Purpose To evaluate the effect of adjuvant radiotherapy (RT) after breast conservation surgery in different breast cancer subtypes in a large, randomized clinical trial with long-term follow-up. ...Patients and Methods Tumor tissue was collected from 1,003 patients with node-negative, stage I and II breast cancer who were randomly assigned in the Swedish Breast Cancer Group 91 Radiotherapy trial between 1991 and 1997 to breast conservation surgery with or without RT. Systemic adjuvant treatment was sparsely used (8%). Subtyping was performed with immunohistochemistry and in situ hybridization on tissue microarrays for 958 tumors. Results RT reduced the cumulative incidence of ipsilateral breast tumor recurrence (IBTR) as a first event within 10 years for luminal A-like tumors (19% v 9%; P = .001), luminal B-like tumors (24% v 8%; P < .001), and triple-negative tumors (21% v 6%; P = .08), but not for human epidermal growth factor receptor 2-positive (luminal and nonluminal) tumors (15% v 19%; P = .6); however, evidence of an overall difference in RT effect between subtypes was weak ( P = .21). RT reduced the rate of death from breast cancer (BCD) for triple-negative tumors (hazard ratio, 0.35; P = .06), but not for other subtypes. Death from any cause was not improved by RT in any subtype. A hypothesized clinical low-risk group did not have a low risk of IBTR without RT, and RT reduced the rate of IBTR as a first event after 10 years (20% v 6%; P = .008), but had no effect on BCD or death from any cause. Conclusion Subtype was not predictive of response to RT, although, in our study, human epidermal growth factor receptor 2-positive tumors seemed to be most radioresistant, whereas triple-negative tumors had the largest effect on BCD. The effect of RT in the presumed low-risk luminal A-like tumors was excellent.
Enfortumab vedotin (EV) is an antibody-drug conjugate (ADC) targeting NECTIN4 (encoded by the
gene) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or ...resistance to EV are unknown. In this study, we sought to (i) examine heterogeneity of
gene expression across molecular subtypes of bladder cancer and (ii) determine whether NECTIN4 expression mediates EV sensitivity or resistance.
Molecular subtyping and
expression data from seven muscle-invasive bladder cancer clinical cohorts (
= 1,915 total specimens) were used to assess
expression across molecular subtypes. The outcome of the transcriptomic analysis was relative
expression in the consensus molecular subtypes of bladder cancer. Expression of
was validated in bladder cancer cell lines. NECTIN4 was stably overexpressed or knocked down in basal and luminal bladder cancer cell lines and EV drug sensitivity assays were performed, as measured by cell proliferation and clonogenic assays.
expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes.
expression is positively correlated with luminal markers
, and
across all cohorts.
expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of
leads to EV resistance.
Sensitivity to EV is mediated by expression of
, which is enriched in luminal subtypes of bladder cancer. These findings may have implications for biomarker development, patient selection, and the inclusion of molecular subtyping in ongoing and future EV clinical trials.
.
Proteogenomic approaches have enabled the generat̲ion of novel information levels when compared to single omics studies although burdened by extensive experimental efforts. Here, we improved a ...data-independent acquisition mass spectrometry proteogenomic workflow to reveal distinct molecular features related to mammographic appearances in breast cancer. Our results reveal splicing processes detectable at the protein level and highlight quantitation and pathway complementarity between RNA and protein data. Furthermore, we confirm previously detected enrichments of molecular pathways associated with estrogen receptor-dependent activity and provide novel evidence of epithelial-to-mesenchymal activity in mammography-detected spiculated tumors. Several transcript–protein pairs displayed radically different abundances depending on the overall clinical properties of the tumor. These results demonstrate that there are differentially regulated protein networks in clinically relevant tumor subgroups, which in turn alter both cancer biology and the abundance of biomarker candidates and drug targets.
