Publications by authors named "Kristina Aaltonen"

24 Publications

  • Page 1 of 1

Anti-tumor effects of rigosertib in high-risk neuroblastoma.

Transl Oncol 2021 Aug 9;14(8):101149. Epub 2021 Jun 9.

Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund, Sweden. Electronic address:

High-risk neuroblastoma has a poor prognosis despite intense treatment, demonstrating the need for new therapeutic strategies. Here we evaluated the effects of rigosertib (ON-01910.Na) in preclinical models of high-risk neuroblastoma. Among several hundred cancer cell lines representing 24 tumor types, neuroblastoma was the most sensitive to rigosertib. Treatment of MYCN-amplified neuroblastoma organoids resulted in organoid disintegration, decreased cell viability, and increased apoptotic cell death. Neuroblastoma response to rigosertib involved G2M cell cycle arrest and decreased phosphorylation of AKT (Ser473) and ERK1/2 (Thr202/Tyr204). Rigosertib delayed tumor growth and prolonged survival of mice carrying neuroblastoma MYCN-amplified PDX tumors (median survival: 31 days, treated; 22 days, vehicle) accompanied with increased apoptosis in treated tumors. We further identified vincristine and rigosertib as a potential promising drug combination treatment. Our results show that rigosertib might be a useful therapeutic agent for MYCN-amplified neuroblastomas, especially in combination with existing agents.
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http://dx.doi.org/10.1016/j.tranon.2021.101149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8207190PMC
August 2021

PAM50 Intrinsic Subtype Profiles in Primary and Metastatic Breast Cancer Show a Significant Shift toward More Aggressive Subtypes with Prognostic Implications.

Cancers (Basel) 2021 Mar 30;13(7). Epub 2021 Mar 30.

Division of Surgery and Division of Oncology, Department of Clinical Sciences, Lund University, SE-223 81 Lund, Sweden.

Background: PAM50 breast cancer intrinsic subtyping adds prognostic information in early breast cancer; however, the role in metastatic disease is unclear. We aimed to identify PAM50 subtypes in primary tumors (PTs) and metastases to outline subtype changes and their prognostic role.

Methods: RNA was isolated from PTs, lymph node metastases (LNMs), and distant metastases (DMs) in metastatic breast cancer patients ( = 140) included in a prospective study (NCT01322893). Gene expression analyses were performed using the Breast Cancer 360 (BC360) assay from Nano-String. The subtype shifts were evaluated using McNemar and symmetry tests, and clinical outcomes were evaluated with log-rank tests and Cox regression.

Results: The PAM50 subtype changed in 25/59 of paired samples between PTs and LNMs ( = 0.002), in 31/61 between PTs and DMs ( < 0.001), and in 16/38 between LNMs and DMs ( = 0.004). Shifts toward subtypes with worse outcomes were the most common. Patients with shifts from the luminal PT to non-luminal DM subtypes had worse progression-free survival compared to patients with a stable subtype (hazard ratio (HR): 2.3; 95% confidence interval (CI): 1.14-4.68, = 0.02).

Conclusion: Strong evidence of PAM50 subtype shifts toward unfavorable subtypes were seen between PTs and metastatic samples. For patients with a shift in subtype from luminal PT to non-luminal DM, a worse prognosis was noted.
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http://dx.doi.org/10.3390/cancers13071592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037951PMC
March 2021

Inhibition of fatty acid synthesis induces differentiation and reduces tumor burden in childhood neuroblastoma.

iScience 2021 Feb 1;24(2):102128. Epub 2021 Feb 1.

Department of Microbiology, Tumor and Cell Biology (MTC), Biomedicum B7, Karolinska Institutet, 171 65 Stockholm, Sweden.

Many metabolic pathways, including lipid metabolism, are rewired in tumors to support energy and biomass production and to allow adaptation to stressful environments. Neuroblastoma is the second deadliest solid tumor in children. Genetic aberrations, as the amplification of the -oncogene, correlate strongly with disease progression. Yet, there are only a few molecular targets successfully exploited in the clinic. Here we show that inhibition of fatty acid synthesis led to increased neural differentiation and reduced tumor burden in neuroblastoma xenograft experiments independently of -status. This was accompanied by reduced levels of the MYCN or c-MYC oncoproteins and activation of ERK signaling. Importantly, the expression levels of genes involved in fatty acid synthesis showed prognostic value for neuroblastoma patients. Our findings demonstrate that inhibition of fatty acid synthesis is a promising pharmacological intervention strategy for the treatment of neuroblastoma independently of -status.
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http://dx.doi.org/10.1016/j.isci.2021.102128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895756PMC
February 2021

Therapeutic targeting of KSP in preclinical models of high-risk neuroblastoma.

Sci Transl Med 2020 09;12(562)

Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, 223 81 Lund, Sweden.

Neuroblastoma is a childhood malignancy with often dismal prognosis; relapse is common despite intense treatment. Here, we used human tumor organoids representing multiple -amplified high-risk neuroblastomas to perform a high-throughput drug screen with approved or emerging oncology drugs. Tumor-selective effects were calculated using drug sensitivity scores. Several drugs with previously unreported anti-neuroblastoma effects were identified by stringent selection criteria. ARRY-520, an inhibitor of kinesin spindle protein (KSP), was among those causing reduced viability. High expression of the KSP-encoding gene was associated with poor outcome in neuroblastoma. Genome-scale loss-of-function screens in hundreds of human cancer cell lines across 22 tumor types revealed that is particularly important for neuroblastoma cell viability. KSP inhibition in neuroblastoma patient-derived xenograft (PDX) cells resulted in the formation of abnormal monoastral spindles, mitotic arrest, up-regulation of mitosis-associated genes, and apoptosis. In vivo, KSP inhibition caused regression of -amplified neuroblastoma PDX tumors. Furthermore, treatment of mice harboring orthotopic neuroblastoma PDX tumors resulted in increased survival. Our results suggested that KSP inhibition could be a promising treatment strategy in children with high-risk neuroblastoma.
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http://dx.doi.org/10.1126/scitranslmed.aba4434DOI Listing
September 2020

The Distribution of Circulating Tumor Cells Is Different in Metastatic Lobular Compared to Ductal Carcinoma of the Breast-Long-Term Prognostic Significance.

