Dominiek Smeets

Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer

BACKGROUND Molecular screening programs use next-generation sequencing (NGS) of cancer gene panels to analyze metastatic biopsies. We interrogated whether plasma could be used as an alternative to metastatic biopsies. PATIENTS AND METHODS The Ion AmpliSeq™ Cancer Hotspot Panel v2 (Ion Torrent), covering 2800 COSMIC mutations from 50 cancer genes was used to analyze 69 tumor (primary/metastases) and 31 plasma samples from 17 metastatic breast cancer patients. The targeted coverage for tumor DNA was ×1000 and for plasma cell-free DNA ×25 000. Whole blood normal DNA was used to exclude germline variants. The Illumina technology was used to confirm observed mutations. RESULTS Evaluable NGS results were obtained for 60 tumor and 31 plasma samples from 17 patients. When tumor samples were analyzed, 12 of 17 (71%, 95% confidence interval (CI) 44% to 90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1 or IDH2 gene. When plasma samples were analyzed, 12 of 17 (71%, 95% CI: 44-90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1, IDH2 and SMAD4. All mutations were confirmed. When we focused on tumor and plasma samples collected at the same time-point, we observed that, in four patients, no mutation was identified in either tumor or plasma; in nine patients, the same mutations was identified in tumor and plasma; in two patients, a mutation was identified in tumor but not in plasma; in two patients, a mutation was identified in plasma but not in tumor. Thus, in 13 of 17 (76%, 95% CI 50% to 93%) patients, tumor and plasma provided concordant results whereas in 4 of 17 (24%, 95% CI 7% to 50%) patients, the results were discordant, providing complementary information. CONCLUSION Plasma can be prospectively tested as an alternative to metastatic biopsies in molecular screening programs.

Mismatch repair deficiency endows tumors with a unique mutation signature and sensitivity to DNA double-strand breaks

DNA replication errors that persist as mismatch mutations make up the molecular fingerprint of mismatch repair (MMR)-deficient tumors and convey them with resistance to standard therapy. Using whole-genome and whole-exome sequencing, we here confirm an MMR-deficient mutation signature that is distinct from other tumor genomes, but surprisingly similar to germ-line DNA, indicating that a substantial fraction of human genetic variation arises through mutations escaping MMR. Moreover, we identify a large set of recurrent indels that may serve to detect microsatellite instability (MSI). Indeed, using endometrial tumors with immunohistochemically proven MMR deficiency, we optimize a novel marker set capable of detecting MSI and show it to have greater specificity and selectivity than standard MSI tests. Additionally, we show that recurrent indels are enriched for the ‘DNA double-strand break repair by homologous recombination’ pathway. Consequently, DSB repair is reduced in MMR-deficient tumors, triggering a dose-dependent sensitivity of MMR-deficient tumor cultures to DSB inducers. DOI: http://dx.doi.org/10.7554/eLife.02725.001

Genetic heterogeneity after first-line chemotherapy in high-grade serous ovarian cancer.

BACKGROUND Most high-grade serous ovarian carcinoma (HGSOC) patients benefit from first-line platinum-based chemotherapy, but progressively develop resistance during subsequent lines. Re-activating BRCA1 or MDR1 mutations can underlie platinum resistance in end-stage patients. However, little is known about resistance mechanisms occurring after a single line of platinum, when patients still qualify for other treatments. METHODS In 31 patients with primary platinum-sensitive HGSOC, we profiled tumours collected during debulking surgery before and after first-line chemotherapy using whole-exome sequencing and single nucleotide polymorphism profiling. RESULTS Besides germline BRCA1/2 mutations, we observed frequent loss-of-heterozygosity in homologous recombination (HR) genes and mutation spectra characteristic of HR-deficiency in all tumours. At relapse, tumours differed considerably from their primary counterparts. There was, however, no evidence of events reactivating the HR pathway, also not in tumours resistant to second-line platinum. Instead, a platinum score of 13 copy number regions, among other genes including MECOM, CCNE1 and ERBB2, correlated with platinum-free interval (PFI) after first-line therapy, whereas an increase of this score in recurrent tumours predicted the change in PFI during subsequent therapy. CONCLUSIONS Already after a single line of platinum, there is huge variability between primary and recurrent tumours, advocating that in HGSOC biopsies need to be collected at relapse to tailor treatment options to the underlying genetic profile. Nevertheless, all primary platinum-sensitive HGSOCs remained HR-deficient, irrespective of whether they became resistant to second-line platinum, further suggesting these tumours qualify for second-line Poly APD ribose polymerase (PARP) inhibitor treatment. Finally, chromosomal instability contributes to acquired resistance after a single line of platinum therapy.

