IJC International Journal of Cancer
Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients Jordan Madic1,2†, Anna Kiialainen3†, Francois-Clement Bidard1,4, Fabian Birzele3, Guillemette Ramey5, Quentin Leroy6, Thomas Rio Frio6, Isabelle Vaucher1, Virginie Raynal2, Virginie Bernard6, Alban Lermine7, Inga Clausen3, Nicolas Giroud3, Roland Schmucki3, Maud Milder1,8, Carsten Horn3, Olivia Spleiss3, Olivier Lantz8,9,10, Marc-Henri Stern2,9, Jean-Yves Pierga1,4,11, Martin Weisser12 and Ronald Lebofsky1 1
Laboratory of Circulating Tumor Biomarkers, SIRIC, Institut Curie, Paris, France Inserm U830, Paris, France 3 Roche Pharma Research and Early Development (pRED), Roche Innovation Center, Basel, Switzerland 4 Department of Medical Oncology, Institut Curie, Paris, France 5 Institut Roche de Recherche et M edecine Translationnelle, Boulogne-Billancourt, Paris, France 6 NGS Platform, ICGex, Institut Curie, Paris, France 7 Inserm U900, Paris, France 8 Inserm CIC BT-507, Paris, France 9 Department of Tumor Biology, Institut Curie, Paris, France 10 Inserm U932, Paris, France 11 University Paris Descartes, Paris, France 12 Roche Pharma Research and Early Development (pRED), Roche Innovation Center, Penzberg, Germany
Early Detection and Diagnosis
2
Circulating tumor DNA (ctDNA) is a new circulating tumor biomarker which might be used as a prognostic biomarker in a way similar to circulating tumor cells (CTCs). Here, we used the high prevalence of TP53 mutations in triple negative breast cancer (TNBC) to compare ctDNA and CTC detection rates and prognostic value in metastatic TNBC patients. Forty patients were enrolled before starting a new line of treatment. TP53 mutations were characterized in archived tumor tissues and in plasma DNA using two next generation sequencing (NGS) platforms in parallel. Archived tumor tissue was sequenced successfully for 31/40 patients. TP53 mutations were found in 26/31 (84%) of tumor samples. The same mutation was detected in the matched plasma of 21/26 (81%) patients with an additional mutation found only in the plasma for one patient. Mutated allele fractions ranged from 2 to 70% (median 5%). The observed correlation between the two NGS approaches (R2 5 0.903) suggested that ctDNA levels data were quantitative. Among the 27 patients with TP53 mutations, CTC count was 1 in 19 patients (70%) and 5 in 14 patients (52%). ctDNA levels had no prognostic impact on time to progression (TTP) or overall survival (OS), whereas CTC numbers were correlated with OS (p 5 0.04) and marginally with TTP (p 5 0.06). Performance status and elevated LDH also had significant prognostic impact. Here, absence of prognostic impact of baseline ctDNA level suggests that mechanisms of ctDNA release in metastatic TNBC may involve, beyond tumor burden, biological features that do not dramatically affect patient outcome.
Key words: circulating tumor DNA, circulating tumor cells, TP53, triple negative breast cancer, next generation sequencing Additional Supporting Information may be found in the online version of this article. † J.M. and A.K. contributed equally to this work Grant sponsor: SiRIC (Site de Recherche Integre contre le Cancer) and Cancerop^ole Ile-de-France; Grant sponsor: Agence Nationale de la Recherche (investissements d’avenir); Grant number: ANR-10EQPX-03 and ANR10-INBS-09-08 DOI: 10.1002/ijc.29265 History: Received 18 July 2014; Accepted 16 Sep 2014; Online 10 Oct 2014 Correspondence to: J.-Y. Pierga, Institut Curie, 26 rue d’Ulm, 75005 Paris, France, Tel.: 133-1-44-32-40-00, Fax: 133-1-53-10-4041, E-mail: [email protected]
C 2014 UICC Int. J. Cancer: 136, 2158–2165 (2015) V
Triple negative breast cancer (TNBC) accounts for approximately 15–20% of all breast cancer cases and are defined by a lack of both estrogen and progesterone receptors expression, and the absence of HER2 amplification.1 Notwithstanding high molecular heterogeneity within this breast cancer subtype, TNBCs frequently display TP53 inactivating mutations, and adverse clinico-pathological features.2 At the metastatic stage, TNBC is associated with worse prognosis than hormone receptor-positive or HER2-amplified metastatic breast cancers. Conventional systemic chemotherapy is the only therapeutic option for most metastatic TNBC patients as there are no validated targetable molecular alterations.3 In the absence of a predictive biomarker of response to chemotherapy, current treatment management consists of radiological evaluation of metastases burden at regular intervals. In this clinical setting, blood-borne dynamic biomarkers with
2159
Madic et al.