Cells 2020 07 17;9(7). Epub 2020 Jul 17.

Department of Clinical Sciences, Division of Surgery, Lund University, SE-223 81 Lund, Sweden.

Background: Invasive lobular carcinoma (ILC) has distinguishing features when compared to invasive ductal carcinoma of no special type (NST). In this study, we explored the distributional and prognostic characteristics of circulating tumor cells (CTCs) in metastatic ILC and NST.

Materials And Methods: Patients were included in an observational trial (ClinicalTrials.gov NCT01322893) with ILC ( = 28) and NST ( = 111). CTC count (number/7.5 mL blood) was evaluated with serial sampling (CellSearch). The primary endpoint was progression-free survival (PFS).

Results: The CTC counts were higher in ILC (median 70) than in NST cases (median 2) at baseline ( < 0.001). The evidence for ≥5 CTCs as a prognostic factor for PFS in ILC was weak, but stronger with higher cut-offs (CTC ≥ 20: hazard ratio (HR) 3.0, = 0.01) (CTC ≥ 80: HR 3.6, = 0.004). In NST, however, the prognostic effect of CTCs ≥5 was strong. Decline in CTC count from baseline to three months was associated with improved prognosis in ILC and NST.

Conclusions: The number of CTCs is higher in ILC than in NST, implying that a higher CTC cut-off could be considered for ILC when applying the CellSearch technique.
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http://dx.doi.org/10.3390/cells9071718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407940PMC
July 2020

Evolution of Estrogen Receptor Status from Primary Tumors to Metastasis and Serially Collected Circulating Tumor Cells.

Int J Mol Sci 2020 Apr 20;21(8). Epub 2020 Apr 20.

Division of Surgery, Department of Clinical Sciences Lund, Lund University, SE-223 81 Lund, Sweden.

Background: The estrogen receptor (ER) can change expression between primary tumor (PT) and distant metastasis (DM) in breast cancer. A tissue biopsy reflects a momentary state at one location, whereas circulating tumor cells (CTCs) reflect real-time tumor progression. We evaluated ER-status during tumor progression from PT to DM and CTCs, and related the ER-status of CTCs to prognosis.

Methods: In a study of metastatic breast cancer, blood was collected at different timepoints. After CellSearch enrichment, CTCs were captured on DropMount slides and evaluated for ER expression at baseline (BL) and after 1 and 3 months of therapy. Comparison of the ER-status of PT, DM, and CTCs at different timepoints was performed using the McNemar test. The primary endpoint was progression-free survival (PFS).

Results: Evidence of a shift from ER positivity to negativity between PT and DM was demonstrated ( = 0.019). We found strong evidence of similar shifts from PT to CTCs at different timepoints ( < 0.0001). ER-positive CTCs at 1 and 3 months were related to better prognosis.

Conclusions: A shift in ER-status from PT to DM/CTCs was demonstrated. ER-positive CTCs during systemic therapy might reflect the retention of a favorable phenotype that still responds to therapy.
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http://dx.doi.org/10.3390/ijms21082885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215368PMC
April 2020

Expression of epithelial-mesenchymal transition-related markers and phenotypes during breast cancer progression.

Breast Cancer Res Treat 2020 Jun 16;181(2):369-381. Epub 2020 Apr 16.

Division of Surgery, Department of Clinical Sciences, Lund, Lund University, Lund, Sweden.

Purpose: The study aimed to investigate expression of epithelial-to-mesenchymal transition (EMT)-related proteins and phenotypes during breast cancer progression and to relate this to patient outcome.

Methods: Protein expression patterns of E-cadherin, N-cadherin, twist, and vimentin were examined by immunohistochemistry on formalin-fixed paraffin-embedded samples from primary tumors (PTs) (n = 419), synchronous lymph node metastases (LNMs) (n = 131) and recurrences (n = 34) from patients included in an observational prospective primary breast cancer study. Markers were evaluated individually and combined as defined EMT phenotypes (epithelial, mesenchymal, partial EMT, and negative). EMT profiles were compared between matched tumor progression stages, and related to clinicopathological data and distant recurrence-free interval (DRFi).

Results: N-cadherin-positivity, vimentin-positivity, mesenchymal and partial EMT phenotypes were associated with more aggressive tumor characteristics such as triple-negative subtype. Single EMT markers and phenotype discordance rates between paired tumor samples were observed in the range of 2-35%. Non-epithelial phenotypes were more frequently identified in recurrences compared to PTs, however, no skewness of expression or phenotype was detected between PTs and matched LNMs or between PTs and matched recurrences (Exact McNemar test). Interestingly, patients with a twist positive PT had shorter DRFi, compared to patients with a twist negative PT (hazard ratio (HR) 2.4, 95% confidence interval (CI) 1.2-5.1, P = 0.02). Essentially, the same effect was seen in multivariable analysis (HR 2.5, 95% CI 0.97-6.6, P = 0.06).

Conclusion: The epithelial phenotype was indicated to be lost between PTs and recurrences as a reflection of tumor progression. Twist status of the PT was related to long-term prognosis warranting further investigation in larger cohorts.
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http://dx.doi.org/10.1007/s10549-020-05627-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188722PMC
June 2020

Serial evaluation of serum thymidine kinase activity is prognostic in women with newly diagnosed metastatic breast cancer.

