Mutation and immune profiling of metaplastic breast cancer: Correlation with survival

The goal of this study is to characterize the genomic and immune profiles of metaplastic breast cancer (MpBC) and identify the association with survival through an analysis of archived tumor tissue. A next-generation sequencing-based mutational assay (Onco-48) was performed for 21 MpBC patients. Clinicopathologic characteristics were captured, including relapse free survival (RFS) and overall survival (OS). Immunohistochemistry (IHC) for CD3, CD4, CD8, and programmed death-ligand 1 (PD-L1) was also performed. Recurrence free survival (RFS) at 5 years was 57% (95% CI 0.34–0.75) and overall survival (OS) at 5 years was 66% (95% CI 0.41–0.82). The most commonly altered genes were TP53 (68.4%, 13/19), PIK3CA (42.1%, 8/19), and PTEN (15.8%, 3/19. For patients with PIK3CA mutations, RFS and OS were significantly worse than for those without (HR 5.6, 95% CI 1.33–23.1 and HR 8.0, 95% CI 1.53–41.7, respectively). Cox regression estimated that PD-L1 expression was associated with worse RFS and OS (HR 1.08, 95% CI 1.01–1.16 and HR 1.05, 95% CI 1.00–1.11, respectively, for an absolute increase in PD-L1 expression of 1%). In conclusion, PIK3CA mutation and PD-L1 expression confer poor prognosis in this cohort of patients with MpBC.


Introduction
Metaplastic breast cancer (MpBC) is a rare malignancy which accounts for 0.05-5% of all breast cancers [1,2]. MpBC is defined by differentiation of the neoplastic epithelium to a nonglandular component, typically either squamous or mesenchymal (e.g. spindle cell, osseous, or chondroid). These cancers are subdivided into groups according to the 2012 WHO Classification of Tumors of the Breast: squamous cell carcinoma, spindle cell carcinoma, mixed squamous and spindle cell carcinoma, spindle cell and mesenchymal, or mesenchymal [3]. The conventional biomarkers of estrogen receptor (ER), progesterone receptor (PR), and human a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 epidermal growth factor receptor 2 (HER2) are usually not expressed in metaplastic breast cancer (i.e., they are "triple negative" breast cancers). Initial gene expression profiling studies demonstrated that MpBC is of basal-like breast cancer [4]. Further analysis has classified MpBC into the "claudin-low" subtype based on mRNA expression profiling [5].
Clinically, MpBC is an aggressive form of breast cancer. Patients present with a more advanced stage and have a greater risk of local recurrence and a worse prognosis compared with conventional invasive ductal carcinoma [6]. The disease is often resistant to chemotherapy, possibly due to complex tumor genetics that results in phenotypically diverse histology and intratumoral heterogeneity [6]. Because of the rarity and heterogeneous nature of metaplastic cancers, there are no randomized controlled trials to inform treatment decisions. Treatment is generally determined by the dominant cell population. Next-generation sequencing (NGS) provides a unique opportunity to understand the underlying biology of cancer. NGS can also assist clinicians in identifying potential biomarkers for risk stratification, targeted therapy, and prediction of response to therapy.
Immune checkpoint inhibitors (ICIs) have shown efficacy in treatment of metastatic TNBC, and immune profiling of tumors may predict efficacy of immunotherapies [7] The first immune checkpoint inhibitor FDA-approved in breast cancer atezolizumab, in combination with nab-paclitaxel in programmed death ligand 1 (PD-L1) positive TNBC, has shown encouraging efficacy [8]. Besides atezolizumab, other ICIs also showed efficacy in TNBCs [7,[9][10][11]. The role of immune check point inhibitors in metaplastic breast cancer is currently undergoing clinical investigation (NCT02834013).
The goal of this study is to understand the genomic and immune profiles of MpBC, and to study the association with clinical outcomes. An individual case of a metastatic MpBC patient carrying a PIK3CA mutation who had an exceptional response to everolimus is also reported here.