What’s new? Circulating tumor DNA (ctDNA) has shown promise as a prognostic biomarker. In this study, the authors compared detection rates and prognostic value of ctDNA versus circulating tumor cells (CTCs) in the plasma of metastatic triple-negative breast cancer (TNBC) patients. The ctDNA was detected more frequently than CTCs. However, while CTC numbers were correlated with prognosis, baseline ctDNA levels were not. This suggests that ctDNA might be more useful in identifying mutations that could provide therapeutic targets than as a prognostic biomarker.
Material and Methods Patients
This study was approved by regional ethics boards. Inclusion criteria were patients treated at the Institut Curie (Paris, France) for metastatic TNBC starting a new line of therapy and written informed consent. No attempt was made to define a target statistical power. However, we considered that the prognostic impact of CTCs and/or ctDNA was previously reported significant in cohorts as small as 40–50 metastatic C 2014 UICC Int. J. Cancer: 136, 2158–2165 (2015) V
breast cancer patients9,15 and so included a similar number of patients in this study. Time to progression (TTP) and overall survival (OS) were defined as the time elapsed between the date of inclusion and the date of documented tumor progression or the date of death, respectively. Tumor response to treatment was retrospectively assessed according to RECIST 1.1 criteria. CTC detection
CTC were detected from 7.5 ml blood samples using the CellsearchV system (Janssen Diagnostics, Raritan, NJ) within 96 hr by experienced technicians, following manufacturer’s instructions. Briefly, EpCAM-enriched cells were stained with phycoerythrin-conjugated antibodies C11 and A53-B/A2 directed against cytokeratins 8, 18 and 19, an allophycocyanin-conjugated antibody HI30 directed against CD45 and nuclear dye 4,6-diamidino-2-phenylindole (DAPI). Following manufacturer instructions, CTCs were defined as nucleated cells lacking CD45 expression and expressing cytokeratin. R
Extraction of DNA from plasma and tissue
Blood samples were processed within 3 hr of draw as described previously.10 Briefly, blood was centrifuged at 820g for 10 min, the supernatant was transferred in sterile 1.5 ml tubes and centrifuged at 16,000g for 10 min to pellet the remaining cellular debris. Plasma was stored at 280 C until further processing. DNA was extracted from 5 ml of plasma using the QIAamp circulating nucleic acid kit (Qiagen), according to the manufacturer’s instructions, and resuspended in 30 ml of AVE buffer. DNAs were extracted from cryopreserved and formalin-fixed paraffin embedded (FFPE) tumor tissues using a classical phenol chloroform protocol and the NucleoSpinV FFPE DNA kit (Macherey Nagel), respectively. The level of cfcDNA in diploid genomeequivalent per ml (GE/ml) of plasma was quantified in each sample using the LINE1 real-time PCR assay (LINE1 PCR) as previously described.16,17 R
Preamplification of plasma DNA
For Illumina sequencing, DNA extracted from plasma samples was preamplified using a set of target specific primers covering all the exons and flanking regions of the TP53 gene.11 Primer sequences are available upon request. The FastStart High Fidelity PCR System (Roche) was used to
Early Detection and Diagnosis
prognostic value may demonstrate clinical utility by allowing the pragmatic management of chemotherapy through multiple repeated blood draws and analyses.4 We recently reported a pooled analysis on the clinical validity of circulating tumor cells (CTCs) count before and during treatment in about 2,000 metastatic breast cancer patients: CTC count was established as a level-of-evidence 1 prognostic dynamic biomarker whose changes during therapy were associated with both progression-free survival and overall survival (OS), whatever the breast cancer subtype.5 However, the clinical utility of non-invasive longitudinal treatment monitoring by repeated CTC count is limited by the low number of detected CTCs, found in only 30–50% of metastatic breast cancer patients. Other blood-borne biomarkers shed by tumor tissue, such as circulating tumor DNA (ctDNA), have been investigated for clinical utility.6 ctDNA represents a fraction of the cell-free circulating DNA (cfcDNA) carrying the same genetic and epigenetic alterations as the patient’s tumor. It has been proposed that tumor DNA enters the bloodstream by tumor necrosis, tumor cell apoptosis and/ or direct secretion.7 Our group as well as others have reported a greater prevalence of ctDNA compared to CTC across multiple cancers both within and between patients, with direct access to molecular signatures.8,9 These factors have led to a growing interest in using ctDNA as a “liquid biopsy” for the molecular characterization and dynamic monitoring of tumors.7,10,11 TP53 mutations are clinically relevant in TNBC since they are present in 60 to >90% of TNBC cases.3,12–14 In this study, we used the high prevalence of TP53 mutations in TNBC to identify and monitor ctDNA. TP53-specific mutations were detected by next-generation sequencing (NGS) in a prospective cohort of metastatic TNBC patients. These results and the prognostic value of ctDNA levels were compared to those obtained with CTC count as well as other clinically accepted biomarkers.
