Harnessing Institutionally Developed Clinical Targeted Sequencing to Improve Patient Survival in Breast Cancer: A Seven-Year Experience

Jiwon Koh; Jinyong Kim; Go-Un Woo; Hanbaek Yi; So Yean Kwon; Jeongmin Seo; Jeong Mo Bae; Jung Ho Kim; Jae Kyung Won; Han Suk Ryu; Yoon Kyung Jeon; Dae-Won Lee; Miso Kim; Tae-Yong Kim; Kyung-Hun Lee; Tae-You Kim; Jee-Soo Lee; Moon-Woo Seong; Sheehyun Kim; Sungyoung Lee; Hongseok Yun; Myung Geun Song; Jaeyong Choi; Jong-Il Kim; Seock-Ah Im

doi:10.4143/crt.2024.296

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Original Article Harnessing Institutionally Developed Clinical Targeted Sequencing to Improve Patient Survival in Breast Cancer: A Seven-Year Experience: Jiwon Koh^1,2, Jinyong Kim^2,3, Go-Un Woo³, Hanbaek Yi³, So Yean Kwon³, Jeongmin Seo³, Jeong Mo Bae¹, Jung Ho Kim¹, Jae Kyung Won¹, Han Suk Ryu^1,2, Yoon Kyung Jeon^1,2, Dae-Won Lee^2,3, Miso Kim^2,3, Tae-Yong Kim^2,3, Kyung-Hun Lee^2,3, Tae-You Kim^2,3, Jee-Soo Lee⁴, Moon-Woo Seong⁴, Sheehyun Kim⁵, Sungyoung Lee⁵, Hongseok Yun⁵, Myung Geun Song⁶, Jaeyong Choi⁷, Jong-Il Kim^2,7, Seock-Ah Im^2,3,; DOI: https://doi.org/10.4143/crt.2024.296
Published online: August 21, 2024

¹Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea

²Cancer Research Institute, Seoul National University, Seoul, Korea

³Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea

⁴Department of Laboratory Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea

⁵Department of Genomic Medicine, Seoul National University Hospital, Seoul, Korea

⁶Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea

⁷Genomic Medicine Institute, Medical Research Center, Seoul National University, Seoul, Korea

Correspondence: Seock-Ah Im, Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea
Tel: 82-2-2072-0850 E-mail: moisa@snu.ac.kr

*Jiwon Koh and Jinyong Kim contributed equally to this work.

• Received: March 23, 2024 • Accepted: August 18, 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abstract

Purpose
Considering the high disease burden and unique features of Asian patients with breast cancer (BC), it is essential to have a comprehensive view of genetic characteristics in this population. An institutional targeted sequencing platform was developed through the Korea Research-Driven Hospitals project and was incorporated into clinical practice. This study explores the use of targeted next-generation sequencing (NGS) and its outcomes in patients with advanced/metastatic BC in the real world.
Materials and Methods
We reviewed the results of NGS tests administered to BC patients using a customized sequencing platform—FiRST Cancer Panel (FCP)—over 7 years. We systematically described clinical translation of FCP for precise diagnostics, personalized therapeutic strategies, and unraveling disease pathogenesis.
Results
NGS tests were conducted on 548 samples from 522 patients with BC. Ninety-seven point six percentage of tested samples harbored at least one pathogenic alteration. The common alterations included mutations in TP53 (56.2%), PIK3CA (31.2%), GATA3 (13.8%), BRCA2 (10.2%), and amplifications of CCND1 (10.8%), FGF19 (10.0%), and ERBB2 (9.5%). NGS analysis of ERBB2 amplification correlated well with human epidermal growth factor receptor 2 immunohistochemistry and in situ hybridization. RNA panel analyses found potentially actionable and prognostic fusion genes. FCP effectively screened for potentially germline pathogenic/likely pathogenic mutation. Ten point three percent of BC patients received matched therapy guided by NGS, resulting in a significant overall survival advantage (p=0.022), especially for metastatic BCs.
Conclusion
Clinical NGS provided multifaceted benefits, deepening our understanding of the disease, improving diagnostic precision, and paving the way for targeted therapies. The concrete advantages of FCP highlight the importance of multi-gene testing for BC, especially for metastatic conditions.
Key words: Breast neoplasms, Next-generation sequencing, Mutation, Copy number variation, Fusion gene, Hereditary cancer, Cancer evolution, Precision diagnosis, Precision medicine

Introduction

The introduction of next-generation sequencing (NGS) technology [1] has brought significant advancement in the field of clinical oncology and precision medicine [2-5]. In 2022, the American Society of Clinical Oncology recommended that all patients with advanced or metastatic solid tumors should undergo somatic genomic testing using multigene panel-based assays [6].

In March 2017, the Republic of Korea became one of the first countries to provide national insurance coverage for NGS testing in patients with advanced or metastatic cancer. Since then, tumor NGS tests have been widely performed, reaching 51,407 insurance claims from 2017 to 2021 [7]; however, panel sequencing on breast cancer (BC) was performed in 2,492 cases, accounting for only 4.8% of all solid tumor NGS tests [7]. BC is one of the most common types of cancer in Korea, accounting for 10.1% of all cancers [8]. Specifically, BC is the most common cancer and the third most common cause of cancer-related death in Korean women [8]. In contrast, the use of NGS as a clinical decision-making tool for BC patients in Korea is limited in both quantity and accessibility.

Korean BC patients exhibit unique characteristics that distinguish them from their Western counterparts [9-11]. Characterized by a younger age at diagnosis, a higher incidence of premenopausal onset, and distinct mutational profiles, these differences underscore the critical need for a dedicated genomic database for Korean patients with BC. A few retrospective studies on the clinical NGS in Korean BC patients have been reported [12,13], however, the numbers of sequenced cases were limited in amount, and the clinical benefit of NGS other than targeted treatment was not fully addressed in detail.

As a part of a national program named Korea Research-Driven Hospitals (KRH) project, Seoul National University Hospital (SNUH) initiated a multidisciplinary approach to develop a customized and institutionally validated targeted sequencing platform. Ever since this institutionally developed NGS panel came into practice in October 2016, BC has been one of the most common types of cancer interrogated by NGS in SNUH, and we established one of the largest genomic datasets of Korean patients with BC.

