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Introduction: Flat epithelial atypia (FEA), classical lobular neoplasia (LN), papillary lesions (PL), radial scars (RS) and atypical ductal hyperplasia (ADH) are lesions of uncertain malignant potential in the breast, which are described as B3 lesions in the B classification system of the European Working Group for Breast Cancer Screening Pathology. Current standard for management of B3 lesions on core needle biopsy (CNB) or vacuum assisted biopsy (VAB), is wide local excision (WE). However, recent consensus-based guidelines no longer recommend WE for all such lesions, but propose surveillance following CNB or VAB to be sufficient in some cases. In the absence of a Belgian guideline on the treatment of B3 lesions, this study was conducted to identify which B3 lesions have the lowest likelihood for breast malignancy and could therefore be spared from WE. Methods: Using data from the Belgian Cancer Registry (BCR), all patients with a new diagnosis of a B3 lesion on CNB or VAB between 2013-2016 and who had a histological follow up with VAB or WE after CNB or WE after VAB were included. Histological follow-up was retrieved from BCR and limited to 12 months following diagnosis. Histology was compared between the first- and follow-up investigation to determine the upgrade risk to ductal carcinoma in situ (DCIS) or invasive breast cancer (IC) according to the type of B3 lesion. Patients with synchronous (pre-) invasive lesions were excluded. Results: Between 2013-2016 there were 812 B3 lesions available for upgrade analysis after initial diagnosis. After CNB 551 lesions had WE or VAB as follow up and after VAB 261 lesions had WE. After primary diagnosis on CNB, the total upgrade risk was 19,0%. There was histological agreement in 57,9% and no B3 lesion or upgrade was reported in 21,8%. Per B3 lesion subtype the upgrade risk to DCIS - IC after diagnosis on CNB was: ADH 17,1%-12,4%, FEA 21,1% - 18,4%, LN 18,9% -21,6%, RS 14,3% - 11,4%, and PL 7,2% - 3,2%. After initial diagnosis on VAB the total upgrade risk was 14,9%. There was histological agreement in 52,9% and no B3 lesion or upgrade was found in 31,4%. Per B3 subtype the upgrade risk to DCIS - IC after diagnosis on VAB was: ADH 17,3%- 2,7%, FEA 11,7%- 5,9%, LN 0,0% - 4,3%, PL 10,4% - 2,1%. We found no upgrade for RS. (Table 1). Conclusions: In a series of B3 lesions with a histological follow-up, we notice that overall upgrade risk is higher for lesions detected on CNB than on VAB: 19,0% vs. 14,9%. The majority of lesions showed histological agreement between initial B3 diagnosis and histological follow-up: 57,9% after CNB and 53,5% after VAB. More investigation is needed to make a proper risk assessment as to which B3 lesions can be followed with regular surveillance. Also, further prospective research is needed to get a better understanding of associated risk factors for upgrade, upgrade risk and lifetime risk of developing breast cancer after diagnosis of a B3 lesion. Table 1: Upgrade risk per B3 subtype after diagnosis on CNB or VABSubtypeNumberCNB followed by VAB or WEDCISICTotal Upgrade riskCNBNumberVAB followed by WEDCISICUpgrade riskVAB followed by WEADH10517,1%12,4%29,5%11017,3%2,7%20,0%FEA3821,1%18,4%39,5%5111,7%5,9%17,6%LN3718,9%21,6%40,5%460%4,3%4,3%PL3367,2%3,2%10,4%4810,4%2,1%12,5%RS3514,3%11,4%25,7%60%0%0%Total551261Upgrade105/55119,0%39/26114,9%B3 lesion319/55157,9%138/26152,9%No B3 lesion120/55121,8%82/26131,4%Result not available7/5511,3%2/2610,8% Citation Format: Nynke Willers, Patrick Neven, Giuseppe Floris, Cecile Colpaert, Eva Oldenburger, Sileny Han, Chantal Van Ongeval, Ann Smeets, Francois Duhoux, Hans Wildiers, Petra Denolf, Isabel De Brabander, Nancy Van Damme, Harlinde De Schutter. The upgrade risk to (pre-)invasive breast cancer for B3 lesions diagnosed on core needle or vacuum assisted biopsy. A Belgian retrospective study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-52.

Contains fulltext : 245198.pdf (Publisher’s version ) (Closed access) High stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC) are associated with pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). Histopathological assessment of sTILs in TNBC biopsies is characterized by substantial interobserver variability, but it is unknown whether this affects its association with pCR. Here, we aimed to investigate the degree of interobserver variability in an international study, and its impact on the relationship between sTILs and pCR. Forty pathologists assessed sTILs as a percentage in digitalized biopsy slides, originating from 41 TNBC patients who were treated with NAC followed by surgery. Pathological response was quantified by the MD Anderson Residual Cancer Burden (RCB) score. Intraclass correlation coefficients (ICCs) were calculated per pathologist duo and Bland-Altman plots were constructed. The relation between sTILs and pCR or RCB class was investigated. The ICCs ranged from -0.376 to 0.947 (mean: 0.659), indicating substantial interobserver variability. Nevertheless, high sTILs scores were significantly associated with pCR for 36 participants (90%), and with RCB class for eight participants (20%). Post hoc sTILs cutoffs at 20% and 40% resulted in variable associations with pCR. The sTILs in TNBC with RCB-II and RCB-III were intermediate to those of RCB-0 and RCB-I, with lowest sTILs observed in RCB-I. However, the limited number of RCB-I cases precludes any definite conclusions due to lack of power, and this observation therefore requires further investigation. In conclusion, sTILs are a robust marker for pCR at the group level. However, if sTILs are to be used to guide the NAC scheme for individual patients, the observed interobserver variability might substantially affect the chance of obtaining a pCR. Future studies should determine the 'ideal' sTILs threshold, and attempt to fine-tune the patient selection for sTILs-based de-escalation of NAC regimens. At present, there is insufficient evidence for robust and reproducible sTILs-guided therapeutic decisions.

PURPOSE: This study evaluated epidemiologic and immune factors associated with pathologic complete response (pCR), breast cancer-specific survival (BCSS) and disease-free survival (DFS) outcomes in inflammatory (IBC) and locally advanced breast cancer (LABC) patients. METHODS: Tumor-infiltrating lymphocytes (TILs) and CD20(+) B-cell frequencies (CD20(+)), and PD-L1 expression on tumor (PD-L1(+)carcinoma cells) and immune (PD-L1(+)TILs) cells were analyzed by immunohistochemistry along with clinicopathologic factors as modifiers of pCR and outcomes in 221 IBC and 162 LABC patients. Analysis included Kaplan–Meier curves and Cox proportional hazard models. RESULTS: IBC and LABC display similar levels of TILs, CD20(+), and combined CD20(+) and PD-L1(+)TILs (CD20(+)PD-L1(+)TILs), while LABC contained more PD-L1(+)TILs and PD-L1(+) carcinoma cells. Absence of lymphovascular involvement, high TILs, PD-L1(+) carcinoma cells, and combined CD20(+) and PD-L1(+) carcinoma cells correlated with pCR in IBC and LABC patients. High PD-L1(+)TILs correlated with pCR only in LABC; less lymph node involvement at diagnosis, CD20(+) and CD20(+)PD-L1(+)TILs correlated with pCR only in IBC (P < 0.04, all comparisons). Achievement of pCR in IBC and LABC patients correlated with BCSS and DFS (P < 0.02). In multivariate analyses, pCR remained an independent prognostic factor of improved DFS in IBC and LABC patients, but of BCSS in only LABC. CD20(+)PD-L1(+)TILs remained an independent prognostic factor of improved DFS and BCSS only in IBC. CONCLUSION: CD20(+)PD-L1(+)TILs are an independent prognostic biomarker of improved outcomes in IBC, but not LABC. Selecting IBC patients by CD20 and PD-L1 status could stratify patients and potentially identify those in whom activating CD20 agents and anti-PD-1/PD-L1 therapy could be explored.

