
==== Front
Rom J Morphol Embryol
Rom J Morphol Embryol
RJME
Romanian Journal of Morphology and Embryology
1220-0522
2066-8279
Academy of Medical Sciences, Romanian Academy Publishing House, Bucharest

39020533
650224195201
10.47162/RJME.65.2.05
Original Paper
NGS mutational status on first diagnostic tissue, liquid biopsy and mastectomy in G2–G3 breast cancer
Ardeleanu Carmen Maria 12
Olinca Maria Victoria 12
Viişoreanu Cristian Gabriel 3
Mureşan Horaţiu Alin 1
Tecuceanu-Vulpe Adriana 1
Manole Georgiana 1
Gune Iulia Elena 1
Gălăţeanu Bianca 4
Ilie-Petrov Andreea-Corina 25
Ultimescu Flavia 126
1 Department of Pathology, OncoTeam Diagnostic, Bucharest, Romania
2 Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania
3 Department of Surgery, Memorial Hospital, Bucharest, Romania
4 Faculty of Biology, University of Bucharest, Romania
5 Department of Surgery, Colţea Clinical Hospital, Bucharest, Romania
6 Department of Pathology, Prof. Dr. Alexandru Trestioreanu Oncology Institute, Bucharest, Romania
Corresponding Author: Andreea-Corina Ilie-Petrov, MD Department of Surgery Colţea Clinical Hospital 1 I.C. Brătianu Avenue, Sector 3 030167 Bucharest Romania + 40721–553 533 andreea.petrov@drd.umfcd.ro
Apr-Jun 2024
30 6 2024
65 2 195201
15 4 2024
18 6 2024
Copyright © 2024, Academy of Medical Sciences, Romanian Academy Publishing House, Bucharest
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This is an open-access article distributed under the terms of a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International Public License, which permits unrestricted use, adaptation, distribution and reproduction in any medium, non-commercially, provided the new creations are licensed under identical terms as the original work and the original work is properly cited.
Breast cancer is one of the more frequently diagnosed cancers leading to death in women, and, like other tumor types, it is heterogeneous in its immunophenotype. It harbors mutations that modify tumor aggressiveness, therapy responses, residual disease, drug resistance, and relapse rates in advanced stages. This study aims to assess the mutational status of G2 and G3 tumors using next-generation sequencing (NGS) on initial tissue biopsies, liquid biopsies, and mastectomy specimens. The histopathological (HP) diagnosis for the 32 selected cases was established via Hematoxylin–Eosin (HE) staining by two observers. For the immunohistochemical (IHC) testing of estrogen receptor (ER), progesterone receptor (PGR) and human epidermal growth factor receptor 2 (HER2), we used the Ventana BenchMark Ultra. Ki67 testing was conducted using Bond-III from Leica. For cases with a score of 2+, gene amplification was assessed by silver-enhanced in situ hybridization (ISH) (SISH; Inform HER2 Dual ISH) on Ventana BenchMark Ultra. NGS analysis was initially performed on biopsies and plasma, and later on mastectomy specimens. After automated deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) extraction, concentrations were measured using the Invitrogen Qubit system. Libraries were created using Oncomine systems, and sequencing and analysis were done with the Ion Torrent system. Most tumors were graded as G3 (19 cases), with Luminal A being the predominant molecular subtype, and a significant number displayed HER2/HER2-low characteristics (24 out of 32 cases). The NGS assessment showed that phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations were the most frequent across all sample types. A significant limitation was the high number of invalid plasma tests due to pre-analytical handling errors or transport issues. Nonetheless, plasma testing (liquid biopsy) proved useful for monitoring tumor evolution and assessing residual disease.

