DOI: https://doi.org/10.21203/rs.3.rs-1259287/v1
Purpose
Mutations in PIK3CA gene occur frequently in patients with breast cancer. Activating PIK3CA mutations have been shown to confer resistance to human epidermal growth factor receptor (HER)2-targeted treatments. In this retrospective study, we investigated whether PIK3CA mutations were correlated with treatment response or duration in HER2-positive (HER2+) breast cancer patients.
Methods
We reviewed the clinical information of patients with HER2 + breast cancer who received HER2-targeted therapy for early stage or metastatic cancers. The pathologic complete response (pCR), progression-free survival (PFS), and overall survival were compared between patients with wild-type PIK3CA (PIK3CAw) and mutated PIK3CA (PIK3CAm), among those selected for the study. Next-generation sequencing was combined with examination of PFS associated with anti-HER2 monoclonal antibody (mAb) treatment.
Results
Data from 90 patients with HER2 + breast cancer were analyzed. Overall, 34 patients had pathogenic PIK3CA mutations (37.8%). The pCR rate of the PIK3CAm group was lower than that of the PIK3CAw group among patients who received neoadjuvant chemotherapy for early stage cancer. In the metastatic setting, the PIK3CAm group showed a significantly shorter mean PFS (mPFS) with first-line anti-HER2 mAb. The mPFS of second-line T-DM1 was lower in the PIK3CAm group than in the PIK3CAw group. Sequencing revealed differences in the Mutational landscape between PIK3CAm and PIK3CAw tumors.
Conclusion
HER2+ breast cancer patients with activating PIK3CA mutations had lower pCR rates and shorter PFS with palliative HER2-targeted therapy than those with wild-type PIK3CA. Our results suggest that HER2+ breast cancer patients with PIK3CA mutations have an unmet clinical need.
From the anti-human epidermal growth factor receptor (HER)2 monoclonal antibody (mAb) and antibody-drug conjugates (ADCs) to novel tyrosine kinase inhibitors, effective targeted therapy has revolutionized the treatment of HER2-positive (HER2+) breast cancer [1, 2]. Although these therapies have greatly improved the prognosis of patients with HER2+ breast cancer, resistance to targeted therapy can develop, with some tumors showing de novo resistance [3]. Continued use of anti-HER2 mAb, trastuzumab beyond progression remains one of the standards of care even after the failure of dual HER2-blockades [4, 5]; however, precise guidelines based on individual tumor biology are still lacking in clinical practice [6, 7].
Results from preclinical and translational studies have highlighted mutations in phosphatidyl inositol-4,5 bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene as a major driver of resistance to HER2-targeted agents [8–10]. The PI3K-alpha catalytic subunit (p110α) acts as a key component in controlling the PI3K/AKT/mTOR signaling pathway [11], which regulates various cellular processes including cell growth, survival, and apoptosis [12]. Tumors with PIK3CA gain-of-function mutations respond poorly to anti-HER2 therapy and are associated with reduced pathological complete response (pCR) rates [13]. Nevertheless, the relationship between palliative treatment response and PIK3CA mutations has not yet been fully characterized. Here, we conducted a retrospective study to determine whether PIK3CA aberrations correlate with the duration of anti-HER2 therapy in patients with HER2 + breast cancer.
Among patients who had received systemic chemotherapy for early or metastatic HER2+ breast cancer at Korea University Anam Hospital, those with available next-generation sequencing (NGS) data and clinical response to treatment were chosen for this study. The clinical characteristics such as age, sex, date of diagnosis, molecular subtype, as well as treatment history, and survival data of each patient were collected from their medical records. This study was approved by the Institutional Review Board (IRB No. 2017AN0401), and individual patient consent was waived.
Mutational analysis of the tumor tissues was performed using targeted NGS. DNA was extracted, purified, and quantified from formalin-fixed paraffin-embedded breast tumor specimens according to the K-MASTER protocol [14]. Using the K-MASTER panel, which allows the detection of variants in 409 representative genes using the HiSeq sequencing platform, we investigated the mutation profile of the collected tissues. After passing the quality control process, the pipeline returned single nucleotide variants (SNVs), copy number variants (CNVs), and genomic fusion data from each sample. Detailed laboratory and bioinformatic protocols are available in the Supplementary Methods section. In this study, the average depth of tumor-targeted sequencing coverage, duplication rate, on-target rate, pass rate (PR) score, and uniformity were 718.07 (range 344.17 – 1128.29), 28.49 (86.65 – 99.14), 93.32 (89.65 – 99.14), 99.27 (69.88 – 100) and 79.62% (69–91%), respectively.
