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Cancer is a highly heterogeneous disease, even within the same patient. Biopsies taken from different regions of a tumor may stand in stark molecular contrast to each other. Therefore, the ability to generate meaningful data from multiple platforms using the same biopsy is crucial for translating multi-omics characterizations into the clinic. However, it is generally a cumbersome and lengthy procedure to generate DNA, RNA and protein material from the same biopsy. The Qiagen AllPrep kit is an accessible, straightforward, and widely used kit in clinics worldwide to process biopsies and generate genomic and transcriptomic data from tumors and we asked if high quality proteomics data could be obtained from the remaining material. Here we develop a procedure for generating deep and quantitatively accurate proteomic information in high throughput from AllPrep flowthrough. With a number of refinements, we obtain in excess of 9,000 quantified proteins, a substantial coverage of the total proteome. We also successfully characterize the signaling state of the tumors using phosphoproteomics. Combining a standard kit with in-depth proteomics will be an attractive approach for clinics seeking to implement multi-omics-based precision oncology.Graphical abstract

Responses to immunotherapy can be very durable but acquired resistance leading to tumor progression often occurs. We investigated a patient with melanoma resistant to anti–programmed death 1 (anti–PD-1) who participated in the CA224-020 clinical trial (NCT01968109) and had further progression after an initial objective response to anti–PD-1 plus anti–lymphocyte activation gene 3. We found consecutive acquisition of beta-2 microglobulin (B2M) loss and impaired Janus kinase 1 (JAK1) signaling that coexisted in progressing tumor cells. Functional analyses revealed a pan T-cell immune escape phenotype, where distinct alterations mediated independent immune resistance to tumor killing by autologous CD8+ tumor-infiltrating lymphocytes (TIL; B2M loss) and CD4+ TILs (impaired JAK1 signaling). These findings shed light on the complexity of acquired resistance to immunotherapy in the post anti–PD-1 setting, indicating that coexisting altered pathways can lead to pan T-cell immune escape.

Supplementary Figure from Coexisting Alterations of MHC Class I Antigen Presentation and IFNγ Signaling Mediate Acquired Resistance of Melanoma to Post–PD-1 Immunotherapy

Patient data

Purpose:We evaluated the clinical benefit of tumor molecular profiling to select treatment in the phase I setting.Experimental Design:Patients with advanced solid cancers and exhausted treatment options referred to a phase I unit were included in a prospective, single-center, single-arm open-label study (NCT02290522). Tumor biopsies were obtained for comprehensive genomic analysis including whole-exome sequencing and RNA sequencing. When possible, patients were treated with regimen matched to the genomic profile. Primary endpoint was progression-free survival (PFS).Results:From May 2013 to January 2017, a total of 591 patients were enrolled, with 500 patients undergoing biopsy. Genomic profiles were obtained in 460 patients and a potential actionable target was identified in 352 (70%) of 500 biopsied patients. A total of 101 patients (20%) received matched treatment based on either gene mutations or RNA expression levels of targets available in early clinical trials or off-label treatment. Objective response according to RECIST1.1 was observed in 15 of 101 patients (0% complete response, 15% partial response), with a median PFS of 12 weeks (95% confidence interval, 9.9–14.4).Conclusions:Our study supports the feasibility of genomic profiling to select patients in the phase I setting and suggests that genomic matching can be beneficial for a minor subset of patients with no other treatment options. Randomized studies may validate this assumption.See related commentary by Ratain, p. 1136

Kaplan-Meier PFS

Potentiel actionable targets divided in cancer subtypes

BACKGROUND: Mutational analysis of circulating tumor DNA (ctDNA) can potentially be used for early detection of recurrence after resection for hepatocellular carcinoma (HCC). Mutations from tumor may be identified in plasma as an early sign of recurrence. We conducted a pilot study investigating if somatic mutations could be detected in plasma in patients undergoing liver resection for HCC and in patients with advanced non-resectable HCC.METHODS AND RESULTS: We prospectively included patients undergoing curative liver resection for HCC. Tumor tissue was investigated with whole exome sequencing and preoperative blood samples were evaluated for ctDNA using targeted next-generation sequencing (NGS) with TruSight Oncology 500 including 523 cancer-associated genes. Subsequently, the method was evaluated in patients with advanced HCC. We included eight patients curatively resected for HCC, where tumor tissue mutations were identified in seven patients. However, only in one patient tumor specific mutations were found in the preoperative blood sample. In all three patients with advanced HCC, tumor mutations were detected in the blood.CONCLUSIONS: In patients with resectable HCC, ctDNA could not be reliably detected using the applied targeted NGS method. In contrast, ctDNA was detected in all patients with advanced HCC. Small tumors, tumor heterogeneity and limited sequencing coverage may explain the lack of detectable ctDNA.

Purpose: TP53 aberration (TP53 mutation and/or 17p deletion) is the most important predictive marker in chronic lymphocytic leukemia (CLL). Although each TP53 aberration is considered an equal prognosticator, the prognostic value of carrying isolated (single-hit) or multiple (multi-hit) TP53 aberrations remains unclear, particularly in the context of targeted agents. Patients and Methods: We performed deep sequencing of TP53 using baseline samples collected from 51 TP53 aberrant patients treated with ibrutinib in a phase II study (NCT01500733). Results: We identified TP53 mutations in 43 patients (84%) and del(17p) in 47 (92%); 9 and 42 patients carried single-hit and multi-hit TP53, respectively. The multi-hit TP53 subgroup was enriched with younger patients who had prior treatments and unmutated immunoglobulin heavy-chain variable region gene status. We observed significantly shorter overall survival, progression-free survival (PFS), and time-to-progression (TTP) in patients with multi-hit TP53 compared with those with single-hit TP53. Clinical outcomes were similar in patient subgroups stratified by 2 or >2 TP53 aberrations. In multivariable analyses, multi-hit TP53 CLL was independently associated with inferior PFS and TTP. In sensitivity analyses, excluding mutations below 1% VAF demonstrated similar outcome. Results were validated in an independent population-based cohort of 112 patients with CLL treated with ibrutinib. Conclusions: In this study, single-hit TP53 defines a distinct subgroup of patients with an excellent long-term response to single-agent ibrutinib, whereas multi-hit TP53 is independently associated with shorter PFS. These results warrant further investigations on prognostication and management of multi-hit TP53 CLL. See related commentary by Bomben et al., p. 4462

T-prolymphocytic leukemia (T-PLL) is a rare, mature T-cell leukemia representing less than 2% of mature lymphocytic leukemias [1,2]. T-PLL is generally resistant to conventional chemotherapy with a...