Sci Rep 2020 03 11;10(1):4484. Epub 2020 Mar 11.

Department of Clinical Sciences, Lund, Division of Surgery, Lund University, Lund, Sweden.

The rapid development of new therapies in metastatic breast cancer (MBC), entails a need for improved prognostic and monitoring tools. Thymidine kinase 1 (TK1) is involved in DNA synthesis and its activity correlates to outcome in cancer patients. The aim of this study was to evaluate serum TK1 activity (sTK1) levels in MBC patients as a tool for prognostication and treatment monitoring. 142 women with MBC scheduled for 1 line systemic treatment were included in a prospective observational study. sTK1 was measured at baseline (BL) and at 1, 3 and 6 months and correlations to progression-free and overall survival (PFS, OS) evaluated. High sTK1 levels (above median) correlated to worse PFS and OS at BL, also after adjusting for other prognostic factors. sTK1 levels were significantly associated with PFS and OS measured from follow-up time points during therapy. Changes from 3 to 6 months during therapy significantly correlated to PFS and OS, whereas early changes did not. We could demonstrate sTK1 level as an independent prognostic factor in patients with newly diagnosed MBC. Changes in sTK1 levels from 3 to 6 months correlated to PFS and OS. Future studies of sTK1 are warranted to further define its clinical utility.
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http://dx.doi.org/10.1038/s41598-020-61416-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066186PMC
March 2020

Detection of circulating tumor cells and circulating tumor DNA before and after mammographic breast compression in a cohort of breast cancer patients scheduled for neoadjuvant treatment.

Breast Cancer Res Treat 2019 Sep 24;177(2):447-455. Epub 2019 Jun 24.

Department of Clinical Sciences Lund, Division of Surgery, Lund University, Lund, Sweden.

Purpose: It is not known if mammographic breast compression of a primary tumor causes shedding of tumor cells into the circulatory system. Little is known about how the detection of circulating biomarkers such as circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) is affected by breast compression intervention.

Methods: CTCs and ctDNA were analyzed in blood samples collected before and after breast compression in 31 patients with primary breast cancer scheduled for neoadjuvant therapy. All patients had a central venous access to allow administration of intravenous neoadjuvant chemotherapy, which enabled blood collection from superior vena cava, draining the breasts, in addition to sampling from a peripheral vein.

Results: CTC and ctDNA positivity was seen in 26% and 65% of the patients, respectively. There was a significant increase of ctDNA after breast compression in central blood (p = 0.01), not observed in peripheral testing. No increase related with breast compression was observed for CTC. ctDNA positivity was associated with older age (p = 0.05), and ctDNA increase after breast compression was associated with high Ki67 proliferating tumors (p = 0.04). CTCs were more abundant in central compared to peripheral blood samples (p = 0.04).

Conclusions: There was no significant release of CTCs after mammographic breast compression but more CTCs were present in central compared to peripheral blood. No significant difference between central and peripheral levels of ctDNA was observed. The small average increase in ctDNA after breast compression is unlikely to be clinically relevant. The results give support for mammography as a safe procedure from the point of view of CTC and ctDNA shedding to the blood circulation. The results may have implications for the standardization of sampling procedures for circulating tumor markers.
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http://dx.doi.org/10.1007/s10549-019-05326-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6661025PMC
September 2019

Longitudinal enumeration and cluster evaluation of circulating tumor cells improve prognostication for patients with newly diagnosed metastatic breast cancer in a prospective observational trial.

Breast Cancer Res 2018 06 8;20(1):48. Epub 2018 Jun 8.

Department of Clinical Sciences Lund, Division of Surgery, Lund University, Medicon Village, SE-223 81, Lund, Sweden.

Background: Circulating tumor cells (CTCs) carry independent prognostic information in patients with metastatic breast cancer (MBC) on different lines of therapy. Moreover, CTC clusters are suggested to add prognostic information to CTC enumeration alone but their significance is unknown in patients with newly diagnosed MBC. We aimed to evaluate whether longitudinal enumeration of circulating tumor cells (CTCs) and CTC clusters could improve prognostication and monitoring of patients with metastatic breast cancer (MBC) starting first-line therapy.

Methods: This prospective study included 156 women with newly diagnosed MBC. CTCs and CTC clusters were detected using CellSearch technology at baseline (BL) and after 1, 3, and 6 months of systemic therapy. The primary end point was progression-free survival (PFS) and the secondary end point overall survival (OS). Median follow-up time was 25 (7-69) months.

Results: There were 79 (52%) and 30 (20%) patients with ≥ 5 CTCs and ≥ 1 CTC cluster at baseline, respectively; both factors were significantly associated with impaired survival. Landmark analyses based on follow-up measurements revealed increasing prognostic hazard ratios for ≥ 5 CTCs and CTC clusters during treatment, predicting worse PFS and OS. Both factors added value to a prognostic model based on clinicopathological variables at all time points and ≥ 5 CTCs and presence of CTC clusters enhanced the model's C-index to > 0.80 at 1, 3, and 6 months. Importantly, changes in CTCs during treatment were significantly correlated with survival and patients with a decline from ≥ 5 CTCs at BL to < 5 CTCs at 1 month had a similar odds ratio for progression to patients with < 5 CTCs at BL and 1 month. Stratification of patients based on CTC count and CTC clusters into four groups (0 CTCs, 1-4 CTCs, ≥ 5 CTCs, and ≥ 1 CTC + CTC clusters) demonstrated that patients with CTC clusters had significantly worse survival compared to patients without clusters.

Conclusions: Longitudinal evaluation of CTC and CTC clusters improves prognostication and monitoring in patients with MBC starting first-line systemic therapy. The prognostic value increases over time, suggesting that changes in CTC count are clinically relevant. The presence of CTC clusters adds significant prognostic value to CTC enumeration alone.