Liquid biopsy was performed in patients with no available NGS data. cfDNA was extracted from plasma samples using the QIAamp Circulating Nucleic Acid Kit, following the manufacturer’s instructions. After cfDNA quantification and purification, targeted sequencing was performed using Axen Cancer Panel 1 (ACP1), which included the exons of 88 cancer-related genes and the intronic regions of the three genes. Detailed laboratory and bioinformatic protocols for cfDNA analysis are available in the Supplementary Methods section.
All the patients included in this study had received at least one additional HER2-targeted therapy during their disease course. pCR was defined as the complete disappearance of invasive breast cancer in both the breast and lymph nodes and was assessed in patients who had received anti-HER2 mAb as neoadjuvant therapy. Response evaluation in the metastatic setting was performed according to Response Evaluation Criteria in Solid Tumors 1.1 [15]. Invasive disease-free survival (iDFS) was defined as the period from the date of curative surgery or the first neoadjuvant therapy to the date of the first relapse (locoregional or distant, not including ductal carcinoma in situ), the date of death in women who died without invasive relapse, or the date of censoring in women who were alive and disease-free. Progression-free survival (PFS) in metastatic or recurrent disease was assessed as the time from the start of treatment to disease progression or death from any cause.
Continuous variables were analyzed using Student’s t-test, while categorical variables were analyzed using the chi-squared test. The median PFS and overall survival (OS) were calculated using the Kaplan–Meier method. Hazard ratios and p-values were assessed using a multivariate Cox regression analysis. IBM SPSS v. 20.0 was used for statistical analyses, and the results were visualized using SPSS v. 4.1.0.
After screening 366 HER2+ breast cancer patients who were treated at the Korea University Anam Hospital from August 2008 to September 2020, 90 patients were selected for this study; their clinical characteristics are summarized in Table 1. Pathogenic mutations in PIK3CA (PIK3CAm) were found in 34 (37.8%) patients, and 57.6% of these mutations were found in the kinase domain (H1047X). The median age of the patients was 50.2 years, and all patients were female, except for one. Estrogen receptor (ER) was positive in 68.9% of the patients. The ER and PIK3CAm status of the analyzed HER2+ breast cancer samples are shown in Supplementary Figure 1. Among the analyzed population, 62 (68.9%) patients were ER positive, and the proportion of patients whose ER status was positive, or negative was similar between PIK3CAm and PIK3CAw patients (67.6% vs. 69.9%). For palliative chemotherapy, patients received multiple lines of anti-HER2 mAbs (trastuzumab and pertuzumab), antibody-drug conjugates (T-DM1), or tyrosine kinase inhibitors (lapatinib) as the disease progressed.
The overall treatment response and duration of treatment for patients with PIK3CAw or PIK3CAm are shown in Figure 1 and summarized in Supplementary Table 2. Among the patients with early stage cancers, 25 received standard neoadjuvant therapy, including single or dual anti-HER2 mAb with trastuzumab or trastuzumab plus pertuzumab. Further, among the patients who received a single anti-HER2 mAb, 57.1% of patients with PIK3CAw achieved pCR (4 of 7), while none of the patients with PIK3CAm (0/3, 0%) achieved pCR. Treatment with the dual anti-HER2 mAbs trastuzumab and pertuzumab improved pCR rates (72.7%) in PIK3CAw patients and PIK3CAm patients (40%). Altogether, 25% of PIK3CAm and 66.7% of PIK3CAw patients were reported to have achieved pCR, and the difference between these two groups was marginally significant (p = 0.09, chi-square test). At the median follow-up of 43.4 months (range 14.2 - 222.2), the iDFS showed no significant difference between the two groups (31.0 months vs. 30.5 months). The treatment duration of anti-HER2 mAb as the palliative first-line and T-DM1 as the second-line were significantly shorter in the PIK3CAm group (mPFS 7.1 vs. 19.1, p = 0.001; mPFS 4.4 vs. 11.0, p = 0.024, respectively; Supplementary Table 1).
