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Background: Breast cancer is known to be a heterogeneous disease both at the clinical and molecular level. In addition, heterogeneity can also exist within a given tumor, and subpopulations can have distinct phenotypic and genomic features. Little is known about the relationship between intra-tumor heterogeneity and prediction of response to treatment. This study aimed at using a combination of immunofluorescence and fluorescence in situ (FISH) technique ("double immunoFISH") to identify intra-tumor heterogeneity in tumors from neo-adjuvant treated patients prior to and after therapy, searching for features predicting the response to neo adjuvant treatment. Material and methods: Twentytwo patients diagnosed with HER2 positive, neo-adjuvant treated breast cancer ((3-4 FEC100 followed by 4 docetaxel plus trastuzumab, 3qw) were selected. Half of the patients had complete response and the others had partial response. By double immunoFISH both phenotypic (ER and HER2 protein) and genomic changes (copy number of HER2 gene and centromere 17) were assessed in the same cells simultaneously on biopsies before and after treatment. The samples were photographed in a Zeiss Axio Imager M1 with 5 fluorescence channels and analyzed with axiovision software. The intensity and localization of HER2 and ER immunofluorescence were semi- quantitatively estimated while the HER2 and centromere 17 FISH signals were counted in 100 cells. Results: The patients with partial response displayed a high grade of cell-to-cell diversity regarding HER2 copy number, nuclear shape and size and the expression of the membrane protein HER2. This was in contrast to the results from the complete responders who showed a reduced diversity and were more frequently ER negative. In the patients with partial response, a higher diversity was seen after treatment. Conclusion: The genomic variability prior to therapy was higher in the partial-responders vs. the complete responders, and the remaining tumor was even more heterogeneous after treatment than prior to treatment. Double immunoFISH is a valuable tool for visualization of both phenotypic and genomic alterations in the same cell in FFPE sections. The cohort will be expanded to explore the diversity further, and the results will be presented. [ABSTRACT FROM AUTHOR]

Breast cancer is the most commonly occurring form of cancer in pregnancy. Among women aged between 25 and 29 years, one in five cases of breast cancer will occur in pregnancy or during the first postnatal year. The prevalence is increasing, presumably because of the increase in maternal age at pregnancy. Interdisciplinary cooperation is needed to ensure a good outcome for both mother and child. The prognosis is good but depends on rapid diagnosis and optimal treatment. In this clinical review article, the most important aspects of breast cancer treatment during pregnancy are reviewed.

Immune checkpoint inhibitors (ICIs) have been introduced in breast cancer (BC) treatment and better biomarkers are needed to predict benefit. Circulating tumor cells (CTCs) are prognostic in BC, but knowledge is limited on CTCs in the context of ICI therapy. In this study, serial sampling of CTCs (CellSearch system) was evaluated in 82 patients with metastatic BC enrolled in two randomized trials investigating ICI plus chemotherapy. Programmed death-ligand 1 (PD-L1) expression on CTCs was also measured. Patients with ≥ 2 CTCs per 7.5 mL at baseline had gene expression profiles in tumor suggestive of increased T-cell activity, including increased tumor inflammation signature (TIS) in both triple-negative (P = 0.010) and hormone receptor-positive (P = 0.024) disease. Patients with luminal A BC had higher CTC levels. The association between CTC status and outcome was most apparent 4 weeks into therapy. PD-L1 expression in CTCs was observed in 6/17 CTC-positive patients and was associated with inferior survival. In conclusion, our study indicates that CTC numbers may inform on tumor immune composition, as well as prognosis. These findings suggest a potential of using CTCs as an accessible biomarker source in BC patients treated with immunotherapy., (© 2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Breast cancer (BC) is a leading cause of cancer-related death worldwide. The diverse nature and heterogeneous biology of BC pose challenges for survival prediction, as patients with similar diagnoses often respond differently to treatment. Clinically relevant BC intrinsic subtypes have been established through gene expression profiling and are implemented in the clinic. While these intrinsic subtypes show a significant association with clinical outcomes, their long-term survival prediction beyond 5 years often deviates from expected clinical outcomes. This study aimed to identify naturally occurring long-term prognostic subgroups of BC based on an integrated multi-omics analysis. This study incorporates a clinical cohort of 335 untreated BC patients from the Oslo2 study with long-term follow-up (>12 years). Multi-Omics Factor Analysis (MOFA+) was employed to integrate transcriptomic, proteomic, and metabolomic data obtained from the tumor tissues. Our analysis revealed three prominent multi-omics clusters of BC patients with significantly different long-term prognoses (p = 0.005). The multi-omics clusters were validated in two independent large cohorts, METABRIC and TCGA. Importantly, a lack of prognostic association to long-term follow-up above 12 years in the previously established intrinsic subtypes was shown for these cohorts. Through a systems-biology approach, we identified varying enrichment levels of cell-cycle and immune-related pathways among the prognostic clusters. Integrated multi-omics analysis of BC revealed three distinct clusters with unique clinical and biological characteristics. Notably, these multi-omics clusters displayed robust associations with long-term survival, outperforming the established intrinsic subtypes., (© 2024. The Author(s).)

Immunotherapies blocking the PD-1/PD-L1 checkpoint show some efficacy in metastatic breast cancer (mBC) but are often hindered by immunosuppressive mechanisms. Understanding these mechanisms is crucial for personalized treatments, with peripheral blood monitoring representing a practical alternative to repeated biopsies. In the present study, we performed a comprehensive mass cytometry analysis of peripheral blood immune cells in 104 patients with HER2 negative mBC and 20 healthy donors (HD). We found that mBC patients had significantly elevated monocyte levels and reduced levels of CD4 + T cells and plasmacytoid dendritic cells, when compared to HD. Furthermore, mBC patients had more effector T cells and regulatory T cells, increased expression of immune checkpoints and other activation/exhaustion markers, and a shift to a Th2/Th17 phenotype. Furthermore, T-cell phenotypes identified by mass cytometry correlated with functionality as assessed by IFN-γ production. Additional analysis indicated that previous chemotherapy and CDK4/6 inhibition impacted the numbers and phenotype of immune cells. From 63 of the patients, fresh tumor samples were analyzed by flow cytometry. Paired PBMC-tumor analysis showed moderate correlations between peripheral CD4 +  T and NK cells with their counterparts in tumors. Further, a CD4 + T cell cluster in PBMCs, that co-expressed multiple checkpoint receptors, was negatively associated with CD4 +  T cell tumor infiltration. In conclusion, the identified systemic immune signatures indicate an immune-suppressed environment in mBC patients who had progressed/relapsed on standard treatments, and is consistent with ongoing chronic inflammation. These activated immuno-suppressive mechanisms may be investigated as therapeutic targets, and for use as biomarkers of response or treatment resistance., (© 2024. The Author(s).)

Background: Most women with advanced breast cancer have skeletal metastases. Radium-223 is an alpha-emitting radionuclide that selectively targets areas of bone metastases., Methods: Two double-blind, placebo-controlled studies of radium-223 were conducted in women with hormone receptor-positive (HR+), bone-predominant metastatic breast cancer. All patients received endocrine therapy (ET), as a single agent of the investigator's choice (Study A) or exemestane + everolimus (Study B). Patients were randomized to receive radium-223 (55 kBq/kg) or placebo intravenously every 4 weeks for six doses. Accrual was halted following unblinded interim analyses per protocol amendments, and both studies were terminated. We report pooled analyses of symptomatic skeletal event-free survival (SSE-FS; primary endpoint), radiologic progression-free survival (rPFS) and overall survival (OS; secondary), and time to bone alkaline phosphatase (ALP) progression (exploratory)., Results: In total, 382 patients were enrolled, and 196 SSE-FS events (70% planned total) were recorded. Hazard ratios (95% confidence intervals) and nominal p values for radium-223 + ET versus placebo + ET were: SSE-FS 0.809 (0.610-1.072), p = 0.1389; rPFS 0.956 (0.759-1.205), p = 0.7039; OS 0.889 (0.660-1.199), p = 0.4410; and time to bone ALP progression 0.593 (0.379-0.926), p = 0.0195. Radium-223- or placebo-related treatment-emergent adverse events were reported in 50.3% versus 35.1% of patients (grade 3/4: 25.7% vs. 8.5%), with fractures/bone-associated events in 23.5% versus 23.9%., Conclusions: In patients with HR+ bone-metastatic breast cancer, numeric differences favoring radium-223 + ET over placebo + ET for the primary SSE-FS endpoint were suggestive of efficacy, in line with the primary outcome measure used in the underlying phase 2 studies. No similar evidence of efficacy was observed for secondary progression or survival endpoints. Adverse events were more frequent with radium-223 + ET versus placebo + ET, but the safety profile of the combination was consistent with the safety profiles of the component drugs. Clinical trial registration numbers Study A: NCT02258464, registered October 7, 2014. Study B: NCT02258451, registered October 7, 2014., (© 2023. The Author(s).)

