PracticeUpdate: Haematology & Oncology

SABCS 2016 27

Tumour tissue, single-CTC exome sequencingmay provide complementary info tomapmetastatic breast tumour heterogeneity T umour tissue and single-circulating tumour cell exome sequencing may provide complementary information Three patients, each with one of the three major breast cancer subtypes, were studied: Patient 1: Oestrogen receptor-negative/human epidermal growth factor receptor 2-positive breast cancer. Samples collected at diagnosis, initially metastatic disease. Patient 2 : Triple-negative breast cancer. Samples collected 2 years after diagnosis. Patient 3 : Oestrogen receptor-positive/human epidermal growth factor receptor 2-negative breast cancer. Samples collected 8 years after diagnosis.

to map tumour heterogeneity in patients with metastatic breast cancer, results of a comparative genomic study of patients with metastatic breast cancer. Michail Ignatiadis, MD, PhD, of Jules Bordet Institut, Brussels, Belgium, explained that he and colleagues set out to determine whether circulating tumour cells can complement metastatic biopsies for genomic analyses. The investigators compared single nucleotide variants and copy number aberrations identi- fied using whole exome sequencing of DNA from frozen tumour tissue from primary and metastatic amplified DNA from circulating tumour cells and normal DNA from three patients with metastatic breast cancer. All samples from the same patient were collected at the same time point. Circulating tumour cell isolation was performed using the CellSearch and DEPArray systems followed by whole genome amplification using the Ampli1 kit. Whole exome sequencing was performed using the Illumina HiSeq2000 with 200X targeted coverage. Reads were aligned using the Burrows-Wheeler aligner. Single nucleotide variants had to be called by both Haplotype Caller (vs reference genome) and Strelka (vs paired normal). Copy number alterations were determined by counting reads in 1MBwindows and comparing tumour/circu- lating tumour cell samples with normal DNA.

First, tumour tissue was compared with circulating tumour cells for single nucleotide variants: Patient 1: Of 77 single nucleotide variants identified in the tumour, 51 were found on at least one of 12 circulating tumour cell samples. Patient 2: Of 62 single nucleotide variants identified in the tumour, 19 were found on at least one of 11 circulating tumour cell samples. Patient 3: Of 225 single nucleotide variants identified in the tumour, 48 were found on at least one of three circulating tumour cell samples.

nucleotide variants with variant allele frac- tions >20% and >40%, respectively (P = 10–12, Fisher exact test). The team then compared tumour tissue and circulating tumour cells for copy number aberrations. As the time from diagnosis of metastatic disease to sample collection increased, significantly higher heterogeneity within circulating tumour cells from the same patient was observed (median P between circulating tumour cells was 86% for patient 1, 84% for patient 2, and 28% for patient 3, P < 0.01). The same observation applied between circulating tumour cells and tumour tissue from the same patient (median P was 78% for patient 1, 67% for patient 2, and 21% for patient 3, P < 10 -4 ). Interestingly, in patient 3, one circulating tu- mour cell was more similar to the metastasis than the other two (P of 53%, 21%, and 21%). When a phylogenetic tree was constructed for patient 3 by combining single nucleotide variants and copy number aberration data, three clones were identified: • One clone with an AKT1 (E17K) and a TP53 (R248W) mutation and an 8p deletion • A second clone with the above profile plus an 8q amplification • A third clone with an AKT1 and an ESR1 (Y537N) mutation and 1p deletion. The metastasis was similar with the first clone. Dr Ignatiadis concluded that tumour tissue and single circulating tumour cell exome sequenc- ing may provide complementary information to map tumour heterogeneity. Further validation for potential clinical applications is needed.

Pairwise concordance of copy number altera- tion profiles of different samples from the same patient was assessed using Spearman correla- tion. Significance of P differences between patients was obtained using the Kruskal-Wallis test. Orthogonal validation for selected single nucleotide variants was performed. Interestingly, by increasing the number of circulating tumour cells analysed, the percent of identified single nucleotide variants from synchronous tumour tissue increased. Single nucleotide variants with high variant allele fraction in tumour tissue were detected significantly more often in circulating tumour cells: 22% of single nucleotide variants with variant allele fractions <20% were found at least once, versus 53% and 74% of single

PracticeUpdate Editorial Team © MedMeetingImages/Todd Buchanan 2016

VOL. 2 • NO. 1 • 2017