Landscape of somatic copy number alterations, mutations, and fusions in selected genes in newly diagnosed glioblastoma patients. The most frequent aberrations occurred in PTEN, CDKN2A/B, EGFR, RB1, and NPAS3, and the most frequent mutations were in PTEN, TP53, NF1, RB1, and EGFR. One NTRK2 fusion and three FGFR3‐TACC3 fusions were identified., Glioblastoma (GBM) is an incurable brain tumor for which new treatment strategies are urgently needed. Next‐generation sequencing of GBM has most often been performed retrospectively and on archival tissue from both diagnostic and relapse surgeries with limited knowledge of clinical information, including treatment given. We sought to investigate the genomic composition prospectively in treatment‐naïve patients, searched for possible targetable aberrations, and investigated for prognostic and/or predictive factors. A total of 108 newly diagnosed GBM patients were included. Clinical information, progression‐free survival, and overall survival (OS) were noted. Tissues were analyzed by whole‐exome sequencing, single nucleotide polymorphism (SNP) and transcriptome arrays, and RNA sequencing; assessed for mutations, fusions, tumor mutational burden (TMB), and chromosomal instability (CI); and classified into GBM subgroups. Each genomic report was discussed at a multidisciplinary tumor board meeting to evaluate for matching trials. From 111 consecutive patients, 97.3% accepted inclusion in this study. Eighty‐six (77%) were treated with radiation therapy/temozolomide (TMZ) and adjuvant TMZ. One NTRK2 and three FGFR3‐TACC3 fusions were identified. Copy number alterations in GRB2 and SMYD4 were significantly correlated with worse median OS together with known clinical variables like age, performance status, steroid dose, and O6‐methyl‐guanine‐DNA‐methyl‐transferase status. Patients with CI‐median or TMB‐high had significantly worse median OS compared to CI‐low/high or TMB‐low/median. In conclusion, performing genomic profiling at diagnosis enables evaluation of genomic‐driven therapy at the first progression. Furthermore, TMB‐high or CI‐median patients had worse median OS, which can support the possibility of offering experimental treatment already at the first line for this group.

Purpose: Investigation of signaling pathways altered by recurrent gene mutations and their clinical impact in a consecutive cohort of patients with newly diagnosed chronic lymphocytic leukemia (CLL). The heterogeneous clinical course and genetic complexity of CLL warrant improved molecular prognostication. However, the prognostic value of recurrent mutations at the time of diagnosis remains unclear. Experimental Design: We sequenced samples from 314 consecutive, newly diagnosed patients with CLL to investigate the clinical impact of 56 recurrently mutated genes assessed by next-generation sequencing. Results: Mutations were identified in 70% of patients with enrichment among IGHV unmutated cases. With 6.5 years of follow-up, 15 mutated genes investigated at the time of diagnosis demonstrated significant impact on time to first treatment (TTFT). Carrying driver mutations was associated with shorter TTFT and poor overall survival. For outcome from CLL diagnosis, the number of signaling pathways altered by driver mutations stratified patients better than the number of driver mutations. Moreover, we demonstrated gradual impact on TTFT with increasing number of altered pathways independent of CLL-IPI risk. Thus, a 25-gene, pathway-based biomarker assessing recurrent mutations refines prognostication in CLL, in particular for CLL-IPI low- and intermediate-risk patients. External validation emphasized that a broad gene panel including low burden mutations was key for the biomarker based on altered pathways. Conclusions: We propose to include the number of pathways altered by driver mutations as a biomarker together with CLL-IPI in prospective studies of CLL from time of diagnosis for incorporation into clinical care and personalized follow-up and treatment.

Responses to immunotherapy can be very durable but acquired resistance leading to tumor progression often occurs. We investigated a patient with melanoma resistant to anti–programmed death 1 (anti–PD-1) who participated in the CA224-020 clinical trial (NCT01968109) and had further progression after an initial objective response to anti–PD-1 plus anti–lymphocyte activation gene 3. We found consecutive acquisition of beta-2 microglobulin (B2M) loss and impaired Janus kinase 1 (JAK1) signaling that coexisted in progressing tumor cells. Functional analyses revealed a pan T-cell immune escape phenotype, where distinct alterations mediated independent immune resistance to tumor killing by autologous CD8+ tumor-infiltrating lymphocytes (TIL; B2M loss) and CD4+ TILs (impaired JAK1 signaling). These findings shed light on the complexity of acquired resistance to immunotherapy in the post anti–PD-1 setting, indicating that coexisting altered pathways can lead to pan T-cell immune escape.

Introduction: Glioblastoma (GBM) is an aggressive brain cancer with a median overall survival of 16-24 months. Evaluation of treatment effect can be difficult as pseudo progression is seen in approximately 15% of patients. Hence, effective treatment can be stopped prematurely and no approved or standard second line treatment exist. Genomic alterations during treatment can cause treatment resistance and treatment failure. In order to investigate the genomic alterations, consecutive tissue samples are wanted. However, this is often not possible due to risk doing surgery. Therefore, liquid biopsies from plasma, containing circulating cell-free DNA (cfDNA) and circulating cell-free tumor DNA (ctDNA) might be an alternative. Based on findings by our group, we aim to improve treatment evaluation and suggest targeted treatment possibilities by investigating the role of cfDNA and ctDNA in a large prospective cohort, included from a neurooncological out-patient clinic. Materials and Methods: Newly diagnosed patients with GBM, a performance status of 0-1 and planned for concurrent chemo/radiation will be eligible. Whole genome sequencing (WGS) will be done on tissue. Peripheral blood will be collected in cell stabilizing Blood Collection Tubes (STRECK) and cfDNA quantified on a Qubit Fluorometer. Samples will be collected before the concurrent treatment and at fixed time points until progression with a total of 8-10 samples per patient. For the ctDNA analyses, a patient specific tumor mutation will be selected based upon WGS from tumor tissue. The identified mutation will be quantified in plasma (ctDNA) by droplet digital PCR (ddPCR). Dual labeled fluorescent probes for the mutation and the wild type loci will be used and PCR reaction mixtures will be run. Mutant allele fractions (AFs) ≥0.001 (0.1%) will be detected. An increase in ctDNA AF will be recorded if the AF increases from non-detectable to detectable levels (AF ≥ 0.001) or increases in two consecutive samples. Results: Inclusion began in June 2022 and by time of abstract deadline, we have 27 patients included. Of these, 16 have completed the concurrent setting with a total of five samples per patient. One patient has died and two have progressed. Hence, 24 patients are still on-treatment and sampling and accrual will continue. The first analyses for cfDNA will be run in the winter of 2022/2023 and results will be presented at the conference. CtDNA analyses are planned for autumn 2023. Expected Impact: The expected impacts of this study are specifically in two clinical areas: treatment evaluation and development of new treatment strategies. We aim to 1) find correlation between cfDNA/ctDNA and the clinical course2) detect a relapse earlier than imaging with MRI and FET/PET analyses3) aid to diagnose pseudo progression4) use ctDNA for detection of clonal selection5) test concordance between mutations detected in tissue and in plasma Citation Format: Dorte Schou Nørøxe, Lise Barlebo Ahlborn, Vincent Fougner, Thomas Urup, Benedikte Hasselbalch, Christina Westmose Yde, Hans Skovgaard Poulsen, Ulrik Lassen. Tracking tumor mutations in ctDNA through repetitive plasma samples in patients with newly diagnosed brain cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1046.