Trial Registration: NCT01322893 . Registered on 25 March 2011.
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http://dx.doi.org/10.1186/s13058-018-0976-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994056PMC
June 2018

The PDGF pathway in breast cancer is linked to tumour aggressiveness, triple-negative subtype and early recurrence.

Breast Cancer Res Treat 2018 Jun 29;169(2):231-241. Epub 2018 Jan 29.

Division of Surgery, Department of Clinical Sciences Lund, Skåne University Hospital, Lund University, SE-221 85, Lund, Sweden.

Purpose: The platelet-derived growth factor (PDGF) signalling pathway is often dysregulated in cancer and PDGF-receptor expression has been linked to unfavourable prognostic factors in breast cancer (e.g. ER negativity, high Ki67 and high grade). This study aimed to evaluate the expression of PDGFRα, PDGFRβ and ligand PDGF-CC in breast cancer in relation to molecular subtypes and prognosis.

Methods: Protein expression of tumour and/or stromal cell PDGFRα, PDGFRβ and PDGF-CC was evaluated in primary tumours (N = 489), synchronous lymph node metastases (N = 135) and asynchronous recurrences (N = 39) using immunohistochemistry in a prospectively maintained cohort of primary breast cancer patients included during 1999-2003. Distant recurrence-free interval (DRFi) was the primary end-point.

Results: High expression of all investigated PDGF family members correlated to increasing Nottingham histopathological grade and high Ki67. Tumour cells displayed high expression of PDGFRα in 20%, and PDGF-CC in 21% of primary tumours, which correlated with the triple-negative subtype (TNBC). Patients with high PDGF-CC had inferior prognosis (P = 0.04) in terms of 5-year DRFi, whereas PDGFRα was up-regulated in lymph node metastasis and recurrences compared to primary tumours. High primary tumour PDGFRα was associated with increased risk of central nervous system (CNS) recurrence.

Conclusions: High PDGFRα and PDGF-CC expression were linked to breast cancer with an aggressive biological phenotype, e.g. the TNBC subtype, and high PDGF-CC increased the risk of 5-year distant recurrence. Tumour cell PDGFRα was significantly up-regulated in lymph node metastases and asynchronous recurrences. Our findings support an active role of the PDGF signalling pathway in tumour progression.
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http://dx.doi.org/10.1007/s10549-018-4664-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945746PMC
June 2018

Circulating tumor cells in patients with advanced urothelial carcinoma of the bladder: Association with tumor stage, lymph node metastases, FDG-PET findings, and survival.

Urol Oncol 2017 10 1;35(10):606.e9-606.e16. Epub 2017 Jul 1.

Department of Urology, Landspitali University Hospital, Reykjavik, Iceland.

Background: There are currently no methods in clinical use that can detect early systemic dissemination of urothelial tumor cells.

Objective: To evaluate measurement of circulating tumor cells (CTCs) as a biomarker for disseminated disease in patients with advanced bladder cancer.

Design, Setting, And Participants: Between March 2013 and October 2015, 88 patients were prospectively included in the study: 78 were scheduled for radical cystectomy (RC) ± perioperative chemotherapy and 10 treated with palliative chemotherapy. The CellSearch CTC test was further assessed in this context by investigating expression of epithelial cell adhesion molecule (EpCAM) in primary tumors obtained at cystectomy from an independent cohort of 409 patients.

Outcome Measurements And Statistical Analysis: Presence of CTCs was tested for association with tumor stage, lymph node metastases, metastatic disease on [18 F]-fluorodeoxyglucose-positron emission tomography (FDG-PET), and cancer-specific and progression-free survival.

Results: CTCs were detected in 17/88 patients (19%). In 61 patients who underwent FDG-PET-computed tomography (CT), a statistically significant association with presence of CTCs was found for radiological metastatic disease but not for normal PET-CT results (12/35 [34%] vs. 2/26 [8%], P = 0.014). After a median follow-up time of 16.5 months (95% CI: 9.6-21.4), presence of CTCs was associated with an increased risk of progression among patients treated with RC with or without perioperative chemotherapy (n = 75, P = 0.049). A multivariate analysis adjusted for clinical tumor stage, clinical lymph node status, and age showed that CTCs were an independent marker of progression (n = 75; hazard ratio = 2.78; 95% CI: 1.005-7.69; P = 0.049) but not of cancer-specific death (P = 0.596). In 409 cystectomised patients, more than 392 (96%) of the bladder tumors expressed EpCAM.

Conclusions: CTCs were present in 19% of patients with advanced urothelial tumors and were associated with metastatic disease on FDG-PET-CT and with increased risk of disease progression after RC. A significant portion of urothelial cancer cells do express EpCAM and can thus be identified using EpCAM-antigen-based CTC detection methods.
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http://dx.doi.org/10.1016/j.urolonc.2017.05.021DOI Listing
October 2017

Molecular characterization of circulating tumor cells from patients with metastatic breast cancer reflects evolutionary changes in gene expression under the pressure of systemic therapy.

Oncotarget 2017 Jul;8(28):45544-45565

Department of Clinical Sciences Lund, Division of Surgery, Lund University, Lund, Sweden.

Resistance to systemic therapy is a major problem in metastatic breast cancer (MBC) that can be explained by initial tumor heterogeneity as well as by evolutionary changes during therapy and tumor progression. Circulating tumor cells (CTCs) detected in a liquid biopsy can be sampled and characterized repeatedly during therapy in order to monitor treatment response and disease progression.Our aim was to investigate how CTC derived gene expression of treatment predictive markers (ESR1/HER2) and other cancer associated markers changed in patient blood samples during six months of first-line systemic treatment for MBC. CTCs from 36 patients were enriched using CellSearch (Janssen Diagnostics) and AdnaTest (QIAGEN) before gene expression analysis was performed with a customized gene panel (TATAA Biocenter).Our results show that antibodies against HER2 and EGFR were valuable to isolate CTCs unidentified by CellSearch and possibly lacking EpCAM expression. Evaluation of patients with clinically different breast cancer subgroups demonstrated that gene expression of treatment predictive markers changed over time. This change was especially prominent for HER2 expression.In conclusion, we found that changed gene expression during first-line systemic therapy for MBC could be a possible explanation for treatment resistance. Characterization of CTCs at several time-points during therapy could be informative for treatment selection.
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http://dx.doi.org/10.18632/oncotarget.17271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542207PMC
July 2017

A FISH-based method for assessment of amplification status in breast cancer circulating tumor cells following CellSearch isolation.