2160
make a reaction mix at the following final concentrations: 0.05 U/ml FastStart High Fidelity Enzyme Blend, 13 FastStart High Fidelity Enzyme Buffer, 200 mM each of dNTPs, 4.5 mM MgCl2 and 5% DMSO. Preamplification reactions were subjected to 15 cycles of amplification (95 C 10 min, 15 cycles of 95 C 15 sec, 60 C 4 min). Following preamplification, 4 ml ExoI (Affymetrix) was added to each reaction and incubated for 15 min at 37 C, then for 15 min at 80 C. The preamplified samples were diluted fivefold in PCR-grade water prior to further manipulation.
Early Detection and Diagnosis
Access array single-plex amplification conditions
One microliter of each preamplified sample was added to 4 ml pre-sample mix containing 0.05 U/ml FastStart High Fidelity Enzyme Blend (Roche), 13 FastStart High Fidelity Enzyme Buffer, 200 mM each of dNTPs, 4.5 mM MgCl2, 5% DMSO and 13 Access Array sample loading solution (Fluidigm). Primer pairs containing 13 AA loading reagent (Fluidigm) were pipetted in the AA inlets at a concentration of 4 mM to achieve a final concentration of 200 nM in the reaction chambers. The AA controller loaded the sample and assay or primer mixes and the BioMark thermal cycled the PCR reactions according to the following: 35 cycles of amplification (50 C 2 min, 70 C 20 min, 95 C 10 min, 10 cycles of 95 C 15 sec, 60 C 30 sec, 72 C 60 sec, 2 cycles of 95 C 15 sec, 80 C 30 sec, 60 C 30 sec, 72 C 60 sec, 8 cycles of 95 C 15 sec, 60 C 30 sec, 72 C 60 sec, 2 cycles of 95 C 15 sec, 80 C 30 sec, 60 C 30 sec, 72 C 60 sec, 8 cycles of 95 C 15 sec, 60 C 30 sec, 72 C 60 sec, 5 cycles of 95 C 15 sec, 80 C 30 sec, 60 C 30 sec, 72 C 60 sec, 1 cycle of 72 C for 3 min). PCR products were harvested and diluted 100-fold. Illumina library construction
DNA extracted from tumor and preamplified plasma DNA were subjected to single-plex amplification on the Access Array microfluidic system (Fluidigm) using target-specific TP53 primers flanked by universal sequences CS1 and CS2 as described in the TAm-Seq protocol.11 The harvested and diluted products were tagged off-chip by PCR with sample indexing primers that were also complementary to Illumina flow-cell sequences PE1 and PE2. All primer sequences are available upon request. The reaction was assembled using the Roche FastStart High Fidelity PCR System as described above except 13 AA sample loading solution was omitted and final primer concentrations were 400 nM. Thermal cycling was performed as follows: 95 C 10 min, 15 cycles of 95 C 15 sec, 60 C 30 sec, 72 C 4 min, 1 cycle of 72 C for 3 min. Barcoded PCR amplicons were run on a Labchip GX (PerkinElmer) to verify the correct amplicon size. Based on the DNA concentrations provided by Labchip, equimolar pools were made. The entire pool was then purified using AMPure XP PCR purification (Beckman Coulter) according to manufacturer recommendations. The purified library was evaluated using a DNA1000 Bioanalyzer chip (Agilent) and quantified using the Qubit dsDNA HS assay kit (Invitrogen).