breast cancer
liquid biopsy
NGS
pathogenic variants
==== Body
pmcIntroduction

Breast cancer (BC)’s intrinsic heterogeneity influences its prognosis and response to treatment. Understanding the genetic underpinnings of this disease, particularly in aggressive G2 and G3 tumors, is crucial for tailoring personalized treatment strategies. Liquid biopsy has become a valuable tool for investigating mutations, tumor burden, and drug-resistance in various malignant tumors, acting as a surrogate for genomic assessment. Specifically, circulating tumor deoxyribonucleic acid (ctDNA) has proven its utility not only as a strong prognostic biomarker [1] but also in identifying new targetable mutations for therapy. Furthermore, next-generation sequencing (NGS) enhances this capability by offering detailed identification of pathogenic variants (PVs), including single nucleotide variants (SNVs), amplifications, and insertions/deletions (indels). Given that initial tissue biopsies sometimes contain tumor fragments too small for molecular methods, liquid biopsy serves as a convenient alternative, being easy to obtain, non-invasive, and effective at capturing ctDNA, ribonucleic acid (RNA), microRNA (miRNA), and exosomes [2]. Nevertheless, certain conditions are necessary to preserve nucleic acids, such as rapid handling and maintaining low temperatures during the transport of samples. These advanced methods enable real-time monitoring of tumor activity, residual disease, and relapses. They also have particularly impacted BC monitoring, where the heterogeneity of the disease and the development of secondary subclones [3] often necessitate a therapeutic switch.

Aim

This study employs NGS to analyze initial core biopsies, liquid biopsies (plasma), and mastectomy specimens. The aim of our study was to provide a detailed evaluation of liquid biopsy’s predictive capabilities in comparison to those of solid tumor specimens. By analyzing these diverse samples, the study seeks to uncover patterns that could guide therapeutic decisions and improve patient outcomes. Furthermore, by utilizing specialized gene panels that target mutations commonly found in advanced stages of BC in both solid and liquid biopsies, we can enhance real-time disease monitoring. This strategy also facilitates the early detection of metastases, residual disease, and the development of drug resistance. While the mutational capacity of BC has been extensively studied in numerous clinical trials, universally accepted parameters for evaluating the effectiveness of liquid biopsy in the ongoing monitoring of BC have yet to be established.

Materials and Methods

Cases were randomly selected from the records of the Department of Surgery, Memorial Hospital, Bucharest, Romania. The study involved female patients who underwent surgical procedures between 2023 and 2024, providing informed consent for inclusion in this prospective study. After applying the selection criteria, the study group consisted of 32 cases of primary untreated BC diagnosed in clinical stages III and IV.

The initial core biopsies, containing both normal and tumoral tissue, were immediately immersed in 10% neutral buffered formalin, fixed for 12 hours, and then automatically processed and paraffin-embedded within four hours. Each core biopsy consisted of 2–5 fragments, measuring 1.2–1.5 cm by 0.2 cm.

Histopathological (HP) diagnosis was conducted on Hematoxylin–Eosin (HE)-stained slides by two observers, and grading was determined using the Nottingham score. Additionally, the extent of tumor-infiltrating lymphocytes (TILs) and the desmoplastic nature of the stroma were assessed.

Immunohistochemical (IHC) testing was performed using the Ventana BenchMark Ultra (Ventana Medical System, Tucson, AZ, USA) for estrogen receptor (ER), progesterone receptor (PGR), and human epidermal growth factor receptor 2 (HER2), and the Bond-III Leica (Newcastle upon Tyne, UK) for Ki67. The specific antibodies used were as follows: anti-ER (Ventana-6F11), anti-PGR (Ventana-16), anti-HER2 (Ventana-4B5), and anti-Ki67 (Leica-MM1). Ready-to-use antibodies were utilized following the manufacturer’s protocols. Results were expressed as percentages for ER, PGR, and Ki67 and as scores ranging from 0 to 3 for HER2 [as per 2018 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines].

Molecular analysis via NGS was conducted on paraffin-embedded biopsies and plasma specimens from 26 out of 32 cases.

RNA and DNA were extracted from solid tumor tissue, while ctDNA and a fraction of circulating free DNA (cfDNA) were analyzed from the plasma specimens.