In this study, we comprehensively reviewed the results of institutionally developed NGS panel assays conducted on individuals with BC at a large tertiary health system in the Republic of Korea over 7 years. We then systemically described the clinical benefits provided by the clinical NGS: understanding pathogenesis, aiding diagnosis, personalizing treatment, and predicting prognosis.

Materials and Methods

1. Study population

Patients aged 20 years or older who were diagnosed with advanced or metastatic BC and underwent NGS in SNUH from October 2016 to July 2023 were enrolled. Staging was determined according to the latest TNM classification by the American Joint Committee on Cancer (AJCC) at the time of diagnosis, and clinical staging was used for the patients who had neoadjuvant therapy. All clinicopathological information was retrieved by reviewing electronic medical records, and patients’ survival data were obtained from the Ministry of Public Administration and Security in the Republic of Korea.

While most of the patients were tested by NGS once, a subset of patients in our study underwent clinical NGS tests more than once; this was accomplished as a part of the KRH Project in some, and the others were performed under the reimbursement policy which allowed the second NGS test at the time of disease progression or treatment refractoriness.

2. FiRST Cancer Panel: institutionally developed, internally validated panel

Medical oncologists, pathologists, bioinformatics specialists, and medical geneticists in SNUH collaborated to develop a customized and institutionally validated targeted sequencing platform. As a result, FiRST Cancer Panel (FCP) service finally came into clinical practice in October 2016. In 2017, FCP was readily incorporated into conditional coverage by the National Health Insurance Service (NHIS) of Korea.

The first panel to be in service was FCP ver. 2.0, which covered 148 genes. FCP service was provided upon clinicians’ requests following patients’ informed consent. The results were then discussed at molecular tumor boards (MTB) held twice a month. Our institution continued to collaborate in upgrading and validating the panel, with the latest version, ver. 4.0, being released in January 2022. The updated version now includes 336 genes by DNA panel and 151 genes by RNA panel. The list of genes in each version and the major updated features are summarized in the S1 Table. Details of targeted sequencing and analytic methods are provided in the Supplementary Method.

3. Immunohistochemistry and ERBB2 in situ hybridization

The results of immunohistochemistry (IHC) were retrospectively reviewed from the medical records and pathology reports. The following antibodies were used for IHC: estrogen receptor (ER; clone 6F11, Novocastra Laboratories), progesterone receptor (PR; clone 16, Novocastra Laboratories), human epidermal growth factor receptor 2 (HER2; clone 4B5, Ventana Medical Systems), and Ki-67 (clone MIB-1, Dako/Agilent). Positive stainings for ER or PR in ≥ 1% of tumor nuclei were interpreted as positive [14].

HER2 IHC was interpreted according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2018 guidelines [15]. ERBB2 in situ hybridization (ISH) was performed for the samples with equivocal (2+) results of HER2 IHC or upon clinicians’ requests. Fluorescent in situ hybridization was performed using PathVysion HER-2 DNA Probe Kit (Abbott Molecular) from 2016 to March 2022, and silver in situ hybridization was performed using INFORM Her2 Dual ISH probe (Ventana Medical Systems) from April 2022 to July 2023. ISH was interpreted according to the ASCO/CAP 2018 guidelines [15].

4. Statistical methods

Categorical variables were summarized with the frequencies and percentages. Continuous variables were represented with the median values and ranges. Categorical variables were compared by chi-square test or Fisher’s exact test and continuous variables were analyzed by Mann-Whitney or Kruskal-Wallis tests, as appropriate. We used the Benjamini-Hochberg method to correct for multiple comparisons when necessary.

Disease-free survival (DFS) was defined as time from curative surgery to recurrence or metastasis. Progression-free survival (PFS) was defined as the time from the initiation of one line of treatment to the progression of disease after that specific treatment. The overall survival (OS) was evaluated in metastatic BC and was designated as the time from the first diagnosis of metastasis to death from any cause. The survival curves were estimated using the Kaplan-Meier method and compared by log-rank test. For live patients, the survival was censored at the latest date of follow-up. A p-value < 0.05 was considered statistically significant.

Results

1. Clinicopathological characteristics of the study population

Clinical NGS was performed using 548 samples from 522 patients with BC. Characteristics of the 522 patients are summarized in Table 1. Only three patients (3/522; 0.6%) were male, and the median age at diagnosis was 47 years (range, 20 to 76 years). 63.8% of the patients were diagnosed with locally advanced stages II-III, while 23.4% presented with de novo stage IV.

Of 548 tumor samples subjected to FCP, the most common histological subtype was invasive ductal carcinoma (IDC) (91.2%), followed by invasive lobular carcinoma (ILC) (3.5%), and rare subtypes including mucinous, metaplastic, and salivary gland type carcinomas. Sequenced tumors were mostly ER+/HER2– BCs (53.1%), followed by triple-negative BCs (TNBCs) (35.4%), ER–/HER2+ BCs (6.4%), and ER+/HER2+ BCs (5.1%). Tissues for the NGS were obtained from various sites, including 45.1% from primary BC, 9.1% from regional, 2.6% from distant lymph nodes, and 43.2% from distant metastatic organs. Within 6 months before the tissue acquisition, 64.1% of the patients received antitumor treatments, including chemotherapy (31.8%), endocrine therapy (29.0%), targeted therapy (17.0%), and immuno-oncologic drugs (2.4%). The median block age of the samples used for NGS tests was 2.3 months, ranging from 0 to 105 months. 52.4% of the tests were performed with samples collected within 6 months.

2. Mutational profiles of clinical NGS with BC

In 548 samples, a total of 1,701 pathogenic alterations in 144 genes were identified (Fig. 1). Ninety-seven point six percent (535/548) of the tested samples harbored at least one pathogenic alteration, and 70.6% had at least one actionable biomarker annotated by OncoKB [16].

The commonly mutated genes were TP53 (56.2%), PIK3CA (31.2%), GATA3 (13.8%), BRCA2 (10.2%), KMT2C (8.8%), PTEN (8.6%), BRCA1 (7.3%), CDH1 (6.4%), ESR1 (5.5%), and ARID1A (5.3%). Recurrent amplifications were observed in CCND1 (10.8%), FGF19 (10.0%), ERBB2 (9.5%), MYC (9.3%), and FGFR1 (8.0%). None of the tested samples was microsatellite instability–high, and the median tumor mutational burden (TMB) was 5.33 per megabase pair (range, 0.0 to 32.79).

