Chitin-mediated blockade of chitinase-like proteins reduces tumor immunosuppression, inhibits lymphatic metastasis and enhances anti-PD-1 efficacy in complementary TNBC models.

BACKGROUND: Chitinase-like proteins (CLPs) play a key role in immunosuppression under inflammatory conditions such as cancer. CLPs are enzymatically inactive and become neutralized upon binding of their natural ligand chitin, potentially reducing CLP-driven immunosuppression. We investigated the efficacy of chitin treatment in the context of triple-negative breast cancer (TNBC) using complementary mouse models. We also evaluated the immunomodulatory influence of chitin on immune checkpoint blockade (ICB) and compared its efficacy as general CLP blocker with blockade of a single CLP, i.e. chitinase 3-like 1 (CHI3L1). METHODS: Female BALB/c mice were intraductally injected with luciferase-expressing 4T1 or 66cl4 cells and systemically treated with chitin in combination with or without anti-programmed death (PD)-1 ICB. For single CLP blockade, tumor-bearing mice were treated with anti-CHI3L1 antibodies. Metastatic progression was monitored through bioluminescence imaging. Immune cell changes in primary tumors and lymphoid organs (i.e. axillary lymph nodes and spleen) were investigated through flow cytometry, immunohistochemistry, cytokine profiling and RNA-sequencing. CHI3L1-stimulated RAW264.7 macrophages were subjected to 2D lymphatic endothelial cell adhesion and 3D lymphatic integration in vitro assays for studying macrophage-mediated lymphatic remodeling. RESULTS: Chitin significantly reduced primary tumor progression in the 4T1-based model by decreasing the high production of CLPs that originate from tumor-associated neutrophils (TANs) and Stat3 signaling, prominently affecting the CHI3L1 and CHI3L3 primary tumor levels. It reduced immunosuppressive cell types and increased anti-tumorigenic T-cells in primary tumors as well as axillary lymph nodes. Chitin also significantly reduced CHI3L3 primary tumor levels and immunosuppression in the 66cl4-based model. Compared to anti-CHI3L1, chitin enhanced primary tumor growth reduction and anti-tumorigenicity. Both treatments equally inhibited lymphatic adhesion and integration of macrophages, thereby hampering lymphatic tumor cell spreading. Upon ICB combination therapy, chitin alleviated anti-PD-1 resistance in both TNBC models, providing a significant add-on reduction in primary tumor and lung metastatic growth compared to chitin monotherapy. These add-on effects occurred through additional increase in CD8α+ T-cell infiltration and activation in primary tumor and lymphoid organs. CONCLUSIONS: Chitin, as a general CLP blocker, reduces CLP production, enhances anti-tumor immunity as well as ICB responses, supporting its potential clinical relevance in immunosuppressed TNBC patients.

The role of adipose tissue and subsequent liver tissue hypoxia in obesity and early stage metabolic dysfunction associated steatotic liver disease.

BACKGROUND: Obesity is linked to several health complication, including Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD). Adipose tissue hypoxia has been suggested as an important player in the pathophysiological mechanism leading to chronic inflammation in obesity, and in the progression of MASLD. The study aims to investigate the effect of progressive obesity on adipose and liver tissue hypoxia. METHODS: Male 8-week-old C57BL/6J mice were fed a high-fat high-fructose diet (HFHFD) or control diet (CD) for 4, 8, 12, 16 and 20 weeks. Serum ALT, AST and lipid levels were determined, and glucose and insulin tolerance testing was performed. Liver, gonadal and subcutaneous adipose tissue was assessed histologically. In vivo tissue pO2 measurements were performed in gonadal adipose tissue and liver under anesthesia. A PCR array for hypoxia responsive genes was performed in liver and adipose tissue. The main findings in the liver were validated in another diet-induced MASLD mice model, the choline-deficient L-amino acid defined high-fat diet (CDAHFD). RESULTS: HFHFD feeding induced a progressive obesity, dyslipidaemia, insulin resistance and MASLD. In vivo pO2 was decreased in gonadal adipose tissue after 8 weeks of HFHFD compared to CD, and decreased further until 20 weeks. Liver pO2 was only significantly decreased after 16 and 20 weeks of HFHFD. Gene expression and histology confirmed the presence of hypoxia in liver and adipose tissue. Hypoxia could not be confirmed in mice fed a CDAHFD. CONCLUSION: Diet-induced obesity in mice is associated with hypoxia in liver and adipose tissue. Adipose tissue hypoxia develops early in obesity, while liver hypoxia occurs later in the obesity development but still within the early stages of MASLD. Liver hypoxia could not be directly confirmed in a non-obese liver-only MASLD mice model, indicating that obesity-related processes such as adipose tissue hypoxia are important in the pathophysiology of obesity and MASLD.

