Prognostic utility of inflammatory indices and circulating nucleic acids for neoadjuvant chemotherapy outcomes in breast cancer: a systematic review

Abstract

Background:

Neoadjuvant chemotherapy (NAC) plays a central role in the management of early breast cancer (BC), offering prognostic information and improving surgical outcomes. Blood-based biomarkers, including inflammatory markers—such as neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR)—and circulating nucleic acids—such as circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), and cfDNA integrity index (cfDI)—have been investigated for their potential to predict treatment response.

Objectives:

To systematically evaluate the prognostic value of inflammatory indices and circulating nucleic acids measured during NAC for predicting pathological complete response (pCR) in early BC.

Design:

Systematic review with qualitative synthesis.

Data sources and methods:

Twenty-four studies were included, examining changes in NLR, PLR, ctDNA, cfDNA, and cfDI during NAC. Study quality was assessed using the Newcastle–Ottawa Scale, and the strength of evidence was evaluated with a qualitative GRADE approach. Due to heterogeneity in biomarker definitions, sampling timepoints, and statistical methods, data were synthesized narratively.

Results:

A reduction in PLR from baseline to mid-NAC (ΔPLR <0) was consistently associated with higher pCR rates. ctDNA clearance, particularly at mid- and post-NAC, was frequently linked to increased pCR and better overall survival. In contrast, findings on NLR dynamics were inconsistent. Evidence for cfDNA and cfDI was limited and mixed, with some studies suggesting lower cfDNA and higher cfDI may be associated with improved outcomes. Variability in biomarker thresholds and timepoints was a common limitation.

Conclusion:

Mid-NAC PLR decrease and ctDNA clearance show moderate evidence as predictors of pCR and may help guide treatment decisions. The prognostic value of NLR, cfDNA, and cfDI remains uncertain and requires further prospective, standardized investigation.

Keywords

biomarkers breast cancer cell-free DNA (cfDNA)chemotherapy circulating tumor DNA (ctDNA)neoadjuvant chemotherapy NLR PLR

Introduction

Neoadjuvant chemotherapy (NAC) plays a central role in the management of breast cancer (BC). Although indication criteria for NAC vary according to immunohistochemistry classification, general benefits include improved surgical outcomes, an increased rate of breast conserving surgery, an early assessment of the malignancy responsiveness, and an increased rate of pathological complete response (pCR) achievement.^1,2 pCR is defined as the absence of cancer cells in the breast and axilla following treatment with chemotherapy.³ Numerous studies have shown that achievement of pCR correlates with better prognosis and survival outcomes in TNBC and HER2+ enriched BC.^4–6 Achieving pCR is the primary goal of neoadjuvant therapy, as it guides surgical planning and informs decisions regarding adjuvant treatment. For these reasons, accurately predicting pCR is paramount.

In recent years, several plasma biomarkers have emerged as potential predictors for outcomes in cancer care. Compared to tissue-based markers, plasma-based markers offer noninvasive and potentially cost-effective methods for prognosticating disease outcomes.^7,8 Among these, inflammatory markers and circulating nucleic acids have gained attention for predicting pCR in BC.

Novel inflammatory markers, such as the neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) can be obtained from complete blood counts and reflect inflammatory status, as well as host immune response and tumor microenvironment activity.^9,10 Elevated NLR and PLRs have been associated with a poor prognosis in several malignancies, including BC.^11,12 Recent studies have also explored NLR and PLR ability to predict the achievement of pCR in patients undergoing NAC, although findings vary.¹³

Circulating nucleic acids, particularly cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) allow for noninvasive cancer genetic and epigenetic assessment from dying cells.¹⁴ Detection of cfDNA is a promising modality for the diagnosis of various cancers. Moreover, these biomarkers have the potential to detect minimal residual disease and monitor response to cancer therapies. Evidence suggests that clearance of ctDNA during NAC in BC has shown promise in predicting pCR, especially in high-risk patients.¹⁵ Dynamic monitoring for actionable mutations may also help predict responses to targeted therapies, thereby aiding in treatment selection for advanced BC.¹⁶

While NLR, PLR, ctDNA, and cfDNA have been extensively studied at baseline and upon completion of NAC, few studies investigate their dynamics during treatment. Measuring these biomarkers early in the course of NAC, such as after the initial chemotherapy cycles, may offer more accurate predictions of treatment response. This could enable earlier identification of patients unlikely to achieve pCR, allowing for timely adjustments to the chemotherapy regimen and potentially sparing unnecessary toxicity. In this systematic review, we aim to evaluate the predictive value of these biomarkers during NAC for pCR in BC.

Methods

Search strategy

A systematic search of articles published up to May 15, 2025 was carried out in three databases: PubMed, Scopus, and Embase. The search strategy encompassed names of inflammatory markers and other keyterms as follows: (“neoadjuvant chemotherapy” OR “NAC” OR “neoadjuvancy”) AND (“pathological complete response” OR “pCR”) AND (“neutrophil-to-lymphocyte ratio” OR “NLR” OR “platelet-to-lymphocyte ratio” OR “PLR” OR “circulating tumor DNA” OR “ctDNA” OR “cDNA” OR “cell-free DNA” OR “cfDNA” OR “Pan-immune inflammation value” OR “PIV” OR “systemic inflammation response index” OR “SIRI”) AND (“breast cancer”). In each database, a list was downloaded containing the results from the search. Each list was imported into Rayyan software (Qatar Foundation). Duplicates were then removed, and two investigators assessed the resulting list of studies, screening for eligible articles to be included, under the “Blind” mode. At the end of this process, a third investigator turned the “Blind” mode off and analyzed the conflicting decisions, resolving these. This study followed the PRISMA guidelines for systematic reviews.¹⁷

PICO strategy

Population, Intervention, Comparison, Outcome (PICO) strategy was as follows: (1) Population: patients with any type of BC who underwent NAC; (2) Intervention: assessment of high values of NLR, PLR, systemic inflammation response index (SIRI), or pan-immune inflammation value (PIV) during NAC or assessment of high dynamics of these inflammatory markers during NAC or assessment of low clearance of cfDNA during NAC or assessment of unfavorable dynamics of ctDNA mutations during NAC; (3) control: assessment of low values of NLR, PLR, SIRI or PIV during NAC or assessment of low dynamics of these inflammatory markers during NAC or assessment of high clearance of cfDNA during NAC or assessment of favorable dynamics of ctDNA mutations during NAC; (4) Outcome: achievement of pCR.

