Presentación de PowerPoint · Recent technological advances have enabled the detection and...
Transcript of Presentación de PowerPoint · Recent technological advances have enabled the detection and...
LIQUID BIOPSY
Miguel Abal Investigador I3SNS
Oncoloxía Médica Traslacional Instituto de Investigación Sanitaria de Santiago (IDIS)
Complexo Hospitalario Universitario de Santiago/SERGAS
www.idisantiago.es
Recent technological advances have enabled the detection and detailed characterization of circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) in blood samples from patients with cancer. Often referred to as a "liquid biopsy," CTCs and ctDNA are expected to provide real-time monitoring of tumor evolution and therapeutic efficacy, with the potential for improved cancer diagnosis and treatment.
A major reason for treatment failures is our inability to monitor tumor evolution and adapt treatment accordingly.
Liquid biopsy
Solid biopsy
Pantel K , and Alix-Panabières C Cancer Res 2013;73:6384-6388
Circulating Tumor Cell (CTC)
• liquid biopsy (clinical tool) • new therapeutic target to prevent and/or eradicate metastasis
We need high sensitive and specific techniques to isolate and quantify CTC
Schematic view of CTC enrichment methods
Pantel & Alix-Panabières. Circulating tumour cells in cancer patients: challenges and perspectives. Trends Mol Med. 2010 Sep;16(9):398-406
The EPISPOT assay procedure.
The microfluidic circulating tumor cell (CTC) chip
Efficient and selective separation of viable CTCs from peripheral whole blood samples, mediated by the interaction of target CTCs with antibody (EpCAM)-coated microposts under precisely controlled laminar flow conditions.
A circulating tumor cell (CTC) selection microfluidic device integrated to an electrokinetic enrichment device for preconcentrating CTCs directly from whole blood to allow for molecular profiling.
Microfluidic devices for CTC capture and characterisation
Tools:
CellTracks® AutoPrep® System
CellTracks Analyzer II®
7.5mL
CK-/CD45 +
CK+/CD45 -
Ferrofluid-nanoparticules/EpCAM CYTOKERATINE CD45 DAPI
The CellSearch™Circulating Tumor Cell Kit is intended for the enumeration of circulating tumor cells (CTC) of epithelial origin (CD45-, EpCAM+, and cytokeratins8, 18+, and/or 19+) in whole blood.
FDA APPROVAL FOR CLINICAL USE
(Cristofanilli et al., N Engl J Med
2004) (Cohen et al., J Clin Oncol
2008) (de Bono et al., Clin Cancer Res
2008)
(Cristofanilli et al., N Engl J Med 2004)
(Cohen et al., J Clin Oncol 2008)
(de Bono et al., Clin Cancer Res 2008)
Progression-free survival (PFS) and overall survival (OS) of metastatic colorectal cancer patients with < three and ≥ three circulating tumor cells (CTCs) in 7.5 mL of blood (A, B) before therapy, (C, D) 1 to 2, 3 to 5, 6 to 12, and 13 to 20 weeks after initiation of therapy, and (E, F) by circulating tumor cell status at baseline and 3 to 5 weeks.
Cohen S J et al. JCO 2008;26:3213-3221
12-weeks
CT
Prediction of therapy response based on CTC-biomarker analysis
Staging
CT
1 month
Cycle 1 Cycle 2 Cycle 3 Cycle 4
Baseline 4-weeks
Anti EpCAM Preamplification
mC
RC
(n=
50
)
RNA extraction qPCR for selected transcripts
vs
21
CTC-markers can predict therapy response more accurately
and earlier than CT imaging
For any technology to be used in the clinic, demonstration of analytic validity (the accuracy of the test to measure the target of interest), clinical validity (the value of the test to predict the clinical outcome), and ultimately clinical utility (ability of the test to lead to improved clinical outcome when treatment choice is informed by test results) is required. A recent study assessed the clinical validity of circulating tumour cell (CTC) quantification for prognostication of patients with metastatic breast cancer by undertaking a pooled analysis of individual patient data (20 studies; approx. 2000 patients). The data confirmed the independent prognostic effect of CTC count on progression-free survival and overall survival. CTC count also improved the prognostication of metastatic breast cancer when added to full clinicopathological predictive models, whereas serum tumour markers do not. (Bidard et al., Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data. Lancet Oncol. 2014;15(4):406-14).
