Introduction
Comprehensive genomic profiling (CGP) is a molecular diagnostic tool, based on next-generation sequencing (NGS) technology, used in the field of cancer research and treatment. CGP identifies various genetic alterations and mutations within the tumor (1). Results from CGP guide oncologists to make decisions about treatment options and select targeted therapies tailored to the specific genetic alterations present within the tumor. CGP examines a broad panel of genes, detecting a wide range of genomic alterations. This includes point mutations (such as PIK3CA p.E453Q and c.1357G>C), insertions/deletions (indels; such as BRCA2 p.E1571Gfs*3 and c.4712_4713delAG), copy number variations (CNVs; such as ERBB2 amplification, copy number:4) and gene fusions (such as TBL1XR1/PIK3CA fusion). It can detect multiple genetic alterations in a single test, reducing the need for multiple individual tests. CGP is an application of NGS that specifically focuses on a detailed analysis of the genetic profile of patients, and can provide a broad overview of the genomic alterations in a tumor. Consequently, precision, efficiency and therapeutic guidance are potential advantages of CGP, which has been advocated for advanced-stage cancers (2-5). A large-sized panel can provide more opportunities for matching patients to targeted therapies or for increased mutation detection in clinical trials, while the use of CGP is limited by its high cost, data complexity and increased turnaround time.
Triple-negative breast cancer (TNBC) is a specific subtype of breast cancer characterized by the absence of three deterministic receptors commonly found in other breast cancers: Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). TNBC cells lack the expression of these three receptors and do not rely on them for growth. TNBC accounts for 10-20% of all breast cancer cases world (6), and is more commonly diagnosed in younger (#x003C;40 years old) women, African-American women and those with a family history of breast cancer (7). TNBC is known for its aggressive behavior and tends to grow and spread quickly, while it is also associated with a higher risk of recurrence and metastasis (8), and ~40% of people with stage I to stage III TNBC will exhibit tumor recurrence after standard treatment (9). Treatment options for TNBC often involve chemotherapy, as endocrine therapies and anti-HER2 therapies that target ER, PR or HER2 are not effective due to the absence of these receptors (8). The prognosis for TNBC varies depending on factors such as the diagnosed stage and response to treatment (10). Due to its aggressiveness, there is an unmet need to identify actionable targets such as AKT1, BRCA1/2, PALB2, ERBB2, PIK3CA and PTEN, for patients with TNBC (11,12).
Research focusing on developing targeted therapies for TNBC continues to improve treatment outcomes and broaden therapeutic options (13,14). For example, biomarker-driven therapies are being applied with the advent of immune checkpoint inhibitors, poly-adenosine-diphosphate-ribose polymerase (PARP) inhibitors, selective PI3K and AKT inhibitors, as well as antibody-drug conjugates, which target immune-enriched, DNA repair-deficient, PI3K/AKT/mTOR-activated and surface antigen-overexpressed TNBC, respectively. However, most umbrella trials for advanced breast cancer are not TNBC-specific, while a recent review highlighted that large-sized panels could identify novel markers, emphasizing the potential of CGP to uncover actionable targets in TNBC (3).
Commercialized CGP assays may be beneficial; however, the size of the panel to be employed to deliver the most optimal coverage of actionable genes for TNBC remains to be explored. Some studies comparing distinct CGP platforms and resulting biomarkers have been conducted for gastric, head and neck squamous cell carcinoma, non-small cell lung cancer, ovarian and prostate cancer, but rarely for TNBC (15,16). In the present study, a subgroup of patients with TNBC from the Veterans General Hospital TAipei-Yung-Ling foundation sinO-canceR (VGH-TAYLOR) study was used (17). TNBC samples were initially assayed using a medium-sized CGP panel, and the remaining specimens of nucleic acid were re-sequenced with a large-sized GCP panel. The aim of the present study was to investigate whether a larger CGP panel offers clinically significant advantages in detecting actionable variants for TNBC management compared with a medium-sized panel.