Genetic variability in drug transport, metabolism or DNA repair affecting toxicity of chemotherapy in ovarian cancer

BackgroundThis study aimed to determine whether single nucleotide polymorphisms (SNPs) in genes involved in DNA repair or metabolism of taxanes or platinum could predict toxicity or response to first-line chemotherapy in ovarian cancer.MethodsTwenty-six selected SNPs in 18 genes were genotyped in 322 patients treated with first-line paclitaxel-carboplatin or carboplatin mono-therapy. Genotypes were correlated with toxicity events (anemia, neutropenia, thrombocytopenia, febrile neutropenia, neurotoxicity), use of growth factors and survival.ResultsThe risk of anemia was increased for variant alleles of rs1128503 (ABCB1, C > T; p = 0.023, OR = 1.71, 95% CI = 1.07-2.71), rs363717 (ABCA1, A > G; p = 0.002, OR = 2.08, 95% CI = 1.32-3.27) and rs11615 (ERCC1, T > C; p = 0.031, OR = 1.61, 95% CI = 1.04-2.50), while it was decreased for variant alleles of rs12762549 (ABCC2, C > G; p = 0.004, OR = 0.51, 95% CI = 0.33-0.81). Likewise, increased risk of thrombocytopenia was associated with rs4986910 (CYP3A4, T > C; p = 0.025, OR = 4.99, 95% CI = 1.22-20.31). No significant correlations were found for neurotoxicity. Variant alleles of rs2073337 (ABCC2, A > G; p = 0.039, OR = 0.60, 95% CI = 0.37-0.98), rs1695 (ABCC1, A > G; p = 0.017, OR = 0.55, 95% CI 0.33-0.90) and rs1799793 (ERCC2, G > A; p = 0.042, OR = 0.63, 95% CI 0.41-0.98) associated with the use of colony stimulating factors (CSF), while rs2074087 (ABCC1, G > C; p = 0.011, OR = 2.09, 95% CI 1.18-3.68) correlated with use of erythropoiesis stimulating agents (ESAs). Homozygous carriers of the rs1799793 (ERCC2, G > A) G-allele had a prolonged platinum-free interval (p = 0.016).ConclusionsOur data reveal significant correlations between genetic variants of transport, hepatic metabolism, platinum related detoxification or DNA damage repair and toxicity or outcome in ovarian cancer.

Expression profiling of budding cells in colorectal cancer reveals an EMT-like phenotype and molecular subtype switching.

Background:Tumour budding, described as the presence of single cells or small clusters of up to five tumour cells at the invasive margin, is established as a prognostic marker in colorectal carcinoma. In the present study, we aimed to investigate the molecular signature of tumour budding cells and the corresponding tumour bulk.Methods:Tumour bulk and budding areas were microdissected and processed for RNA-sequencing. As little RNA was obtained from budding cells, a special low-input mRNA library preparation protocol was used. Gene expression profiles of budding as compared with tumour bulk were investigated for established EMT signatures, consensus molecular subtype (CMS), gene set enrichment and pathway analysis.Results:A total of 296 genes were differentially expressed with an FDR <0.05 and a twofold change between tumour bulk and budding regions. Genes that were upregulated in the budding signature were mainly involved in cell migration and survival while downregulated genes were important for cell proliferation. Supervised clustering according to an established EMT gene signature categorised budding regions as EMT-positive, whereas tumour bulk was considered EMT-negative. Furthermore, a shift from CMS2 (epithelial) to CMS4 (mesenchymal) was observed as tumour cells transit from the tumour bulk to the budding regions.Conclusions:Tumour budding regions are characterised by a phenotype switch compared with the tumour bulk, involving the acquisition of migratory characteristics and a decrease in cell proliferation. In particular, most tumour budding signatures were EMT-positive and switched from an epithelial subtype (CMS2) in the tumour bulk to a mesenchymal subtype (CMS4) in budding cells.

