Breast cancer is the second most common cancer in the world and, by far, the most frequent cancer among women with an estimated 1.67 million new cancer cases diagnosed in 2012 (25% of all cancers). It is estimated that by 2030 the global burden of breast cancer will increase to over 2 million new cases per year. [1,3] 

Breast cancer in India: Breast cancer accounts for 25% to 31% of all cancers in women in India [1].  According to GLOBOCAN (WHO), for the year 2012, 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 the world (second: China - 47984 deaths and third: US - 43909 deaths ).. [2]   

Once a patient is diagnosed with breast cancer, she is given the standard of care treatment after which follow up is usually done by serial imaging techniques for monitoring the treatment response 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 up recurrent disease but most often too late to institute meaningful therapy to significantly prolong survival.  In spite of an exponential increase in our understanding of tumor biology over recent decades, and the availability of technologies to characterize tumors, whether and when an individual cancer will metastasize/ recur/ relapse remains unknown.

There is therefore an urgent need for biomarkers that measure

ü  Tumor burden with high sensitivity and specificity.

ü  Response to therapy

ü  Early detection of relapse

Investigations are now focusing on blood-based assays that detect and characterize circulating tumor cells or circulating tumor DNA (a component of cell-free DNA). These minimally invasive, ’liquid biopsies’ can be performed at multiple intervals to monitor disease and tailor cancer therapy. Research has shown the feasibility of using circulating tumor DNA to monitor tumor dynamics since circulating tumor DNA had sensitivity superior to that of circulating tumor cells.

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.

Advances in sequencing technologies have enabled the rapid identification of somatic genomic alterations in individual tumors, and these can be used to design personalized assays for the monitoring of circulating tumor DNA.

We propose 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.

State of art: Evaluating the levels of tumor markers like Alpha-fetoprotein (AFP), Anaplastic lymphoma kinase (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), Epidermal growth factor receptor (EGFR), Lactate dehydrogenase (LDH), Neuron-specific enolase (NSE), Prostate-specific antigen (PSA), NMP22 are routinely used for detection of various cancers.

These available tumor markers do not predict response to chemotherapy and recurrence of cancer. Also, these markers are mostly cancer specific.

With the advent of high-throughput technology like NGS, it is now possible to identify somatic alterations in the recurrently mutated genes in the circulating tumor DNA in individual cancer patients. The next-generation sequencing (NGS) approach holds a number of potential advantages over traditional methods, including the ability to fully sequence large numbers of genes (hundreds to thousands) in a single test and simultaneously detect deletions, insertions, copy number alterations, translocations, and exome-wide base substitutions (including known “hot-spot mutations”) in all known cancer-related genes. Continuing advances in NGS technology will lower the overall cost, speed the turnaround time, increase the breadth of genome sequencing, detecting epigenetic markers and other important genomic parameters. Targeted deep sequencing of plasma DNA provides a cost-effective alternative for high-throughput analysis and may overcome limitations of initial tumor-tissue assessment by virtue of allowing for the direct identification of mutations in plasma. We have collected the data on 50 hotspot genes that are routinely mutated in various cancers. Among these 50 hotspot genes, around 15 genes are found to be commonly mutated in at-least three out of top seven cancers in India.

S J Dawson et.al compared the radiographic imaging of tumors with the assay of circulating tumor DNA, CA 15-3, and circulating tumor cells in 30 women with metastatic breast cancer who were receiving systemic therapy. Circulating tumor DNA was successfully detected in 29 of the 30 women (97%) in whom somatic genomic alterations were identified. Somatic mutations were identified by tagged-amplicon deep sequencing22 for PIK3CA and TP53 or paired-end whole-genome sequencing. Circulating tumor DNA levels showed a greater dynamic range, and greater correlation with changes in tumor burden, than did CA 15-3 or circulating tumor cells (2).

We plan to sequence 44 genes from Qiagen- Human Breast Cancer Gene Read DNAseq Targeted Panel. The Human Breast Cancer GeneRead DNAseq Targeted Panel is a collection of multiplexed PCR primer assays for targeted enrichment of the coding (exonic) regions of the 44 genes most commonly mutated in human breast cancer samples.

Alternative cancer gene panels are

Also, the Illumina Trusight Cancer gene panel shares 23 common genes with the Cosmic-Cancer census gene list of 522 top mutated genes in various cancers.

