Exploring the utility of circulating tumor DNA in neoadjuvant setting
- Conditions
- Breast Cancer
- Registration Number
- CTRI/2016/09/007308
- Lead Sponsor
- DEPARTMENT OF BIOTECHNOLOGY
- Brief Summary
Breast cancer is the second most common cancer in the worldand, by far, the most frequent cancer among women with an estimated 1.67million new cancer cases diagnosed in 2012 (25% of all cancers). It isestimated that by 2030 the global burden of breast cancer will increase to over2 million new cases per year. [1,3]
**Breast cancer in India**: Breast cancer accounts for 25% to 31% ofall cancers in women in India [1]. According to GLOBOCAN (WHO), for the year2012, incidence of Breast cancer in India was **144937** and an estimated **70218**women died in India due to breast cancer, more than any other country in theworld (second: China - 47984 deaths and third: US - 43909 deaths).. [2]
Once a patient is diagnosed withbreast cancer, she is given the standard of care treatment after which followup is usually done by serial imaging techniques for monitoring the treatmentresponse and relapse of tumor. Monitoring of treatment response is essential:-
Ø to avoid continuing ineffective therapies,
Ø to prevent unnecessary side effects,
Ø to determine the benefit of new therapeutics.
Imaging techniques do pick uprecurrent disease but most often too late to institute meaningful therapy tosignificantly prolong survival. In spiteof an exponential increase in our understanding of tumor biology over recentdecades, and the availability of technologies to characterize tumors, whetherand when an individual cancer will metastasize/ recur/ relapse remains unknown.
There is therefore an urgent needfor biomarkers that measure
ü Tumor burden with high sensitivity and specificity.
ü Response to therapy
ü Early detection of relapse
Investigations are now focusing onblood-based assays that detect and characterize circulating tumor cells orcirculating tumor DNA (a component of cell-free DNA). These minimally invasive,’liquid biopsies’ can be performed at multiple intervals to monitor disease andtailor cancer therapy. Research has shown the feasibility of using circulatingtumor DNA to monitor tumor dynamics since circulating tumor DNA had sensitivitysuperior to that of circulating tumor cells.
Evaluation of circulatingbiomarkers, specifically circulating tumor DNA (ctDNA), can provide informationabout the molecular characteristics of a patient’s tumor from a noninvasiveblood draw, thus aiding the clinical management of this disease.
Advances in sequencing technologieshave enabled the rapid identification of somatic genomic alterations inindividual tumors, and these can be used to design personalized assays for themonitoring of circulating tumor DNA.
Wepropose to identify the biomarkers/ mutations derived from tumor tissue andsubsequently use these markers (in a patient specific manner) to monitortherapy and relapse by quantifying the same mutations in peripheral blood.
**State of art:** Evaluatingthe levels of tumor markers like Alpha-fetoprotein (AFP), Anaplastic lymphomakinase (ALK), BCR-ABL, Beta-2-microglobulin (B2M), Bladder tumor antigen (BTA),CA 15-3, CA 19-9, CA 27-29, CA 125, Carcinoembryonic antigen (CEA), Epidermalgrowth factor receptor (EGFR), Lactate dehydrogenase (LDH), Neuron-specificenolase (NSE), Prostate-specific antigen (PSA), NMP22 are routinely used fordetection of various cancers.
These available tumormarkers do not predict response to chemotherapy and recurrence of cancer. Also,these markers are mostly cancer specific.
With the advent ofhigh-throughput technology like NGS, it is now possible to identify somaticalterations in the recurrently mutated genes in the circulating tumor DNA inindividual cancer patients. The next-generation sequencing (NGS) approach holdsa number of potential advantages over traditional methods, including theability to fully sequence large numbers of genes (hundreds to thousands) in asingle test and simultaneously detect deletions, insertions, copy numberalterations, translocations, and exome-wide base substitutions (including known“hot-spot mutationsâ€) in all known cancer-related genes. Continuing advances inNGS technology will lower the overall cost, speed the turnaround time, increasethe breadth of genome sequencing, detecting epigenetic markers and otherimportant genomic parameters. Targeted deep sequencing of plasma DNA provides acost-effective alternative for high-throughput analysis and may overcomelimitations of initial tumor-tissue assessment by virtue of allowing for thedirect identification of mutations in plasma. We have collected the data on 50hotspot genes that are routinely mutated in various cancers. Among these 50hotspot genes, around 15 genes are found to be commonly mutated in at-leastthree out of top seven cancers in India.
