Mariana Fitarelli-Kiehl

Brigham and Women's Hospital

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G. Mike Makrigiorgos

Dana-Farber Cancer Institute

Fangyan Yu

Brigham and Women's Hospital

Wai Yie Leong

INTI International University

Viktor A. Adalsteinsson

Broad Institute

Heather A. Parsons

Dana-Farber Cancer Institute

Nikhil Wagle

Harvard University

Nancy U. Lin

Harvard University

Pedro Exman

Instituto do Câncer do Estado de São Paulo

Ann H. Partridge

Harvard University

Kan Xiong

Broad Institute

Cooperative Institutions

Dana-Farber Cancer Institute

Harvard University

Broad Institute

Hospital de Clínicas de Porto Alegre

Universidade Federal do Rio Grande do Sul

Brigham and Women's Hospital

Dana-Farber Brigham Cancer Center

Massachusetts Institute of Technology

Massachusetts General Hospital

Boston University

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Sensitive Detection of Minimal Residual Disease in Patients Treated for Early-Stage Breast Cancer

Clinical Cancer Research (2020)

Heather A. Parsons Justin Rhoades Sarah C. Reed Gregory Gydush Priyanka Ram

Abstract Purpose: Existing cell-free DNA (cfDNA) methods lack the sensitivity needed for detecting minimal residual disease (MRD) following therapy. We developed a test for tracking hundreds of patient-specific mutations to detect MRD with a 1,000-fold lower error rate than conventional sequencing. Experimental Design: We compared the sensitivity of our approach to digital droplet PCR (ddPCR) in a dilution series, then retrospectively identified two cohorts of patients who had undergone prospective plasma sampling and clinical data collection: 16 patients with ER+/HER2− metastatic breast cancer (MBC) sampled within 6 months following metastatic diagnosis and 142 patients with stage 0 to III breast cancer who received curative-intent treatment with most sampled at surgery and 1 year postoperative. We performed whole-exome sequencing of tumors and designed individualized MRD tests, which we applied to serial cfDNA samples. Results: Our approach was 100-fold more sensitive than ddPCR when tracking 488 mutations, but most patients had fewer identifiable tumor mutations to track in cfDNA (median = 57; range = 2–346). Clinical sensitivity was 81% (n = 13/16) in newly diagnosed MBC, 23% (n = 7/30) at postoperative and 19% (n = 6/32) at 1 year in early-stage disease, and highest in patients with the most tumor mutations available to track. MRD detection at 1 year was strongly associated with distant recurrence [HR = 20.8; 95% confidence interval, 7.3–58.9]. Median lead time from first positive sample to recurrence was 18.9 months (range = 3.4–39.2 months). Conclusions: Tracking large numbers of individualized tumor mutations in cfDNA can improve MRD detection, but its sensitivity is driven by the number of tumor mutations available to track.

Minimal Residual Disease

10.1158/1078-0432.ccr-19-3005

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Citations (152)

The breast cancer immunophenotype of TP53-p.R337H carriers is different from that observed among other pathogenic TP53 mutation carriers

Familial Cancer (2015)

Mariana Fitarelli-Kiehl Juliana Giacomazzi Patrícia Santos-Silva Márcia Silveira Graudenz Edenir Inêz Palmero

Immunophenotyping

Li–Fraumeni syndrome

10.1007/s10689-015-9779-y

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Citations (10)

Supplementary Tables from Sensitive Detection of Minimal Residual Disease in Patients Treated for Early-Stage Breast Cancer

Heather A. Parsons Justin Rhoades Sarah C. Reed Gregory Gydush Priyanka Ram

Supplementary Tables

Minimal Residual Disease

10.1158/1078-0432.22478900.v1

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Supplementary Figure 3 from Sensitive Detection of Minimal Residual Disease in Patients Treated for Early-Stage Breast Cancer

Heather A. Parsons Justin Rhoades Sarah C. Reed Gregory Gydush Priyanka Ram

Overview schematic of MRD assay. MRD assay workflow from characterizing a primary tumor to determining if a blood draw contains evidence of residual disease.

Minimal Residual Disease

10.1158/1078-0432.22478924.v1

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Supplementary Figure 13 from Sensitive Detection of Minimal Residual Disease in Patients Treated for Early-Stage Breast Cancer

Heather A. Parsons Justin Rhoades Sarah C. Reed Gregory Gydush Priyanka Ram

Patients who were recurrence free after treatment for early stage breast cancer. cfDNA time points for all patients who did not experienced recurrence. Those marked with poor detection limits had an estimated detection limit worse than 1/1,000. End points represent time of last follow up or death.

