Karen Page

Genomic analysis of circulating cell-free DNA infers breast cancer dormancy

Biomarkers in breast cancer to monitor minimal residual disease have remained elusive. We hypothesized that genomic analysis of circulating free DNA (cfDNA) isolated from plasma may form the basis for a means of detecting and monitoring breast cancer. We profiled 251 genomes using Affymetrix SNP 6.0 arrays to determine copy number variations (CNVs) and loss of heterozygosity (LOH), comparing 138 cfDNA samples with matched primary tumor and normal leukocyte DNA in 65 breast cancer patients and eight healthy female controls. Concordance of SNP genotype calls in paired cfDNA and leukocyte DNA samples distinguished between breast cancer patients and healthy female controls (P < 0.0001) and between preoperative patients and patients on follow-up who had surgery and treatment (P = 0.0016). Principal component analyses of cfDNA SNP/copy number results also separated presurgical breast cancer patients from the healthy controls, suggesting specific CNVs in cfDNA have clinical significance. We identified focal high-level DNA amplification in paired tumor and cfDNA clustered in a number of chromosome arms, some of which harbor genes with oncogenic potential, including USP17L2 (DUB3), BRF1, MTA1, and JAG2. Remarkably, in 50 patients on follow-up, specific CNVs were detected in cfDNA, mirroring the primary tumor, up to 12 yr after diagnosis despite no other evidence of disease. These data demonstrate the potential of SNP/CNV analysis of cfDNA to distinguish between patients with breast cancer and healthy controls during routine follow-up. The genomic profiles of cfDNA infer dormancy/minimal residual disease in the majority of patients on follow-up.

Influence of plasma processing on recovery and analysis of circulating nucleic acids.

Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp® DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (ρ = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA.

Detection of HER2 amplification in circulating free DNA in patients with breast cancer

Background:Human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in 20–25% of breast cancers. This study investigated circulating free DNA (cfDNA) for detection of HER2 gene amplification in patients with breast cancer.Methods:Circulating free DNA was extracted from plasma of unselected patients with primary breast cancer (22 before surgery and 68 following treatment), 30 metastatic patients and 98 female controls using the QIAamp Blood DNA Mini Kit (Qiagen). The ratio of HER2 to an unamplified reference gene (contactin-associated protein 1 (CNTNAP1)) was measured in cfDNA samples by quantitative PCR (qPCR) using SK-BR-3 cell line DNA as a positive control.Results:We validated the qPCR assay with DNA extracted from 23 HER2 3+ and 40 HER2-negative tumour tissue samples; the results agreed for 60 of 63 (95.2%) tumours. Amplification was detected in cfDNA for 8 of 68 patients following primary breast cancer treatment and 5 of 30 metastatic patients, but was undetected in 22 patients with primary breast cancer and 98 healthy female controls. Of the patients with amplification in cfDNA, 10 had HER2 3+ tumour status by immunohistochemistry.Conclusions:The results demonstrate for the first time the existence of amplified HER2 in cfDNA in the follow-up of breast cancer patients who are otherwise disease free. This approach could potentially provide a marker in patients with HER2-positive breast cancer.

The Importance of Careful Blood Processing in Isolation of Cell‐Free DNA

Abstract:  In healthy individuals, the source of cell‐free plasma DNA is predominantly apoptotic, whereas, increased plasma DNA integrity is seen in cancer patients. Therefore, it is important to carefully isolate absolutely “cell‐free” plasma DNA. Plasma DNA from 30 healthy females was analyzed using 4 PCR amplicons of increasing size, comparing standard blood processing with additional centrifugation steps prior to DNA extraction. Cellular DNA contamination, indicated by positive amplicons >300 bp was eliminated only after the extra centrifugation step. This highlights the importance of careful processing in preparation of cell‐free plasma DNA as a tool for cancer detection and we recommend the use of a microcentrifuge spin, prior to DNA extraction.

Hide and seek: tell-tale signs of breast cancer lurking in the blood

Breast cancer treatment is improving due to the introduction of new drugs, guided by molecular testing of the primary tumour for mutations/oncogenic drivers (e.g. HER2 gene amplification). However, tumour tissue is not always available for molecular analysis, intra-tumoural heterogeneity is common and the “cancer genome” is known to evolve with time, particularly following treatment as resistance develops. After resection, those patients with only residual micrometastases are likely to be cured but those with radiologically detectable overt disease are not. Thus, the discovery of blood test(s) that could (1) alert clinicians to early primary or recurrent disease and (2) monitor response to treatment could impact significantly on mortality. Towards this, we and others have focused on molecular profiling of circulating nucleic acids isolated from plasma, both cell-free DNA (cfDNA) and microRNAs, and the relationship of these to circulating tumour cells (CTCs). This review considers the utility of each as circulating biomarkers in breast cancer with particular emphasis on the bioinformatic tools available to support molecular profiling.