Introduction: Inflammatory breast cancer (IBC) is a rare but aggressive form of breast cancer (BC) in which the (prognostic) role of stromal tumour-infiltrating lymphocytes (sTIL) and the peripheral circulating immune cells in patients with residual disease (RD) after neo-adjuvant chemotherapy (NACT) is not clearly established. Methodology: To describe the evolution of sTIL and some peripheral inflammation markers (Neutrophil-to-lymphocyte ratio, Platelet-to-lymphocyte ratio and Lymphocyte-to-monocyte ratio) after NACT in IBC, we retrospectively collected clinicopathological variables for 125 stage III IBC patients. sTILs were scored by three different researchers on an H&amp, E slide of the mastectomy specimen. A cohort of subtype-matched non-IBC breast cancer patients (nIBC) treated with NACT was included for comparison. Results: There was no significant difference in the pre- and posttreatment sTIL scores between IBC and nIBC and in both groups the number of sTIL was significantly lower after NACT. However, the IBC phenotype did correlate with a stronger decrease of sTIL after NACT (OR: 0.25, 95% CI: 0.073–0.76, p = 0.018). The change in the peripheral immune markers was not significantly different between IBC and nIBC. After NACT, 75 patients had residual disease. In this group, a high number of sTIL before NACT (HR: 0.23, 95% CI: 0.05–1.02, p = 0.05) was prognostic for a longer OS, while a low number of sTIL after NACT (HR: 0.33, 95% CI: 0.11–0.98, p = 0.046) and a low residual cancer cellularity (HR: 0.20, 95% CI: 0.08–0.52, p &lt, 0.001) was associated with a longer DFS. Conclusions: IBC is associated with a significantly stronger decrease of sTIL after NACT compared to nIBC. Furthermore, a high number of sTIL after NACT was associated with a worse prognosis in IBC.

Histopathological assessment of ductal carcinoma in situ, a nonobligate precursor of invasive breast cancer, is characterized by considerable interobserver variability. Previously, post hoc dichotomization of multicategorical variables was used to determine the "ideal" cutoffs for dichotomous assessment. The present international multicenter study evaluated interobserver variability among 39 pathologists who performed upfront dichotomous evaluation of 149 consecutive ductal carcinomas in situ. All pathologists independently assessed nuclear atypia, necrosis, solid ductal carcinoma in situ architecture, calcifications, stromal architecture, and lobular cancerization in one digital slide per lesion. Stromal inflammation was assessed semiquantitatively. Tumor-infiltrating lymphocytes were quantified as percentages and dichotomously assessed with a cutoff at 50%. Krippendorff's alpha (KA), Cohen's kappa and intraclass correlation coefficient were calculated for the appropriate variables. Lobular cancerization (KA = 0.396), nuclear atypia (KA = 0.422), and stromal architecture (KA = 0.450) showed the highest interobserver variability. Stromal inflammation (KA = 0.564), dichotomously assessed tumor-infiltrating lymphocytes (KA = 0.520), and comedonecrosis (KA = 0.539) showed slightly lower interobserver disagreement. Solid ductal carcinoma in situ architecture (KA = 0.602) and calcifications (KA = 0.676) presented with the lowest interobserver variability. Semiquantitative assessment of stromal inflammation resulted in a slightly higher interobserver concordance than upfront dichotomous tumor-infiltrating lymphocytes assessment (KA = 0.564 versus KA = 0.520). High stromal inflammation corresponded best with dichotomously assessed tumor-infiltrating lymphocytes when the cutoff was set at 10% (kappa = 0.881). Nevertheless, a post hoc tumor-infiltrating lymphocytes cutoff set at 20% resulted in the highest interobserver agreement (KA = 0.669). Despite upfront dichotomous evaluation, the interobserver variability remains considerable and is at most acceptable, although it varies among the different histopathological features. Future studies should investigate its impact on ductal carcinoma in situ prognostication. Forthcoming machine learning algorithms may be useful to tackle this substantial diagnostic challenge. ispartof: MODERN PATHOLOGY vol:33 issue:3 pages:354-366 ispartof: location:United States status: published

Introduction Inflammatory breast cancer (IBC) is an uncommon, but aggressive form of breast cancer that accounts for a disproportionally high fraction of breast cancer related mortality. The aim of this study was to explore the peripheral immune response and the prognostic value of blood-based biomarkers, such as the neutrophil-to-lymphocyte ratio (NLR), in a large IBC cohort. Patients & methods We retrospectively identified 127 IBC patients and collected lab results from in-hospital medical records. The differential count of leukocytes was determined at the moment of diagnosis, before any therapeutic intervention. A cohort of early stage (n = 108), locally advanced (n = 74) and metastatic breast cancer patients (n = 41) served as a control population. Results The NLR was significantly higher in IBC compared to an early stage breast cancer cohort, but no difference between IBC patients and locally advanced breast cancer patients was noted. In the metastatic setting, there was also no significant difference between IBC and nIBC. However, a high NLR (>4.0) remained a significant predictor of worse outcome in IBC patients (HR: 0.49; 95% CI: 0.24–1.00; P = .05) and a lower platelet-lymphocyte ratio (PLR) (≤210) correlated with a better disease-free survival (DFS) (HR: 0.51; 95% CI: 0.28–0.93; P = .03). Conclusion Patients with a high NLR (>4.0) have a worse overall prognosis in IBC, while the PLR correlated with relapse free survival (RFS). Since NLR and PLR were not specifically associated with IBC disease, they can be seen as markers of more extensive disease., Highlights • There is no association between NLR or PLR and inflammatory breast cancer (IBC). • IBC and non-inflammatory breast cancer elicit a similar peripheral immune response. • A high neutrophil-to-lymphocyte ratio (NLR) is associated with a worse overall survival in IBC. • A high platelet-lymphocyte ratio (PLR) correlates with a worse disease-free survival in IBC. • An elevated PLR or NLR should be seen as a marker of more extensive disease in IBC.

Introduction: Inflammatory breast cancer (IBC) is a rare, but aggressive form of breast cancer. Increasing evidence indicates that immune cells in the tumor micro-environment play an important role in IBC progression: infiltration with stromal Tumor Infiltrating Lymphocytes (sTIL) is associated with a better response to neo-adjuvant chemotherapy (NACT) and longer overall survival in IBC patients. However, the prognostic role of sTIL in patients without a complete pathological response (pCR) after NACT remains unclear. In this study we evaluated the effect of NACT on sTIL in IBC and locally advanced non-inflammatory breast cancer (LABC) and the prognostic impact of sTIL in IBC. Methodology: In this retrospective case-control study we evaluated sTIL in patients with IBC (n=60) and LABC (n= 134) that received anthracyclin-taxane based NACT. Tumor tissue was sampled as part of the routine work-up, before (diagnostic biopsy) and after (resection specimen) NACT. sTIL scoring was performed on Haematoxylin & Eosin stained 5-µm sections of formalin-fixed paraffin-embedded tumor tissue by two different researchers according to the recommendations by the International TILs Working Group. In case of discrepancy, the sample was scored by a third observer in order to obtain a consensus score. sTIL difference between pre-treatment and post-treatment specimen was called δsTIL. Cellularity of the residual cancer after NACT was evaluated according to the MD Anderson Residual Cancer Burden (RCB) method. Results: Most of the IBC patients presented with a hormone receptor (HR) positive carcinoma (n=35/60, 58.3%) and 25 patients had pCR after NACT (42%); the latter patients had less than 1% sTIL in the tumor bed area. Besides having histologically more poorly differentiated tumors (P= 0.033), no significant clinicopathological differences between the IBC and LABC cohort were observed. There was no significant difference in the pre-treatment median sTIL score between IBC (12.5%, range: 1% - 80%) and LABC (10%, range: 1% - 85%). Furthermore, both in the IBC (median δsTIL: -4.5%, P= 0.01) and in the LABC (median δsTIL: -1%, P= 0.06) cohort the number of sTIL was lower after NACT. This decrease was significantly greater in the IBC cohort (P= 0.04). In a multivariate model - including HR and HER2 status, histological differentiation grade, nodal status and IBC/LABC phenotype - IBC disease correlated with a stronger decrease of sTILs after NACT (OR: 0.31, 95% CI 0.10 - 0.89, P= 0.04). As we previously demonstrated, higher sTIL score before NACT was associated with better overall survival (OS) (P= 0.006) in IBC. IBC patients without pCR, but with a stronger decrease (> 4.5 %) of the number of sTIL after NACT had a better OS (P= 0.04) and disease-free survival (DFS) (P= 0.04). There was a significant correlation between δsTIL and both higher sTIL score before NACT (P< .001) and lower residual cancer cellularity (P= 0.02). However, in a multivariate model - including sTIL before NACT, δsTIL and residual cancer cellularity - both a strong decrease of sTIL (HR: 0.32; 95% CI 0.10 - 1.00; P= 0.05) and a low residual cancer cellularity (HR: 0.22; 95% CI 0.07 - 0.68; P= 0.009) were independent predictors of a better DFS. Conclusion: Both the IBC and LABC cohort had the same number of sTIL before NACT. However, the IBC phenotype was associated with a stronger decrease of sTIL after NACT independent of molecular subtype and nodal stage. Furthermore, a strong decrease in sTIL was an independent predictor of better prognosis in IBC. Citation Format: Christophe Van Berckelaer, Leonie Vercauteren, Iris Vermeiren, Glenn Broeckx, Steven Van Laere, Luc Dirix, Cecile Colpaert, Peter Van Dam. The prognostic role of tumor-infiltrating lymphocytes after treatment with neo-adjuvant chemotherapy in inflammatory breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-13.