The PFS of palliative HER2-directed therapy was assessed using Kaplan–Meier analysis (Figure 2). After a median 36.4 months of total follow-up period (range 3.5 - 206.7), the median PFS of the patients who were treated with palliative chemotherapy with anti-HER2 mAb as the first-line treatment was 51.8 months (95% CI: 6.4 – 22.4) in the PIK3CAw group and 7.5 months (95% CI: 2.5 – 12.5) in the PIK3CAm group (p < 0.001, Mantel–Cox Log Rank test), with a hazard ratio (HR) of 4.602 (95% CI 2.057 – 10.514, p < 0.001, Cox regression analysis; Figure 2A). When we compared the HR based on the mutational status of PIK3CA among the patients who received anti-HER2 mAbs, the HR of patients with PIK3CAm was the highest (HR 8.181, 95% CI 2.780 – 24.075, p = 0.001) (Figure 2B). However, the median PFS in first-line treatment with anti-HER2 mAb was not influenced by ER positivity (HR 0.761, p = 0.577; Supplementary Figure 2A). The risk of progression was higher in patients with PIK3CAm, regardless of the ER status (Supplementary Figure 2B).
The PFS for T-DM1, which is usually prescribed as the second line of treatment after anti-HER2 mAb treatment, also significantly differed in the patients with PIK3CAw and PIK3CAm (3.5 months vs. 11.8 months, HR 3.018, 95% CI 1.054 – 8.644, p = 0.04; Figure 2C). The median PFS of lapatinib, usually prescribed as a later line of treatment combined with capecitabine, showed no differences between two groups (7.8 months vs. 6.2 months, HR 1.189, 95% CI 0.247 – 5.732, p = 0.767; Figure 2D).
Although the data were often censored, the median OS of the patients without PIK3CA mutation was 135.9 months and the median OS of the patients with PIK3CA mutation was 137.1 months, and this difference was not statistically significant (HR 1.755, 95% CI 0.553 – 5.571, p = 0.33; Supplementary Figure 3A). When we compared the OS among patients with PIK3CA mutations in different regions, the helical domain (E542X or E545X), kinase domain (H1047X), other minor sites, and patients with PIK3CA wild type, the median OS in patients with mutations in the helical domain was the shortest (HR 84.368, 95% CI 6.031 – 1191.384, p = 0.006; Supplementary Figure 3B).
We analyzed the mutation profiles of the available tumor tissues by targeted sequencing using a cancer panel (Figure 3). Among the 34 PIK3CAm patients, TP53 was the most frequently co-occurring pathogenic mutation (74%), followed by ERBB2 (35%), and MSH3 (29%; Figure 3A). ALK and BRCA2 mutations co-occurred with PIK3CAm in 24% and 21% of patients, respectively, some of which are known to be clinically pathogenic. BRCA1 was not among the top 20 mutated genes in the PIK3CAm group. In the 56 patients with PIK3CAw, alterations in TP53, NOTCH1, and ERBB2 were frequently observed (58%, 28%, and 26%, respectively; Figure 3B). BRCA2 and BRCA1 mutations were identified in 19% and 17% of the PIK3CAw patients, respectively.
We compared the mutational landscapes in tumor tissues of patients with different tumor statuses. A total of 67 patients whose primary tumor tissue was sequenced and 23 patients who underwent NGS at their recurrent time points were included in the analysis (Figure 4). Figure 4A shows the mutational landscape of primary HER2+ cancer cells. TP53 was the most frequently mutated gene in primary tumor tissue (58%), followed by PIK3CA (33%), and MSH3 (30%). Upon sequencing patients’ recurrent tumor tissue or blood cfDNA, TP53, ERBB2, and NOTCH1 were revealed to be the three most frequently altered genes (78%, 52%, and 48%, respectively), whereas mutation of PIK3CA was determined as fourth most common genetic mutation (Figure 4B).
