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Up to thirty percent of patients with early breast cancer (BC) will eventually develop metastases. This results in an ongoing search for treatments to improve the long-term survival of these patients. Immune checkpoint inhibitors (ICI) are one promising type of therapeutic targets and have shown good results in malignancies as melanoma and non-small cell lung cancer. In BC, and especially in triple negative BC (TNBC), a clinical benefit for the addition of ICI to standard therapy has been shown in both the metastatic and early stage of disease. However, only a small subset of patients experience durable and long-term disease control by ICI, with many tumors unlikely to respond. In addition, measurable anti-tumor activity may appear slower in immune therapies than in cytotoxic therapies and lesions may enlarge before shrinking.Therefore, we designed a window-of-opportunity trial (BioKey) to evaluate the short-term effects of the ICI pembrolizumab in patients with early BC with biomarker determination by comparing multiple markers pre-treatment and at surgery.Furthermore, a better understanding of the tumor immune microenvironment (TIME) of TNBC and the other molecular subtypes of BC tumors is paramount to identify and implement new biomarkers in ICI BC treatment. Changes related to intratumoral immunity and proliferation may provide evidence for biological activity of pembrolizumab.The vast majority of studies investigating ICI in patients with BC have focused on TNBC. In our retrospective study, we were able to show that the TIME is very similar between TNBC and HR-negative HER2-positive BC tumors by performing an in-depth characterization of the TIME. A comparative analysis of TNBC and HER2-positive BC revealed no significant differences regarding sTIL and the majority of the investigated immune cell subpopulations (CD3, CD4, CD8, CD73, PD-1, PD-L1). We nevertheless identified significantly higher levels of CD68+ and FOXP3+ cells in TNBC as compared to HER2-positive tumors. Additionally, we performed a similar comparison using publicly available transcriptomics data. Altogether, the results show a comparable TIME in both groups, with possible implications for the use of ICI in patients with HR-negative HER2-positive breast tumors.BioKey was a single center, prospective, open-label, non-randomized study in patients with a non-metastatic newly diagnosed primary invasive carcinoma of the breast. All patients were treated with one intravenous administration of 200 mg of pembrolizumab at 10 ± 4 days before surgery. The study consisted of 2 cohorts with a total of 54 patients; cohort A included 39 patients who were scheduled for upfront surgery and cohort B included 15 patients with estimated residual tumor size of at least 10 mm on imaging after neo-adjuvant chemotherapy. Patients were monitored carefully for the development of adverse events, which were actively reviewed at 10 ± 4 days and 30 ± 7 days after administration of the study medication.Single-cell transcriptome, T-cell receptor and proteome profiling was used to compare paired pre- versus on-treatment biopsies. Pre-treatment, one-third of tumors contained PD1-expressing T-cells, which clonally expanded upon anti-PD1. This expansion mainly involved CD8+ T-cells with increased expression of cytotoxic-activity, immune-cell homing and exhaustion markers, as well as CD4+ T-cells with improved T-helper-1 and follicular-helper activity. We designated these as patients with clonotype expansion or 'expanders' (E), whereas patients lacking clonotype expansion were considered 'non-expanders' (NE). Pre-treatment, immunoregulatory dendritic cells and several macrophage phenotypes expressing PD-L1, and cancer cells exhibiting MHC class I/II expression correlated positively with T-cell expansion. We depicted an immune landscape consisting of various immunophenotypes and associated gene sets being either positively or negatively correlated with T-cell expansion following anti-PD1. Our data suggest novel biomarkers or potential immunotherapeutic modalities to improve clinical benefit from immune checkpoint blockade.Finally, we investigated the TIME of the expander phenotype with central pathology. We observed that i) higher levels of sTIL, PD-1+ sTIL, PD-L1+ and FOXP3+ cells in the pre-treatment biopsies, ii) higher levels of sTIL, PD-1+ sTIL, PD-L1+, CD3+, CD4+, CD8+, CD68+, FOXP3+ cells and Ki67 in the post-treatment surgical specimen, as well as, iii) higher delta values (surgical specimen - biopsy) of sTIL, CD3+, CD4+, CD8+, CD68+ and Ki67 are associated with the expander phenotype. We were able to detect associations between various cell populations of the tumor immune microenvironment evaluated before and after a single dose of pembrolizumab and the T-cell expansion phenotype previously defined at the single-cell level.In a chapter we discuss the immune related adverse events (irAEs) after a single dose of pembrolizumab. Twenty-two patients (41%) experienced at least one adverse event in the follow-up period. In general, the irAEs were mild (grade I or II), and in most cases, no intervention was needed. The observed AEs were common AEs in the context of ICI and were mainly related to gastrointestinal disorders, skin and subcutaneous disorders and hyperthyroidism.Two patients developed late irAEs, which they reported 115 and 101 days after administration of the study medication. The first patient was diagnosed with sarcoidosis and uveitis and the second patient with severe hepatitis. Both patients initially developed hyperthyroidism which later evolved into hypothyroidism. Our observations reinforce the importance of clinical vigilance for late irAEs after ICI discontinuation and imply that merely a single dose of ICI can have a long-lasting immunomodulating effect.In conclusion, we observed that one in three patients demonstrated pronounced clonotype expansion of PD-1-expressing proliferative T-cells along CD8+ or CD4+ trajectories in the post-treatment samples after one dose of pembrolizumab. We hypothesize that this T cell clonotype expansion is a mechanism of response to pembrolizumab. This pronounced clonotype expansion was not limited to patients with TNBC, but was also observed in patients with HR-positive HER2 positive and HR positive BC.Clonotype expansion is clearly measurable via single-cell profiling. The immunohistochemistry data showed associations between the clonotype expansion status, predefined based on single cell profiling, and levels of sTIL and expression of various other immune markers. Nonetheless, we were not able to predict the clonotype expansion status of patients by immunohistochemistry alone. A new neo-adjuvant clinical trial has been designed and will start soon to allow the validation of our findings and to evaluate if T-cIell clonotype expansion is indeed associated with clinical benefit to ICI.