Multiple independent studies have shown that tumor-infiltrating lymphocytes (TIL) are prognostic in breast cancer with potential relevance for response to immune-checkpoint inhibitor therapy. Although many groups are currently evaluating TIL, there is no standardized system for diagnostic applications. This study reports the results of two ring studies investigating TIL conducted by the International Working Group on Immuno-oncology Biomarkers. The study aim was to determine the intraclass correlation coefficient (ICC) for evaluation of TIL by different pathologists. A total of 120 slides were evaluated by a large group of pathologists with a web-based system in ring study 1 and a more advanced software-system in ring study 2 that included an integrated feedback with standardized reference images. The predefined aim for successful ring studies 1 and 2 was an ICC above 0.7 (lower limit of 95% confidence interval (CI)). In ring study 1 the prespecified endpoint was not reached (ICC: 0.70; 95% CI: 0.62-0.78). On the basis of an analysis of sources of variation, we developed a more advanced digital image evaluation system for ring study 2, which improved the ICC to 0.89 (95% CI: 0.85-0.92). The Fleiss' kappa value for

SCOPUS: le.j, info:eu-repo/semantics/published

Background Inflammatory breast cancer (IBC) is a rare and rapidly progressive form of invasive breast cancer. The aim of this study was to explore the clinical evolution, stromal tumour-infiltrating lymphocytes (sTIL) infiltration and programmed death-ligand 1 (PD-L1) expression in a large IBC cohort. Patients and methods Data were collected prospectively from patients with IBC as part of an international collaborative effort since 1996. In total, 143 patients with IBC starting treatment between June 1996 and December 2016 were included. Clinicopathological variables were collected, and sTIL were scored by two pathologists on standard H&E stained sections. PD-L1 expression was assessed using a validated PD-L1 (SP142) assay. A validation cohort of 64 patients with IBC was used to test our findings. Results Survival outcomes of IBC remained poor with a 5-year overall survival (OS) of 45.6%. OS was significantly better in patients with primary non-metastatic disease who received taxane-containing (neo)adjuvant therapy (P = 0.01), had a hormone receptor-positive tumour (P = 0.001) and had lower cN stage at diagnosis (P = 0.001). PD-L1 positivity on immune cells (42.9%) was higher in IBC than in non-IBC in both our patient samples and the validation cohort. Furthermore, PD-L1 expression predicted pCR (P = 0.002) and correlated with sTIL infiltration (P

INTRODUCTION: Inflammatory breast cancer (IBC) is a rare and aggressive type of locally advanced breast cancer. The hallmark of IBC is the presence of E-Cadherin positive tumor emboli within the dermal and parenchymal lymphatic vessels. These tumor emboli are thought to be responsible for the clinical presentation by the physical obstruction of draining lymph vessels. In addition, the intravascular growth of tumor emboli may account for the typical diffuse growth pattern and the fast local spread associated with IBC. Tumor emboli can result from encircling lymphovasculogenesis or lymphovascular invasion and little is known about the infiltrating immune cells in these emboli. This is the first study to investigate the immune infiltrate in the tumor emboli of patients with IBC. METHODOLOGY: To analyze the immune composition of the emboli, four slides of 24 IBC patients were stained according to a validated protocol and scored using the VISIOPHARM® software that makes virtual multiplexing possible after the alignment of different digital slides. We used four validated antibodies: CD79α (B-cell lineage), CD8 (cytotoxic T-cells), FOXP3 (Tregs) and CD163 (Tumor associated macrophages; TAMs), combined with a standard H&E stained section to mark the tumor area and tumor emboli on pretreatment biopsy sections. RESULTS: Half of the IBC patients presented with a hormone receptor positive carcinoma (54.2% - n= 13/24) and 8 out of 21 patients with initially localized disease achieved complete pathological response (pCR) after neo-adjuvant chemotherapy (NACT). Our results are summarized in table 1. Table 1: For every staining we report the number of patients that have infiltrated emboli, the median density in the invasive carcinoma (IC), the median density in the emboli (E) and the ratio between the invasive tumor and emboli in table 1.# patientsDensity (IC) #/μm2Density (E) #/μm2Ratio Density E/ICP-valueCD812/24 (50 %)19.62.40.11< .001CD16319/23 (83 %)35.412.80.43.02CD79α11/24 (46 %)18.23.50.31.01FOXP34/23 (17 %)5.10.30.04.003 Patients with more immune cells in the invasive carcinoma (IC) also had more immune cells in the emboli (E). This correlation was significant for all stainings (Table 1). CD163+ macrophages are the most abundant immune cells in emboli, especially when compared to the invasive carcinoma, whereas CD8+ cells were uncommon in the emboli. The density of CD8+ cells in the invasive carcinoma or emboli did not predict pCR. Interestingly, patients with a higher IC/E CD8 density ratio achieved more often pCR (Kruskal-Wallis χ = 3.6, P = 0.05). Only 20.8% (n= 5/24) had invasive carcinoma directly surrounding the intravascular emboli, however, median overall survival in these patients was significantly shorter (P= 0.003). CONCLUSIONS: This is the first study looking at infiltrating immune cells in IBC. Although based on a small discovery cohort of only 24 patients, we showed that there is a strong correlation between the density of immune cells in the emboli and the density in the invasive carcinoma. Furthermore, we demonstrate that TAMs are the predominant immune cell type in IBC emboli. Cytotoxic T cells are less common and having more CD8+ cells in the invasive carcinoma compared to the emboli was associated with pCR. Patients with invasive carcinoma directly surrounding the emboli had a worse prognosis. Citation Format: Christophe Van Berckelaer, Charlotte Rypens, Peter Vermeulen, Steven Van Laere, Peter Van Dam, Cecile Colpaert. Infiltrating immune cells in the tumor emboli of inflammatory breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-08.