ctDNA and CTC in metastatic triple negative breast cancer
454 sequencing library construction
PCR primers covering all the exons and flanking regions of the TP53 gene were designed using Primer3 (http://frodo.wi. mit.edu/) and ordered from Microsynth AG. Primer sequences are available upon request. Ten nanograms of tumor DNA were amplified with 0.2 mM primers, 0.04 U/ml Fast Start High Fidelity polymerase (Roche) and 250 mM dNTPs in 13 High Fidelity Buffer 2 (Roche) in a reaction volume of 15 ml at 95 C for 5 min, 25 cycles of 95 C for 45 sec, 68 or 60 C for 45 sec and 72 C for 1 min, followed by 72 C for 5 min. The PCR product was then diluted 1:10 in H2O. One microliter of the diluted PCR product was subjected to another round of amplification with 0.4 mM indexing primers containing the adapter sequences needed for 454 sequencing (Microsynth), 0.05 U/ml Fast Start High Fidelity polymerase (Roche) and 200 mM dNTPs in 13 High Fidelity Buffer 2 (Roche) in a reaction volume of 15 ml at 95 C for 3 min, 25 cycles of 95 C for 15 sec, 60 C for 45 sec and 72 C for 1 min, followed by 72 C for 8 min. The PCR products were analyzed on DNA1000 chips (Agilent) on the Agilent 2100 Bioanalyzer and pooled equimolarly. Purified PCR product pools were quantified using the Quant-iT PicoGreen dsDNA assay kit (Invitrogen), diluted to 1 3 109 molecules/microliter and pooled at equal volumes as required. Plasma samples were processed similarly and up to 7 ml of cfcDNA was used for the initial PCR reaction. Illumina and 454 sequencing
Paired-end 150 bp read length sequencing, followed by an index read, was performed on an Illumina HiSeq2500 according to manufacturer’s instructions. 454 sequencing was performed on the 454 GS Junior sequencer (Roche) according to the manufacturer’s instructions. Sequencing data analysis
Following Illumina sequencing, reads were aligned to the human genome using Novoalign (version 3) and Samtools mpileup was used to summarize read alignment information per base on the human reference genome (version hg19).18 Base distribution (reference and non-reference calls) was computed for each position of TP53 that was covered by more than 1,000 reads. All differences between reference and reads sequenced for a sample that occurred in >1% of the read population were used for further analysis. The obtained 454 reads were aligned against the chr17:7564597–7591356 sequence (hg19) and the variants in this region were called using the GS Amplicon Variant Analyzer (AVA) software v2.6 and v2.7 (Roche). Analysis results were verified using in house scripts. Quantification of ctDNA
ctDNA level in diploid GE/ml of plasma was calculated by multiplying the level of cfcDNA measured by LINE1 PCR by C 2014 UICC Int. J. Cancer: 136, 2158–2165 (2015) V
2161
Figure 1. Sample flowchart.
the percentage of mutant TP53 fraction measured by sequencing.