Selection criteria

The following inclusion criteria were used: (1) population of any type of BC receiving NAC; (2) analysis of one of the selected inflammatory markers during NAC in relation to pCR or analysis of circulating nucleic acid during NAC in relation to pCR; (3) studies written in English; (4) manuscripts and conference abstracts. The following exclusion criteria were used: (1) studies without assessment of biomarkers during NAC; (2) studies without original data; (3) if multiple studies investigated the same population, the one providing fewer details was excluded; (4) case reports, reviews, expert opinions, and studies involving animals.

Data extraction

The following data were extracted by two independent investigators: first author, year of publication, country of origin, type of disease, chemotherapy regimen, assessed biomarkers, phase of NAC when biomarkers were assessed, number of patients, prospective or retrospective nature, single institution or multicentric fashion, outcomes, follow-up time.

Quality evaluation

The Newcastle–Ottawa Scale (NOS) was employed for quality evaluation. Two independent investigators assigned scores in the various domains. Scores ⩾7 were considered to represent high-quality studies and low risk of bias. Discrepancies of the assigned scores were resolved by a third investigator. We also performed a qualitative GRADE assessment to evaluate the overall strength of evidence for each biomarker.

Results

Selection methods

The preliminary search of databases yielded 544 articles. Following the deduplication process, 252 articles were excluded, and the remaining 292 were examined based on titles and abstracts. This assessment resulted in the eligibility of 35 publications, including full-text articles and conference abstracts. Eventually, after considerations and exclusion of studies with overlapping populations, 23 studies were included for this systematic review. One additional study not captured by the search strategy was included after a thorough consideration, totaling 24 included studies.^18–41 The details of the selection process are depicted in Figure 1. Figure 2 illustrates the time points at which blood samples were collected for biomarker analysis across the included studies, as well as the key findings.

Figure 1.

PRISMA flowchart outlining the selection process.

Figure 2.

Schematic illustration of various time points of biomarkers drawn during NAC.

Characteristics of included studies

The collection of included studies comprises a total of 1867 early BC patients who underwent NAC and had outcomes assessed based on biomarkers collected during NAC. Nine out of the 24 studies were conducted in China, 5 in the United States, 2 in Korea, 1 in France, 1 in Greece, 1 in Spain, 1 in Italy, 1 in Brazil, and 3 in multiple countries. Fifteen studies were conducted in single institutions, seven were multicenter, and two studies do not clarify whether a single institution or multicenter approach was used. Two of the studies used the same population: one to assess the prognostic impact of ctDNA and the second to assess the prognostic impact of cfDNA. These studies were named Magbanua (I) et al.²² and Magbanua (II) et al.,²³ respectively. Two studies conducted in China investigated serial ctDNA in 32 and 193 early BC patients. They were named Zhou (I) et al.¹⁹ and Zhou (II) et al.³⁵ The complete study characteristics are described in Table 1.

Table 1.

Included studies.

