ProTarget: a Danish Nationwide Clinical Trial on Targeted Cancer Treatment based on genomic profiling - a national, phase 2, prospective, multi-drug, non-randomized, open-label basket trial.

BACKGROUND: An increasing number of trials indicate that treatment outcomes in cancer patients with metastatic disease are improved when targeted treatments are matched with druggable genomic alterations in individual patients (pts). An estimated 30-80% of advanced solid tumors harbor actionable genomic alterations. However, the efficacy of personalized cancer treatment is still scarcely investigated in larger, controlled trials due to the low frequency and heterogenous distribution of druggable alterations among different histologic tumor types. Therefore, the overall effect of targeted cancer treatment on clinical outcomes still needs investigation. STUDY DESIGN/METHODS: ProTarget is a national, non-randomized, multi-drug, open-label, pan-cancer phase 2 trial aiming to investigate the anti-tumor activity and toxicity of currently 13 commercially available, EMA-approved targeted therapies outside the labeled indication for treatment of advanced malignant diseases, harboring specific actionable genomic alterations. The trial involves the Danish National Molecular Tumor Board for confirmation of drug-variant matches. Key inclusion criteria include a) measurable disease (RECIST v.1.1), b) ECOG performance status 0-2, and c) an actionable genomic alteration matching one of the study drugs. Key exclusion criteria include a) cancer type within the EMA-approved label of the selected drug, and b) genomic alterations known to confer drug resistance. Initial drug dose, schedule and dose modifications are according to the EMA-approved label. The primary endpoint is objective response or stable disease at 16 weeks. Pts are assigned to cohorts defined by the selected drug, genomic alteration, and tumor histology type. Cohorts are monitored according to a Simon's two-stage-based design. Response is assessed every 8 weeks for the first 24 weeks, then every 12 weeks. The trial is designed similar to the Dutch DRUP and the ASCO TAPUR trials and is a partner in the Nordic Precision Cancer Medicine Trial Network. In ProTarget, serial fresh tumor and liquid biopsies are mandatory and collected for extensive translational research including whole genome sequencing, array analysis, and RNA sequencing. DISCUSSION: The ProTarget trial will identify new predictive biomarkers for targeted treatments and provide new data and essential insights in molecular pathways involved in e.g., resistance mechanisms and thereby potentially evolve and expand the personalized cancer treatment strategy. PROTOCOL VERSION: 16, 09-MAY-2022. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04341181. Secondary Identifying No: ML41742. EudraCT No: 2019-004771-40.

A phase II study of daily encorafenib in combination with biweekly cetuximab in patients with BRAF V600E mutated metastatic colorectal cancer: the NEW BEACON study.

BACKGROUND: Patients with BRAF V600E mutated metastatic colorectal cancer (mCRC) have a poor prognosis. The introduction of BRAF targeted therapy with encorafenib and weekly administered cetuximab have shown improved survival with a median progression free survival (PFS) of 4.3 months. However, a regimen with cetuximab given every second week may have comparable efficacy and is more convenient for patients. While BRAF targeted therapy is a new standard therapy in pre-treated patients with BRAF V600E mutated mCRC, resistance invariably occurs and is an emerging challenge. The aim of this study is to investigate the efficacy and tolerability of cetuximab given every second week in combination with daily encorafenib and to explore the correlation between markers of resistance and outcome. METHODS: The study is an open label, single arm, phase II study, investigating the efficacy and tolerability of cetuximab given every second week in combination with encorafenib in patients with BRAF V600E mutated mCRC. Furthermore, we will be investigating mechanisms of response and resistance against BRAF targeted therapy though comprehensive genomic profiling on tumor tissue and blood for circulating tumor DNA analysis. A total of 53 patients (19 + 34 in two steps) will be included according to Simon's optimal two stage design. The primary end point of the study is 2 months PFS rate. DISCUSSION: By combining BRAF inhibitor with cetuximab given every second week we can halve the number of visits in the hospital compared to the currently approved regimen with weekly cetuximab. This seems particularly relevant in a group of patients with a median overall survival of 9.3 months. Resistance after initial response to targeted therapy can be either adaptive (e.g., epigenetic, or transcriptomic alterations) or acquired (selective genetic alterations - e.g., activating de novo mutations) resistance. It is of great importance to untangle these complex mechanisms of resistance in patients with BRAF V600E mutated mCRC to improve treatment strategies in the future potentially even further. TRIAL REGISTRATION: EU Clinical Trial Register, Eudract no. 2020-003283-10 . Registered on 11 November 2020.

