Uncovering the molecular and cellular mechanisms of metastatic dormancy in luminal breast cancer

Resumen

Uncovering the molecular and cellular mechanisms of metastatic dormancy in luminal breast cancer INTRODUCTION Breast cancer (BC) is the most common cancer in women and it represents the most common cause of cancer-related death in women worldwide . Advances in early detection, prevention, risk stratification, and therapeutic strategies as well as supportive care for patients have resulted in important improvements in mortality and reduction of cancer relapse. However, around 20% of the patients will present metastatic disease. Advanced breast cancer is still an incurable disease with a median overall survival (OS) of ∼3 years, while the 5-year survival of only ∼25% . Thus, the deeper understanding of cancer biology, inter- and intra-tumor heterogeneity, and as well as, the biology behind the progression of tumor cells toward metastasis is urgent. To date, evidence suggests that both intrinsic properties of breast cancer cells and host organ microenvironment participate actively to this matter . In general, detectable distant breast cancer metastases occur years, or even decades, after primary tumor diagnosis. Metastatic lesions are supposed to originate from disseminated tumor cells that underwent a period of dormancy . Nevertheless, the molecular factors that promote the formation of detectable metastasis from disseminated tumor cells are largely unknown. Nowadays, the genomic revolution has transformed the landscape of cancer, leading possible an improvement in understanding of tumor biology. To try to explore BC capability in producing metastases, a molecular analysis consisting on transcriptome study was performed among a retrospective cohort of metastatic breast cancer patients. Paired tissue samples from primary and metastatic tumor were available to be analyzed. We focused in PAM50 intrinsic subtype, genes that have been found to play an important role in breast cancer. And finally, we explored several genes signatures related with immune system and chromosomal instability. HYPOTHESIS Most biological changes occur during metastatic progression of breast cancer. Thus, we hypothesized that there is a different biology behavior between primary and metastatic breast cancer. For this reason, we proposed a transcriptomic approach in paired primary and metastatic tissues from a cohort of metastatic breast cancer patients. OBJECTIVES Aim 1. To characterize metastatic disease and analyze the PAM50 subtypes between PT and MT Aim 2. To analyzed the gene expression changes between PT and MT Aim 3. To explore the role of chromosomal instability in the metastasis process Aim 4. To explore the implication and changes of immune-system gene expression between both setting RESULTS A total of 123 patients were included in this analysis. The median age at breast cancer diagnosis was 52.5 years (range, 28–90). In primary disease, the distribution of classical subtypes defined by immunohistochemical was: 73.17% (n= 90) Luminal, 15.45% (n= 19) HER2-positive, and 9.76% (n= 12) triple-negative BC. No significant differences were observed in metastatic disease. Fourteen patients (11.38%) presented with de novo metastatic disease. Median follow-up and OSmet were 76.5 and 84 months, respectively. However, the change of molecular intrinsic subtype observed between primary tumor (PT) versus metastatic tumor (MT) was statically significant in luminal A (39% versus 26%) and HER2-enriched [HER2-E] (11.4% versus 22%) tumors. In the first case, patients who presented a luminal A tumor in PT, only in a 44.7% maintained a luminal A profile in the metastasis, switching to luminal B (40.4%) or HER2-E (14.9%) of the cases. Interestingly, we observed that the 8 patients, whose tumors changed from luminal A/B in PT to HER2-E in metastatic disease showed an increase in FGFR4 expression but not ERBB2 expression. Metastatic tumors were enriched in Luminal B, HER2-E, and proliferation signatures, in contrast, Luminal A and normal-like signatures were found significantly less expressed in metastatic than in primary tumors. When we analyzed cancer-related genes, we found that metastases were enriched for genes involved in survival and migration, cell cycle, and DNA repair. Furthermore, genes involved in response to hormone stimulus, differentiation, and chromatin regulation were downregulated. We also evaluated the ability in primary versus metastatic tumors of different signatures and each single gene to predict overall survival in the metastatic disease (OSmet). Interestingly, no signatures and genes consistently predicted OSmet in both primary and metastatic disease. In our cohort, in primary tumor, basal-like signature was found to be associated with worse outcome, (HR = 1.50, p= 0.007), meanwhile the VEGF/Hypoxia signature was associated with a better prognosis (HR = 0.65, p= 0.016). In metastatic disease, proliferation was found to be associated with worse outcome (HR = 1.40, P = 0.047). Among 105 genes, only one gene (GATA3) consistently predicted favorable outcome in both settings. Finally, to evaluate if the gene expression changes observed in metastatic tissues is a reflection of tumor evolution over time, we plotted the magnitude of change of the expression of each signature versus time to relapse (TTR). The results revealed a positive correlation between TTR and HER2-E (corr = 0.324, p < 0.001), luminal B (corr = 0.27, p= 0.004), Proliferation score (corr = 0.291, P = 0.002), and ROR-P (corr = 0.295, p= 0.001). In contrast, normal-like and luminal A signatures showed a negative correlation with TTR (corr = −0.285, p= 0.002; corr = −0.219, p= 0.019, respectively). Gene-by-gene analysis showed a positive correlation between TTR and the magnitude of change of genes implicated in cell proliferation, mitogenesis, and differentiation biological process. In contrast, a negative correlation was observed with genes that participate in cell-to-cell adhesion, regulation of DNA damage repair, tumor suppression, mammary gland development, and that attenuate cell migration. To better characterize differences between primary tumors and metastases, further molecular analyses were planned. As chromosomal instability (CIN) is one of the most consistent characteristics of human tumors, we decided to analyze it in our cohort of patients. To evaluate the role of CIN in the process of metastasis, we used a validated gene expression signature of chromosomal instability (CIN70) (Carter SL, et al. Nature Genetics. 