Mutational landscape of inflammatory breast cancer.

BACKGROUND: Inflammatory breast cancer (IBC) is the most pro-metastatic form of BC. Better understanding of its enigmatic pathophysiology is crucial. We report here the largest whole-exome sequencing (WES) study of clinical IBC samples. METHODS: We retrospectively applied WES to 54 untreated IBC primary tumor samples and matched normal DNA. The comparator samples were 102 stage-matched non-IBC samples from TCGA. We compared the somatic mutational profiles, spectra and signatures, copy number alterations (CNAs), HRD and heterogeneity scores, and frequencies of actionable genomic alterations (AGAs) between IBCs and non-IBCs. The comparisons were adjusted for the molecular subtypes. RESULTS: The number of somatic mutations, TMB, and mutational spectra were not different between IBCs and non-IBCs, and no gene was differentially mutated or showed differential frequency of CNAs. Among the COSMIC signatures, only the age-related signature was more frequent in non-IBCs than in IBCs. We also identified in IBCs two new mutational signatures not associated with any environmental exposure, one of them having been previously related to HIF pathway activation. Overall, the HRD score was not different between both groups, but was higher in TN IBCs than TN non-IBCs. IBCs were less frequently classified as heterogeneous according to heterogeneity H-index than non-IBCs (21% vs 33%), and clonal mutations were more frequent and subclonal mutations less frequent in IBCs. More than 50% of patients with IBC harbored at least one high-level of evidence (LOE) AGA (OncoKB LOE 1-2, ESCAT LOE I-II), similarly to patients with non-IBC. CONCLUSIONS: We provide the largest mutational landscape of IBC. Only a few subtle differences were identified with non-IBCs. The most clinically relevant one was the higher HRD score in TN IBCs than in TN non-IBCs, whereas the most intriguing one was the smaller intratumor heterogeneity of IBCs.

Immunohistochemical characterisation of the immune landscape in primary uveal melanoma and liver metastases.

BACKGROUND: The immune landscape of uveal melanoma liver metastases (UMLM) has not been sufficiently studied. METHODS: Immune cell infiltrates (ICIs), PD-1 and PD-L1 were characterised in 62 UMLM and 28 primary uveal melanomas (PUM). ICI, PD-1 and PD-L1 were scored as: (1) % tumoral area occupied by tumour-infiltrating lymphocytes or macrophages (TILs, TIMs) and (2) % perTumoral (perT) area. ICIs and other variables including histopathologic growth patterns (HGPs), replacement and desmoplastic, of UMLM were analysed for their prognostic value. RESULTS: ICIs recognised by haematoxylin-eosin-saffron (HES) and IHC (e.g., T cells (CD3), B cells (CD20). Macrophages (CD68), (CD163), were primarily localised to the perT region in PUM and UMLM and were more conspicuous in UMLM. HES, CD3, CD4, FoxP3, CD8, CD20, PD-1 TILs were scant (<5%). TIMs were more frequent, particularly in UMLM than in PUM. Both CD68+ TIMs and HGPs remained significant on multivariate analysis, influencing overall (OS) and metastasis-specific overall survival (MSOS). CD68 + , CD163+ and CD20+ perT infiltrates in UMLM predicted increased OS and MSOS on univariate analysis. CONCLUSIONS: TILs and PD-L1 have no predictive value in PUM or UMLM. CD68+ and CD163+TIMs, CD20+ perT lymphocytes, and HGPs are important prognostic factors in UMLMs.

Can medical imaging identify the histopathological growth patterns of liver metastases?