Extensive genomic analysis in patients with KRAS-mutated solid tumors shows high frequencies of concurrent alterations and potential targets but has limited clinical impact.

BACKGROUND: This study aimed to investigate the distribution and frequency of concurrent alterations in different cancers across KRAS subtypes and in different KRAS subtypes across cancers, and to identify potentially actionable targets and patients who received targeted treatment matched to their genomic profile (GP). MATERIALS AND METHODS: In this descriptive and single-center study, we included 188 patients with solid tumors harboring KRAS mutations in codon 12, 13, 61, 117, or 146, referred to the Phase 1 Unit, Rigshospitalet, Copenhagen, Denmark from mid-2016 to 2020. Genomic co-alterations were detected with whole-exome sequencing, RNA sequencing, SNP array, and mRNA expression array on fresh biopsies. The study is part of the Copenhagen Prospective Personalized Oncology study (NCT02290522). RESULTS: The majority of patients had colorectal cancer (60.1%), non-small cell lung cancer (11.2%), or pancreatic cancer (10.6%). Most tumors were KRAS-mutated in codon 12 or 13 (93.7%) including G12D (27.1%), G12V (26.6%), G12C (11.7%), and G13D (11.2%). A total of 175 different co-alterations were found, most frequently pathogenic APC and TP53 mutations (55.9% and 46.4%, respectively) and high expression of CEACAM5 (73.4%). Different cancers and KRAS subtypes showed different patterns of co-alterations, and 157 tumors (83.5%) had potentially actionable targets with varying evidence of targetability (assessed using ESMO Scale for Clinical Actionability of molecular Targets). Of the 188 patients included in the study, 15 (7.4%) received treatment matched to their GP (e.g., immunotherapy and synthetic lethality drugs), of whom one had objective partial response according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. CONCLUSION: Performing extensive genomic analysis in patients with known KRAS-mutated solid tumors may contribute with information to the genomic landscape of cancers and identify targets for immunotherapy or synthetic lethality drugs, but currently appears to have overall limited clinical impact, as few patients received targeted therapy matched to their GP.

Ultra-fast detection and quantification of nucleic acids by amplification-free fluorescence assay.

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.

Immunotherapy drives mesenchymal tumor cell state shift and TME immune response in glioblastoma patients.

BACKGROUND: Glioblastoma is a highly aggressive type of brain tumor for which there is no curative treatment available. Immunotherapies have shown limited responses in unselected patients, and there is an urgent need to identify mechanisms of treatment resistance to design novel therapy strategies. METHODS: Here we investigated the phenotypic and transcriptional dynamics at single-cell resolution during nivolumab immune checkpoint treatment of glioblastoma patients. RESULTS: We present the integrative paired single-cell RNA-seq analysis of 76 tumor samples from patients in a clinical trial of the PD-1 inhibitor nivolumab and untreated patients. We identify a distinct aggressive phenotypic signature in both tumor cells and the tumor microenvironment in response to nivolumab. Moreover, nivolumab-treatment was associated with an increased transition to mesenchymal stem-like tumor cells, and an increase in TAMs and exhausted and proliferative T cells. We verify and extend our findings in large external glioblastoma dataset (n = 298), develop a latent immune signature and find 18% of primary glioblastoma samples to be latent immune, associated with mesenchymal tumor cell state and TME immune response. Finally, we show that latent immune glioblastoma patients are associated with shorter overall survival following immune checkpoint treatment (P = .0041). CONCLUSIONS: We find a resistance mechanism signature in one fifth of glioblastoma patients associated with a tumor-cell transition to a more aggressive mesenchymal-like state, increase in TAMs and proliferative and exhausted T cells in response to immunotherapy. These patients may instead benefit from neuro-oncology therapies targeting mesenchymal tumor cells.

Nivolumab reaches brain lesions in patients with recurrent glioblastoma and induces T-cell activity and upregulation of checkpoint pathways.