Onco Targets Ther 2016 16;9:7095-7103. Epub 2016 Nov 16.

Division of Surgery, Department of Clinical Sciences Lund, Lund University, Lund; Department of Surgery, Skåne University Hospital, Malmö, Sweden.

Introduction: Amplification of the () proto-oncogene occurs in 10%-15% of primary breast cancer, leading to an activated HER-2 receptor, augmenting growth of cancer cells. Tumor classification is determined in primary tumor tissue and metastatic biopsies. However, malignant cells tend to alter their phenotype during disease progression. Circulating tumor cell (CTC) analysis may serve as an alternative to repeated biopsies. The Food and Drug Administration-approved CellSearch system allows determination of the HER-2 protein, but not of the gene. The aim of this study was to optimize a fluorescence in situ hybridization (FISH)-based method to quantitatively determine amplification in breast cancer CTCs following CellSearch-based isolation and verify the method in patient samples.

Methods: Using healthy donor blood spiked with human epidermal growth factor receptor 2 (HER-2)-positive breast cancer cell lines, SKBr-3 and BT-474, and a corresponding negative control (the HER-2-negative MCF-7 cell line), an in vitro CTC model system was designed. Following isolation in the CellSearch system, CTC samples were further enriched and fixed on microscope slides. Immunocytochemical staining with cytokeratin and 4',6-diamidino-2'-phenylindole dihydrochloride identified CTCs under a fluorescence microscope. A FISH-based procedure was optimized by applying the HER2 IQFISH pharmDx assay for assessment of amplification status in breast cancer CTCs.

Results: A method for defining the presence of amplification in single breast cancer CTCs after CellSearch isolation was established using cell lines as positive and negative controls. The method was validated in blood from breast cancer patients showing that one out of six patients acquired CTC amplification during treatment against metastatic disease.

Conclusion: amplification status of CTCs can be determined following CellSearch isolation and further enrichment. FISH is superior to protein assessment of status in predicting response to HER-2-targeted immunotherapy in breast cancer patients. This assay has the potential of identifying patients with a shift in status who may benefit from treatment adjustments.
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http://dx.doi.org/10.2147/OTT.S118502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5117892PMC
November 2016

Prognostic impact of circulating tumor cell apoptosis and clusters in serial blood samples from patients with metastatic breast cancer in a prospective observational cohort.

BMC Cancer 2016 07 8;16:433. Epub 2016 Jul 8.

Department of Surgery, Skåne University Hospital, SE-214 28, Malmö, Sweden.

Background: Presence of circulating tumor cells (CTCs) is a validated prognostic marker in metastatic breast cancer. Additional prognostic information may be obtained by morphologic characterization of CTCs. We explored whether apoptotic CTCs, CTC clusters and leukocytes attached to CTCs are associated with breast cancer subtype and prognosis at base-line (BL) and in follow-up (FU) blood samples in patients with metastatic breast cancer scheduled for first-line systemic treatment.

Methods: Patients with a first metastatic breast cancer event were enrolled in a prospective observational study prior to therapy initiation and the CellSearch system (Janssen Diagnostics) was used for CTC enumeration and characterization. We enrolled patients (N = 52) with ≥5 CTC/7.5 ml blood at BL (median 45, range 5-668) and followed them with blood sampling for 6 months during therapy. CTCs were evaluated for apoptotic changes, CTC clusters (≥3 nuclei), and leukocytes associated with CTC (WBC-CTC, ≥1 CTC + ≥1 leukocytes) at all time-points by visual examination of the galleries generated by the CellTracks Analyzer.

Results: At BL, patients with triple-negative and HER2-positive breast cancer had blood CTC clusters present more frequently than patients with hormone receptor-positive cancer (P = 0.010). No morphologic characteristics were associated with prognosis at BL, whereas patients with apoptotic CTCs or clusters in FU samples had worse prognosis compared to patients without these characteristics with respect to progression-free (PFS) and overall survival (OS) (log-rank test: P = 0.0012 or lower). Patients with apoptotic or clustered CTCs at any time-point had impaired prognosis in multivariable analyses adjusting for number of CTCs and other prognostic factors (apoptosis: HROS = 25, P < 0.001; cluster: HROS = 7.0, P = 0.006). The presence of WBC-CTCs was significantly associated with an inferior prognosis in terms of OS at 6 months in multivariable analysis.

Conclusions: Patients with a continuous presence of apoptotic or clustered CTCs in FU samples after systemic therapy initiation had worse prognosis than patients without these CTC characteristics. In patients with ≥5 CTC/7.5 ml blood at BL, morphologic characterization of persistent CTCs could be an important prognostic marker during treatment, in addition to CTC enumeration alone. Clinical Trials (NCT01322893), registration date 21 March 2011.
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http://dx.doi.org/10.1186/s12885-016-2406-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938919PMC
July 2016

Systemic Monocytic-MDSCs Are Generated from Monocytes and Correlate with Disease Progression in Breast Cancer Patients.

PLoS One 2015 20;10(5):e0127028. Epub 2015 May 20.

Center for Molecular Pathology, Jan Waldenströms gata 59, Skåne University Hospital (SUS), Lund University (LU), 20502 Malmö, Sweden.