Background: Immune checkpoint inhibitors have shown minimal clinical activity in hormone receptor-positive metastatic breast cancer (HR + mBC). Doxorubicin and low-dose cyclophosphamide are reported to induce immune responses and counter regulatory T cells (Tregs). Here, we report the efficacy and safety of combined programmed cell death protein-1/cytotoxic T-lymphocyte-associated protein 4 blockade concomitant with or after immunomodulatory chemotherapy for HR + mBC., Methods: Patients with HR + mBC starting first-/second- line chemotherapy (chemo) were randomized 2:3 to chemotherapy (pegylated liposomal doxorubicin 20 mg/m 2 every second week plus cyclophosphamide 50 mg by mouth/day in every other 2-week cycle) with or without concomitant ipilimumab (ipi; 1 mg/kg every sixth week) and nivolumab (nivo; 240 mg every second week). Patients in the chemo-only arm were offered cross-over to ipi/nivo without chemotherapy. Co-primary endpoints were safety in all patients starting therapy and progression-free survival (PFS) in the per-protocol (PP) population, defined as all patients evaluated for response and receiving at least two treatment cycles. Secondary endpoints included objective response rate, clinical benefit rate, Treg changes during therapy and assessment of programmed death-ligand 1 (PD-L1), mutational burden and immune gene signatures as biomarkers., Results: Eighty-two patients were randomized and received immune-chemo (N=49) or chemo-only (N=33), 16 patients continued to the ipi/nivo-only cross-over arm. Median follow-up was 41.4 months. Serious adverse events occurred in 63% in the immune-chemo arm, 39% in the chemo-only arm and 31% in the cross-over-arm. In the PP population (N=78) median PFS in the immune-chemo arm was 5.1 months, compared with 3.6 months in the chemo-only arm, with HR 0.94 (95% CI 0.59 to 1.51). Clinical benefit rates were 55% (26/47) and 48% (15/31) in the immune-chemo and chemo-only arms, respectively. In the cross-over-arm (ipi/nivo-only), objective responses were observed in 19% of patients (3/16) and clinical benefit in 25% (4/16). Treg levels in blood decreased after study chemotherapy. High-grade immune-related adverse events were associated with prolonged PFS. PD-L1 status and mutational burden were not associated with ipi/nivo benefit, whereas a numerical PFS advantage was observed for patients with a high Treg gene signature in tumor., Conclusion: The addition of ipi/nivo to chemotherapy increased toxicity without improving efficacy. Ipi/nivo administered sequentially to chemotherapy was tolerable and induced clinical responses., Trial Registration Number: ClinicalTrials.gov Identifier: NCT03409198., Competing Interests: Competing interests: JAK has in the last 5 years received research support from Bristol Myers Squibb, F. Hoffmann-La Roche, NanoString, and NEC OncoImmunity and has previously received advisory board/lecture honoraria from pharmaceutical companies, including Bristol Myers Squibb. CQ has received honoraria for advisory board from AstraZeneca. BG has received honoraria for advisory boards from Eli Lilly, Gilead, Daiichi Sankyo, Roche, and Pierre Fabre. LJ has received lecture honoraria from Pfizer, Novartis, and AstraZeneca. AG has received travel grants or honoraria for advisory boards from Lilly, Daiichi Sankyo, Seagen, Pfizer, and AstraZeneca. HGR has received research support from Illumina and NanoString. OCL has over the last 2 years received honoraria for work as statistical advisor for Novartis. All other authors declare no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Breast cancer is the most common cancer among women in Norway. Nine out of ten will become long-term survivors. Being cancer-free does not necessarily mean feeling healthy, and many experience troublesome late effects, such as fatigue, pain and fear of recurrence. General practitioners represent the most important medical support for the majority of these women. This clinical review article summarises up-to-date knowledge about late effects after breast cancer treatment. Non-pharmacological interventions can have a positive effect on many of the most common late effects.

Immune checkpoint inhibitors have shown efficacy against metastatic triple-negative breast cancer (mTNBC) but only for PD-L1 positive disease. The randomized, placebo-controlled ALICE trial ( NCT03164993 , 24 May 2017) evaluated the addition of atezolizumab (anti-PD-L1) to immune-stimulating chemotherapy in mTNBC. Patients received pegylated liposomal doxorubicin (PLD) and low-dose cyclophosphamide in combination with atezolizumab (atezo-chemo; n = 40) or placebo (placebo-chemo; n = 28). Primary endpoints were descriptive assessment of progression-free survival in the per-protocol population (>3 atezolizumab and >2 PLD doses; n = 59) and safety in the full analysis set (FAS; all patients starting therapy; n = 68). Adverse events leading to drug discontinuation occurred in 18% of patients in the atezo-chemo arm (7/40) and in 7% of patients in the placebo-chemo arm (2/28). Improvement in progression-free survival was indicated in the atezo-chemo arm in the per-protocol population (median 4.3 months versus 3.5 months; hazard ratio (HR) = 0.57; 95% confidence interval (CI) 0.33-0.99; log-rank P = 0.047) and in the FAS (HR = 0.56; 95% CI 0.33-0.95; P = 0.033). A numerical advantage was observed for both the PD-L1 positive (n = 27; HR = 0.65; 95% CI 0.27-1.54) and PD-L1 negative subgroups (n = 31; HR = 0.57, 95% CI 0.27-1.21). The progression-free proportion after 15 months was 14.7% (5/34; 95% CI 6.4-30.1%) in the atezo-chemo arm versus 0% in the placebo-chemo arm. The addition of atezolizumab to PLD/cyclophosphamide was tolerable with an indication of clinical benefit, and the findings warrant further investigation of PD1/PD-L1 blockers in combination with immunomodulatory chemotherapy., (© 2022. The Author(s).)

Multigene assays for molecular subtypes and biomarkers can aid management of early invasive breast cancer. Using RNA-sequencing we aimed to develop single-sample predictor (SSP) models for clinical markers, subtypes, and risk of recurrence (ROR). A cohort of 7743 patients was divided into training and test set. We trained SSPs for subtypes and ROR assigned by nearest-centroid (NC) methods and SSPs for biomarkers from histopathology. Classifications were compared with Prosigna in two external cohorts (ABiM, n = 100 and OSLO2-EMIT0, n = 103). Prognostic value was assessed using distant recurrence-free interval. Agreement between SSP and NC for PAM50 (five subtypes) was high (85%, Kappa = 0.78) for Subtype (four subtypes) very high (90%, Kappa = 0.84) and for ROR risk category high (84%, Kappa = 0.75, weighted Kappa = 0.90). Prognostic value was assessed as equivalent and clinically relevant. Agreement with histopathology was very high or high for receptor status, while moderate for Ki67 status and poor for Nottingham histological grade. SSP and Prosigna concordance was high for subtype (OSLO-EMIT0 83%, Kappa = 0.73 and ABiM 80%, Kappa = 0.72) and moderate and high for ROR risk category (68 and 84%, Kappa = 0.50 and 0.70, weighted Kappa = 0.70 and 0.78). Pooled concordance for emulated treatment recommendation dichotomized for chemotherapy was high (85%, Kappa = 0.66). Retrospective evaluation suggested that SSP application could change chemotherapy recommendations for up to 17% of postmenopausal ER+/HER2-/N0 patients with balanced escalation and de-escalation. Results suggest that NC and SSP models are interchangeable on a group-level and nearly so on a patient level and that SSP models can be derived to closely match clinical tests., (© 2022. The Author(s).)

Background: Cancer patient pathways (CPPs) were implemented in Norway to reduce unnecessary waiting times, regional variations, and to increase the predictability of cancer care for the patients. This study aimed to determine if 70% of cancer patients started treatment within the recommended time frames, and to identify potential delays., Methods: Patients registered with a colorectal, lung, breast, or prostate cancer diagnosis at the Cancer Registry of Norway in 2015-2016 were linked with the Norwegian Patient Registry and Statistics Norway. Adjusting for sociodemographic variables, multivariable quantile (median) regressions were used to examine the association between place of residence and median time to start of examination, treatment decision, and start of treatment., Results: The study included 20 668 patients. The proportions of patients who went through the CPP within the recommended time frames were highest among colon (84%) and breast (76%) cancer patients who underwent surgery and lung cancer patients who started systemic anticancer treatment (76%), and lowest for prostate cancer patients who underwent surgery (43%). The time from treatment decision to start of treatment was the main source of delay for all cancers. Travelling outside the resident health trust prolonged waiting time and was associated with a reduced odds of receiving surgery and radiotherapy for lung and rectal cancer patients, respectively., Conclusions: Achievement of national recommendations of the CCP times differed by cancer type and treatment. Identified bottlenecks in the pathway should be targeted to decrease waiting times. Further, CPP guidelines should be re-examined to determine their ongoing relevance., (© 2022. The Author(s).)

The PAM50 gene expression subtypes and the associated risk of recurrence (ROR) score are used to predict the risk of recurrence and the benefits of adjuvant therapy in early-stage breast cancer. The Prosigna assay includes the PAM50 subtypes along with their clinicopathological features, and is approved for treatment recommendations for adjuvant hormonal therapy and chemotherapy in hormone-receptor-positive early breast cancer. The Prosigna test utilizes RNA extracted from macrodissected tumor cells obtained from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, RNA extracted from fresh-frozen (FF) bulk tissue without macrodissection is widely used for research purposes, and yields high-quality RNA for downstream analyses. To investigate the impact of the sample preparation approach on ROR scores, we analyzed 94 breast carcinomas included in an observational study that had available gene expression data from macrodissected FFPE tissue and FF bulk tumor tissue, along with the clinically approved Prosigna scores for the node-negative, hormone-receptor-positive, HER2-negative cases (n = 54). ROR scores were calculated in R; the resulting two sets of scores from FFPE and FF samples were compared, and treatment recommendations were evaluated. Overall, ROR scores calculated based on the macrodissected FFPE tissue were consistent with the Prosigna scores. However, analyses from bulk tissue yielded a higher proportion of cases classified as normal-like; these were samples with relatively low tumor cellularity, leading to lower ROR scores. When comparing ROR scores (low, intermediate, and high), discordant cases between the two preparation approaches were revealed among the luminal tumors; the recommended treatment would have changed in a minority of cases.