2006). We found a global significantly enrichment in the metastatic setting (p= 2.11 e-3). Moreover, this increased in expression of CIN70 signature was especially present among Luminal A subtype (p= 6.74 e-4). In fact, when we focus the analysis in Luminal A patients from primary tumor, we observed that the increase in expression was higher in patients whose tumors switched the subtype in the metastasis form Luminal A to Luminal B or HER2-E (p= 2.18 e-8). Chromosomal instability was related with tumor phenotype. The most aggressive subtypes as Basal-like and HER2E showed the highest expression of CIN followed by Luminal B, while Luminal A presented less chromosomal instability expression in both primary and metastatic disease (p= 1.57 e-12 and p= 3.63 e-13, respectively). Furthermore, CIN signature presented a positive correlation with RORP (corr = 0.82, p= 6.7 e-51) and Proliferation signature (corr = 0.812, p= 3.41 e-49). And, a negative correlation was observed with CESP signature (corr = -0.36, p= 1.79 e-24). According with these data, the highest expression of CIN signature was associated with shorter disease-free survival (p= 8.79 e-4). Lastly, we explore how the tumor immune microenvironment influences tumor progression. We performed an analysis of 6 immune-related genes (CD4, CD8A, CD45, CTLA4, PDCD1 and CD274) and specific immune signatures. We found that Basal-like group showed the highest expression followed by HER2-E, while Luminal subtypes presented less immune-genes related expression in both early and advanced disease. We investigated both innate immune cell (dendritic cells, mast cells, macrophages, NK cells, and neutrophils) and adaptive immune cells (B, T helper 1, T helper 2, T ). In our cohort, innate immune signature was significantly higher expressed in metastatic tissue vs primary samples (p= 6.1e-1). Interestingly, we found upregulated the subpopulations cells of dendritic cells (p= 9.68e-6), Th2 (p= 0.006), and Th1 (p=0.024). When we evaluated the signatures across PAM50 subtypes, we found a significant upregulation of innate immune system in Luminal A (p= 1.19e-4) and HER2-E (0.002) in metastatic setting. We also tested the ability of immune signatures to predict prognosis in primary versus metastatic setting. Of note, no single gene consistently predicted prognosis neither in primary nor metastatic disease. Furthermore, no single gene predicted disease-free survival (DFS) when measured on primary tumor. But, in metastatic disease, CD274, CD8A and CD4 were found to be associated with better outcomes in terms of overall survival (OS) (HR= 0.51, p= 0.003; HR= 0.63, p= 0.026; HR= 0.72, p= 0.039; respectively). According with these data, all innate immune signatures were associated with good prognosis in terms of OS in the metastatic group (HR<1, p< 0.05). CONCLUSIONS Most biological changes occurring during metastatic progression of breast cancer are largely unknown nowadays. Here, we compared a transcriptomic analysis in paired primary and metastatic tissues from a cohort of metastatic breast cancer patients. Our results showed different biology behavior between both primary and metastatic breast cancer. Although intrinsic subtype was largely maintained during metastatic progression, luminal/HER2-negative tumors acquire a luminal B or HER2-E profile during metastatic progression, likely reflecting tumor evolution and/or acquisition of estrogen-independency. Moreover, chromosomal instability could contribute to transformation and changes in cancer phenotypes during metastasis process. Moreover, we detected differences in the immune microenvironment between primary and metastatic lesions and across different molecular subtypes. Our results suggest that immune system has different implications in primary and metastatic tumors with different prognostic value. This knowledge could lead to different strategies for the immunotherapy treatment of breast cancer subtypes. Finally, our study highlights the importance of molecular characterization of metastatic disease. mRNA expression profiles provide a new tool to explore the distinct nature of breast cancer subtypes. DISCUSSION Here, we explored RNA-based expression differences between paired primary and metastatic breast tumors, leading to the following observations: (I) intrinsic molecular subtype is largely maintained during metastatic recurrence, except for luminal A disease, in which the conversion to luminal B and HER2-enriched is present in about 55% of the cases; (II) metastatic tissues show higher expression of proliferative and lower expression of luminal-related genes compared to primary tumors, except for basal-like disease, which seems to be very stable from a RNA-based perspective; (III) different biological processes can predict overall survival from recurrence when evaluated in primary versus metastatic disease; (IV) an intriguing relationship seems to exist between the time taken to develop detectable metastases and the aggressiveness of the tumor, indicating that a tumor might evolve towards a more aggressive phenotype as time evolves. Chromosomal instability provides cells plasticity, due to this plasticity the cells could acquire new properties to develop metastasis during dormancy status and induce the change in breast cancer subtypes. CIN could be related with phenotypic adaptation and produce tumor evolution. Patients diagnosed with Luminal A breast cancer which tumor switched to luminal B or HER2E in the metastasis presented an increased in CIN expression. Furthermore, CIN70 was related with more aggressive cancer subtypes and represented a poor prognosis signature in terms of DFS. When immune system was evaluated, the results in our cohort, suggest that metastatic tumors presented more immunoreactivity than primary tumors. Basal-like tumors followed by HER2-E were more immunogenic than luminal breast cancer subtypes. This proposes that luminal tumors would be immunologically silent. There was a robust association between immune cell infiltration of metastatic tumors and favorable patient outcomes. Except Th2 cells, T cell subset has been widely described as promoting metastasis and it was related to worse overall survival.