Myeloid-derived suppressor cells (MDSCs) are highly immunosuppressive myeloid cells, which increase in cancer patients. The molecular mechanism behind their generation and function is unclear. Whereas granulocytic-MDSCs correlate with poor overall survival in breast cancer, the presence and relevance of monocytic-MDSCs (Mo-MDSCs) is unknown. Here we report for the first time an enrichment of functional blood Mo-MDSCs in breast cancer patients before they acquire a typical Mo-MDSC surface phenotype. A clear population of Mo-MDSCs with the typical cell surface phenotype (CD14(+)HLA-DR(low/-)CD86(low/-)CD80(low/-)CD163(low/-)) increased significantly first during disease progression and correlated to metastasis to lymph nodes and visceral organs. Furthermore, monocytes, comprising the Mo-MDSC population, from patients with metastatic breast cancer resemble the reprogrammed immunosuppressive monocytes in patients with severe infections, both by their surface and functional phenotype but also at their molecular gene expression profile. Our data suggest that monitoring the Mo-MDSC levels in breast cancer patients may represent a novel and simple biomarker for assessing disease progression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127028PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4439153PMC
April 2016

A novel method for downstream characterization of breast cancer circulating tumor cells following CellSearch isolation.

J Transl Med 2015 Apr 21;13:126. Epub 2015 Apr 21.

Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.

Background: Enumeration of circulating tumor cells (CTCs) obtained from minimally invasive blood samples has been well established as a valuable monitoring tool in metastatic and early breast cancer, as well as in several other cancer types. The gold standard technology for detecting CTCs in blood against a backdrop of millions of leukocytes is the FDA-approved CellSearch system (Janssen Diagnostics), which relies on EpCAM-based immunomagnetic separation. Secondary characterization of these cells could enable treatment selection based on specific targets in these cells, as well as providing a real time window into the metastatic process and offering unique insights into tumor heterogeneity. The objective of this study was to develop a method for downstream characterization of CTCs following isolation with the CellSearch system.

Methods: An in vitro CTC model system focusing on clinically useful treatment predictive biomarkers in breast cancer, specifically the estrogen receptor α (ERα) and the human epidermal growth factor receptor 2 (HER2), was established using healthy donor blood spiked with breast cancer cell lines MCF7 (ERα(+)/HER2(-)) and SKBr3 (ERα(-)/HER2(+)). Following CTC isolation by CellSearch, the captured CTCs were further enriched and fixed on a microscope slide using the in-house-developed CTC-DropMount technique.

Results: The recovery rate of CTCs after CellSearch Profile analysis and CTC-DropMount was 87%. A selective and consistent triple-immunostaining protocol was optimized. Cells positive for DAPI, cytokeratin (CK) 8, 18 and 19, but negative for the leukocyte-specific marker CD45, were classified as CTCs and subsequently analyzed for ERα and HER2 expression. The method was verified in breast cancer patient samples, thus demonstrating its clinical relevance.

Conclusions: Our results show that it is possible to ascertain the status of important predictive biomarkers expressed in breast cancer CTCs using the newly developed CTC-DropMount technique. Downstream characterization of multiple biomarkers using a standard fluorescence microscope demonstrates that important clinical and biological information may be obtained from a single patient blood sample following either CellSearch epithelial or profile analyses.

Trial Registration: Clinical Trials NCT01322893.
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http://dx.doi.org/10.1186/s12967-015-0493-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4409738PMC
April 2015

Association between insulin-like growth factor-1 receptor (IGF1R) negativity and poor prognosis in a cohort of women with primary breast cancer.

BMC Cancer 2014 Nov 3;14:794. Epub 2014 Nov 3.

Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Medicon Village, SE-223 81 Lund, Sweden.

Background: Resistance towards endocrine therapy is a great concern in breast cancer treatment and may partly be explained by the activation of compensatory signaling pathways. The aim of the present study was to investigate if the insulin-like growth factor-1 receptor (IGF1R) signaling pathway was activated or deregulated in breast cancer patients and to explore if any of the markers were prognostic, with or without adjuvant tamoxifen. This signaling pathway has been suggested to cause estrogen independent cell growth and thus contribute to resistance to endocrine treatment in estrogen receptor (ER) positive breast cancer.

Methods: The protein expression of IGF1R, phosphorylated Mammalian Target of Rapamycin (p-mTOR) and phosphorylated S6 ribosomal protein (p-S6rp) were investigated by immunohistochemistry using tissue microarrays in two patient cohorts. Cohort I (N = 264) consisted of mainly postmenopausal women with stage II breast cancer treated with tamoxifen for 2 years irrespective of ER status. Cohort II (N = 206) consisted of mainly medically untreated, premenopausal patients with node-negative breast cancer. Distant disease-free survival (DDFS) at 5 years was used as end-point for survival analyses.

Results: We found that lower IGF1R expression was associated with worse prognosis for tamoxifen treated, postmenopausal women (HR = 0.70, 95% CI = 0.52 - 0.94, p = 0.016). The effect was seen mainly in ER-negative patients where the prognostic effect was retained after adjustment for other prognostic markers (adjusted HR = 0.49, 95% CI = 0.29 - 0.82, p = 0.007). Expression of IGF1R was associated with ER positivity (p < 0.001) in the same patient cohort.

Conclusions: Our results support previous studies indicating that IGF1R positivity reflects a well differentiated tumor with low metastatic capacity. An association between lack of IGF1R expression and worse prognosis was mainly seen in the ER-negative part of Cohort I. The lack of co-activation of downstream markers (p-mTOR and p-S6rp) in the IGF1R pathway suggested that the prognostic effect was not due to complete activation of this pathway. Thus, no evidence could be found for a compensatory function of IGF1R signaling in the investigated cohorts.
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http://dx.doi.org/10.1186/1471-2407-14-794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4232733PMC
November 2014

The three receptor tyrosine kinases c-KIT, VEGFR2 and PDGFRα, closely spaced at 4q12, show increased protein expression in triple-negative breast cancer.