We examined the sequencing results in the context of anti-HER2 treatment duration (Figure 5). A total of 45 patients who underwent palliative first-line anti-HER2 mAb treatment and whose tumor tissues were available for NGS were included in the analysis. The median number of reported SNVs was 16 (range, 5–27) and the median number of pathologic SNVs was 3 (range, 0–6). The PFS of palliative first-line anti-HER2 mAb therapy was compared between subgroups within the median SNVs or pathological SNVs. The median PFS was 11.2 months (SD 15.4, range 0.5 ~ 51.8) in patients with fewer SNVs and 10.8 months (SD 12.0, range 0.7 ~ 39.9) in patients with more SNVs (p = 0.72). Among the patients with pathologic SNVs, patients with more pathologic variants showed shorter PFS (mPFS 8.7m, SD 11.0, range 2.7 ~ 39.9) than patients with fewer pathologic variants (mPFS 14.4m, SD 15.4, range 0.5 ~ 51.8), but the difference was not statistically significant (p = 0.097).
In this study, we retrospectively analyzed treatment duration and clinical response to HER2-directed treatment based on PIK3CA mutations in patients with HER2+ breast cancer. Patients with pathogenic PIK3CA mutations had lower pCR rates after neoadjuvant therapy and significantly shorter PFS with anti-HER2 monoclonal antibody therapy or ADC than those with wild-type PIK3CA in a palliative setting. The PFS with lapatinib was similar in both the groups. In addition, clinically targeted sequencing revealed that TP53 was the most frequently co-occurring mutation in HER2+/PIK3CAm breast cancer tissues. Patients whose tumor tissues had a higher number of pathological SNVs tended to show shorter PFS with palliative anti-HER2 mAb treatment.
Anti-HER2 mAbs, including trastuzumab and pertuzumab, have several antitumor effects, including inhibition of signal transduction through HER, induction of antibody-dependent cellular cytotoxicity in tumor cells with HER2 amplification [16], and inhibition of HER2 extracellular domain cleavage [17]. Most importantly, downregulation of intracellular signaling through the PI3K/AKT/mTOR pathway [18] is the main cellular event after the binding of trastuzumab to domain IV or pertuzumab to domain II of HER2. However, constitutive activation of the PI3K/AKT/mTOR pathway is known to drive resistance to HER2-directed therapy, and pathologic mutations in PIK3CA are found in approximately 23–30% of HER2+ breast-cancer patients [19].
The significance of PIK3CAm in primary tumors of early stage breast cancer has been addressed in several prospective clinical trials. In a pooled analysis of 967 patients who were undergoing chemotherapy and received neoadjuvant treatment with either trastuzumab, or lapatinib, or both [13], the pCR rate was significantly lower in the PIK3CAm group than in the wild-type group (16.2% vs. 29.6%; P < 0.001). In the NeoALTTO trial, which compared the efficacy of dual therapy with trastuzumab and lapatinib to lapatinib alone, patients with PIK3CAm had poorer outcomes in all treatment groups (pCR rate, 28.6% vs. 53.1%, p = 0.012) [20]. Similar results were reported in the TBCRC006 trial, which evaluated neoadjuvant lapatinib and trastuzumab with hormonal therapy and without chemotherapy, and the pCR rate was significantly lower in patients with PIK3CA mutations or low PTEN levels (4% vs. 39%, p=0.006) [21]. In the era of dual blockade using trastuzumab and pertuzumab, a retrospective analysis from the NeoSphere study showed an inferior pCR rate in patients with HER2+/PIK3CAm tumors [22]. Our data from Korean patients support these previous findings, suggesting that patients with PIK3CAm have significant unmet needs despite receiving dual HER2-directed neoadjuvant treatment as the standard of care.
In a retrospective analysis of adjuvant trastuzumab treatment in combination with chemotherapy, the presence of PIK3CAm did not change the prognosis of the patients who participated in the ShortHER or FinHER trials [23, 24], with a 5-year DFS rate of 90.6% for PIK3CAm and 86.2% for PIK3CAw, p = 0.417 [23]. However, a previous study reported that patients who retained the initial PIK3CAm after neoadjuvant therapy had worse DFS after surgical resection, implying a possible role of PI3KCAm in predicting worse clinical outcomes in early breast cancer [25].