Response to androgen receptor signaling inhibitors (ARSI) varies widely in metastatic castration resistant prostate cancer (mCRPC). To improve treatment guidance, biomarkers are needed. We use ...whole-genomics (WGS; n = 155) with matching whole-transcriptomics (WTS; n = 113) from biopsies of ARSI-treated mCRPC patients for unbiased discovery of biomarkers and development of machine learning-based prediction models. Tumor mutational burden (q < 0.001), structural variants (q < 0.05), tandem duplications (q < 0.05) and deletions (q < 0.05) are enriched in poor responders, coupled with distinct transcriptomic expression profiles. Validating various classification models predicting treatment duration with ARSI on our internal and external mCRPC cohort reveals two best-performing models, based on the combination of prior treatment information with either the four combined enriched genomic markers or with overall transcriptomic profiles. In conclusion, predictive models combining genomic, transcriptomic, and clinical data can predict response to ARSI in mCRPC patients and, with additional optimization and prospective validation, could improve treatment guidance.
Adjuvant radiotherapy is the standard of care after breast-conserving surgery for primary breast cancer, despite a majority of patients being over- or under-treated. In contrast to adjuvant endocrine ...therapy and chemotherapy, no diagnostic tests are in clinical use that can stratify patients for adjuvant radiotherapy. This study presents the development and validation of a targeted gene expression assay to predict the risk of ipsilateral breast tumor recurrence and response to adjuvant radiotherapy after breast-conserving surgery in primary breast cancer.
Fresh-frozen primary tumors from 336 patients radically (clear margins) operated on with breast-conserving surgery with or without radiotherapy were collected. Patients were split into a discovery cohort (N = 172) and a validation cohort (N = 164). Genes predicting ipsilateral breast tumor recurrence in an Illumina HT12 v4 whole transcriptome analysis were combined with genes identified in the literature (248 genes in total) to develop a targeted radiosensitivity assay on the Nanostring nCounter platform. Single-sample predictors for ipsilateral breast tumor recurrence based on a k-top scoring pairs algorithm were trained, stratified for estrogen receptor (ER) status and radiotherapy. Two previously published profiles, the radiosensitivity signature of Speers et al., and the 10-gene signature of Eschrich et al., were also included in the targeted panel.
Derived single-sample predictors were prognostic for ipsilateral breast tumor recurrence in radiotherapy-treated ER+ patients (AUC 0.67, p = 0.01), ER+ patients without radiotherapy (AUC = 0.89, p = 0.02), and radiotherapy-treated ER- patients (AUC = 0.78, p < 0.001). Among ER+ patients, radiotherapy had an excellent effect on tumors classified as radiosensitive (p < 0.001), while radiotherapy had no effect on tumors classified as radioresistant (p = 0.36) and there was a high risk of ipsilateral breast tumor recurrence (55% at 10 years). Our single-sample predictors developed in ER+ tumors and the radiosensitivity signature correlated with proliferation, while single-sample predictors developed in ER- tumors correlated with immune response. The 10-gene signature negatively correlated with both proliferation and immune response.
Our targeted single-sample predictors were prognostic for ipsilateral breast tumor recurrence and have the potential to stratify patients for adjuvant radiotherapy. The correlation of models with biology may explain the different performance in subgroups of breast cancer.
Immunotherapy has been less successful in treating prostate cancer than other solid tumors. We sought to better understand the immune landscape in prostate cancer and identify immune-related ...biomarkers and potential therapeutic targets.
We analyzed gene expression data from 7826 prospectively collected prostatectomy samples (2013-2016), and 1567 retrospective samples with long-term clinical outcomes, for a total of 9393 samples, all profiled on the same commercial clinical platform in a CLIA-certified lab. The primary outcome was distant metastasis-free survival (DMFS). Secondary outcomes included biochemical recurrence-free survival (bRFS), prostate cancer-specific survival (PCSS), and overall survival (OS). All statistical tests were two-sided.