Phylogenetic analysis of metastatic progression in breast cancer using somatic mutations and copy number aberrations

Several studies using genome-wide molecular techniques have reported various degrees of genetic heterogeneity between primary tumours and their distant metastases. However, it has been difficult to discern patterns of dissemination owing to the limited number of patients and available metastases. Here, we use phylogenetic techniques on data generated using whole-exome sequencing and copy number profiling of primary and multiple-matched metastatic tumours from ten autopsied patients to infer the evolutionary history of breast cancer progression. We observed two modes of disease progression. In some patients, all distant metastases cluster on a branch separate from their primary lesion. Clonal frequency analyses of somatic mutations show that the metastases have a monoclonal origin and descend from a common ‘metastatic precursor’. Alternatively, multiple metastatic lesions are seeded from different clones present within the primary tumour. We further show that a metastasis can be horizontally cross-seeded. These findings provide insights into breast cancer dissemination.

Chromosomal instability in cell-free DNA as a highly specific biomarker for detection of ovarian cancer in women with adnexal masses

Purpose: Chromosomal instability is a hallmark of ovarian cancer. Here, we explore copy-number alteration (CNA) profiling in cell-free DNA as a potential biomarker to detect malignancy in patients presenting with an adnexal mass. Experimental Design: We prospectively enrolled 68 patients with an adnexal mass, of which 57 were diagnosed with invasive or borderline carcinoma and 11 with benign disease. Cell-free DNA was extracted from plasma and analyzed by low-coverage whole-genome sequencing. Results: Patterns of chromosomal instability were detectable in cell-free DNA using 44 healthy individuals as a reference. Profiles were representative of those observed in matching tumor tissue and contained CNAs enriched in two large datasets of high-grade serous ovarian cancer (HGSOC). Quantitative measures of chromosomal instability, referred to as genome-wide z-scores, were significantly higher in patients with ovarian carcinoma than in healthy individuals or patients with benign disease. Cell-free DNA testing improved malignancy detection (AUC 0.89) over serum CA-125 (AUC 0.78) or the risk of malignancy index (RMI, AUC 0.81). AUC values of cell-free DNA testing even further increased for HGSOC patients specifically (AUC 0.94). At a specificity of 99.6%, a theoretical threshold required for ovarian cancer screening, sensitivity of cell-free DNA testing was 2- to 5-fold higher compared with CA-125 and RMI testing. Conclusions: This is the first study evaluating the potential of cell-free DNA for the diagnosis of primary ovarian cancer using chromosomal instability as a read-out. We present a promising method to increase specificity of presurgical prediction of malignancy in patients with adnexal masses. Clin Cancer Res; 23(9); 2223–31. ©2016 AACR.

Molecular characterization of 7 new established cell lines from high grade serous ovarian cancer

Cancer cell lines are good in vitro models to study molecular mechanisms underlying chemoresistance and cancer recurrence. Recent works have demonstrated that most of the available ovarian cancer cell lines are most unlikely high grade serous (HGSOC), the major type of epithelial ovarian cancer. We aimed at establishing well characterized HGSOC cell lines, which can be used as optimal models for ovarian cancer research. We successfully established seven cell lines from HGSOC and provided the major genomic alterations and the transcriptomic landscapes of them. They exhibited different gene expression patterns in the key pathways involved in cancer resistance. Each cell line harbored a unique TP53 mutation as their corresponding tumors and expressed cytokeratins 8/18/19 and EpCAM. Two matched lines were established from the same patient, one at diagnosis and being sensitive to carboplatin and the other during chemotherapy and being resistant. Two cell lines presented respective BRCA1 and BRCA2 mutations. To conclude, we have established seven cell lines and well characterized them at genomic and transcriptomic levels. They are optimal models to investigate the molecular mechanisms underlying the progression, chemo resistance and recurrence of HGSOC.