Discussion
To achieve the goal of personalized and precision medicine, CGP has been recognized as a key tool that can potentially transform cancer risk prediction, detection, diagnosis, treatment and monitoring. CGP can augment clinical decision-making in the form of either in vitro diagnostics (IVD) or companion diagnostics. Most importantly, the continuous price reduction across small- to large-sized CGP panels has improved patient access (35). Consequently, therapy assignments for newly diagnosed breast cancers and monitoring of patients who are in treatment are expected to benefit from CGP.
In the context of CGP, actionable genes refer to specific genetic alterations or mutations in a patient's (tumor) DNA that can guide therapeutic decisions or clinical interventions. These genetic changes are considered ‘actionable’ because they suggest potential treatments or clinical strategies that can directly impact the patient's care. This may include targeting specific mutations with precision medicine therapies, selecting clinical trials based on the genomic profile, such as basket or umbrella trials, or monitoring the patient for disease progression. Actionable genes typically include those that are associated with known therapies (such as targeted therapies and immunotherapies, including PARP inhibition for pathogenic BRCA1/2 mutations), have established clinical guidelines for intervention based on their presence, or may offer prognostic or predictive value for disease outcomes, guiding treatment options (1-5). In the present study, the performance of medium- to large-sized gene panels in a TNBC patient cohort was compared. Both OCP and TSO500 are used worldwide, but there are few direct comparison studies for breast cancer actionable genes in the literature (18,19,36,37).
The four HER2-positive breast cancer cases provided an opportunity to evaluate the association between clinical HER2-status and NGS-based ERBB2 CNVs. None of these cases were reported as HER2-amplified by OCP while two of three HER2-amplified cases reported by TSO500 coincided with the four clinically HER2-positive breast cancers. A higher number of HER2-gained breast cancers were identified by TSO500, indicating the potential for anti-HER2 targeting therapy. As NTRK translocation was not listed as the targeted alteration of OCP, the detectability of structure variants (CNV and fusion) was not compared further.
In the present study, the genes and variants reported from either OCP v3 or TSO500 when genomic, cDNA and protein coordinates were used for variant sorting were detailed. This subset represented the most actionable variants revealed by medium- and large-sized CGP. AKT1 is an intra-cellular kinase and is predominantly altered in breast and endometrial cancer. The AKT1 E17K is the most common alteration across various tumor types, including breast and gynecological cancers (38,39). The CAPItello-291 phase III trial demonstrated that capivasertib (AZD5363) and fulvestrant combination therapy resulted in markedly longer progression-free survival compared with the placebo and fulvestrant groups (40). The three AKT1 E17K-mutant cases in the present study were identified by both panels.
There were two BRCA1 (S405* and R1203*) and one BRCA2 (S521*) truncating mutations, and reflex germline testing should precede if PARP inhibitor was considered for these patients, as only germline BRCA1/2 mutations are actionable in breast cancer (41). The BRCA1 c.5470-1G>A SNP has been recognized as pathogenic by both the Breast Cancer Information Core and Consortium of Investigators of Modifiers of BRCA1/2 (42,43). The BRCA2 S521* truncating mutation impairs nuclear localization of BRCA2, which is essential for normal BRCA2 function (44). Despite common truncating and frameshift mutations (five for BRCA1 and three for BRCA2) called by both panels, only TSO500 revealed more suspicious variants (eight variants among 9 cases for BRCA1 and 13 variants among 21 subjects), while OCP was flawed by spurious mutations (BRCA1: S1180fs and T1376fs; and BRCA2: S538fs, T598fs, S973fs, E33*, T912fs, S3041fs and S3147fs).
PALB2 K353fs is a truncating mutation in a tumor suppressor gene and therefore is likely oncogenic. The phase II TBCRC 048 study also indicated the predictive power of the germline PALB2 mutation for metastatic breast cancer, as a high response rate (82%) was observed when olaparib, a PARP inhibitor, was used (45). In the present study, TSO500 identified three additional variants in 5 cases while OCP reported 1 false positive case with up to four variants (D1125fs, N368fs, S357fs and N342fs). Tumor-only sequencing made differentiating germline from somatic BRCA1/2 and PALB2 mutations challenging. However, LOH-germline inference calculator and the somatic-germline-zygosity algorithms helped distinguish these mutations, with one somatic and eight germline BRCA1/2 mutations identified (46).
