Epic Sciences

Single Cell Pheno-Genomics

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

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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.

  1. Slide prep: Upon patient blood sample receipt at Epic Sciences, whole blood is lysed and nucleated cells are deposited onto microscope slides.
  2. 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.
  3. 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:

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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.

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2) Coordinates Recorded

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

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3) CTC Relocation and Isolation

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

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4) CTC Transferred to PCR Plate

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

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5) Whole Genome Amplified

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

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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.

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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:

  1. Samples are processed and CTCs are identified for recovery.
  2. Using the unique cell ID# and the associated XY coordinates, CTCs are re-located and recovered from slide surface.
  3. Cells are lysed, their genomes amplified, and constructed into libraries.
  4. Libraries are target enriched by probe capture for pan-cancer gene exonic regions.
  5. Following enrichment, libraries are sequenced.
  6. 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