10.1158/1078-0432.22478939.v1

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Supplementary Data from Sensitive Detection of Minimal Residual Disease in Patients Treated for Early-Stage Breast Cancer

Heather A. Parsons Justin Rhoades Sarah C. Reed Gregory Gydush Priyanka Ram

Supplementary Methods

Minimal Residual Disease

10.1158/1078-0432.22478954.v1

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Supplementary Figure 15 from Sensitive Detection of Minimal Residual Disease in Patients Treated for Early-Stage Breast Cancer

Heather A. Parsons Justin Rhoades Sarah C. Reed Gregory Gydush Priyanka Ram

MRD testing results in early stage breast cancer cohort. (A) Detection limits versus the number of SNVs tracked for each cfDNA sample. (B) Comparison of fingerprint size distributions between patients that were MRD+ at any time point and those that were always MRD-, split by distant recurrence status. The Mann-Whitney U test was used to determine whether MRD+ patients tracked significantly more mutations than MRD- patients. (C) Tumor fractions for MRD+ samples from patients with distant recurrence split by the minimum fingerprint size required to still detect MRD.

Minimal Residual Disease

10.1158/1078-0432.22478933.v1

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Supplementary Figure 14 from Sensitive Detection of Minimal Residual Disease in Patients Treated for Early-Stage Breast Cancer

Heather A. Parsons Justin Rhoades Sarah C. Reed Gregory Gydush Priyanka Ram

Patients who experienced local recurrence only after treatment for early stage breast cancer. cfDNA time points for all patients who experienced local recurrence only. End points represent time of last follow up or death.

Minimal Residual Disease

10.1158/1078-0432.22478936

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Supplementary Figure 9 from Sensitive Detection of Minimal Residual Disease in Patients Treated for Early-Stage Breast Cancer

Heather A. Parsons Justin Rhoades Sarah C. Reed Gregory Gydush Priyanka Ram

Technical performance of the MRD Assay. All samples from early stage breast cancer cohort. Includes plasma cfDNA and buffy coat gDNA samples. Samples from the same patient and probed with the same panel are represented independently. (A) On target fraction calculated from Picard CollectHsMetrics and using pct_selected_bases. (B) Coefficient of variation of duplex fragment depth across fingerprint sites. (C) Fold 80 base penalty score for duplex fragment depth across fingerprint sites. (D, E) Error rate comparison between conventional sequencing (requiring Q20 base quality and removing duplicate fragments) and our duplex sequencing with added filters (see methods). (F) Fraction of total duplexes after downsampling raw reads. We considered a sample to be saturated if it still contained at least 95% of its duplexes after downsampling to 80% of its raw reads.

Duplex (building)

Coefficient of variation

10.1158/1078-0432.22478903

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Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53

Genetics and Molecular Biology (2016)

Mariana Fitarelli-Kiehl Gabriel S. Macedo Rosane Paixão Schlatter Patrícia Koehler‐Santos Úrsula da Silveira Matte

Germline mutations in the TP53 gene are associated with Li-Fraumeni and Li-Fraumeni-Like Syndromes, characterized by increased predisposition to early-onset cancers. In Brazil, the prevalence of the TP53-p.R337H germline mutation is exceedingly high in the general population and in cancer-affected patients, probably as result of a founder effect. Several genotyping methods are used for the molecular diagnosis of LFS/LFL, however Sanger sequencing is still considered the gold standard. We compared performance, cost and turnaround time of Sanger sequencing, PCR-RFLP, TaqMan-PCR and HRM in the p.R337H genotyping. The performance was determined by analysis of 95 genomic DNA samples and results were 100% concordant for all methods. Sequencing was the most expensive method followed by TaqMan-PCR, PCR-RFLP and HRM. The overall cost of HRM increased with the prevalence of positive samples, since confirmatory sequencing must be performed when a sample shows an abnormal melting profile, but remained lower than all other methods when the mutation prevalence was less than 2.5%. Sequencing had the highest throughput and the longest turnaround time, while TaqMan-PCR showed the lowest turnaround and hands-on times. All methodologies studied are suitable for the detection of p.R337H and the choice will depend on the application and clinical scenario.

Sanger sequencing

TaqMan

COLD-PCR

High Resolution Melt

Li–Fraumeni syndrome

10.1590/1678-4685-gmb-2014-0351

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Citations (12)