The value of CTC enumeration for treatment decision making in metastatic breast cancer was prospectively tested in the Southwest Oncology Group (SWOG) S0500 clinical trial. The SWOG trial evaluated the benefit of an early change in chemotherapy for patients with persistently increased CTCs at first follow-up after starting first-line chemotherapy. Of 595 evaluable patients, 123 patients with persistently elevated CTCs on day 21 of therapy were randomized to either continue the same treatment or to switch to an alternative chemotherapy of physician's choice. In this trial, an early switch to an alternative chemotherapy did not increase overall survival (OS). Although CTCs were strongly prognostic, the absence of a survival benefit from changing treatment based on elevated CTC counts suggests that earlier detection of relapse can only be important when a more effective treatment is available: Switching from one ineffective therapy to another ineffective therapy does not change outcome. Instead, changing treatment based on CTC molecular characterization might be a more promising approach to test.
DEP Array semi-conductor chip
Silicon Biosystems Patented Technology:
Moving DEP Cages
+ + + + - -
-
nDEP Cage nDEP Cage
+ + + + - -
Non-uniform electric field
generated by the chip electrodes (cross section)
Cell trapping by DEP cages
cage-move
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Moving DEP Cages Enables Outstanding Performance in Single-Cell Sorting
Main
chamber
Parking Recovery
1. Inject, trap and image all cells
2. Move all cells of interest
into Parking chamber
3. Move separately
to Recovery chamber
and flush
Homogeneous pools of cells from FFPE
300 pure cells tumor vs. stromal from FFPE tissue
Direct multiplex PCR
Deep Sequencing
data analysis
100% purity
Digitalization improves resolution
Sequencing
Quantitation
T GG TA CA G T T
10
T NA TA G G AC T
20
AA TG GG AA AA
30
TTTA AA G TGC
40
AAC CA G T CT G
50
AG T CAA CA G A
60
T TT CT T CCAA
70
T TA NGT T GAC
80
AG GT GTAGG T
90
C C TAC TA A TA
100
C T GT AC C TA T
110
A GC TT TAT GT
120
C CACA
AG AT T T
130
C TAT GA GTA T
140
C T GAT CA TA C
150
T GT C TTAC T T
160
TG ATAA A AC C
170
T CCAANT C CC
180
NC T AT CA T T T
190
T T GGT T TC CA
200
T C T T CC T GGC
210
AA AC T CA T T T
220
C TT C TA A TA C
230
T GTA T CA T C T
240
GC TC C T GT A
AT
250
C TA AT A GA GC
260
T TC C T T TA GT
270
T GC C CCC C T A
280
T C TT TA T T GT
290
GA C GA GG GGT
300
C GT T GC CA A A
310
GA GT GA TC T G
320
A GGGA A GT TA
330
A A GG ATAC A G
340
T T C C T TGT CT
350
AT C GGC TC C T
360
GCT T C TGAG A
370
GG GA
AGT T GT T
380
GTC TC TA C C C
390
C AGA CCT GAA
400
GC T CT C T T C T
410
GGT GGG GC T G
420
T TG GC T CT GG
430
T C T GC TC T GA
440
A GAA A AT T CC
450
C T GGCC T TC C
460
CTT GT AGGAA
470
GGC CA G AT CT
480
T CCC TAA A AA
490
AT TA GC C
CT GT
500
CAC TCAGT AC
510
A A T C T T T CAT
520
T TG GT GT CCT
530
TCCT TT C CAC
540
AT T T C CA AC A
550
GC C CT T TT TC
560
CT AGGGGC CC
570
T GC AA T T T CT
580
GGC TAT GT GC
590
C C T TC TT T GC
600
CACAA T TGAA
610
AC AC TT AACA
620
AT CT
Model 3100
BC 1.2.d.4
50_50F_E03_09.ab1
50_50F
Lane 9
Signal G:515 A:390 T:445 C:290
DT3100POP6{BD}v2.mob
Points 592 to 11557 Base 1: 592
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Spacing: 13.65
TTT CT CT
630
GGT T CCTAAA
640
AT T GC CT CT C
650
T GCAT CA TT A
660
T GGTAG CT GA
670
AT TT G TT AC T
680
T GGC TCA TT G
690
CT TCAGC C AA
700
AAC T CTT G CC
710
TT AT GG C C GG
720
GTC C TCC CAC
730
T CCCT GAC AT
740