One patient harbored two mutations in ERBB2 (L811V and L839R) and another with the L725S mutation was recognized by both platforms, none of which were among the oncogenic mutations approved by U.S. Food and Drug Administration for the use of neratinib, a pan-HER kinase inhibitor that binds irreversibly to the ATP-kinase domain of HER2 inhibiting the downstream phosphorylation of AKT and MAPK (47). TSO500 identified three additional ERBB2 mutations (P1140A, V743_M744insHV and Y742_A745dup). It is important to note that TSO500 detected more complex mutations (such as Y742_A745dup and V743_M744insHV, both classified as VUS) than did OCP.
Certain PIK3CA hotspot mutations have been identified from the SOLAR-1 trial for the usage of alpelisib, a selective PI3K-α inhibitor, namely C420R, E542K, E545A, E545D, E545G, E545K, Q546E, Q546R, H1047L, H1047R and H1047Y (48). Our previous study also observed double mutations of PIK3CA among the Taiwanese population (21). Only four cases with E726K, G1049R, N345I and Q546R mutations were outside the hotspot region, while two cases with D350N or D725N were co-mutant with other hotspots, which were only reported by TSO500. Consequently, both platforms identified the same number of PIK3CA-altered breast cancer cases.
Conversely, selective PI3K-β inhibitors, GSK2636771 and AZD8186, are ATP competitors and have shown preclinical antitumor response and durability for PTEN-deficient solid tumors including TNBC (49). The PTEN c.1026+1G>A SNP has been reported as clinically pathogenic at least twice (Invitae Clinical Genomics Group and ClinGen PTEN Variant Curation Expert Panel), with germline origin (50,51). The remaining five consensual variants (E150*, E43fs, P38fs, R130* and Q245*) were truncating mutations; TSO500 identified two additional cases with PTEN mutations (G127_G129del and V290Sfs*8) and OCP called one inappropriate variant due to misalignment (c.386G>A).
Quite a few TP53 alterations were identified by both OCP and TSO500. Despite not being currently druggable, TP53 may act as an independent prognostic factor for early and advanced-stage cancer with a wide range of mutational frequency (52). It has been argued that all TP53 mutations from tumor-only sequencing are somatic, and rarely representative of the germline Li-Fraumeni syndrome (53). TSO500 identified more variants than OCP did, while the latter reported three cases with V73fs in homopolymer region and one case with misaligned V73fs mutation, both of which were spurious.
A total of 272 variants were identified among actionable genes and TP53 from at least one panel. Interestingly, the concordance rate from original reports was low at slightly more than one-third (34.6%). With extensive bioinformatics analyses and manual curation, three-fifths of concordance (58.9%) were achieved after exclusion of unfiltered polymorphisms, low-VAF variants and those beyond the targeted scope of OCP. Among inconsistent variants, some were identical after manual inspections, further indicating the necessity of analytical abilities and domain knowledge in genomic nomenclature (54). Furthermore, an additional 62 variants were revealed by TSO500, indicating missed opportunities by OCP, which may be explained by fundamental discrepancies in sequencing technology. The accuracy of the amplicon-based OCP vs. the hybrid capture-based TSO500 panel can be influenced by their underlying methodologies. Amplicon-based methods utilize PCR amplification with sequence-specific primers to enrich target regions. By contrast, hybrid capture sequencing employs hybridization-based capture to enrich for desired genomic regions. Hybrid capture may offer a more robust and accurate approach, especially for clinical applications (55).
In the context of TNBC research, a VAF cut-off of #x003C;10% is used to filter out low-confidence or likely spurious SNVs identified through NGS of tumor samples (56,57). The threshold may be beneficial to eliminate technical, background noise and subclonal mutations, as tumor samples can be contaminated with normal cells, and low-level DNA damage or degradation can introduce background noise that interferes with accurate SNV calling. In addition, tumors are often composed of multiple subclones with distinct genetic profiles. SNVs present in minor subclones may have VAFs #x003C;10%, making them difficult to distinguish from background noise (58). A VAF cut-off of 5-10% is commonly used in various cancer genomics studies, including those focused on TNBC. This range is considered to achieve a reasonable balance between sensitivity (detecting true SNVs) and specificity (excluding false positives) (59).