Employing the Ion Torrent platform (Thermo Fisher Scientific, Waltham, MA, USA), the study identified various genomic alterations, including point mutations, SNVs, indels, copy number variations (CNVs) and fusions, as also specified in various studies [4]. Sequencing in the 26 DNA samples yielded an average of 75 532 978 bases per patient, with a mean of 92% per patient, achieving a Phred quality (Q) score of ≥Q20. The mean read length was 123 base pairs (bp) (Figure 1).

Figure 1 Summary metrics of an NGS run: the mean values of each parameter per patient are reported (Ion Torrent Platform, Thermo Fisher Scientific). NGS: Next-generation sequencing; Q: Quality

Liquid biopsies were analyzed using the 52-gene panel Pan Cancer and for solid tumors was used Oncomine Focus Assay Panel, both from Thermo Fisher Scientific.

DNA and RNA were extracted from tissue samples using RecoverAll™ Total Nucleic Acid Isolation kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Liquid biopsy samples were centrifuged, and cell-free total nucleic acid was extracted using MagMAX™ Cell-Free Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA). The concentration of DNA and RNA was assessed using Qubit RNA HS Assay kit, Qubit 1X double-stranded DNA (dsDNA) HS Assay and Qubit 4 Fluorometer (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Afterwards, RNA from both tissue and liquid biopsy samples was reverse transcribed using SuperScript™ IV VILO™ Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). Libraries for tissue samples were created using Oncomine Focus Assay Chef-Ready Library (Thermo Fisher Scientific, Waltham, MA, USA), while those for liquid biopsy samples were prepared using Oncomine™ Pan-Cancer Cell-Free Assay (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing of libraries was performed using Ion GeneStudio™ S5 Prime sequencer (Thermo Fisher Scientific, Waltham, MA, USA), and the resulting sequences were analyzed using Ion Reporter™ software (Thermo Fisher Scientific, Waltham, MA, USA). The PVs and variants of uncertain significance (VUS) were then selected for further analysis.

Results

The parameters analyzed for the female patients in this study included age, HP appearance, immunophenotype, molecular surrogate types and gene mutations identified by NGS in the initial core biopsy, liquid biopsy (plasma), and mastectomy specimen of each subject. All findings were summarized in Table 1.

Table 1 Analysis of initial core biopsies, mastectomy specimens, and plasma in female breast cancer patients: summarized findings

CN

	Age [years]

	Initial core biopsies

	Mastectomy specimens

	Plasma

	
HP

	G

	MT

	IHC

	HER2

	MUT

	HP (+)

	IHC

	MUT

	MUT

	
1.

	64

	IDC

	G3

	LB

	ER/PGR (+)

Ki67 55%

	(1+)

	PV (-)

	None

	N/A

	UPV

	
2.

	58

	IDC

	G3

	LB-l

	ER 95%

PGR 10%

Ki67 45%

	(3+)

	NDD

	IDC G3

	ER 98%

PGR (-)

HER2 (3+)

Ki67 65%

	HER2

FGFR4

(NGS-A)

	N/R

	
3.

	42

	IDC

	G2

	LA

	ER/PGR (+)

Ki67 12%

	(1+)

	PIK3CA PV

	IDC G1

	ER/PGR (+)

Ki67 15%

	PIK3CA PV*

	Invalid

	
4.

	39

	IDC

	G3

	LB

	ER/PGR (+)

Ki67 30%

	(2+)

SISH-A

	NDD

	None

	N/A

	N/A

	
5.

	54

	IDC

	G2

	LA

	ER/PGR (+)

Ki67 10%

	(1+)

	PIK3CA PV

(AR-A)

	IDC G1

	ER/PGR (+)

Ki67 5%

HER2 (-)

	N/A

	PIK3CA PV

	
6.

	49

	IDC

	G3

	LB

	ER/PGR (+)

Ki67 30%

	(2+)

SISH-NA

	PIK3CA PV

	ILC G2

	ER (+)

PGR (+/-)

Ki67 30%

HER2 (1+)

	PIK3CA PV**

	UPV

	
7.