We found distinct genetic alterations in each BC subtype. TP53 mutations were the most frequent in TNBC tumors (83.5%), followed by 76.2% of HER2+ and 33.7% of ER+/HER2– tumors. ESR1 mutations were found exclusively in the ER+/HER2– tumors (30/291; Fisher’s exact test, p < 0.001). Of note, ESR1 mutations were significantly enriched in BCs with prior exposure to aromatase inhibitors (AI) (22.6%, 21/93 in prior AI-treated BCs compared to 4.5%, 9/198 in the patients without prior AI exposure; Fisher exact test, p < 0.001) (S2 Fig). Pathogenic alterations of the homologous recombination repair–associated genes were found in 129 (23.6%) tumors and were significantly enriched in TNBC (56/194, 28.9%) compared to the other subtypes (73/354, 20.6%) (chi-square, p=0.030). We found no significant difference in TMB among BC subtypes (Kruskal-Wallis, p=0.114).

We found 40 tumors with pathogenic BRCA1 mutations of which 32 (80.0%) were confirmed as germline variants by blood tests. BRCA1 mutations were significantly enriched in the TNBC subtype (29/40, 72.5%; chi-square, p < 0.001) compared to ER+/HER2– tumors (11/40, 27.5%). None of the BCs with BRCA1 mutations were HER2+. Among 56 tumors with BRCA2 mutations, 80.4% (45/56) were confirmed as germline. Sixty-seven point nine percentage (38/56) of BRCA2 mutated samples were ER+/HER2–, 23.2% (13/56) were TNBC, and 8.9% (5/56) were HER2+. We also found tumors harboring other germline likely pathogenic/pathogenic mutations involving BC predisposing genes: PALB2 (2/82, 2.4%), TP53 (2/82, 2.4%), and PTEN (1/82, 1.2%). Mutational profiles of the BCs with germline predispositions are summarized in the S3 Fig.

To gain further insights into the distinctive genetic characteristics of Korean BC patients, we conducted a comparative analysis of genetic alterations between our dataset and a Western dataset reported by Razavi et al. [17] from Memorial Sloan Kettering Cancer Center (MSKCC). Key findings are described in the S4 Table. Significant enrichments in the patients from SNUH included TP53 mutations (56.2% in SNUH vs. 35.5% in MSKCC, adjusted p < 0.001), BRCA1 mutations (7.3% in SNUH vs. 1.4% in MSKCC, adjusted p < 0.001), and BRCA2 mutations (10.2% in SNUH vs. 3.3% in MSKCC, adjusted p < 0.001). MAP3K1, CDH1, FOXA1, PIK3CA, ERBB2, ESR1 mutations, and CCND1, FGF19, and FGFR1 amplifications were more frequently found in the MSKCC cohort (adjusted p < 0.05).

3. Diagnostic performance of FCP in determining the HER2 status

ERBB2 amplification is by far the most important copy number variation (CNV) in BC in terms of therapeutic and prognostic implications. To assess the diagnostic performance of the NGS panel in determining HER2 status, we compared the conventionally defined HER2 status by IHC and ISH with ERBB2 amplifications by FCP (Table 2). The concordance rate was 96.7% with only 18 samples (3.3%) having discordant results between the two methods, with the sensitivity, specificity, positive predictive, and negative predictive values of 74.6%, 99.6%, 95.9%, and 96.8%, respectively. The ERBB2 copy number (CN) estimated by NGS were significantly higher in the samples with HER2 IHC 3+ compared to 2+ (Mann-Whitney, p=0.001) (Fig. 2A) and showed significant correlation when compared to the ERBB2 CN assessed by ISH method (p=0.042; Pearson coefficient=0.594) (Fig. 2B).

4. Identifying gene fusions with clinical implications

Since the introduction of ver. 4.0 in 2022, the RNA panel gave us a better view of fusion events in patients with BC. In our study population, fusion genes were identified in nine patients (S5 Table). ETV6::NTRK3 fusion was detected in a patient with secretory carcinoma, which has both diagnostic and therapeutic implications. Other potentially targetable fusions included TRIM56::BRAF and RBPMS::NRG1 fusions. Although partner genes have not been reported in the literature, these fusion products were expected to have intact tyrosine kinase and epidermal growth factor–like domains respectively, thus potentially targetable.

We also found a patient with ER+/HER2– ILC harboring ESR1::NBAT1 fusion, which was predicted to have a truncated ligand binding domain (LBD) of ESR1 (Fig. 3A). The patient (case No. 225) experienced disease progression with endocrine therapy, possibly suggesting secondary endocrine resistance. We also report the first detailed human cases of BCL2L14::ETV6 fusion with aggressive TNBC and clinical resistance to conventional chemotherapy (Fig. 3B). Case No. 205 was a 30-year-old female diagnosed with locally advanced TNBC who received window of opportunity trial chemoimmunotherapy with olaparib and durvalumab followed by standard care of neoadjuvant chemotherapy doxorubicin+cyclophosphamide followed by docetaxel, and then curative surgery. Despite the partial clinical response before surgery, this patient could not achieve a pathologic complete response and relapsed with multiple lung metastases with DFS of 1 year and 3 months. Case No. 296 was a 43-year-old premenopausal woman who initially presented with extensively metastatic BC involving the axial skeleton. Despite palliative chemotherapy with docetaxel and cisplatin, the disease rapidly progressed after 3 months with diffuse leptomeningeal seeding and bone metastases. After she received sacituzumab govitecan as salvage treatment for 3 months, the leptomeningeal seeding and extensive bone metastases were improved.

5. Bridge to the genetic counseling

With the release of FCP ver. 4.0 in early 2022, we began suggesting additional work-up and genetic counseling through clinical genomic medicine specialists when reporting possible germline pathogenic mutations in cancer susceptibility genes. The suggestions were made in 11.7% (21/180) tests and resulted in the actual clinical actions in 17 patients. Eleven patients were confirmed as the carriers of germline likely pathogenic/pathogenic variants and received genetic counseling. The other six patients were found to have somatic mutations, obviating the need for additional testing of family members.

