Blood-based genomics of triple-negative breast cancer progression in patients treated with neoadjuvant chemotherapy

E Ortolan, V Appierto, M Silvestri, R Miceli, S Veneroni, S Folli, G Pruneri, A Vingiani, A Belfiore, V Cappelletti, M Vismara, F Dell'Angelo, L De Cecco, G V Bianchi, F G de Braud, M G Daidone, S Di Cosimo, E Ortolan, V Appierto, M Silvestri, R Miceli, S Veneroni, S Folli, G Pruneri, A Vingiani, A Belfiore, V Cappelletti, M Vismara, F Dell'Angelo, L De Cecco, G V Bianchi, F G de Braud, M G Daidone, S Di Cosimo

Abstract

Background: As neoadjuvant chemotherapy (NAC) is increasingly used in triple-negative breast cancer (TNBC), we investigated the value of circulating tumor DNA (ctDNA) for patient monitoring prior, during, and after NAC, and circulating tumor cells (CTCs) for disease characterization at clinical progression.

Materials and methods: Forty-two TNBC patients undergoing NAC were prospectively enrolled. Primary tumor mutations identified by targeted-gene sequencing were validated and tracked in 168 plasma samples longitudinally collected at multiple time-points by droplet digital polymerase chain reaction. At progression, plasma DNA underwent direct targeted-gene assay, and CTCs were collected and analyzed for copy number alterations (CNAs) by low-pass whole genome sequencing.

Results: ctDNA detection after NAC was associated with increased risk of relapse, with 2-year event-free survival estimates being 44.4% [95% confidence interval (CI) 21.4%-92.3%] versus 77.4% (95% CI 57.8%-100%). ctDNA prognostic value remained worthy even after adjusting for age, residual disease, systemic inflammatory indices, and Ki-67 [hazard ratio (HR) 1.91; 95% CI 0.51-7.08]. During follow-up, ctDNA was undetectable in non-recurrent cases with the unique exception of one showing a temporary peak over eight samples. Conversely, ctDNA was detected in 8/11 recurrent cases, and predated the clinical diagnosis up to 13 months. Notably, recurrent cases without ctDNA developed locoregional, contralateral, and bone-only disease. At clinical progression, CTCs presented chromosome 10 and 21q CNAs whose network analysis showed connected modules including HER/PI3K/Ras/JAK signaling and immune response.

Conclusion: ctDNA is not only associated with but is also predictive of prognosis in TNBC patients receiving NAC, and represents an exploitable tool, either alone or with CTCs, for personalized TNBC management.

Keywords: circulating tumor DNA; circulating tumor cells; neoadjuvant chemotherapy; prognosis; triple-negative breast cancer.

Conflict of interest statement

Disclosure GCP received honoraria from Roche, GSK, Genomic Health, and Hybrid Genetics, and FGdB declares receiving speakers bureau honoraria from BMS, Eli Lilly, Roche, Amgen, AstraZeneca, Gentili, Fondazione Menarini, Novartis, MSD, Ignyta, Bayer, Noema SRL, ACCMED, DEPHAFORUM SRL, Nadirex, Roche, Biotechspert Ltd., PriME Oncology, Pfizer, and is a consultant/advisory board member for BMS, Tiziana Life Sciences, Celgene, Novartis, Servier, Phanm Research Associated, Daiichi Sankyo, Ignyta, Amgen, Pfizer, Roche, Teogarms, and Pierre Fabre. All other authors declare no conflicts of interest.

Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

Figures

Figure 1
Figure 1
CONSORT diagram showing patients included in each analysis and reasons for their exclusion. Green shading: patients with primary tumor bearing at least one mutation. Burgundy shading: patients with overall ctDNA assessment: and according to different treatment time points and follow-up. ctDNA, circulating tumor DNA; NGS, next-generation sequencing; TNBC, triple-negative breast cancer.
Figure 2
Figure 2
Matched primary tumor and plasma mutations. The heatmap shows the mutations detected in both primary tumor tissue and plasma samples (green), in just primary tumor tissue (burgundy) or not tested in plasma (olive) for the limited amount of plasma samples. Information on clinical stage, i.e. tumor size (T), and nodal status (N), grade, Ki-67, and germinal BRCA status is also reported. ctDNA, circulating tumor DNA; NAC, neoadjuvant chemotherapy.
Figure 3
Figure 3
Kaplan–Meier event-free survival. The curves represent event-free survival according to post-NAC ctDNA status. Number of patients at risk and censored are shown at the bottom of the figure. ctDNA, circulating tumor DNA; NAC, neoadjuvant chemotherapy.
Figure 4
Figure 4
Event-free survival plot among individual patients with or without detectable ctDNA during the study. For each patient, times of surgical resection and relapse are indicated by a yellow line and a light blue asterisk, respectively. Censored patients did not develop an unfavorable event at the time of data collection. ctDNA, circulating tumor DNA.
Figure 5
Figure 5
Copy number alterations (CNAs) detected in CTCs from relapsed cases. (A) The bar plot shows the distribution of CNAs along chromosomes considering all the CTCs collected at time of relapse. Green and burgundy colors refer to amplifications and deletions, respectively. (B) The heatmap reports patients in the column and the top 20 altered chromosome arms on the rows. Green and burgundy colors refer to amplifications and deletions, respectively; 21q (43%), 10p (41%) and 10q (41%) were the most commonly altered arms.

