We previously identified in a single-center study a 76-gene prognostic signature for lymph node-negative (LNN) breast cancer patients. The aim of this study was to validate this gene signature in an ...independent more diverse population of LNN patients from multiple institutions.
Using custom-designed DNA chips we analyzed the expression of the 76 genes in RNA of frozen tumor samples from 180 LNN patients who did not receive adjuvant systemic treatment.
In this independent validation, the 76-gene signature was highly informative in identifying patients with distant metastasis within 5 years (hazard ratio, HR, 7.41; 95% CI, 2.63 to 20.9), even when corrected for traditional prognostic factors in multivariate analysis (HR, 11.36; 95% CI, 2.67 to 48.4). The actuarial 5- and 10-year distant metastasis-free survival were 96% (95% CI, 89% to 99%) and 94% (95% CI, 83% to 98%), respectively, for the good profile group and 74% (95% CI, 64% to 81%) and 65% (53% to 74%), respectively for the poor profile group. The sensitivity for 5-yr distant metastasis-free survival was 90%, and the specificity was 50%. The positive and negative predictive values were 38% (95% CI, 29% to 47%) and 94% (95% CI, 86% to 97%), respectively. The 76-gene signature was confirmed as a strong prognostic factor in subgroups of estrogen receptor-positive patients, pre- and postmenopausal patients, and patients with tumor sizes 20 mm or smaller. The subgroup of patients with estrogen receptor-negative tumors was considered too small to perform a separate analysis.
Our data provide a strong methodologic and clinical multicenter validation of the predefined prognostic 76-gene signature in LNN breast cancer patients.
The androgen receptor (AR) has potential clinical relevance in metastatic breast cancer (mBC) since it might be a treatment target and has been associated with endocrine resistance. A ...minimal‐invasive way to determine AR expression on metastatic tumor cells is by characterization of circulating tumor cells (CTCs). Here, we assessed AR mRNA expression in CTCs (CTC‐AR) and in matched primary tumor samples from mBC patients representing different breast cancer subtypes. In addition, we explored CTC‐AR‐status in relation to outcome on endocrine therapy. AR, and 92 AR or estrogen receptor (ER) related genes, were measured in CellSearch‐enriched CTCs from 124 mBC patients and in 52 matched FFPE primary tissues using quantitative reverse‐transcriptase PCR. AR in CTCs was considered positive if the expression was 1 standard deviation higher than the expression measured in 11 healthy blood donors. A total of 31% of the mBC patients had AR‐positive (AR+) CTCs. 58% of the matched CTC and primary tumor samples were discordant with respect to AR status, observing both switches from AR+ to AR‐negative (AR‐) and vice versa. There was no statistically significant difference in progression‐free survival for patients treated with ER‐targeting drugs and CTC‐AR‐status (13 AR+/ 37 AR‐ cases, p = 0.28). Thus, AR can be determined in RNA isolated from CTCs, with in our set 31% AR‐positive samples. Given the discordance between AR status in CTC samples and corresponding primary tumors, determination of AR expression in CTCs might be a promising tool to select mBC patients for AR inhibiting agents.
What's new?
Some patients with metastatic breast cancer respond well to drugs that target the androgen receptor (AR). Biomarkers are needed to identify these patients, but testing for AR on the primary tumor is not predictive enough. Here, the authors analyzed whether circulating tumor cells could provide better information about AR expression. Primary tumors and circulating tumor cells differed in AR expression in 58% of cases, they found, and CTCs are known to better represent the metastases. One of the next steps will also be to test a larger cohort to see whether patients with AR expressing CTCs respond less well to ER‐targeting therapy.
Published prognostic gene signatures in breast cancer have few genes in common. Here we provide a rationale for this observation by studying the prognostic power and the underlying biological ...pathways of different gene signatures.
Gene signatures to predict the development of metastases in estrogen receptor-positive and estrogen receptor-negative tumors were identified using 500 re-sampled training sets and mapping to Gene Ontology Biological Process to identify over-represented pathways. The Global Test program confirmed that gene expression profilings in the common pathways were associated with the metastasis of the patients.
The apoptotic pathway and cell division, or cell growth regulation and G-protein coupled receptor signal transduction, were most significantly associated with the metastatic capability of estrogen receptor-positive or estrogen-negative tumors, respectively. A gene signature derived of the common pathways predicted metastasis in an independent cohort. Mapping of the pathways represented by different published prognostic signatures showed that they share 53% of the identified pathways.