Purpose: Presence of disseminated tumour cells (DTCs) in the bone marrow (BM) has been described as a surrogate of residual disease in patients with early breast cancer (EBC). PADDY (Pooled Analysis of DTC Detection in Early Breast Cancer) is a large international analysis of pooled data that aimed to assess the prognostic impact of DTCs in patients with EBC., Experimental Design: Individual patient data were collected from 11 centres. Patients with EBC and available follow-up data in whom BM sampling was performed at the time of primary diagnosis before receiving any anticancer treatment were eligible. DTCs were identified by antibody staining against epithelial cytokeratins. Multivariate Cox regression was used to compare the survival of DTC-positive versus DTC-negative patients., Results: In total, 10,307 patients were included. Of these, 2814 (27.3%) were DTC-positive. DTC detection was associated with higher tumour grade, larger tumour size, nodal positivity, oestrogen receptor and progesterone receptor negativity, and HER2 positivity (all p < 0.001). Multivariate analyses showed that DTC detection was an independent prognostic marker for overall survival, disease-free survival and distant disease-free survival with hazard ratios (HR) and 95% confidence intervals (CI) of 1.23 (95% CI: 1.06-1.43, p = 0.006), 1.30 (95% CI: 1.12-1.52, p < 0.001) and 1.30 (95% CI: 1.08-1.56, p = 0.006), respectively. There was no association between locoregional relapse-free survival and DTC detection (HR 1.21; 95% CI 0.68-2.16; p = 0.512)., Conclusions: DTCs in the BM represent an independent prognostic marker in patients with EBC. The heterogeneous metastasis-initiating potential of DTCs is consistent with the concept of cancer dormancy., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ADH, FAT and SYB report a grant from Exact Sciences. TNF received personal fees from AstraZeneca, Novartis, Roche, Pfizer, Esai, Tesaro, Teva, Celgene, Daichi Sankyo, MSD, Menarini. PAF reports grants from Cepheid, Novartis and Biontech, personal fees from Novartis, Roche, Pfizer, Celgene, Daiichi-Sankyo, TEVA, Astra Zeneca, Merck Sharp & Dohme, Myelo Therapeutics, Macrogenics, Eisai, Puma and Lilly. CAK is a member of the SAB of HiberCell, New York. All other authors declare no potential conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Low steady-state levels of active tamoxifen metabolites have been associated with inferior treatment outcomes. In this retrospective analysis of 406 estrogen receptor-positive breast cancer (BC) patients receiving adjuvant tamoxifen as initial treatment, we have associated our previously reported thresholds for the two active metabolites, Z-endoxifen and Z-4-hydroxy-tamoxifen (Z-4OHtam), with treatment outcomes in an independent cohort of BC patients. Among all patients, metabolite levels did not affect survival. However, in the premenopausal subgroup receiving tamoxifen alone (n = 191) we confirmed an inferior BC -specific survival in patients with the previously described serum concentration threshold of Z-4OHtam ≤ 3.26 nm (HR = 2.37, 95% CI = 1.02-5.48, P = 0.039). The 'dose-response' survival trend in patients categorized to ordinal concentration cut-points of Z-4OHtamoxifen (≤ 3.26, 3.27-8.13, > 8.13 nm) was also replicated (P-trend log-rank = 0.048). Z-endoxifen was not associated with outcome. This is the first study to confirm the association between a published active tamoxifen metabolite threshold and BC outcome in an independent patient cohort. Premenopausal patients receiving 5-year of tamoxifen alone may benefit from therapeutic drug monitoring to ensure tamoxifen effectiveness., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

In the bone marrow (BM) microenvironment, where breast cancer (BC) disseminated tumour cells (DTCs) can remain dormant for decades, NG2 + /Nestin + mesenchymal stem cells (MSCs) promote hematopoietic stem cell quiescence. Here, we reveal that periarteriolar BM-resident NG2 + /Nestin + MSCs can also instruct BC DTCs to enter dormancy. NG2 + /Nestin + MSCs produce TGFβ2 and BMP7 and activate a quiescence pathway dependent on TGFBRIII and BMPRII, which via p38-kinase result in p27 induction. Genetic depletion of MSCs or conditional knock-out of TGFβ2 in MSCs using an NG2-Cre ER driver led to bone metastatic outgrowth of otherwise dormant p27 + /Ki67 - DTCs. Also ER + BC patients without systemic recurrence displayed higher frequency of TGFβ2 and BMP7 detection in the BM. Our results provide a direct proof that HSC dormancy niches control BC DTC dormancy and suggest that aging or extrinsic factors that affect the NG2 + /Nestin + MSC niche homeostasis may result in a break from dormancy and BC bone relapse.

A subset of breast cancer patients benefits from preoperative bevacizumab and chemotherapy, but validated predictive biomarkers are lacking. Here, we aimed to evaluate tissue-based angiogenesis markers for potential predictive value regarding response to neoadjuvant bevacizumab treatment in breast cancer. In this randomized 1:1 phase II clinical trial, 132 patients with large or locally advanced HER2-negative tumors received chemotherapy ± bevacizumab. Dual Factor VIII/Ki-67 immunohistochemical staining was performed on core needle biopsies at baseline and week 12. Microvessel density (MVD), proliferative microvessel density (pMVD; Factor VIII/Ki-67 co-expression), glomeruloid microvascular proliferation (GMP), and a gene expression angiogenesis signature score, were studied in relation to pathologic complete response (pCR), clinico-pathologic features and intrinsic molecular subtype. We found that high baseline MVD (by median) significantly predicted pCR in the bevacizumab-arm (odds ratio 4.9, P = 0.012). High pMVD, presence of GMP, and the angiogenesis signature score did not predict pCR, but were associated with basal-like (P ≤ 0.009) and triple negative phenotypes (P ≤ 0.041). pMVD and GMP did also associate with high-grade tumors (P ≤ 0.048). To conclude, high baseline MVD significantly predicted response to bevacizumab treatment. In contrast, pMVD, GMP, and the angiogenesis signature score, did not predict response, but associated with aggressive tumor features, including basal-like and triple-negative phenotypes.

Purpose: Antiangiogenic therapy using bevacizumab has proven effective for a number of cancers; however, in breast cancer (BC), there is an unmet need to identify patients who benefit from such treatment., Patients and Methods: In the NeoAva phase II clinical trial, patients (N = 132) with large (≥ 25 mm) human epidermal growth factor receptor 2 (HER2)-negative primary tumors were randomly assigned 1:1 to treatment with neoadjuvant chemotherapy (CTx) alone or in combination with bevacizumab (Bev plus CTx). The ratio of the tumor size after relative to before treatment was calculated into a continuous response scale. Tumor biopsies taken prior to neoadjuvant treatment were analyzed by reverse-phase protein arrays (RPPA) for expression levels of 210 BC-relevant (phospho-) proteins. Lasso regression was used to derive a predictor of tumor shrinkage from the expression of selected proteins prior to treatment., Results: We identified a nine-protein signature score named vascular endothelial growth factor inhibition response predictor (ViRP) for use in the Bev plus CTx treatment arm able to predict with accuracy pathologic complete response (pCR) (area under the curve [AUC] = 0.85; 95% CI, 0.74 to 0.97) and low residual cancer burden (RCB 0/I) (AUC = 0.80; 95% CI, 0.68 to 0.93). The ViRP score was significantly lower in patients with pCR ( P < .001) and in patients with low RCB ( P < .001). The ViRP score was internally validated on mRNA data and the resultant surrogate mRNA ViRP score significantly separated the pCR patients ( P = .016). Similarly, the mRNA ViRP score was validated ( P < .001) in an independent phase II clinical trial (PROMIX)., Conclusion: Our ViRP score, integrating the expression of nine proteins and validated on mRNA data both internally and in an independent clinical trial, may be used to increase the likelihood of benefit from treatment with bevacizumab combined with chemotherapy in patients with HER2-negative BC., Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/cci/author-center. Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments). Mads H. HaugenPatents, Royalties, Other Intellectual Property: Patent application 62/969770 to USPTOOle Christian LingjærdeConsulting or Advisory Role: NovartisThomas HatschekConsulting or Advisory Role: Roche, Pfizer, Pierre Fabre Research Funding: Roche, Pfizer Travel, Accommodations, Expenses: RocheAnne-Lise Børresen-DaleEmployment: Arctic Pharma AS, PubGene Stock and Other Ownership Interests: Arctic Pharma ASGordon B. MillsStock and Other Ownership Interests: Catena, SignalChem, Tarveda Therapeutics, ImmunoMET Honoraria: Nuevolution: AstraZeneca, Tarveda Therapeutics, Tesaro, Symphogen, PDX Pharmacy, ImmunoMET, Lilly Consulting or Advisory Role: AstraZeneca, SignalChem, Tarveda Therapeutics, Symphogen, Takeda/Millennium, PDX Pharmacy, ImmunoMET, Lilly, Turbine, ION Pharma, Zentalis Research Funding: Adelson Medical Research Foundation, AstraZeneca, NanoString Technologies, Breast Cancer Research Foundation, Karus Therapeutics, Pfizer, Prospect Creek Foundation, Tarveda Therapeutics, Ions Pharmaceuticals, ImmunoMET Patents, Royalties, Other Intellectual Property: HRD assay to Myriad Genetics, DSP technology patent with Nanostring Travel, Accommodations, Expenses: AstraZeneca, Pfizer, Symphogen, Chrysalis Biomedical Advisors, ImmunoMET, Michigan Primary Care ConsortiumGunhild M. MælandsmoPatents, Royalties, Other Intellectual Property: Patent application submitted for a nine-protein/gene panel predicting response to anti VEGF therapies in combination with chemotherapyOlav EngebraatenPatents, Royalties, Other Intellectual Property: Patent application pending for a biomarker for antibody drug conjugates, Patent application submitted for a nine-protein/gene panel predicting response to anti VEGF therapies in combination with chemotherapy No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

In patients with presumed pancreatic ductal adenocarcinoma (PDAC), biomarkers that may open for personalised, risk-adapted treatment are lacking. The study analysed the impact of CTCs-presence on the patterns of recurrence and survival in 98 patients resected for PDAC with 5-10 years of follow-up. Preoperative samples were analysed by the CellSearch ® system for EpCAM+/DAPI+/CK+/CD45-CTCs. CTCs were detected in 7 of the 98 patients. CTCs predicted a significantly shorter median disease-free survival (DFS) of 3.3 vs. 9.2 months and a median cancer specific survival (CSS)of 6.3 vs. 18.5 months. Relapse status was confirmed by imaging for 87 patients. Of these, 58 patients developed distant metastases (DM) and 29 developed isolated local recurrence (ILR) as the first sign of cancer relapse. All patients with CTCs experienced DM. pN-status and histological grade >2 were other independent risk factors for DM, but only CTCs predicted significantly shorter cancer-specific, disease-free and post-recurrence survival. Preoperative parameters did not affect clinical outcome. We conclude that CTC presence in resected PDAC patients predicted early distant metastasis and impaired survival. Preoperative CTCs alone or in combination with histopathological factors may guide initial treatment decisions in patients with resectable PDAC in the future.