Author	Year	Country	Type of disease	Chemotherapy regimen	Biomarkers	Time point of biomarker collection	Outcome	Results	Number of patients	Study design (prospective or retrospective)	Institution type (single institution or multicentric)	Follow-up time	pCR definition
Gao et al.¹⁸	2023	China	Invasive breast cancer	Six to eight cycles of NAC, consisting either of four cycles of anthracyclines plus cyclophosphamide followed by four cycles of taxanes, or six cycles of a combination regimen with docetaxel, anthracyclines, and cyclophosphamide	NLR	At every second cycle of NAC (i.e., the second, fourth, sixth, and eighth cycles)	OS, pCR	Patients without a pathological complete response (non-pCR) exhibited a gradual rise in NLR during NAC, whereas those achieving pCR maintained stable levels. On average, non-pCR patients showed a +0.032 increase in log-transformed NLR every two cycles, a statistically significant difference (p = 0.024)	176	Retrospective	Single institution	43.2 months	ypT0/ ypN0
Zhou et al.¹⁹	2021	China	Locally invasive BC	n/a	ctDNA	NR (during chemotherapy)	DFS (primary endpoint), but only pCR for peri-neoadjuvant ctDNA analysis (secondary endpoint)	ctDNA was undetectable during and after chemotherapy in 21 patients (67.7%), including four who achieved pCR. Among the 22 patients with baseline ctDNA detection, its persistence during treatment was linked to poorer pathological response (Miller-Payne grade, p = 0.0244)	Thirty-two patients were enrolled, but only 30 had sufficient blood-derived DNA at baseline, during, and after chemotherapy for analysis	Prospective	Single institution	2 years	n/a
Chen et al.³⁰	2024	USA	TNBC	Standard anthracycline-based NAC for four cycles	ctDNA, CTC	Twelve weeks after starting NAC (mid-treatment, post four cycles)	pCR, OS, RFS	ctDNA clearance at mid-NAC was significantly linked to pCR (p = 0.02); 58% of patients who cleared ctDNA achieved pCR, whereas none with detectable mid-NAC ctDNA (n = 6) did. While baseline and mid-treatment ctDNA levels alone did not predict pCR, clearance at mid-NAC remained a significant marker among those initially ctDNA-positive (p = 0.02). Furthermore, mid-NAC ctDNA positivity was strongly associated with poorer long-term outcomes. Five-year OS was 33.3% in ctDNA-positive vs 77.4% in ctDNA-negative patients (p = 0.0002), and recurrence-free survival was 33.3% vs 85.7%, respectively (p = 0.0034). Baseline ctDNA status was not associated with differences in survival outcomes	37	Prospective	Single institution	4 years	No evidence of invasive tumor in the breast or axillary lymph nodes in the surgical specimens
Zhou et al.³⁵	2022	Austria, Sweden	Early BC HR+, HER−, TNBC	Anthracycline/cyclophosphamide followed by taxane	ctDNA	Following four chemotherapy cycles and 12 weeks of aromatase inhibitor therapy	pCR, RCB	At mid-NAC, ctDNA was detectable in 25 of 63 patients (39.7%) and was significantly associated with higher RCB, reflecting a poorer response. Among the 31 patients with detectable ctDNA at this timepoint, 96.8% did not achieve pCR, with only one patient reaching pCR	193	Prospective randomized Phase 2 trial	Multicentric	5 years	ypT0/is ypN0
Kim et al.³⁶	2017	South Korea	Locally invasive BC	Adrimycin/cyclophosphamide combination chemotherapy and taxane	ctDNA	After first cycle of chemotherapy	pCR	In two of the three patients who achieved pCR, ctDNA became undetectable after just one NAC cycle. Of the 15 patients in total, 3 achieved pCR while 12 had residual disease. Notably, none of the patients with persistent ctDNA after the first cycle achieved pCR, highlighting its prognostic significance	12	Prospective	Single institution	3 years	Absence of residual tumor cells after NAC
Riva et al.³¹	2017	France	Non-metastatic TNBC	Anthracycline/taxane-based NAC	ctDNA, TP53 mutation	Prior to the second chemotherapy cycle, approximately 2–3 weeks after the first	pCR, DFS, OS	pCR was achieved in 42% of patients, with no significant association between pCR and ctDNA detection at baseline or any subsequent timepoint. Similarly, clinical response showed no correlation with ctDNA levels or their changes over time. However, ctDNA positivity after one NAC cycle was significantly linked to poorer outcomes, including shorter DFS (p < 0.001), shorter OS (p = 0.006), and increased risk of metastatic relapse (p = 0.002). Baseline ctDNA status did not correlate with survival or recurrence	46, 40 TP53 mutated tumors analyzed	Prospective	Single institution	36 months	ypT0/isN0
Dan et al.³⁷	2023	China	Invasive ductal breast carcinoma	Taxanes-, anthracycline-, cyclophosphamide-based regimens	PLR	Starting after two NAC cycles and then every two cycles, up to a total of four	pCR	Of 257 patients, 75 (29.1%) achieved pCR after NAC. A significantly higher pCR rate was observed in those with a decrease in PLR (ΔPLR <0) compared to those with stable or increased PLR (ΔPLR ⩾0)	257	Retrospective	Single institution	5 years	Lack of invasive tumor in the breast and nodes
Cavallone et al.³⁸	2020	Canada, United States	TNBC	Patients received standard neoadjuvant chemotherapy with either anthracycline/taxane-based or taxane-only regimens. Treatments included variations of AC followed by weekly paclitaxel, taxane-first followed by AC, taxane monotherapy, or combined ACTax regimens such as AC plus docetaxel or carboplatin/paclitaxel	ctDNA	Following one NAC cycle, at mid-treatment (between anthracycline and taxane phases), and after one cycle of the second regimen	RFS, OS, pCR	ctDNA detection was linked to poorer RFS (p = 0.046) and OS (p = 0.043). At the first NAC cycle (T1), ctDNA was detected in 17 of 24 patients (71%), but this early detection was not significantly associated with RFS (p = 0.071) or OS (p = 0.13)	26	Prospective	Multicentric	63 months post-diagnosis, 55 months post-surgery	n/a
Cirmena et al.³⁹	2022	Italy	Invasive BC	NAC based on anthracyclines and taxanes (epirubicin followed by 12 cycles of paclitaxel). For HER2-positive cases, trastuzumab was given concurrently with paclitaxe	cfDI(cfDI)	After completing the epirubicin-based NAC cycles, just before the first paclitaxel dose (T1)	pCR	No significant difference in cfDNA fragment concentrations was observed between pCR and non-pCR patients at baseline (T0) or after anthracycline treatment (T1)	51	Prospective	Single institution	6 years	ypT0 ypN0 or ypT0/is ypN0
Giro et al.⁴⁰	2024	Brazil	Localized and/or locally advanced BC	n/a	cfDNA, cfDI	Every 2 weeks during NAC before surgical ressection	DFS, pCR	cfDNA integrity levels did not significantly change between pre-NAC and post-first NAC cycle (p = 0.7904). However, higher integrity levels after treatment initiation were significantly associated with pCR (p = 0.045) and longer DFS (p = 0.0371)	Thirty-eight patients were included in the intention-to-treat analysis, of whom 27 completed the full neoadjuvant protocol	Prospective	Single institution	4 years	n/a
Wang et al.⁴¹	2019	China	Locally advanced BC (invasive ductal carcinoma)	Treatment regimens included combinations such as docetaxel with epirubicin and cyclophosphamide; epirubicin plus cyclophosphamide followed by biweekly paclitaxel; epirubicin and cyclophosphamide followed by paclitaxel with trastuzumab; fluorouracil combined with epirubicin and cyclophosphamide; and a regimen of docetaxel, carboplatin, and trastuzumab	cfDNA, cfDI from two repetitive elements (Alu and LINE1)	Following the second cycle in four-cycle NAC regimens, or the fourth cycle in eight-cycle regimens	pCR, tumor recurrence	Patients who achieved pCR showed a progressive rise in cfDI levels throughout NAC	29	Prospective	Single institution	6 months	n/a
Fiste et al.²⁹	2024	Greece	Non-metastatic BC	Standard cytotoxic chemotherapy regimens, administered alone or in combination with immunotherapy for TNBC or anti-HER2 agents for HER2-positive disease, as appropriate	NLR and PLR	NLR and PLR were assessed at mid-NAC, with Δ1NLR and Δ1PLR representing changes from baseline to mid-treatment, and Δ2NLR and Δ2PLR indicating changes from baseline to post-treatment	pCR,	Δ1NLR was not significantly linked to pCR (p = 0.710), whereas an increase in Δ1PLR was significantly associated with reduced pCR likelihood (p = 0.042)	107	Retrospective	Multicentric	6 years	n/a
Du et al.²⁸	2024	China	Invasive ductal carcinoma, invasive lobular carcinoma, and other types	Patients received one of three NACT regimens: (1) doxorubicin plus cyclophosphamide followed by docetaxel; (2) docetaxel with carboplatin; or (3) docetaxel alone, all given every 3 weeks. HER2-positive patients also received trastuzumab concurrently	ctDNA	Baseline, after two cycles of NAC, and at the completion of NAC	pCR (primary endpoint), DFS	Patients who achieved pCR exhibited a significantly greater decline in ctDNA levels during NAC compared to non-pCR patients (p < 0.001)	231	Retrospective	Single institution	Follow-up ranged from 14 to 88 months, with a mean of 40 months and a median of 34 months	No histological evidence of malignancy in the regional lymph nodes of primary or metastatic BC
Butler et al.²⁷	2019	USA	Locally advanced BC	Regimens varied according to ER/HER2 status and ISPY2-TRIAL participation. NAC typically included sequential paclitaxel followed by AC. Some patients also received targeted agents like pertuzumab, TDM1, ganitumab, trastuzumab, ganetespib, or trebananib as part of their treatment.	ctDNA and cfDNA	Before initiating NAC, before each NAC infusion, and during follow-up ranging from 350 to 1150 days after NAC initiation	pCR	Patients who achieved pCR showed rapid ctDNA decline during NAC, while non-pCR patients frequently had persistent ctDNA. ctDNA became undetectable during treatment in those who responded, either with pCR or tumor shrinkage	10	Prospective	Single institution	1–3 years	Total absence of detectable disease after neoadjuvant therapy
