Gert Van den Eynden

Increased Angiogenesis and Lymphangiogenesis in Inflammatory versus Noninflammatory Breast Cancer by Real-Time Reverse Transcriptase-PCR Gene Expression Quantification

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.

Tumor Lymphangiogenesis in Inflammatory Breast Carcinoma: A Histomorphometric Study

Purpose: At the time of diagnosis, metastatic dissemination of tumor cells via the lymphatic system has occurred in nearly all patients with inflammatory breast cancer (IBC). The objective of this study was twofold: (a) to determine which is the most suitable marker of lymph vessels in primary breast tumors and (b) to compare histomorphometric lymph vessel variables in IBC and non-IBC. Experimental Design: Serial sections of 10 IBCs and 10 non-IBCs were immunostained for D2-40, LYVE-1, podoplanin, and PROX-1. Relative lymph vessel area, lymph vessel perimeters, and counts and lymphatic endothelial cell proliferation (LECP) were then measured in D2-40/Ki-67 double-immunostained sections of 10 normal breast tissues, 29 IBCs, and 56 non-IBCs. Results: D2-40 was the most suitable antibody for staining peritumoral and intratumoral lymph vessels. D2-40-stained intratumoral lymph vessels were present in 80% of non-IBCs and 82.8% of IBCs (P = 0.76). In non-IBC, lymph vessels located in the tumor parenchyma were smaller and less numerous than those at the tumor periphery (P < 0.0001) whereas in IBC, intratumoral and peritumoral variables were not significantly different. The mean relative tumor area occupied by lymph vessels was larger in IBC than in non-IBC (P = 0.01). LECP at the tumor periphery was higher in IBC than in non-IBC: median LECP was 5.74% in IBC versus 1.83% in non-IBC (P = 0.005). Conclusions: The high LECP in IBC suggests that lymphangiogenesis contributes to the extensive lymphatic spread of IBC.

Nuclear Factor-κB Signature of Inflammatory Breast Cancer by cDNA Microarray Validated by Quantitative Real-time Reverse Transcription-PCR, Immunohistochemistry, and Nuclear Factor-κB DNA-Binding

Purpose: Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer with high metastatic potential. In a previous study, we showed that IBC is a different form of breast cancer compared with non-IBC by cDNA microarray analysis. A list of 756 genes with significant expression differences between IBC and non-IBC was identified. In-depth functional analysis revealed the presence of a high number of nuclear factor-κB (NF-κB) target genes with elevated expression in IBC versus non-IBC. This led to the hypothesis that NF-κB contributes to the phenotype of IBC. The aim of the present study was to further investigate the role of NF-κB in IBC. Experimental Design: Immunohistochemistry and NF-κB DNA-binding experiments were done for all NF-κB subunits (RelA, RelB, cRel, NFkB1, and NFkB2) using IBC and non-IBC specimens. Transcriptionally active NF-κB dimers were identified by means of coexpression analysis. In addition, quantitative real-time reverse transcription-PCR for eight NF-κB target genes, selected upon a significant, 3-fold gene expression difference between IBC and non-IBC by cDNA microarray analysis, was done. Results: We found a significant overexpression for all of eight selected NF-κB target genes in IBC compared with non-IBC by quantitative real-time reverse transcription-PCR. In addition, we found a statistically elevated number of immunostained nuclei in IBC compared with non-IBC for RelB (P = 0.038) and NFkB1 (P < 0.001). Immunohistochemical data were further validated by NF-κB DNA-binding experiments. Significant correlations between immunohistochemical data and NF-κB DNA binding for RelA, RelB, NFkB1, and NFkB2 were found. Transcriptionally active NF-κB dimers, composed of specific combinations of NF-κB family members, were found in 19 of 44 IBC specimens compared with 2 of 45 non-IBC specimens (P < 0.001). In addition, we found evidence for an estrogen receptor (ER)–mediated inhibition of the NF-κB signaling pathway. NF-κB target genes were significantly elevated in ER− versus ER+ breast tumors. Also, the amount of immunostained nuclei for RelB (P = 0.025) and NFkB1 (P = 0.031) was higher in ER− breast tumors versus ER+ breast tumors. Conclusions: The NF-κB transcription factor pathway probably contributes to the phenotype of IBC and possibly offers new options for treatment of patients diagnosed with this aggressive form of breast cancer.