Patient selection
A total of 21 cases of MpBC in patients who were diagnosed and treated from 1996 to 2014 were retrospectively identified. The eligibility criteria were pathological diagnosis of MpBC and availability of paraffin-embedded tumor tissue for analysis. The patient characteristics, disease characteristics, treatment history and survival data were collected. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All tumor specimens were identified through a City of Hope IRB-approved retrospective protocol from patients consented to City of Hope Biorepository Protocol IRB 07047 (COH does not provide a separate approval number). Written informed consent was obtained from all participants of this study.

Pathology review
The archived tissue block from 20 surgical specimen and 1 metastatic biopsy were obtained. Hematoxylin and eosin (H&E) and immunohistochemical (IHC) stained slides were reviewed by two designated breast pathologists to confirm the diagnosis and morphological subtypes. The diagnosis of MpBC were based on the WHO 2012 Criteria [3]. In detail, MpBC were defined by the presence of squamous and/or mesenchymal (spindle or matrix-producing) elements, with or without coexisting ductal carcinoma in situ (DCIS) or conventional mammarytype invasive carcinoma. In cases lacking a conventional epithelial component, the diagnosis was confirmed using IHC staining, including low and high molecular weight cytokeratins and P63.

Immune profiling
Tumor infiltrating lymphocyte (TIL) quantification was performed using the International TIL Working Group Criteria [12]. Immunohistochemistry for CD3, CD4, CD8, and immune cell PD-L1 was performed. For PD-L1 testing, Ventana PD-L1 clone SP263 was used. A total of > 1% of PD-L1 expression on immune cell and > 1+ by IHC was considered positive in this study [13,14].

Clinicopathologic analysis
Patient characteristics were captured including age, race, stage, chemotherapy and radiation therapy history, relapse free survival (RFS), and overall survival (OS). RFS was defined as date of surgery to date of first relapse, and OS was defined as date of surgery to date of death.

Statistical analysis
Categorical variables were reported as counts (percent). Continuous data were reported as median (range). Descriptive survival analysis was performed using the Kaplan-Meier method, testing with the log-rank test. Estimates were reported along with their 95% confidence interval. Inference was drawn from Cox proportional-hazards regression modeling. Individual predictors were tested for significant differences using the Wald test. Statistical programming was done in the R programming language, version 3.4.1 [15]. Survival was estimated using the Survival add-on package, and plotted using the Survminer package [16,17].

Mutations and survival analysis
Of the 49 cancer-driver genes studied, the most commonly altered genes in this cohort of  (Fig 1  and S1 Table). In a univariate analysis, the association of TP53, PIK3CA, and PTEN mutations and breast cancer specific survival was determined. The median follow-up time of patients alive at date of analysis was 7.3 years (range 4.3-17.9). Recurrence free survival (RFS) at 5 years was 57% (95% CI 0.34-0.75) and overall survival (OS) at 5 years was 66% (95% CI 0.41-0.82). For patients with PIK3CA mutations, RFS and OS were significantly worse than for those without (HR 5.6, 95% CI 1.33-23.1 and HR 8.0, 95% CI 1.53-41.7, respectively) (Fig 2). The other mutations were underpowered to be conclusive: TP53 mutation associated with non-statistically significant reduction in hazard for RFS and OS (HR 0.66, 95% CI 0.16-2.63 and HR 0.56, 95% CI 0.13-2.34, respectively); PTEN was associated with non-statistically significant reduction in RFS and increase in hazard for OS (HR 0.79, 95% CI 0.10-6.44 and HR 1.54, 95% CI 0.17-13.8, respectively).

Immune profiling of metaplastic breast cancer and survival analysis
Representative Immunohistochemistry (IHC) for CD3, CD4, CD8, and PD-L1 is shown in Fig  3 and Fig). When dichotomizing the data based on best

