Background: We have previously demonstrated how transcriptional pathway activity and the molecular subtypes of breast cancer metastases significantly influence patient post-relapse survival. Here we extend our analysis to determine whether the prognostic information provided by gene expression signatures in primary breast tumours is also relevant in the metastatic setting. Specifically, we test the research versions of the Genomic Grade Index (GGI), Mammaprint, Recurrence score (RS) and PAM50 gene signatures along with our own cell-cycle based classifier (CCS). Methods: 287 patients with morphologically confirmed loco-regional or distant breast cancer relapse were enrolled in the Swedish multicenter TEX trial from December 2002 until June 2007. Of these, sufficient tumour RNA for gene expression profiling was obtained from metastatic tissue by fine needle aspiration from 111 patients (totalling 120 relapse biopsies). Gene signatures were applied as described in the original research articles and their relationship to short (1.5 year) and long-term (5 year) post-relapse survival was assessed using likelihood ratio, Kaplan-Meier and Cox regression analysis. Results: As anticipated from an aggressive metastatic cohort, the majority of samples (> 70%) were classified into intermediate or high risk groups by all signatures. In both short and long-term survival analysis only PAM50 provided statistically significant prognostic information (short: LRχ2 = 14.7, p = 0.005 and long: LRχ2 = 13.2, p = 0.010), with the cell cycle score signature displaying a prognostic trend in long-term survival only (LRχ2 = 5.2, p = 0.074). Kaplan-Meier curves and Cox regression analysis suggest that the strength of both signatures resides in their ability to select a group of low-risk patients with better long-term survival. Conclusions: Our findings demonstrate the prognostic utility of the multi-level PAM50 and to a lesser extent, cell cycle score signatures in predicting survival of patients with metastatic breast cancer. Simpler binary gene expression signatures (GGI and Mammaprint) do not appear to capture the same prognostic information and as such may have limited utility in a metastatic setting. Short and long term survival Likehood Ratios for five gene expression signatures in metastatic breast cancer Short term survival (1.5 year)Long term survival (5 year)Gene SignatureLRχ2P-valueLRχ2P-valueGGI1.30.2510.50.500Mammaprint1.70.1900.60.427RS3.90.1434.40.110CCS2.80.2425.20.074PAM5014.70.00513.20.010GGI: Genomic grade index; RS: Recurrence score; CCS: Cell cycle score Citation Format: Tobin NP, Lundberg A, Lindström LS, Harrell JC, Egyhazi Brage S, Frostvik Stolt M, Einbeigi Z, Loman N, Malmberg M, Perou CM, Bergh J, Hatschek T. Multi-level gene expression signatures provide significant prognostic information in metastatic breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-16.
Background Better tools are needed to identify breast cancer patients at very low risk of dying from their disease. We applied a previously specified 'Ultralow risk' threshold for the FDA-cleared MammaPrint 70-gene expression score to predict long-term absence of breast cancer-specific mortality in a Swedish randomized trial of breast cancer patients treated with tamoxifen (Tam) versus not and followed for more than 25 years. Methods Between 1976-1990 the Stockholm Tamoxifen (STO) trial enrolled and randomized node negative breast cancer patients with tumor size less than 30 mm to receive 2 years of Tam versus not, without regard to hormone receptor status. In the Tam-treated arm, patients without relapse at 2 years were further randomized to receive 3 additional years of Tam versus no additional endocrine therapy. From the original STO randomized trial cohort, about half (778 cases) had remaining formalin-fixed paraffin-embedded primary tumor blocks for additional tumor characterization and MammaPrint analysis. In this validation dataset, which now has >25 years follow-up, breast cancer-specific survival was assessed between MammaPrint scored 'Ultralow risk', 'Low risk' or 'High risk' categories by Kaplan-Meier analyses and multivariate Cox proportional hazard modeling, adjusting for treatment, age, period of diagnosis, tumor size, grade, receptor (ER, PR, HER2) and Ki-67 status. Results: In this unscreened patient population MammaPrint scored 15% of patients as 'Ultra-low risk', 43% as 'Low risk' and 42% as 'High risk'. At 20 years, a statistically significant difference in survival between risk categories was seen for all patients (log rank, P=0.0001), the Tam treated arm (log rank, P=0.0088) and the untreated arm (log rank, P=0.014). Ultra-low risk patients have significantly lower risk of disease-specific death relative to Low and High risk patients in our multivariate Cox analysis. Among Ultra-low risk patients there were no deaths out to 15 years in the Tam-treated arm; by 20 years, disease-specific survival in Tam-treated and untreated arms were 94% and 90%. The majority of Tam-treated patients received only 2 years of treatment, with ∼35% going on to receive 5 years of treatment. All Ultra-low risk cases were HR+HER2-. 78% were luminal A by PAM50; but only 31% of luminal A were Ultra-low risk. Invasive ductal (no-special-type) carcinomas were the most frequent, but lobular, tubular, invasive papillary and invasive cribriform subtypes were enriched and mucinous types were absent. Conclusions: Node-negative, T<3cm breast cancer cases designated as 'Ultralow risk' by the 70-gene MammaPrint assay have minimal risk of metastatic death within the first 15 years if given a short (2+ year) course of adjuvant Tam. While a small but very late (>15 y) metastatic recurrence risk exists if left untreated, 'Ultralow risk' status could identify women for whom lumpectomy alone and a short course of adjuvant endocrine therapy is sufficient. Given the high frequency (∼50%) of such 'Ultralow risk' tumors detected in modern screening cohorts (e.g. MINDACT), a substantial fraction of newly diagnosed women could benefit from more targeted and less aggressive local and adjuvant therapy. Citation Format: Esserman LJ, Thompson CK, Yau C, van 't Veer LJ, Borowsky AD, Tobin NP, Nordenskjöld B, Fornander T, Stål O, Benz CC, Lindström LS. Identification of tumors with an indolent disease course: MammaPrint ultralow signature validation in a retrospective analysis of a Swedish randomized tamoxifen trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-09-01.