Park et al.²⁶	2018	Korea	TNBC	Patients received four cycles of Adriamycin and cyclophosphamide, followed by four cycles of either cisplatin or docetaxel, prior to surgery	Circulating cf DNA and TILs	At baseline (before chemotherapy) and after four cycles of AC-CFD	Correlation between cfDNA levels and pCR; EFS	cfDNA levels significantly decreased from baseline (239 ± 68 ng/mL) to post-NAC (210 ± 66 ng/mL, p = 0.001). Baseline cfDNA levels >264 ng/mL were associated with a higher relapse risk (p = 0.029). However, no significant differences in baseline, mid-NAC, or changes in cfDNA levels were found between pCR and non-pCR patients	72	Prospective	Single institution	33.6 median f/u time	n/a
Chung et al.²⁵	2022	Taiwan	Stage II and III primary, operable TNBC	Patients received four cycles of anthracycline plus cyclophosphamide, followed by four cycles of taxane with platinum, each administered every 3 weeks	NLR PLR Percentage change in PLR	NLR and PLR were measured before and after the first NAC cycle to assess changes, with the nomogram using pre-treatment values. PLR percentage change was based on these two time points	pCR	A decrease in PLR after the first NAC cycle was significantly associated with higher pCR rates in multivariate analysis (p = 0.02), while NLR change was not a significant predictor. A nomogram incorporating five significant variables, including PLR change, was developed to predict pCR	88	Retrospective	Single institution	n/a	Absence of invasive disease in the breast or axillary lymph nodes
Ciriaco et al.²¹	2022	Spain	stage I/III HER2+ or TN infiltrating breast carcinoma	NAC followed institutional protocols, primarily using anthracyclines and taxanes. Platinum was added for some TNBC cases, and HER2-positive patients received targeted anti-HER2 therapies	ctDNA	Assessments were conducted at baseline (pre-NAC), mid-NAC, post-NAC (pre-surgery), and postoperatively at 10 weeks, 6 months, and 12 months	pCR	Among patients with cCR after NAC, NMD ctDNA status accurately identified pCR in 14 of 15 cases (93.3%), with an overall predictive accuracy of 89.5%. Over 12 months of follow-up, none of the 15 NMD patients had detectable ctDNA in 40 plasma samples, and no recurrences were reported	20	Prospective	Single institution	12 months follow-up after surgery for ctDNA monitoring	Lack of invasive residual disease in the breast and axillary lymph nodes following NAC
Dong et al.²⁰	2025	China	Stage II/III resectable BC	NAC regimens included EC-T, EC-TH, TEC, AC-TH, and EC-TCb, combining epirubicin or doxorubicin with cyclophosphamide, followed by paclitaxel—with additions like trastuzumab or capecitabine depending on the regimen	ctDNA	At three timepoints: before NAC (P1), during NAC after the first cycle (P2), and post-NAC before surgery (P3)	pCR	Undetectable ctDNA at mid-NAC (P2) and post-treatment (P3) was strongly linked to better treatment response and survival. ctDNA clearance predicted pCR with 80% sensitivity at P2 and 90% at P3	Seventy-three patients were included, with ctDNA data available for 63 at P1, 42 at P2, and 40 at P3	Retrospective	Single institution	The median follow-up was 58.2 months, with an interquartile range of 55.2–85.9 months	YpT0/is ypN0
Liu et al.³²	2023	China	Operable stage II and III BC	Patients received anthracycline- and taxane-based NAC. HER2-positive cases were treated with trastuzumab or dual HER2 blockade (trastuzumab plus pertuzumab). Regimens included EC, EC-T(H/HP), T(H)-EC, TEC, T(HP), TC(H), and TCbH(P), combining anthracyclines, taxanes, cyclophosphamide, and/or platinum with targeted HER2 therapy	ctDNA	ctDNA blood samples were collected at four points: before starting chemotherapy (T0), after the first and before the second NAC cycle (T1), at mid-treatment evaluation (T2), and post-NAC before surgery (T3)	pCR, DFS	All patients who achieved pCR had undetectable ctDNA at both T2 and T3, with no disease progression, whereas persistent ctDNA at these points was linked to non-pCR, underscoring the predictive value of serial ctDNA monitoring. Patients who became ctDNA-negative at T1, T2, or T3 had recurrence and metastasis risks similar to those negative at baseline (T0), indicating late clearance still provides clinical benefit. Notably, ctDNA clearance by T1–T3 was associated with longer DFS, and clearance at T3 correlated with improved survival, supporting its role as a prognostic biomarker	A total of 269 patients were prospectively enrolled, with 246 deemed eligible. Tumor DNA sequencing was conducted on biopsies from 243 patients. ctDNA analysis was performed on 216 blood samples from 56 patients, 50 of whom completed all four time points	Prospective	Multicentric	The median follow-up for survival analysis was 898 days, ranging from 97 to 1765 days	yp-T0/is, ypN0
Rothe et al.²⁴	2019	Belgium, Australia, UK, Switzerland, Italy, Brazil, Canada, Japan, Germany, USA	HER2-overexpressed and/or amplified primary BC	Anti-HER2 agents (lapatinib and/or trastuzumab) were administered alone for 6 weeks, followed by 12 weeks of combined therapy with paclitaxel	ctDNA and TILs	Plasma for ctDNA analysis was collected at baseline (pre-NAC), at week 2 of NAC, and preoperatively	pCR, EFS	ctDNA was detected in 41% of patients at baseline, dropping to 20% by week 2 of NAC and 5% before surgery, showing a trend toward clearance during treatment. The highest pCR rates were seen in HER2-enriched tumors with undetectable baseline ctDNA, while persistent ctDNA at baseline and week 2 was linked to the lowest pCR rates. However, ctDNA detection at week 2 or pre-surgery was not significantly associated with pCR, likely due to small sample sizes at those timepoints	Sixty-nine patients with evaluable baseline ctDNA results were included in this analysis, selected from the broader NeoALTTO trial cohort of 455 patients	Retrospective	Multicentric	Median follow-up of 6.64 years	Lack of tumor cells in the breast and lymph nodes
Magbanua (I) et al.²²	2021	USA	High-risk early BC (stage II/III)	Patients received paclitaxel followed by anthracyclines, with standard NAC either alone or combined with the AKT inhibitor MK-2206 as part of the I-SPY 2 TRIAL	ctDNA	Samples were collected pre-treatment, at 3 weeks after starting paclitaxel (T1), at 12 weeks between paclitaxel and anthracycline phases (T2), and post-NAC before surgery (T3)	pCR	ctDNA positivity declined throughout NAC—from 73% at baseline (T0) to 35% at T1 (3 weeks after starting paclitaxel), 14% at T2 (between paclitaxel and anthracycline phases), and 9% post-NAC (T3). At T1, residual disease was present in 83% of patients with persistent ctDNA vs 52% in those who cleared it, a significant difference (p = 0.012). ctDNA positivity at T1 and T2 was also significantly associated with increased risk of metastatic recurrence	84	Retrospective analysis	Multicentric	Median follow-up of 4.8 years	Lack of residual cancer in the breast and lymph nodes after NAC
Ma et al.³³	2019	China	Locally advanced BC	EC4-T4 regimen NCT	CTCs and cfDNA	At biopsy, after the first NAC cycle, and following the final NAC cycle	pCR	After the first NAC cycle, high-response patients (those achieving pCR) had significantly higher cfDI than low-response patients. Total cfDNA levels across all timepoints showed no correlation with treatment outcomes	45	n/a	n/a	n/a	n/a
Lucci et al.³⁴	2023	USA	TNBC	n/a	ctDNA	At four time points: pre-NAC, mid-NAC (12 weeks after starting), post-NAC, and post-surgery	pCR	ctDNA was detected in 90% (27/30) of patients at diagnosis, and by mid-NAC, 76% (19/25) of those initially ctDNA-positive had achieved early clearance. This early ctDNA clearance was significantly associated with pCR (p = 0.0304)	37 patients	n/a	n/a	n/a	Lack of invasive tumor in the breast and axillary lymph nodes in surgical specimens
Magbanua (II) et al.²³	2024	USA	Early-stage hormone receptor-positive, HER2-negative, and triple-negative BC	T-AC regimens, administered with or without an investigational agent	cfDNA	At 3 weeks after treatment start and at 12 weeks, between the paclitaxel and AC phases	RCB/ pCR and DRFS	pCR (RCB-0) was achieved in 15.2% of HR-positive/HER2-negative patients and 24.6% of TNBC patients. In the HR-positive/HER2-negative group, cfDNA concentration showed no significant correlation with RCB at any timepoint (p = 0.29). In TNBC, higher cfDNA at 3 weeks (T1) correlated with lower RCB at surgery (r = −0.24, p = 0.011), and pCR patients had higher T1 cfDNA than those with extensive residual disease (p = 0.034). DRFS events occurred in 22.5% of HR-positive/HER2-negative and 32.3% of TNBC patients. In the HR-positive/HER2-negative group, high cfDNA shedding at baseline (T0) was associated with worse DRFS (HR 2.12, p = 0.037), remaining significant after adjustment in multivariable analysis (HR 2.41, p = 0.02). No significant DRFS differences were observed in TNBC at any timepoint	283	Retrospective	Multicentric	For the HR-positive/HER2-negative group, follow-up averaged 3.10 years (range 0.46–7.6), and for the TNBC group, 3.12 years (range 0.31–7.91)	RCB-0