Results Patient characteristics
A total of 40 metastatic triple negative breast cancer (TNBC) patients were enrolled in the study between Dec 2010 and April 2013. The entire patient flow chart is displayed in Figure 1 and patient characteristics are shown in Supporting Information Table S1. Identification of mutated TP53 alleles in tumor and plasma of TNBC patients
To assess the feasibility of the detection of ctDNA by NGS, DNA extracted from primary and metastatic tumor tissues and corresponding plasma samples were processed according to the TAm-Seq method followed by Illumina sequencing11 and, alternatively, by 454 sequencing. Two panels of target specific primers optimized for the respective sequencing platforms covering all exons of TP53 and flanking untranslated regions were used. For 454 sequencing of plasma DNA, only the amplicon where the mutation was detected in the tumor was assayed except for nine patients where all amplicons C 2014 UICC Int. J. Cancer: 136, 2158–2165 (2015) V
were analyzed. Variants detected at
Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients Jordan Madic1,2†, Anna Kiialainen3†, Francois-Clement Bidard1,4, Fabian Birzele3, Guillemette Ramey5, Quentin Leroy6, Thomas Rio Frio6, Isabelle Vaucher1, Virginie Raynal2, Virginie Bernard6, Alban Lermine7, Inga Clausen3, Nicolas Giroud3, Roland Schmucki3, Maud Milder1,8, Carsten Horn3, Olivia Spleiss3, Olivier Lantz8,9,10, Marc-Henri Stern2,9, Jean-Yves Pierga1,4,11, Martin Weisser12 and Ronald Lebofsky1 1
Laboratory of Circulating Tumor Biomarkers, SIRIC, Institut Curie, Paris, France Inserm U830, Paris, France 3 Roche Pharma Research and Early Development (pRED), Roche Innovation Center, Basel, Switzerland 4 Department of Medical Oncology, Institut Curie, Paris, France 5 Institut Roche de Recherche et M edecine Translationnelle, Boulogne-Billancourt, Paris, France 6 NGS Platform, ICGex, Institut Curie, Paris, France 7 Inserm U900, Paris, France 8 Inserm CIC BT-507, Paris, France 9 Department of Tumor Biology, Institut Curie, Paris, France 10 Inserm U932, Paris, France 11 University Paris Descartes, Paris, France 12 Roche Pharma Research and Early Development (pRED), Roche Innovation Center, Penzberg, Germany
Early Detection and Diagnosis
2
Circulating tumor DNA (ctDNA) is a new circulating tumor biomarker which might be used as a prognostic biomarker in a way similar to circulating tumor cells (CTCs). Here, we used the high prevalence of TP53 mutations in triple negative breast cancer (TNBC) to compare ctDNA and CTC detection rates and prognostic value in metastatic TNBC patients. Forty patients were enrolled before starting a new line of treatment. TP53 mutations were characterized in archived tumor tissues and in plasma DNA using two next generation sequencing (NGS) platforms in parallel. Archived tumor tissue was sequenced successfully for 31/40 patients. TP53 mutations were found in 26/31 (84%) of tumor samples. The same mutation was detected in the matched plasma of 21/26 (81%) patients with an additional mutation found only in the plasma for one patient. Mutated allele fractions ranged from 2 to 70% (median 5%). The observed correlation between the two NGS approaches (R2 5 0.903) suggested that ctDNA levels data were quantitative. Among the 27 patients with TP53 mutations, CTC count was 1 in 19 patients (70%) and 5 in 14 patients (52%). ctDNA levels had no prognostic impact on time to progression (TTP) or overall survival (OS), whereas CTC numbers were correlated with OS (p 5 0.04) and marginally with TTP (p 5 0.06). Performance status and elevated LDH also had significant prognostic impact. Here, absence of prognostic impact of baseline ctDNA level suggests that mechanisms of ctDNA release in metastatic TNBC may involve, beyond tumor burden, biological features that do not dramatically affect patient outcome.
Key words: circulating tumor DNA, circulating tumor cells, TP53, triple negative breast cancer, next generation sequencing Additional Supporting Information may be found in the online version of this article. † J.M. and A.K. contributed equally to this work Grant sponsor: SiRIC (Site de Recherche Integre contre le Cancer) and Cancerop^ole Ile-de-France; Grant sponsor: Agence Nationale de la Recherche (investissements d’avenir); Grant number: ANR-10EQPX-03 and ANR10-INBS-09-08 DOI: 10.1002/ijc.29265 History: Received 18 July 2014; Accepted 16 Sep 2014; Online 10 Oct 2014 Correspondence to: J.-Y. Pierga, Institut Curie, 26 rue d’Ulm, 75005 Paris, France, Tel.: 133-1-44-32-40-00, Fax: 133-1-53-10-4041, E-mail: [email protected]
C 2014 UICC Int. J. Cancer: 136, 2158–2165 (2015) V
Triple negative breast cancer (TNBC) accounts for approximately 15–20% of all breast cancer cases and are defined by a lack of both estrogen and progesterone receptors expression, and the absence of HER2 amplification.1 Notwithstanding high molecular heterogeneity within this breast cancer subtype, TNBCs frequently display TP53 inactivating mutations, and adverse clinico-pathological features.2 At the metastatic stage, TNBC is associated with worse prognosis than hormone receptor-positive or HER2-amplified metastatic breast cancers. Conventional systemic chemotherapy is the only therapeutic option for most metastatic TNBC patients as there are no validated targetable molecular alterations.3 In the absence of a predictive biomarker of response to chemotherapy, current treatment management consists of radiological evaluation of metastases burden at regular intervals. In this clinical setting, blood-borne dynamic biomarkers with
2159
Madic et al.