We also compared these 44 genes with the top 1000 Breast cancer genes reported in COSMIC database (according to the number of samples mutated) and found that there are 19 common genes between the two lists.

Key words: circulating, chromatin, response, breast cancer

3. AIMS AND OBJECTIVES

1. To identify the mutations in the 44 genes from Qiagen Breast Cancer Gene Read DNAseq Targeted Panel and subtract these mutations from the background normal tissue.

2. To characterize these identified mutations.

3. To use these mutations in blood to monitor response to therapy and to evaluate whether 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 for neoadjuvant chemotherapy we will first obtain the tumor biopsy samples from these patients biopsy in strict accordance with ethical committee guidance. Simultaneously blood and buccal mucosal samples will also be collected from patients.      

Medical history of the consenting patients will be obtained and serological test for HIV, HCV, and HBsAg will be done. One or two tumor tissue samples of approximately 1 cm size will be collected using appropriate biopsy punch/RNAse-free sterile forceps.. After histopathological examination, specimens will be transported on ice to sequencing laboratory in AQIX RS-1/ RNA later biopsy storage and transport medium. 30ml blood samples will be collected every 3 weeks prior to each cycle of chemotherapy when it is once per 3-week chemotherapy regimen. 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 3 weeks. In the weekly regimen, this will be done prior to 1^st cycle, 4^th cycle, 7^th cycle, 10^th cycle and after 12^th cycle. Blood and buccal swab specimens will be collected in appropriate containers with EDTA/ Acid citrate dextrose/ suitable media and shipped on ice to the sequencing laboratory.

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.

Total 02 biopsies will be performed; First when the patients are diagnosed with Breast cancer who are planned for neoadjuvant chemotherapy and another at the end of the planned neoadjuvant chemotherapy at the time of surgery.

The peripheral blood for germline mutation analysis will be collected at the time of accrual and first progression.

Sequencing: Next generation sequencing will be done using the Illumina HiSeq 1000 system according to the established protocol. Only High Quality (HQ) sequence information will be used for analysis. The HQ metric is assessed by

a) <5% Adapter contamination

b) Even (~25% of each base) Base distribution of sample

c) Q>30% and

d) Acceptable Kmer values and GC content (as defined by standard softwares such as FasQC, etc). A minimum of 3 GB, paired–end sequence information, passing the quality standards mentioned before, will be used for the analysis.

Library Construction: DNA Exome: A total of 1-3 μg DNA will be used for the library preparation. Genomic DNA will be prepared from the blood and tumor tissue by using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Canada). After centrifugation of DNA as per the Qiagen kit protocol, purification by microdialysis through DNeasy membrane will be done. DNA quality will be assessed by spectrophotometry (260/280 and 260/230) and gel electrophoresis before library construction. The DNA library will be prepared by following the indexed pair end library protocol (Illumina Inc., USA). Briefly, the DNA will be subjected to end-repair, and phosphorylation by T4 DNA polymerase, Klenow DNA Polymerase, and T4 polynucleotide kinase respectively in a single reaction, and then 3’ A overhangs generation by Klenow fragment (3’ to 5’ exon minus), and ligated to Illumina PE adapters, which contain 5’ T overhangs. PCR products will be purified on Qiaquick MinElute columns (Qiagen) and the DNA quality will be assessed and quantified using an Agilent DNA 1000 series II assay and Nanodrop 7500 spectrophotometer (Nanodrop, USA).

Exome capture: Exome capture will be done by using TrueSeq Exome Enrichment Kit from Illumina Inc or SureSelect Target Enrichment Kit from Agilent, according to the manufacturer’s instructions. The validation of exome capture will be assessed post-facto based on the parameters of “coverage” and “depth”, with the expectation being greater than 90% of the manufacturer claims at 20-100X depth.

Isolation and Quantification of Circulating Tumor DNA: 30ml blood samples will be collected every 3 weeks prior to each cycle of chemotherapy when it is once per 3-week chemotherapy regimen. 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 3 weeks. In the weekly regimen, this will be done prior to 1^st cycle, 4^th cycle, 7^th cycle, 10^th cycle and after 12^th cycle.    

The blood samples collected in EDTA tubes will be processed within 1 hour after collection and will be centrifuged to separate the plasma from the peripheral-blood cells. Circulating tumor DNA will be extracted from aliquots (2 ml) of plasma and normal DNA will be extracted from the buffy coat with the use of the QIAamp circulating nucleic acid kit (Qiagen).