We show that divergent gene sets classifying patients for the same clinical endpoint represent similar biological processes and that pathway-derived signatures can be used to predict prognosis. Furthermore, our study reveals that the underlying biology related to aggressiveness of estrogen receptor subgroups of breast cancer is quite different.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Tumors of germline
mutated carriers show homologous recombination (HR) deficiency (HRD), resulting in impaired DNA double-strand break (DSB) repair and high sensitivity to PARP inhibitors. Although ...this therapy is expected to be effective beyond germline
mutated carriers, a robust validated test to detect HRD tumors is lacking. In this study, we therefore evaluated a functional HR assay exploiting the formation of RAD51 foci in proliferating cells after
irradiation of fresh breast cancer tissue: the recombination REpair CAPacity (RECAP) test.
Fresh samples of 170 primary breast cancer were analyzed using the RECAP test. The molecular explanation for the HRD phenotype was investigated by exploring
deficiencies, mutational signatures, tumor-infiltrating lymphocytes (TIL), and microsatellite instability (MSI).
RECAP was completed successfully in 148 of 170 samples (87%). Twenty-four tumors showed HRD (16%), whereas six tumors were HR intermediate (HRi; 4%). HRD was explained by
deficiencies (mutations, promoter hypermethylation, deletions) in 16 cases, whereas seven HRD tumors were non-BRCA related. HRD tumors showed an increased incidence of high TIL counts (
= 0.023) compared with HR proficient (HRP) tumors and MSI was more frequently observed in the HRD group (2/20, 10%) than expected in breast cancer (1%;
= 0.017).
RECAP is a robust functional HR assay detecting both
-deficient and
-proficient HRD tumors. Functional assessment of HR in a pseudo-diagnostic setting is achievable and produces robust and interpretable results.
Purpose: We previously discovered an extracellular matrix (ECM) gene cluster associated with resistance to first-line tamoxifen therapy
of patients with metastatic breast cancer. In this study, we ...determined whether the six individual ECM genes collagen 1A1
( COL1A1 ), fibronectin 1 ( FN1 ), lysyl oxidase ( LOX ), secreted protein acidic cysteine-rich ( SPARC ), tissue inhibitor of metalloproteinase 3 ( TIMP3 ), and tenascin C ( TNC ) were associated with treatment response, prognosis, or both.
Experimental Design: In 1,286 primary breast tumors, mRNA expression (quantitative real-time PCR) was related to clinicopathologic factors and
disease outcome in univariate and multivariate analysis including traditional factors.
Results: TIMP3, FN1, LOX , and SPARC expression levels (continuous variables) were significantly associated with distant metastasis-free survival (MFS) in 680
lymph node–negative untreated patients ( P < 0.03). Using a calculated linear prognostic score, these patients were evenly divided into five prognostic groups with
a significant difference in 10-year MFS of ∼40% between the two extreme prognostic groups. Furthermore, high TNC expression as continuous variable was associated with ( a ) shorter MFS in 139 estrogen receptor–positive and lymph node–positive patients who received adjuvant tamoxifen therapy (hazard
ratio, 1.53; P = 0.001), and ( b ) no clinical benefit (odds ratio, 0.81; P = 0.035) and shorter progression-free survival (hazard ratio, 1.19; P = 0.002) in 240 patients in whom recurrence was treated with tamoxifen as first-line monotherapy. These results were also
significant in multivariate analyses.
Conclusion: FN1, LOX, SPARC , and TIMP3 expression levels are associated with the prognosis of patients with breast cancers, whereas TNC is associated with resistance
to tamoxifen therapy. Further validation and functional studies are necessary to determine the use of these ECM genes in decisions
regarding treatment and whether they can serve as targets for therapy.