Background: Inflammatory breast cancer (IBC) is a rare and aggressive type of locally advanced breast cancer. A 79-gene signature, reported by our lab, is shaped by specific immune response programs and discriminates between IBC and non-IBC (nIBC). However, it remains an enigma how infiltrating immune cells are able to determine the IBC phenotype. Furthermore, the presence of immune cells like FOXP3+ Tregs and CD8+ cytotoxic T cells is associated with outcome in proliferative subtypes of breast cancer and the interaction between these cells plays a role in the functional immune response. Therefore, we aimed to assess the spatial associations between immune cells in IBC. Additionally, we used deep-learning to examine interactions between cancer and immune cells. Methodology: Immunostainings (Hematoxylin-DAB, H-DAB) were done according to well-validated protocols for CD8 (cytotoxic T-cells), FOXP3 (Tregs) and CD163 (tumor-associated macrophages, TAMs) in a large population of 134 IBC patients. All slides were digitalized and evaluated using VISIOPHARM® software, allowing virtual multiplexing. We quantified the number of DAB+ immune cells and each positive immune cell was located using XY coordinates. Spatial co-localization was examined using statistics developed for ecological studies based on point pattern and quadrant analysis. TILs were scored according to the TIL working group guidelines on H&E slides. Tumor cell coordinates were collected using a deep-learning algorithm applied to the CD8 stained slide. To perform deep-learning, we aligned two consecutive slides: one PanCK stained slide and one slide stained for CD8 (H-DAB). Using virtual multiplexing and the PanCK staining, we determined the tumor regions on the H-DAB stained slide. Subsequently, 18 images were incorporated to train the algorithm with more than 150.000 iterations (Deeplabv3+), after which the algorithm was evaluated in a test set of 12 images, approved and applied to all images to locate the tumor cells. Results: Most of the patients presented with a hormone receptor (HR) positive carcinoma (60.6%, n= 82/132). The presence of distant disease, HR status and TIL score were associated with overall survival (OS), but the density of the different immune cells or the CD8/FOXP3 ratio was not. However, using an effector index we demonstrated that patients with more FOXP3+ cells in a radius of 30 μm surrounding a CD8+ cell had a significant worse outcome (Median OS: 2.7 vs. 6.3 years, P= .01) and this remained significant in a multivariate model (HR: 2.85, P< .001). Complete pathological response (pCR) after neo-adjuvant chemotherapy was achieved by 28.7% (n= 27/94) of the patients with initially localized disease. Infiltration with CD8+ T cells (P= .005), TAMs (P= .008) and TILs (P= .002) predicted pCR, but a likelihood ratio test showed no difference between a model using CD8+ cells, TAMs or TILs. Interestingly, pCR was less often achieved in patients that had colocalization of FOXP3+ cells near the tumor cells (P= .003). This colocalization was determined using a Morista-Horn index and independent of the number of Tregs, CD8+ cells or TILs. The deep-learning algorithm had a mean dice coefficient in the test set of 0.83, indicating a good overlap between the tumor area determined by the AI on the CD8 slides and the area on the PanCK stained slides. Conclusions: We have created a deep learning algorithm that adequately detects IBC tumor cells on H-DAB stained images and show, in a large cohort of 132 patients, that the negative impact of Tregs appears to depend on the spatial arrangement. While solely the number of Tregs is not associated with pCR or OS, patients with FOXP3+ Tregs that cluster together near CD8+ cytotoxic T cells had a worse outcome and pCR was achieved more often in patients with less Tregs near the tumor cells. Citation Format: Christophe Van Berckelaer, Charlotte Rypens, Steven Van Laere, Koen Marien, Pieter-Jan van Dam, Peter Vermeulen, Luc Dirix, Mark Kockx, Cecile Colpaert, Peter van Dam. The spatial interactions between FOXP3+ Tregs, CD8+ cytotoxic T cells and tumor cells predict response to therapy and prognosis in inflammatory breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-04.

Recent studies suggest that anaplastic lymphoma kinase (ALK) could serve as a therapeutic target for inflammatory breast cancer (IBC) and that strategies targeting ALK should be considered for evaluation in clinical trials. Reverse phase protein arays revealed activation of the ALK receptor tyrosine kinase and biochemically-linked downstream signalling molecules in pre-clinical models of IBC. ALK genetic abnormalities have been reported in 80% (20/25) of IBC patients with a high prevalence of ALK alterations in basal-like IBC (Robertson F. et al., 2013). However, ALK protein expression has not been studied in human IBC samples. Immunohistochemical detection of the ALK protein is increasingly being used as a screening tool to test samples for ALK rearrangements. The biological premise is that the genetic abnormality of the ALK gene leads to overexpression of the ALK protein and therefore overactivity of the ALK tyrosine kinase; since this kinase is the target of crizotinib, it makes sense to assess the drug target directly. Formalin fixed paraffin embedded tissue samples from pre-treatment surgical biopsies of 79 consecutive IBC patients were immunohistochemically (IHC) tested for ALK protein over-expression using a validated IHC test (NCL-ALK, clone 5A4) with a sensitivity of 93% and a specificity of 100% when the Vysis ALK-FISH break apart test is used as a gold standard. Stained tissue sections were evaluated using the IHC histoscore proposed by Thunnissen E. et al., 2012, assessing both the proportion of tumor cells showing cytoplasmic staining and the staining intensity. Cytoplasmic staining of appendiceal ganglion cells was used as an on-slide external positive control; weak to moderate cytoplasmic staining of macrophages was used as an internal positive control. In 75 of the 79 tissue samples none of the tumor cells showed any ALK immunoreactivity. One tumor showed moderate cytoplasmic staining in a few cells ( These results demonstrate that ALK protein overexpression is not a feature of IBC. Although genetic abnormalities of ALK without protein overexpression are not excluded, our results show that ALK IHC cannot be used to identify IBC patients eligible for enrollment in clinical trials evaluating ALK targeted therapeutics. Citation Format: Cecile Colpaert, Melike Marsan, Peter Vermeulen, Luc Dirix, Steven Van Laere, Inflammatory Breast Cancer International Consortium. Anaplastic lymphoma kinase (ALK) protein overexpression is not a feature of inflammatory breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-39.

Objectives: Given the crucial role of pathology reporting in the management of breast cancers, we aimed to investigate the quality and variability of breast cancer pathology reporting in Belgium. Materials and methods: Detailed information on non-molecular and molecular parameters was retrieved from the pathology protocols available at the Belgian Cancer Registry for 10,007 breast cancers diagnosed in Belgium in 2008. Results: Substantial underreporting was shown for several clinically relevant non-molecular parameters, such as lymphovascular invasion. High-volume laboratories performed only slightly better than others, and analyses at the individual laboratory level showed clear inter-laboratory variability in reporting for all volume categories. Information on ER/PR and HER2 IHC was mentioned in respectively 91.7% and 90.8% of evaluative cases. HER2 ISH data were available for 78.5% of the cases judged to be 2+ for HER2 IHC. For cases with different specimens analysed, discordance between these specimens was highest for HER2, followed by PR. For HER2, results obtained from different laboratories were even less concordant. In addition, inter-laboratory differences were noted in the used ER/PR scoring systems, the proportion of ER-/PR+ cases, and the relation between histological grade and ER/PR positivity. Data on Ki67 were only available for 43.8% of the investigated cases, and showed inconsistent use of cut-off values. Conclusion: Breast pathology reporting in Belgium in 2008 was suboptimal and showed considerable inter-laboratory variability. Synoptic reporting has been proposed as a facilitator towards increased reporting quality and harmonization, but the lack of aligned informatics remains a major hurdle in its concrete implementation. (C) 2014 Elsevier Ltd. All rights reserved.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

Purpose: Inflammatory breast cancer is a distinct and aggressive form of locally advanced breast cancer with unique clinical and pathological features. Recently, histologic evidence of intense angiogenesis was found in inflammatory breast cancer specimens. The aim of this study was to confirm the angiogenic phenotype of inflammatory breast cancer and to investigate its potential to induce lymphangiogenesis. Experimental Design: Real-time quantitative reverse transcriptase-PCR was used to measure levels of mRNA of tumor angiogenesis and lymphangiogenesis-related factors [vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, Flt-1, KDR, Flt-4, Ang-1, Ang-2, Tie-1, Tie-2, cyclooxygenase-2, fibroblast growth factor-2 (FGF-2), Egr-1, Prox-1, and LYVE-1] in tumor specimens of 16 inflammatory breast cancer and 20 noninflammatory breast cancer patients. Tissue microarray technology and immunohistochemistry were used to study differential protein expression of some of the angiogenic factors in inflammatory breast cancer and noninflammatory breast cancer. Active lymphangiogenesis was further assessed by measuring lymphatic endothelial cell proliferation. Results: Inflammatory breast cancer specimens had significantly higher mRNA expression levels than noninflammatory breast cancer specimens of the following genes: KDR (P = 0.033), Ang-1, (P = 0.0001), Tie-1 (P = 0.001), Tie-2 (P = 0.001), FGF-2 (P = 0.002), VEGF-C (P = 0.001), VEGF-D (P = 0.012), Flt-4 (P = 0.001), Prox-1 (P = 0.005), and LYVE-1 (P = 0.013). High mRNA levels of FGF-2 and cyclooxygenase-2 corresponded to increased protein expression by immunohistochemistry. Inflammatory breast cancer specimens contained significantly higher fractions of proliferating lymphatic endothelial cells than noninflammatory breast cancer specimens (P = 0.033). Conclusions: Using real-time quantitative reverse transcriptase-PCR and immunohistochemistry, we confirmed the intense angiogenic activity in inflammatory breast cancer and demonstrated the presence of active lymphangiogenesis in inflammatory breast cancer. This may help explain the high metastatic potential of inflammatory breast cancer by lymphatic and hematogenous route. Both pathways are potential targets for the treatment of inflammatory breast cancer.