References

    1. Haddad T.C., Goetz M.P. Landscape of neoadjuvant therapy for breast cancer. Ann Surg Oncol. 2015;22:1408–1415.
    1. Rastogi P., Anderson S.J., Bear H.D. Preoperative chemotherapy: updates of National Surgical Adjuvant Breast and Bowel Project Protocols B-18 and B-27. J Clin Oncol. 2008;26:778–785.
    1. Cortazar P., Zhang L., Untch M. Pathological complete response and long-term clinical benefit in breast cancer: the CTNeoBC pooled analysis. Lancet. 2014;384(9938):164–172.
    1. Loibl S., von Minckwitz G., Untch M., Denkert C. Predictive factors for response to neoadjuvant therapy in breast cancer. Oncol Res Treat. 2014;37:563–568.
    1. Untch M., Konecny G.E., Paepke S., von Minckwitz G. Current and future role of neoadjuvant therapy for breast cancer. Breast. 2014;23:526–537.
    1. Lindenberg M.A., Miquel-Cases A., Retèl V.P. Imaging performance in guiding response to neoadjuvant therapy according to breast cancer subtypes: a systematic literature review. Crit Rev Oncol Hematol. 2017;112:198–207.
    1. Masuda N., Lee S.J., Ohtani S. Adjuvant capecitabine for breast cancer after preoperative chemotherapy. N Engl J Med. 2017;376(22):2147–2159.
    1. Denduluri N., Chavez-Mac Gregor M., Telli M.L. Selection of optimal adjuvant chemotherapy and targeted therapy for early breast cancer: ASCO Clinical Practice Guideline Focused Update. J Clin Oncol. 2018;36(23):2433–2443.
    1. Appierto V., Di Cosimo S., Reduzzi C., Pala V., Cappelletti V., Daidone M.G. How to study and overcome tumor heterogeneity with circulating biomarkers: the breast cancer case. Semin Cancer Biol. 2017;44:106–116.
    1. Kilgour E., Rothwell D.G., Brady G., Dive C. Liquid biopsy-based biomarkers of treatment response and resistance. Cancer Cell. 2020;37(4):485–495.
    1. Bidard F.C., Michiels S., Riethdorf S. Circulating tumor cells in breast cancer patients treated by neoadjuvant chemotherapy: a meta-analysis. J Natl Cancer Inst. 2018;110(6):560–567.
    1. Garcia-Murillas I., Schiavon G., Weigelt B. Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. Sci Transl Med. 2015;7:302ra133.
    1. Riva F., Bidard F.C., Houy A. Patient specific circulating tumor DNA detection during neoadjuvant chemotherapy in triple-negative breast cancer. Clin Chem. 2017;63:691–699.
    1. McDonald B.R., Contente-Cuomo T., Sammut S.J. Personalized circulating tumor DNA analysis to detect residual disease after neoadjuvant therapy in breast cancer. Sci Transl Med. 2019;11(504):eaax7392.
    1. Li S., Lai H., Liu J. Circulating tumor DNA predicts the response and prognosis in patients with early breast cancer receiving neoadjuvant chemotherapy. JCO Precis Oncol. 2020;4:244–257.
    1. Garcia-Murillas I., Chopra N., Comino-Méndez I. Assessment of molecular relapse detection in early-stage breast cancer. JAMA Oncol. 2019;5(10):1473–1478.
    1. Coombes R.C., Page K., Salari R. Personalized detection of circulating tumor DNA antedates breast cancer metastatic recurrence. Clin Cancer Res. 2019;25(14):4255–4263.
    1. Di Cosimo S., Appierto V., Silvestri M. Primary tumor somatic mutations in the blood of women with ductal carcinoma in situ of the breast. Ann Oncol. 2020;31(3):435–437.
    1. Rothé F., Silva M.J., Venet D. Circulating tumor DNA in HER2-amplified breast cancer: a translational research substudy of the NeoALTTO phase III trial. Clin Cancer Res. 2019;25(12):3581–3588.
    1. Heinze G., Schemper M. A solution to the problem of monotone likelihood in Cox regression. Biometrics. 2001;57(1):114–119.
    1. Li Z., Zhang X., Hou C. Comprehensive identification and characterization of somatic copy number alterations in triple-negative breast cancer. Int J Oncol. 2020;56(2):522–530.
    1. Radovich M., Jiang G., Hancock B.A. Association of circulating tumor DNA and circulating tumor cells after neoadjuvant chemotherapy with disease recurrence in patients with breast cancer: preplanned secondary analysis of the BRE12-158 randomized clinical trial. JAMA Oncol. 2020;6(9):1410–1415.
    1. Kim J.Y., Park D., Son D.S. Circulating tumor DNA shows variable clonal response of breast cancer during neoadjuvant chemotherapy. Oncotarget. 2017;8(49):86423–86434.
    1. Lo Y.M., Zhang J., Leung T.N., Lau T.K., Chang A.M., Hjelm N.M. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet. 1999;64:218–224.
    1. Forshew T., Murtaza M., Parkinson C. Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci Transl Med. 2012;4:136ra168.
    1. Dawson S.J., Tsui D.W., Murtaza M. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med. 2013;368:1199–1209.
    1. Razavi P., Li B.T., Brown D.N. High-intensity sequencing reveals the sources of plasma circulating cell-free DNA variants. Nat Med. 2019;25(12):1928–1937.
    1. Reduzzi C., Motta R., Bertolini G. Development of a protocol for single-cell analysis of circulating tumor cells in patients with solid tumors. Adv Exp Med Biol. 2017;994:83–103.

Source: PubMed

Sponzoři a spolupracovníci

Zdravotní podmínky

Drogové intervence

3
Předplatit