Glioblastoma (GBM) is an aggressive brain tumor with poor prognosis. Although immunotherapy is being explored as a potential treatment option for patients with GBM, it is unclear whether systemic immunotherapy can reach and modify the tumor microenvironment in the brain. We evaluated immune characteristics in patients receiving the anti-PD1 immune checkpoint inhibitor Nivolumab one week prior to surgery, compared to control patients receiving salvage resection without prior Nivolumab treatment. We observed saturating levels of Nivolumab bound to intratumorally- and tissue-resident T cells in the brain, implicating saturating levels of Nivolumab reaching brain tumors. Following Nivolumab treatment, significant changes in T-cell activation and proliferation were observed in the tumor resident T-cell population, and peripheral T cells upregulated chemokine receptors related to brain homing. A strong Nivolumab-driven upregulation in compensatory checkpoint inhibition molecules, TIGIT, LAG-3, TIM-3 and CTLA-4 was observed, potentially counteracting the treatment effect. Finally, tumor-reactive tumor-infiltrating lymphocytes (TILs) were found in a subset of Nivolumab-treated patients with prolonged survival, and neoantigen-reactive T cells were identified in both TILs and blood. This indicates a systemic response towards GBM in a subset of patients, which was further boosted by Nivolumab, with T-cell responses towards tumor-derived neoantigens. Our study demonstrates that Nivolumab does reach the GBM tumor lesion and enhances antitumor T-cell responses both intratumorally and systemically. However, various anti-inflammatory mechanisms mitigate the clinical efficacy of the anti-PD1 treatment.

Dose escalation study of a personalized peptide-based neoantigen vaccine (EVX-01) in patients with metastatic melanoma.

BACKGROUND: Neoantigens can serve as targets for T cell-mediated antitumor immunity via personalized neopeptide vaccines. Interim data from our clinical study NCT03715985 showed that the personalized peptide-based neoantigen vaccine EVX-01, formulated in the liposomal adjuvant, CAF09b, was safe and able to elicit EVX-01-specific T cell responses in patients with metastatic melanoma. Here, we present results from the dose-escalation part of the study, evaluating the feasibility, safety, efficacy, and immunogenicity of EVX-01 in addition to anti-PD-1 therapy. METHODS: Patients with metastatic melanoma on anti-PD-1 therapy were treated in three cohorts with increasing vaccine dosages (twofold and fourfold). Tumor-derived neoantigens were selected by the AI platform PIONEER and used in personalized therapeutic cancer peptide vaccines EVX-01. Vaccines were administered at 2-week intervals for a total of three intraperitoneal and three intramuscular injections. The study's primary endpoint was safety and tolerability. Additional endpoints were immunological responses, survival, and objective response rates. RESULTS: Compared with the base dose level previously reported, no new vaccine-related serious adverse events were observed during dose escalation of EVX-01 in combination with an anti-PD-1 agent given according to local guidelines. Two patients at the third dose level (fourfold dose) developed grade 3 toxicity, most likely related to pembrolizumab. Overall, 8 out of the 12 patients had objective clinical responses (6 partial response (PR) and 2 CR), with all 4 patients at the highest dose level having a CR (1 CR, 3 PR). EVX-01 induced peptide-specific CD4+ and/or CD8+T cell responses in all treated patients, with CD4+T cells as the dominating responses. The magnitude of immune responses measured by IFN-γ ELISpot assay correlated with individual peptide doses. A significant correlation between the PIONEER quality score and induced T cell immunogenicity was detected, while better CRs correlated with both the number of immunogenic EVX-01 peptides and the PIONEER quality score. CONCLUSION: Immunization with EVX-01-CAF09b in addition to anti-PD-1 therapy was shown to be safe and well tolerated and elicit vaccine neoantigen-specific CD4+and CD8+ T cell responses at all dose levels. In addition, objective tumor responses were observed in 67% of patients. The results encourage further assessment of the antitumor efficacy of EVX-01 in combination with anti-PD-1 therapy.

Neoepitope load, T cell signatures and PD-L2 as combined biomarker strategy for response to checkpoint inhibition immunotherapy.

Immune checkpoint inhibition for the treatment of cancer has provided a breakthrough in oncology, and several new checkpoint inhibition pathways are currently being investigated regarding their potential to provide additional clinical benefit. However, only a fraction of patients respond to such treatment modalities, and there is an urgent need to identify biomarkers to rationally select patients that will benefit from treatment. In this study, we explore different tumor associated characteristics for their association with favorable clinical outcome in a diverse cohort of cancer patients treated with checkpoint inhibitors. We studied 29 patients in a basket trial comprising 12 different tumor types, treated with 10 different checkpoint inhibition regimens. Our analysis revealed that even across this diverse cohort, patients achieving clinical benefit had significantly higher neoepitope load, higher expression of T cell signatures, and higher PD-L2 expression, which also correlated with improved progression-free and overall survival. Importantly, the combination of biomarkers serves as a better predictor than each of the biomarkers alone. Basket trials are frequently used in modern immunotherapy trial design, and here we identify a set of biomarkers of potential relevance across multiple cancer types, allowing for the selection of patients that most likely will benefit from immune checkpoint inhibition.

