1139601 research-article20222022 TAM0010.1177/17588359221139601Therapeutic Advances in Medical OncologyN Ciriaco, E Zamora Therapeutic Advances in Medical Oncology Original Research Clearance of ctDNA in triple-negative and Ther Adv Med Oncol 2022, Vol. 14: 1­–12 HER2-positive breast cancer patients during DOI: 10.1177/ https://doi.org/10.1177/17588359221139601 https://doi.org/10.1177/17588359221139601 17588359221139601 neoadjuvant treatment is correlated with © The Author(s), 2022. Article reuse guidelines: sagepub.com/journals- pathologic complete response permissions Nikaoly Ciriaco , Esther Zamora , Santiago Escrivá-de-Romaní , Ignacio Miranda Gómez, José Jiménez Flores , Cristina Saura, Hillary Sloane, Anna Starus, Johannes Fredebohm, Lucy Georgieva, Graham Speight, Frederick Jones, Santiago Ramón y Cajal, Martín Espinosa-Bravo and Vicente Peg Correspondence to: Vicente Peg Abstract Universidad Autónoma de Barcelona, Barcelona, Background: Although the standard of care is to perform surgery of primary breast cancer Spain (BC) after neoadjuvant chemotherapy (NAC), for certain patients achieving clinical complete Pathology Department, Vall d’Hebron University response (cCR) and pathologic complete response (pCR), omission of surgical treatment may Hospital, Paseo Vall be an option. Levels of circulating tumor DNA (ctDNA) during and after therapy could identify d’Hebron 119-129, Barcelona 08035, Spain patients achieving minimal residual disease. In this study, we evaluated whether ctDNA Spanish Biomedical clearance during NAC could be a correlate to effective response in human epidermal growth Research Network Centre in Oncology (CIBERONC), factor receptor 2 positive (HER2+) and triple-negative (TN) BC patients. Madrid, Spain. Methods: A prospective study was conducted to identify patient-specific PIK3CA and TP53 vicente.peg@vallhebron. cat. mutations in tissue using next-generation sequencing, which could then be used to track the Nikaoly Ciriaco presence/absence of mutations prior to, during, and following NAC using Sysmex SafeSEQ Pathology Department, Hospital del Mar, technology. All patients underwent a surgical excision after NAC, and pCR was assessed. Barcelona, Spain Results: A total of 29 TN and HER2+ BC patients were examined and 20 that carried Universidad Autónoma de Barcelona, Barcelona, mutations in the PIK3CA and/or TP53 genes were recruited. Overall, 19 of these 20 patients Spain harbored at least one tumor-specific mutation in their plasma at baseline. After NAC, 15 Esther Zamora Universidad Autónoma patients (75.0%) achieved pCR according to the histopathologic evaluation of the surgical de Barcelona, Barcelona, specimen, and 15 patients (75.0%) had a cCR; 18 of 20 patients (90.0%) had concordant pCR Spain Breast Cancer Group, and cCR. The status of ‘no mutation detected’ (NMD) following NAC in cCR patients correctly Vall d’Hebron Institute identified the pCR in 14 of 15 patients (93.33%), as well as correctly ruled out pCR in three of Oncology (VHIO), Barcelona, Spain patients, with an accuracy of 89.47%. During the 12-month follow-up after surgery, 40 plasma Medical Oncology samples collected from 15 patients all showed no detectable ctDNA (NMD), and no patient Department, Vall d’Hebron University Hospital, recurred. Barcelona, Spain Conclusion: These findings prompt further research of the value of ctDNA for non-invasive Santiago Escrivá- de-Romaní prediction of clinical/pathological response, raising the possibility of sparing surgery following Breast Cancer Group, NAC in selected BC patients. Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain Keywords: breast cancer, circulating tumor DNA, liquid biopsy, neoadjuvant therapy, Medical Oncology Department, Vall d’Hebron pathologic complete response University Hospital, Barcelona, Spain Ignacio Miranda Gómez Received: 13 May 2022; revised manuscript accepted: 31 October 2022. Radiology Department, Vall d’Hebron University Hospital, Barcelona, Spain Introduction It is also the second cause of cancer-related death José Jiménez Flores Breast cancer (BC) is the most commonly diag- in developed countries.1 The use of neoadjuvant Molecular Oncology Lab. Vall d’Hebron Institute nosed malignancy in women worldwide and chemotherapy (NAC) has increased over the of Oncology (VHIO), accounts for almost one out of four cancer cases. years, not only in locally advanced BC, but also in Barcelona, Spain journals.sagepub.com/home/tam 