Purpose: A fibrotic focus, the scar-like area found in the center of an invasive breast tumor, is a prognostic parameter associated with an expansive growth pattern, hypoxia, and (lymph)angiogenesis. Little is known about the molecular pathways involved. Experimental Design: Sixty-five patients were selected of whom microarray data of the tumor and H&E slides for histologic analysis were available. The growth pattern and the presence and size of a fibrotic focus were assessed. Differences in biological pathways were identified with global testing. The correlations of growth pattern and fibrotic focus with common breast cancer signatures and with clinicopathologic variables and survival were investigated. Results: Tumors with a large fibrotic focus showed activation of Ras signaling and of the hypoxia-inducible factor-1α pathway. Furthermore, unsupervised hierarchical cluster analysis with hypoxia- and (lymph)angiogenesis-related genes showed that hypoxia-inducible factor-1α, vascular endothelial growth factor A, and carbonic anhydrase 9 were overexpressed. The presence of a fibrotic focus, especially a large fibrotic focus, was associated with the basal-like subtype (P = 0.009), an activated wound-healing signature (P = 0.06), and a poor-prognosis 76-gene signature (P = 0.004). The presence of a fibrotic focus (P = 0.02) and especially of a large fibrotic focus (P = 0.004) was also associated with early development of distant metastasis. Conclusions: Our results sustain the hypothesis that hypoxia-driven angiogenesis is essential in the biology of a fibrotic focus. Ras and Akt might play a role as downstream modulators. Our data furthermore suggest that vascular endothelial growth factor A does not only drive angiogenesis but also lymphangiogenesis in tumors with a fibrotic focus. Our data also show an association between the presence of a fibrotic focus and infaust molecular signatures.

Purpose To determine the role of magnetic resonance (MR) mammography in detection and assessment of extent of tumors with extensive intraductal component (EIC+). Material and methods In a prospective study, 233 consecutive women with a suspicious lesion underwent preoperative MR mammography and 209 invasive ductal carcinomas were detected. We studied the prediction of intraductal spread on mammography (MX), ultrasound (US) and MR. We compared the size of the total lesion on MX, US and MR and correlated it with histopathology. Enhancement patterns on MR were described. Results Of 209 invasive ductal carcinomas, 50 were EIC+ (24%). MX predicted intraductal spread in EIC+ carcinomas in 48.5%, US in 34.2% and MR in 68%. Compared to MX and US, MR was best in assessment of total tumor size. On MR, ductal spread in EIC+ tumors presented as ductal or linear enhancement, long spicules, a regional enhancing area or nodules adjacent to a mass. Conclusion MR had the highest sensitivity to predict intraductal spread and was superior in assessing total tumor size.

Completion axillary lymph node dissection (cALND) is the golden standard if breast cancer involves the sentinel lymph node (SLN). However, most non-sentinel lymph nodes (NSLN) are not involved, cALND has a considerable complication rate and does not improve outcome. We here present and validate our predictive model for positive NSLNs in the cALND if the SLN is positive. Consecutive early breast cancer patients from one center undergoing cALND for a positive SLN were included. We assessed demographic and clinicopathological variables for NSLN involvement. Uni- and multivariate analysis was performed. A predictive model was built and validated in two external centers. 21.9% of 470 patients had at least one involved NSLN. In univariate analysis, seven variables were significantly correlated with NSLN involvement: tumor size, grade, lymphovascular invasion (LVI), number of positive and negative SLNs, size of SLN metastasis and intraoperative positive SLN. In multivariate analysis, LVI, number of negative SLNs, size of SLN metastasis and intraoperative positive pathological evaluation were independent predictors for NSLN involvement. The calculated risk resulted in an AUC of 0.76. Applied to the external data, the model was accurate and discriminating for one (AUC = 0.75) and less for the other center (AUC = 0.58). A discriminative predictive model was constructed to calculate the risk of NSLN involvement in case of a positive SLN. External validation of our model reveals differences in performance when applied to data from other institutions concluding that such a predictive model requires validation prior to use. (C) 2014 Elsevier Ltd. All rights reserved

Purpose: Lymph node (LN) lymphangiogenesis has recently been shown to be important in the premetastatic niche of sentinel LNs. To study its role in the further metastatic spread of human breast cancer, we investigated the association of angiogenesis and lymphangiogenesis in sentinel LN metastases with the presence of nonsentinel LN metastases in breast cancer patients with a positive sentinel LN. Experimental Design: Angiogenesis and lymphangiogenesis—quantified as endothelial cell proliferation fraction (ECP%) and lymphatic ECP fraction (LECP%)—were assessed in sentinel LN metastases of 65 T1/T2 patients with breast cancer using CD34/Ki67 and D2-40/Ki67 immunohistochemical double stains. Correlations were analyzed between nonsentinel LN status, LECP%, and other clinicopathologic variables (number of involved sentinel LNs, size of the primary tumor and LN metastasis, presence of lymphovascular invasion in the primary tumor, and of extracapsular growth in the sentinel LN metastasis). Results: Thirty seven out of 65 patients (56.9%) had at least one involved nonsentinel LN. Size of the sentinel LN metastasis (P = 0.001), lymphovascular invasion (P = 0.02), extracapsular growth (P = 0.02), and LECP% (P = 0.01) were correlated with a positive nonsentinel LN status. The multivariate logistic regression model retained high LECP% (odds ratios = 4.2, P = 0.01) and the presence of extracapsular growth (odds ratios = 3.38, P = 0.04) as independently associated with the presence of nonsentinel LN metastases. Conclusions: Increased sentinel LN metastasis lymphangiogenesis is associated with metastatic involvement of nonsentinel axillary LNs. These are the first data sustaining the hypothesis that sentinel LN lymphangiogenesis is involved in further metastatic spread of human breast cancer.

Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer with poor prognosis. Multimodal treatment including neo-adjuvant chemotherapy is the treatment of choice for IBC patients, with pathological complete response (pCR) being one of the most important prognostic factors. Tumor infiltrating lymphocytes (TILs) are an independent predictor of response to neo-adjuvant chemotherapy in breast cancer (Denkert C. et al., 2010). In a search for multigene predictors of pCR in IBC using DNA micro-arrays, a 107 gene signature was found that distinguished between responders and non-responders. This signature was enriched for immunity-related genes showing that in IBC, as in non-IBC, response to neo-adjuvant chemotherapy is associated with immunity related processes (Bertucci F. et al., 2014). Standard H&E stained sections of formalin-fixed paraffin-embedded pre-treatment tumor tissue of 77 IBC patients were used to evaluate the presence of TILs according to the Denkert method: mononuclear cell infiltrates were considered to be intratumoral TILs when making direct contact with the tumor cells, while stromal TILs are mononuclear cells being present in the tumor stroma without making direct contact with tumor cells. Intratumoral TILs were observed in 10,4% (8/77) of the patients. Stromal TILs (strTILs ) were present in all tumors: in 35% (27/77) strTILs occupied less than 5% of the tumor stroma, in 62 % (48/77) strTILs occupied between 5 and 50% of the tumor stroma and 2,6% (2/77) were "lymphocyte predominant breast cancers (LPBC)" with strTILS occupying more than 50% of the tumor stroma. One LPBC was HER2 positive, the other one was triple negative. There was a significant association of strTILs with response to neo-adjuvant chemotherapy: pCR was obtained in 13% of the patients with strTILs Tertiary lymphoid structures - lymphoid follicles with germinal centres, known to be associated with better clinical outcomes (Gu-Trantien C. et al., 2013) - were detected in 2.6% of the patients (2/77); both patients had a disease free survival of more than 3 years. This study shows that IBC tumors do not contain more TILs than non-IBC tumors and that, as in non-IBC, there is a positive association of stromal TILs with pCR in IBC. Further validation of these results and further study of lymphocyte subsets in IBC is warranted. Citation Format: Cecile Colpaert, Melike Marsan, Peter Vermeulen, Luc Dirix, Steven Van Laere, Inflammatory Breast Cancer International Consortium. Tumor infiltrating lymphocytes in inflammatory breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-20.