GC TGTCAT CA
750
TT T
T CTT CTA G
760
TGT AG CT GCT
770
GGTC CCAATG
780
C TTT T AAAA T
790
A GT C TTAC AA
800
T CT GGG TTCG
810
C AT TCTGGAC
820
CAA C A NGGTT
830
CT GNCA TC C A
840
AT T TT T A CCT
850
ATA GNGAGTC
860
GNATTA CC GG
870
GA T CCTT TAN
880
A GT C
CGA CCTG
890
CA GGCAT GC A
900
A CCTT GGCGT
910
A TT AT GGGN
Model 3100
BC 1.2.d.4
50_50F_E03_09.ab1
50_50F
Lane 9
Signal G:515 A:390 T:445 C:290
DT3100POP6{BD}v2.mob
Points 592 to 11557 Base 1: 592
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Fri, Apr 28, 2000 7:36 PM
Tue, Apr 4, 2000 6:31 AM
Spacing: 13.65
Cancer Mutation Panel for CTCs
Ampli1- WGA
Gene CTC WBCpool
MU27 PIK3CA Ex 9 mut wt wt wt wt wt wt wt DO wt wt wt wt wt wt wt
Ex 20 mut mut mut mut mut mut mut mut mut mut DO wt DO wt wt wt
Her2 CNV+ NA n n n n n n DO n n n n NA n n
pool
MU22 PIK3CA Ex 9 mut wt wt wt wt wt wt wt wt wt wt DO wt wt wt
Ex 20 mut mut mut mut mut mut mut mut mut mut mut mut wt wt wt
Her2 CNV+ NA n n n n n n n n DO DO NA n n
pool
MU28 PIK3CA Ex 9 mut wt wt wt DO DO wt wt wt wt wt wt
Ex 20 mut wt wt wt wt wt wt wt wt wt wt wt
Her2 CNV+ NA n n DO n n n DO NA n NA
MU09 PIK3CA Ex 9 mut wt wt wt wt wt wt wt wt wt
Ex 20 mut mut mut mut mut wt wt wt wt wt
Her2 CNV+ a a a a a a NA NA NA
pool
MU18 PIK3CA Ex 9 mut wt mut wt wt wt wt wt
Ex 20 mut mut wt wt wt wt wt wt
Her2 CNV+ DO NA n n DO NA n
MU37 PIK3CA Ex 9 mut mut mut wt wt wt wt
Ex 20 mut wt wt wt wt wt wt
Her2 CNV+ a a a a NA n
MU23 PIK3CA Ex 9 mut mut mut wt DO wt wt wt
Ex 20 mut wt wt wt DO wt wt wt
Her2 CNV+ n DO n n NA n n
MU21 PIK3CA Ex 9 mut wt wt wt wt wt wt
Ex 20 mut wt wt wt wt wt wt
Her2 CNV+ a a a NA n n
MU29 PIK3CA Ex 9 mut wt wt wt DO DO
Ex 20 mut wt wt wt wt wt
Her2 CNV+ n n n n NA
MU05 PIK3CA Ex 9 mut wt wt wt
Ex 20 mut wt wt wt
Her2 CNV+ a NA NA
CTC Characterization by DEPArray™ and Ampli1™ yield Actionable Information
Source: Division of Oncogenomics, Pathology Dept., Uni Regensburg
PI3K-inhibitor
Lapatinib Herceptin
Gene CTC WBCpool
MU27 PIK3CA Ex 9 mut wt wt wt wt wt wt wt DO wt wt wt wt wt wt wt
Ex 20 mut mut mut mut mut mut mut mut mut mut DO wt DO wt wt wt
Her2 CNV+ NA n n n n n n DO n n n n NA n n
pool
MU22 PIK3CA Ex 9 mut wt wt wt wt wt wt wt wt wt wt DO wt wt wt
Ex 20 mut mut mut mut mut mut mut mut mut mut mut mut wt wt wt
Her2 CNV+ NA n n n n n n n n DO DO NA n n
pool
MU28 PIK3CA Ex 9 mut wt wt wt DO DO wt wt wt wt wt wt
Ex 20 mut wt wt wt wt wt wt wt wt wt wt wt
Her2 CNV+ NA n n DO n n n DO NA n NA
MU09 PIK3CA Ex 9 mut wt wt wt wt wt wt wt wt wt
Ex 20 mut mut mut mut mut wt wt wt wt wt
Her2 CNV+ a a a a a a NA NA NA
pool
MU18 PIK3CA Ex 9 mut wt mut wt wt wt wt wt
Ex 20 mut mut wt wt wt wt wt wt
Her2 CNV+ DO NA n n DO NA n
MU37 PIK3CA Ex 9 mut mut mut wt wt wt wt
Ex 20 mut wt wt wt wt wt wt
Her2 CNV+ a a a a NA n
MU23 PIK3CA Ex 9 mut mut mut wt DO wt wt wt
Ex 20 mut wt wt wt DO wt wt wt
Her2 CNV+ n DO n n NA n n
MU21 PIK3CA Ex 9 mut wt wt wt wt wt wt
Ex 20 mut wt wt wt wt wt wt
Her2 CNV+ a a a NA n n
MU29 PIK3CA Ex 9 mut wt wt wt DO DO
Ex 20 mut wt wt wt wt wt
Her2 CNV+ n n n n NA
MU05 PIK3CA Ex 9 mut wt wt wt
Ex 20 mut wt wt wt
Her2 CNV+ a NA NA
“if CTCs can be isolated from cancer patients as viable cells that can be genotyped and functionally characterized over the course of therapy, they have the potential to identify treatments that most effectively target the evolving mutational profile of the primary tumor “ (Yu et al., Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility. Science. 2014 July 11; 345(6193): 216–220).