Background: Pancreatic and periampullary carcinoma are aggressive tumours where preoperative assessment is challenging. Disseminated tumour cells (DTC) in the bone marrow (BM) are associated with impaired prognosis in a variety of epithelial cancers. In a cohort of patients with presumed resectable pancreatic and periampullary carcinoma, we evaluated the frequency and the potential prognostic impact of the preoperative presence of DTC, defined as cytokeratin-positive cells detected by immunocytochemistry (ICC)., Methods: Preoperative BM samples from 242 patients selected for surgical resection of presumed resectable pancreatic and periampullary carcinoma from 09/2009 to 12/2014, were analysed for presence of CK-positive cells by ICC. The median observation time was 21.5 months. Overall survival (OS) and disease-free survival (DFS) were calculated by Kaplan-Meier and Cox regression analysis., Results: Successful resections of malignant tumours were performed in 179 of the cases, 30 patients resected had benign pancreatic disease based on postoperative histology, and 33 were deemed inoperable intraoperatively due to advanced disease. Overall survival for patients with resected carcinoma was 21.1 months (95% CI: 18.0-24.1), for those with benign disease OS was 101 months (95% CI: 69.4-132) and for those with advanced disease OS was 8.8 months (95% CI: 4.3-13.3). The proportion of patients with detected CK-positive cells was 6/168 (3.6%) in resected malignant cases, 2/31 (6.5%) in advanced disease and 4/29 (13.8%) in benign disease. The presence of CK-positive cells was not correlated to OS or DFS, neither in the entire cohort nor in the subgroup negative for circulating tumour cells (CTC)., Conclusions: The results indicate that CK-positive cells may be present in both patients with malignant and benign diseases of the pancreas. Detection of CK-positive cells was not associated with differences in prognosis for the entire cohort or any of the subgroups analysed., Trial Registration: clinicaltrials.gov ( NCT01919151 ).

Antiangiogenic drugs are potentially a useful supplement to neoadjuvant chemotherapy for a subgroup of patients with human epidermal growth factor receptor 2 (HER2) negative breast cancer, but reliable biomarkers for improved response are lacking. Here, we report on a randomized phase II clinical trial to study the added effect of bevacizumab in neoadjuvant chemotherapy with FEC100 (5-fluorouracil, epirubicin and cyclophosphamide) and taxanes (n = 132 patients). Gene expression from the tumors was obtained before neoadjuvant treatment, and treatment response was evaluated by residual cancer burden (RCB) at time of surgery. Bevacizumab increased the proportion of complete responders (RCB class 0) from 5% to 20% among patients with estrogen receptor (ER) positive tumors (P = .02). Treatment with bevacizumab was associated with improved 8-year disease-free survival (P = .03) among the good responders (RCB class 0 or I). Patients treated with paclitaxel (n = 45) responded better than those treated with docetaxel (n = 21; P = .03). Improved treatment response was associated with higher proliferation rate and an immune phenotype characterized by high presence of classically activated M1 macrophages, activated NK cells and memory activated CD4 T cells. Treatment with bevacizumab increased the number of adverse events, including hemorrhage, hypertension, infection and febrile neutropenia, but despite this, the ECOG status was not affected., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: Cancer patient pathways (CPPs) were implemented in 2015 to reduce waiting time, regional variation in waiting time, and to increase the predictability of cancer care for the patients. The aims of this study were to see if the national target of 70% of all cancer patients being included in a CPP was met, and to identify factors associated with CPP inclusion., Methods: All patients registered with a colorectal, lung, breast or prostate cancer diagnosis at the Cancer Registry of Norway in the period 2015-2016 were linked with the Norwegian Patient Registry for CPP information and with Statistics Norway for sociodemographic variables. Multivariable logistic regression examined if the odds of not being included in a CPP were associated with year of diagnosis, age, sex, tumour stage, marital status, education, income, region of residence and comorbidity., Results: From 2015 to 2016, 30,747 patients were diagnosed with colorectal, lung, breast or prostate cancer, of whom 24,429 (79.5%) were included in a CPP. Significant increases in the probability of being included in a CPP were observed for colorectal (79.1 to 86.2%), lung (79.0 to 87.3%), breast (91.5 to 97.2%) and prostate cancer (62.2 to 76.2%) patients (p < 0.001). Increasing age was associated with an increased odds of not being included in a CPP for lung (p < 0.001) and prostate cancer (p < 0.001) patients. Colorectal cancer patients < 50 years of age had a two-fold increase (OR = 2.23, 95% CI: 1.70-2.91) in the odds of not being included in a CPP. The odds of no CPP inclusion were significantly increased for low income colorectal (OR = 1.24, 95%CI: 1.00-1.54) and lung (OR = 1.52, 95%CI: 1.16-1.99) cancer patients. Region of residence was significantly associated with CPP inclusion (p < 0.001) and the probability, adjusted for case-mix ranged from 62.4% in region West among prostate cancer patients to 97.6% in region North among breast cancer patients., Conclusions: The national target of 70% was met within 1 year of CPP implementation in Norway. Although all patients should have equal access to CPPs, a prostate cancer diagnosis, older age, high level of comorbidity or low income were significantly associated with an increased odds of not being included in a CPP.

Objective: We evaluated the prognostic impact of circulating tumor cells (CTCs) for patients with presumed resectable pancreatic and periampullary cancers., Summary of Background Data: Initial treatment decisions for this group are currently taken without a reliable prognostic marker. The CellSearch system allows standardized CTC-testing and has shown excellent specificity and prognostic value in other applications., Methods: Preoperative blood samples from 242 patients between September 2009 and December 2014 were analyzed. One hundred seventy-nine patients underwent tumor resection, of whom 30 with stage-I tumors and duodenal cancer were assigned to the low-risk group, and the others to the high-risk group. Further 33 had advanced disease, 30 benign histology. Observation ended in December 2016. Cancer-specific survival (CSS) and disease-free survival (DFS) were calculated by log-rank and Cox regression., Results: CTCs (CTC-positive; ≥1 CTC/7.5 mL) were detected in 6.8% (10/147) of the high-risk patients and 6.2% (2/33) with advanced disease. No CTCs (CTC-negative) were detected in the low-risk patients or benign disease. In high-risk patients, median CSS for CTC-positive versus CTC-negative was 8.1 versus 20.0 months (P < 0.0001), and DFS 4.0 versus 10.5 months (P < 0.001). Median CSS in advanced disease was 7.7 months. Univariate hazard ratio (HR) of CTC-positivity was 3.4 (P < 0.001). In multivariable analysis, CTC-status remained independent (HR: 2.4, P = 0.009) when corrected for histological type (HR: 2.7, P = 0.030), nodal status (HR: 1.7, P = 0.016), and vascular infiltration (HR: 1.7, P = 0.001)., Conclusion: Patients testing CTC-positive preoperatively showed a detrimental outcome despite successful tumor resections. Although the low CTC-rate seems a limiting factor, results indicate high specificity. Thus, preoperative analysis of CTCs by this test may guide treatment decisions and warrants further testing in clinical trials.

How mixtures of immune cells associate with cancer cell phenotype and affect pathogenesis is still unclear. In 15 breast cancer gene expression datasets, we invariably identify three clusters of patients with gradual levels of immune infiltration. The intermediate immune infiltration cluster (Cluster B) is associated with a worse prognosis independently of known clinicopathological features. Furthermore, immune clusters are associated with response to neoadjuvant chemotherapy. In silico dissection of the immune contexture of the clusters identified Cluster A as immune cold, Cluster C as immune hot while Cluster B has a pro-tumorigenic immune infiltration. Through phenotypical analysis, we find epithelial mesenchymal transition and proliferation associated with the immune clusters and mutually exclusive in breast cancers. Here, we describe immune clusters which improve the prognostic accuracy of immune contexture in breast cancer. Our discovery of a novel independent prognostic factor in breast cancer highlights a correlation between tumor phenotype and immune contexture.

Background: In 2015, Norway implemented cancer patient pathways to reduce waiting times for treatment. The aims of this paper were to describe patterns in waiting time and their association with patient characteristics for colorectal, lung, breast and prostate cancers., Methods: National, population-based data from 2007 to 2016 were used. A multivariable quantile regression examined the association between treatment period, age, stage, sex, place of residence, and median waiting times., Results: Reduction in median waiting times for radiotherapy among colorectal, lung and prostate cancer patients ranged from 14 to 50 days. Median waiting time for surgery remained approximately 21 days for both colorectal and breast cancers, while it decreased by 7 and 36 days for lung and prostate cancers, respectively. The proportion of lung and prostate cancer patients with metastatic disease at the time of diagnosis decreased, while the proportion of colorectal patients with localised disease and patients with stage I breast cancer increased (p < 0.001). After adjusting for case-mix, a patient's place of residence was significantly associated with waiting time for treatment (p < 0.001), however, differences in waiting time to treatment decreased over the study period., Conclusions: Between 2007 and 2016, Norway experienced improved stage distributions and consistently decreasing waiting times for treatment. While these improvements occurred gradually, no significant change was observed from the time of cancer patient pathway implementation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: Increased focus on quality indicators and the use of clinical registries for breast cancer for real world studies have shown higher compliance to recommended therapy and better survival. In 2010, the European Society of Breast Cancer Specialist (EUSOMA) proposed quality indicators (QI) covering diagnosis, treatment and follow-up. To become a EUSOMA certified Breast Cancer Unit, 14 specified quality indicators, in addition to other requirements, need to be met. To evaluate the compliance and results of recommended treatment in breast cancer care in Norway and to improve the quality of epidemiological data, the Cancer Registry of Norway (CRN) in cooperation with the Norwegian Breast Cancer Group (NBCG) developed the Norwegian Breast Cancer Registry (NBCR). The objective of this study is to assess the feasibility of using the NBCR for estimating the EUSOMA QI individually for all hospitals diagnosing and treating breast cancer in Norway., Methods: To provide researchers with high quality cancer data as well as for the purpose of national cancer statistics, the CRN employs a cancer registry system to 1) longitudinal capture data from all patients from all medical entities that diagnose and/or treat cancer patients (e.g., pathology, radiology and clinical departments) in Norway; 2) curate data, i.e. validate the correctness of collected data, and assemble the validated cancer data as cancer cases; 3) provide data for analytics and presentation. Estimates for 10 EUSOMA QI were calculated at national and hospital level. To compare hospitals, a summary score of QIs was defined for each hospital., Results: All hospitals currently treating breast cancer patients have the technical ability to submit data to the NBCR for estimation of QIs defined by EUSOMA. Data from pathology and surgery are of high quality. However, data from oncological and radiological departments are incomplete, but improving. This currently hinders three QIs from being calculated. QI on benign to malign diagnosis needs to be calculated at the individual Breast Centre. Over time the adherence to guidelines have improved and the hospital variation for the respective QI have decreased. Two hospitals met all minimum standard on ten QIs in year 2016 and one hospital did not meet one minimum standard, but met all other targets., Conclusion: The NBCR has since 2012 published annual reports on breast cancer care and for the year 2016 measured 10 of 14 QI defined by EUSOMA. Increased compliance of recommended treatment in Norway has been observed during the years the registry has been active., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

