Murine breast cancer models relying on intraductal tumor cell inoculations are attractive because they allow the study of breast cancer from early ductal carcinoma in situ to metastasis. Using a ...fully immunocompetent 4T1-based intraductal model for triple-negative breast cancer (TNBC) we aimed to investigate the immunological responses that guide such intraductal tumor progression, focusing on the prominent role of macrophages.
Intraductal inoculations were performed in lactating female mice with luciferase-expressing 4T1 mammary tumor cells either with or without additional RAW264.7 macrophages, mimicking basal versus increased macrophage-tumor cell interactions in the ductal environment. Imaging of 4T1-derived luminescence was used to monitor primary tumor growth and metastases. Tumor proliferation, hypoxia, disruption of the ductal architecture and tumor immune populations were determined immunohistochemically. M1- (pro-inflammatory) and M2-related (anti-inflammatory) cytokine levels were determined by Luminex assays and ELISA to investigate the activation state of the macrophage inoculum. Levels of the metastatic proteins matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) as well as of the immune-related disease biomarkers chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) were measured by ELISA to evaluate disease progression at the protein level.
Mice intraductally co-injected with macrophages showed severe splenomegaly with faster ductal breakthrough of tumor cells and increased metastases in axillary lymph nodes and lungs. These mice showed higher M1-related cytokines in the early disease stages (at 1 to 3 weeks post-inoculation) due to the pro-inflammatory nature of RAW264.7 macrophages with increased Ly6G-positive neutrophils and decreased anti-inflammatory macrophages in the tumor microenvironment. However, upon metastasis (at 5 weeks post-inoculation), a prominent increase in M2-related cytokine levels was detected and established a tumor microenvironment with similar immune populations and cytokine responses as in mice which received only 4T1 tumor cells. The observed tumor-associated immune responses and the increased metastasis were associated with significantly induced local and systemic levels of MMP-9, VEGF, CHI3L1 and LCN2.
The current experimental study with an innovative immunocompetent intraductal model for TNBC pinpoints towards a metastasis-supporting M1 to M2 macrophage polarization in the mammary ducts mediated by 4T1-derived signaling. We propose to explore this process as immunotherapeutic target.
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.
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.
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.
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.
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).
The high LECP in IBC suggests that lymphangiogenesis contributes to the extensive lymphatic spread of IBC.
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.
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.
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).
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.
A large cell neuroendocrine carcinoma of the ovary is presented because of tubal metastases with peculiar growth patterns: pagetoid and serous tubal intraepithelial carcinoma–like. The possibility ...that a tubal intraepithelial carcinoma could represent a metastasis should be considered by pathologists.
Introduction. Lysosomal storage disorders (LSDs) are rare diseases with more than 50 different entities described today. The spectrum of phenotypes varies from severe to lethal and early-onset ...disease to mild and late onset. Recognition of the clinical signs and diagnostic workup is challenging and requires expertise. Diagnosis relies on finding abnormal metabolites in urine and serum followed by further enzymatic or molecular analysis. Routine histological examination of the foetal and placental tissues frequently shows vacuolisation, providing a readily available important clue to the diagnosis. Case Report. A third child of consanguineal parents showed several dysmorphic features and a complicated neonatal period with eventual demise in the early postneonatal period due to respiratory failure. An LSD was suspected based on clinical presentation, urine metabolite excretion, skeletal radiograph, and vacuolisation in lymphocytes and placental tissues on, respectively, blood smear and routine histological examination. Homozygosity mapping favoured galactosialidosis. The diagnosis was confirmed by massive parallel sequencing, revealing a single nucleotide variation in the CTSA gene (c.265A>C, p.Ser89Arg). Discussion. Histological placental examination may be either the first clue or complimentary evidence in recognizing LSDs. It is important to recognize these clues as it may prompt further investigation and facilitate earlier recognition of the disease.
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.
Angiogenesis and lymphangiogenesis--quantified as endothelial cell proliferation fraction (ECP%) and lymphatic ECP fraction (LECP%)--were assessed in sentinel LN metastases of 65 T(1)/T(2) 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).
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.
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.
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&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 < 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.
Despite the indisputable link between smoking and the increased risk for lung cancer, the inclusion of this factor in prognostic survival analysis has been scarce. Important clinical questions ...regarding the smoking status are the basis of this study and are as follow: what is the prognostic benefit of having been a non-smoker or having stopped smoking prior to developing lung cancer and what is the prognostic benefit of smoking cessation at the time of diagnosis of lung cancer? Cigarette smoking status of 311 patients operated for non-small cell lung cancer (NSCLC) by a single surgeon was determined based on two independent questionnaires taken prospectively prior to lung operation. Of all patients analysed, 169 (54.3%) were current smokers, 25 (8.0%) were non-smokers, 82 (26.4%) were former smokers and 35 (11.3%) were recent quitters. A Cox multiple regression model was used to test the prognostic value of smoking status on survival together with other relevant clinicopathological factors. For overall survival, older age (P = 0.011), presence of lymph node metastases (P < 0.001) and current smoking (P = 0.001) were independent predictors of poor prognosis, while non-smokers (relative risk = 0.447, 95% confidence interval = 0.206–0.970, P = 0.042), former smokers (relative risk = 0.543, 95% confidence interval = 0.350–0.843, P = 0.006) and recent quitters (relative risk = 0.340, 95% confidence interval = 0.164–0.705, P = 0.004) had a significant better prognosis compared to current smokers (referent group). Similar results were obtained for disease-free survival. These results indicate that smoking cessation is beneficial for lung cancer patients at any time point prior to lung operation and current smoking at the time of operation is associated with poor prognosis.
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