We studied the presence of lymphangiogenesis in lymph node (LN) metastases of breast cancer. Lymph vessels were present in 52 of 61 (85.2%) metastatically involved LNs vs 26 of 104 (25.0%) uninvolved LNs (P

Breast cancer can metastasize via lymphatic and hematogenous pathways. Hypoxia and (lymph)angiogenesis are closely related processes that play a pivotal role in the tumor progression and metastasis. The aim of this study was to compare expression of hypoxia and (lymph)angiogenesis-related genes between primary breast tumors and metastases in different tissues.A gene list of 269 hypoxia and (lymph)angiogenesis-related genes was composed and validated using Onto-Express, Pathway-express and Ingenuity software. The expression of these genes was compared in microarray data of 62 samples of primary tumors and metastases of 31 patients with breast cancer retrieved from Gene Expression Omnibus. Similarity between samples was investigated using unsupervised hierarchical clustering analysis, principal component analysis and permutation testing. Differential gene expression between primary tumors and metastases and between metastases from different organs was analyzed using Kruskall-Wallis and Mann-Whitney statistics.Unsupervised hierarchical cluster analysis demonstrated that hypoxia and (lymph)angiogenesis-related gene expression was more similar between samples from the same patient, than between samples from the same organ. Principal component analysis indicated that 22.7% and 7.0% of the total variation in the gene list was respectively patient and organ related. When differences in gene expression were studied between different organs, liver metastases seemed to differ most from the other secondary sites. Some of the best characterized molecules differentially expressed were VEGFA, PDGFRB, FGF4, TIMP1, TGFB-R1 and collagen 18A1 (precursor of endostatin). To confirm the results of these experiments at the protein level, immunohistochemical experiments were performed with antibodies for VEGFA and MMP-2.Our results suggest that hypoxia and (lymph)angiogenesis-related gene expression is more dependent on the characteristics of the primary tumor than on the characteristics of the organs that bear the metastasis. However, when different organs are compared, the expression in liver metastases differs most from other metastatic sites and primary tumors, possibly due to organ-specific angiogenic and lymphangiogenic responses to metastasis-related hypoxia.

Inactivation of TGF-beta/SMAD4 signaling was postulated to play an important role in breast cancer development. Even though SMAD4 is located on 18q21, a region frequently lost in breast cancers, point mutations of SMAD4 were rarely observed, implying that biallelic inactivation of SMAD4 was not necessary in the process. In this study, a novel homozygous deletion of SMAD4 was identified in breast cancer cell line SW527 during a screening of 31 breast cancer cell lines. As several breast cancer cell lines were shown to contain SMAD4 homozygous deletion, we sought to develop a reliable method to access such lesions in archived primary tumor specimens. First, a DNA quantification method was developed to measure as few as 5 copies of DNA templates so that the amount of genomic DNA isolated by laser-capture microdissection can be accurately determined. Next, accurate DNA quantitation allowed sufficient DNA templates to be included in the homozygous deletion assay for the robust amplification of SMAD4 genetic markers. Two out of 24 primary infiltrative ductal carcinomas (IDC) with 18q allelic imbalance were determined to contain SMAD4 homozygous deletions, and these samples are also negative for Smad4 protein expression by immunohistochemistry. Our data suggest that biallelic inactivation of SMAD4 through homozygous deletion does occur in a small percentage of IDCs, and support the hypothesis that inactivation of TGF-beta/SMAD4 signaling plays in a role in the development of a subset of IDC.

Recently, peritumoural (lympho)vascular invasion, assessed on haematoxylin-eosin (HE)-stained slides, was added to the St Gallen criteria for adjuvant treatment of patients with operable breast cancer (BC). New lymphatic endothelium-specific markers, such as D2-40, make it possible to distinguish between blood (BVI) and lymph vessel invasion (LVI). The aim of this prospective study was to quantify and compare BVI and LVI in a consecutive series of patients with BC. Three consecutive sections of all formalin-fixed paraffin-embedded tissue blocks of 95 BC resection specimens were (immuno)histochemically stained in a fixed order: HE, anti-CD34 (pan-endothelium) and anti-D2-40 (lymphatic endothelium) antibodies. All vessels with vascular invasion were marked and relocated on the corresponding slides. Vascular invasion was assigned LVI (CD34 [plus sign in circle] or [minus sign in circle]/D2-40 [plus sign in circle]) or BVI (CD34 [plus sign in circle]/D2-40 [minus sign in circle]) and intra- (contact with tumour cells or desmoplastic stroma) or peritumoural. The number of vessels with LVI and BVI as well as the number of tumour cells per embolus were counted. Results were correlated with clinico-pathological variables. Sixty-six (69.5%) and 36 (37.9%) patients had, respectively, LVI and BVI. The presence of 'vascular' invasion was missed on HE in 20% (peritumourally) and 65% (intratumourally) of cases. Although LVI and BVI were associated intratumourally (P=0.02), only peritumoural LVI, and not BVI, was associated with the presence of lymph node (LN) metastases (p(peri)=0.002). In multivariate analysis, peritumoural LVI was the only independent determinant of LN metastases. Furthermore, the number of vessels with LVI was larger than the number of vessels with BVI (P=0.001) and lymphatic emboli were larger than blood vessel emboli (P=0.004). We demonstrate that it is possible to distinguish between BVI and LVI in BC specimens using specific lymphatic endothelium markers. This is important to study the contribution of both processes to BC metastasis. Furthermore, immunohistochemical detection of lymphovascular invasion might be of value in clinical practice.

We validated with univariate and multivariate (Cox) analysis, the prognostic value of the mitotic activity index (MAI), the fibrotic focus (FF) and other prognosticators in 448 patients with lymph node-negative (LN-) invasive breast cancer55 years without adjuvant systemic treatment (72.5 months median follow-up, range 4-119). Of these patients, 24.8% developed distant and 1.6% loco-regional recurrence. FF showed excellent inter-observer reproducibility (kappa = 0.93). Strong prognosticators were MAI, grade, nuclear atypia, FF and the St. Gallen criterion (SG). The subgroup with excellent survival selected by SG was only 16% of all patients, implying over-treatment of more than 70% of all LN- patients when using SG as adjuvant therapy selection criterion. If MAI10, 13% showed distant metastases, contrasting with 41% if MAIor = 10. FF was prognostic in the ductal and mixed ductal cancers, but not in the lobular and other subtype cancers. Patients with invasive (mixed) ductal cancers with FF absent, FF1/3 or FF1/3 of the tumour area, had distant metastasis rates of 17%, 35% and 48%; in MAI10 and FF absent, FF1/3 or FF1/3, metastasis rates were 11%, 13% and 42% and if MAIor = 10, metastasis rates were 31%, 48% and 50%, respectively. In the 12 patients with MAI10 and a large FF1/3, event-free survival was similar to patients with MAIor = 10. With multiple regression MAI10 versusor = 10 is the strongest prognosticator (also stronger than the SG). The FF may be important as it has additional prognostic value to the MAI in the small subgroup of invasive ductal or mixed-ductal breast cancer patients with combined MAI10 and an FF1/3 of the tumour area.