Substantial efforts have been made to identify biomarkers for resistance to anti-HER2 treatment in metastatic HER2+ breast cancer. In preclinical studies, breast cancer cell lines with BRAF, KRAS, and PIK3CA mutations were less sensitive to trastuzumab than the wild-type [26], and drug susceptibility also differed depending on the mutation site in PIK3CA [27]. Genome-wide loss-of-function genetic screens have also identified that reduced ARID1A expression confers resistance to HER2-targeted therapy [19]. Nevertheless, treatment for patients with HER2+ breast cancer is still a “one-size-fits-all” approach based on positive results from key trials. In a retrospective analysis of the CLEOPATRA study, PIK3CA mutation status had the greatest prognostic impact on PFS in palliative first-line therapy (docetaxel, trastuzumab, and pertuzumab) [28]. Biomarker analysis from clinical trials assessing T-DM1 and lapatinib in the later lines of treatment had similar consequences [29, 30].
Our study has several limitations, including its retrospective nature. First, the use of HER2 antibodies was switched from single to dual initial trastuzumab therapy combined with pertuzumab in both the early and metastatic settings. Thus, the number of patients in each treatment group was divided according to the genotype, resulting in a small sample size. Second, due to the retrospective nature of our study, we could not control for other potential prognostic factors, such as the levels of HER2 protein, HER2 and HER3 mRNA, soluble HER2, or the PIK3CA genotype. In addition, access to tissue biopsies is limited in some patients with metastatic cancers. Considering that 8–10% of patients are known to acquire PIK3CA mutations in a metastatic setting [31], our data should be interpreted with caution.
In our study, patients with PIK3CA mutations showed weakened responses to single or dual HER2 antibodies combined with taxane and subsequent T-DM1. Different genomic landscapes were identified according to the PIK3CA status. Thus, there is a significant unmet need for these patients, which may be addressed by targeting the aberrantly activated PI3K/AKT/mTOR pathway. Given the promise of the FDA-approved α-specific PI3K inhibitor alpelisib for the treatment of HR+/HER2- advanced stage cancers, studies examining the clinical utility of targeting PIK3CA as well as HER2 are warranted.
In a retrospective analysis, pathogenic aberration of PIK3CA was associated with a lower rate of pCR after neoadjuvant therapy and shorter PFS after palliative HER2-targeted therapy. Patients with activating PIK3CA mutations in HER2+ breast cancer have substantially unmet clinical needs and require a more individualized therapeutic approach.
Acknowledgements
We thank the patients and their families, nurses, and trial coordinators, who helped with this trial.
Conflicts of interest
The authors declare that they have no conflicts of interest.
Funding
This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea (Grant number: HI17C2206).
Author contributions
All authors contributed to the conception and design of the study. Data were collected by Ju Won Kim and Ah Reum Lim. Ju Won Kim performed the data analysis and figure visualization. Ju Won Kim and Kyong Hwa Park wrote the first draft of the manuscript and all authors commented on the previous version of the manuscript. All authors have read and approved the final manuscript.
Table 1. Patient clinical characteristics
|
Total (N = 90) |
PIK3CAm (n = 34) |
PIK3CAw (n = 56) |
p-value |
|
|
|
|
|
Age at diagnosis, median (range, years) |
|
|
|
0.307 |
|
50.2 (27.7 – 86.5) |
51.70 (27.7 – 86.5) |
49.24 (31.1 – 73.5) |
|
Sex |
|
|
|
1.000 |
Female |
89 (98.9%) |
34 (100%) |
55 (98.2%) |
|
Male |
1 (1.1%) |
0 (0%) |
1 (1.8%) |
|
ER status |
|
|
|
1.000 |
Positive |
62 (68.9%) |
23 (67.7%) |
39 (69.6%) |
|
Negative |
28 (31.1%) |
11 (32.3%) |
17 (30.4%) |
|
Samples for NGS |
|
|
|
0.198 |
Tumor tissue - Primary site |
65 (72.2%) |
23 (64.7%) |
42 (75.0%) |
|
Tumor tissue - Metastatic site |
15 (16.7%) |
6 (17.6%) |
9 (16.1%) |
|
Blood - plasma |
10 (11.1%) |
5 (14.7%) |
5 (8.9%) |
|
PIK3CAm |
|
|
|
|
H1047X (Kinase domain) |
19 (21.1%) |
19 (57.6%) |
- |
|
E542X or E545X (Helical domain) |
4 (4.4%) |
4 (12.1%) |
- |
|
Others |
9 (11.1%) |
10 (30.3%) |
- |
|
ER; estrogen receptor, NGS; Next-generation sequencing
Supplementary Table 2 is not available with this version.