Ki67 assessment in breast cancer: an update Penault-Llorca, Frederique; Radosevic-Robin, Nina
Pathology,
February 2017, 2017-Feb, 2017-02-00, 20170201, 2017-02, Volume:
49, Issue:
2
Journal Article
Peer reviewed
Although immunohistochemical detection of the Ki67 antigen has been used for many years to assess cancer proliferation, this marker is still not recommended for routine use in clinical management of ...breast cancer. The major reason for this situation is a lack of a standardised procedure for Ki67 assessment as well as persistence of several issues of debate with regards to the Ki67 assay interpretation and the marker’s clinical utility. Nowadays Ki67 assessment is principally used for estimation of prognosis and guiding the decision on adjuvant treatment choice, as well as for prediction of response to neoadjuvant treatment in ER+/HER2– breast cancer. In ER–/HER2+ and ER–/HER2– tumours, high post-neoadjuvant Ki67 index is associated with unfavourable prognosis.
We review here the elements impacting analytical validity of the Ki67 immunohistochemical assay, the evidence of its clinical utility and the current recommendations for its use in breast cancer management.
Tumour-infiltrating lymphocytes (TILs) have been demonstrated to significantly influence prognosis and response to therapy of invasive breast cancer (IBC). Thus, it has been suggested that TIL ...density or/and immunophenotype could serve as biomarkers for selection of IBC patients for immunotherapy. However, much less is known about significance of TILs in breast ductal carcinoma in situ (DCIS).
We retrospectively investigated TIL density and immunophenotype in 96 pure DCIS and 35 microinvasive carcinomas (miCa). TIL density was assessed on H&E-stained breast biopsy sections as the percentage of tumour stromal area occupied by TILs, and classified into 4 grades: 0 (0%-9%), 1 (10-29%), 2 (30-49%) and 3 (50%-100%). TIL immunophenotype was assessed by immunohistochemistry for CD8, CD4, FoxP3, CD38 or CD20.
Compared to pure DCIS, miCa contained significantly more cases with TIL density grade 3 (p = 0.028). Concordantly, CD8+, CD4+ and CD38+ cells were more numerous in miCa than in pure DCIS. In the pure DCIS subgroup with TIL density grades 2 and 3, all TIL subpopulations were more numerous than in the pure DCIS with TIL density grades 0 and 1, however the ratio between T-lymphocytes (CD8+ and CD4+) and B-lymphocytes (CD20+) was significantly lower (p = 0.029). On the other side, this ratio was significantly higher in miCa, in comparison with pure DCIS having TIL density grades 2 and 3 (p = 0.017). By cluster analysis of tumour cell pathobiological features we demonstrated similarity between miCa and the pure DCIS with TIL density grades 2 and 3. The only significant difference between those two categories was in the ratio of T- to B-TILs, higher in miCa.
Results indicate that TIL density level can distinguish 2 biologically different DCIS subgroups, one of which (DCIS with ≥30% TILs, the TIL-rich DCIS) is like miCa. Similarity of TIL-rich pure DCIS and miCa as well as the role of B-lymphocytes in DCIS invasiveness are worth further investigating with regards to the potential development of immunotherapy-based prevention of DCIS progression.
Vasculogenic mimicry (VM) formed by aggressive tumor cells to create vascular networks connected with the endothelial cells, plays an important role in breast cancer progression. WISP2 has been ...considered as a tumor suppressor protein; however, the relationship between WISP2 and VM formation remains unclear. We used the in vitro tube formation assay and in vivo immunohistochemical analysis in a mouse model, and human breast tumors were used to evaluate the effect of WISP2 on VM formation. Here we report that WISP2 acts as a potent inhibitor of VM formation in breast cancer. Enforced expression of WISP2 decreased network formation while knockdown of WISP2 increased VM. Mechanistically, WISP2 increased retention of oncogenic activators YAP/TAZ in cytoplasm, leading to decreased expression of the angiogenic factor CYR61. Studies using an in vivo mouse model and human breast tumors confirmed the in vitro cell lines data. In conclusion, our results indicate that WISP2 may play a critical role in VM and highlight the critical role of WISP2 as a tumor suppressor.
Introduction: Patient-derived xenografts (PDX) can be used to explore tumour pathophysiology and could be useful to better understand therapeutic response in breast cancer. PDX from mammary tumours ...are usually made from metastatic tumours. Thus, PDX from primary mammary tumours or after neoadjuvant treatment are still rare. This study aims to assess the feasibility to establish xenografts from tumour samples of patients with triple negative or luminal B breast cancer in neoadjuvant, adjuvant or metastatic setting.