AC, Adriamycin (Doxorubicin) + Cyclophosphamide; AC-CFD, AC neoadjuvant chemotherapy; BC, breast cancer; cCR, clinical complete response; cfDI, cell-free DNA integrity; cfDNA, cell-free DNA; CTC, circulating tumor cell; ctDNA, circulating tumor DNA; DFS, disease-free survival; DRFS, distant recurrence-free survival; EC, Epirubicin + Cyclophosphamide; EC-T(H/HP), Epirubicin + Cyclophosphamide followed by Taxane + Trastuzumab (H) and/or Pertuzumab (HP); EFS, event-free survival; HR+, hormone receptor positive; NAC, neoadjuvant chemotherapy; NLR, neutrophil-to-lymphocyte ratio; NMD, absence of mutations in ctDNA; OS, overall survival; pCR, pathologic complete response; PLR, platelet-to-lymphocyte ratio; RCB, residual cancer burden; RFS, relapse-free survival; T-AC, Taxane and anthracycline/cyclophosphamide; TCbH(P), Taxane + Carboplatin + Trastuzumab + Pertuzumab; TC(H), Taxane + Cyclophosphamide + Trastuzumab; TDM1, Trastuzumab Emtansine; TEC, Docetaxel + Epirubicin + Cyclophosphamide; T(H)-EC, Taxane (with or without Trastuzumab) followed by Epirubicin + Cyclophosphamide; T(HP), Taxane + Trastuzumab ± Pertuzumab; TIL, tumor-infiltrating lymphocytes; TNBC, triple negative breast cancer; UK, United Kingdom; USA, United States.