What’s new? Circulating tumor DNA (ctDNA) has shown promise as a prognostic biomarker. In this study, the authors compared detection rates and prognostic value of ctDNA versus circulating tumor cells (CTCs) in the plasma of metastatic triple-negative breast cancer (TNBC) patients. The ctDNA was detected more frequently than CTCs. However, while CTC numbers were correlated with prognosis, baseline ctDNA levels were not. This suggests that ctDNA might be more useful in identifying mutations that could provide therapeutic targets than as a prognostic biomarker.
Material and Methods Patients
This study was approved by regional ethics boards. Inclusion criteria were patients treated at the Institut Curie (Paris, France) for metastatic TNBC starting a new line of therapy and written informed consent. No attempt was made to define a target statistical power. However, we considered that the prognostic impact of CTCs and/or ctDNA was previously reported significant in cohorts as small as 40–50 metastatic C 2014 UICC Int. J. Cancer: 136, 2158–2165 (2015) V
breast cancer patients9,15 and so included a similar number of patients in this study. Time to progression (TTP) and overall survival (OS) were defined as the time elapsed between the date of inclusion and the date of documented tumor progression or the date of death, respectively. Tumor response to treatment was retrospectively assessed according to RECIST 1.1 criteria. CTC detection
CTC were detected from 7.5 ml blood samples using the CellsearchV system (Janssen Diagnostics, Raritan, NJ) within 96 hr by experienced technicians, following manufacturer’s instructions. Briefly, EpCAM-enriched cells were stained with phycoerythrin-conjugated antibodies C11 and A53-B/A2 directed against cytokeratins 8, 18 and 19, an allophycocyanin-conjugated antibody HI30 directed against CD45 and nuclear dye 4,6-diamidino-2-phenylindole (DAPI). Following manufacturer instructions, CTCs were defined as nucleated cells lacking CD45 expression and expressing cytokeratin. R
Extraction of DNA from plasma and tissue
Blood samples were processed within 3 hr of draw as described previously.10 Briefly, blood was centrifuged at 820g for 10 min, the supernatant was transferred in sterile 1.5 ml tubes and centrifuged at 16,000g for 10 min to pellet the remaining cellular debris. Plasma was stored at 280 C until further processing. DNA was extracted from 5 ml of plasma using the QIAamp circulating nucleic acid kit (Qiagen), according to the manufacturer’s instructions, and resuspended in 30 ml of AVE buffer. DNAs were extracted from cryopreserved and formalin-fixed paraffin embedded (FFPE) tumor tissues using a classical phenol chloroform protocol and the NucleoSpinV FFPE DNA kit (Macherey Nagel), respectively. The level of cfcDNA in diploid genomeequivalent per ml (GE/ml) of plasma was quantified in each sample using the LINE1 real-time PCR assay (LINE1 PCR) as previously described.16,17 R
Preamplification of plasma DNA
For Illumina sequencing, DNA extracted from plasma samples was preamplified using a set of target specific primers covering all the exons and flanking regions of the TP53 gene.11 Primer sequences are available upon request. The FastStart High Fidelity PCR System (Roche) was used to
Early Detection and Diagnosis
prognostic value may demonstrate clinical utility by allowing the pragmatic management of chemotherapy through multiple repeated blood draws and analyses.4 We recently reported a pooled analysis on the clinical validity of circulating tumor cells (CTCs) count before and during treatment in about 2,000 metastatic breast cancer patients: CTC count was established as a level-of-evidence 1 prognostic dynamic biomarker whose changes during therapy were associated with both progression-free survival and overall survival (OS), whatever the breast cancer subtype.5 However, the clinical utility of non-invasive longitudinal treatment monitoring by repeated CTC count is limited by the low number of detected CTCs, found in only 30–50% of metastatic breast cancer patients. Other blood-borne biomarkers shed by tumor tissue, such as circulating tumor DNA (ctDNA), have been investigated for clinical utility.6 ctDNA represents a fraction of the cell-free circulating DNA (cfcDNA) carrying the same genetic and epigenetic alterations as the patient’s tumor. It has been proposed that tumor DNA enters the bloodstream by tumor necrosis, tumor cell apoptosis and/ or direct secretion.7 Our group as well as others have reported a greater prevalence of ctDNA compared to CTC across multiple cancers both within and between patients, with direct access to molecular signatures.8,9 These factors have led to a growing interest in using ctDNA as a “liquid biopsy” for the molecular characterization and dynamic monitoring of tumors.7,10,11 TP53 mutations are clinically relevant in TNBC since they are present in 60 to >90% of TNBC cases.3,12–14 In this study, we used the high prevalence of TP53 mutations in TNBC to identify and monitor ctDNA. TP53-specific mutations were detected by next-generation sequencing (NGS) in a prospective cohort of metastatic TNBC patients. These results and the prognostic value of ctDNA levels were compared to those obtained with CTC count as well as other clinically accepted biomarkers.