// Dorte Schou Noroxe 1 , 2 , Olga Ostrup 3 , Christina Westmose Yde 3 , Lise Barlebo Ahlborn 3 , Finn Cilius Nielsen 3 , Signe Regner Michaelsen 1 , Vibeke Andree Larsen 4 , Jane Skjoth-Rasmussen 5 , Jannick Brennum 5 , Petra Hamerlik 6 , Hans Skovgaard Poulsen 1 , 2 and Ulrik Lassen 2 1 Department of Radiation Biology, Rigshospitalet, 2100 Copenhagen, Denmark 2 Department of Oncology, Rigshospitalet, 2100 Copenhagen, Denmark 3 Center for Genomic Medicine, Rigshospitalet, 2100 Copenhagen, Denmark 4 Department of Neuroradiology, Rigshospitalet, 2100 Copenhagen, Denmark 5 Department of Neurosurgery, Rigshospitalet, 2100 Copenhagen, Denmark 6 Danish Cancer Society, 2100 Copenhagen, Denmark Correspondence to: Dorte Schou Noroxe, email: anne.dorte.schou.noeroexe@regionh.dk Keywords: glioblastoma; liquid biopsy; cell-free DNA; fragment length; base-pair Received: March 10, 2019 Accepted: May 29, 2019 Published: July 09, 2019 ABSTRACT Background: Glioblastoma (GB) is an incurable brain cancer with limited treatment options. The aim was to test the feasibility of using cell-free DNA (cfDNA) to support evaluation of treatment response, pseudo-progression and whether progression could be found before clinical and/or radiologic progression. Results: CfDNA fluctuated during treatment with the highest levels before diagnostic surgery and at progression. An increase was seen in 3 out of 4 patients at the time of progression while no increase was seen in 3 out of 4 patients without progression. CfDNA levels could aid in 3 out of 3 questionable cases of pseudo-progression. Methods: Eight newly diagnosed GB patients were included. Blood samples were collected prior to diagnosis, before start and during oncologic treatment until progression. Seven patients received concurrent radiotherapy/Temozolomide with adjuvant Temozolomide with one of the patients included in a clinical trial with either immunotherapy or placebo as add-on. One patient received radiation alone. CfDNA concentration was determined for each blood sample. Conclusions: It was feasible to measure cfDNA concentration. Despite the limited cohort size, there was a good tendency between cfDNA and treatment course and -response, respectively with the highest levels at progression.

Selecting patients for early clinical trials is a challenging process and clinicians lack sufficient tools to predict overall survival (OS). Circulating cell-free DNA (cfDNA) has recently been shown to be a promising prognostic biomarker. The aim of this study was to investigate whether baseline cfDNA measurement could improve the prognostic information of the Royal Marsden Hospital (RMH) score. Solid tumour patients referred for phase I trials were included in the Copenhagen Personalized Oncology (CoPPO) programme. Baseline characteristics were collected prospectively, including the RMH prognostic score, Eastern Cooperative Oncology Group (ECOG) performance status and concentration of cfDNA per millilitre plasma. Cox proportional hazards model was used to assess the prognostic value of baseline variables. Plasma cfDNA concentration was quantifiable in 302 patients out of a total of 419 included in the study period of 2 years and 5 months. The RMH score was confirmed to be associated with OS. Cell-free DNA was shown to be an independent prognostic marker of OS and improved the risk model, including RMH, performance status and age. Furthermore, both plasma cfDNA concentration and RMH score were associated with treatment allocation (p

BackgroundCopy-number variations (CNVs) have important clinical implications for several diseases and cancers. The clinically relevant CNVs are hard to detect because CNVs are common structural variations that define large parts of the normal human genome. CNV calling from short-read sequencing data has the potential to leverage available cohort studies and allow full genomic profiling in the clinic without the need for additional data modalities. Questions regarding performance of CNV calling tools for clinical use and suitable sequencing protocols remain poorly addressed, mainly because of the lack of good reference data sets.MethodsWe reviewed 50 popular CNV calling tools and included 11 tools for benchmarking in a unique reference cohort encompassing 39 whole genome sequencing (WGS) samples paired with analysis by the current clinical standard —SNP-array based CNV calling. Additionally, for nine of these samples we performed whole exome sequencing (WES) performed, in order to address the effect of sequencing protocol on CNV calling. Furthermore, we included Gold Standard reference sample NA12878, and tested 12 samples with CNVs confirmed by multiplex ligation-dependent probe amplification (MLPA).ResultsTool performance varied greatly in the number of called CNVs and bias for CNV lengths. Some tools had near-perfect recall of CNVs from arrays for some samples, but poor precision. Filtering output by CNV ranks from tools did not salvage precision. Several tools had better performance patterns for NA12878, and we hypothesize that this is the result of overfitting during the tool development.ConclusionsWe suggest combining tools with the best recall: GATK gCNV, Lumpy, DELLY, and cn.MOPS. These tools also capture different CNVs. Further improvements in precision requires additional development of tools, reference data sets, and annotation of CNVs, potentially assisted by the use of background panels for filtering of frequently called variants.