1 Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). Therapeutic Advances in Medical Oncology Volume 14 Cristina Saura early high-risk tumors. The availability of NAC in Based on these results, our working hypothesis Medical Oncology Department, Vall d’Hebron therapeutic modalities enables more immediate was that the detection and quantification of University Hospital, treatment of local and micrometastatic disease. ctDNA from patients receiving NAC may be a Barcelona, Spain Hillary Sloane Hence, a greater number of breast-conserving complementary method to assess pCR along with Anna Starus surgeries may be possible in the future by assess- current imaging techniques. This should allow for Johannes Fredebohm Frederick Jones ing the efficacy of effective NAC treatment by future identification of patients without infiltrat- Sysmex Inostics, Inc., visualizing tumor shrinkage by imaging, as well as ing or residual disease that could eventually avoid Baltimore, MD, USA by other sensitive molecular techniques. In many surgery. Thus, we performed longitudinal track- Sysmex Inostics GmbH, Hamburg, Germany cases, pathologic complete responses (pCR) can ing of plasma TP53 and PIK3CA mutations in Lucy Georgieva be achieved, both in the breast and axilla, which HER2+ and TN BC patients to examine ctDNA Graham Speight significantly improves disease-free and overall clearance during NAC as a correlate to effective Oxford Gene Technology, Oxford, UK survival.2,3 The likelihood of achieving pCR after response to treatment, as benchmarked by clini- Santiago Ramón y Cajal NAC depends on tumor molecular subtype. cal complete response (cCR) and pCR. We also Universidad Autónoma de Triple-negative (TN) and human epidermal analyzed serial samples after surgery in a subset of Barcelona, Barcelona, Spain Pathology Department, growth factor receptor 2 positive (HER2+) BC patients to explore the possible re-emergence of Vall d’Hebron University patients present a higher rate of pCR (up to 60%) ctDNA over time and whether continued ctDNA Hospital, Barcelona, Spain as compared to luminal BC patients after NAC.4,5 surveillance over time might also identify patients Spanish Biomedical Research Network Centre who would later recur radiologically. in Oncology (CIBERONC), Currently, the standard of care for primary BC Madrid, Spain after NAC is to perform surgery to completely Martín Espinosa-Bravo Breast Cancer Unit, Vall remove any residual disease that may remain in Materials and methods d’Hebron University non-pCR cases or to diagnose/confirm a pCR. Hospital, Barcelona, Spain However, if a pCR could be diagnosed with suffi- Study design and patients cient certainty using non-invasive procedures, a We prospectively recruited patients diagnosed with surgical intervention might hypothetically be with- BC and scheduled to receive NAC in a Spanish out benefit and cause unnecessary harm for the Hospital Cancer center from 2018 to 2019. Eligible patient.6,7 Breast imaging methods are not able to patients were enrolled upon histologically con- accurately estimate residual disease after NAC; firmed stage I/III HER2+ or TN infiltrating breast hence, they cannot replace the pathologic diagno- carcinoma. The pilot cohort was subsequently sis of the surgical excision specimen.8,9 A recent stratified to select those patients having specific meta-analysis that assessed the diagnostic accuracy PIK3CA and/or TP53 mutations detected in the of image-guided minimally invasive biopsy tech- biopsied tissue; this was determined by performing niques in predicting breast pCR after NAC con- next-generation sequencing (NGS) of the patients’ cluded that they are not accurate enough to locally primary tumor sample. TP53 and/or PIK3CA visualize residual disease pathology in the tissue.10 mutations identified in the tumor sample were used to generate ‘bespoke’ or tumor-informed Liquid biopsy has been shown to be a very powerful ctDNA liquid biopsy assays customized for each non-invasive technology that provides valuable sup- patient using SafeSEQ technology, that were then plementary information in terms of prognosis assess- used to track the presence/absence of these specific ment, drug resistance, and individualized treatment.11 mutations in each patient’s plasma samples for Circulating tumor DNA (ctDNA) is fragmented longitudinal tracking of ctDNA during NAC. genomic DNA (fgDNA) resulting from apoptosis Radiological assessment (mammography, ultra- and necrosis of tumor cells that can be detected in sound, and magnetic resonance imaging), as part blood by the presence of somatic mutations.12 In BC, of the routine clinical practice, and mutation integrated molecular analyses revealed that TP53 and detection in plasma samples were carried out at PIK3CA are the most frequently mutated genes, baseline prior to NAC, at treatment mid-point, each with a mutation frequency >35%.13 Moreover, and at post-treatment immediately prior to sur- several forward-looking studies have provided semi- gery, as well as at three time-points post-surgery nal clinical evidence that levels of ctDNA in general, (10 weeks, 6 months, and 12 months). The NAC as well as PIK3CA and TP53 mutations in particular, regimens were administered according to the hos- detected during NAC and post-surgery can identify pital’s protocol, based on anthracyclines and taxa- patients that exhibit effective responses versus those nes. In some TN patients, the regimens also showing minimal residual disease14–18 and can be included platinum agents and, in HER2+ patients, used to predict tumor response to NAC and progno- treatments included was specific antibody thera- sis in early BC.19–21 pies associated with HER2 signaling pathway 2 journals.sagepub.com/home/tam N Ciriaco, E Zamora et al. blockade. All patients underwent a surgical exci- Coulter, Brea (CA), USA) and dilution. A second sion after NAC, and pCR was assessed. The study PCR in which sample-specific index sequences was approved by the medical ethical committee of were attached served to generate and then discrimi- the Hospital Universitario Vall d’Hebron nate UID families. Index PCR products were puri- (PR(AG)204/2016) on 30 September 2016. All fied in a pooled format using QIAquick (Qiagen) patients provided oral and written informed con- followed by one round of AMPure XP bead purifi- sent for sample acquisition for research purposes. cation (Beckman Coulter). DNA samples were then quantified using a Bioanalyzer (Agilent, Santa Clara (CA), USA) and sequenced on NextSeq 550 Targeted sequencing analysis of tissue and instrument (Illumina, San Diego (CA), USA). plasma DNA Sequencing data were analyzed using proprietary Tumor tissue samples from all patients were ini- software packages developed by Sysmex Inostics. tially examined for mutations in both the PIK3CA Mutation analyses and individual mutation calls and TP53 genes using the NGS-based SureSeq were performed with a positive mutation call technology (Oxford Gene Technology) after isola- [mutation detected, MD9 or negative mutation call tion of DNA using Qiasymphony DNA Tissue Kits (no mutation detected, NMD)] for each patient (Qiagen, Hilden, Germany) following the manu- sample time point. Positive mutation calls for each facturer’s instructions. For liquid biopsy assays, tumor informed mutation that were reported in blood was collected in Streck® Cell-Free DNA mutant molecules [i.e. copies)/ml of plasma (MM/ BCT tubes and processed to plasma using a dou- ml)]. The positive call thresholds (cutoffs) for each ble-spin protocol in which the blood was centri- tumor-informed mutation detected in plasma were fuged twice to obtain 8–9 ml of plasma; samples determined individually; these were set as mutant were then stored at −80°C until DNA extraction. molecule values at 3× the value of the background DNA was extracted with the QIAamp Circulating established for each mutation position (Limit of Nucleic Acid Kit (Qiagen) using extended lysis of Blank) and above a minimum threshold of 3 MM 60 min and analyzed using the Plasma SafeSEQ (copies) detected in a patient’s sample. Any values technology (Sysmex Inostics, Inc., Hamburg, falling below both mutations-specific and sample Germany). Personalized SafeSEQ liquid biopsy level calling thresholds failed ‘Mutation Detected’ assays were designed custom to each patient based call criteria by the software program and were thus on the tumor mutations identified by the SureSeq designated as ‘No Mutations Detected (NMD). NGS assay. To examine each mutation in PIK3CA and TP53 that were found in the patient’s tumor sample in liquid biopsy, patient sample specific Statistical analysis multiplex reactions were designed at the research The primary outcome measure was pCR, as laboratories at Sysmex Inostics Germany to detect defined by the absence of invasive residual disease the specific mutations previously identified in