	54

	IDC

	G2

	LA

	ER/PGR (+)

Ki67 12%

	(-)

	PV (-)

	G2

	ER/PGR (+)

HER2 (+)

SISH-NA

	N/A

	UPV

	
8.

	70

	IDC

	G3

	LB

	ER/PGR (-)

Ki67 30%

	(-)

	PV (-)

	None

	N/A

	UPV

	
9.

	61

	IDC

	G3

	BAS-LTN

	ER/PGR (-)

Ki67 40%

	(-)

	VUS ( RET )

	G2

	ER/PGR (-)

Ki67 40%

HER2 (-)

PD-L1 1%

	VUS ( RET )

	VUS ( TP53 )

	
10.

	70

	IDC

	G2

	LB-l

	ER/PGR (+)

Ki67 30%

	(2+)

SISH-A

	PIK3CA PV

	G2

	ER/PGR (+)

HER2 2(+)

Ki67 40%

	PIK3CA PV*

	PIK3CA PV

	
11.

	46

	MTS

IDC

	G3

	LB

	ER/PGR (+)

Ki67 60%

	(1+)

	PV (-)

	None

	N/A

	UPV

	
12.

	64

	IDC

	G2

	LA

	ER/PGR (+)

Ki67 12%

	(1+)

	PV (-)

	None

	N/A

	UPV

	
13.

	54

	IDC

	G3

	HER2-ENR

	ER/PGR (-)

Ki67 12%

	(3+)

	PIK3CA PV

	None

	N/A

	Invalid

	
14.

	45

	IDC

	G2

	LA

	ER/PGR (+)

Ki67 14%

	(1+)

	PIK3CA PV

	G2

	ER/PGR (+)

HER2 (+)

Ki67 20%

	N/A

	Invalid

	
15.

	43

	MTS

IDC

	G3

	LB

	ER/PGR (+)

Ki67 25%

	(1+)

	AKT PV

	G2

	ER/PGR (+)

HER2 (-)

Ki67 10%

	N/A

	N/A

	
16.

	38

	MTS

IDC

	G2

	LB-l

	ER/PGR (+)

Ki67 25%

	(2+)

SISH-A

	PV (-)

	G2

	ER (+)

PGR (-)

HER2 (3+)

Ki67 20%

	N/A

	Invalid

	
17.

	45

	IDC

	G2

	LA

	ER/PGR (+)

Ki67 10%

	(1+)

	PIK3CA PV

	None

	N/A

	Invalid

	
18 #

	58

	IDC

	G2

	LA

	ER/PGR (+)

Ki67 17%

	(-)

	PV (-)

	G2

	ER/PGR (+)

HER2 (-)

Ki67 10%

	N/A

	UPV

	
19 #

	43

	IDC

	G3

	LB

	ER/PGR (+)

Ki67 30%

	(-)

	PV (-)

	None

	ER/PGR (+)

HER2 (-)

Ki67 30%

	N/A

	Invalid

	
20.

	49

	IDC

	G2

	LA

	ER/PGR (+)

Ki67 18%

	(1+)

	AKT PV

AMP FGFR

	None

	N/A

	N/A

	
21.

	62

	ILC

	G2

	LA

	ER/PGR (+)

Ki67 10%

	(-)

	PV (-)

AMP MYC

	None

	N/A

	UPV

	
22.

	86

	IDC

	G3

	TN

	ER/PGR (-)

Ki67 60%

	(1+)

	NDD

	None

	N/A

	N/A

	
23.

	51

	IDC

	G3

	LB

	ER/PGR (+)

Ki67 45%

	(-)

(1+)

	VUS ( SMO )

FGFR3

	None

	ER (+)

PGR (+/-)

HER2 (1+)

Ki67 30%

	N/A

	UPV

	
24.

	47

	ILC

	G2

	LA

	ER/PGR (+)

Ki67 5%

	(-)

	NDD

	None

	N/A

	PV (-)

	
25.