Purpose: The purpose of this study was to identify mechanisms of resistance to BRAF targeted therapy using proteomics together with genomics and transcriptomics in patients with BRAFV600E mutated solid tumors. Experimental procedures: A total of nine patients with BRAFV600E mutated advanced solid tumors (5 with colorectal cancer, 2 with neuroendocrine carcinoma, 1 with cholangiocarcinoma and 1 with breast cancer) treated with BRAF targeted therapy (BRAF inhibitor in combination with either MEK inhibitor and/or EGRF antibody) as part of the Copenhagen Prospective Personalized Oncology study, were included in this study. Tumor biopsies at baseline and at disease progression were analyzed with whole exome/genome sequencing (WES/WGS), transcriptomics (RNA sequencing) and proteomics. Genomic variants were analyzed together with changes in protein expression. Three filtering steps were used to identify potential resistance mechanisms from the proteomics measurements. Proteins were filtered for 1) proteins with a high shift in abundance between baseline and progression 2) proteins with known associations to the patient’s primary cancer based on text-mining and 3) proteins in pathways where BRAF is also involved. Results: Alterations conferring resistance were identified in 2 out of 9 patients when comparing data from WES/WGS and RNA sequencing at baseline and at disease progression (one patient with PTBP2-BRAF fusion and one with NRAS mutation). Genomic or transcriptomic alterations conferring resistance were not detected in the remaining 7 patients. Notably, there was not a single protein shared between all 9 patients after our filtering, but when comparing overlap on pathway level, six pathways related to RAF and MAPK signaling were affected in all 9 patients. Particularly, paradoxical activation of RAF signaling seems to be an interesting candidate to explain resistance, where formation of RAF dimer structures happens as a response to treatment with BRAF inhibitors, selectively inhibiting BRAF monomer structures (i.e. BRAFV600E). Conclusion: With a multiomic approach using proteomics together with genomics and transcriptomics, potential mechanisms of resistance were detected in all patients at disease progression, where resistance to BRAF targeted therapies had occurred. Six mechanisms of resistance were shared independently of diagnosis and BRAF inhibitor combination regimes. The potential of integrating proteomics with genomics and transcriptomics is promising and may potentially guide therapy for future patients with treatment resistant BRAFV600E mutated solid tumors. Citation Format: Martina Eriksen, Annelaura Bach Nielsen, Filip Mundt, Josephine Kerzel Duel, Matthias Mann, Ulrik Lassen, Christina Westmose Yde, Camilla Qvortrup, Martin Højgaard, Iben Spanggaard, Kristoffer Staal Rohrberg. Multiomics detect potential mechanisms of resistance to BRAF targeted therapy in patients with BRAFV600E mutated solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3254.

Simple Summary Copy-number variations (CNVs) have important clinical implications for several diseases and cancers. We reviewed 50 popular CNV calling tools and included 11 tools for benchmarking in a reference cohort encompassing 39 whole genome sequencing (WGS) samples paired current clinical standard—SNP-array based CNV calling. For nine samples we also performed whole exome sequencing (WES), to address the effect of sequencing protocol on CNV calling. Furthermore, we included Gold Standard reference sample NA12878, and tested 12 samples with CNVs confirmed by multiplex ligation-dependent probe amplification (MLPA). Tool performance varied greatly in the number of called CNVs and bias for CNV lengths. Some tools had near-perfect recall of CNVs from arrays for some samples, but poor precision. We suggest combining the best tools also based on different methodologies: GATK gCNV, Lumpy, DELLY, and cn.MOPS. Abstract Copy-number variations (CNVs) have important clinical implications for several diseases and cancers. Relevant CNVs are hard to detect because common structural variations define large parts of the human genome. CNV calling from short-read sequencing would allow single protocol full genomic profiling. We reviewed 50 popular CNV calling tools and included 11 tools for benchmarking in a reference cohort encompassing 39 whole genome sequencing (WGS) samples paired current clinical standard—SNP-array based CNV calling. Additionally, for nine samples we also performed whole exome sequencing (WES), to address the effect of sequencing protocol on CNV calling. Furthermore, we included Gold Standard reference sample NA12878, and tested 12 samples with CNVs confirmed by multiplex ligation-dependent probe amplification (MLPA). Tool performance varied greatly in the number of called CNVs and bias for CNV lengths. Some tools had near-perfect recall of CNVs from arrays for some samples, but poor precision. Several tools had better performance for NA12878, which could be a result of overfitting. We suggest combining the best tools also based on different methodologies: GATK gCNV, Lumpy, DELLY, and cn.MOPS. Reducing the total number of called variants could potentially be assisted by the use of background panels for filtering of frequently called variants.

Two types of clinically important nucleic acid biomarkers, microRNA (miRNA) and circulating tumor DNA (ctDNA) were detected and quantified from human serum using an amplification-free fluorescence hybridization assay. Specifically, miRNAs hsa-miR-223-3p and hsa-miR-486-5p with relevance for rheumatoid arthritis and cancer related mutations BRAF and KRAS of ctDNA were directly measured. The required oligonucleotide probes for the assay were rationally designed and synthesized through a novel "clickable" approach which is time and cost-effective. With no need for isolating nucleic acid components from serum, the fluoresence-based assay took only 1 hour. Detection and absolute quantification of targets was successfully achieved despite their notoriously low abundance, with a precision down to individual nucleotides. Obtained miRNA and ctDNA amounts showed overall a good correlation with current techniques. With appropriate probes, our novel assay and signal boosting approach could become a useful tool for point-of-care measuring other low abundance nucleic acid biomarkers.

Genetic testing of BRCA1/2 includes screening for single nucleotide variants and small insertions/deletions and for larger copy number variations (CNVs), primarily by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). With the advent of next-generation sequencing (NGS), it has become feasible to provide CNV information and sequence data using a single platform. We report the use of NGS gene panel sequencing on the Illumina MiSeq platform and JSI SeqPilot SeqNext software to call germline CNVs in BRCA1 and BRCA2. For validation 18 different BRCA1/BRCA2 CNVs previously identified by MLPA in 48 Danish breast and/or ovarian cancer families were analyzed. Moreover, 120 patient samples previously determined as negative for BRCA1/BRCA2 CNVs by MLPA were included in the analysis. Comparison of the NGS data with the data from MLPA revealed that the sensitivity was 100%, whereas the specificity was 95%. Taken together, this study validates a one-step bioinformatics work-flow to call germline BRCA1/2 CNVs using data obtained by NGS of a breast cancer gene panel. The work-flow represents a robust and easy-to-use method for full BRCA1/2 screening, which can be easily implemented in routine diagnostic testing and adapted to genes other than BRCA1/2.