The presence of disseminated tumour cells in the bone marrow is inversely related to circulating free DNA in plasma in breast cancer dormancy

Background:The aim of this study was to gain insight into breast cancer dormancy by examining different measures of minimal residual disease (MRD) over time in relation to known prognostic factors.Methods:Sixty-four primary breast cancer patients on follow-up (a median of 8.3 years post surgery) who were disease free had sequential bone marrow aspirates and blood samples taken for the measurement of disseminated tumour cells (DTCs), circulating tumour cells (CTCs) by CellSearch and qPCR measurement of overlapping (96-bp and 291-bp) amplicons in circulating free DNA (cfDNA).Results:The presence of CTCs was correlated with the presence of DTCs measured by immunocytochemistry (P=0.01) but both were infrequently detected. Increasing cfDNA concentration correlated with ER, HER2 and triple-negative tumours and high tumour grade, and the 291-bp amplicon was inversely correlated with DTCs measured by CK19 qRT-PCR (P=0.047).Conclusion:Our results show that breast cancer patients have evidence of MRD for many years after diagnosis despite there being no overt evidence of disease. The inverse relationship between bone marrow CK19 mRNA and the 291-bp amplicon in cfDNA suggests that an inverse relationship between a measure of cell viability in the bone marrow (DTCs) and cell death in the plasma occurs during the dormancy phase of breast cancer.

Whole Genome Sequence Analysis Suggests Intratumoral Heterogeneity in Dissemination of Breast Cancer to Lymph Nodes

Background Intratumoral heterogeneity may help drive resistance to targeted therapies in cancer. In breast cancer, the presence of nodal metastases is a key indicator of poorer overall survival. The aim of this study was to identify somatic genetic alterations in early dissemination of breast cancer by whole genome next generation sequencing (NGS) of a primary breast tumor, a matched locally-involved axillary lymph node and healthy normal DNA from blood. Methods Whole genome NGS was performed on 12 µg (range 11.1–13.3 µg) of DNA isolated from fresh-frozen primary breast tumor, axillary lymph node and peripheral blood following the DNA nanoball sequencing protocol. Single nucleotide variants, insertions, deletions, and substitutions were identified through a bioinformatic pipeline and compared to CIN25, a key set of genes associated with tumor metastasis. Results Whole genome sequencing revealed overlapping variants between the tumor and node, but also variants that were unique to each. Novel mutations unique to the node included those found in two CIN25 targets, TGIF2 and CCNB2, which are related to transcription cyclin activity and chromosomal stability, respectively, and a unique frameshift in PDS5B, which is required for accurate sister chromatid segregation during cell division. We also identified dominant clonal variants that progressed from tumor to node, including SNVs in TP53 and ARAP3, which mediates rearrangements to the cytoskeleton and cell shape, and an insertion in TOP2A, the expression of which is significantly associated with tumor proliferation and can segregate breast cancers by outcome. Conclusion This case study provides preliminary evidence that primary tumor and early nodal metastasis have largely overlapping somatic genetic alterations. There were very few mutations unique to the involved node. However, significant conclusions regarding early dissemination needs analysis of a larger number of patient samples.

The role of ctDNA detection and the potential of the liquid biopsy for breast cancer monitoring

ABSTRACT Introduction: Recent advances in deep amplicon sequencing have enabled rapid assessment of somatic mutations and structural changes in multiple cancer genes in DNA isolated from tumour tissues and circulating cell-free DNA (cfDNA). This cfDNA is under investigation as a ‘liquid biopsy’ for the real time monitoring of patients with cancer in a growing number of research studies and clinical trials. Areas covered: Here we will provide a brief overview of the potential clinical utility of cfDNA profiling for detection and monitoring of patients with breast cancer. The review was conducted in English using PubMed and search terms including ‘breast cancer’, ‘plasma DNA’, ‘circulating cell free DNA’ and ‘circulating tumour DNA’. Expert commentary: Liquid biopsies through circulating tumor DNA (ctDNA) enable monitoring of patients with breast cancer. The challenge ahead will be to incorporate cfDNA mutation profiling into routine clinical practice to provide patients with the most appropriate and timely treatment.

Determination of Breast Cancer Dormancy: Analysis of Circulating Free DNA Using SNP 6.0 Arrays

New biomarkers are needed in breast cancer to monitor minimal residual disease. Specific point mutations, promoter methylation and loss of heterozygosity have been demonstrated previously in paired tumor and circulating cell-free DNA (cfDNA) isolated from plasma. Moreover, acquired alterations unique to cfDNA have also been found, suggesting disease progression. This prompted us to characterize the “circulating” breast cancer genome, thus testing the hypothesis that cfDNA acts as a surrogate liquid biopsy of breast cancer. This was achieved using Affymetrix SNP 6.0 technology and bioinformatics to map single nucleotide polymorphism (SNP) and copy number variation (CNV), comparing cfDNA with matched normal leucocyte and primary tumor DNA in breast cancer patients and paired normal leucocytes and cfDNA in healthy female controls. Our results show that concordance of SNP genotype calls in paired leucocytes and cfDNA can distinguish between primary breast cancer patients and healthy controls (p < 0.0001), and between pre-surgical breast cancer patients and patients on follow-up after surgery and treatment (p = 0.0016). In 50 patients on follow-up, a significant difference (p = 0.0006) was seen between cfDNA samples taken an average of 3 years apart, suggesting disease progression. Considering CNVs, amplification was observed in matched tumor and cfDNA at multiple loci on different chromosome arms but was quiet or absent in normal DNA. Many of these tumor-specific CNVs contributed significantly to disease through binary logistic regression analysis. Furthermore these CNVs remained detectable in cfDNA up to 12 years after diagnosis and treatment despite no other evidence of disease. Taken together these cfDNA results suggest breast cancer dormancy in the majority of the patients on follow-up.