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Non-primary breast tumors are a very rare phenomenon. It occurs most often in patients with a history of malignancies. The symptoms similar to primary breast carcinoma. To conduct this study we first got Ethical approval by The Research Ethics Committee UZ/KU Leuven. Next, we searched the pathology database. We further gathered the used immunohistocytochemistry (IHC) markers, age at diagnosis of metastasis, survival, follow-up and type of therapy. We further examined the literature for single center retrospective reviews and included 7 studies. Our dataset, consisting of 118 patients, was the largest among these studies. In our center, the average age at diagnosis of metastasis was 61 years. The most frequently observed tumor was Diffuse Large B-cell Lymphoma (DLBCL), which occurred in 24 different patients, accounting for 20% of cases. Second most frequent was malignant melanoma with 16 different patients (14%). This was followed by ovarian carcinoma, which occurred in 9 cases (8%). The other studies showed some differences in tumor types, such as more frequent lung adenocarcinomas than ovarian carcinoma, which might have been due to referral bias in our center. Histological diagnosis can be tricky and this highlights the importance of sufficient clinical history and IHC. Survival is overall poor and therapeutic options are mostly systemic, reflecting late stage metastatic disease. Both are however very heterogenous in different tumor types.

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Background: High levels of stromal tumor infiltrating lymphocytes (TILs) in breast cancer (BC) patients treated with neoadjuvant therapy is predictive of higher levels of pathological complete response (pCR). In this study we evaluated whether the predictive value of TILs is influenced by body mass index (BMI) in HER2+ BC patients treated with neoadjuvant chemotherapy (NACT). Methods: TILs scoring at baseline and post NACT were done on a retrospective series of nonunderweight HER2+ BC patients from 2007-2015 (N = 120). Relevant clinic-pathological information was retrieved from the MBC database of UZL, missing data were collected from medical files of the patients. All patients received anti-HER2 therapy. All variables were considered as categorical (BMI: lean vs overweight vs obese, different predefined cut offs were used for TILs). Descriptive statistical analyzes were performed using the Fisher exact test. Associations between clinicopathological variables and pCR were assessed using Firth logistic regression models. Results: 52 (43%) patients were lean, 44 (37%) overweight and 24 (20%) obese. Overall, pCR was achieved in 53 (44%) of the patients. Median TILs at baseline and post NACT was 8.16 and 2.68, respectively. High TILs were associated with pCR in all BMI categories and regardless of ER status. A decline of mean TILs after neoadjuvant treatment was greater in tumors achieving pCR (16.93% to 2.05%) as compared to tumors with residual disease. The highest rate of pCR was established in overweight patients with ER- BC (20 of 29, 69%). In obese patients a much lower rate of pCR (6 of 24, 25%) was seen compared to lean (21 of 52, 40%) and overweight (26 of 44, 59%) patients. However, no interactions were observed after multivariate analyses using categorical values. Conclusion: High level of TILs are a strong predictor of pCR in HER2+ BC regardless of ER status. The interaction between TILs and BMI deserve further study in HER2+ BC treated with neoadjuvant chemotherapy.