tis- both in the breast and axillary lymph nodes fol- sue.22 For certain patients, other specific mutations lowing NAC. The ctDNA mutation results detected in patients’ tumor samples were also ana- (undetectable; NMD/detectable; MD) from each lyzed alongside of PIK3CA or TP53. Prior to detec- patient’s plasma sample obtained at the baseline/ tion of ctDNA in patient plasma samples, DNA pre-surgery were then compared to the sample(s) was isolated from plasma and quantified using obtained after NAC. The accuracy of the test as LINE-1 qPCR as described previously.23 Patient- well as positive predictive value and negative pre- customized multiplexes assay was designed and dictive value (NPV) were calculated for the com- QCed on fgDNA to a mean size of ~160 bp using bination of ctDNA positive and cCR using the the Covaris M220 instrument (Covaris LLC, Diagnostic test evaluation calculator (MedCalc Woburn (MA), USA). Background noise was Statistical Software Version 20.027, MedCalc determined for each multiplex using 2× 10,000 Software bv, Ostend, Belgium). genomic equivalents of genomic DNA (NIST, RM8398). Each multiplex contained primers attached to unique identifiers (UIDs), which served Results as molecular barcodes and used for error correction according to the Safe-SeqS principle.22 Specifically, Patient and tumor characteristics UID PCR was performed with 13 cycles followed A total of 29 BC patients scheduled to receive by bead-based purification of patient-specific NAC were prospectively enrolled and histologi- amplicons using AMPure XP beads (Beckman cally examined; nine patients were excluded from journals.sagepub.com/home/tam 3 Therapeutic Advances in Medical Oncology Volume 14 Figure 1. Study flowchart. further study because of the absence of TP53 or Response to NAC PIK3CA mutations in tissue, as assessed by NGS In all, 15 patients (75.0%) had a pCR after NAC (Figure 1). Within the study cohort (N = 20), all according to the histopathologic evaluation of patients had at least one TP53 mutation detected surgical specimen. A pCR occurred in 10 out of and five patients also had a PIK3CA mutation. 12 patients (83.3%) with TN tumors and 5 out of Patient-specific SafeSEQ assays were generated 8 patients (62.5%) with HER2+ tumors (Table 2). for all of the TP53 and PIK3CA mutations that Based on clinical examination and imaging assess- were detected in tissue to detect these mutations ment, 15 patients (75.0%) had a cCR; 18 out of in plasma during NAC. In addition, three patients 20 patients (90.0%) had concordant pCR and (32, 33, and 34) – each having a single TP53 cCR. At mid-treatment, 15 (75.0%) plasma sam- mutation detected in their tumors – also had ples had non-detectable ctDNA (12 cCR patients accompanying mutations in the following genes versus 3 non-cCR patients). Immediately prior to (NTRK1, NTRK3, HER2/ERBB2, GRM3, surgery, 17 patients (85.0%) had non-detectable AKT3, FGF10, and FANCF); patient-specific ctDNA (15 cCR patients versus 2 non-cCR SafeSEQ plasma assays were also prepared to patients). Patients showing a good response to detect the presence of these additional mutations NAC had ctDNA drops in the middle of the in plasma. The clinical and pathological charac- treatment (examples in Figure 2). teristics, tumor mutations identified, and plasma mutations detected in baseline plasma samples prior to NAC are all shown in Table 1. Overall, Accuracy of pre-surgery ctDNA + cCR for there were 12 TN and 8 HER2+ BC patients pCR determination enrolled in the pilot cohort. The median (range) Table 3 summarizes the performance of the combi- age of patients was 57 (38–88) years. Overall, 19 nation of a ctDNA-negative result and achieving of these 20 patients (overall percent agree- cCR (ctDNA + cCR) in predicting pCR after NAC. ment = 95%) showed at least one tumor-specific The status ctDNA and cCR correctly identified the mutation detected in their plasma (ctDNA posi- pCR in 14 out of 15 patients (93.33%), whereas tive) at baseline. there was one false-positive case (patient 7), one 4 journals.sagepub.com/home/tam N Ciriaco, E Zamora et al. Table 1. Baseline clinical and tumor characteristics and ctDNA status. Patient ID Tumor subtype Tissue mutation Plasma mutation 1 TN TP53 p.R273P TP53 p.R273P (351.8 MM; 58.6 MM/mL) 4 TN TP53 p.R110Pfs*39, TP53 p.R290Pfs*55, TP53 p.R110Pfs*39 (70.9 MM; 12.9 MM/mL), TP53 TP53 p.R175H and PIK3CA p.R310H p.R290Pfs*55 (NMD), TP53 p.R175H (11.2 MM; 