Inflammatory markers

Four studies assessed the novel inflammatory markers. One study focused on analyzing NLR every 2 weeks during NAC.¹⁸ This study found significant associations between stable NLR values during NAC and pCR achievement, as well as between increasing NLR trends and failure to achieve pCR. One study³⁷ investigated PLR and the difference in values (ΔPLR) between the post-second NAC cycle and baseline. A statistically significant correlation between ΔPLR <0 and achievement of pCR is reported. Two studies examined both dynamics NLR and PLR during NAC.^25,29 One of these assessed NLR (ΔNLR) and ΔPLR from baseline to mid-NAC, with ΔPLR <0 having a significant correlation with pCR achievement. Similarly, Chung et al.²⁵ analyzed the percentage variation of each inflammatory index after the first cycle of NAC and compared it to the baseline values, finding a significant association between negative percentage PLR variation and pCR. Neither of the two studies had significant findings for NLR variations. No study evaluated survival according to mid-NAC biomarker quantification or dynamics.

Circulating nucleic acids

A total of 20 studies examined the presence of circulating nucleic acids during NAC.

Circulating tumor DNA

Fifteen studies investigated the effect of ctDNA on post-NAC outcomes. Eight studies reported that persistent ctDNA by mid-NAC is associated with lack of or significantly reduced rates of pCR.^{19,24,28,30,32,34–36} Conversely, two studies reported early clearance of ctDNA at mid-NAC and that all patients with pCR had complete clearance of ctDNA during NAC.^22,27 Similarly, one study concluded that the subgroup of patients who had clearance of ctDNA and no pCR by the end of NAC had similar outcomes, compared to those who achieved pCR.²² In contrast, one study found no association between the clearance of ctDNA during NAC and achievement of pCR.³¹ Other outcomes were also explored, with three studies reporting worse overall survival (OS) for patients with positive ctDNA detection during mid-NAC^20,30,31 and one study failing to find such an association.³⁸ Three studies investigated disease-free survival (DFS) in relation to ctDNA measurement during NAC, finding improved DFS among patients who cleared ctDNA after one cycle of NAC. Two of these studies reported a strong correlation between the persistence of ctDNA during NAC and higher rates of disease recurrence and metastasis.^22,32

Cell-free DNA

Five studies investigated the effect of cfDNA or cell-free DNA integrity (cfDI) during NAC on post-NAC outcomes. Three studies didn’t find correlations between cfDNA concentrations and pCR achievement.^26,33,41 In contrast, one study reported a significant correlation between higher cfDNA concentration and lower pCR rates when cfDNA is measured 3 weeks after NAC onset. Giro et al.⁴⁰ performed an analysis of cfDI after the first NAC cycle and found significantly higher levels of cfDI in patients with pCR rates and superior DFS. Wang et al.⁴¹ reported that cfDI is useful to monitor responses to NAC and that a gradual increase over the course of NAC is correlated to better outcomes.

Quality assessment

The results of the Newcastle–Ottawa quality assessment are presented in Tables 2 and 3. Some of the retrospective studies scored fewer points in the selection domain due to a lack of disclosure regarding whether the outcome of interest was known at the study’s onset. In the comparability domain, the main cause of score loss was the lack of multivariate analysis for the parameters. With regard to the outcome domain, many studies lost points due to a lack of clarity with respect to the assessment of outcome by an independent, blind assessment, or record linkage.

Table 2.

Newcastle–Ottawa assessment for the included studies.

Study	Selection	Comparability	Outcome	NOS score
Gao et al.¹⁸	***	**	***	8
Zhou (I) et al.¹⁹	****	**	**	8
Chen et al.³⁰	****	**	***	9
Zhou (II) et al.³⁵	****	**	***	9
Kim et al.³⁶	***	*	**	6
Riva et al.³¹	***	*	**	6
Dan et al.³⁷	****	*	**	7
Cavallone et al.³⁸	***	*	***	7
Cirmena et al.³⁹	***	*	**	6
Giro et al.⁴⁰	***	*	**	6
Wang et al.⁴¹	***	*	**	6
Fiste et al.²⁹	***	*	**	6
Du et al.²⁸	***	*	***	7
Butler et al.²⁷	****	*	***	8
Park et al.²⁶	****	**	***	9
Chung et al.²⁵	****	**	***	9
Ciriaco et al.²¹	****	*	***	8
Dong et al.²⁰	***	*	***	7
Liu et al.³²	****	**	***	9
Rothe et al.²⁴	***	*	***	7
Magbanua (I) et al.²²	***	**	***	8
Magbanua (II) et al.²³	***	**	***	8
Ma et al.³³	***	*	**	6
Lucci et al.³⁴	***	*	**	6

Each * represents a point in a specific domain.

NOS, Newcastle-Ottawa Scale

Table 3.

Qualitative GRADE assessment for various biomarkers.

Outcome	Number of studies	Combined risk of bias	Inconsistency	Indirectness	Imprecision	Certainty of evidence
ΔPLR predicting pCR	3	Moderate	Low	Low	Moderate	Moderate
ΔNLR predicting pCR	3	Moderate	Low	Low	Moderate	Low
ctDNA clearance predicting pCR	15	Moderate	Low	Low	Moderate	Moderate
cfDNA concentration predicting pCR	4	Moderate	High	Low	High	Low
cfDI predicting pCR	2	Moderate	Low	Moderate	High	Low

cfDI, cell-free DNA integrity; cfDNA, cell-free DNA; NLR, neutrophil-to-lymphocyte ratio; pCR, pathologic complete response; PLR, platelet-to-lymphocyte ratio.