Importantly, seven out of 11 aforementioned patients who harbored germline likely pathogenic/pathogenic BRCA1/2 mutations were not eligible for insurance reimbursed germline mutations tests under the current Korean health policy. The results of FCP in these seven patients prompted accurate genetic diagnosis and adequate clinical management. One example with germline predisposed BC initially identified by FCP was a 67-year-old woman who presented with metastatic ER+/HER2– BC after 5.5 years of disease-free interval. Clinical NGS revealed two PALB2 mutations along with TP53 and PIK3CA mutations. Two PALB2 mutations were found in the different sequencing reads (Fig. 4A), suggesting bi-allelic inactivation of PALB2. This result prompted confirmatory germline testing, which revealed the germline origin of the PALB2 H276fs mutation. The patient was enrolled in a clinical trial to receive matched therapy with a poly(ADP-ribose) polymerase (PARP) inhibitor olaparib and had a remarkable metabolic response of multiple bone metastasis after 3 months (Fig. 4B) and currently maintaining stable disease by Response Evaluation Criteria in Solid Tumor ver. 1.1 for 1 year and 4 months.

6. Clues to the pathogenesis and clonal evolution of BC

Out of 522 patients, 24 patients (4.6%) were tested twice, and one patient underwent sequencing of multiple lesions. Among the 24 patients with serial tests, clinically meaningful differences were observed in five patients (5/24, 20.8%). Two patients acquired ESR1 mutations following endocrine therapy (AI), and the other two patients exhibited intertumoral heterogeneity. The remaining patient was an interesting case that acquired somatic BRCA1 reversion mutations in the absence of prior PARP inhibitor therapy (Fig. 4C).

Multi-regional sequencing was performed on a patient diagnosed with a rare mixed ER+/HER2– IDC and metaplastic carcinoma (spindle cell carcinoma) (Fig. 5). The spindle cell carcinoma component of the resected breast tumor and the IDC component of the metastatic axillary lymph nodes were separately taken for NGS. Both tumors shared PIK3CA, PTEN, and KMT2C mutations, indicating a potential shared ancestral origin despite their morphological differences. Interestingly, distinctive genetic variations were also identified; two different TP53 mutations were detected in the two components, whereas TERT promoter mutation and amplifications of JAK1 and CDK4 were exclusively present within the spindle cell carcinoma component. Additional NGS was done in brain metastasis in which the genetic profiles were identical to the spindle cell carcinoma of the breast, suggesting evidence of clonal evolution in rare cancer.

7. Supplementary test for the accurate diagnosis of rare BC subtype

FCP can aid in diagnosing rare types of BC, like sarcomas, by detecting specific genetic alterations. We have experienced a 53-year-old woman who was initially diagnosed with metaplastic carcinoma with heterologous mesenchymal differentiation by histopathologic analysis (S6 Fig.). FCP was performed and revealed mutations in SUZ12 and NF1. Complete loss of H3K27me3 was confirmed by IHC, leading to a revised diagnosis of malignant peripheral nerve sheath tumor (MPNST).

8. Broadening the therapeutic options including matched therapy and clinical trials

Of 522 patients, 53 (10.2%) received matched therapies or participated in clinical trials based on biomarkers identified from NGS data across various lines of treatment (Table 3). Six patients (1.1%) were enrolled in two trials. Eligible alterations for matched therapy and trials included BRCA1 or BRCA2 mutations (3.6%), ERBB2 amplification (2.7%), PIK3CA mutation (2.1%), high TMB (1.0%), alterations of homologous recombination (HR)–related genes (0.8%), FGFR1 alteration (0.6%), AKT1 mutation (0.4%), and ERBB2 mutation (0.2%). Median PFS and OS starting from day 1 of treatment in this population were 4.7 months and 39.2 months, respectively (Fig. 6A and B).

To determine if FCP-guided matched therapy improved survival in BC, we compared OS from the first diagnosis of metastatic disease in patients who underwent matched therapy versus those who did not. Notable differences in OS were observed between the groups (log-rank, p=0.022) (Fig. 6C), indicating that NGS utilization offers tangible benefits to BC patients. One example was a 56-year-old patient (case No. 372) with ER+/HER2– metastatic BC who experienced disease progression over 10 years despite various treatments. After undergoing FCP, a somatic ATM mutation was detected. Consequently, the patient entered a talazoparib trial and showed a partial response within three months (Fig. 6D), remaining progression-free for 16.3 months.

We further investigated the prognostic factors from the FCP results of metastatic BCs (S7 Fig). It was evident that the presence of a TP53 mutation correlated significantly with adverse outcomes of metastatic BCs (log-rank, p < 0.001). Among the TNBC population, mutations in HR-related genes were linked to a more favorable prognosis with marginal significance (log-rank, p=0.080). This trend might stem from the available therapeutic options for DNA damage repair pathway including PARP inhibitors among metastatic TNBCs with HR-related gene mutations compared to the rest (28.1% vs. 5.7%; Fisher’s exact, p=0.001).

Discussion

The comprehensive clinical advantages of FCP within a major tertiary healthcare institution are highlighted in this study. While previous studies mainly captured the broad landscape of genetic alterations, we focused on the direct translation of NGS findings into enhanced clinical outcomes.

We confirmed previously reported unique characteristics of East Asian BC patients including a significantly higher frequency of TP53 mutations [5,18] and a higher prevalence of germline BRCA1/2 mutations [19], especially BRCA2 [20]. The lower frequencies of PIK3CA, CDH1, FOXA1, and ERBB2 mutations observed in our study may be attributed to the lower prevalence of ER+ BCs and ILCs in our population, compared to those reported in the MSKCC dataset [17]. ESR1 mutations were detected in 5.5% of our study population, which is lower compared to the MSKCC dataset but consistent with the 6.1% reported by a recent study from a tertiary hospital in Korea. This observation may also be due to the epidemiological differences in Asian BC patients, characterized by younger age, a higher proportion of premenopausal patients, and consequently, less frequent use of AIs in adjuvant endocrine therapy [21]. This is corroborated by our subgroup analysis, which revealed that ESR1 mutations were more prevalent among patients with prior AI exposure.[20]. The lower frequencies of PIK3CA, CDH1, FOXA1, and ERBB2 mutations observed in our study may be attributed to the lower prevalence of ER+ BCs and ILCs in our population, compared to those reported in the MSKCC dataset [17]. ESR1 mutations were detected in 5.5% of our study population, which is lower compared to the MSKCC dataset but consistent with the 6.1% reported by a recent study from a tertiary hospital in Korea. This observation may also be due to the epidemiological differences in Asian BC patients, characterized by younger age, a higher proportion of premenopausal patients, and consequently, less frequent use of AIs in adjuvant endocrine therapy [21]. This is corroborated by our subgroup analysis, which revealed that ESR1 mutations were more prevalent among patients with prior AI exposure.