2.0 MM/ mL) 5 HER2+ TP53 p.R290Pfs*55 TP53 p.R290Pfs*55 (259.5 MM; 89.9 MM/mL) 6 TN TP53 p.Q144* TP53 p.Q144* (90.9 MM; 15.1 MM/mL) 7 TN TP53 p.R110P TP53 p.R110P (1850.1 MM; 308.4 MM/mL) 9 TN TP53 p.R273L TP53 p.R273L (112.7 MM; 18.8 MM/mL) 10 HER2+ TP53 p.V173L and PIK3CA p.H1047R TP53 p.V173L (NMD) and PIK3CA p.H1047R (4.6 MM; 0.8 MM/mL) 11 TN TP53 p.R175H TP53 p.R175H (15.3 MM; 7.7 MM/mL) 12 HER2+ TP53 p.M237I and PIK3CA p.E542K TP53 p.M237I (13.7 MM; 6.9 MM/mL) and PIK3CA p.E542K (39.2 MM; 19.6 MM/mL) 14 TN TP53 p.C176F TP53 p.C176F (67.2 MM; 11.2 MM/mL) 17 TN TP53 p.N131Tfs*39 TP53 p.N131Tfs*39 (1168.5 MM; 194.8 MM/mL) 19 TN TP53 p.Y163* TP53 p.Y163* (58.5 MM; 9.7 MM/mL) 26 HER2+ TP53 p.D281V and PIK3CA p.H1047R TP53 p.D281V (39.4 MM; 8.8 MM/mL) and PIK3CA p.H1047R (47.7 MM; 10.6 MM/mL) 28 TN TP53 p.R273H TP53 p.R273H (31.7 MM; 8.1 MM/mL) 29 HER2+ TP53 p.K139_V143del TP53 p.K139_V143del (1.0 MM; 0.2 MM/mL) 30 HER2+ TP53 p.R213*, TP53 p.N131Tfs*39 and TP53 p.R213* (9.9 MM; 1.7 MM/mL), TP53 p.N131Tfs*39 PIK3CA p.H1047R (NMD) and PIK3CA p.H1047R (13.3 MM; 2.2 MM/mL) 31 TN TP53 p.S127F TP53 p.S127F (2157.4 MM; 365.7 MM/mL) 32 HER2+ NTRK1 p.Ser433Cys, AKT3 p.Leu262Val, NTRK1 p.Ser433Cys (NMD), AKT3 L262V (1.3 MM; NTRK3 p.Glu474Lys, TP53 p.Glu285Lys, 0.2 MM/mL), NTRK3 E474K (1.5 MM; 0.3 MM/mL), TP53 ERBB2 p.Ile989Met, GRM3 p.Asp97His E285K (2.7 MM; 0.5 MM/mL), GRM3 D97H (2.3 MM; and GRM3 p.Arg101Thr 0.4 MM/mL) and GRM3 D101T (2.3 MM; 0.4 MM/mL) 33 TN TP53 A74fs and FGF10 F146fs TP53 A74fs (12.2 MM; 3.4 MM/mL) and FGF10 F146fs (8.1 MM; 2.3 MM/mL) 34 HER2+* FANCF p.Leu241Arg and TP53 NMD c.375 + 1dupG *Patient ID 34 had 90% of estrogen receptor-positive cells. ctDNA, circulating tumor DNA; HER2+, human epidermal growth factor receptor 2 positive; MM, mutant molecules; NMD, no mutation detected; TN, triple negative. false-negative case (patient 32), and three true- Post-surgery ctDNA status and recurrence negative cases (patients 10, 26, and 31). This To assess whether any of the 15 patients achieving resulted in an accuracy of 89.5%, a false-positive cCR showed any detectable ctDNA during a sur- rate of 3.8%, and a NPV of 75.0%. veillance period following surgery, blood journals.sagepub.com/home/tam 5 Therapeutic Advances in Medical Oncology Volume 14 Table 2. Responses to NAC and results of the ctDNA analyses performed in the middle of and after the treatment. Patient ID cCR Mid-NAC plasma Pre-surgery plasma pCR 1 Yes NMD NMD Yes 4 Yes NMD NMD Yes 5 Yes TP53 p.R290Pfs*55 (201.9 MM; NMD Yes 33.6 MM/mL) 6 Yes NMD NMD Yes 7 No NMD TP53 p.R110P (1 MM; 0.2 MM/ Yes mL) 9 Yes NMD NMD Yes 10 No NMD NMD No 11 Yes TP53 p.R175H (43.7 MM; 7.3 MM/ NMD Yes mL) 12 Yes NMD NMD Yes 14 Yes NMD NMD Yes 17 Yes NMD NMD Yes 19 No NMD NMD No 26 No TP53 p.D281V (17.7 MM; 3.0 TP53 p.D281V (1.2 MM; 0.2 No MM/mL) and PIK3CA p.H1047R MM/mL) and PIK3CA p.H1047R (19.3 MM; 3.2 MM/mL) (0.6 MM; 0.1 MM/mL) 28 Yes NMD NMD Yes 29 Yes TP53 p.K139_V143del (0.6 MM; NMD Yes 0.1 MM/mL) 30 Yes NMD NMD Yes 31 No TP53 p.S127F (2.0 MM; 0.3 TP53 p.S127F (5.5 MM; 1.0 No MM/mL) MM/mL) 32 Yes NMD NMD No 33 Yes NMD NMD Yes 34 Yes NMD NMD Yes Patients that did not achieve cCR are highlighted in boldface type. cCR, clinical complete response; ctDNA, circulating tumor DNA; MM, mutant molecules; NAC, neoadjuvant chemotherapy; NMD, no mutation detected; pCR, pathologic complete response. collections from these patients were performed at detectable ctDNA at any point during the three time points after surgery (4 weeks, 6 months, 12-month follow-up period. In addition, no and 12 months) and ctDNA testing was performed ctDNA was detected after 24 months of follow-up using the same patient-specific tissue-informed in 13 out of 15 patients (two patients were not SafeSEQ assays used to monitor each patient dur- available for blood collection; data not shown) ing NAC. As shown in Table 4, in 40 samples col- achieving cCR, and none of these patients were lected from these 15 patients, none showed any found to have recurrent disease. Only two patients 6 journals.sagepub.com/home/tam N Ciriaco, E Zamora et al. Figure 2. Representative plots of longitudinal ctDNA tracking. Two patients (patients 5 and 17) showed complete clearance of TP53 mutations in plasma before surgery, which associated with cCR to NAC. cCR, clinical complete response; ctDNA, circulating tumor DNA; NAC, neoadjuvant chemotherapy. Table 3. Performance of the status of ctDNA and cCR ctDNA