The results of the qualitative GRADE assessment are displayed in Table 3. The combined risk of bias was assessed for each outcome; if the majority, but not all, of the studies had a high-quality grade according to the NOS, the combined risk of bias was classified as moderate. Inconsistency was evaluated by comparing results across studies for each biomarker. Indirectness was judged based on how closely the included studies matched the target population, intervention, and outcome. Studies that lacked subtype-specific analysis or reported surrogate endpoints (rather than pCR or survival) were considered to have moderate indirectness. Imprecision was assessed by considering sample size and the presence or absence of confidence intervals or statistical power calculations. Studies with small cohorts or wide variability in results were downgraded for imprecision. Certainty of evidence was then categorized into high, moderate, low, or very low, depending on the cumulative impact of the aforementioned domains.

Discussion

In recent years, the introduction of inflammatory indices obtained from the plasma, as well as circulating nucleic acids, gained attention for their prognostic ability. A number of challenges surround the implementation of these components into clinical practice, including the absence of specific thresholds for novel inflammatory markers and a lack of a standardization for ctDNA and cfDNA measurement.^42,43 Moreover, identifying the most appropriate timing for biomarker measurement is challenging, as various time points reflect different stages in patient care. Therefore, a thorough understanding of biomarker dynamics is essential. The number of studies assessing biomarkers during BC NAC has increased in recent years, and a systematic review of this topic is timely.^44,45

This study aimed to investigate some biomarker measurements and their dynamics during NAC in early BC patients. A total of 24 studies were included in an analysis that assessed NLR, PLR, ctDNA, and cfDNA as predictive biomarkers for NAC outcomes. The studies encompassed various types of BC, and a number of different NAC regimens were used.

NLR and PLR are two novel inflammatory markers that were investigated in this study. The rationale for their use as predictors of disease outcomes is the central role of inflammation in driving tumoral microenvironment and tumor cell proliferation.⁴³ Lymphocytes, neutrophils, and platelets are the three components involved in NLR and PLR and intricate interactions between these cells seem to suggest the reasons for the utility of these biomarkers in cancer prognosis. There is evidence that circulating BC cells use pro-tumor neutrophils to invade tissues and metastasize.⁴⁶ Moreover, tumor cells secrete G-CSF, which contributes to neutrophilia.¹⁰ Platelets have been implicated in shielding tumor cells from immune recognition and are known to be activated by a number of chemokines released by malignant cells.⁴⁷ CD4 and CD8 lymphocytes have a crucial role in tumor cell destruction through immunosurveillance.⁴⁸ These factors are thought to be some of the reasons accounting for the poor prognosis represented by higher NLR and PLR in cancer.

In our systematic review, the variation of PLR values from baseline to mid-NAC measurement appears to have a predictive value for achievement of pCR, with three studies reporting a negative variation associated with microscopic complete response.^25,29,37 Unfortunately, the parameter used to define the odds ratio in one of the studies differed from the others, rendering a meta-analysis unfeasible.²⁵ The data on NLR variation are less robust and more conflicting, with two studies reporting a lack of significance association between a negative variation and pCR^25,29 and one study reporting increasing NLR as a significant predictor for lack of pCR.¹⁸ Although the implementation of PLR and NLR is hampered by a lack of standardized cutoffs, with multiple studies using different cutoffs, the variation or ΔNLR and ΔPLR is a useful method for their use in clinical practice since studies generally compare Δ <0 versus >0. Even though Dan et al.³⁷ ruled ΔPLR as an independent prognostic factor for pCR, more studies should investigate this parameter. Unfortunately, none among the included studies carried out an assessment of mid-NAC PLR, NLR or their dynamics in relation to survival.

To the best of our knowledge, this is the first study to investigate the dynamics of inflammatory biomarkers in NAC in a systematic manner. However, pretreatment NLR and PLR have been studied, as well as ΔNLR and ΔPLR for the difference post-NAC–pre-NAC. A 2021 meta-analysis that pooled patients with all types of BC failed to find a significant association between pretreatment NLR and pCR achievement.⁴⁹ Another study also failed to find any difference when all BC subtypes were considered, nonetheless found a significant association between lower NLR values and higher pCR achievement for postmenopausal Luminal B/HER2− patients.⁵⁰ A 2022 study investigated the dynamic variations of NLR and PLR before and after NAC and found a significant association between ΔNLR <0 and achievement of pCR.⁵¹ No association between ΔPLR >0 or <0 and pCR was found. This contrasts with the findings of studies included in this systematic review, suggesting that the prognostic value of ΔNLR and ΔPLR may differ depending on whether the final biomarker value is measured during or after NAC. Further research is needed to clarify these differences.

cfDNA and ctDNA are other biomarkers that gained relevance in the context of cancer outcome prediction. cfDNA represents genetic material shed from cells in the event of cell death. A small fraction of cfDNA consists of ctDNA, which is released by tumor cells undergoing necrosis or apoptosis. The concentration of ctDNA is generally proportional to tumor burden.^52,53 The detection of ctDNA through liquid biopsy is useful for identifying and monitoring tumor mutations in a minimally invasive manner.³⁰ In the context of BC’s high heterogeneity, the search for ctDNA can provide a more efficient genetic map of the tumor compared to traditional biopsies. Furthermore, one of the advantages of ctDNA monitoring is the ability to identify disease at a molecular level before radiologic evidence is present, prompting preemptive interventions.³⁰ The clearance of ctDNA has been described as an important emerging tool in monitoring disease response to therapies, with a lack of clearance having associations with worse disease outcomes.¹⁵ An interesting method to study cfDNA and ctDNA simultaneously is the examination of cfDI, which is the ratio of larger to smaller DNA fragments from the same genetic locus.⁴⁰ While larger fragments represent the total cfDNA, shorter ones represent the estimated ctDNA.⁵⁴ In contrast to the traditional methods for ctDNA identification, cfDI doesn’t depend on previous genetic sequencing, representing a faster and less costly process. cfDI also has the potential to estimate ctDNA during BC NAC.