In the preceding decades, molecular characterization has revolutionized breast cancer (BC) research and therapeutic approaches. Presented herein, an unbiased analysis of breast tumor proteomes, inclusive of 9995 proteins quantified across all tumors, for the first time recapitulates BC subtypes. Additionally, poor-prognosis basal-like and luminal B tumors are further subdivided by immune component infiltration, suggesting the current classification is incomplete. Proteome-based networks distinguish functional protein modules for breast tumor groups, with co-expression of EGFR and MET marking ductal carcinoma in situ regions of normal-like tumors and lending to a more accurate classification of this poorly defined subtype. Genes included within prognostic mRNA panels have significantly higher than average mRNA-protein correlations, and gene copy number alterations are dampened at the protein-level; underscoring the value of proteome quantification for prognostication and phenotypic classification. Furthermore, protein products mapping to non-coding genomic regions are identified; highlighting a potential new class of tumor-specific immunotherapeutic targets.

Background: Chemotherapeutic agents such as anthracyclines and taxanes are commonly used in the neoadjuvant setting. Bevacizumab is an antibody which binds to vascular endothelial growth factor A (VEGFA) and inhibits its receptor interaction, thus obstructing the formation of new blood vessels., Methods: A phase II randomized clinical trial of 123 patients with Her2-negative breast cancer was conducted, with patients treated with neoadjuvant chemotherapy (fluorouracil (5FU)/epirubicin/cyclophosphamide (FEC) and taxane), with or without bevacizumab. Serial biopsies were obtained at time of diagnosis, after 12 weeks of treatment with FEC ± bevacizumab, and after 25 weeks of treatment with taxane ± bevacizumab. A time course study was designed to investigate the genomic landscape at the three time points when tumor DNA alterations, tumor percentage, genomic instability, and tumor clonality were assessed. Substantial differences were observed with some tumors changing mainly between diagnosis and at 12 weeks, others between 12 and 25 weeks, and still others changing in both time periods., Results: In both treatment arms, good responders (GR) and non-responders (NR) displayed significant difference in genomic instability index (GII) at time of diagnosis. In the combination arm, copy number alterations at 25 loci at the time of diagnosis were significantly different between the GR and NR. An inverse aberration pattern was also observed between the two extreme response groups at 6p22-p12 for patients in the combination arm. Signs of subclonal reduction were observed, with some aberrations disappearing and others being retained during treatment. Increase in subclonal amplification was observed at 6p21.1, a locus which contains the VEGFA gene for the protein which are targeted by the study drug bevacizumab. Of the 13 pre-treatment samples that had a gain at VEGFA, 12 were responders. Significant decrease of frequency of subclones carrying gains at 17q21.32-q22 was observed at 12 weeks, with the peak occurring at TMEM100, an ALK1 receptor signaling-dependent gene essential for vasculogenesis. This implies that cells bearing amplifications of VEGFA and TMEM100 are particularly sensitive to this treatment regime., Conclusions: Taken together, these results suggest that heterogeneity and subclonal architecture influence the response to targeted treatment in combination with chemotherapy, with possible implications for clinical decision-making and monitoring of treatment efficacy., Trial Registration: NCT00773695 . Registered 15 October 2008.

Cancers elicit an immune response by modifying the microenvironment. The immune system plays a pivotal role in cancer recognition and eradication. While the potential clinical value of infiltrating lymphocytes at the tumor site has been assessed in breast cancer, circulating cytokines - the molecules coordinating and fine-tuning immune response - are still poorly characterized. Using two breast cancer cohorts (MicMa, n = 131, DCTB, n = 28) and the multiplex Luminex platform, we measured the levels of 27 cytokines in the serum of breast cancer patients prior to treatment. We investigated the cytokine levels in relation to clinicopathological characteristics and in perspective of the tumor infiltrating immune cells predicted from the bulk mRNA expression data. Unsupervised clustering analysis of the serum cytokine levels in the MicMa cohort identified a cluster of pro-inflammatory, pro-angiogenic, and Th2-related cytokines which was associated with poor prognosis. Notably high levels of platelet derived growth factor BB (PDGF) reflected a more aggressive tumor phenotype and larger tumor size. A significant positive correlation between serum levels of interferon gamma-induced protein 10 (IP10) and its mRNA expression at the tumor site suggested that tumor-IP10-production may outflow to the bloodstream. High IP10 serum levels were associated with a worse prognosis. Finally, we found serum levels of both PDGF and IP10 associated with enrichment scores of specific tumor infiltrating immune cells. Our study suggests that monitoring cytokine circulating levels in breast cancer could be used to characterize breast cancers and the immune composition of their microenvironment through readily available biological material.

Background: The presence of disseminated tumor cells (DTCs) in bone marrow (BM) is an independent prognostic factor in early breast cancer but does not uniformly predict outcome. Tumor cells can persist in a quiescent state over time, but clinical studies of markers predicting the awakening potential of DTCs are lacking. Recently, experiments have shown that NR2F1 (COUP-TF1) plays a key role in dormancy signaling., Methods: We analyzed the NR2F1 expression in DTCs by double immunofluorescence (DIF) staining of extra cytospins prepared from 114 BM samples from 86 selected DTC-positive breast cancer patients. Samples collected at two or more time points were available for 24 patients. Fifteen samples were also analyzed for the proliferation marker Ki67., Results: Of the patients with detectable DTCs by DIF, 27% had ≥ 50% NR2F1 high DTCs, chosen a priori as the cut-off for "dormant profile" classification. All patients with systemic relapse within 12 months after BM aspiration carried ≤ 1% NR2F1 high DTCs, including patients who transitioned from having NR2F1 high -expressing DTCs in previous BM samples. Of the patients with serial samples, half of those with no relapse at follow-up had ≥ 50% NR2F1 high DTCs in the last BM aspiration analyzed. Among the 18 relapse-free patients at the time of the last DTC-positive BM aspiration with no subsequent BM analysis performed, distant disease-free intervals were favorable for patients carrying ≥ 50% NR2F1 high DTCs compared with those with predominantly NR2F1 low DTCs (p = 0.007, log-rank). No survival difference was observed by classification according to Ki67-expressing DTCs (p = 0.520)., Conclusions: Our study translates findings from basic biological analysis of DTC dormancy to the clinical situation and supports further clinical studies of NR2F1 as a marker of dormancy.

A high concentration of circulating vascular endothelial growth factor (VEGF) in cancer patients is associated with an aggressive tumor phenotype. Here, serum levels of 27 cytokines and blood cell counts were assessed in breast cancer patients receiving neoadjuvant chemotherapy with or without bevacizumab (Bev) in a randomized cohort of 132 patients with non-metastatic HER2-negative tumors. Cytokine levels were determined prior to treatment and at various time-points. The cytotoxic chemotherapy regimen of fluorouracil, epirubicin, and cyclophosphamide (FEC) had a profound impact on both circulating white blood cells and circulating cytokine levels. At the end of FEC treatment, the global decrease in cytokine levels correlated with the drop in white blood cell counts and was significantly greater in the patients of the Bev arm for cytokines, such as VEGF-A, IL-12, IP-10 and IL-10. Among patients who received Bev, those with pathological complete response (pCR) exhibited significantly lower levels of VEGF-A, IFN-γ, TNF-α and IL-4 than patients without pCR. This effect was not observed in the chemotherapy-only arm. Certain circulating cytokine profiles were found to correlate with different immune cell types at the tumor site. For the Bev arm patients, the serum cytokine levels correlated with higher levels of cytotoxic T cells at the end of the therapy regimen, which was indicative of treatment response. The higher response rate for Bev-treated patients and stronger correlations between serum cytokine levels and infiltrating CD8T cells merits further investigation.