Importance: The frequency of cancers with indolent behavior has increased with screening. We asked whether an ultralow risk threshold on a multigene classifier would identify women whose cancers had an indolent course over 2 decades of follow-up, and which features were most predictive of outcome. Methods: An ultralow risk threshold of the FDA-cleared MammaPrint 70-gene expression score was set to predict long-term absence of breast cancer-specific mortality in the absence of systemic therapy. The Stockholm Tamoxifen (STO) trial conducted between 1976 and 1990, where postmenopausal women with clinically detected node-negative breast cancers <3cm were randomized to receive tamoxifen versus not, was used for validation. Immunohistochemistry markers (n=727) and Agilent microarrays for MammaPrint risk scoring (n=652) were performed from formalin-fixed paraffin-embedded primary tumor blocks. Recursive partitioning was performed using the rpart package in R to select variables and construct a regression tree that best predicts 20-year breast cancer specific survival. Input variables include: age, period of diagnosis, grade, hormone receptor status, HER2 and Ki69 status, 70-gene risk categories (high, low but not ultra, or ultralow), treatment arm and tumor size; and cross-validation was used to select the final regression tree model. Results: In this trial conducted in the era before mammographic screening, 58% and 42% were MammaPrint low and high risk, respectively, while 15% were above the ultralow threshold. In the tamoxifen treated arm, women with tumors above the ultralow threshold had no deaths at 15 years and their 20-year disease-specific survival rates of 97%; whereas if untreated, their survival rates were 94%. Recursive partitioning identified the ultralow threshold classification as the first primary split in the model. Once the indolent tumors were partitioned out, among women with tumors below the ultralow threshold, the next most prognostic feature was size, where patients with tumors >20mm have worse breast cancer specific survival. The last split in the model divides the patients with tumors ≤20mm into 70-gene high risk vs low but not ultralow risk groups. Conclusions and Relevance: A threshold of the 70-gene MammaPrint assay can identify patients with indolent disease whose long-term risk of death from breast cancer after surgery alone is exceedingly low. This threshold emerged as the most prognostic variable, followed by tumor size, and mammaprint high vs. low but not ultralow in our recursive partitioning analysis. This suggests that finding indolent tumors early at a small size may not have much impact on patient outcome. Determining the presence of an ultralow risk breast cancer may prevent overtreatment. Conversely, once the indolent tumors are taken out of consideration, both biology and size impact outcome, and finding these tumors at a small size is likely still important and supports screening in this postmenopausal node negative population. Citation Format: Esserman LJ, Yau C, Thompson CK, van't Veer LJ, Borowsky AD, Hoadley KA, Tobin NP, Nordenskjöld B, Fornander T, Stål O, Benz CC, Lindström LS. Identification of breast cancers with an indolent disease course: 70 gene indolent threshold validation in a Swedish randomized trial of tamoxifen vs. not, with 20 year outcomes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD7-02.