PLoS One 2014 15;9(7):e102176. Epub 2014 Jul 15.

Division of Surgery, Department of Clinical Sciences Lund, Skåne University Hospital, Lund, Sweden.

Background: Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer with poor prognosis and no targeted therapy available. Receptor tyrosine kinases (RTKs) are emerging targets in anticancer therapy and many RTK-inhibiting drugs are currently being developed. The aim of this study was to elucidate if there is a correlation between the protein expression of three RTKs c-KIT, VEGFR2 and PDGFRα, their gene copy number, and prognosis in TNBC compared to non-TNBC.

Methods: Tumor tissue samples from patients diagnosed with primary breast cancer were stained with immunohistochemistry (IHC) for protein assessment, and with fluorescence in situ hybridization (FISH) for gene copy number determination. Breast cancer mortality (BCM), measured from the date of surgery to death, was used as endpoint.

Results: The cohort included 464 patients, out of which 34 (7.3%) had a TNBC. High expression of the three RTKs was more common in TNBC compared to non-TNBC: c-KIT 49% vs. 10% (P<0.001), PDGFRα 32% vs. 19% (P = 0.07) and VEGFR2 32% vs. 6% (P<0.001). The odds ratio (OR) of c-KIT, VEGFR2 and PDGFRα positivity, adjusted for tumor characteristics, was 6.8, 3.6 and 1.3 times higher for TNBC than for non-TNBC. 73.5% of the TNBC had high expression of at least one of the three investigated receptors, compared to 30.0% of the non-TNBC (P<0.001). Survival analysis showed no significant difference in BCM for TNBC patients with high vs. low c-KIT, PDGFRα or VEGFR2 protein expression. 193 (42%) tumors were evaluated with FISH. No correlation was seen between increased gene copy number and TNBC, or between increased gene copy number and high protein expression of the RTK.

Conclusion: c-KIT, VEGFR2 and PDGFRα show higher protein expression in TNBC compared to non-TNBC. Further investigation clarifying the importance of these RTKs in TNBC is encouraged, as they are possible targets for anticancer therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102176PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4098911PMC
March 2015

Transurethral bladder tumor resection can cause seeding of cancer cells into the bloodstream.

J Urol 2015 Jan 1;193(1):53-7. Epub 2014 Jul 1.

Department of Urology, Clinical Sciences, Lund University, Skåne University Hospital, Malmö, Sweden. Electronic address:

Purpose: Transurethral bladder tumor resection is the initial diagnostic procedure for bladder cancer. Hypothetically tumor resection could induce seeding of cancer cells into the circulation and subsequent metastatic disease. In this study we ascertain whether transurethral bladder tumor resection induces measurable seeding of cancer cells into the vascular system.

Materials And Methods: Patients newly diagnosed with suspected invasive bladder cancer and planned for transurethral resection of bladder tumor in 2012 to 2013 were enrolled in the study. Before transurethral bladder tumor resection a vascular surgeon placed a venous catheter in the inferior vena cava via the femoral vein. Blood samples were drawn before and during the resection from the inferior vena cava and a peripheral vein, and analyzed for circulating cancer cells using the CellSearch® system. The number of circulating tumor cells identified was compared in preoperative and intraoperative blood samples.

Results: The circulating tumor cell data on 16 eligible patients were analyzed. In 6 of 7 positive inferior vena cava samples (86%) the number of circulating tumor cells was increased intraoperatively (28 vs 9, 28 vs 0, 28 vs 5, 3 vs 0, 4 vs 0, 1 vs 0), and results were similar, although less conclusive, for the corresponding peripheral vein samples.

Conclusions: Our study confirms that tumor cells can be released into the circulation during transurethral bladder tumor resection. It is currently unknown whether this will increase the risk of metastatic disease.
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http://dx.doi.org/10.1016/j.juro.2014.06.083DOI Listing
January 2015

No evidence for shedding of circulating tumor cells to the peripheral venous blood as a result of mammographic breast compression.

Breast Cancer Res Treat 2013 Sep 29;141(2):187-95. Epub 2013 Aug 29.

Medical Radiation Physics, Department of Clinical Sciences, Lund University, Skåne University Hospital Malmö, 205 02, Malmö, Sweden,

This pilot study aimed to investigate whether mammographic compression procedures might cause shedding of tumor cells into the circulatory system as reflected by circulating tumor cell (CTC) count in peripheral venous blood samples. From March to October 2012, 24 subjects with strong suspicion of breast malignancy were included in the study. Peripheral blood samples were acquired before and after mammography. Enumeration of CTCs in the blood samples was performed using the CellSearch(®) system. The pressure distribution over the tumor-containing breast was measured using thin pressure sensors. The median age was 66.5 years (range, 51-87 years). In 22 of the 24 subjects, breast cancer was subsequently confirmed. The difference between the average mean tumor pressure 6.8 ± 5.3 kPa (range, 1.0-22.5 kPa) and the average mean breast pressure 3.4 ± 1.6 kPa (range, 1.5-7.1 kPa) was statistically significant (p < 0.001), confirming that there was increased pressure over the tumor. The median pathological tumor size was 19 mm (range, 9-30 mm). Four subjects (17 %) were CTC positive before compression and two of these (8 %) were also CTC positive after compression. A total of seven CTCs were isolated with a mean size of 8 × 6 μm(2) (range of the longest diameter, 5-12 μm). The study supports the view that mammography is a safe procedure from the point of view of tumor cell shedding to the peripheral blood.
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http://dx.doi.org/10.1007/s10549-013-2674-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3785181PMC
September 2013

Co-targeting of the PI3K pathway improves the response of BRCA1 deficient breast cancer cells to PARP1 inhibition.