Background: We conducted a meta-analysis in nonmetastatic breast cancer patients treated by neoadjuvant chemotherapy (NCT) to assess the clinical validity of circulating tumor cell (CTC) detection as a prognostic marker., Methods: We collected individual patient data from 21 studies in which CTC detection by CellSearch was performed in early breast cancer patients treated with NCT. The primary end point was overall survival, analyzed according to CTC detection, using Cox regression models stratified by study. Secondary end points included distant disease-free survival, locoregional relapse-free interval, and pathological complete response. All statistical tests were two-sided., Results: Data from patients were collected before NCT (n = 1574) and before surgery (n = 1200). CTC detection revealed one or more CTCs in 25.2% of patients before NCT; this was associated with tumor size (P < .001). The number of CTCs detected had a detrimental and decremental impact on overall survival (P < .001), distant disease-free survival (P < .001), and locoregional relapse-free interval (P < .001), but not on pathological complete response. Patients with one, two, three to four, and five or more CTCs before NCT displayed hazard ratios of death of 1.09 (95% confidence interval [CI] = 0.65 to 1.69), 2.63 (95% CI = 1.42 to 4.54), 3.83 (95% CI = 2.08 to 6.66), and 6.25 (95% CI = 4.34 to 9.09), respectively. In 861 patients with full data available, adding CTC detection before NCT increased the prognostic ability of multivariable prognostic models for overall survival (P < .001), distant disease-free survival (P < .001), and locoregional relapse-free interval (P = .008)., Conclusions: CTC count is an independent and quantitative prognostic factor in early breast cancer patients treated by NCT. It complements current prognostic models based on tumor characteristics and response to therapy.

Aim: To determine the value of mammography and breast ultrasound (US) in predicting outcomes in HER2 positive breast cancer patients (pts) within Neo-ALTTO trial., Patients and Methods: Mammography and US were required at baseline, week 6 and surgery. Two independent blinded investigators reviewed the measurements and assigned the corresponding response category. Pts showing complete or partial response according to RECIST (v1.1) were classified as responders. The association between imaging response at week 6 or prior to surgery was evaluated with respect to pathological complete response (pCR) and event-free Survival (EFS)., Results: Of the 455 pts enrolled in the trial, 267 (61%) and 340 (77%) had evaluable mammography and US at week 6; 248 (56%) and 309 (70%) pts had evaluable mammography and US prior to surgery. At week 6, 32% and 43% of pts were classified as responders by mammography and US, respectively. pCR rates were twice as high for responders than non-responders (week 6: 46% versus 23% by US, p < 0.0001; 41% versus 24% by mammography, p = 0.007). Positive and negative predictive values of mammography and US prior to surgery were 37% and 35%, and 82% and 70%, respectively. No significant correlation was found between response by mammography and/or US at week 6/surgery and EFS., Conclusions: Mammography and US were underused in Neo-ALTTO although US had the potential to assess early response whereas mammography to detect residual disease prior to surgery. Our data still emphasise the need for further imaging studies on pts treated with neoadjuvant HER2-targeted therapy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Controversies exist as to whether the genetic polymorphisms of the enzymes responsible for the metabolism of tamoxifen can predict breast cancer outcome in patients using adjuvant tamoxifen. Direct measurement of concentrations of active tamoxifen metabolites in serum may be a more biological plausible and robust approach. We have investigated the association between CYP2D6 genotypes, serum concentrations of active tamoxifen metabolites, and long-term outcome in tamoxifen treated breast cancer patients., Methods: From an original observational study comprising 817 breast cancer patients, 99 women with operable breast cancer were retrospectively included in the present study. This cohort of patients were adjuvantly treated with tamoxifen, had provided serum samples suitable for measuring tamoxifen metabolites, and were relapse-free at 3 years after the primary treatment commenced. The median follow-up time from this entry point to breast cancer death was 13.9 years. Patients were CYP2D6 genotyped and grouped into four CYP2D6 phenotype groups (Ultra rapid, extensive, intermediate, and poor metabolizers). Tamoxifen and nine metabolites were quantified in serum (n = 86) and compared with CYP2D6 phenotype groups and outcome., Results: Breast cancer patients with low concentrations of Z-4-hydroxy-tamoxifen (Z-4OHtam; ≤ 3.26 nM) had a breast cancer-specific survival (BCSS) of 60% compared to 84% in patients with Z-4OHtam concentrations > 3.26 nM (p = 0.020, log-rank hazard ratio (HR) = 3.56, 95% confidence interval (CI) = 1.14-11.07). For patients with Z-4-hydroxy-N-desmethyl-tamoxifen (Z-endoxifen) levels ≤ 9.00 nM BCSS was 57% compared to 84% for patients with concentrations > 9.00 nM (p = 0.029, HR = 3.73, 95% CI = 1.05-13.22). Low concentrations of Z-4OHtam and Z-endoxifen were associated with poorer survival also after adjusting for clinically relevant variables (HR = 4.27, 95% CI = 1.35-13.58, and HR = 3.70, 95% CI = 1.03-13.25, respectively). Overall survival analysis showed similar survival differences for both active metabolites. The Antiestrogen Activity Score showed comparable effects, but did not improve the prognostic information., Conclusions: Patients with Z-4OHtam and Z-endoxifen concentrations lower than 3.26 nM or 9.00 nM, respectively, showed an adverse outcome. Our results suggest that direct measurement of active tamoxifen metabolite concentrations could be of clinical value. Validation in larger study cohorts is warranted.

Background: The aim of this study was to investigate the prognostic value of the PAM50 intrinsic subtypes and risk of recurrence (ROR) score in patients with early breast cancer and long-term follow-up. A special focus was placed on hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) pN0 patients not treated with chemotherapy., Methods: Patients with early breast cancer (n = 653) enrolled in the observational Oslo1 study (1995-1998) were followed for distant recurrence and breast cancer death. Clinicopathological parameters were collected from hospital records. The primary tumors were analyzed using the Prosigna® PAM50 assay to determine the prognostic value of the intrinsic subtypes and ROR score in comparison with pathological characteristics. The primary endpoints were distant disease-free survival (DDFS) and breast cancer-specific survival (BCSS)., Results: Of 653 tumors, 52.2% were classified as luminal A, 26.5% as luminal B, 10.6% as HER2-enriched, and 10.7% as basal-like. Among the HR+/HER2- patients (n = 476), 37.8% were categorized as low risk by ROR score, 22.7% as intermediate risk, and 39.5% as high risk. Median follow-up durations for BCSS and DDFS were 16.6 and 7.1 years, respectively. Multivariate analysis showed that intrinsic subtypes (all patients) and ROR risk classification (HR+/HER2- patients) yielded strong prognostic information. Among the HR+/HER2- pN0 patients with no adjuvant treatment (n = 231), 53.7% of patients had a low ROR, and their prognosis at 15 years was excellent (15-year BCSS 96.3%). Patients with intermediate risk had reduced survival compared with those with low risk (p = 0.005). In contrast, no difference in survival between the low- and intermediate-risk groups was seen for HR+/HER2- pN0 patients who received tamoxifen only. Ki-67 protein, grade, and ROR score were analyzed in the unselected, untreated pT1pN0 HR+/HER2- population (n = 171). In multivariate analysis, ROR score outperformed both Ki-67 and grade. Furthermore, 55% of patients who according to the PREDICT tool ( http://www.predict.nhs.uk/ ) would be considered chemotherapy candidates were ROR low risk (33%) or luminal A ROR intermediate risk (22%)., Conclusions: The PAM50 intrinsic subtype classification and ROR score improve classification of patients with breast cancer into prognostic groups, allowing for a more precise identification of future recurrence risk and providing an improved basis for adjuvant treatment decisions. Node-negative patients with low ROR scores had an excellent outcome at 15 years even in the absence of adjuvant therapy.

Breast cancers exhibit genome-wide aberrant DNA methylation patterns. To investigate how these affect the transcriptome and which changes are linked to transformation or progression, we apply genome-wide expression-methylation quantitative trait loci (emQTL) analysis between DNA methylation and gene expression. On a whole genome scale, in cis and in trans, DNA methylation and gene expression have remarkably and reproducibly conserved patterns of association in three breast cancer cohorts (n = 104, n = 253 and n = 277). The expression-methylation quantitative trait loci associations form two main clusters; one relates to tumor infiltrating immune cell signatures and the other to estrogen receptor signaling. In the estrogen related cluster, using ChromHMM segmentation and transcription factor chromatin immunoprecipitation sequencing data, we identify transcriptional networks regulated in a cell lineage-specific manner by DNA methylation at enhancers. These networks are strongly dominated by ERα, FOXA1 or GATA3 and their targets were functionally validated using knockdown by small interfering RNA or GRO-seq analysis after transcriptional stimulation with estrogen.