	45

	MTS

IDC

	G3

	TN

	ER/PGR (-)

TRPS1 (+)

	(-)

	PV (-)

	None

	ER/PGR (-)

HER2 (-)

Ki67 90%

	N/A

	PV (-)

AMP FGFR1

	
26.

	58

	MTS

IDC

	G3

	LB

	ER/PGR (+)

Ki67 25%

	(1+)

	PV (-)

	None

	N/A

	UPV

	
27.

	56

	MTS

IDC

	G3

	LB

	ER (+)

PGR (-)

Ki67 35%

	(1+)

	VUS ( MYC )

	None

	ER (+)

PGR (-)

HER2 (1+)

Ki67 8%

	N/A

	UPV

	
28.

	69

	IDC

	G2

	LA

	ER/PGR (+)

Ki67 15%

	(1+)

	NDD***

	G1

	NDD

	N/A

	N/A

	
29.

	29

	IDC

	G3

	LB-l

	ER/PGR (+)

Ki67 60%

	(3+)

	AMP ERBB2

	None

	ER 5%

PGR (-)

HER2 (3+)

Ki67 20%

	N/A

	Invalid

	
30.

	42

	IDC

	G3

	LB-l

	ER/PGR (+)

Ki67 60%

	(3+)

	AMP ERBB2

CCND1

	None

	ER (+)

PGR (-)

HER2 (2+)

Ki67 5%

	N/A

	Invalid

	
31 #

	63

	IDC

	G3

	HER2-ENR

LB-l

	ER/PGR (-)

Ki67 80%

	(3+)

	TP53 PV

AMP HER2

	None

	NDD p

	N/A

	UPV

	
32 p

	55

	IDC

	G3

	BAS-LTN

	ER/PGR (-)

Ki67 80%

PD-L1 5%

	(1+)

	NDD

	None

	N/A

	BRCA1

BRCA2

UPV

	
AMP: Amplified; AR-A: Amplified androgen receptor; BAS-LTN: Basal-like triple negative; BRCA1/2: Breast cancer 1/2; CCND1: Cyclin D1; CN: Case No.; ER: Estrogen receptor; ERBB2: Erb-B2 receptor tyrosine kinase 2; FGFR1/3/4: Fibroblast growth factor receptor 1/3/4; G: Histological grade; G1: Well differentiated; G2: Moderately differentiated; G3: Poorly differentiated; HER2: Human epidermal growth factor receptor 2; HER2-ENR: HER2 enriched; HP: Histopathology; HP (+): Other histopathological observations; IDC: Invasive ductal carcinoma; IHC: Immuno-histochemistry; ILC: Invasive lobular carcinoma; LA: Luminal A; LB: Luminal B; LB-l: Luminal B-like; MT: Molecular types; MTS: Metastasized tumor; MUT: Mutations; N/A: Not available; N/R: Not received; NDD: Not determined; NGS: Next-generation sequencing; NGS-A: NGS amplified; PD-L1: Programmed death-ligand 1; PGR: Progesterone receptor; PIK3CA: Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha; PV: Pathogenic variant; SISH: Silver-enhanced in situ hybridization (ISH); SISH-A: Amplified by SISH; SISH-NA: Non-amplified by SISH; TN: Triple negative; TP53: Tumor protein p53; TRPS1: Transcriptional repressor GATA binding 1; UPV: Undetectable pathogenic variant; VUS: Variants of uncertain significance. *The same mutation as the one identified in the other specimen; **Other mutation, different from the one identified in the other specimen; ***Tumor exhausted (insufficient material); #The patient underwent chemoradiotherapy; pThe patient had a pathological complete response

All 32 cases underwent initial core biopsy, with 26 having plasma analyzed, and 20 cases having mastectomy specimens tested. Patients ranged in age from 38 to 86 years, with an average age of 56.2 years. Prior to participation, all had undergone clinical and imaging examinations and ethical approval had been obtained from the institution’s Ethics Committee. Among the 32 studied cases, 30 were classified as no special type (NST) – invasive ductal carcinomas of not otherwise specified (NOS) ductal type, while two were classical invasive lobular carcinomas. The majority of cases were histopathologically graded as G3 according to the Nottingham classification (19 cases), with the remaining cases classified as G2 (13 cases). Notably, seven cases exhibited lymph node invasion or distant metastases at the time of the initial core biopsy. Among these cases, there were five Luminal B (LB), one triple negative (TN), and one Luminal A (LA); histological G3 was observed in five cases. Also, noteworthy was the pronounced desmoplasia of the stroma, with a 2 or 3 score, which was associated with a generally reduced percentage of TILs.