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A significant effort is being made in the identification of prognostic markers to improve the risk stratification of invasive micropapillary breast carcinoma (IMPC). Expression of BCL-2is normally associated an improved overall prognosis. However, it remains unclear whether this applies also to IMPC. Recently, it was observed that tumor-infiltrating lymphocytes (TILs) in IMPC are a negative prognostic influence. The aim of this study is to evaluate the reproducibility in the assessment of BCL-2 expression and stromal TILs measurement in IMPC. Secondly we aim at validate BCL-2 expression and stromal TILs measurement as independent prognostic markers in IMPC. A total of 132 female patients with IMPC were included in the study for retrospective analysis and pathology review was performed on all cases. A tissue micro array (TMA) was created to evaluate the amount of BCL-2 using immunohistochemical staining with an BCL-2 antibody. The results were graded independently by 2 different readers in a semiquantative manner. We also examined these patients on the percentage of TILs by 3 different observers, following the guidelines as set by Salgado et al. Afterwards these results were compared to assess reproducibility of the different scoring system between readers. Subsequently TILs scores were compared to breast cancer surrogate subtypes, Lymphovascular invasion (LVI), perineural invasion (PNI) and tertiary lymphoid aggregates (TLA) status. We observed an excellent correlation between the BCL-2 scores of the 2 readers. We were able to determine that the readers had an ICC of 0.776 on resection specimens (RS) and an ICC of 0.753 on core needle biopsies (CNB). When we compared the TILs scores on a CNB and a RS in the same patient, we found a poor correlation for reader 1 and 2. We did find a good correlation for CNB and RS in reader 3 with an ICC of 0.803. The ANOVA test shows that there is a significant difference in the scores for each breast cancer surrogate subtype with a p-value of 0.026 (α = 0.05). We could not establish a significant difference in TIL scoring for patients with LVI vs. patients without LVI and no significant difference between the patients with PNI and without PNI (p = 0.36). However we did find a significant difference in TIL scores between patients with TLA (19.89%) and the group without TLA (14.65%) with a p value of 0.02. we ascertained a significant correlation between the presence of LVI and the presence of TLA in our patient group In this study we were able to determine BCL-2 staining as a highly reproducible test, independent of the interpreter’s experience level. TILs score was also found to be a reproducible test for RS and CNB, with potential for further research to be done and guidelines to be created about how to standardize TIL grading in IMPC specifically. We found a significant difference in TILs scoring between different breast cancer surrogate subtypes. No significant correlation was found between TILs score and the presence of LVI nor between the TIL score and PNI, however a significant correlation between TIL score and TLA was found. We also observed a strong relationship between the presence of LVI and TLA. The next step will be to evaluate the value of BCL-2 and TILs as independent prognostic factors.

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Literature review HER2 is known to be overexpressed in approximately 15% to 20% of all breast cancers. To guarantee proper high quality testing in daily clinical practice, ASCO/CAP have developed guidelines. Three different versions have been written since 2007, aiming to generalize and standardize analytical aspects and interpretation of HER2 testing. The Latest version, published in 2018, aims to optimize identification of HER2 positive patients. Although proven to be fit for purpose for identification of this specific subgroup of patients, It remains unclear whether these guidelines are the golden standard for identification of a new group of patient, so called HER-low. Recently,multiple different therapy options have been approved by the FDA for HER2-low patients. The 2018 ASCO/CAP guideline also updated its recommendation regarding FISH reflex testing. Reflex testing for HER2 IHC 3+ cases would no longer be supported by guidelines. Objectives According to recent guidelines published in 2018 by ASCO/CAP, in situ hybridization diagnostic tests with the aim to validate IHC 3+ cases are no longer recommended. These changes imply that pathologists are no longer able to rely on ISH methods to validate their initial evaluation concerning HER2 IHC 3+ cases. Given this paradigmatic switch in the interpretation of the HER2 IHC, research is needed to assess whether current ASCO/CAP guidelines are still fit for purpose to correctly identify this novel category of patients. In this scenario Belgian pathologists will have to be proficient in accurately scoring the lower range of HER2 protein expression and distinguish HER2-LOW patients from HER2 true negative ones. Materials and methods Based on a consensus provided by expert pathologists, we performed a modified Delphi study and Cases able to reach a consensus score were eligible for inclusion in a HER2 training scheme. A training scheme was designed to include 42 cases of which 32 would have to be scored by participants themselves. 61 participants were included in the study. Results Out of 102 total cases scored according to the ASCO/CAP guidelines of 2007 and 2018, in a first round of consensus, experts were able to reach a consensus for 41 and 24 cases respectively. In general we observed a reduction in the size of the HER2 IHC 1+ subgroup when comparing results obtained from both guidelines.. using the ASCO/CAP 2007 guidelines our participants reached an accuracy of 78.9% of all assessments (N=473). Using the ASCO/CAP 2018 guidelines accuracy was found to be 74.1% meaning 1405 out of 1897 cases evaluated using the ASCO/CAP 2018 guidelines were concordant with the consensus score. For both guidelines, scoring accuracy was found to be approaching 100% for HER2 IHC 3+ while scoring accuracy for HER2 LOW was found to be below 80%. Across all cohorts, all assessments and both guidelines, deviating results were found to be dominated by overestimation. Breast pathology experience, familiar primary method/assay of scoring, screen resolution as well as type of laboratory affiliated with participants did no significantly influence obtained data. conclusion While current guidelines have proven to be fit for purpose with the aim of identifying patients to be HER2 positive or negative, using these guidelines to correctly identify HER2 LOW patients will not be desirable due to the extent of discordant results.