Unsupervised hierarchical clustering of hallmark pathways demonstrated an immune-related tumor cluster. Increased estimated immune content scores based on immune-specific genes from the literature were associated with worse bRFS (hazard ratio HR = 1.26 95% confidence interval CI = 1.12 to 1.42; P < .001), DMFS (HR = 1.34 95% CI = 1.13 to 1.58; P < .001), PCSS (HR = 1.53 95% CI = 1.21 to 1.92; P < .001), and OS (HR = 1.27 95% CI = 1.07 to 1.50; P = .006). Deconvolution using Cibersort revealed that mast cells, natural killer cells, and dendritic cells conferred improved DMFS, whereas macrophages and T-cells conferred worse DMFS. Interestingly, while PD-L1 was not prognostic, consistent with its low expression in prostate cancer, PD-L2 was expressed at statistically significantly higher levels (P < .001) and was associated with worse bRFS (HR = 1.17 95% CI = 1.03 to 1.33; P = .01), DMFS (HR = 1.25 95% CI = 1.05 to 1.49; P = .01), and PCSS (HR = 1.45 95% CI = 1.13 to 1.86; P = .003). PD-L2 was strongly associated with immune-related pathways on gene set enrichment analysis suggesting that it is playing an important role in immune modulation in clinical prostate cancer samples. Furthermore, PD-L2 was correlated with radiation response pathways, and also predicted response to postoperative radiation therapy (PORT) on multivariable interaction analysis (P = .03).
In the largest study of its kind to date, these results illustrate the complex relationship between the tumor-immune interaction, prognosis, and response to radiotherapy, and nominate PD-L2 as a potential novel therapeutic target in prostate cancer, potentially in combination with radiotherapy.
Most patients with early-stage breast cancer are treated with adjuvant radiotherapy (RT) after breast-conserving surgery (BCS) to prevent locoregional recurrence (LRR). However, no genomic tools are ...used currently to select the optimal RT strategy.
We profiled the transcriptome of primary tumors on a clinical grade assay from the SweBCG91-RT trial, in which patients with node-negative breast cancer were randomly assigned to either whole-breast RT after BCS or no RT. We derived a new classifier, Adjuvant Radiotherapy Intensification Classifier (ARTIC), comprising 27 genes and patient age, in three publicly available cohorts, then independently validated ARTIC for LRR in 748 patients in SweBCG91-RT. We also compared previously published genomic signatures for ability to predict benefit from RT in SweBCG91-RT.
ARTIC was highly prognostic for LRR in patients treated with RT (hazard ratio HR, 3.4; 95% CI, 2.0 to 5.9;
< .001) and predictive of RT benefit (
= .005). Patients with low ARTIC scores had a large benefit from RT (HR, 0.33 95% CI, 0.21 to 0.52,
< .001; 10-year cumulative incidence of LRR, 6%
21%), whereas those with high ARTIC scores benefited less from RT (HR, 0.73 95% CI, 0.44 to 1.2,
= .23; 10-year cumulative incidence of LRR, 25%
32%). In contrast, none of the eight previously published signatures were predictive of benefit from RT in SweBCG91-RT.
ARTIC identified women with a substantial benefit from RT as well as women with a particularly elevated LRR risk in whom whole-breast RT was not sufficiently effective and, thus, in whom intensified treatment strategies such as tumor-bed boost, and possibly regional nodal RT, should be considered. To our knowledge, ARTIC is the first classifier validated as predictive of benefit from RT in a phase III clinical trial with patients randomly assigned to receive or not receive RT.
The effects of radiotherapy (RT) on the basis of the presence of stromal tumor infiltrating lymphocytes (TILs) have not been studied. The purpose of this study was to analyze the association of TILs ...with the effect of postoperative RT on ipsilateral breast tumor recurrence (IBTR) in a large randomized trial.
In the SweBCT91RT (Swedish Breast Cancer Group 91 Radiotherapy) trial, 1,178 patients with breast cancer stage I and II were randomly assigned to breast-conserving surgery plus postoperative RT or breast-conserving surgery only and followed for a median of 15.2 years. Tumor blocks were retrieved from 1,003 patients. Stromal TILs were assessed on whole-section hematoxylin-eosin-stained slides using a dichotomized cutoff of 10%. Subtypes were scored using immunohistochemistry on tissue microarray. In total, 936 patients were evaluated.