Regarding molecular types, the current utility lies in the St. Gallen [5] surrogate types, established immunohistochemically based on positivity for ER/PGR, HER2, and Ki67, as determined through high-precision molecular transcriptomic analyses. Thus, within the study group, according to World Health Organization (WHO) criteria [6], 11 cases were classified as LA-like, eight cases as LB-like (LB-l) (HER2-negative), six cases as LB-l (HER2-positive), two cases as HER2-positive (non-luminal), and five cases as TN. All cases underwent IHC testing, which specified the hormonal expression percentage and HER2 expression; the latter was also scored for HER2-low cases [2+ score non-amplified by in situ hybridization (ISH) and 1+ score]. The proportion of HER2-low cases in the initially tested biopsies was similar to that reported in the literature [7] for advanced or metastatic tumors. Among the eight cases tested on mastectomy specimens, five cases showed reduced HER2 expression, either from 1+ to 0 or from 2+ to 1+. In 25 cases, the initial biopsies and the plasma were tested by NGS in parallel, and the results were quite heterogeneous.

HP and IHC features of invasive BC are illustrated in Figure 2A, 2B, 2C, 2D, 2E, 2F.

Analyzing PVs by NGS in the initial biopsies revealed that seven cases had phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) PV, and two cases had AKT PV (~35%); occasionally, PIK3CA PV was associated with amplified androgen receptor (AR) or fibroblast growth factor receptor (FGFR), a pattern also observed with AKT PV. In one case, the tumor protein p53 (TP53) PV was associated with HER2 amplification, and in another case, breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) mutations were detected in plasma.

Figure 2 Illustration of HP and IHC features in invasive BC. HP features are illustrated in the first row: (A) Invasive BC NST (invasive ductal carcinoma NOS) – G2 (×200); (B) Invasive BC NST (invasive ductal carcinoma NOS) – G3 (×400); (C) Invasive lobular BC – G2 (×400). IHC features are illustrated in the second row: (D) ER positive, strong diffuse nuclear expression in tumor cells (×200); (E) PGR positive, strong diffuse nuclear expression in tumor cells (×200); (F) HER2 positive (3+ score) according to ASCO/CAP recommendations (×400). HE staining: (A–C). ASCO/CAP: American Society of Clinical Oncology/College of American Pathologists; BC: Breast cancer; ER: Estrogen receptor; HE: Hematoxylin–Eosin; HER2: Human epidermal growth factor receptor 2; HP: Histopathological; IHC: Immunohistochemical; NOS: Not otherwise specified; NST: No special type; PGR: Progesterone receptor

Discussions

The initial case analysis included primary core biopsies and plasma. Subsequently, mastectomy specimens were included, with or without plasma from patients who had received therapy, since these mastectomies were performed after chemoradiotherapy.

The molecular testing of the analyzed plasma batch has thus far yielded heterogeneous results, largely due to disruptive factors such as the inability to obtain liquid biopsy samples, inadequate preservation of liquid biopsy samples, patient refusal for collection, and delayed mastectomy. Standard NGS protocols on the Ion Torrent system were examined and found to be applicable under our laboratory conditions. Suitable protocols were employed for both paraffin-embedded tissue specimens and plasma samples, encompassing those obtained from initial core biopsies and patients’ plasma pre-treatment, as well as those derived from mastectomy specimens and patients’ plasma post-therapy.