Meningiomas are rare in children. They are highly complex, harboring unique clinical and pathological characteristics, and many occur in patients with neurofibromatosis type 2. Hereby, we present a case of a two-year-old boy presented with a diagnostically challenging intraventricular tumor. It was incompletely resected 6 times over 14 months but kept progressing and was ultimately deemed unresectable. Histologically, the tumor was initially classified as schwannoma, but extensive international review concluded it was most likely an atypical meningioma, WHO grade II. Comprehensive genomic profiling revealed a TFG-ROS1 fusion, suggesting that ROS1-signaling pathway alterations were driving the tumor growth. In light of this new information, the possibility of a diagnosis of inflammatory myofibroblastic tumor was considered; however the histopathological results were not conclusive. This specific molecular finding allowed the potential use of precision medicine and the patient was enrolled in the AcSé phase 2 trial with crizotinib (NCT02034981), leading to a prolonged partial tumor response which is persisting since 14 months. This case highlights the value of precision cancer medicine in children.

Genomic screening of cancer patients for predisposing variants is traditionally based on age at onset, family history and type of cancer. Whereas the clinical guidelines have proven efficient in identifying families exhibiting classical attributes of hereditary cancer, the frequency of patients with alternative presentations is unclear. We identified and characterized germline variants in 636 patients with advanced solid cancer using whole exome sequencing. Pathogenic and likely pathogenic germline variants among 168 genes associated with hereditary cancer were considered. These variants were identified in 17.8% of the patients and within a wide range of cancer types. In particular, patients with mesothelioma, ovarian cancer, cervical cancer, urothelial cancer, and cancer of unknown primary origin displayed high frequencies of pathogenic variants. Variants were predominantly found in DNA-repair pathways and about half were within genes involved in homologous recombination repair. Twenty-two BRCA1 and BRCA2 germline variants were identified in 12 different cancer types, of which 10 (45%) were not previously identified in these patients based on the current clinical guidelines. Loss of heterozygosity and somatic second hits were identified in several of the affected genes, supporting possible causality for cancer development. A potential treatment target based on the pathogenic germline variant could be suggested in 25 patients (4%). The study demonstrates a high frequency of pathogenic germline variants in the homologous recombination pathway in patients with advanced solid cancers. We infer that genetic screening in this group of patients may reveal high-risk families and identify patients with potential PARP inhibitor sensitive tumors.

Introduction: Glioblastoma (GBM) is a heterogenous cancer with a high degree of intratumoral subclonality. We foresee that future treatment will be a combination of non-targeted and targeted therapies against molecular features of each patient. In order to accommodate the personalized treatment strategy, it is therefore pivotal to know the dynamics of tumor alterations during treatment. Today, this is only possible by consecutive intracranial tissue samples for each patient and hence is a difficult discipline. Materials and methods: We included 30 patients from the Copenhagen Glioblastoma Cohort who had undergone relapse surgery. Each sample had a tumor cell content > 10%. The majority of patients 24/30 (80%) were treated with concurrent Temozolomide (TMZ)/radiation and adjuvant TMZ. Whole exome sequencing was performed in primary and corresponding relapse tumors focusing on GBM-related mutations. Results: It was possible to identify the same specific mutation in 16/30 (53%) patients before and after treatment, with a total of 22 shared mutations identified. In 7 (23%) relapse samples, mutations were called but none were shared, suggesting temporal tumorogenesis and in the remaining 7 (23%) relapse samples, no mutations were identified. Progression-free survival (PFS) in the three groups was 6.6, 7.8 and 10.1 months, respectively. Corresponding overall survival (OS) was 15.0, 18.0 and 17.8 months, respectively. The results indicate a better PFS, but not OS, in the group with no mutations in the relapse sample. Six patients had three surgeries performed; one diagnostic surgery followed by two relapse surgeries. In one patient, a BRAF mutation (c.1786G>C, p.G596R) was identified at diagnosis, not present at the first relapse but reappeared in the second relapse, most probably as the sign for development of resistance. This was also the case in two other patients with a BRCA- and a PTEN mutation, respectively. In two other patients, the same TP53 (c.814G>A, p.R175H) and EGFR (c.866C>T, p.A289V) mutations, respectively, were identified in all three samples. In the last patient, no mutations were identified in the diagnostic and first relapse sample, but two mutations were identified in the second relapse sample, demonstrating tumor evolution. Conclusion: We show that mutation dynamics in GBM differ between patients, and frequencies of driver mutations vary during the tumor progression. It was possibly to identify the same mutation during treatment in the same patient. These observations can be used to design future targeted trials and potentially be used for evaluation of treatment response in the individual patient. The presence of mutations in the diagnostic and relapse samples, and specifically shared mutations, indicate potential treatment failure and/or presence of specifically malignant mutations. Citation Format: Dorte Schou Noeroexe, Olga Oestrup, Christina Westmose Yde, Finn Cilius Nielsen, Jane Skjøth-Rasmussen, Petra Hamerlik, Hans Skovgaard Poulsen, Ulrik Lassen. Dynamics of tumor mutations in paired samples from glioblastoma patients during treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 444.

Since the online publication of the above article, the authors have noted errors in subfigures 1c and 3b. Therefore, new images of the original immmunocytochemistry stainings have been obtained for Fig. 1c, and the Western blots for siRNA-mediated FYN knockdown in Fig. 3b were repeated. The amended versions of Figs. 1c and 3b are now provided.

// Lise Barlebo Ahlborn 1, 2 , Ida Viller Tuxen 1 , Florent Mouliere 3 , Savvas Kinalis 2 , Ane Y. Schmidt 2 , Kristoffer Staal Rohrberg 1 , Eric Santoni-Rugiu 4 , Finn Cilius Nielsen 2 , Ulrik Lassen 1 , Christina Westmose Yde 2 , Olga Oestrup 2 and Morten Mau-Sorensen 1 1 The Phase I Unit, Department of Oncology, Rigshospitalet, Copenhagen University, Copenhagen, Denmark 2 Center for Genomic Medicine, Rigshospitalet, Copenhagen University, Copenhagen, Denmark 3 Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom 4 Department of Pathology, Rigshospitalet, Copenhagen University, Copenhagen, Denmark Correspondence to: Lise Barlebo Ahlborn, email: lise.barlebo.ahlborn@regionh.dk Keywords: BRAF inhibitor; circulating tumor DNA; mutant allele fraction; early phase study; solid cancer Received: June 04, 2018 Accepted: July 29, 2018 Published: August 24, 2018 ABSTRACT Purpose: We evaluated longitudinal tracking of BRAF V600E in circulating cell-free DNA (cfDNA) as a marker of treatment response to BRAF inhibitor (BRAFi) combination therapies in non-melanoma solid tumors included in the Copenhagen Prospective Personalized Oncology (CoPPO) program. Experimental design: Patients with BRAF V600E-mutated tumors were treated with combination therapies including BRAFi. Quantification of mutant cfDNA from plasma was determined and correlated to clinical outcomes. Exome sequencing was performed to identify possible resistance mutations. Results: Twenty-three patients had BRAF-mutated tumors out of 455 patients included in CoPPO and 17 started BRAFi combination (EGFRi/MEKi) therapy. Tumor responses were achieved in 8 out of 16 evaluable patients and the median overall- and progression-free survival (OS and PFS) was 15 and 4.8 months, respectively. Longitudinal measurements of BRAF V600E-mutant cfDNA indicated disease progression prior to radiological evaluation and a reduction in the mutant fraction of more than 50% after 4 and 12 weeks of therapy was associated with a significantly longer PFS (p=0.003 and p=0.029) and OS (p=0.029 and p=0.017). Furthermore, the baseline mutant fraction and total level of cfDNA positively correlated with tumor burden (p=0.026 and p=0.024). Finally, analysis of cfDNA at progression revealed novel mutations potentially affecting the MAPK pathway. Conclusion: BRAFi combination therapies showed a response rate of 50% in BRAF V600E-mutated non-melanoma tumors. The fraction of BRAF-mutant cfDNA represent a sensitive indicator for clinical outcomes with plasma collected at week 4 and 12 as crucial time points for monitoring response and disease progression.