Purpose: Chemotherapy-induced alterations to gene expression are due to transcriptional reprogramming of tumor cells or subclonal adaptations to treatment. The effect on whole-transcriptome mRNA expression was investigated in a randomized phase II clinical trial to assess the effect of neoadjuvant chemotherapy with the addition of bevacizumab. Experimental Design: Tumor biopsies and whole-transcriptome mRNA profiles were obtained at three fixed time points with 66 patients in each arm. Altogether, 358 specimens from 132 patients were available, representing the transcriptional state before treatment start, at 12 weeks and after treatment (25 weeks). Pathologic complete response (pCR) in breast and axillary nodes was the primary endpoint. Results: pCR was observed in 15 patients (23%) receiving bevacizumab and chemotherapy and 8 patients (12%) receiving only chemotherapy. In the estrogen receptor-positive patients, 11 of 54 (20%) treated with bevacizumab and chemotherapy achieved pCR, while only 3 of 57 (5%) treated with chemotherapy reached pCR. In patients with estrogen receptor-positive tumors treated with combination therapy, an elevated immune activity was associated with good response. Proliferation was reduced after treatment in both treatment arms and most pronounced in the combination therapy arm, where the reduction in proliferation accelerated during treatment. Transcriptional alterations during therapy were subtype specific, and the effect of adding bevacizumab was most evident for luminal-B tumors. Conclusions: Clinical response and gene expression response differed between patients receiving combination therapy and chemotherapy alone. The results may guide identification of patients likely to benefit from antiangiogenic therapy. Clin Cancer Res; 23(16); 4662-70. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: Two randomized trials recently demonstrated that regional nodal irradiation (RNI) could reduce the risk of recurrence in early breast cancer; however, these trials were conducted in the pretrastuzumab era. Whether these results are applicable to human epidermal growth factor receptor 2 (HER2)-positive breast cancer patients treated with anti-HER2-targeted therapy is unknown., Methods: This retrospective analysis was performed on patients with node-positive breast cancer who were enrolled in the Adjuvant Lapatinib and/or Trastuzumab Treatment Optimization phase III adjuvant trial and subjected to BCS. The primary objective of the present study was to examine the effect of RNI on disease-free survival (DFS). A multivariable cox regression analysis adjusted for number of positive lymph nodes, tumor size, grade, age, hormone receptors status, presence of macrometastatis, treatment arm, and chemotherapy timing was carried out to investigate the relationship between RNI and DFS., Results: One thousand six hundred sixty-four HER2-positive breast cancer patients were included, of whom 878 (52.8%) had received RNI to the axillary, supraclavicular, and/or internal mammary lymph nodes. Patients in the RNI group had higher nodal burden and more frequently had tumors larger than 2 cm. At a median follow-up of 4.5 years, DFS was 84.3% in the RNI group and 88.3% in the non-RNI group. No differences in regional recurrence (0.9 % vs 0.6 %) or in overall survival (93.6% vs 95.3%) were observed between the two groups. After adjustment in multivariable analysis, there was no statistically significant association between RNI and DFS (hazard ratio = 0.96, 95% confidence interval = 0.71 to 1.29)., Conclusions: Our analysis did not demonstrate a DFS benefit of RNI in HER2-positive, node-positive patients treated with adjuvant HER2-targeted therapy. The benefit of RNI in HER2-positive breast cancer needs further testing within randomized clinical trials., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objective: The aim of this study was to analyse the survival impact of primary tumor nodal status (N0/N+) in patients with resectable colorectal liver metastases (CLM), and to determine the value of circulating and disseminated tumor cells (CTCs/DTCs) in this setting., Methods: In this prospective study of patients undergoing resection of CLM from 2008 to 2011, peripheral blood was analyzed for CTCs using the CellSearch System ® , and bone marrow was sampled for DTC analyses just prior to hepatic resection. The presence of one or more tumor cells was scored as CTC/DTC-positive. Following resection of the primary tumor, the lymph nodes (LNs) were examined by routine histopathological examination., Results: A total of 140 patients were included in this study; 38 patients (27.1%) were negative at the primary colorectal LN examination (N0). CTCs were detected in 12.1% of all patients; 5.3% of patients in the N0 group and 14.7% of patients in the LN-positive (N+) group (p = 0.156), with the LN-positive group (N+) consisting of both N1 and N2 patients. There was a significant difference in recurrence-free survival (RFS) when analysing the N0 group versus the N+ group (p = 0.007) and CTC-positive versus CTC-negative patients (p = 0.029). In multivariate analysis, CTC positivity was also significantly associated with impaired overall survival (OS) [p = 0.05], whereas DTC positivity was not associated with survival., Conclusion: In this cohort of resectable CLM patients, 27% had primary N0 colorectal cancer. Assessment of CTC in addition to nodal status may contribute to improved classification of patients into high- and low-risk groups, which has the potential to guide and improve treatment strategies.

Cancer cells can have different patterns of exon usage of individual genes when compared to normal tissue, suggesting that alternative splicing may play a role in shaping the tumor phenotype. The discovery and identification of gene variants has increased dramatically with the introduction of RNA-sequencing technology, which enables whole transcriptome analysis of known, as well as novel isoforms. Here we report alternative splicing and transcriptional events among subtypes of invasive ductal carcinoma in The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA) cohort. Alternative exon usage was widespread, and although common events were shared among three subtypes, ER+ HER2-, ER- HER2-, and HER2+, many events on the exon level were subtype specific. Additional RNA-seq analysis was carried out in an independent cohort of 43 ER+ HER2- and ER- HER2- primary breast tumors, confirming many of the exon events identified in the TCGA cohort. Alternative splicing and transcriptional events detected in five genes, MYO6, EPB41L1, TPD52, IQCG, and ACOX2 were validated by qRT-PCR in a third cohort of 40 ER+ HER2- and ER- HER2- patients, showing that these events were truly subtype specific.

The tumor microenvironment is now widely recognized for its role in tumor progression, treatment response, and clinical outcome. The intratumoral immunological landscape, in particular, has been shown to exert both pro-tumorigenic and anti-tumorigenic effects. Identifying immunologically active or silent tumors may be an important indication for administration of therapy, and detecting early infiltration patterns may uncover factors that contribute to early risk. Thus far, direct detailed studies of the cell composition of tumor infiltration have been limited; with some studies giving approximate quantifications using immunohistochemistry and other small studies obtaining detailed measurements by isolating cells from excised tumors and sorting them using flow cytometry. Herein we utilize a machine learning based approach to identify lymphocyte markers with which we can quantify the presence of B cells, cytotoxic T-lymphocytes, T-helper 1, and T-helper 2 cells in any gene expression data set and apply it to studies of breast tissue. By leveraging over 2,100 samples from existing large scale studies, we are able to find an inherent cell heterogeneity in clinically characterized immune infiltrates, a strong link between estrogen receptor activity and infiltration in normal and tumor tissues, changes with genomic complexity, and identify characteristic differences in lymphocyte expression among molecular groupings. With our extendable methodology for capturing cell type specific signal we systematically studied immune infiltration in breast cancer, finding an inverse correlation between beneficial lymphocyte infiltration and estrogen receptor activity in normal breast tissue and reduced infiltration in estrogen receptor negative tumors with high genomic complexity., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no competing interests.

The use of trastuzumab in patients with breast cancer that overexpresses human epidermal growth factor receptor 2 has significantly improved treatment outcomes. However, a substantial proportion of this patient group still experiences progression of the disease after receiving the drug. Evaluation of the changes in expression of the human epidermal growth factor receptors could be of interest. Monoclonal antibodies against the extracellular domain of the human growth factor receptors, 2, 3, and 4, have been raised, and specific and sensitive immunoassays have been established. Sera from healthy individuals (Nordic Reference Interval Project and Database) were analyzed in the human epidermal growth factor receptor 2 assay (N = 805) and the human epidermal growth factor receptor 3 and 4 assays (N = 114), and reference limits were calculated. In addition, sera from 208 individual patients with breast cancer were tested in all three assays. Finally, the human epidermal growth factor receptor 2 assay was compared with a chemiluminescent immunoassay for serum human epidermal growth factor receptor 2/neu. Reference values were as follows: human epidermal growth factor receptor 2, <2.5 µg/L; human epidermal growth factor receptor 3, <2.8 µg/L; and human epidermal growth factor receptor 4, <1.8 µg/L. There were significant differences in human epidermal growth factor receptor 2 and human epidermal growth factor receptor 3 serum levels between the patients with tissue human epidermal growth factor receptor 2-positive and tissue human epidermal growth factor receptor 2-negative ( p = 0.0026, p = 0.000011) tumors, but not in the serum levels of human epidermal growth factor receptor 4 ( p = 0.054). There was good agreement between the in-house human epidermal growth factor receptor 2 assay and the chemiluminescent immunoassay. Our new specific antibodies for all the three human epidermal growth factor receptors may prove valuable in the development of novel anti-human epidermal growth factor receptor targeted therapies with sensitive immunoassays for measuring serum levels of the respective targets and in monitoring established treatment.

Background: Breast cancer is a heterogeneous disease at the clinical and molecular level. In this study we integrate classifications extracted from five different molecular levels in order to identify integrated subtypes., Methods: Tumor tissue from 425 patients with primary breast cancer from the Oslo2 study was cut and blended, and divided into fractions for DNA, RNA and protein isolation and metabolomics, allowing the acquisition of representative and comparable molecular data. Patients were stratified into groups based on their tumor characteristics from five different molecular levels, using various clustering methods. Finally, all previously identified and newly determined subgroups were combined in a multilevel classification using a "cluster-of-clusters" approach with consensus clustering., Results: Based on DNA copy number data, tumors were categorized into three groups according to the complex arm aberration index. mRNA expression profiles divided tumors into five molecular subgroups according to PAM50 subtyping, and clustering based on microRNA expression revealed four subgroups. Reverse-phase protein array data divided tumors into five subgroups. Hierarchical clustering of tumor metabolic profiles revealed three clusters. Combining DNA copy number and mRNA expression classified tumors into seven clusters based on pathway activity levels, and tumors were classified into ten subtypes using integrative clustering. The final consensus clustering that incorporated all aforementioned subtypes revealed six major groups. Five corresponded well with the mRNA subtypes, while a sixth group resulted from a split of the luminal A subtype; these tumors belonged to distinct microRNA clusters. Gain-of-function studies using MCF-7 cells showed that microRNAs differentially expressed between the luminal A clusters were important for cancer cell survival. These microRNAs were used to validate the split in luminal A tumors in four independent breast cancer cohorts. In two cohorts the microRNAs divided tumors into subgroups with significantly different outcomes, and in another a trend was observed., Conclusions: The six integrated subtypes identified confirm the heterogeneity of breast cancer and show that finer subdivisions of subtypes are evident. Increasing knowledge of the heterogeneity of the luminal A subtype may add pivotal information to guide therapeutic choices, evidently bringing us closer to improved treatment for this largest subgroup of breast cancer.

Context: Chronic fatigue (CF) in breast cancer (BC) survivors is multifactorial and may be caused by immune activation triggered by BC or its treatment. In the Neoadjuvant Avastin in Breast Cancer study, BC patients received neoadjuvant chemotherapy (FEC100→taxane) ± bevacizumab, a monoclonal antibody with fatigue as a potential side effect., Objectives: To examine fatigue levels and prevalence of CF before and during chemotherapy and at follow-up, and their associations with C-reactive protein (CRP) and clinical variables., Methods: Eighty-four HER2-negative patients with cT2-4N0-3M0 BC responded to questionnaires and had CRP measured before treatment (T0), after FEC100 (T1), after taxanes before surgery (T2), and at two-year follow-up (T3)., Results: The prevalence of CF increased from 8% at T0 to 36% at T3, P < 0.0001. Fatigue levels peaked during chemotherapy from 12.0 at T0 to 20.0 at T2, and declined to 16.7 at T3, P < 0.001. Women with CF at T3 had higher fatigue levels at T0, T2, and T3 than those without CF (P ≤ 0.01). Psychological distress (P = 0.03) and pain (P = 0.04) at T3 were associated with CF at T3. Only psychological distress remained a significant predictor in multivariate analysis. CRP increased from T0 to T1 (P < 0.01) and declined to baseline values at T3, but changes were not associated with bevacizumab treatment. No association was found between bevacizumab or CRP, and fatigue levels or CF., Conclusion: Neither bevacizumab treatment nor low-grade systemic inflammation as measured by CRP was associated with the increased fatigue levels and raised prevalence of CF, observed during and after BC therapy. Increased fatigue levels at baseline and psychological distress at T3 were associated with CF at T3., (Copyright © 2017 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.)