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

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.

Personalized therapy with peptide-based neoantigen vaccine (EVX-01) including a novel adjuvant, CAF®09b, in patients with metastatic melanoma.

The majority of neoantigens arise from unique mutations that are not shared between individual patients, making neoantigen-directed immunotherapy a fully personalized treatment approach. Novel technical advances in next-generation sequencing of tumor samples and artificial intelligence (AI) allow fast and systematic prediction of tumor neoantigens. This study investigates feasibility, safety, immunity, and anti-tumor potential of the personalized peptide-based neoantigen vaccine, EVX-01, including the novel CD8+ T-cell inducing adjuvant, CAF®09b, in patients with metastatic melanoma (NTC03715985). The AI platform PIONEERTM was used for identification of tumor-derived neoantigens to be included in a peptide-based personalized therapeutic cancer vaccine. EVX-01 immunotherapy consisted of 6 administrations with 5-10 PIONEERTM-predicted neoantigens as synthetic peptides combined with the novel liposome-based Cationic Adjuvant Formulation 09b (CAF®09b) to strengthen T-cell responses. EVX-01 was combined with immune checkpoint inhibitors to augment the activity of EVX-01-induced immune responses. The primary endpoint was safety, exploratory endpoints included feasibility, immunologic and objective responses. This interim analysis reports the results from the first dose-level cohort of five patients. We documented a short vaccine manufacturing time of 48-55 days which enabled the initiation of EVX-01 treatment within 60 days from baseline biopsy. No severe adverse events were observed. EVX-01 elicited long-lasting EVX-01-specific T-cell responses in all patients. Competitive manufacturing time was demonstrated. EVX-01 was shown to be safe and able to elicit immune responses targeting tumor neoantigens with encouraging early indications of a clinical and meaningful antitumor efficacy, warranting further study.

Adoptive cell therapy with tumor-infiltrating lymphocytes supported by checkpoint inhibition across multiple solid cancer types.

BACKGROUND: Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) has shown remarkable results in malignant melanoma (MM), while studies on the potential in other cancer diagnoses are sparse. Further, the prospect of using checkpoint inhibitors (CPIs) to support TIL production and therapy remains to be explored. STUDY DESIGN: TIL-based ACT with CPIs was evaluated in a clinical phase I/II trial. Ipilimumab (3 mg/kg) was administered prior to tumor resection and nivolumab (3 mg/kg, every 2 weeks ×4) in relation to TIL infusion. Preconditioning chemotherapy was given before TIL infusion and followed by low-dose (2 10e6 international units (UI) ×1 subcutaneous for 14 days) interleukin-2 stimulation. RESULTS: Twenty-five patients covering 10 different cancer diagnoses were treated with in vitro expanded TILs. Expansion of TILs was successful in 97% of recruited patients. Five patients had sizeable tumor regressions of 30%-63%, including two confirmed partial responses in patients with head-and-neck cancer and cholangiocarcinoma. Safety and feasibility were comparable to MM trials of ACT with the addition of expected CPI toxicity. In an exploratory analysis, tumor mutational burden and expression of the alpha-integrin CD103 (p=0.025) were associated with increased disease control. In vitro tumor reactivity was seen in both patients with an objective response and was associated with regressions in tumor size (p=0.028). CONCLUSION: High success rates of TIL expansion were demonstrated across multiple solid cancers. TIL ACTs were found feasible, independent of previous therapy. Tumor regressions after ACT combined with CPIs were demonstrated in several cancer types supported by in vitro antitumor reactivity of the TILs. TRIAL REGISTRATION NUMBERS: NCT03296137, and EudraCT No. 2017-002323-25.

A Comparison of Tools for Copy-Number Variation Detection in Germline Whole Exome and Whole Genome Sequencing Data.

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.

The Number of Signaling Pathways Altered by Driver Mutations in Chronic Lymphocytic Leukemia Impacts Disease Outcome.

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.

Copenhagen Prospective Personalized Oncology (CoPPO)-Clinical Utility of Using Molecular Profiling to Select Patients to Phase I Trials.

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.

Cell-free DNA in newly diagnosed patients with glioblastoma - a clinical prospective feasibility study.

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.

High frequency of pathogenic germline variants within homologous recombination repair in patients with advanced cancer.

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.

Breast cancer cells with acquired antiestrogen resistance are sensitized to cisplatin-induced cell death.

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.

Circulating tumor DNA as a marker of treatment response in BRAF V600E mutated non-melanoma solid tumors.

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.