So far, all initial biopsies and mastectomy specimens have been subjected to IHC analysis. Following perioperative chemoradiotherapy (PCT), notably in Cases Nos. 2, 6, 16, 23, 29, and 30, a reduction or absence of PGR was observed, while ER remained positive. PGR, recognized as an activated gene, a target of ER, and dependent on estrogen, influences the function of ERα [8]. Furthermore, the absence of PGR post-therapy serves as a negative prognostic factor, warranting the repetition of IHC testing on the mastectomy specimens [9].

Cases with HER2-low also exhibited post-therapeutic changes, with some becoming completely negative on the mastectomy specimen (Cases Nos. 3, 5, 15), or shifting from a 2+ to 1+ score (Case No. 6). In two cases, an unusual therapy response was observed: one with a HER2 3+ score (Case No. 31) and another with HER2-low (Case No. 32), both achieving a pathological complete response (pCR), despite some authors associating pCR primarily with a HER2 3+ score in early BC [10]. Furthermore, it is noteworthy that among the five cases of TN BC, two exhibited pCR, with the complete disappearance of tumor tissue (Cases Nos. 31 and 32), which rendered the repetition of IHC tests unfeasible.

Regarding PVs and VUS, the paraffin-embedded tissue samples provided the most reliable results, indicating consistent PVs between the core biopsy and mastectomy specimens. PVs also remained consistent between the initial core biopsy and plasma specimens, except for one isolated case (Case No. 6), where the mutation was undetectable in plasma but retained in the mastectomy tissue. However, definitive conclusions cannot be drawn yet as not all the planned mastectomies have been performed. Notably, plasma determinations yielded results consistent with those from the initial core biopsies in four out of six parallel tests. Among the two discordant cases involving paraffin-embedded samples, one harbored a PIK3CA PV (Case No. 6, G3, LB–HER2 negative), and the other exhibited a TP53 PV (Case No. 31, G3, LB–HER2 enriched), both of which had undetectable PVs in plasma; both were G3 tumors. Of the five tested mastectomies, three displayed PIK3CA PV (Cases Nos. 3, 6 and 10), and one showed fibroblast growth factor receptor 4 (FGFR4) and HER2 amplifications (Case No. 2, G3, LB-l).

It is also evident that the sensitivity of liquid biopsy to preservation methods is much higher compared to paraffin-embedded tissue. While NGS procedures were successfully performed on all tissue samples, including biopsies and mastectomies, plasma samples yielded invalid results in eight out of 26 tests. This was likely due to specimens being collected outside of our laboratory, where conditions or transport time may have been inadequate, leading to RNA degradation. Additionally, PVs were undetectable in 13 out of 26 plasma samples tested. This could be attributed to either structural alterations of the RNA or due to a suboptimal concentration of the tested mutations.

The phosphatidylinositol-3-kinase (PI3K) plays a role in proliferation and apoptosis processes, with its mutations often activating these processes in malignant tumors. A study on early BC discovered PIK3CA mutations in 26.7% of cases, notably associated with positive G1/G2 hormone receptor and HER2-negative cases [11]. Within our study group, the most prevalent mutation in terms of frequency was PIK3CA. This mutation is also present in approximately 30% of early-stage HER2-positive tumors [12], inducing resistance to HER2-targeted agents. Recognized as a “driver” somatic mutation, it holds predictive value for response to inhibitors of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway [13]. In our study cohort, this mutation was detected in seven out of 26 cases analyzed, accounting for approximately 25%. Among these, two cases also had amplified HER2 (one HER2-negative and one HER2-positive); the five TN cases had no PIK3CA mutations, maintaining a similar proportion to other studies [14]. Additionally, two cases of AKT PV mutations were identified; both mutations occur within the PI3K/AKT/mTOR pathway and can be induced by different growth factors such as HER2, fibroblast growth factor receptor 1 (FGFR1) and insulin-like growth factor 1R (IGF-1R) [15]; in our study group, consistent results were observed between the initial core biopsy and mastectomy specimens. The mutations identified in the tested cases were E545K in exon 9 and H1047R in exon 20 of the PIK3CA gene.