S J Dawson et.al comparedthe radiographic imaging of tumors with the assay of circulating tumor DNA, CA15-3, and circulating tumor cells in 30 women with metastatic breast cancer whowere receiving systemic therapy. Circulating tumor DNA was successfullydetected in 29 of the 30 women (97%) in whom somatic genomic alterations wereidentified. Somatic mutations were identified by tagged-amplicon deepsequencing22 for PIK3CA and TP53 or paired-end whole-genome sequencing.Circulating tumor DNA levels showed a greater dynamic range, and greatercorrelation with changes in tumor burden, than did CA 15-3 or circulating tumorcells (2).
We plan to sequence 44 genesfrom Qiagen- Human Breast Cancer Gene Read DNAseq Targeted Panel. The HumanBreast Cancer GeneRead DNAseq Targeted Panel is a collection of multiplexed PCRprimer assays for targeted enrichment of the coding (exonic) regions of the 44genes most commonly mutated in human breast cancer samples.
Alternative cancer genepanels are
Also, the Illumina TrusightCancer gene panel shares 23 common genes with the Cosmic-Cancer census genelist of 522 top mutated genes in various cancers.
We also compared these 44genes with the top 1000 Breast cancer genes reported in COSMIC database(according to the number of samples mutated) and found that there are 19 commongenes between the two lists.
| | | | |
| --- | --- | --- | --- |
|**The Human Breast Cancer GeneRead DNAseq Targeted Panel (Qiagen)**
|**ACVR1B**
**EP300**
**ITCH**
**PIK3CA (p110α)**
|**AKT1**
**ERBB2 (HER2)**
**MAP2K4**
**PIK3R1**
|**ATM**
**ERBB3**
**MAP3K1**
**PPM1L**
|**BAP1**
**ESR1 (ERα)**
**MDM2**
**PTEN**
|**BRCA1**
**EXOC2**
**MLL3**
**PTGFR**
|**BRCA2**
**EXT2**
**MUC16**
**RB1**
|**CBFB**
**FBXO32**
**MYC**
**SEPT.9**
|**CCND1**
**FGFR1**
**NCOR1**
**TP53**
|**CDH1**
**FGFR2**
**NEK2**
**TRAF5**
|**CDKN2A(p16INK4)**
**GATA3**
**PBRM1**
**WEE1**
|**EGFR**
**IRAK4**
**PCGF2 (RNF110)**
**ZBED4**
**Key words:** circulating, chromatin, response, breastcancer
**3. AIMS AND OBJECTIVES**
1. To identify the mutations in the 44 genes from Qiagen Breast CancerGene Read DNAseq Targeted Panel and subtract these mutations from thebackground normal tissue.
2. To characterize these identified mutations.
3.To use these mutations in blood to monitor response to therapy and to evaluatewhether tumor burden relates quantitatively to circulating tumor DNA.
**4. DESIGN OF THE STUDY**
Basic Research, Technology Development.
**5.STUDY METHODOLOGY**
**Tissue and blood sample collection:** Once the patients are diagnosed with Breast cancer who are planned forneoadjuvant chemotherapy we will first obtain the tumor biopsy samples from thesepatients biopsy in strict accordance with ethical committee guidance.Simultaneously blood and buccal mucosal samples will also be collected frompatients.
Medical history of the consenting patients will be obtained and serologicaltest for HIV, HCV, and HBsAg will be done. One or two tumor tissue samples ofapproximately 1 cm size will be collected using appropriate biopsypunch/RNAse-free sterile forceps.. After histopathological examination,specimens will be transported on ice to sequencing laboratory in AQIX RS-1/ RNAlater biopsy storage and transport medium. 30ml blood samples will be collected every 3 weeksprior to each cycle of chemotherapy when it is once per 3-week chemotherapyregimen. Patients who are on once per week chemotherapy regimen (for e.g.weekly paclitaxel X 12) will also have their blood samples drawn once every 3weeks. In the weekly regimen, this will be done prior to 1st cycle,4th cycle, 7th cycle, 10th cycle and after 12thcycle.Blood and buccal swab specimens will be collected inappropriate containers with EDTA/ Acid citrate dextrose/ suitable media andshipped on ice to the sequencing laboratory.
A repeat biopsy and blood sample will be collected from the same patientsat the end of planned neoadjuvant chemotherapy at the time of surgery.
Total 02 biopsies will be performed; First when the patients are diagnosedwith Breast cancer who are planned for neoadjuvant chemotherapy and another atthe end of the planned neoadjuvant chemotherapy at the time of surgery.
The peripheral blood for germline mutationanalysis will be collected at the time of accrual and first progression.