Increased Sentinel Lymph Node Lymphangiogenesis is Associated with Nonsentinel Axillary Lymph Node Involvement in Breast Cancer Patients with a Positive Sentinel Node

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.

Vessel co-option mediates resistance to anti-angiogenic therapy in liver metastases

The efficacy of angiogenesis inhibitors in cancer is limited by resistance mechanisms that are poorly understood. Notably, instead of through the induction of angiogenesis, tumor vascularization can occur through the nonangiogenic mechanism of vessel co-option. Here we show that vessel co-option is associated with a poor response to the anti-angiogenic agent bevacizumab in patients with colorectal cancer liver metastases. Moreover, we find that vessel co-option is also prevalent in human breast cancer liver metastases, a setting in which results with anti-angiogenic therapy have been disappointing. In preclinical mechanistic studies, we found that cancer cell motility mediated by the actin-related protein 2/3 complex (Arp2/3) is required for vessel co-option in liver metastases in vivo and that, in this setting, combined inhibition of angiogenesis and vessel co-option is more effective than the inhibition of angiogenesis alone. Vessel co-option is therefore a clinically relevant mechanism of resistance to anti-angiogenic therapy and combined inhibition of angiogenesis and vessel co-option might be a warranted therapeutic strategy.

Standardized evaluation of tumor-infiltrating lymphocytes in breast cancer : results of the ring studies of the international immuno-oncology biomarker working group

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 <60 vs ≥60% TIL improved from 0.45 (ring study 1) to 0.63 in RS2 and the mean concordance improved from 88 to 92%. This large international standardization project shows that reproducible evaluation of TIL is feasible in breast cancer. This opens the way for standardized reporting of tumor immunological parameters in clinical studies and diagnostic practice. The software-guided image evaluation approach used in ring study 2 may be of value as a tool for evaluation of TIL in clinical trials and diagnostic practice. The experience gained from this approach might be applicable to the standardization of other diagnostic parameters in histopathology.

Tumor-infiltrating lymphocyte composition, organization and PD-1/ PD-L1 expression are linked in breast cancer

ABSTRACT The clinical relevance of tumor-infiltrating lymphocytes (TIL) in breast cancer (BC) has been clearly established by their demonstrated correlation with long-term positive outcomes. Nevertheless, the relationship between protective immunity, observed in some patients, and critical features of the infiltrate remains unresolved. This study examined TIL density, composition and organization together with PD-1 and PD-L1 expression in freshly collected and paraffin-embedded tissues from 125 patients with invasive primary BC. Tumor and normal breast tissues were analyzed using both flow cytometry and immunohistochemistry. TIL density distribution is a continuum with 25% of tumors identified as TIL-negative at a TIL density equivalent to normal breast tissues. TIL-positive tumors (75%) were equally divided into TIL-intermediate and TIL-high. Tumors had higher mean frequencies of CD4+ T cells and CD19+ B cells and a lower mean frequency of CD8+ T cells compare with normal tissues, increasing the CD4+/CD8+ T-cell ratio. Tertiary lymphoid structures (TLS), principally located in the peri-tumoral stroma, were detected in 60% of tumors and correlated with higher TIL infiltration. PD-1 and PD-L1 expression were also associated with higher TIL densities and TLS. TIL density, TLS and PD-L1 expression were correlated with more aggressive tumor characteristics, including higher proliferation and hormone receptor negativity. Our findings reveal an important relationship between PD-1/PD-L1 expression, increased CD4+ T and B-cell infiltration, TIL density and TLS, suggesting that evaluating not only the extent but also the nature and location of the immune infiltrate should be considered when evaluating antitumor immunity and the potential for benefit from immunotherapies.