Hypoxia and angiogenesis are important factors in breast cancer progression. Little is known of hypoxia and angiogenesis in lymph node metastases of breast cancer. The aim of this study was to quantify hypoxia, by hypoxia-induced marker expression levels, and angiogenesis, by endothelial cell proliferation, comparing primary breast tumours and axillary lymph node metastases. Tissue sections of the primary tumour and a lymph node metastasis of 60 patients with breast cancer were immunohistochemically stained for the hypoxia-markers carbonic anhydrase 9 (CA9), hypoxia-inducible factor-1alpha (Hif-1alpha) and DEC-1 and for CD34/Ki-67. Endothelial cell proliferation fraction (ECP%) and tumour cell proliferation fraction (TCP%) were assessed. On haematoxylin-eosin stain, the growth pattern and the presence of a fibrotic focus were assessed. Hypoxia-marker expression, ECP% and TCP% in primary tumours and in lymph node metastases were correlated to each other and to clinico-pathological variables. Median ECP% and TCP% in primary tumours and lymph node metastases were comparable (primary tumours: ECP%=4.02, TCP%=19.54; lymph node metastases: ECP%=5.47, TCP%=21.26). ECP% correlated with TCP% (primary tumours: r=0.63, P0.001; lymph node metastases: r=0.76, P0.001). CA9 and Hif-1alpha expression were correlated (primary tumours P=0.005; lymph node metastases P0.001). In primary tumours, CA9 and Hif-1alpha expression were correlated with DEC-1 expression (P=0.05), presence of a fibrotic focus (P0.007) and mixed/expansive growth pattern (P0.001). Primary tumours and lymph node metastases with CA9 or Hif-1alpha expression had a higher ECP% and TCP% (P0.003); in primary tumours, mixed/expansive growth pattern and fibrotic focus were characterised by higher ECP% (P=0.03). Furthermore, between primary tumours and lymph node metastases a correlation was found for ECP%, TCP%, CA9 and Hif-1alpha expression (ECP% r=0.51, P0.001; TCP r=0.77, P0.001; CA9 and Hif-1alpha P0.001). Our data demonstrate that the growth of breast cancer lymph node metastases is angiogenesis dependent and that angiogenesis and hypoxia in the primary tumour predict angiogenesis and hypoxia in the lymph node metastases. Together with previous findings in breast cancer liver metastases, which grow in 96% of cases angiogenesis independently, these data suggest that both the intrinsic growth characteristics and angiogenic potential of breast cancer cells and the site-specific tumour microenvironment determine angiogenesis and hypoxia in breast cancer.

Introduction Identification of breast cancer patients at risk for postoperative distant relapse is an important clinical issue. Existing pathological markers can predict disease recurrence only to a certain extent, and there is a need for more accurate predictors. Methods Using 'counting alleles', a novel experimental method, we determined allelic status of chromosomes 8p and 18q in a case-control study with 65 early stage, node negative, invasive ductal carcinomas (IDCs). The association between allelic imbalance (AI) of both chromosomal markers and distant relapses was examined. Results Eighty percent of tumors contained 8pAI and sixty-eight percent of tumors contained 18qAI. However, none of the tumor samples retained both chromosome 8p and 18q alleles. More importantly, tumors with 8pAI but not 18qAI were more likely to have distant relapse compared to tumors with 18qAI but not 8pAI. Conclusion Our finding suggests that differential allelic loss of chromosomes 8p and 18q may represent subtypes of early stage IDC with different tumor progression behaviors.

Although angiogenesis is a prerequisite for the growth of most human solid tumours, alternative mechanisms of vascularisation can be adopted. We have previously described a non-angiogenic growth pattern in liver metastases of colorectal adenocarcinomas (CRC) in which tumour cells replace hepatocytes at the tumour-liver interface, preserving the liver architecture and co-opting the sinusoidal blood vessels. The aim of this study was to determine whether this replacement pattern occurs during liver metastasis of breast adenocarcinomas (BC) and whether the lack of an angiogenic switch in such metastases is due to the absence of hypoxia and subsequent vascular fibrinogen leakage. The growth pattern of 45 BC liver metastases and 28 CRC liver metastases (73 consecutive patients) was assessed on haematoxylin- and eosin-stained tissue sections. The majority of the BC liver metastases had a replacement growth pattern (96%), in contrast to only 32% of the CRC metastases (P

The enhancing area surrounding breast carcinoma on MR mammography is correlated with findings from pathological examination. We studied 194 patients with breast cancer who underwent preoperative MR mammography. Of all malignant lesions presenting with an enhancing surrounding area on MR mammography, morphologic features including long spicules, a ductal pattern, diffuse enhancement or nodules were evaluated and compared with histopathological examination. A double breast coil was used; we performed a 3D FLASH sequence with contiguous coronal slices of 2 mm, before and after injection of 0.2 mmol/kg GD-DTPA, and subtraction images were obtained. In total, 297 malignant lesions were detected at MR mammography and 101 of them had one or more types of enhancing surrounding area. In 49 of the 53 cancers with long spicules and in 49 of the 55 cancers with surrounding ductal pattern of enhancement, pathological examination showed in situ and/or invasive carcinoma. Multiple nodules adjacent to the carcinoma were seen in 20 patients and corresponded with six cases of invasive and ten cases of ductal in situ carcinoma. A diffuse enhancing area next to a mass was seen in ten patients and consisted of carcinoma in all cases: seven in situ and three invasive carcinomas. Enhancing areas including long spicules, a ductal pattern, noduli, or diffuse enhancement surrounding a carcinoma corresponded with in situ or invasive extension of the carcinoma in 92.5, 89, 80 and 100% of cases, respectively.

Inflammatory breast cancer (IBC) is an aggressive subtype of breast cancer with poor prognosis. The mechanisms responsible for the aggressive clinical evolution are incompletely understood. We constructed a tissue microarray (TMA) and validated its use in translational IBC research. Differential expression of proteins that might play a role in causing the IBC phenotype was studied.A TMA containing 34 IBC and 41 non-stage matched non-IBC tumours was constructed. Five core biopsies were taken for each IBC and three cores for each non-IBC tumour. The TMA was validated using three approaches: (1) the excellent concordance between immunohistochemical results of the initial pathological examination and the results obtained with the TMA for ER, PR and HER2/neu (kappa0.74); (2) the known differential expression between IBC and non-IBC for four bio-markers in IBC (ER, PR, p53 and HER2/neu) was confirmed ( p0.01); (3) the HER2/neu status using three different antibodies (CB11, TAB250 and HercepTest) was highly concordant (kappa0.75). Furthermore, the overexpression of E-Cadherin and RhoC GTPase in IBC ( p0.05) was confirmed. We did not find a differential expression pattern for carbonic anhydrase IX (CA IX) and EGFR.Using different approaches, we have validated the use of our TMA for studying differential protein expression in IBC and non-IBC. We confirm the overexpression of E-Cadherin and RhoC GTPase in IBC. The lack of differential expression for CA IX and EGFR might suggest the pathways are equally utilised in both types of breast cancer.

Purpose. To assess the value of local staging with preoperative magnetic resonance imaging (MRI) in patients with suspect breast lesions and the effect on therapeutic approach. Materials and methods. Two hundred and four consecutive women with suspect breast lesions on clinical examination (CE) and/or mammography (MX) and/or ultrasound (US) underwent preoperative contrast-enhanced MRI. Detection of multifocal, multicentric and bilateral breast cancer by all three imaging modalities was evaluated. Results of preoperative breast MRI were discussed with the treating surgeons. The type of therapeutic change after preoperative MRI was marked on a questionnaire (none, additional fine needle aspiration, core biopsy, open biopsy, wider excision, mastectomy) and considered ‘necessary’ or ‘unnecessary’ using final histopathological results as gold standard. Results. In 170 patients, breast cancer was diagnosed. MRI detected 96% of multifocal disease and 95% of multicentric disease, whereas MX depicted 37 and 18%, and US 41 and 9% of them, respectively. All bilateral breast cancers were seen on MRI; both MX and US detected 56%. Findings of more extensive disease and unsuspected multiple breast cancer foci identified on MRI only, changed the therapeutic approach correctly in 30.6% of breast cancer patients. Nine unnecessary wider excisions and three unnecessary FNA/core biopsies were performed because MRI overestimated the number or size of malignant lesions. Conclusion. Preoperative breast MRI is an important adjunct to conventional imaging in the loco-regional staging of breast cancer and a useful tool in treatment planning.

The value of the fibrotic focus (FF) as a marker of intra-tumoral hypoxia in invasive breast carcinoma was assessed by studying its relationship with the expression of the hypoxia-induced carbonic anhydrase IX (CA IX), angiogenesis indices and prognosis. CA IX expression was immunohistochemically detected in 2 independent study groups, totaling 184 patients, and correlated with tumor characteristics, angiogenesis related parameters and patient outcome by univariate analysis. CA IX immunostaining scores in carcinoma cells and in tumoral fibroblasts were significantly higher in expansively growing tumors (p = 0.0001 and p < 10(-4), respectively), containing an FF (p = 0.0004 and p < 10(-4)) and showing high histological grade (p = 0.016 and p = 0.0006). Microvessel density, quantified by Chalkley counting, was correlated with CA IX expression both in the carcinoma cells and in the fibroblasts (p = 0.0076 and p = 0.0025) and with the presence and relative size of an FF (p = 0.006). The fraction of proliferating endothelial cells was positively correlated with CA IX scores in the fibroblasts (r = 0.4, p = 0.02) and with the presence of an FF (p = 0.02). CA IX scores in the fibroblasts--and to a lesser extent in the carcinoma cells--were associated with a higher relapse rate (p = 0.006) and a worse overall survival (p = 0.003). The highest CA IX immunostaining scores were found in the fibroblasts of large FF occupying more than one-third of the tumor. A large FF was associated with worse overall survival in a consecutive patient group (p = 0.01) and with shorter disease-free (p = 0.02) and overall survival (p = 0.0005) in T1-2N0 breast cancer patients. The strong association of CA IX expression with the presence of an FF shows that the latter is a marker of intra-tumoral hypoxia. FF is useful as a surrogate marker of hypoxia-driven ongoing angiogenesis and is associated with a higher relapse rate and a worse overall survival.