A case of PI3K-targeted therapy for metaplastic breast cancer
We identified an exceptional responder treated with mTORC1 inhibitor everolimus. A 49-year-old woman underwent a right modified radical mastectomy for a stage II high grade MpBC in March 2011. She received standard adjuvant chemotherapy with doxorubicin, cyclophosphamide and paclitaxel followed by radiation. In September 2013, a solitary lung nodule was identified on routine chest x-ray and was resected via thoracotomy, which confirmed a 2.5 cm metastatic MpBC (Fig 4). FoundationOne1 genomic test identified TP53 p190del, PIK3CA C420R, CDK6 amplification, MYCL1 amplification, and JUN amplification. Patient received capecitabine with poor response. Restaging PET-CT in February 2015 demonstrated multiple bilateral pulmonary nodules with high standardized uptake value (SUV) of 7.6-16.7 (Fig 5A). Everolimus (10mg daily) was initiated in February 2015 with significantly reduced cough and shortness of breath, and improved performance status within a week. A restaging PET-CT in April 2015 confirmed marked improvement of pulmonary metastases (Fig 5B).
The patient was continued on treatment until December 2015 and achieved 8 months of disease control by PET-CT imaging (Fig 5C-5F).

Discussion
Chemotherapy remains the treatment of choice for MpBC and the prognosis is extremely poor with a median overall survival of 8 months in patients with metastatic MpBC [18,19]. There is an urgent need to identify novel molecular targets and develop clinical trials for treatment of this rare disease. In this study, we identified an association between PIK3CA mutation and poor RFS and OS in a cohort of patients with MpBC. We reported a patient with metastatic MpBC with an exceptional response to everolimus, a PI3K-mTOR targeted therapy. The current findings are hypothesis-generating and may indicate a role of PI3K-targeted therapy in treating MpBC. NGS can provide unique insight into the underlying biology of MpBC and may inform clinical decision making. In our study, PIK3CA mutation was found in 42.1% of MpBC patients. Other studies have reported a similar finding of 23-48% of PIK3CA mutations in MpBCs [20][21][22]. In a recent study by Ng et al., PIK3CA (29%), PIK3R1 (11%), ARID1A (11%), FAT1 (11%), and PTEN (11%) were identified a cohort of 35 MpBCs (23). Compared with invasive ductal TNBCs, MpBCs significantly more frequently harbored mutations in PI3K/AKT/mTOR pathway-related (57% vs. 22%) and canonical Wnt pathway-related (51% vs. 28%) genes [23]. Collectively, these studies provide evidence of heterogeneity of MpBC and indicate that PIK3CA/ AKT/mTOR pathway could serve as an important target for therapy. Although TP53 is one of the most commonly identified mutations in human breast cancer, targeting p53 has been challenging [24]. PIK3CA mutations are common in breast cancer with a reported rate of 25-40% of all breast cancers. The PIK3CA mutation C420R is an activating mutation predicted to be oncogenic. PIK3CA encodes the p110-alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K), and this pathway is involved in cell growth, proliferation, differentiation, motility and survival. Activating mutations may predict sensitivity to inhibitors of PI3K, AKT, or mTOR, including mTOR inhibitors everolimus and temsirolimus. In a subset analysis of a phase I study, metastatic MpBC treated with mTOR inhibitor temsirolimus plus liposomal doxorubicin and bevacizumab had an encouraging response rate of 42% [25,26]. Moulder et al. recently reported a response rate of 25% in 23 patients with metastatic MpBC treated with six different temsirolimus-based chemotherapy regimens [19]. A prospective clinical trial is needed to verify this finding.  [28,29]. In our study, 46.7% of MpBCs have >1% of PD-L1 expression and tumor PD-L1 expression is associated with poor 5-year RFS and OS. PD-L1 has been shown to be a predictive biomarker for response to immune checkpoint inhibitors [30]. Our study provides additional evidence that immune checkpoint inhibitors may play an important role in the treatment of MpBC.
Due to the rarity of MpBC, the current study is limited by its small sample size, retrospective nature, single center experience and limited 49-gene panel. A larger sized study with inclusion of whole exome sequencing and RNA sequencing will be required to verify the current finding. We are currently working with the Oncology Research Information Exchange Network (ORIEN) to build a genomic database of MpBC.

Conclusion
In this study, we report a unique cohort of MpBC with genomic and immune characterization. PIK3CA mutation and PD-L1 expression were associated with worse survival in this cohort of patients with MpBC. The case of exceptional response to everolimus reported here demonstrates how next-generation sequencing can identify effective therapies in patients with uncommon breast cancer histologies, for whom evidenced-based treatments are currently lacking.