Altogether, 670 (71%) of patients had TILs less than 10%. In a multivariable regression analysis with IBTR as dependent variable and RT, TILs, subtype, age, and grade as independent variables, RT (hazard ratio HR, 0.42; 95% CI, 0.29 to 0.61;
< .001), high TILs (HR, 0.61; 95% CI, 0.39 to 0.96,
= .033) grade (3
1; HR, 2.17; 95% CI, 1.08 to 4.34;
= .029), and age (≥ 50
< 50 years; HR, 0.55; 95% CI, 0.38 to 0.80;
= .002) were predictive of IBTR. RT was significantly beneficial in the low TILs group (HR, 0.37; 95% CI, 0.24 to 0.58;
< .001) but not in the high TILs group (HR, 0.58; 95% CI, 0.28 to 1.19;
= .138). The test for interaction between RT and TILs was not statistically significant (
= .317).
This study shows that high values of TILs in the primary tumor independently seem to reduce the risk for an IBTR. Our findings further suggest that patients with breast cancer with low TILs may derive a larger benefit from RT regarding the risk of IBTR.
Prostate cancer is a highly heterogeneous disease, understanding the crosstalk between complex genomic and epigenomic alterations will aid in developing targeted therapeutics. We demonstrate that, ...even though snail family transcriptional repressor 2 (SNAI2) is frequently amplified in prostate cancer, it is epigenetically silenced in this disease, with dynamic changes in SNAI2 levels showing distinct clinical relevance. Integrative clinical data from 18 prostate cancer cohorts and experimental evidence showed that gene fusion between transmembrane serine protease 2 (TMPRSS2) and ETS transcription factor ERG (ERG) (TMPRSS2–ERG fusion) is involved in the silencing of SNAI2. We created a silencer score to evaluate epigenetic repression of SNAI2, which can be reversed by treatment with DNA methyltransferase inhibitors and histone deacetylase inhibitors. Silencing of SNAI2 facilitated tumor cell proliferation and luminal differentiation. Furthermore, SNAI2 has a major influence on the tumor microenvironment by reactivating tumor stroma and creating an immunosuppressive microenvironment in prostate cancer. Importantly, SNAI2 expression levels in part determine sensitivity to the cancer drugs dasatinib and panobinostat. For the first time, we defined the distinct clinical relevance of SNAI2 expression at different disease stages. We elucidated how epigenetic silencing of SNAI2 controls the dynamic changes of SNAI2 expression that are essential for tumor initiation and progression and discovered that restoring SNAI2 expression by treatment with panobinostat enhances dasatinib sensitivity, indicating a new therapeutic strategy for prostate cancer.
Here, the authors define, for the first time, the distinct clinical relevance of SNAI2 expression at different disease stages of prostate cancer (PC). They elucidated how the epigenetic silencing of SNAI2 controls the dynamic changes in SNAI2 expression that are essential for tumor initiation and progression. Importantly, restoring SNAI2 expression by LBH589 (HDACi) enhances dasatinib sensitivity, indicating a new therapeutic strategy for PC.
It is of highest importance to find proteins responsible for breast cancer dissemination, for use as biomarkers or treatment targets. We established and performed a combined nontargeted LC–MS/MS and ...a targeted LC–SRM workflow for discovery and validation of protein biomarkers. Eighty breast tumors, stratified for estrogen receptor status and development of distant recurrence (DR ± ), were collected. After enrichment of N-glycosylated peptides, label-free LC–MS/MS was performed on each individual tumor in triplicate. In total, 1515 glycopeptides from 778 proteins were identified and used to create a map of the breast cancer N-glycosylated proteome. Based on this specific proteome map, we constructed a 92-plex targeted label-free LC–SRM panel. These proteins were quantified across samples by LC–SRM, resulting in 10 proteins consistently differentially regulated between DR+/DR– tumors. Five proteins were further validated in a separate cohort as prognostic biomarkers at the gene expression level. We also compared the LC–SRM results to clinically reported HER2 status, demonstrating its clinical accuracy. In conclusion, we demonstrate a combined mass spectrometry strategy, at large scale on clinical samples, leading to the identification and validation of five proteins as potential biomarkers for breast cancer recurrence. All MS data are available via ProteomeXchange and PASSEL with identifiers PXD001685 and PASS00643.
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