The PIK3CA mutation is regarded as a driver mutation in the progression of malignant tumors and is associated with the development of anti-drug resistance, particularly in anti-HER2 treatment. However, recent evidence suggests that in ER/PGR-positive BC, it may serve as a favorable prognostic factor for the first decade following initial diagnosis [16, 17]. Although PIK3CA gene mutations are sometimes associated with other genetic disorders, such as HER2 gene amplifications, this correlation was not observed in our cases.

Others genetic alterations observed in our cohort included AKT and TP53 PVs. Notably, TP53 mutation was identified in just one case, contrasting with findings from other studies where this mutation is typically reported as the most prevalent [18]. Other associated or isolated disorders included amplifications of Erb-B2 receptor tyrosine kinase 2 (ERBB2), FGFR1, and MYC, which were detected in paraffin-embedded tissue samples, and BRCA1 and BRCA2 mutations found in plasma specimens. These latter mutations were only detected in the plasma of a single TN case (No. 32), despite being reported as the prevailing mutations in TN BC [19]. Another significant observation was FGFR1 amplification exclusively detected in plasma, while two TN cases tested negative for PVs.

The findings highlight the dynamic nature of BC genetics, wherein mutations such as PIK3CA not only shape initial tumor characteristics but also adapt in response to treatment pressures. The study also delves into the potential clinical applications of liquid biopsies, despite existing limitations related to sample handling and stability. Another noteworthy observation regarding plasma results is the number of undetectable mutations (in seven out of 26 plasma samples tested). These findings can be attributed to the fact that ctDNA constitutes only a small fraction of cfDNA [20]. Additionally, there were instances of invalid tests in plasma, underscoring the necessity for enhanced techniques to improve the reliability of these tests, which could significantly impact clinical practices.

NGS analysis revealed that PIK3CA mutations were highly prevalent, detected across various molecular types within the sample sets. Although discrepancies between tissue and plasma results occurred in some cases, overall, the findings remained largely consistent. The study noted a high rate of invalid plasma tests, primarily attributed to sample handling or transport issues, underscoring the sensitivity of liquid biopsy to pre-analytical conditions. Nevertheless, plasma testing retains its value for tumor monitoring and assessing residual disease [21], showing promise for screening in BC [22].

In summary, despite encountering some challenges with sample validity, liquid biopsies demonstrated promise in non-invasively reflecting the current genetic status of the BC. This method has the potential to revolutionize how clinicians monitor treatment efficacy and detect early indications of relapse or resistance to therapy.

Conclusions

The findings indicate a prevalence of PIK3CA mutations within the study group, potentially representing a characteristic feature of our geographical region (Romania); additionally, the study observed a predominance of PIK3CA and AKT mutations in cases with HER2-low expression. Identified through NGS, these mutations could serve as therapeutic targets in BC from the disease’s onset, as evidenced by their presence in initial core biopsies, liquid biopsies (plasma), and mastectomy specimens among tested cases, with liquid biopsy holding potential in cancer monitoring by detecting ctDNA in residual disease and predicting therapy response. This comprehensive NGS analysis underscores the importance of understanding the genetic landscape of BC at multiple treatment stages, as the ability to detect and monitor significant mutations can guide personalized treatment strategies and potentially lead to better patient outcomes, highlighting the need for further research and improvements in liquid biopsy techniques to fully realize the potential of genetic profiling in clinical oncology. Future studies should focus on longitudinal tracking of tumor genetics in individual patients to better comprehend the evolution of resistance mechanisms and metastatic potential, while expanding the use of liquid biopsies could provide more detailed insights into tumor heterogeneity and enable real-time monitoring of therapeutic efficacy.

Conflict of interests

The authors declare no conflict of interests regarding the research, authorship, and/or publication of this article.

Source of funding

The study was sustained by the project ONCOGUARD PN-III-P2-2.1-PTE-2021-0663.
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