mutations detected during NAC. Both in predicting pCR after NAC. patients showed a significant increase in ctDNA, with patient 7 presenting the TP53 p.R110P Measure mutation at 397.7 MM/ml, and patient 10 pre- Accuracy 89.5% senting both TP53 p.V173L (8.4 MM; 1.5 MM/ ml) and PIK3CA p.H1047R (6.9 MM; 1.2 MM/ False-positive rate 3.8% ml) mutations. It was unfortunate that these patients were unavailable for ctDNA surveillance PPV 93.3% monitoring during the period shortly after surgery NPV 75.0% when positive detection of these mutations might have provided an early signal of recurrent disease. cCR, clinical complete response; ctDNA, circulating tumor DNA; NPV, negative predictive value; pCR, pathologic complete response; PPV, positive predictive value. Discussion The effectiveness of NAC treatment regimens for BC has improved during recent years and has experienced recurrent disease after longer clinical achieved high rates of pCR, particularly in HER2+ follow-up: patient 7 (time until recurrence, and TN tumors.24,25 These observations have 22 months), who did not achieve cCR and had a prompted the notion that patients that achieve ctDNA-positive result after NAC, and patient 10 cCR following NAC might be spared breast sur- (time until recurrence, 27 months), who did not gery, as forgoing surgery would potentially reduce achieve neither cCR nor pCR, but had NMD after surgical complications, improve quality of life, NAC. Unfortunately, these two patients were not and decrease healthcare costs. This idea of de- available for any follow-up blood collection to escalation of breast surgery in BC patients with an undergo ctDNA testing during the 12-month fol- apparent complete response to NAC is gaining low-up period after surgery. However, blood sam- recognition as an alternative approach to patient ples were obtained in patients 7 and 10 after management. However, there are current chal- recurrence, and ctDNA testing revealed the same lenges with accurately determining the status of journals.sagepub.com/home/tam 7 Therapeutic Advances in Medical Oncology Volume 14 Table 4. Surveillance ctDNA testing results for the 15 BC patients achieving cCR after NAC*. Patient ID 10 weeks after surgery 6 months after surgery 12 months after surgery 1 NMD NMD NMD 4 NMD NMD NMD 5 NMD NMD NMD 6 NMD NMD NMD 9 NMD NMD NMD 11 NMD NMD NMD 12 NMD NMD NMD 14 NMD NA NMD 17 NMD NA NA 28 NMD NMD NMD 29 NMD NMD NMD 30 NMD NMD NMD 32 NMD NMD NMD 33 NMD NMD NA 34 NMD NMD NA *Allbut one patient (patient 32) also achieved pCR. BC, breast cancer; cCR, clinical complete response; ctDNA, circulating tumor DNA; MM, mutant molecules; NA, patient sample not available; NAC, neoadjuvant chemotherapy; NMD, no mutation detected; pCR, pathologic complete response. residual disease at the end of neoadjuvant treat- period, and furthermore did not present resur- ment using the current clinico-radiological evalu- gence of tumor mutation in plasma. These obser- ations or image-guided biopsy techniques.8,9,26 vations are in accord with the conclusion that, in Therefore, surgery after NAC is still considered this patient, residual disease was minimal and did the standard of care for primary BC, either to not spread to other organs before the surgery or completely remove residual disease in non-pCR disease was eliminated during NAC. With longer cases or to confirm a pCR. follow-up, an eventual reappearance of the patient’s specific mutation in plasma ctDNA may In this pilot study, we identified certain HER2+ indicate microscopic occult disease before its and TN BC patients showing clearance of ctDNA detection/visualization by imaging techniques.27 mutations as a potential correlate of complete response to NAC. In fact, 14 out of 15 patients In the future, if liquid biopsy negative results were meeting the criteria of absence of plasma muta- considered as a correlate of pCR in the decision- tions plus cCR based on clinical examination and making process, patients may not undergo sur- imaging assessment had a proven pCR according gery but instead would undergo an intensive to the histopathological evaluation of the surgical follow-up (frequent imaging and/or liquid biopsy specimen (PPV = 93.3%). This observation leads surveillance). In case of minimal residual disease, to the tenable conclusion that these patients could this might be addressed by radiotherapy and/or have safely avoided breast surgery after NAC. adjuvant treatment according