Purpose: We evaluated the clinical benefit of tumor molecular profiling to select treatment in the phase I setting. Experimental Design: Patients with advanced solid cancers and exhausted treatment options referred to a phase I unit were included in a prospective, single-center, single-arm open-label study (NCT02290522). Tumor biopsies were obtained for comprehensive genomic analysis including whole-exome sequencing and RNA sequencing. When possible, patients were treated with regimen matched to the genomic profile. Primary endpoint was progression-free survival (PFS). Results: From May 2013 to January 2017, a total of 591 patients were enrolled, with 500 patients undergoing biopsy. Genomic profiles were obtained in 460 patients and a potential actionable target was identified in 352 (70%) of 500 biopsied patients. A total of 101 patients (20%) received matched treatment based on either gene mutations or RNA expression levels of targets available in early clinical trials or off-label treatment. Objective response according to RECIST1.1 was observed in 15 of 101 patients (0% complete response, 15% partial response), with a median PFS of 12 weeks (95% confidence interval, 9.9–14.4). Conclusions: Our study supports the feasibility of genomic profiling to select patients in the phase I setting and suggests that genomic matching can be beneficial for a minor subset of patients with no other treatment options. Randomized studies may validate this assumption. See related commentary by Ratain, p. 1136

Aromatase inhibitors (AIs) are used for treatment of estrogen receptor α (ER)-positive breast cancer; however, resistance is a major obstacle for optimal outcome. This preclinical study aimed at identifying potential new treatment targets in AI-resistant breast cancer cells. Parental MCF-7 breast cancer cells and four newly established cell lines, resistant to the AIs exemestane or letrozole, were used for a functional kinase inhibitor screen. A library comprising 195 different compounds was tested for preferential growth inhibition of AI-resistant cell lines. Selected targets were validated by analysis of cell growth, cell cycle phase distribution, protein expression, and subcellular localization. We identified 24 compounds, including several inhibitors of Aurora kinases e.g., JNJ-7706621 and barasertib. Protein expression of Aurora kinase A and B was found upregulated in AI-resistant cells compared with MCF-7, and knockdown studies showed that Aurora kinase A was essential for AI-resistant cell growth. In AI-resistant cell lines, the clinically relevant Aurora kinase inhibitors alisertib and danusertib blocked cell cycle progression at the G2/M phase, interfered with chromosome alignment and spindle pole formation, and resulted in preferential growth inhibition compared with parental MCF-7 cells. Even further growth inhibition was obtained when combining the Aurora kinase inhibitors with the antiestrogen fulvestrant. Our study is the first to demonstrate that Aurora kinase A and B may be treatment targets in AI-resistant cells, and our data suggest that therapy targeting both ER and Aurora kinases may be a potent treatment strategy for overcoming AI resistance in breast cancer.

Tamoxifen‑resistant breast cancer is a major clinical problem and new treatment strategies are highly warranted. In this study, the multitargeting kinase inhibitors sorafenib and nilotinib were investigated as potential new treatment options for tamoxifen‑resistant breast cancer. The two compounds inhibited cell growth, reduced expression of total estrogen receptor α (ER), Ser118-phosphorylated ER, FOXA1 and AIB1 and resensitized tamoxifen‑resistant cells to tamoxifen. The ER downmodulator fulvestrant exerted strong growth inhibition of tamoxifen‑resistant cells and addition of sorafenib and nilotinib could not further suppress growth, showing that sorafenib and nilotinib exerted growth inhibition via ER. In support of this, estradiol prevented sorafenib and nilotinib mediated growth inhibition. These results demonstrate that sorafenib and nilotinib act via ER and ER-associated proteins, indicating that these kinase inhibitors in combination with tamoxifen may be potential new treatments for tamoxifen‑resistant breast cancer.

Resistance to antiestrogens is a major clinical problem in current breast cancer treatment and development of new treatment strategies for these tumors is highly prioritized. In this study, we have investigated the effects of 1α,25-dihydroxyvitamin D3 on the proliferation of tamoxifen-resistant cells. Further, we have investigated on a molecular level the effects of vitamin D on NFkB signaling in tamoxifen-resistant breast cancer cells. Parental human breast cancer MCF-7 cells and four tamoxifen-resistant sublines have been used to investigate the effects of 1α,25-dihydroxyvitamin D3 on cell proliferation using a colorimetric method, gene expression using quantitative PCR, protein phosphorylation using Western blot analysis and cellular localization of proteins using immunofluorescence microscopy. We found that 1α,25-dihydroxyvitamin D3 is able to strongly decrease the growth of both tamoxifen-sensitive and -resistant breast cancer cells and that this antiproliferative effect of 1α,25-dihydroxyvitamin D3 might be mediated via inhibition of the NFκB pathway. We found that 1α,25-dihydroxyvitamin D3 stimulates the gene expression of IkB, an NFκB-inhibiting protein, and that cells pretreated with 1α,25-dihydroxyvitamin D3 have a decreased sensitivity to TNFα stimulation. Further, we show that 1α,25-dihydroxyvitamin D3 treatment strongly decreases the TNFα-induced translocation of p65 into the nucleus. This manuscript reports novel findings regarding the effects of 1α,25-dihydroxyvitamin D3 on NFκB signaling in tamoxifen-resistant breast cancer cells and suggests that vitamin D might be interesting for further evaluation as a new strategy to treat antiestrogen-resistant breast cancers.