The isolation of viable CTCs is technically challenging, success associated with unmanipulated CTCs. CTCs proliferated best as tumor spheres when cultured in serum-free media supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) under hypoxic conditions (4% O2). Nonadherent culture conditions were critical, because CTCs senesced after a few cell divisions in adherent monolayer culture
CONCLUSION single-cell analyses have provided higher-resolution evidence of intratumor heterogeneity with the finding of substantial clonal diversity and subclonal heterogeneity, such that no two individual tumor cells are genetically identical. Beyond spatial heterogeneity, solid tumors also exhibit temporal heterogeneity, evolving over time under selection pressure from treatment. Thus, there is an increased appreciation that the management of metastatic disease should rely on analysis of contemporary tumor tissue rather than on the primary tumor diagnosed years ago. However, obtaining serial samples of metastatic tissue is impractical and complicated by spatial heterogeneity and sampling bias. Analysis of circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) thus holds appeal and promise for noninvasive real-time assessment of tumor molecular profiles during the course of disease. Evaluation of CTCs and ctDNA may enable more sensitive monitoring of treatment efficacy and thereby guide drug selection, even potentially in the adjuvant setting where no such tools exist today.
Miguel Abal Translational Medical Oncology (IDIS) Complexo Hospitalario Universitario de Santiago de Compostela (SERGAS) Trav. Choupana s/n 15706 Santiago de Compostela (Spain) phone. +34 981955073 e-mail: [email protected]
Fragments of DNA are shed into the bloodstream from dying cells during cellular turnover or other forms of cell death.
More than 90% of healthy individuals having less than 25 ng cfDNA per mL.
cfDNA in the circulation is typically fragmented to 160 to 180 bp in length, corresponding to nucleosome-protected DNA observed in apoptotic cells.
In certain conditions, including inflammation, exercise, or tissue injury, cfDNA levels can be substantially higher.
ctDNA
ctDNA may be derived from primary tumors, metastatic lesions, or CTCs. The fraction of ctDNA that is tumor derived in patients with cancer has a
variable contribution ranging from <0.1% to >10% of the DNA molecules. The variability in levels of ctDNA is not well understood and is thought to be
affected by tumor burden, stage, cellular turnover, accessibility to the circulation, and factors affecting blood volume.
Although patients with similar tumor types may have varying absolute levels of ctDNA at the time of diagnosis, the relative levels of ctDNA within an individual have been shown to correlate with tumor burden and response to therapy.
Subject 11 had a sigmoid adenocarcinoma and two liver metastases that were treated with systemic chemotherapy before surgery (Chemotherapy 1). The subject underwent a sigmoid colectomy, left hepatic lobectomy and RFA of a solitary right hepatic lesion (Surgery 1). Imaging studies at 2 months showed recurrence in the liver, and the subject underwent a right hepatectomy (Surgery 2). Given the high risk of recurrence, chemotherapy was reinitiated (Chemotherapy 2). At 8 months, imaging showed three recurrent liver lesions and a suspicious celiac lymph node. The subject underwent RFA of these lesions and resection of the celiac node (Surgery 3). After surgery, the subject received additional chemotherapy (Chemotherapy 3); however, later imaging revealed multiple pulmonary metastases.
Technologies for ctDNA analysis
BEAMing Up Personalized Medicine: Detecting Cancer-Driving Mutations in Circulating DNA
BEAMing (Beads, Emulsification, Amplification, and Magnetics)