Purpose: Prognostic factors are useful in order to identify early-stage breast cancer patients who might benefit from adjuvant treatment. The metastasis-promoting protein S100A4 has previously been associated with poor prognosis in breast cancer patients. The protein is expressed in diverse subcellular compartments, including the cytoplasm, extracellular space, and nucleus. Nuclear expression is an independent predictor of poor outcome in several cancer types, but the significance of subcellular expression has not yet been assessed in breast cancer., Methods: Nuclear and cytoplasmic expression of S100A4 was assessed by immunohistochemistry in prospectively collected tumor samples from early-stage breast cancer patients using tissue microarrays., Results: In patients not receiving adjuvant systemic therapy, nuclear or cytoplasmic expression was found in 44/291 tumors (15%). Expression of either nuclear or cytoplasmic S100A4 was associated with histological grade III, triple-negative subtype, and Ki-67-expression. Patients with S100A4-positive tumors had inferior metastasis-free and overall survival compared to S100A4-negative. When expression was analyzed separately, nuclear S100A4 was a significant predictor of outcome, while cytoplasmic was not. In patients who received adjuvant treatment 23/300 tumors (8%) were S100A4-positive, but no tumors displayed nuclear staining alone. S100A4-expression was strongly associated with histological grade III and triple-negative subtype. Although not significant, metastasis-free and overall survival was numerically reduced in patients with S100A4-positive tumors., Conclusion: S100A4-expression was associated with poor outcome in early-stage breast cancer, but the low percentage of positive tumors and the modest survival differences imply that the clinical utility in selection of patients for adjuvant treatment is limited.

Breast cancer patients with Luminal A disease generally have a good prognosis, but among this patient group are patients with good prognosis that are currently overtreated with adjuvant chemotherapy, and also patients that have a bad prognosis and should be given more aggressive treatment. There is no available method for subclassification of this patient group. Here we present a DNA methylation signature (SAM40) that segregates Luminal A patients based on prognosis, and identify one good prognosis group and one bad prognosis group. The prognostic impact of SAM40 was validated in four independent patient cohorts. Being able to subdivide the Luminal A patients may give the two-sided benefit of identifying one subgroup that may benefit from a more aggressive treatment than what is given today, and importantly, identifying a subgroup that may benefit from less treatment.

Background: Single-cell micro-metastases of solid tumors often occur in the bone marrow. These disseminated tumor cells (DTCs) may resist therapy and lay dormant or progress to cause overt bone and visceral metastases. The molecular nature of DTCs remains elusive, as well as when and from where in the tumor they originate. Here, we apply single-cell sequencing to identify and trace the origin of DTCs in breast cancer., Results: We sequence the genomes of 63 single cells isolated from six non-metastatic breast cancer patients. By comparing the cells' DNA copy number aberration (CNA) landscapes with those of the primary tumors and lymph node metastasis, we establish that 53% of the single cells morphologically classified as tumor cells are DTCs disseminating from the observed tumor. The remaining cells represent either non-aberrant "normal" cells or "aberrant cells of unknown origin" that have CNA landscapes discordant from the tumor. Further analyses suggest that the prevalence of aberrant cells of unknown origin is age-dependent and that at least a subset is hematopoietic in origin. Evolutionary reconstruction analysis of bulk tumor and DTC genomes enables ordering of CNA events in molecular pseudo-time and traced the origin of the DTCs to either the main tumor clone, primary tumor subclones, or subclones in an axillary lymph node metastasis., Conclusions: Single-cell sequencing of bone marrow epithelial-like cells, in parallel with intra-tumor genetic heterogeneity profiling from bulk DNA, is a powerful approach to identify and study DTCs, yielding insight into metastatic processes. A heterogeneous population of CNA-positive cells is present in the bone marrow of non-metastatic breast cancer patients, only part of which are derived from the observed tumor lineages.

Glycosylation and related processes play important roles in cancer development and progression, including metastasis. Several studies have shown that N-glycans have potential diagnostic value as cancer serum biomarkers. We have explored the significance of the abundance of particular serum N-glycan structures as important features of breast tumour biology by studying the serum glycome and tumour transcriptome (mRNA and miRNA) of 104 breast cancer patients. Integration of these types of molecular data allows us to study the relationship between serum glycans and transcripts representing functional pathways, such as metabolic pathways or DNA damage response. We identified tri antennary trigalactosylated trisialylated glycans in serum as being associated with lower levels of tumour transcripts involved in focal adhesion and integrin-mediated cell adhesion. These glycan structures were also linked to poor prognosis in patients with ER negative tumours. High abundance of simple monoantennary glycan structures were associated with increased survival, particularly in the basal-like subgroup. The presence of circulating tumour cells was found to be significantly associated with several serum glycome structures like bi and triantennary, di- and trigalactosylated, di- and trisialylated. The link between tumour miRNA expression levels and N-glycan production is also examined., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Background: The presence of circulating tumor cells (CTCs) is negatively associated with survival after resection of colorectal liver metastases (CLM). The current study aimed to determine the prognostic value of CTCs and disseminated tumor cells (DTCs) at the time of surgery and the prognostic value of CTCs at follow-up assessment, for patients scheduled to undergo two-stage hepatectomy with portal vein embolization (PVE) for CLM., Methods: Samples were collected at surgery (blood and bone marrow) and at follow-up assessment (blood) for the period 2008 through 2011. In this study, CTCs were detected with the CellSearch system, and DTCs were detected using standard immunocytochemical analysis., Results: Of 24 patients, 18 completed both stages, and no patients were lost to follow-up. The median overall survival (OS) was 37 months, and the median recurrence-free survival (RFS) was 7 months. At surgery, CTCs were found in nine patients (38 %), and their presence was associated with reduced OS (p < 0.001) and RFS (p = 0.006). Follow-up CTC status was available for 11 patients. All eight patients with positive CTC status experienced recurrence. Two of three patients with negative CTC status remained recurrence free. In seven patients (32 %), DTCs were detected but were not associated with OS or RFS., Conclusions: The presence of CTCs at surgery is associated with worse OS and RFS for patients undergoing two-stage hepatectomy with PVE for CLM. Analysis of CTCs should be explored further for their potential to assist in treatment decisions and monitoring for CLM patients.

WRAP53 protein controls intracellular trafficking of DNA repair proteins, the telomerase enzyme, and splicing factors. Functional loss of the protein has been linked to carcinogenesis, premature aging and neurodegeneration. The aim of this study was to investigate the prognostic significance of WRAP53 protein expression in breast cancer. A tissue microarray was constructed from primary breast tumors and immunostained by a polyclonal WRAP53 antibody to assess the protein expression pattern. Two different patient cohorts with long term follow-up were studied; a test- and a validation set of 154 and 668 breast tumor samples respectively. Breast cancer patients with tumor cells lacking the expression of WRAP53 in the nucleus had a significantly poorer outcome compared to patients with tumor cells expressing this protein in the nuclei (HR = 1.95, 95%CI = 1.09-3.51, p = 0.025). Nuclear localization of WRAP53 was further shown to be an independent marker of prognosis in multivariate analysis (HR = 2.57, 95%CI = 1.27-5.19, p = 0.008), and also significantly associated with better outcome in patients with TP53 mutation. Here we show that the sub-cellular localization of the WRAP53 protein has a significant impact on breast cancer survival, and thus has a potential as a clinical marker in diagnostics and treatment.

Background: Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of the human transcriptome. Different patterns of exon usage have been observed when comparing normal tissue to cancers, suggesting that variant transcripts may play a role in the tumor phenotype., Methods: Ribonucleic acid-sequencing (RNA-seq) data from breast cancer samples was used to identify an intronic start variant transcript of Acyl-CoA oxidase 2, ACOX2 (ACOX2-i9). Difference in expression between Estrogen Receptor (ER) positive and ER negative patients was assessed by the Wilcoxon rank sum test, and the findings validated in The Cancer Genome Atlas (TCGA) breast cancer dataset (BRCA). ACOX2-i9 expression was also assessed in cell lines using both quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analysis. Knock down by short hairpin RNA (shRNA) and colony formation assays were used to determine whether ACOX2-i9 expression would influence cellular fitness. The effect of ACOX2-i9 expression on patient survival was assessed by the Kaplan-Meier survival function, and association to clinical parameters was analyzed using a Fisher exact test., Results: The expression and translation of ACOX2-i9 into a 25 kDa protein was demonstrated in HepG2 cells as well as in several breast cancer cell lines. shRNA knock down of the ACOX2-i9 variant resulted in decreased cell viability of T47D and MDA-MB 436 cells. Moreover, expression of ACOX2-i9 was shown to be estrogen regulated, being induced by propyl pyrazoletriol and inhibited by tamoxifen and fulvestrant in ER+ T47D and Mcf-7 cells, but not in the ER- MDA-MB 436 cell line. This variant transcript showed expression predominantly in ER-positive breast tumors as assessed in our initial set of 53 breast cancers and further validated in 87 tumor/normal pairs from the TCGA breast cancer dataset, and expression was associated with better outcome in ER positive patients., Conclusions: ACOX2-i9 is specifically enriched in ER+ breast cancers where expression of the variant is associated with improved outcome. These data identify variant ACOX2 as a potential novel therapeutic biomarker in ER+ breast tumors.