BACKGROUND: Tumor mutational load (TML) is the number of nonsynonymous mutations in a tumor sample. TML has proved predictive of response to immune therapy (IT) in other TML-high tumors. Research in TML in glioblastoma (GBM) is limited and has to our knowledge not been done prospectively in paired samples before and after treatment. MATERIALS AND METHODS: Over a 2-year period from February 2016 to March 2018, 27 patients with newly diagnosed GBM were included. All patients had one biopsy at diagnosis and another when having secondary surgery due to progression. Three patients had three surgeries. We noted clinical and pathological data. Blood samples were extracted and whole exome sequencing has been performed. We are now investigating TML including clonal and subclonal mutations, mutations in mismatch repair genes and the aberrant cell fraction. TML will be reported as per Megabase exome and will be adjusted for coverage in each sample. RESULTS: All the included patients had primary GBM with isocitrate dehydrogenase (IDH) wild type. O-6-methylguanine-DNA methyltransferase (MGMT) was methylated in 6 patients (22%) and the majority of patients have been treated with chemo/radiation with Temozolomide. Since the patients were included from our daily clinical oncological department, the clinical characteristics resembled the average GBM patient according to age, performance status, treatment, progression-free survival and overall survival. CONCLUSION: Analyses are forthgoing and will be ready at the time of the SNO-meeting. An agreement of how to report TML is strongly needed and TML should be included in future trials in GBM.

Introduction: Estrogen is the main driver of growth of the majority of breast cancers and many patients benefit from endocrine therapy. However, both innate and acquired resistance is a major problem. In order to study resistance mechanisms and to explore targeted treatment options for endocrine resistant breast cancer, we have developed in vitro cell culture models based on the estrogen receptor (ER) positive and estrogen responsive human breast cancer cell lines MCF-7 and T47D. Methods: Panels of MCF-7 and T47D sublines with acquired resistant to tamoxifen, fulvestrant and aromatase inhibitors were established by selection of colonies surviving long term treatment. Long-term estrogen deprived (LTED) MCF-7/S9 cells were developed by step-wise serum reduction until transfer to serum-free medium. The endocrine resistant sublines were analysed with respect to expression and function of the ER and the HER system, with respect to response to 195 kinase inhibitors and to second-line endocrine therapy. Results: ER expression was reduced in tamoxifen and fulvestrant resistant MCF-7 sublines, unchanged in tamoxifen resistant T47D sublines, whereas fulvestrant resistant T47D sublines were ER negative. In general, ER was functional in the resistant MCF-7 cells. Both tamoxifen and fulvestrant resistant MCF-7 sublines expressed increased level of HER1, the activated form of HER3 and activated ERK1/2, whereas HER4 was severely decreased. In fulvestrant resistant T47D sublines, HER1 and HER4 expression were decreased whereas HER2 expression was highly upregulated. The kinase inhibitor screen disclosed HER receptors as common hits in the tamoxifen and fulvestrant resistant MCF-7 sublines. Regarding T47D, c-Src and Aurora were common hits for tamoxifen and fulvestrant resistant sublines. Tamoxifen resistant MCF-7 and T47D sublines were severely growth inhibited by ER knockdown with either siRNA or fulvestrant, but complete growth arrest required combined treatment with e.g ERK1/2 inhibitor. LTED MCF-7 cells were resistant to aromatase inhibitor treatment and sensitive to both tamoxifen and fulvestrant. Conclusions: In our large panels of MCF-7 and T47D derived endocrine resistant sublines, it was a general finding that ER is the main driver of growth of tamoxifen resistant tumor cells, and also of growth of LTED MCF-7 cells. The ER down modulator fulvestrant inhibited growth of tamoxifen resistant sublines but complete growth arrest required combined treatment with e.g. a kinase inhibitor targeting the activated growth promoting pathways in the resistant cells. In fulvestrant resistant sublines, ER played only a minor role in MCF-7 sublines and no role in T47D sublines. The HER system played a major role in fulvestrant resistant MCF-7 cells, whereas Aurora B and c-Src were new candidate targets for fulvestrant resistant T47D cells. These cell culture models reflect clinical findings and will be available for collaborative studies, e.g. to discover new predictive markers to select targeted therapy for resistant tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-02.

Resistance to tamoxifen is a major clinical challenge in the treatment of breast cancer; however, it is still unclear which signaling pathways are the major drivers of tamoxifen-resistant growth. To characterize resistance mechanisms, we have generated different tamoxifen-resistant breast cancer cell lines from MCF-7. In this model, we investigated whether signaling from human epidermal growth factor receptors (HERs), their downstream kinases, or from the estrogen receptor α (ERα) was driving tamoxifen-resistant cell growth. Increased expression of EGFR and increased phosphorylation of HER3 were observed upon acquisition of tamoxifen resistance, and the extracellular activated kinase (ERK) signaling pathway was highly activated in the resistant cells. The EGFR inhibitor gefitinib and the ERK pathway inhibitor U0126 resulted in partial and preferential growth inhibition of tamoxifen-resistant cells. All the tamoxifen-resistant cell lines retained ERα expression but at a lower level compared to that in MCF-7. Importantly, we showed via ERα knockdown that the tamoxifen-resistant cells were dependent on functional ERα for growth and we observed a clear growth stimulation of resistant cell lines with clinically relevant concentrations of tamoxifen and 4-OH-tamoxifen, indicating that tamoxifen-resistant cells utilize agonistic ERα stimulation by tamoxifen for growth. The tamoxifen-resistant cells displayed high phosphorylation of ERα at Ser118 in the presence of tamoxifen; however, treatment with U0126 neither affected the level of Ser118 phosphorylation nor expression of the ERα target Bcl-2, suggesting that ERK contributes to cell growth independently of ERα in our cell model. In support of this, combined treatment against ERα and ERK signaling in resistant cells was superior to single-agent treatment and as effective as fulvestrant treatment of MCF-7 cells. Together, these findings demonstrate that ERα is a major driver of growth in tamoxifen-resistant cells supported by HER/ERK growth signaling, implying that combined targeting of these pathways may have a clinical potential for overcoming tamoxifen resistance.