Gene Expression Profiles Associated with the Presence of a Fibrotic Focus and the Growth Pattern in Lymph Node–Negative 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.

Comprehensive Profiling of the Androgen Receptor in Liquid Biopsies from Castration-resistant Prostate Cancer Reveals Novel Intra-AR Structural Variation and Splice Variant Expression Patterns

BACKGROUND Expression of the androgen receptor splice variant 7 (AR-V7) is associated with poor response to second-line endocrine therapy in castration-resistant prostate cancer (CRPC). However, a large fraction of nonresponding patients are AR-V7-negative. OBJECTIVE To investigate if a comprehensive liquid biopsy-based AR profile may improve patient stratification in the context of second-line endocrine therapy. DESIGN, SETTING, AND PARTICIPANTS Peripheral blood was collected from patients with CRPC (n=30) before initiation of a new line of systemic therapy. We performed profiling of circulating tumour DNA via low-pass whole-genome sequencing and targeted sequencing of the entire AR gene, including introns. Targeted RNA sequencing was performed on enriched circulating tumour cell fractions to assess the expression levels of seven AR splice variants (ARVs). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Somatic AR variations, including copy-number alterations, structural variations, and point mutations, were combined with ARV expression patterns and correlated to clinicopathologic parameters. RESULTS AND LIMITATIONS Collectively, any AR perturbation, including ARV, was detected in 25/30 patients. Surprisingly, intra-AR structural variation was present in 15/30 patients, of whom 14 expressed ARVs. The majority of ARV-positive patients expressed multiple ARVs, with AR-V3 the most abundantly expressed. The presence of any ARV was associated with progression-free survival after second-line endocrine treatment (hazard ratio 4.53, 95% confidence interval 1.424-14.41; p=0.0105). Six out of 17 poor responders were AR-V7-negative, but four carried other AR perturbations. CONCLUSIONS Comprehensive AR profiling, which is feasible using liquid biopsies, is necessary to increase our understanding of the mechanisms underpinning resistance to endocrine treatment. PATIENT SUMMARY Alterations in the androgen receptor are associated with endocrine treatment outcomes. This study demonstrates that it is possible to identify different types of alterations via simple blood draws. Follow-up studies are needed to determine the effect of such alterations on hormonal therapy.

Relapse-Free Survival in Breast Cancer Patients Is Associated with a Gene Expression Signature Characteristic for Inflammatory Breast Cancer

Purpose: We hypothesize that a gene expression profile characteristic for inflammatory breast cancer (IBC), an aggressive form of breast cancer associated with rapid cancer dissemination and poor survival, might be related to tumor aggressiveness in non-IBC (nIBC). Experimental Design: RNA from 17 IBC samples and 40 nIBC samples was hybridized onto Affymetrix chips. A gene signature predictive of IBC was identified and applied onto 1,157 nIBC samples with survival data of 881 nIBC samples. Samples were classified as IBC-like or nIBC-like. The IBC signature classification was compared with the classifications according to other prognostically relevant gene signatures and clinicopathologic variables. In addition, relapse-free survival (RFS) was compared by the Kaplan-Meyer method. Results: Classification according to the IBC signature is significantly (P < 0.05) associated with the cell-of-origin subtypes, the wound healing response, the invasive gene signature, the genomic grade index, the fibroblastic neoplasm signature, and the 70-gene prognostic signature. Significant associations (P < 0.01) were found between the IBC signature and tumor grade, estrogen receptor status, ErbB2 status, and patient age at diagnosis. Patients with an IBC-like phenotype show a significantly shorter RFS interval (P < 0.05). Oncomine analysis identified cell motility as an important concept linked with the IBC signature. Conclusions: We show that nIBC carcinomas having an IBC-like phenotype have a reduced RFS interval. This suggests that IBC and nIBC show comparable phenotypic traits, for example augmented cell motility, with respect to aggressive tumor cell behavior. This observation lends credit to the use of IBC to study aggressive tumor cell behavior.