In this systematic review, clearance of ctDNA appears to have a strong association with pCR achievement, according to the majority of the included studies. Riva et al.³¹ was the only study that didn’t find an association between clearance of ctDNA and pCR, however, a positive ctDNA after one cycle of NAC was significantly correlated with shorter DFS and OS. In fact, one study noted that patients who had clearance of ctDNA by the end NAC but failed to achieve pCR had a similar risk of metastatic recurrence as patients who cleared ctDNA and had pCR.²² Taken in conjunction, these factors suggest an intrinsic benefit of ctDNA clearance irrespective of pCR achievement. Also, according to three studies, one investigating all subtypes of BC and two investigating triple-negative BC, clearance of ctDNA at mid-NAC and post-NAC is associated with increased OS.^20,30,31 However, one study focusing on triple-negative BC failed to find this association.³⁸ Unfortunately, a meta-analysis to pool the results was not possible. Therefore, it is premature to draw definitive conclusions about this association. More studies should investigate the clearance of ctDNA at mid-NAC and survival with larger cohorts and with attention to the possible difference in results between the diverse BC subtypes. Our findings for the use of ctDNA clearance as a predictor for pCR and survival are consistent with studies investigating neoadjuvancy in other malignancies such as colorectal and lung.^55–57

Another interesting use of ctDNA dynamics is the serial assessments of gene mutations. One study performed a dynamic assessment of a panel of genes and their mutations between mid-NAC and post-NAC for prediction of pCR.²¹ The findings seem to suggest that persistence of mutations of ctDNA in post-NAC period or arise of new mutations from mid-NAC to post-NAC are correlated with lack of pCR achievement. More studies should be conducted to confirm this association. If confirmed, these results could support the incorporation of serial mutation profiling into routine monitoring during NAC. Our study also assessed the cfDNA and cfDI as possible predictors for NAC outcomes. One study²³ found a significant correlation between higher cfDNA concentrations 3 weeks after NAC onset and lower rates of pCR achievement. In contrast, three of the included studies, one focusing on triple negative disease and the two others on all subtypes, seem to point that cfDNA concentrations during NAC don’t have a significant association with pCR achievement.^26,33,41 One of these studies found that although changes in cfDNA concentrations during NAC were not prognostic for pCR, baseline cfDNA concentrations below a certain cutoff were independently associated with higher pCR rates and longer event-free survival (EFS).²⁶ The small number of studies and conflicting results with regard to pCR rates make it challenging to draw any conclusion. Very few studies of other malignancies have evaluated cfDNA concentration during NAC, therefore, a comparison with other diseases is unfeasible. CfDI was assessed by two of the included studies, both of which associated higher integrity with improved pCR rates and survival outcomes.^40,41 This is biologically plausible, as tumor-derived DNA is typically more fragmented than DNA from non-malignant cells.⁵⁸ Therefore, higher integrity may reflect decreased tumor activity or burden. To the best of our knowledge, there are no studies that evaluated cfDI during NAC in other malignancies. Before more studies emerge, it is challenging to make any conclusions.

The implication for an accurate prediction of treatment outcome during NAC could possibly be an early change in regimen and modifications in the treatment plan. The advantages offered by this prospect include sparing the patient from additional chemotherapy cycles that wouldn’t accomplish pCR by the end of NAC. Another advantage is to avoid additional toxicity. NLR and PLR are inexpensive, cost-effective, and easily obtainable from complete blood count.⁵⁹ As previously discussed, their applicability is limited by the lack of a consensus surrounding cutoff values. However, ΔNLR and ΔPLR are interesting tools to measure variation between time points and may be applied during NAC. Circulating nucleic acids are promising biomarkers for the prediction of therapy outcomes. Although one systematic review concluded that ctDNA is not a cost-effective tool for BC screening, no cost-effective analysis has been performed for its use in BC neoadjuvancy. Health economic studies investigating ctDNA during NAC are warranted in the future. CfDI has been recognized as a cost-effective and less expensive method, although its applicability is more limited than ctDNA.^40,60

This study has a number of limitations, including the lack of data for meta-analyses performance, inclusion of retrospective and single-institution studies, and a relatively small total number of patients. Many studies lacked separate analyses for the different subtypes of BC, making it challenging to make conclusions for specific subtypes. Furthermore, the qualitative GRADE assessment revealed moderate to low certainty of evidence. Therefore, our findings should be interpreted carefully. More prospective studies, with larger cohorts, investigating the different subtypes of disease are needed in the future.

Nonetheless, we believe that this systematic review has some strengths, including the high quality of a majority of the included studies and the introduction of a topic that may impact clinical practice in the near future. Furthermore, the gathering of data presented in our systematic review may prompt additional studies to be carried out.

Conclusion

In conclusion, the negative variation of PLR from baseline to mid-NAC period and a clearance of ctDNA from baseline to mid-NAC period appear to have a moderate level of evidence to predict pCR. A negative prediction may prompt changes in the therapeutic plan. More studies should be conducted to validate these findings. Additional studies should also be carried out to investigate the predictive value for NAC outcomes of the following: variation of NLR levels, cfDNA concentration during NAC and cfDI during NAC. Future research should consider the heterogeneity of BC and include analyses stratified by subtype.

Supplemental Material

sj-docx-1-tam-10.1177_17588359251386765 – Supplemental material for Prognostic utility of inflammatory indices and circulating nucleic acids for neoadjuvant chemotherapy outcomes in breast cancer: a systematic review

Supplemental material, sj-docx-1-tam-10.1177_17588359251386765 for Prognostic utility of inflammatory indices and circulating nucleic acids for neoadjuvant chemotherapy outcomes in breast cancer: a systematic review by Silvio Matsas, Anderson Ruiz Simões, Luiza Giuliani Schmitt, Yara Abdou, Stephen Kimani, Kishore Karri and Auro del Giglio in Therapeutic Advances in Medical Oncology

Footnotes

Acknowledgements

The authors gratefully acknowledge the support provided by the Centro de Estudos de Hematologia e Oncologia (CEPHO) in the development of this project.

Declarations

ORCID iDs

Luiza Giuliani Schmitt

Auro del Giglio

Supplemental material

The PRISMA checklist for systematic reviews was filled and is available as a Supplemental File.

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