In addition to single nucleotide variations and indels, we were able to identify oncogenic amplifications and deletions of crucial tumor suppressor genes. We found that the NGS-based determination of ERBB2 amplification—the most important CNV in BCs—is a reliable method, as similarly suggested by a recent report [22]. The specificity, positive predictive, and negative predictive values were sufficiently high, and the CN estimates of ERBB2 by NGS and ISH showed significantly tight associations. One limitation of the FCP-based test was the limited sensitivity. FCP failed to detect ERBB2 amplification in 16 HER2+ samples and the latest ver. 4.0 of FCP was responsible for only two of discordances. Further calibration of FCP are expected to improve the predictive performance of our panel.

Though genomic instability represented by CNVs and genomic rearrangements has been considered the hallmark of BC genomes [23,24], recurrent genetic fusions in BCs are less understood compared to other cancers. We examined the fusion genes in BCs by incorporating the RNA panel into FCP. ESR1 fusions with multiple partner genes have been recently reported [25], and disruption of LBD is thought to be associated with endocrine resistance. BCL2L14::ETV6 fusion was first reported in patients with aggressive TNBC in 2020 [26] and was known to show paclitaxel resistance in vitro. However, detailed clinical features of the TNBC patients harboring BCL2L14::ETV6 fusions had not been reported. Here, we reported for the first time, the detailed human cases of BCL2L14::ETV6 fusion with aggressive TNBC resistant to taxane-based chemotherapy. Our findings suggest that the identification of fusion genes through clinical NGS can assist in the stratification of BC patients for prognosis and prediction of treatment response.

FCP played a crucial role in bridging patients to genetic counseling for BC. The actual benefit provided by this multidisciplinary approach is of great importance in the Republic of Korea, for there exist strict governmental regulations regarding the genetic tests such as germline BRCA1/2 tests. In Korea, the criteria for germline BRCA1/2 testing encompass (1) family history of breast, ovarian, pancreatic, or prostate cancer; (2) BC diagnosis before age 40; (3) TNBC diagnosis before age 60; (4) bilateral BC; (5) coexisting ovarian or pancreatic cancer; and (6) male patients. More than half of the patients to whom the MTB recommended genetic evaluation were not eligible for germline tests under current health policy. This suggests that clinical tumor-only NGS tests can also act as a screening tool for identifying potential germline pathogenic variants, especially in ER-positive metastatic BCs diagnosed after the age of 40 who are not a candidate for the germline BRCA1/2 testing because of the limited insurance coverage.

Identifying certain types of genetic changes can aid in accurately diagnosing rare BCs such as sarcomas [27]. Fortunately, FCP assisted in identifying these genetic changes, making it an invaluable tool in the fight against these fatal diseases. The described case of MPNST highlights the value of clinical NGS as a supplementary diagnostic tool in daily practice.

Korea provided selective national healthcare coverage for clinical NGS testing in cases of locally advanced, metastatic, or recurrent malignant neoplasm until November 2023. For this reason, our study reflects a greater prevalence of advanced breast cancer cases. We proved that FCP-guided matched therapy improved survival in metastatic BC patients, highlighting its clinical benefit. It is worth noting that our institution provided matched therapy to a higher percentage of patients with BC compared to previous reports from Korea [12,13]. This can be attributed to regular multidisciplinary discussions during the MTB and our continuous efforts to improve the custom NGS panel. Hence, our findings demonstrate that collaborative decisions in clinical practice, informed by NGS results, optimize the use of this assay.

Taken together, our seven-year experience with the custom-made FCP panel underscores that clinical NGS brings multifaceted benefits, deepening our understanding of the disease, improving diagnostic precision, and paving the way for targeted therapeutic interventions. The concrete advantages of FCP in our institution highlight the importance of multi-gene testing for BC, especially for those with metastatic conditions. Therefore, we conclude that clinical NGS is essential for better management of BC patients and should be included in the national reimbursement system shortly.

Electronic Supplementary Material

Supplementary materials are available at Cancer Research and Treatment website (https://www.e-crt.org).

crt-2024-296_S1_Table.xlsx

crt-2024-296_S2_Fig.pdf

crt-2024-296_S3_Fig.pdf

crt-2024-296_S4_Table.xlsx

crt-2024-296_S5_Table.xlsx

crt-2024-296_S6_Fig.pdf

crt-2024-296_S7_Fig.pdf

crt-2024-296_Supplementary_Method.pdf

NOTES

Ethical Statement

This study was approved by the institutional review board (IRB) of SNUH (No 1509-047-702). An informed consent process was carried out when performing the clinical next-generation sequencing tests.

Author Contributions

Conceived and designed the analysis: Im SA.

Collected the data: Koh J, Kim J, Woo GU, Yi H, Kwon SY, Im SA.

Contributed data or analysis tools: Koh J, Kim J, Seo J, Bae JM, Won JK, Ryu HS, Jeon YK, Lee DW, Kim M, Kim TY, Lee KH, Kim TY, Lee JS, Seong MW, Kim S, Lee S, Yun H, Song MG, Choi J, Kim JI, Im SA.

Performed the analysis: Koh J, Kim J, Im SA.

Wrote the paper: Koh J, Kim J, Im SA.