Methods: XENOBREAST is a single-centre and prospective study. This feasibility pilot trial aims to produce xenografts from tumour samples of patients with triple negative or luminal B breast cancer. Patient enrolment is expected to take 3 years: 85 patients will be enrolled and followed for 28 months. Additional blood samples will be taken as part of the study. Surgical specimens from post-NAC surgery, primary surgery or surgical excision of the metastases will be collected to establish PDX. Histomolecular characteristics of the established PDX will be investigated and compared with the initial histomolecular profile of the collected tumours to ensure that they are well-established.
Ethics and dissemination: XENOBREAST belongs to category 2 interventional research on the human person. This study has been approved by the Sud Méditerranée IV - Montpellier ethics committee. It is conducted notably in accordance with the Declaration of Helsinki and General Data Protection Regulation (GDPR). Study data and findings will be published in peer-reviewed medical journals. We also plan to present the study and all data at national congresses and conferences.
Registration: ClinicalTrials.gov ID
NCT04133077; registered on October 21, 2019.
Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway are common and therapeutically targeted in triple-negative ...breast cancer (TNBC). However, activation of another EGFR family member human epidermal growth factor receptor 3 (HER3) (or ErbB3) may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.
Abstract
To find metastatic recurrence biomarkers of triple-negative breast cancer (TNBC) treated by neoadjuvant chemotherapy and anti-EGFR antibodies (NAT), we evaluated tumor genomic, ...transcriptomic, and immune features, using MSK-IMPACT assay, gene arrays, Nanostring technology, and TIL assessment on H&E. Six patients experienced a rapid fatal recurrence (RR) and other 6 had later non-fatal recurrences (LR). Before NAT, RR had low expression of 6 MHC class I and 13 MHC class II genes but were enriched in upregulated genes involved in the cell cycle-related pathways. Their TIL number before NAT in RR was very low (<5%) and did not increase after treatment. In post-NAT residual tumors, RR cases showed high expression of
SOX2
and
CXCR4
. Our results indicate that high expression of cell cycle genes, combined with cold immunological phenotype, may predict strong TNBC resistance to NAT and rapid progression after it. This biomarker combination is worth validation in larger studies.
Introduction
Triple negative breast cancer (TNBC) is highly heterogeneous, but still most of the patients are treated by the anthracycline/taxane-based neoadjuvant therapy (NACT). Tumor-infiltrating ...lymphocytes (TILs) are a strong predictive and prognostic biomarker in TNBC, however are not always available. Peripheral blood counts, which reflect the systemic inflammatory/immune status, are easier to obtain than TILs. We investigated whether baseline white cell or platelet counts, as well as, Neutrophil-to-Lymphocyte Ratio (NLR) or Platelet-to-Lymphocyte Ratio (PLR) could replace baseline TILs as predictive or prognostic biomarkers in a series of TNBC treated by standard NACT.
Patients and Methods
One hundred twenty patients uniformly treated by FEC/taxane NACT in a tertiary cancer care center were retrospectively analyzed. The presence of pathological complete response (pCR: ypT0/Tis, ypN0) or the presence of pCR and/small residual disease (ypT0/Tis/T1ab, ypN0) were considered as good responses in data analysis. Baseline/pre-NACT blood count, NLR, PLR and TILs were evaluated as predictors of response, distant recurrence rate and distant recurrence-free survival (DRFS).
Results
TILs ≥30% and ≥1.5% were best predictors of pCR and distant recurrence risk, respectively (p = 0.007, p = 0.012). However, in this cohort, pCR status was not significantly associated with recurrence. Only the ensemble of patients with pCR and small residual disease had lower recurrence risk and longer survival DRFS (p = 0.042, p = 0.024, respectively) than the rest of the cohort (larger residual disease). The only parameter which could predict the pCR/small residual disease status was PLR: patients with values lower than 133.25 had significantly higher chance of reaching that status after NACT (p = 0.045). However, no direct correlation could be established between baseline PLR and metastatic recurrence. No correlation either was found between TIL and individual blood counts, or between TILs and NLR or PLR.
Conclusion
In this cohort, TILs retained their pCR predictive value; however PLR was a better predictor of the ensemble of responses which had good outcome in terms of less distant recurrences or longer DRFS (pCR or small residual disease). Thus, baseline PLR is worth further, prospective investigation together with baseline TILs, as it might indicate a good TNBC response to NACT when TILs are unavailable.
The role of tumour infiltrating lymphocytes (TILs) as a biomarker in non-invasive breast cancer is unclear. This meta-analysis assessed the prognostic impact of TIL levels in patients with ...non-invasive breast cancer.