Background: We have previously demonstrated that gene expression signatures and Ki67 stratify the same breast tumour into opposing good/poor prognosis groups in approximately 20% of cases. Given this, we hypothesized that the combination of a clinically relevant gene signature and IHC markers may provide more prognostic information than either classifier alone. We tested this hypothesis in a large independent cohort of Swedish breast cancer patients with long-term follow-up data. Methods: We assessed Ki67, ER, PR, HER2 and the research versions of the Genomic Grade Index (GGI), Mammaprint, cell-cycle score (CCS), Recurrence Score (RS) and PAM50 gene expression classifiers on matching TMA and microarray data in a Swedish breast cancer cohort of 623 patients. Change in likelihood-ratio (Δ LR-χ2) was used to first determine the additional prognostic information provided by gene expression signatures beyond that provided by 1) Ki67 alone and 2) Ki67 plus ER, PR and HER2, grouped to form the IHC molecular subtypes. Secondly and conversely, we then determined the additional prognostic information provided by Ki67/IHC subtypes beyond gene expression signatures. Results: Representative images from Ki67/gene signature contrast groups show tumours with high levels of Ki67 expression that are classified as good prognosis by gene signatures and conversely, tumours with low Ki67 that are classified into poor prognosis groups by gene signatures. In all patients (n=623), the majority of signatures provided statistically significant information beyond that of Ki67 alone, however only RS and PAM50 remained significant in the presence of the IHC subtypes (Δ LR-χ2 RS= 11.7 and PAM50 = 15.4; P = 0.002 and 0.004, respectively). Conversely, IHC subtypes added prognostic information beyond gene signatures whilst Ki67 alone did not, a notable exception to this was PAM50. Conclusions: In general, a combination of the IHC subtypes with gene signatures provides more prognostic information than either classifier alone when considering all breast cancer patients. Subsequent analyses will focus on patient subgroups including ER positive, node positive and ER positive, node negative groups, along with validation of our work in a second dataset of 253 patients. Change in likelhood ratio with the addition of gene expression signatures to Ki67/IHC subgroups and vice-versa All Patients All PatientsSig. added to Ki67:Sig. Δ LRχ2P-valueSig. added to IHC subtypesSig. Δ LRχ2P-valueGGI6.00.014GGI2.50.108Mammaprint6.30.011Mammaprint1.10.279RS20.8< 0.001RS11.70.002CCS1.70.409CCS2.00.360PAM5025.0< 0.001PAM5015.40.004 Ki67 added to sig.:Ki67 Δ LRχ2P-valueIHC added to sig.:IHC Δ LRχ2P-valueGGI1.60.205GGI14.90.001Mammaprint1.60.199Mammaprint15.30.001RS0.50.477RS12.60.005CCS4.10.041CCS16.10.001PAM502.30.13PAM506.10.107Sig.: Gene expression signature; GGI: Genomic grade index; RS: Recurrence score; CCS: Cell cycle score. Citation Format: Lundberg A, Lindström LS, Falato C, Carlson JW, Foukakis T, Czene K, Bergh J, Tobin NP. Gene expression signatures and immunohistochemical subtypes add prognostic value to each other [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-07.
Background: Proliferation, migration, and stem cell (SC) activity have all been implicated in the development and progression of breast cancer. Growing evidence suggests a relationship between these cellular characteristics, which are tightly regulated by complex signalling pathways. The oncogene cyclin D1 is an essential cell cycle protein that regulates G1 to S phase transition and is overexpressed in many breast cancers. Despite the proliferation activating properties of cyclin D1 it has been linked to a less malignant phenotype. Aim: The aim is to investigate the relationship between migration, proliferation and SC activity in breast cancer and to determine the molecular mechanisms that control these processes focusing on cyclin D1. Methods and Results: To establish a link between proliferation and migration two breast cancer cell lines (MDAMB231 and MDAMB468) were quiesced and proliferation assessed by Ki67 and migration by transwell migration assays. G0/G1 cells displayed an increased migratory capacity (2.9 ±0.15 fold) compared to cycling cells. Next we downregulated cyclin D1 in actively cycling cells using siRNA which resulted in significantly decreased proliferation and the novel observation of increased migration (1.7±0. 19 fold). Having established a link between proliferation and migration the next aim was to investigate relationships with SC activity. To achieve this, cells were sorted following Hoechst 33342 and 7AAD labelling into three populations G0/G1, S, and G2/M. SC-like activity was assessed using a non-adherent mammosphere culture system where single cells are seeded at a density of 500 cells per cm2 for cultured for 7 days. The data clearly demonstrated that G0/G1 cells had increased migratory capacity and also SC-like activity (1.5±0.2 fold) compared to actively cycling cells. Interestingly, down-regulation of cyclin D1 increased both migratory and SC-like activity with most significant effects observed in G0/G1 and S phase of the cell cycle indicating that cyclin D1 is a key protein in the regulation of these cellular processes. When downregulating the cyclin D1 associated kinases CDK4/6 no effect on migration or SC activity was observed suggesting that cyclin D1 affects these cellular processes independent of CDK activation. Finally, when analysing a large set of premenopausal breast cancers we observed an inverse, proliferation-independent link between cyclin D1 and cancer recurrence as well as tumour size (p=0.03), validating that the protein might abrogate infiltrative and malignant behaviour in vivo. Conclusion: These novel functions of cyclin D1 illustrates the interplay between tumor proliferation and migration and SC activity and may explain the lessened malignant behaviour in cyclin D1 high breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-04-01.
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