to the standard Moreover, the patient that achieved cCR, but did practice. By contrast, patients that do not achieve not achieve a pCR (patient 32) did not showed cCR after NAC (five in our study; see patients ID detectable ctDNA after NAC and did not experi- in Table 3) should undergo breast surgery regard- ence disease recurrence during the follow-up less of the presence or absence of plasma 8 journals.sagepub.com/home/tam N Ciriaco, E Zamora et al. mutations detected. Moreover, three of five Accordingly, the overall percent agreement patients not achieving cCR had plasma mutations between tissue and plasma mutations in this study detected prior to surgery, which has been associ- was 95%, much higher than the observed with ated with poorer prognosis,28,29 and, in fact, two other techniques,31,32 even when low levels of on these patients (ID 7 and ID 10) relapsed. ctDNA are usually not detectable in early dis- Regrettably, both of these patients were not avail- ease.33 Another explanation for the presence of low able for ctDNA testing during the 4-week, levels of (clinically irrelevant) mutant molecules 6-month, and 12-month follow-up period post- could be the occurrence of clonal hematopoiesis of surgery, and both patients showed markedly indeterminate potential, which has been reported increase ctDNA levels at 24 months after recur- to be frequent for the TP53 gene.34,35 rence. Although patient 10 showed clearance of both TP53 and PIK3CA mutations in ctDNA The results of this pilot study should be inter- from baseline, the same mutations re-appeared preted within the context of its strengths and limi- after recurrence at 24 months. Similarly, patient 7 tations. An important limitation of this study is its had NMD at mid-NAC but again the baseline small sample size, although this patient cohort is mutation in the TP53 gene was present at the end fairly representative of a typical neoadjuvant pop- of the treatment. These observations raise the ulation. A second limitation of this study is that possibility that these mutations may indicate the post-surgery follow-up time was restricted to underlying resistance mechanisms of the tumor.30 12 months, so it is not possible to rule out any other potential recurrence in the future, as the The detection of ctDNA in plasma regardless of recurrence window of HER2+ and TN BC is achieving cCR and pCR in five cases prompts the estimated to be 5 years, or even more in the case need to rigorously establish a clinically relevant of luminal tumors.36 Given these limitations (only level of ctDNA that triggers heightened suspicion two patients had recurred at the writing of this of molecular residual disease (MRD). Moreover, manuscript), ctDNA levels could not be assessed clinical ctDNA cutoffs that comprise qualitative as a prognostic factor for recurrence. In addition, judgments of ‘MRD positive’ versus ‘MRD nega- we were unable to perform post-surgery surveil- tive’ are likely to require more sophisticated algo- lance blood testing of all patients and at all of the rithms to be developed that integrate both scheduled time points during the follow-up. patient-specific mutations, as well as sample-level tumor mutational burden to gain greater certain- In summary, the SafeSEQ NGS technology iden- ties of an individual patient’s risk of recurrence/ tified 19/20 (95%) of BC patients with detectable relapse or cure in response treatment. In this study, mutations in plasma at baseline, enabling the vast we have provided a framework of longitudinal test- majority of patients to be tracked via liquid biopsy ing in a small cohort of BC patients to begin estab- during NAC. Clearance of ctDNA positivity and lishing a clinically relevant cutoff using SafeSEQ achieving cCR after NAC had favorable correla- technology. Further trials with greater numbers of tion with effective pathological response. patients will be required to ascertain both the fre- Moreover, the presence of clearly detectable quency of testing, qualitative result, and quantities ctDNA levels is likely to be associated with the of ctDNA that will serve of a useful adjunct to lack of clinical/pathological complete response in determine both disease clearance and risk of dis- BC patients. These findings prompt further stud- ease recurrence. In managing the patient using ies with larger patient cohorts to investigate the ctDNA results, it will be critical to establish both added value of ctDNA to predict