Systematic literature search was performed to identify studies assessing local recurrence in patients with non-invasive breast cancer according to TIL levels (high vs. low). Subgroup analyses per local recurrence (invasive and non-invasive) were performed. Secondary objectives were the association between TIL levels and non-invasive breast cancer subtypes, age, grade and necrosis. Odds ratios (ORs) and 95% confidence intervals (CI) were extracted from each study and a pooled analysis was conducted with random-effect model.
Seven studies (N = 3437) were included in the present meta-analysis. High-TILs were associated with a higher likelihood of local recurrence (invasive or non-invasive, N = 2941; OR 2.05; 95%CI, 1.03–4.08; p = 0.042), although with a lower likelihood of invasive local recurrence (N = 1722; OR 0.69; 95%CI, 0.49–0.99; p = 0.042). High-TIL levels were associated with triple-negative (OR 3.84; 95%CI, 2.23–6.61; p < 0.001) and HER2-positive (OR 6.27; 95%CI, 4.93–7.97; p < 0.001) subtypes, high grade (OR 5.15; 95%CI, 3.69–7.19; p < 0.001) and necrosis (OR 3.09; 95%CI, 2.33–4.10; p < 0.001).
High-TIL levels were associated with more aggressive tumours, a higher likelihood of local recurrence (invasive or non-invasive) but a lower likelihood of invasive local recurrence in patients with non-invasive breast cancer.
•The prognostic role of TILs in non-invasive breast cancer is unknown.•We observed an association between high-TILs and local recurrence.•High-TILs were associated with triple-negative and HER2+ subtypes, high grade and necrosis.•Non-invasive lesions with aggressive characteristics may also be more immunogenic.•Patients with low-TILs showed a higher likelihood of presenting an invasive recurrence.
The hypermetabolic nature of cancer cells and their increased reliance on "aerobic glycolysis", as originally described by Otto Warburg and colleagues, are considered metabolic hallmarks of cancer ...cells. BRCA1 is a major tumor suppressor in breast cancer and it was implicated in numerous pathways resulting in anticarcinogenic functions. The objective of our study was to address specific contributions of BRCA1 to the metabolic features of cancer cells, including the so-called "Warburg effect". To get a comprehensive approach of the role of BRCA1 in tumor cell metabolism, we performed a global transcriptional and metabolite profiling in a BRCA1-mutated breast cancer cell line transfected or not by wild-type BRCA1. This study revealed that BRCA1 induced numerous modifications of metabolism, including strong inhibition of glycolysis while TCA cycle and oxidative phosphorylation tended to be activated. Regulation of AKT by BRCA1 in both our cell model and BRCA1-mutated breast tumors was suggested to participate in the effect of BRCA1 on glycolysis. We could also show that BRCA1 induced a decrease of ketone bodies and free fatty acids, maybe consumed to supply Acetyl-CoA for TCA cycle. Finally increased activity of antioxidation pathways was observed in BRCA1-transfected cells, that could be a consequence of ROS production by activated oxidative phosphorylation. Our study suggests a new function for BRCA1 in cell metabolic regulation, globally resulting in reversion of the Warburg effect. This could represent a new mechanism by which BRCA1 may exert tumor suppressor function.
Radiation therapy (RT), a novel approach to boost the anticancer immune response, has been progressively evaluated in the neoadjuvant setting in breast cancer (BC).
We aimed to evaluate ...immunity-related indicators of response to neoadjuvant chemoradiation therapy (NACRT) in BC for better treatment personalization.
We analyzed data of the first 42 patients included in the randomized phase 2 Neo-APBI-01 trial comparing standard neoadjuvant chemotherapy (NACT) and NACRT regimen in locally advanced triple-negative (TN) and luminal B (LB) subtype BC. Clinicopathological parameters, blood counts and the derived parameters, total tumor-infiltrating lymphocytes (TILs) and their subpopulation, as well as
mutation status, were assessed as predictors of response.
Twenty-one patients were equally assigned to each group. The pathologic complete response (pCR) was 33% and 38% in the NACT and NACRT groups, respectively, with a dose-response effect. Only one LB tumor reached pCR after NACRT. Numerous parameters associated with response were identified, which differed according to the assigned treatment. In the NACRT group, baseline hemoglobin of ≥13 g/dL and body mass index of <26 were strongly associated with pCR. Higher baseline neutrophils-to-lymphocytes ratio, total TILs, and T-effector cell counts were favorable for pCR.
This preliminary analysis identified LB and low-TIL tumors as poor responders to the NACRT protocol, which delivered RT after several cycles of chemotherapy. These findings will allow for amending the selection of patients for the trial and help better design future trials of NACRT in BC.
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