**Sequencing:** Next generation sequencing willbe done using the Illumina HiSeq 1000 system according to the establishedprotocol. Only High Quality (HQ) sequence information will be used foranalysis. The HQ metric is assessed by
a) <5% Adapter contamination
b) Even (~25% of each base) Base distribution ofsample
c) Q>30% and
d) Acceptable Kmer values and GC content (as definedby standard softwares such as FasQC, etc). A minimum of 3 GB, paired–endsequence information, passing the quality standards mentioned before, will beused for the analysis.
**Library Construction: DNA Exome:** A total of 1-3 μg DNA will be used for the library preparation. Genomic DNAwill be prepared from the blood and tumor tissue by using the Qiagen DNeasyBlood and Tissue Kit (Qiagen, Canada). After centrifugation of DNA as per theQiagen kit protocol, purification by microdialysis through DNeasy membrane willbe done. DNA quality will be assessed by spectrophotometry (260/280 and260/230) and gel electrophoresis before library construction. The DNA librarywill be prepared by following the indexed pair end library protocol (IlluminaInc., USA). Briefly, the DNA will be subjected to end-repair, andphosphorylation by T4 DNA polymerase, Klenow DNA Polymerase, and T4polynucleotide kinase respectively in a single reaction, and then 3’ Aoverhangs generation by Klenow fragment (3’ to 5’ exon minus), and ligated toIllumina PE adapters, which contain 5’ T overhangs. PCR products will bepurified on Qiaquick MinElute columns (Qiagen) and the DNA quality will beassessed and quantified using an Agilent DNA 1000 series II assay and Nanodrop7500 spectrophotometer (Nanodrop, USA).
**Exome capture:** Exome capture will bedone by using TrueSeq Exome Enrichment Kit from Illumina Inc or SureSelectTarget Enrichment Kit from Agilent, according to the manufacturer’sinstructions. The validation of exome capture will be assessed post-facto basedon the parameters of “coverage†and “depthâ€, with the expectation being greaterthan 90% of the manufacturer claims at 20-100X depth.
**Isolation and Quantification of Circulating Tumor DNA**: 30ml bloodsamples will be collected every 3 weeks prior to each cycle of chemotherapywhen it is once per 3-week chemotherapy regimen. Patients who are on once perweek chemotherapy regimen (for e.g. weekly paclitaxel X 12) will also havetheir blood samples drawn once every 3 weeks. In the weekly regimen, this willbe done prior to 1st cycle, 4th cycle, 7thcycle, 10th cycle and after 12th cycle.
The blood samples collected in EDTA tubes will beprocessed within 1 hour after collection and will be centrifuged to separatethe plasma from the peripheral-blood cells. Circulating tumor DNA will beextracted from aliquots (2 ml) of plasma and normal DNA will be extracted fromthe buffy coat with the use of the QIAamp circulating nucleic acid kit(Qiagen).
- Detailed Description
Not available
Recruitment & Eligibility
- Status
- Completed
- Sex
- All
- Target Recruitment
- 20
- •Patients just diagnosed with breast cancer and planned for NACT.
- •Patients willing to provide written informed consent to participate in the study.
- •Patients whose tumors have any receptor phenotype.
- •Patients willing to receive the usual standard treatment in NACT setting.
Patients with metastatic disease will not be included.
Study & Design
- Study Type
- Observational
- Study Design
- Not specified
- Primary Outcome Measures
Name Time Method Evaluation of circulating biomarkers, specifically circulating tumor DNA (ctDNA), can provide information about the molecular characteristics of a patient’s tumor from a noninvasive blood draw, thus aiding the clinical management of this disease. First biopsy and blood sample will be collected when the patients are planned for neoadjuvant chemotherapy. | A repeat biopsy and blood sample will be collected from the same patients at the end of planned neoadjuvant chemotherapy at the time of surgery. To identify the biomarkers/ mutations derived from tumor tissue and subsequently use these markers (in a patient specific manner) to monitor therapy and relapse by quantifying the same mutations in peripheral blood. First biopsy and blood sample will be collected when the patients are planned for neoadjuvant chemotherapy. | A repeat biopsy and blood sample will be collected from the same patients at the end of planned neoadjuvant chemotherapy at the time of surgery.
- Secondary Outcome Measures
Name Time Method trying to identify the cluster of mutations occurring in the original tumor and their repetition in circulating tumor DNA. Identification of such biomarkers/ mutations derived from tumor tissue can be subsequently used to monitor therapy and relapse by quantifying the same mutations in peripheral blood.
Trial Locations
- Locations (1)
Tata Memorial centre
🇮🇳Mumbai, MAHARASHTRA, India
Tata Memorial centre🇮🇳Mumbai, MAHARASHTRA, IndiaDr Sudeep GuptaPrincipal investigator02224177201sudeepgupta04@yahoo.com