Conflicts of Interest

Jiwon Koh reports receiving consultation fees from DCGen. Co., Ltd. Seock-Ah Im reports advisory role for AstraZeneca, Novartis, Roche/Genentech, Eisai, Pfizer, Amgen, Hanmi, Lilly, MSD, Daiichi Sankyo, and received research grants through institution from AstraZeneca (Inst), Pfizer (Inst), Roche/Genentech (Inst), Daewoong Pharmaceutical (Inst), Eisai (Inst), Boryung Pharmaceuticals (Inst).

Funding

This study supported by the Ministry of Health and Welfare, Republic of Korea [grant number: HR14C0003].

Fig. 1.

The genetic profile of breast cancer patients unveiled by FiRST Cancer Panel. The overall distribution of commonly detected mutations, copy number variations, fusions, as well as microsatellite instability scores and tumor mutational burden are presented alongside clinicopathological features. CTx, chemotherapy; ET, endocrine therapy; ER, estrogen receptor; IDC, invasive ductal carcinoma; HER2, human epidermal growth factor receptor 2; ILC, invasive lobular carcinoma; IO, immuno-oncology; MSI, microsatellite instability; TMB, tumor mutational burden; TNBC, triple-negative BCs; Tx, therapy.

Fig. 2.

ERBB2 copy number assessment by FiRST Cancer Panel compared to human epidermal growth factor receptor 2 (HER2) immunohistochemistry and in situ hybridization. (A) ERBB2 copy numbers were higher in tumors with HER2 immunohistochemistry (IHC) 3+ compared to HER2 IHC 2+. (B) ERBB2 copy number assessed by FiRST Cancer Panel showed a significant correlation with the results of HER2 in situ hybridization. CN, copy number; ISH, in situ hybridization; NGS, next-generation sequencing.

Fig. 3.

Fusion genes identified by FiRST Cancer Panel. (A) The novel ESR1::NBAT fusion gene is expected to cause the distruption of ligand binding domain of ESR1. (B) Fusion gene structures and clinical course of the patients harboring BCL2L14::ETV6 fusions are presented Case No. 205 was a 30-year-old female diagnosed with locally advanced triple-negative breast cancer. The lesions in the right breast, right axillary lymph nodes, and internal mammary lymph nodes (arrows in the first image) decreased after the neoadjuvant treatment with olaparib+durvalumab window of opportunity treatment followed by standard neoadjuvant therapy consisting of doxorubicin+ cyclophosphamide followed by docetaxel (arrow in the second image). However, the disease recurred with lung, pleura, and mediastinal lymph nodes (arrows in the third image) metastases after 1 year and 3 months. Case No. 296 was a 43-year-old premenopausal woman who initially presented with extensively metastatic breast cancer involving the axial skeleton. Despite palliative chemotherapy with docetaxel and cisplatin, the disease rapidly progressed after 3 months in bone and leptomeningeal seeding, however, extensive bone metastases showed metabolic response after 4 cycles of Sacituzumab Govitecan. CTx, chemotherapy; SG, Sacituzumab Govitecan.

Fig. 4.

Utilization of FiRST Cancer Panel for screening germline predisposition and cancer evolution. (A) Biallelic inactivation of PALB2 revealed by FiRST Cancer Panel (FCP) prompted germline confirmatory testing. The patient was confirmed to harbor germline PALB2 pathogenic mutation and enrolled in a clinical trial using a poly(ADP-ribose) polymerase inhibitor (PARPi), resulting in a remarkable clinical response (B). (C) Sequential FCP tests using samples from two different time points showed a BRCA1 reversion mutation. VAF, variant allele frequency.

Fig. 5.

Genetic evolution model of a rare breast cancer by multi-region sequencing. A rare mixed invasive ductal carcinoma and spindle cell carcinoma was subject to multi-region sequencing, where we found evidence of divergent evolution from a common ancestor. AC, adriamycin+cyclophosphamide; DFS, disease-free survival; IDC, invasive ductal carcinoma; LN, lymph node; PD, progressive disease; PFS, progression-free survival.

Fig. 6.

Matched therapy guided by FiRST Cancer Panel. (A, B) Progression-free survival and overall survival (OS) from the day 1 of the matched therapy are presented. (C) Better OS from metastasis diagnosis was observed in patients who received matched therapy compared to those who did not. (D) A remarkable response to poly(ADP-ribose) polymerase inhibitor (PARPi) was observed in a patient with somatic ATM mutation identified by FiRST Cancer Panel. C1D1, cycle 1 day 1; EOT, end of treatment; OS, overall survival; PFS, progression-free survival.

Table 1.

Clinicopathological characteristics of patients and samples in the study

	No. (%)
No. of patients	522 (100)
Sex
Female	519 (99.4)
Male	3 (0.6)
Age at diagnosis (yr), median (range)	47 (20-76)
Menopausal status
Pre or perimenopause	319 (61.1)
Postmenopause	197 (37.7)
Oophorectomy	3 (0.6)
Male	3 (0.6)
Parity	342 (65.5)
Family history of breast cancer	104 (19.9)
Bilateral breast cancer	50 (9.6)
Synchronous	24 (48.0)
Metachronous	26 (52.0)
Initial stage^a)
Stage 0	3 (0.6)
Stage I	62 (11.9)
Stage II	170 (32.6)
Stage III	163 (31.2)
De novo stage IV	122 (23.4)
Data not available	2 (0.4)
Relapsed with metastatic disease	330 (63.2)
No. of samples	548 (100)
Panel version
V2	38 (6.9)
V3	32 (5.8)
V3.1	102 (18.6)
V3.2	99 (18.1)
V3.3	96 (17.5)
V4	181 (33.0)
Block age (mo)
< 3	286 (42.2)
≥ 3 and < 6	56 (10.2)
≥ 6 and < 12	55 (10.0)
≥ 12	151 (27.6)
Diagnosis
Invasive ductal carcinoma	500 (91.2)
Invasive lobular carcinoma	19 (3.5)
Others	29 (5.3)
Subtypes
ER+/HER2–	291 (53.1)
ER+/HER2+	28 (5.1)
ER–/HER2+	35 (6.4)
TNBC	194 (35.4)
Specimen type
Biopsy	336 (61.3)
Surgery	210 (38.3)
Cytology	2 (0.4)
Source site of the tissue used for NGS
Breast	247 (45.1)
LN, regional	50 (9.1)
LN, distant	14 (2.6)
Bone	25 (4.6)
Brain	10 (1.8)
Liver	89 (16.2)
Lung/Pleura	44 (8.0)
Skin or soft tissue	62 (11.3)
Others	7 (1.3)
Treatment within 6 months prior to tissue acquisition
No treatment	197 (35.9)
Treatment	351 (64.1)
Hormone therapy	159 (29.0)
Chemotherapy	174 (31.8)
Targeted therapy	93 (17.0)
Immuno-oncologic drugs	13 (2.4)
Line of treatment at the time of NGS request
Adjuvant	56 (10.2)
1st line for metastatic BC	194 (35.4)
2nd line for metastatic BC	136 (24.8)
3rd line for metastatic BC	75 (13.7)
4th or later line for metastatic BC	87 (15.9)
Biomarkers found in NGS
Presence of actionable biomarkers (by OncoKB)	387 (70.6)
No. of actionable biomarkers in a sample
1	252 (46.0)
2	112 (20.4)
3	21 (3.8)
4	1 (0.2)
5	1 (0.2)