Despite being one of the pioneers to directly compare two commercialized targeted panels, there were some limitations to the present study. First, sequencing was not conducted concurrently, and the 1-2 year delay of the TSO500 analysis following OCP may have introduced some bias from nucleic acid degradation, despite all samples being stored under temperature-controlled conditions. Second, variants called by each panel were considered based on the standard or formal algorithm of each platform (Ion Reporter/Oncomine Knowledgebase Reporter for OCP and PierianDx for TSO500), which limited the comparability between distinct platforms. For unbiased comparison, the same aligner, caller and annotator should be applied. However, browser extensible data files were unavailable from manufacturers to confirm the jointly interrogated regions. On the other hand, all commercial CGP solutions are under regulation as either laboratory developed tests or IVD, and practically these pre-set algorithms should not be modified arbitrarily to enhance reproducibility. Despite this, the present study conducted an exhaustive bioinformatic analysis from BAM files to dissect the conflicting results from the same samples. Third, NTRK is not within the targeted fusion genes of OCP; therefore, a comparison with TSO500 was not possible. Moreover, NTRK sequence variants, amplifications or fusions are not actionable for breast cancer. As larotrectinib and entrectinib are approved in a number of countries, NTRK fusion as a tumor-agnostic marker is important, and its detection should be incorporated into CGP for breast cancer (60). Fourth, as most cases are triple negative phenotypically, a cohort of ~100 patients with TNBC is suitable for a comparative sequencing panel study. However, if the goal is to perform more detailed subgroup analyses or identify rare mutations, a larger cohort may be more appropriate. In the future, conducting a prospective and parallel study across distinct CGP platforms could reveal whether larger sequencing panels justify their higher costs by improving the detection of actionable mutations, refining novel therapeutic strategies, and characterizing the complex genomic landscape of TNBC. Furthermore, it would provide critical insights to optimize genomic profiling for other breast cancer subtypes, advancing both research and clinical care.
As an observational study, the present study could not directly assess the impact of CGP on treatment outcomes, as no intervention was performed. During the study period, the compassionate use of alpelisib (a selective PI3K inhibitor) benefited nine patients, and the out-of-pocket use of olaparib (a PARP inhibitor) benefited two patients, demonstrating the actionability of the testing. Since May 2024, NGS, specifically whole-exome sequencing for BRCA1/2, has been reimbursed in Taiwan for patients with stage II or higher TNBC. The Regulations of Special Medical Techniques of Taiwan require that all NGS testing results (including both whole-exome sequencing and targeted panels) be submitted to the National Health Research Institutes Biobank. It is anticipated that cost-effective implementation of larger NGS panels in routine clinical practice will be possible in the future, once sufficient data from NGS results and accompanying clinical outcomes are available.
In conclusion, the present study comparing medium- and large-sized sequencing panels in a cohort of patients with TNBC may provide critical insights into the balance between actionable gene findings, cost and complexity. TSO500, the larger panel, detected more variants than OCP did, even from the same set of ESCAT-defined actionable genes and TP53. Conversely, a proportion of inconsistent variants could be manually curated and were identical with aliasing coordinates or starting positions. Finally, there were variants detectable only by TSO500, indicating potential and fundamental differences in sequencing technology, bioinformatic algorithms and variant filtering. The value of a large-sized panel in clinical usage is ascertained, considering the beneficiaries of PARP inhibition for the higher number of BRCA1/2 and PALB2 mutations detected, as indicated by the National Comprehensive Cancer Network and ESMO guidelines (61,62). Given the experience from the present study, the updated Oncomine Comprehensive Assay Plus with >500 genes profiled is anticipated in future studies (63). The results could guide panel selection for precision oncology, ensuring optimal clinical outcomes while maximizing resource efficiency. This approach would be particularly impactful in refining strategies for TNBC management.