Background Small fragments of tumor DNA can be found in the circulation of cancer patients, providing a noninvasive access to tumor material (liquid biopsy). Analysis of circulating tumor DNA (ctDNA) has been used for diagnosis, treatment decisions, and detection of therapy resistance, including in patients with tumors inaccessible for biopsy, making ctDNA an important alternative source of tumor material. Immediate separation of plasma is widely used in standard isolation of cell-free DNA to ensure high quality plasma DNA. However, these procedures are labor intensive and logistically challenging in a clinical setting. Here we investigate the concordance between standard blood collection for molecular analysis using immediate separation of plasma, compared to the use of collection tubes allowing for delayed processing. Methods In this study, we measured the fractional abundance of tumor specific mutations (BRAF p.V600E and PIK3CA p.H1047R) in ctDNA isolated from blood samples collected in either cell-stabilizing Cell-Free DNA BCT tubes (delayed processing within 72 hours) or standard K3EDTA tubes (immediate processing within 15 minutes). Twenty-five blood sample pairs (EDTA/BCT) were collected from patients with advanced solid cancers enrolled in early clinical trials. Results Concordance in the fractional abundance of mutations in ctDNA isolated from blood collected in either K3EDTA or BCT tubes from patients with different solid cancers was observed. Conclusions This study indicates that BCT tubes are preferable for collection of circulating DNA in a clinical setting due to the favorable storage and shipping conditions.

Adenoid cystic carcinoma (AdCC) is a malignant salivary gland tumor. To date, no cases of AdCC in first-degree relatives have been reported in the literature. We present a 50-year-old female (Case 1) and this patients' father (Case 2), both of whom were diagnosed with AdCC of the minor salivary glands. Histology of Case 1 demonstrated a tubulocribriform AdCC whereas Case 2 primarily was an AdCC of solid type. Both cases harbored the MYB-NFIB gene fusion as demonstrated by FISH and RNA-sequencing. After filtering and selection of putative deleterious variants, whole exome sequencing identified 18 germline variants in common between Case 1 and Case 2. However, none of the variants were associated with AdCC or other head and neck cancers. To our knowledge, we present the first potential case of familial AdCC. The presented genetic data may contribute to further investigations of the underlying genetic mechanisms for AdCC susceptibility.

Antiestrogens are currently used for treating breast cancer patients who have estrogen receptor–positive tumors. However, patients with advanced disease will eventually develop resistance to the drugs. Therefore, compounds effective on antiestrogen-resistant tumors will be of great importance for future breast cancer treatment. In this study, we have investigated the effect of the chemotherapeutic compound cisplatin using a panel of antiestrogen-resistant breast cancer cell lines established from the human breast cancer cell line MCF-7. We show that the antiestrogen-resistant cells are significantly more sensitive to cisplatin-induced cell death than antiestrogen-sensitive MCF-7 cells and we show that cisplatin induces cell death by activating both the caspase and lysosomal death pathways. The antiestrogen-resistant cell lines express lower levels of antiapoptotic Bcl-2 protein compared with parental MCF-7 cells. Our data show that Bcl-2 can protect antiestrogen-resistant breast cancer cells from cisplatin-induced cell death, indicating that the reduced expression of Bcl-2 in the antiestrogen-resistant cells plays a role in sensitizing the cells to cisplatin treatment. [Mol Cancer Ther 2007;6(6): 1869–76]

Pilocytic astrocytoma (PA) is one of the most common brain cancers among children and activation of the Mitogen-Activated Protein Kinase (MAPK) pathway is considered the hallmark. In the majority of cases, oncogenic BRAF fusions or BRAF V600E mutations are observed, while RAF1 or NF1 alterations are more rarely found. However, in some cases, no apparent cancer driver events can be identified. Here, we describe a novel fusion between the transcription factor nuclear factor 1A (NFIA) and Raf-1 proto-oncogene (RAF1) in a 5-year old boy with PA. The novel fusion was identified as part of a comprehensive genomic tumor profiling. We show that the NFIA:RAF1 fusion results in constitutive Raf1 kinase activity, leading to activation of downstream MEK1/2 cascade and increased proliferation of cancer cells. The NFIA:RAF1 fusion displayed distinct subcellular localization towards the plasma membrane indicative of Raf-1 activation, in contrast to both wild type NFIA and Raf-1, which were localized in the nucleus and cytoplasm, respectively. In conclusion, our data support the existence of rare oncogenic RAF1 fusions with constitutive Raf-1 activity. This highlights the need for broad genetic testing in order to refine diagnostics of PA and to unravel potential treatment options, e.g. with MEK inhibitors.

Tamoxifen resistance is a major clinical problem in the treatment of estrogen receptor a-positive breast tumors. It is, at present, unclear what exactly causes tamoxifen resistance. For decades, chlorpromazine has been used for treating psychotic diseases, such as schizophrenia. However, the compound is now also recognized as a multitargeting drug with diverse potential applications, for example, it has antiproliferative properties and it can reverse resistance toward antibiotics in bacteria. Furthermore, chlorpromazine can reverse multidrug resistance caused by overexpression of P-glycoprotein in cancer cells. In this study, we have investigated the effect of chlorpromazine on tamoxifen response of human breast cancer cells. We found that chlorpromazine worked synergistically together with tamoxifen with respect to reduction of cell growth and metabolic activity, both in the antiestrogen-sensitive breast cancer cell line, MCF-7, and in a tamoxifen-resistant cell line, established from the MCF-7 cells. Tamoxifen-sensitive and tamoxifen-resistant cells were killed equally well by combined treatment with chlorpromazine and tamoxifen. This synergistic effect could be prevented by addition of estrogen, suggesting that chlorpromazine enhances the effect of tamoxifen through an estrogen receptor-mediated mechanism. To investigate this putative mechanism, we applied biophysical techniques to simple model membranes in the form of unilamellar liposomes of well-defined composition and found that chlorpromazine interacts strongly with lipid bilayers of different composition leading to increased permeability. This implies that chlorpromazine can change influx properties of membranes hence suggesting that chlorpromazine may be a promising chemosensitizing compound for enhancing the cytotoxic effect of tamoxifen. Udgivelsesdato: September

Protein kinase CK2 is involved in cell proliferation and survival, and found overexpressed in virtually all types of human cancer, including breast cancer. We demonstrate that inhibition of CK2 with 2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole (DMAT), a potent and specific CK2 inhibitor, results in caspase-mediated killing of human breast cancer cells with acquired resistance to antiestrogens, while DMAT fails to kill parental MCF-7 cells. The antiestrogen resistant breast cancer cells express reduced levels of Bcl-2 compared to MCF-7 cells. Reduced Bcl-2 protein level is also found in a tamoxifen resistant human breast tumor grown as a xenograft. We show that re-expression of Bcl-2 partially rescues antiestrogen resistant MCF-7 sublines from DMAT-induced cell death. In summary, our data suggest a novel role of CK2 in antiestrogen resistance.