Single Cell Pheno-Genomics

Single cell analysis is essential to detecting and understanding cancer heterogeneity.

The Epic Sciences' platform analyzes all nucleated cells within a blood sample at single cell resolution. Cells from a patient’s blood sample are deposited on replicate glass slides and compared for morphological features, expression of biomarkers and nuclear integrity, using immunofluorescent staining. Guided by cell phenotype and marker expression, CTCs are individually recovered from the slide surface. Their genomes are amplified (WGA) and analyzed by next generation sequencing (NGS) for the presence of point mutations, copy number alterations, genome wide chromosomal instability, ploidy or genome wide scarring.

Slide prep: Upon patient blood sample receipt at Epic Sciences, whole blood is lysed and nucleated cells are deposited onto microscope slides.
Cell staining and CTC identification: Cells are stained for immunofluorescence to identify CTCs and other biomarkers in the study. Coordinates of all CTCs on the slide are preserved.
Genomics Analysis: Coverslips are removed and cell coordinates are used to relocate CTCs. Once relocated, cells are picked and placed into individual wells of an assay-ready 96 well plate.

Whole Genome Copy Number Variation (CNV)

Capability

Characterize CNV and genomic instability in subclonal CTC populations to establish disease heterogeneity.
Genome wide identification of specific oncogene amplification or tumor suppressor deletion.

Epic Sciences’ CNV assay service consists of:

1) CTC Identified

Samples are processed by standard procedures and CTCs are identified (see standard enumeration process for details) for recovery guided by morphology and marker expression.

2) Coordinates Recorded

Using the unique cell ID number and the associated XY coordinates, CTCs are relocated and recovered from the slide surface.

3) CTC Relocation and Isolation

Cells are lysed. Their genomes are amplified and are then constructed into libraries.

4) CTC Transferred to PCR Plate

Libraries are sequenced using a HiSeq 2500. Reads are trimmed and aligned.

5) Whole Genome Amplified

Aligned reads are binned into genomic regions and normalized for variance, read number and WBC control.

6) Library Prep and CNV Assay

CNV profiles are clustered to measure # of clonal populations per patient and associated with cell morphology and biomarker expression to characterize CTC clonality.

Example data

Example data showing the clonal evolution: CTCs from a single patient were identified and characterized through our CNV assay. The distinct clonal species and their relationship to each other are shown below.

Targeted Resequencing Assay

Capability

Detection of CTC sub-clonal populations harboring actionable mutations to monitor sensitivity to targeted therapies through the course of treatment.
Sequence only genes of interest, focus on those that are drug targets.

Epic Sciences’ targeted resequencing assay service consists of:

Samples are processed and CTCs are identified for recovery.
Using the unique cell ID# and the associated XY coordinates, CTCs are re-located and recovered from slide surface.
Cells are lysed, their genomes amplified, and constructed into libraries.
Libraries are target enriched by probe capture for pan-cancer gene exonic regions.
Following enrichment, libraries are sequenced.
Reads are aligned and analyzed by single nucleotide variant calling pipeline to characterize the presence on SNV and INDELs in each CTC.

Example data

Comparison of called variant function (left) and impact (right) across all patient CTCs: Moderate and high impact clonal and subclonal alterations detected across all patient CTCs (below).

Gene	Mutatioin	%CTCs	Cosmic ID	Impact	Comment
MLL3	p.Glyu838Ser/c.2512G>A	71.9%		Moderate	~20% in crc associated with tumor progression and microsatellite instability
SSX2	p.Awe174Gly/c.520A>G	31.3%		Moderate
MLL3	p.Tyr816fs/c.2447dupA	25.0%	289942	High
ATM	p.Val1534Leu/c.4600G>C	18.8%		Moderate	Homologous recombination deficiency ~ PARP inhibitors
EIF4A2	p.Gln118*/c.352C>T	9.4%		High	Co-occurance with MLL3 clonal alteration
NOTCH2	pAsn46Ser/c.137A>G	9.4%		Moderate
KRAS	p.Gly12Asp/c.35G.A	6.3%	521	Moderate	Associated with resistance to EGFR TKIs
EML4	p.Glu130fs/c.387delA	6.3%	1408084	High
ABL2	p.His544fs/c.1630delC	3.1%		High
PIK3CA	p.Pro27fs/c.80delC	3.1%		High	Resistance to EFFR-TKIs
TP53	p.Cys229*/c.687T>A	3.1%	45394	High	Tumor suppressor inactivation
BRCA1	pLys654fs/c.1961delA	3.1%	1383519	High	Homologous recombination deficiency ~ PARP inhibitors

Every time Epic’s AR-V7 test is administered the healthcare system saves about $15,000 per year of life extended. In other words approximately $7,000 to $9,000 per patient tested.
Of the roughly 30 million cells in a typical blood sample, about 5 are cancer cells on their way to form tumors elsewhere in the body. Modern cancer diagnostics technology can't see them.
We spend about $100 billion a year on cancer drugs. But because we’re not matching the right drug to the right patient and their disease in a personalized way, about 3/4ths of that drug spend is wasted.
The Epic approach answers a critical question for a doctor: When is a patient resistant? By answering that question accurately and precisely we increase patient life and save the system money at the same time.
Today, Epic Sciences is embedded in clinical trials with 48 leading producers of oncology drugs and 35 of the top academic cancer hospitals.
It takes only 7 minutes to acquire an entire image of 6 million stained cells on a 1 x 3 slide. Then that image, which is about 20GB of data, gets sent up to the cloud where the algorithms run and find the rare events in about 2 more minutes.