2160
make a reaction mix at the following final concentrations: 0.05 U/ml FastStart High Fidelity Enzyme Blend, 13 FastStart High Fidelity Enzyme Buffer, 200 mM each of dNTPs, 4.5 mM MgCl2 and 5% DMSO. Preamplification reactions were subjected to 15 cycles of amplification (95 C 10 min, 15 cycles of 95 C 15 sec, 60 C 4 min). Following preamplification, 4 ml ExoI (Affymetrix) was added to each reaction and incubated for 15 min at 37 C, then for 15 min at 80 C. The preamplified samples were diluted fivefold in PCR-grade water prior to further manipulation.
Early Detection and Diagnosis
Access array single-plex amplification conditions
One microliter of each preamplified sample was added to 4 ml pre-sample mix containing 0.05 U/ml FastStart High Fidelity Enzyme Blend (Roche), 13 FastStart High Fidelity Enzyme Buffer, 200 mM each of dNTPs, 4.5 mM MgCl2, 5% DMSO and 13 Access Array sample loading solution (Fluidigm). Primer pairs containing 13 AA loading reagent (Fluidigm) were pipetted in the AA inlets at a concentration of 4 mM to achieve a final concentration of 200 nM in the reaction chambers. The AA controller loaded the sample and assay or primer mixes and the BioMark thermal cycled the PCR reactions according to the following: 35 cycles of amplification (50 C 2 min, 70 C 20 min, 95 C 10 min, 10 cycles of 95 C 15 sec, 60 C 30 sec, 72 C 60 sec, 2 cycles of 95 C 15 sec, 80 C 30 sec, 60 C 30 sec, 72 C 60 sec, 8 cycles of 95 C 15 sec, 60 C 30 sec, 72 C 60 sec, 2 cycles of 95 C 15 sec, 80 C 30 sec, 60 C 30 sec, 72 C 60 sec, 8 cycles of 95 C 15 sec, 60 C 30 sec, 72 C 60 sec, 5 cycles of 95 C 15 sec, 80 C 30 sec, 60 C 30 sec, 72 C 60 sec, 1 cycle of 72 C for 3 min). PCR products were harvested and diluted 100-fold. Illumina library construction
DNA extracted from tumor and preamplified plasma DNA were subjected to single-plex amplification on the Access Array microfluidic system (Fluidigm) using target-specific TP53 primers flanked by universal sequences CS1 and CS2 as described in the TAm-Seq protocol.11 The harvested and diluted products were tagged off-chip by PCR with sample indexing primers that were also complementary to Illumina flow-cell sequences PE1 and PE2. All primer sequences are available upon request. The reaction was assembled using the Roche FastStart High Fidelity PCR System as described above except 13 AA sample loading solution was omitted and final primer concentrations were 400 nM. Thermal cycling was performed as follows: 95 C 10 min, 15 cycles of 95 C 15 sec, 60 C 30 sec, 72 C 4 min, 1 cycle of 72 C for 3 min. Barcoded PCR amplicons were run on a Labchip GX (PerkinElmer) to verify the correct amplicon size. Based on the DNA concentrations provided by Labchip, equimolar pools were made. The entire pool was then purified using AMPure XP PCR purification (Beckman Coulter) according to manufacturer recommendations. The purified library was evaluated using a DNA1000 Bioanalyzer chip (Agilent) and quantified using the Qubit dsDNA HS assay kit (Invitrogen).
