A diagnostic tissue biopsy provides a genomic snapshot limited to the primary tumor. However, in advanced cancers, biopsies from metastatic and relapse sites are difficult to perform, is an invasive procedure and can have sampling bias. Liquid biopsy offers new possibilities for inoperable patients and for monitoring therapy.[14] Circulating tumor (ctDNA) can be measured in the blood samples of breast cancer patients. It is a minimally invasive approach for disease monitoring and evaluation of response to the treatment.[14] Studies have shown that metastatic cancer can harbour a different mutational profile following relapse as compared to the primary tumor.[15] Moreover, tumors at primary and metastatic sites may have spatial heterogeneity, with individual metastases possessing different resistant mechanisms.[14,15] However, ctDNA released into circulation following tumor cell death is a mixture of tumor DNA from heterogenous metastatic sites and from primary tumor.[15] It represents the tumor heterogeneity in a metastatic setting. It can be measured in blood samples of patients and is easily processed as compared to tumor biopsy and hence can be used to serially monitor response to treatment and relapse. [14,15] In the current study, we propose to use personalised ctDNA as a biomarker for monitoring response to systemic therapy with or without RT, as a part of an already approved clinical trial, in metastatic breast cancer patients presenting to our tertiary cancer centre. The additive value of ctDNA mutation profiles to stratify patients with residual disease post treatment will be used to predict response and survival. We propose that the ctDNA dynamics assessed at different time points will be useful to assess the patient outcome and survival. Based on the ctDNA dynamics, further effective treatments can be formulated for MBC patients earlier as compared to clinical or radiological progression.

Study Rationale

Metastatic breast cancer patients are composed of heterogenous groups presenting with totally different metastatic patterns and prognostic outcomes based on the sites of metastasis and hence require distinct strategies for treatment. The current study aims to examine the role of personalized ctDNA as a predictive biomarker for response and outcome in systemic treatment followed by RT for denovo metastatic breast cancers. It is an attempt to explore if additive value of ctDNA can stratify patients based on the treatment outcome.

In the current study we would be observing the effect of RT after systemic therapy on the survival in MBC and comparing it with only systemic therapy. Evaluation of Ct DNA at different timepoints during RT would help to further prognosticate these patients based on response to treatment. The effect of RT on the ctDNA profiling has never been studied and hence it is a research question which we would like to explore. We hypothesize that RT given to ablate the primary tumor in MBC patients will subsequently lead to eradication of ctDNA mutations in blood and subsequent follow up ctDNA assessments in these patients can show whether the mutations remain absent or develop sooner as compared to the standard arm patients who do not receive RT.

Materials and Methods

Study type/design:  Phase III randomized controlled trial, Two arm. (Part of the Phase III randomized controlled clinical trial approved by IEC on 01.05.22 and CTRI registered Ref No. CTRI/2022/06/043551)

Methodology of the ctDNA testing panel

Custom panel design: Targeted Sequencing panel is made based on the known genes (n=74) relevant in breast cancer metastasis and with pathways that are actionable.

ct DNA extraction will be done using standardized kits meant for the purpose and ctDNA will be quantified. Library preparation, provenance testing, next generation sequencing and bioinformatics pipelines will be designed.  In brief, an aliquot of each ctDNA library will be used as input into the associated patient-specific multiplex polymerase chain reaction. The resulting amplicon products will be uniquely barcoded, followed by pooling and ultra-high depth NGS Sequencing will be performed on a standard NGS Platform like Illumina. Rapid Run with 50 cycles of paired-end reads using the Illumina Paired End v2 kit with an average read depth of >90,000x per amplicon can be used. A previously validated cutoff of ≥2 variants detected was used as criteria for ctDNA positivity. The cutoff can be chosen based on a previously defined confidence threshold necessary to achieve high specificity of >99.8% while maintaining high sensitivity.

WBCs DNA Extraction: The WBC DNA obtained from blood samples will be used for germ line mutation detection.

ct- DNA extraction and quantification. A total of 300 plasma samples from 60 patients will be analyzed in this study, with each patient having 4 samples in Arm A and 6 samples in Arm B available. The mean volume of plasma processed will be 3.3 mL (range: 1–6 mL). The entire volume of plasma will be used for ctDNA extraction using the QIAamp Circulating Nucleic Acid kit (Qiagen) and eluted into 50 μL DNA Suspension Buffer (Sigma). Each ctDNA sample will be quantified by Quant-iT High Sensitivity dsDNA Assay Kit (Invitrogen). The mean DNA yield isolated from plasma samples will be identified (ng/mL).

ct- DNA library preparation. Up to 66 ng or 20,000 genome equivalents (mean: 26.6 ng; range: 1–66 ng) of ctDNA from each plasma sample will be used as input into library preparation, as described previously [7]. The ctDNA will be end-repaired, A-tailed, and ligated with custom adapters. The purified ligation product will be amplified for 20 cycles and purified using Ampure XP beads (Agencourt/Beckman Coulter).

Provenance check. Genotyping of a set of 45 single nucleotide polymorphism (SNPs) will be used to confirm provenance of DNA samples. A concordance of >85% of SNP genotypes between samples indicate that samples originated from the same individual.

Bioinformatics pipeline. All paired-end reads will be merged using Pear software. Bases that did not match in forward and reverse reads or had a low-quality score were filtered out to minimize sequencing errors. Merged reads were mapped to the hg19 reference genome with Novoalign version 2.3.4 (http://www.novocraft.com/). Quality control (QC) will be performed that checks for a wide list of statistics per sample, including total numbers of reads, mapped reads, on-target reads, number of failed targets and average error rate. Target amplicons with <5,000 high quality reads will be considered to have failed Q.

Plasma variant calling and quantification. For high confidence variants, the variant allele frequency (VAF) will be calculated as the ratio of observed mutant reads by total reads. The number of mutant molecules per mL of plasma was calculated as follows:

ctDNA extracted (ng)   x 1000 pg     x haploid genome equivalent (hGE) x Variant Allele Fraction (VAF)

             1 ng                3.3 pg per hGE                      Plasma volume for extraction (mL)

The mean mutant molecules will be calculated by dividing the total number of mutant molecules by the number of targets detected.