Increased APOBEC3B mRNA levels are associated with a hypermutator phenotype and poor prognosis in ER-positive breast cancer patients. In addition, a 29.5 kb deletion polymorphism of APOBEC3B, ...resulting in an APOBEC3A-B hybrid transcript, has been associated with an increased breast cancer risk and the hypermutator phenotype. Here we evaluated whether the APOBEC3B deletion polymorphism also associates with clinical outcome of breast cancer. Copy number analysis was performed by quantitative PCR (qPCR) in primary tumors of 1,756 Dutch breast cancer patients. The APOBEC3B deletion was found in 187 patients of whom 16 carried a two-copy deletion and 171 carried a one-copy deletion. The prognostic value of the APOBEC3B deletion for the natural course of the disease was evaluated among 1,076 lymph-node negative (LNN) patients who did not receive adjuvant systemic treatment. No association was found between APOBEC3B copy number values and the length of metastasis-free survival (MFS; hazard ratio (HR) = 1.00, 95% confidence interval (CI) = 0.90-1.11, P = 0.96). Subgroup analysis by ER status also did not reveal an association between APOBEC3B copy number values and the length of MFS. The predictive value of the APOBEC3B deletion was assessed among 329 ER-positive breast cancer patients who received tamoxifen as the first-line therapy for recurrent disease and 226 breast cancer patients who received first-line chemotherapy for recurrent disease. No association between APOBEC3B copy number values and the overall response rate (ORR) to either tamoxifen (odds ratio (OR) = 0.88, 95% CI = 0.69-1.13, P = 0.31) or chemotherapy (OR = 0.97, 95% CI = 0.71-1.33, P = 0.87) was found. Thus, in contrast to APOBEC3B mRNA levels, the APOBEC3B deletion polymorphism has neither a prognostic nor a predictive value for breast cancer patients. Although a correlation exists between APOBEC3B copy number and mRNA expression, it is relatively weak. This suggests that other mechanisms exist that may affect and therefore determine the prognostic value of APOBEC3B mRNA levels.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Breast cancer (BC) consists of multiple subtypes defined by various molecular characteristics, for instance, estrogen receptor (ER) expression. Methods for visualizing BC include mammography, MR ...imaging, ultrasound, and nuclear medicine-based methods such as (99m)Tc-sestamibi and (18)F-FDG PET, unfortunately all lacking specificity. Peptide receptor scintigraphy and peptide receptor radionuclide therapy are successfully applied for imaging and therapy of somatostatin receptor-expressing neuroendocrine tumors using somatostatin receptor radioligands. On the basis of a similar rationale, radioligands targeting the gastrin-releasing peptide receptor (GRP-R) might offer a specific method for imaging and therapy of BC. The aim of this study was to explore the application of GRP-R radioligands for imaging and therapy of BC by introducing valid preclinical in vitro and in vivo models.
GRP-R expression of 50 clinical BC specimens and the correlation with ER expression was studied by in vitro autoradiography with the GRP-R agonist (111)In-AMBA. GRP-R expression was also analyzed in 9 BC cell lines applying (111)In-AMBA internalization assays and quantitative reverse transcriptase polymerase chain reaction. In vitro cytotoxicity of (177)Lu-AMBA was determined on the GRP-R-expressing BC cell line T47D. SPECT/CT imaging and biodistribution were studied in mice with subcutaneous and orthotopic ER-positive T47D and MCF7 xenografts after injection of the GRP-R antagonist (111)In-JMV4168.
Most of the human BC specimens (96%) and BC cell lines (6/9) were found to express GRP-R. GRP-R tumor expression was positively (P = 0.026, χ(2)(4) = 12,911) correlated with ER expression in the human BC specimens. Treatment of T47D cells with 10(-7) M/50 MBq of (177)Lu-AMBA resulted in 80% reduction of cells in vitro. Furthermore, subcutaneous and orthotopic tumors from both BC cell lines were successfully visualized in vivo by SPECT/CT using (111)In-JMV4168; T47D tumors exhibited a higher uptake than MCF7 xenografts.
Targeting GRP-R-expressing BC tumors using GRP-R radioligands is promising for nuclear imaging and therapy, especially in ER-positive BC patients.