ctDNA and CTC in metastatic triple negative breast cancer
454 sequencing library construction
PCR primers covering all the exons and flanking regions of the TP53 gene were designed using Primer3 (http://frodo.wi. mit.edu/) and ordered from Microsynth AG. Primer sequences are available upon request. Ten nanograms of tumor DNA were amplified with 0.2 mM primers, 0.04 U/ml Fast Start High Fidelity polymerase (Roche) and 250 mM dNTPs in 13 High Fidelity Buffer 2 (Roche) in a reaction volume of 15 ml at 95 C for 5 min, 25 cycles of 95 C for 45 sec, 68 or 60 C for 45 sec and 72 C for 1 min, followed by 72 C for 5 min. The PCR product was then diluted 1:10 in H2O. One microliter of the diluted PCR product was subjected to another round of amplification with 0.4 mM indexing primers containing the adapter sequences needed for 454 sequencing (Microsynth), 0.05 U/ml Fast Start High Fidelity polymerase (Roche) and 200 mM dNTPs in 13 High Fidelity Buffer 2 (Roche) in a reaction volume of 15 ml at 95 C for 3 min, 25 cycles of 95 C for 15 sec, 60 C for 45 sec and 72 C for 1 min, followed by 72 C for 8 min. The PCR products were analyzed on DNA1000 chips (Agilent) on the Agilent 2100 Bioanalyzer and pooled equimolarly. Purified PCR product pools were quantified using the Quant-iT PicoGreen dsDNA assay kit (Invitrogen), diluted to 1 3 109 molecules/microliter and pooled at equal volumes as required. Plasma samples were processed similarly and up to 7 ml of cfcDNA was used for the initial PCR reaction. Illumina and 454 sequencing
Paired-end 150 bp read length sequencing, followed by an index read, was performed on an Illumina HiSeq2500 according to manufacturer’s instructions. 454 sequencing was performed on the 454 GS Junior sequencer (Roche) according to the manufacturer’s instructions. Sequencing data analysis
Following Illumina sequencing, reads were aligned to the human genome using Novoalign (version 3) and Samtools mpileup was used to summarize read alignment information per base on the human reference genome (version hg19).18 Base distribution (reference and non-reference calls) was computed for each position of TP53 that was covered by more than 1,000 reads. All differences between reference and reads sequenced for a sample that occurred in >1% of the read population were used for further analysis. The obtained 454 reads were aligned against the chr17:7564597–7591356 sequence (hg19) and the variants in this region were called using the GS Amplicon Variant Analyzer (AVA) software v2.6 and v2.7 (Roche). Analysis results were verified using in house scripts. Quantification of ctDNA
ctDNA level in diploid GE/ml of plasma was calculated by multiplying the level of cfcDNA measured by LINE1 PCR by C 2014 UICC Int. J. Cancer: 136, 2158–2165 (2015) V
2161
Figure 1. Sample flowchart.
the percentage of mutant TP53 fraction measured by sequencing.
Results Patient characteristics
A total of 40 metastatic triple negative breast cancer (TNBC) patients were enrolled in the study between Dec 2010 and April 2013. The entire patient flow chart is displayed in Figure 1 and patient characteristics are shown in Supporting Information Table S1. Identification of mutated TP53 alleles in tumor and plasma of TNBC patients
To assess the feasibility of the detection of ctDNA by NGS, DNA extracted from primary and metastatic tumor tissues and corresponding plasma samples were processed according to the TAm-Seq method followed by Illumina sequencing11 and, alternatively, by 454 sequencing. Two panels of target specific primers optimized for the respective sequencing platforms covering all exons of TP53 and flanking untranslated regions were used. For 454 sequencing of plasma DNA, only the amplicon where the mutation was detected in the tumor was assayed except for nine patients where all amplicons C 2014 UICC Int. J. Cancer: 136, 2158–2165 (2015) V
were analyzed. Variants detected at