The rs1800716 variant in CYP2D6 is associated with an increased double endometrial thickness in postmenopausal women on tamoxifen

BACKGROUND Tamoxifen remains important in the treatment and prevention of estrogen receptor-positive breast cancer. In postmenopausal women, it can lead to endometrial changes such as cystic appearances, hyperplasia, polyps and endometrial cancer. Tamoxifen is metabolized by cytochrome P450 (CYP450) enzymes to the more active metabolite endoxifen. Several genetic variants in the CYP450 enzymes reduce tamoxifen metabolism, leading to reduced endoxifen levels. We hypothesize that carriers of these variants, which are established poor metabolizers of tamoxifen, do not have the typical tamoxifen-induced increase in endometrial thickness. We test the association between genetic variability in CYP450 enzymes and the increase in double endometrial thickness (DET) as measured through transvaginal ultrasound (TVU). PATIENTS AND METHODS We carried out a retrospective study on postmenopausal tamoxifen users for which germline DNA was available and at least one DET measurement was made between January 2000 and October 2011. Genotyping of 33 single nucleotide polymorphisms in CYP450 genes involved in tamoxifen metabolism was carried out using Sequenom MassARRAY. The association between these variants and TVU outcome (DET ≥5 mm) was assessed by proportional hazards regression. RESULTS Data were available for 184 women: 47 with a DET of <5 mm on all ultrasounds and 137 with a DET of ≥5 mm on at least one ultrasound. The rs1800716 variant in CYP2D6 showed a statistically significant association with DET. In particular, mutant carriers of rs1800716 had an increased chance of having a DET of ≥5 mm (P = 0.0022, false discovery rate 0.0179). None of the other variants were associated with DET. CONCLUSION Although mutant carriers of rs1800716 are characterized by reduced CYP2D6 enzyme activity and by low levels of endoxifen, we observed that mutant alleles of rs1800716 were associated with an increased chance of having a DET of ≥5 mm in postmenopausal women on tamoxifen. We conclude that the increase in endometrial thickness seen under tamoxifen cannot be used as a marker for favorable genotypes. CLINICAL TRIAL NUMBER B32220084284.

Phospholipase C gamma 1 (PLCG1) R707Q mutation is counterselected under targeted therapy in a patient with hepatic angiosarcoma.

Hepatic angiosarcoma is a rare and aggressive vascular neoplasm. Pathogenic driver mutations are largely unknown. We present the case of a patient with recurrent hepatic angiosarcoma, who initially showed good response to sunitinib, followed by progression. Using comprehensive molecular techniques, we explored the potential mechanisms of resistance. By low-read-depth whole-genome sequencing, the comparison of copy number aberrations (CNAs) of the primary tumor to the skin metastatic lesion that developed after progression on sunitinib, revealed high-level amplification of the 4q11-q13.1 region (containing KIT, PDGFRA and VEGFR2 genes) that was sustained in both lesions. Whole exome sequencing on the germline, primary and metastatic tumor DNAs, resulted in 27 confirmed mutations, 19 of which (including TP53 mutation) presented in both primary and metastatic lesions. One mutation, ZNF331 frameshift deletion, was detected only in the primary tumor. In contrast, seven other mutations, including phospholipase C-gamma1 (PLCG1) R707Q mutation, were found only in the metastatic tumor, indicating selection of cells with the resistant genotype under sunitinib pressure. Our study supports the notion that PLCG1-R707Q mutation may confer VEGFR2-independent signaling and may thus cause resistance against VEGF(R)-directed therapies. This case illustrates also the advantages of using next-generation technologies in identifying individualized targeted therapy.