In a previous study, we detected a significant association between phosphoserine aminotransferase 1 (PSAT1) hyper-methylation and mRNA levels to outcome to tamoxifen treatment in recurrent disease. ...We here aimed to study the association of PSAT1 protein levels to outcome upon tamoxifen treatment and to obtain more insight in its role in tamoxifen resistance. A cohort of ER positive, hormonal therapy naïve primary breast carcinomas was immunohistochemically (IHC) stained for PSAT1. Staining was analyzed for association with patient's time to progression (TTP) and overall response on first-line tamoxifen for recurrent disease. PSAT1 mRNA levels were also assessed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR; n = 161) and Affymetrix GeneChip (n = 155). Association of PSAT1 to biological pathways on tamoxifen outcome were assessed by global test. PSAT1 protein and mRNA levels were significantly associated to poor outcome to tamoxifen treatment. When comparing PSAT1 protein and mRNA levels, IHC and RT-qPCR data showed a significant association. Global test results showed that cytokine and JAK-STAT signaling were associated to PSAT1 expression. We hereby report that PSAT1 protein and mRNA levels measured in ER positive primary tumors are associated with poor clinical outcome to tamoxifen.
The androgen receptor splice variant (AR‐V) 7 in circulating tumor cells (CTCs) is a predictor for resistance to anti‐AR‐targeted treatment, but not to taxane‐based chemotherapy in metastatic ...castration‐resistant prostate cancer (mCRPC). In this study, we investigated whether the presence of two constitutively active variants (AR‐V3, AR‐V7) and two other conditionally activated variants (AR‐V1, AR‐V9) vs full‐length androgen receptor (AR‐FL) measured in CTCs from patients with mCRPC were associated with outcome to therapy with the taxane cabazitaxel. Blood was collected at baseline and after two cycles of cabazitaxel from 118 mCRPC patients starting cabazitaxel in a prospective phase II trial. CellSearch‐enriched CTCs were enumerated and in parallel characterized for the presence of the AR‐Vs by reverse transcription quantitative polymerase chain reaction. Correlations with CTC and prostate‐specific antigen response to cabazitaxel as well as associations with overall survival (OS) were investigated. All AR‐Vs were frequently present and co‐expressed at frequencies of 31–48% at baseline and at 19–40% after two cycles of cabazitaxel. No specific directions of change in the measured variants were detected between the start of treatment and after two cycles of cabazitaxel. No associations between the presence of AR‐V3 and AR‐V7 and outcome to cabazitaxel were observed. While a reduction in CTCs to < 5 CTCs during treatment (CTC5‐response) was less often observed in patients with AR‐V9‐positive CTCs at baseline (P = 0.004), the CTC5‐adjusted detection of AR‐V1 after two cycles of cabazitaxel was an independent prognostic factor for OS HR 2.4 (95% CI 1.1–5.1, P = 0.03). These novel findings are expected to contribute to more personalized treatment approaches in mCRPC patients.
We investigated whether the presence of AR splice variants measured in CTCs from mCRPC patients was associated with outcome to cabazitaxel therapy. Whereas the presence of AR‐FL and the constitutively active AR‐V3 and AR‐V7 were not associated with outcome to this therapy, the presence of the conditionally activated AR‐V9 and AR‐V1 associated with a negative CTC response rate during treatment and decreased overall survival after two cycles of cabazitaxel, respectively.
Intrapatient tumour heterogeneity is likely a major determinant of clinical outcome in cancer patients. To assess heterogeneity in a minimally invasive manner, methods to perform single circulating ...tumour cell (CTC) genomics at high resolution are necessary. However, due to the rarity of CTCs, development of such methods is challenging. Here, we developed a modular single CTC analysis pipeline to assess intrapatient heterogeneity by copy number (CN) profiling. To optimize this pipeline, spike‐in experiments using MCF‐7 breast cancer cells were performed. The VyCAP puncher system was used to isolate single cells. The quality of whole genome amplification (WGA) products generated by REPLI‐g and Ampli1™ methods, as well as the results from the Illumina Truseq and the Ampli1™ LowPass library preparation techniques, was compared. Moreover, a bioinformatic pipeline was designed to generate CN profiles from single CTCs. The optimal combination of Ampli1™ WGA and Illumina Truseq library preparation was successfully validated on patient‐derived CTCs. In conclusion, we developed a novel modular pipeline to isolate single CTCs and subsequently generate detailed patient‐derived CN profiles that allow assessment of intrapatient heterogeneity in future studies.
Here, we present a technical study on the development of a single circulating tumour cell (CTC) analysis pipeline to assess intrapatient heterogeneity by copy number profiling. We compared two whole genome amplification methods, two library preparation methods and three quality control assays for single‐cell analysis after CellSearch enrichment and VyCAP punching. The established modular pipeline was applied to blood‐derived single cells from patients with metastatic breast or prostate cancer.
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