pCR, aiding in durability of neoadjuvant response by determining determining which patients might be safely spared consistency of negative ctDNA test results as well surgery after NAC. If this strategy is confirmed to as a higher risk of recurrence triggered by consecu- be clinically useful, an approach of ‘watch and tive positive and ascending values of ctDNA in wait’ (including serial plasma testing) could be plasma. Here, the serial sampling after the surgery implemented in selected minimal-risk patients. was reassuring, as none of these five patients expe- rienced elevations of ctDNA levels during the fol- low-up. Interpretation of the results at exceedingly Declarations low levels of ctDNA of detection should be done with caution, and bearing in mind that SafeSEQ is Ethics approval and consent to participate an ultra-sensitive technique able to detect as few as The study protocol was approved by the medical three mutants in 20,000 total DNA molecules22 ethical committee of the Hospital Universitario journals.sagepub.com/home/tam 9 Therapeutic Advances in Medical Oncology Volume 14 Vall d’Hebron on 30/09/2016 (ID PR(AG) Acknowledgements 204/2016). All patients provided written informed The authors thank all the patients and their fami- consent. lies who participated in this study. They thank Anabel Herrero, PhD, on behalf of Springer Consent for publication Healthcare Communications, for providing med- All authors have read the manuscript and ical writing support for this manuscript. This approved its submission to Therapeutic Advances medical writing assistance was funded by Sysmex in Medical Oncology. Inostics, Inc. Author contribution(s) Funding Nikaoly Ciriaco: Conceptualization; Data cura- The authors disclosed receipt of the following tion, Formal analysis; Writing – original draft; financial support for the research, authorship, Writing – review & editing. and/or publication of this article: This study was supported by a grant from Sysmex Inostics, Inc. Esther Zamora: Conceptualization; Writing – The sponsor coordinated data collection from original draft; Writing – review & editing. study centers, and funded the statistical analysis Santiago Escrivá-de-Romaní: Conceptu­ and medical writing assistance. alization; Writing – original draft; Writing – review & editing. Competing interests E.Z. has received fees as consultant, participated Ignacio Miranda Gómez: Data curation; in advisory boards or received travel grants from Writing – original draft; Writing – review & Roche/Genentech, Eisai Europe, and Daiichi editing. Sankyo/AstraZeneca; S.E.R has received fees as José Jiménez Flores: Data curation. consultant, participated in advisory boards or had an investigator role from Daiichi Sankyo/ Cristina Saura: Conceptualization; Writing – AstraZeneca, Pfizer, Roche, Seagen, Byondis, original draft; Writing – review & editing. Lilly, MedSIR, and Synthon; C.S has served as Hillary Sloane: Formal analysis; Methodology; consultant, participated in advisory boards or Writing – original draft. received travel grants from Byondis, AstraZeneca, Daiichi Sankyo, Eisai, Exact Sciences, Exeter Anna Starus: Formal analysis; Methodology; Pharma, F. Hoffmann – La Roche Ltd, MediTech, Writing – original draft. Merck Sharp & Dohme, Novartis, Pfizer, Philips, Johannes Fredebohm: Formal analysis; Pierre Fabre, Puma biotechnology, Sanofi- Methodology; Writing – review & editing. Aventis, SeaGen and Zymeworks; V.P. has received fees as consultant, participated in advi- Lucy Georgieva: Formal analysis; Methodology; sory boards or received travel grants from Sysmex, Writing – original draft. Roche, Merck Sharp & Dohme, AstraZeneca, Graham Speight: Formal analysis; Metho­ Bayer and Exact Sciences. dology; Writing – original draft; Writing – review The remaining authors declared no potential con- & editing. flicts of interest with respect to the research, Frederick Jones: Conceptualization; Metho­ authorship, and/or publication of this article. dology; Writing – original draft; Writing – review & editing. Availability of data and materials Data supporting the results presented in this Santiago Ramón y Cajal: Writing – original study are available from the corresponding author draft; Writing – review & editing. upon reasonable request. Martín Espinosa-Bravo: Conceptualization; Data curation; Writing – original draft; Writing – ORCID iDs review & editing. 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