Cancer Lett 2012 Jun 20;319(2):232-241. Epub 2012 Jan 20.

Department of Oncology, Clinical Sciences, Lund University, Lund, Sweden; CREATE Health Strategic Center for Translational Cancer Research, Lund University, Lund, Sweden. Electronic address:

Although pre-clinical and clinical studies on PARP1 inhibitors, alone and in combination with DNA-damaging agents, show promising results, further ways to improve and broaden the scope of application of this therapeutic approach are warranted. To this end, we have investigated the possibility of improving the response of BRCA1 mutant breast cancer cells to PARP1 inhibition by co-targeting the PI3K pathway. Human breast cancer cell lines with or without the expression of BRCA1 and/or PTEN were treated with PARP1 and PI3K inhibitors as single agents and in combination. PARP1 inhibition induced DNA damage conferring a G2/M arrest and decrease in viability, paralleled by the induction of apoptosis. PI3K inhibition alone caused a G1 arrest and decreased cell growth. Most importantly, sequential combination of PARP and PI3K inhibitors interacted synergistically to significantly decrease growth compared to PARP inhibition alone. Global transcriptional profiling revealed that this decrease in growth was associated with down-regulation of macromolecule biosynthesis and the induction of apoptosis. Taken together, these results suggest an improved treatment strategy for BRCA1-mutant and possibly also triple-negative breast cancers with similar molecular defects.
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http://dx.doi.org/10.1016/j.canlet.2012.01.015DOI Listing
June 2012

Increased gene copy number of KIT and VEGFR2 at 4q12 in primary breast cancer is related to an aggressive phenotype and impaired prognosis.

Genes Chromosomes Cancer 2012 Apr 14;51(4):375-83. Epub 2011 Dec 14.

Department of Oncology, Clinical Sciences and CREATE Health Strategic Center for Translational Cancer Research, Lund University, Lund, Sweden.

Triple-negative breast cancer (TNBC) is associated with poor prognosis and no targeted treatments are available for TNBC. Drugs inhibiting tyrosine kinases, such as vascular endothelial growth factor receptor 2 (VEGFR2) and KIT, have shown some promising results for patients with TNBC. The aim of the study was to investigate whether gains and/or amplifications of VEGFR2 and KIT, located at 4q12, occur in TNBC. Fluorescence in situ hybridization (FISH) was used to quantify gene copy numbers of VEGFR2 and KIT in 83 primary human breast cancers including 31 TNBCs. Gains were defined as ≥ 4 gene copies in >40% of the cancer cells, whereas amplification was defined as CEP >2 in more than 10% of the cancer cells. A tumor was considered FISH positive for KIT and/or VEGFR2 if it displayed copy number gain and/or amplification. Ten (32%) of the TNBCs were VEGFR2 FISH positive and nine (29%) were KIT FISH positive, whereas non-TNBCs were FISH positive for VEGFR2 and KIT in nine (18%) cases for both genes, but no significant difference between TNBCs and non-TNBCs was found. FISH positivity for VEGFR2 and KIT was significantly correlated (χ(2) test, P < 0.001), and significantly related to ER negativity and high Nottingham histological grade (NHG). A significantly worse 5-year breast cancer specific survival (BCSS) was seen for FISH positive cases. Increased copy number of VEGFR2 and KIT thus has the potential of functioning as a novel predictive biomarker for selected targeted therapy particularly in the difficult-to-treat TNBC patient category.
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http://dx.doi.org/10.1002/gcc.21922DOI Listing
April 2012

Laser capture microdissection (LCM) and whole genome amplification (WGA) of DNA from normal breast tissue --- optimization for genome wide array analyses.

BMC Res Notes 2011 Mar 18;4:69. Epub 2011 Mar 18.

Department of Oncology, Clinical Sciences, Lund, Lund University, Barngatan 2B, SE-221 85 Lund, Sweden.

Background: Laser capture microdissection (LCM) can be applied to tissues where cells of interest are distinguishable from surrounding cell populations. Here, we have optimized LCM for fresh frozen normal breast tissue where large amounts of fat can cause problems during microdissection. Since the amount of DNA needed for genome wide analyses, such as single nucleotide polymorphism (SNP) arrays, is often greater than what can be obtained from the dissected tissue, we have compared three different whole genome amplification (WGA) kits for amplification of DNA from LCM material. In addition, the genome wide profiling methods commonly used today require extremely high DNA quality compared to PCR based techniques and DNA quality is thus critical for successful downstream analyses.

Findings: We found that by using FrameSlides without glass backing for LCM and treating the slides with acetone after staining, the problems caused by excessive fat could be significantly decreased. The amount of DNA obtained after extraction from LCM tissue was not sufficient for direct SNP array analysis in our material. However, the two WGA kits based on Phi29 polymerase technology (Repli-g® (Qiagen) and GenomiPhi (GE Healthcare)) gave relatively long amplification products, and amplified DNA from Repli-g® gave call rates in the subsequent SNP analysis close to those from non-amplified DNA. Furthermore, the quality of the input DNA for WGA was found to be essential for successful SNP array results and initial DNA fragmentation problems could be reduced by switching from a regular halogen lamp to a VIS-LED lamp during LCM.

Conclusions: LCM must be optimized to work satisfactorily in difficult tissues. We describe a work flow for fresh frozen normal breast tissue where fat is inclined to cause problems if sample treatment is not adapted to this tissue. We also show that the Phi29-based Repli-g® WGA kit (Qiagen) is a feasible approach to amplify DNA of high quality prior to genome wide analyses such as SNP profiling.
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http://dx.doi.org/10.1186/1756-0500-4-69DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068970PMC
March 2011
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