BC, breast cancer; ER, estrogen receptor; HER2, human epidermal receptor 2; LN, lymph node; NGS, next-generation sequencing; TNBC, triple-negative breast cancer.

^a) Clinical stage for the patients who received neoadjuvant chemotherapy, and pathologic staging for those who received upfront surgery.

Table 2.

Comparison between conventional assessment of HER2 status and NGS-based ERBB2 amplification detection

	HER2 status		Total	p-value
	Negative	Positive	Total	p-value
ERBB2 copy number
No amplification	483 (99.6)	16 (25.4)	499 (91.1)	< 0.001
Amplification	2 (0.4)	47 (74.6)	49 (8.9)
Total	485 (88.5)	63 (11.5)	548 (100)

Values are presented as number (%). HER2, human epidermal growth factor receptor 2; NGS, next-generation sequencing.

Table 3.

Matched therapy and clinical trial enrollment guided by clinical sequencing

	No. (%) (n=522)
No. of matched therapy/clinical trials	53 (10.1)
1	47 (9.0)
2	6 (1.1)
Line of treatment at the matched therapy/clinical trials
Adjuvant	2 (0.4)
1st line	5 (1.0)
2nd line	19 (3.6)
3rd line	13 (2.5)
4th or later line	20 (3.8)
Target gene of the matched therapy/clinical trials
BRCA1 or BRCA2 mutation	19 (3.6)
ERBB2 amplification	14 (2.7)
PIK3CA mutation	11 (2.1)
High TMB	5 (1.0)
Alterations of HRR-related genes	4 (0.8)
FGFR1 alteration	3 (0.6)
AKT1 mutation	2 (0.4)
ERBB2 mutation	1 (0.2)
Total	522 (100)

HRR, homologous recombination repair; TMB, tumor mutational burden.

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15. Wolff AC, Hammond ME, Allison KH, Harvey BE, Mangu PB, Bartlett JM, et al. Human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline focused update. J Clin Oncol. 2018;36:2105–22. PubMed
16. Chakravarty D, Gao J, Phillips SM, Kundra R, Zhang H, Wang J, et al. OncoKB: a precision oncology knowledge base. JCO Precis Oncol. 2017;2017:PO.17.00011.PubMed
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Harnessing Institutionally Developed Clinical Targeted Sequencing to Improve Patient Survival in Breast Cancer: A Seven-Year Experience

DOI: https://doi.org/10.4143/crt.2024.296

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Abstract
Introduction
Materials and Methods
Results
Discussion
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REFERENCES

Harnessing Institutionally Developed Clinical Targeted Sequencing to Improve Patient Survival in Breast Cancer: A Seven-Year Experience

Fig. 1. The genetic profile of breast cancer patients unveiled by FiRST Cancer Panel. The overall distribution of commonly detected mutations, copy number variations, fusions, as well as microsatellite instability scores and tumor mutational burden are presented alongside clinicopathological features. CTx, chemotherapy; ET, endocrine therapy; ER, estrogen receptor; IDC, invasive ductal carcinoma; HER2, human epidermal growth factor receptor 2; ILC, invasive lobular carcinoma; IO, immuno-oncology; MSI, microsatellite instability; TMB, tumor mutational burden; TNBC, triple-negative BCs; Tx, therapy.

Fig. 2. ERBB2 copy number assessment by FiRST Cancer Panel compared to human epidermal growth factor receptor 2 (HER2) immunohistochemistry and in situ hybridization. (A) ERBB2 copy numbers were higher in tumors with HER2 immunohistochemistry (IHC) 3+ compared to HER2 IHC 2+. (B) ERBB2 copy number assessed by FiRST Cancer Panel showed a significant correlation with the results of HER2 in situ hybridization. CN, copy number; ISH, in situ hybridization; NGS, next-generation sequencing.

Fig. 3. Fusion genes identified by FiRST Cancer Panel. (A) The novel ESR1::NBAT fusion gene is expected to cause the distruption of ligand binding domain of ESR1. (B) Fusion gene structures and clinical course of the patients harboring BCL2L14::ETV6 fusions are presented Case No. 205 was a 30-year-old female diagnosed with locally advanced triple-negative breast cancer. The lesions in the right breast, right axillary lymph nodes, and internal mammary lymph nodes (arrows in the first image) decreased after the neoadjuvant treatment with olaparib+durvalumab window of opportunity treatment followed by standard neoadjuvant therapy consisting of doxorubicin+ cyclophosphamide followed by docetaxel (arrow in the second image). However, the disease recurred with lung, pleura, and mediastinal lymph nodes (arrows in the third image) metastases after 1 year and 3 months. Case No. 296 was a 43-year-old premenopausal woman who initially presented with extensively metastatic breast cancer involving the axial skeleton. Despite palliative chemotherapy with docetaxel and cisplatin, the disease rapidly progressed after 3 months in bone and leptomeningeal seeding, however, extensive bone metastases showed metabolic response after 4 cycles of Sacituzumab Govitecan. CTx, chemotherapy; SG, Sacituzumab Govitecan.

