1, MFSD3, FAF2, TLCD1, GPAA1, CYC1, ASAP1, JPH1, LDB3, and KDELR3, were identified as PGSs through which we could accurately separate UM samples with shorter MFS from those with longer MFS. By combining the DNA methylation data set and MFSGs, we further identified 265 MFSGs, which contained CpG sites that significantly hyper- or hypo-methylated in UM samples compared with control samples. Functional enrichment analysis and pathway crosstalk analysis of those genes indicated significant enrichment of cancer-related pathways. In conclusion, we identified an 11-gene-based prognostic signature and several gene biomarkers for UM metastasis, which should be helpful for selecting an appropriate treatment method for specific patients with UM.

Programmed cell death ligand-1 expression in tumor and immune cells is associated with better patient outcome and decreased tumor-infiltrating lymphocytes in uveal melanoma

Programmed cell death-1/ligand (PD-1/PD-L1) interaction negatively regulates T cell activity. PD-L1 expression in tumor cells, antigen-presenting cells, and lymphocytes of the tumor microenvironment is associated with response to treatment with PD-1/PD-L1 inhibitors, but there is still debate on the cutoff value that correlates with responders. In uveal melanoma (UM), 40% of patients will develop liver metastases and, amongst them, 90% will succumb to their disease. The aim of this study was to analyze PD-L1 expression as a prognostic marker and as a possible therapeutic target for UM. Sixty-seven enucleated eyes from UM patients with relevant clinical information were analyzed. Univariate and multivariate analysis were used to evaluate association of PD-L1 with survival. PD-L1 expression was positive relatively to tumor cells, immune cells, and the tumor and tumor-infiltrating immune cell group scoring in 46, 34 and 55% of the cases, respectively. On univariate analysis, tumor cells and the tumor and tumor-infiltrating immune cell group PD-L1 expression was associated with a longer metastasis-free survival (P = 0.04 and P = 0.007). However, on multivariate analysis, only the tumor and tumor-infiltrating immune cell group positivity was associated with longer metastasis-free survival (P = 0.01). Furthermore, tumor cells and the tumor and tumor-infiltrating immune cell group PD-L1 expression was associated with decreased tumor-infiltrating lymphocytes (P = 0.02). PD-L1, when expressed in uveal melanoma, is associated with better patient outcome and decreased tumor-infiltrating lymphocytes. These results support the consideration of anti-PD-1/PD-L1 therapy in uveal melanoma. To determine the best cutoff value, further studies from patients enrolled in clinical trials treated with PD-1/PD-L1 inhibitors are necessary.

Loss of circadian clock gene expression is associated with tumor progression in breast cancer

Several studies suggest a link between circadian rhythm disturbances and tumorigenesis. However, the association between circadian clock genes and prognosis in breast cancer has not been systematically studied. Therefore, we examined the expression of 17 clock components in tumors from 766 node-negative breast cancer patients that were untreated in both neoadjuvant and adjuvant settings. In addition, their association with metastasis-free survival (MFS) and correlation to clinicopathological parameters were investigated. Aiming to estimate functionality of the clockwork, we studied clock gene expression relationships by correlation analysis. Higher expression of several clock genes (e.g., CLOCK, PER1, PER2, PER3, CRY2, NPAS2 and RORC) was found to be associated with longer MFS in univariate Cox regression analyses (HR<1 and FDR-adjusted P < 0.05). Stratification according to molecular subtype revealed prognostic relevance for PER1, PER3, CRY2 and NFIL3 in the ER+/HER2- subgroup, CLOCK and NPAS2 in the ER-/HER2- subtype, and ARNTL2 in HER2+ breast cancer. In the multivariate Cox model, only PER3 (HR = 0.66; P = 0.016) and RORC (HR = 0.42; P = 0.003) were found to be associated with survival outcome independent of established clinicopathological parameters. Pairwise correlations between functionally-related clock genes (e.g., PER2-PER3 and CRY2-PER3) were stronger in ER+, HER2- and low-grade carcinomas; whereas, weaker correlation coefficients were observed in ER- and HER2+ tumors, high-grade tumors and tumors that progressed to metastatic disease. In conclusion, loss of clock genes is associated with worse prognosis in breast cancer. Coordinated co-expression of clock genes, indicative of a functional circadian clock, is maintained in ER+, HER2-, low grade and non-metastasizing tumors but is compromised in more aggressive carcinomas.

Abstract 1190: Gene expression profiling identified FYN as an important gene in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy .

Abstract To elucidate the molecular mechanisms of tamoxifen resistance in breast cancer we performed microarray profiling and identified 366 genes with altered expression in 4 tamoxifen-resistant (TamRs) vs. the parental tamoxifen-sensitive MCF-7/S0.5 cell lines. Most of these genes were functionally linked to cell growth, proliferation, death and control of gene expression and included FYN, PRKCα, ITPR1, DPYD, DACH1, GBP1 and PRLR. Increased expression of FYN was observed in TamR cells and treatment with FYN-specific interfering RNA or a specific Src family kinase (SFK) inhibitor restored the anti-proliferative effect of tamoxifen in these cells. TamR cells also expressed increased levels of phosphorylated breast cancer antiestrogen resistance protein 1 (pBCAR1), an important downstream SFK substrate in the MAPK pathway. Evaluation of the association of subcellular localization of FYN in cancer tissue and clinical endpoints was made in 2 cohorts of ER+ breast cancer patients, one with advanced disease (N=47) and the other with early breast cancer (N=76). The result showed that plasma membrane-associated FYN expression in the cancer cells strongly correlated with longer progression-free survival compared to FYN expression not associated with the plasma membrane (p&amp;lt;0.0002) in the advanced disease cohort. Similarly, in the early breast cancer patients, plasma membrane associated FYN expression was associated with longer metastasis-free survival (p&amp;lt;0.003) and overall survival (p&amp;lt;0.0005) independent of tumor size, grade and node status. Our results indicate that FYN plays an important role in tamoxifen resistance, and its subcellular location in breast tumor cells may be an important novel biomarker of response to endocrine therapy in breast cancer. Citation Format: Daniel Elias Roro, Henriette Vever, Henrik Ditzel. Gene expression profiling identified FYN as an important gene in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1190. doi:10.1158/1538-7445.AM2013-1190

Abstract P5-04-02: Tumor Associated High Endothelial Venules (HEV) Predict Lymphocyte Infiltration and Breast Cancer Patients Clinical Outcome

Abstract Even if lymphocyte presence within tumor tissues and its impact on cancer patient prognosis have been well documented past few years, the type of tumor vessels governing infiltration of effector cells into tumors remain to be determined. High endothelial venules (HEV) are specialized vessels for lymphocyte recruitment physiologically present in secondary lymphoidorgans also found in chronic inflammation that may impact lymphocyte recruitment and cancer patient's clinical outcome. We recently identified HEV like vessels in the peritumoral stroma of human solid tumors including melanomas, breast, colon, ovarian, and lung carcinomas. In order to better analyse the functional consequence of HEV presence within human solid tumors, we quantified HEV and tumor infiltrating lymphocytes by immunohistochemistry on serial tumor sections of 146 breast cancer patients. We demonstrate that HEV like vessels density within tumor stroma is an important predictor of CD3+, CD8+ T and B lymphocyte tumor infiltration suggesting an active role played by these vessels in peripheral blood lymphocyte migration into tumors. The breast tumor infiltrating lymphocyte phenotype associated with HEV presence was further characterized by large scale flow cytometry analysis in 30 freshly operated breast tumors. Breast tumors with a high density of HEV (HEVhi) have increased numbers of effector memory T cells (CD45RA-CD62L-) that display an activated mature phenotype (CD69+, Granzyme A+, Granzyme B+, perforin+). T cells with a naïve (CD45RA+CD62L+) and a central memory (CD45RA-CD62L+) phenotype are also specifically increased within HEVhi tumors as compared to HEVlo tumors. Finally, we analyzed the prognostic value of HEV in a retrospective cohort of 146 invasive breast cancer patients and demonstrate that HEV density within tumor stroma is an independent predictor of good clinical outcome. Indeed, HEVhi patients have a significantly longer metastasis free survival (MFS) (p&amp;lt; 0,004), disease free survival (DFS) (P&amp;lt;0,012) and overall survival (OS) (P&amp;lt;0,023) than HEVlo patients. HEV like vessels, through the recruitment of high number of T lymphocytes and B lymphocytes from the periphery could limit the dissemination of primary breast tumors and prevent metastatic recurrence. Therefore, signals and cells that stimulate HEV differentiation within tumor stroma represent important solid tumors therapeutic target to modulate immune infiltration and patient's clinical outcome. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-04-02.

Characterization of triple-negative breast cancer utilizing microarray-based gene-expression profiling

e22014 Background: Triple-negative breast cancer (TNBC) is characterized by lack of expression of estrogen receptor (ER), progesterone receptor (PR) and HER-2. This makes it one of the most challenging subgroups for clinical decision-making. Methods: We analyzed gene-expression profiles of 200 node-negative breast cancer patients utilizing the Affymetrix HG-U133A array. After performing an unsupervised hierarchical cluster analysis using 2579 genes selected for variable expression within our dataset, we constructed metagenes for five dominant cluster (basal-like, luminal, proliferation, T-cell, B-cell). The interrelation of the median expression of these metagenes between TNBC and those cancer specimens expressing at least one of those receptors was analysed with the Wilcoxon-Mann-Whitney test. An intrinsic gene list was used to define basal-like breast cancer (BLBC). Sensitivity and specificity of TNBC to correctly classify BLBC was calculated. Metastasis-free survival (MFS) at 5 years was calculated according to Kaplan Meier. Results: Of the 200 node-negative breast cancer patients, 33 (16.5%) were triple-negative and 20 (10%) basal-like. Sensitivity of TNBC to correctly predict BLBC was 100%, specificity was 93.1%. TNBC showed an overexpression of the basal-like metagene (P=7.905e-13), an inverse relation with the luminal metagene (p=1.151e-12) and had higher proliferation and higher expression of T-cell (P=6.316e-5) and B-cell (P=3.551e-6) metagene. A higher expression of the B-cell metagene was associated with longer MFS in both TNBC (P=0.048) and BLBC (P=0.041). Conclusions: Characterization of TNBC reveals marked differences in gene-expression. Overexpression of mRNA transcripts related to the humoral immune system might serve as a protective factor in this particular subgroup. No significant financial relationships to disclose.