To investigate the use of MRI in preoperative characterization of invasive lobular breast cancer (ILC) and in detection of multifocal/multicentric disease. We retrospectively reviewed T1-weighted FLASH 3D precontrast and postcontrast MR images together with subtraction images of 26 women with histopathologically proven invasive lobular cancer. Two experienced radiologists described tumor patterns of ILC independently. MR findings of unifocal, multifocal, single quadrant and multiquadrant disease were correlated with results of other imaging techniques and compared with histopathological findings as gold standard. Most ILC presented on MRI as a single spiculated/irregular, inhomogeneous mass (pattern 1, n=12) or as a dominant lesion surrounded by multiple small enhancing foci (pattern 2, n=8). Multiple small enhancing foci with interconnecting enhancing strands (pattern 3) and an architectural distortion (pattern 4) were both described in three cases. There was one case of a focal area of inhomogeneous enhancement (pattern 5) and one normal MR examination (pattern 6). Unifocal and multifocal lesions were identified on MRI in four patients with normal conventional imaging. In nine women, multiple additional lesions or more extensive multiquadrant disease were correctly identified only on MRI. MRI may play an important role in the evaluation of patients with ILC, which is often difficult to diagnose on clinical examination and conventional imaging and more likely occur in multiple sites and in both breasts. However, false-negative MR findings do occur in a small percentage of ILC.

Serum and plasma concentrations of vascular endothelial growth factor (sVEGF and pVEGF), serum concentrations of interleukin 6 (IL-6), and VEGF platelet load (VEGF/pl) in the blood of healthy controls (n = 26), breast cancer patients with locoregional disease (n = 31), and patients with progressive advanced disease (n = 73) have been compared. The 95th percentile values for the control population were 250 pg/mL for sVEGF, 30 pg/mL for pVEGF, and 1.6 pg/mL for IL-6. The 95th percentile value of the calculated VEGF/pl was 1.0 pg/10(6) platelets in the control population. Serum VEGF concentrations correlated with platelet number in all the groups. Patients with thrombocytosis had a median sVEGF concentration of 833 pg/mL, compared to 249 pg/mL in other patients (P = 0.018). Serum IL-6 levels correlated with sVEGF levels and with the calculated VEGF/pl. Serum IL-6 concentration was significantly higher in patients with breast cancer compared to healthy controls (P < 0.0001). Median IL-6 serum levels were nearly 10 times higher in patients with metastatic breast cancer as compared to the those with locoregional disease (6.0 pg/mL versus 0.7 pg/mL, respectively). Plasma VEGF and the VEGF/pl were also significantly different in the 3 groups. The ratio between sVEGF and pVEGF tended to be smaller in the metastatic breast cancer group compared to the patients with locoregional disease (median, 7.5 versus 10.1, respectively; P = 0.066), suggestive of more intravasal platelet degranulation in the former group. Serum IL-6 level is the most discriminative factor separating healthy controls and the locoregional and metastatic breast cancer patient groups. These results suggest a role for tumor-derived IL-6 in regulating VEGF expression in platelets and their precursors and also confirm the role of circulating platelets in the storage of VEGF.

Plasma levels of D-dimer are elevated in cancer patients. Activation of the extrinsic coagulation system and the fibrinolytic cascade within a tumour is thought to be related with growth, invasion and metastasis. We have investigated the relationship between these markers of fibrin metabolism, standard clinicopathological variables and serum levels of angiogenic cytokines in three cohorts: group A (n=30) consisted of 30 healthy female volunteers, group B (n=23) of consecutive patients with operable breast cancer and group C (n=84) of patients with untreated or progressive metastatic breast cancer. Plasma D-dimers, fibrinogen, IL-6, vascular endothelial growth factor and calculated vascular endothelial growth factor load in platelets are clearly increased in patients with breast cancer. D-dimers were increased in nearly 89% of patients with progressive metastatic disease. The level of D-dimers was positively correlated with tumour load (P

Early distant relapse occurs in a minority of node-negative breast cancer patients. Whether this poor prognosis can be predicted by the features of the primary tumour, or by the presence of occult metastases in the "negative" lymph nodes (LNs), remains a matter of debate. One hundred and four T(1-2)N(0)M(0) breast carcinoma patients were divided into two groups: group 1 (44%) showing early distant relapse with a median disease-free survival of 25 months, and group 2 (56%) showing no evidence of disease after a median follow-up of 91.5 months. All patients had received locoregional treatment only. All tumours were evaluated for medial/lateral location, histological type, size, grade, mitotic activity, fibrotic focus, necrosis, angiogenesis, growth pattern, and lymphatic vessel permeation. The haematoxylin and eosin-stained slides of all axillary LNs were revised and two additional levels were cut from each paraffin block for cytokeratin immunohistochemistry. In 24 patients (23%), occult metastases were found. These consisted of single cells or small clusters (SCs) in the marginal sinus in 17 patients (16%) and of larger colonies of cells in seven patients (7%). All detected metastases were smaller than 2 mm in diameter (micrometastases). There was no significant correlation between the presence of occult LN metastases (SCs or colonies) and the prognostically important features of the primary tumour. Early metastatic disease was significantly correlated with larger tumour size (p=0.02), higher histological grade (p=0.0008), mitotic activity (p

// Francois Bertucci 1, 2, 3 , Pascal Finetti 1 , Cecile Colpaert 4 , Emilie Mamessier 1 , Maxime Parizel 4 , Luc Dirix 5 , Patrice Viens 1, 2, 3 , Daniel Birnbaum 1 , Steven van Laere 5 1 Departement d’Oncologie Moleculaire, “Equipe Labellisee Ligue Contre le Cancer”, Centre de Recherche en Cancerologie de Marseille (CRCM), Institut Paoli-Calmettes, INSERM UMR1068, CNRS UMR725, Marseille, France 2 Departement d’Oncologie Medicale, CRCM, Institut Paoli-Calmettes, Marseille, France 3 Faculte de Medecine, Aix-Marseille Universite, Marseille, France 4 Department of Pathology, GZA Hospitals Sint-Augustinus, Antwerp, Belgium 5 Center for Oncological Research (CORE), Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp Belgium Correspondence to: Francois Bertucci, e-mail: bertuccif@ipc.unicancer.fr Keywords: chemotherapy, inflammatory breast cancer, immune response, PDL1, survival Received: January 23, 2015 Accepted: March 23, 2015 Published: April 11, 2015 ABSTRACT We retrospectively analyzed PDL1 mRNA expression in 306 breast cancer samples, including 112 samples of an aggressive form, inflammatory breast cancer (IBC). PDL1 expression was heterogeneous, but was higher in IBC than in non-IBC. Compared to normal breast samples, PDL1 was overexpressed in 38% of IBC. In IBC, PDL1 overexpression was associated with estrogen receptor-negative status, basal and ERBB2-enriched aggressive subtypes, and clinico-biological signs of anti-tumor T-cell cytotoxic response. PDL1 overexpression was associated with better pathological response to chemotherapy, independently of histo-clinical variables and predictive gene expression signatures. No correlation was found with metastasis-free and overall specific survivals. In conclusion, PDL1 overexpression in IBC correlated with better response to chemotherapy. This seemingly counterintuitive correlation between expression of an immunosuppressive molecule and improved therapeutic response may be resolved if PDL1 expression is viewed as a surrogate marker of a strong antitumor immune response among patients treated with immunogenic chemotherapy. In such patients, PDL1 inhibition could protect activated T-cells or reactivate inhibited T-cells and improve the therapeutic response, notably when associated with immunogenic chemotherapy.