Fig. 4. Utilization of FiRST Cancer Panel for screening germline predisposition and cancer evolution. (A) Biallelic inactivation of PALB2 revealed by FiRST Cancer Panel (FCP) prompted germline confirmatory testing. The patient was confirmed to harbor germline PALB2 pathogenic mutation and enrolled in a clinical trial using a poly(ADP-ribose) polymerase inhibitor (PARPi), resulting in a remarkable clinical response (B). (C) Sequential FCP tests using samples from two different time points showed a BRCA1 reversion mutation. VAF, variant allele frequency.

Fig. 5. Genetic evolution model of a rare breast cancer by multi-region sequencing. A rare mixed invasive ductal carcinoma and spindle cell carcinoma was subject to multi-region sequencing, where we found evidence of divergent evolution from a common ancestor. AC, adriamycin+cyclophosphamide; DFS, disease-free survival; IDC, invasive ductal carcinoma; LN, lymph node; PD, progressive disease; PFS, progression-free survival.

Fig. 6. Matched therapy guided by FiRST Cancer Panel. (A, B) Progression-free survival and overall survival (OS) from the day 1 of the matched therapy are presented. (C) Better OS from metastasis diagnosis was observed in patients who received matched therapy compared to those who did not. (D) A remarkable response to poly(ADP-ribose) polymerase inhibitor (PARPi) was observed in a patient with somatic ATM mutation identified by FiRST Cancer Panel. C1D1, cycle 1 day 1; EOT, end of treatment; OS, overall survival; PFS, progression-free survival.

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Harnessing Institutionally Developed Clinical Targeted Sequencing to Improve Patient Survival in Breast Cancer: A Seven-Year Experience

	No. (%)
No. of patients	522 (100)
Sex
Female	519 (99.4)
Male	3 (0.6)
Age at diagnosis (yr), median (range)	47 (20-76)
Menopausal status
Pre or perimenopause	319 (61.1)
Postmenopause	197 (37.7)
Oophorectomy	3 (0.6)
Male	3 (0.6)
Parity	342 (65.5)
Family history of breast cancer	104 (19.9)
Bilateral breast cancer	50 (9.6)
Synchronous	24 (48.0)
Metachronous	26 (52.0)
Initial stage^a)
Stage 0	3 (0.6)
Stage I	62 (11.9)
Stage II	170 (32.6)
Stage III	163 (31.2)
De novo stage IV	122 (23.4)
Data not available	2 (0.4)
Relapsed with metastatic disease	330 (63.2)
No. of samples	548 (100)
Panel version
V2	38 (6.9)
V3	32 (5.8)
V3.1	102 (18.6)
V3.2	99 (18.1)
V3.3	96 (17.5)
V4	181 (33.0)
Block age (mo)
< 3	286 (42.2)
≥ 3 and < 6	56 (10.2)
≥ 6 and < 12	55 (10.0)
≥ 12	151 (27.6)
Diagnosis
Invasive ductal carcinoma	500 (91.2)
Invasive lobular carcinoma	19 (3.5)
Others	29 (5.3)
Subtypes
ER+/HER2–	291 (53.1)
ER+/HER2+	28 (5.1)
ER–/HER2+	35 (6.4)
TNBC	194 (35.4)
Specimen type
Biopsy	336 (61.3)
Surgery	210 (38.3)
Cytology	2 (0.4)
Source site of the tissue used for NGS
Breast	247 (45.1)
LN, regional	50 (9.1)
LN, distant	14 (2.6)
Bone	25 (4.6)
Brain	10 (1.8)
Liver	89 (16.2)
Lung/Pleura	44 (8.0)
Skin or soft tissue	62 (11.3)
Others	7 (1.3)
Treatment within 6 months prior to tissue acquisition
No treatment	197 (35.9)
Treatment	351 (64.1)
Hormone therapy	159 (29.0)
Chemotherapy	174 (31.8)
Targeted therapy	93 (17.0)
Immuno-oncologic drugs	13 (2.4)
Line of treatment at the time of NGS request
Adjuvant	56 (10.2)
1st line for metastatic BC	194 (35.4)
2nd line for metastatic BC	136 (24.8)
3rd line for metastatic BC	75 (13.7)
4th or later line for metastatic BC	87 (15.9)
Biomarkers found in NGS
Presence of actionable biomarkers (by OncoKB)	387 (70.6)
No. of actionable biomarkers in a sample
1	252 (46.0)
2	112 (20.4)
3	21 (3.8)
4	1 (0.2)
5	1 (0.2)

	HER2 status		Total	p-value
	Negative	Positive	Total	p-value
ERBB2 copy number
No amplification	483 (99.6)	16 (25.4)	499 (91.1)	< 0.001
Amplification	2 (0.4)	47 (74.6)	49 (8.9)
Total	485 (88.5)	63 (11.5)	548 (100)

	No. (%) (n=522)
No. of matched therapy/clinical trials	53 (10.1)
1	47 (9.0)
2	6 (1.1)
Line of treatment at the matched therapy/clinical trials
Adjuvant	2 (0.4)
1st line	5 (1.0)
2nd line	19 (3.6)
3rd line	13 (2.5)
4th or later line	20 (3.8)
Target gene of the matched therapy/clinical trials
BRCA1 or BRCA2 mutation	19 (3.6)
ERBB2 amplification	14 (2.7)
PIK3CA mutation	11 (2.1)
High TMB	5 (1.0)
Alterations of HRR-related genes	4 (0.8)
FGFR1 alteration	3 (0.6)
AKT1 mutation	2 (0.4)
ERBB2 mutation	1 (0.2)
Total	522 (100)

Table 1. Clinicopathological characteristics of patients and samples in the study

BC, breast cancer; ER, estrogen receptor; HER2, human epidermal receptor 2; LN, lymph node; NGS, next-generation sequencing; TNBC, triple-negative breast cancer.

Clinical stage for the patients who received neoadjuvant chemotherapy, and pathologic staging for those who received upfront surgery.

Table 2. Comparison between conventional assessment of HER2 status and NGS-based ERBB2 amplification detection

Values are presented as number (%). HER2, human epidermal growth factor receptor 2; NGS, next-generation sequencing.

Table 3. Matched therapy and clinical trial enrollment guided by clinical sequencing

HRR, homologous recombination repair; TMB, tumor mutational burden.