The histopathological growth patterns (HGPs) of liver metastases of colorectal cancer and of several other tumor types predict outcome of patients in multiple studies. The HGPs of liver metastases have a prognostic but also a predictive value with one of the growth patterns, the replacement growth pattern, related to resistance to systemic treatment. Given that the HGP can only be assessed in a reliable manner when a surgical resection of the metastasis has been performed, this biomarker cannot be exploited to the full. For example, HGPs can at this moment, not be used to decide whether patients with liver metastatic breast or colorectal cancer will benefit or not from locoregional treatment, such as surgery or radiotherapy, and from peri-operative systemic treatment. In this review we highlight studies that suggest that the HGPs of liver metastases can be identified by medical imaging. Although still to be confirmed by a prospective multicenter approach, some studies indeed achieve a high accuracy in predicting the HGPs by applying radiomic algorithms on CT- or MR-images of liver metastases. This is an important step towards a treatment planning of patients with liver metastatic cancer that takes into account the biology and the progression kinetics of the metastases.

L1CAM and laminin vascular network: Association with the high-risk replacement histopathologic growth pattern in uveal melanoma liver metastases.

The replacement histopathologic growth pattern (rHGP) in melanoma liver metastases connotes an aggressive phenotype (vascular co-option; angiotropic extravascular migratory spread) and adverse prognosis. Herein, replacement and desmoplastic HGP (dHGP) were studied in uveal melanoma liver metastases (MUM). In particular, L1CAM and a "laminin vascular network" were detected at the advancing front of 14/20 cases (p = 0.014) and 16/20 cases (p = 6.4e-05) rHGPs, respectively, but both were absent in the dHGP (8/8 cases) (p = 0.014, and p = 6.3e-05, respectively). L1CAM highlighted progressive extension of angiotropic melanoma cells along sinusoidal vessels in a pericytic location (pericytic mimicry) into the hepatic parenchyma. An inverse relationship between L1CAM expression and melanin index (p = 0.012) suggested differentiation toward an amelanotic embryonic migratory phenotype in rHGP. Laminin labeled the basement membrane zone interposed between sinusoidal vascular channels and angiotropic melanoma cells at the advancing front. Other new findings: any percentage of rHGP and pure rHGP had a significant adverse effect on metastasis-specific overall survival (p = 0.038; p = 0.0064), as well as predominant rHGP (p = 0.0058). Pure rHGP also was associated with diminished metastasis-free survival relative to dHGP (p = 0.040), possibly having important implications for mechanisms of tumor spread. In conclusion, we report for the first time that L1CAM and a laminin vascular network are directly involved in this high-risk replacement phenotype. Further, this study provides more detailed information about the adverse prognostic effect of the rHGP in MUM.

The circular RNome of primary breast cancer.

Circular RNAs (circRNAs) are a class of RNAs that is under increasing scrutiny, although their functional roles are debated. We analyzed RNA-seq data of 348 primary breast cancers and developed a method to identify circRNAs that does not rely on unmapped reads or known splice junctions. We identified 95,843 circRNAs, of which 20,441 were found recurrently. Of the circRNAs that match exon boundaries of the same gene, 668 showed a poor or even negative (R < 0.2) correlation with the expression level of the linear gene. In silico analysis showed only a minority (8.5%) of circRNAs could be explained by known splicing events. Both these observations suggest that specific regulatory processes for circRNAs exist. We confirmed the presence of circRNAs of CNOT2, CREBBP, and RERE in an independent pool of primary breast cancers. We identified circRNA profiles associated with subgroups of breast cancers and with biological and clinical features, such as amount of tumor lymphocytic infiltrate and proliferation index. siRNA-mediated knockdown of circCNOT2 was shown to significantly reduce viability of the breast cancer cell lines MCF-7 and BT-474, further underlining the biological relevance of circRNAs. Furthermore, we found that circular, and not linear, CNOT2 levels are predictive for progression-free survival time to aromatase inhibitor (AI) therapy in advanced breast cancer patients, and found that circCNOT2 is detectable in cell-free RNA from plasma. We showed that circRNAs are abundantly present, show characteristics of being specifically regulated, are associated with clinical and biological properties, and thus are relevant in breast cancer.

Coagulation/fibrinolysis and circulating tumor cells in patients with advanced breast cancer.

PURPOSE: To evaluate the relationship between circulating tumor cells (CTCs) and standard coagulation tests in both a discovery and a validation cohort of patients with advanced breast cancer. METHODS: In a retrospective (n = 77) and a prospective (n = 92) study of patients with progressive advanced breast cancer, CTC count, platelet number, fibrinogen level, D-dimers, prothrombin time, and activated partial thromboplastin time were measured. The association between these coagulation studies and CTC count was analyzed. The impact of these measurements on overall survival (OS) was assessed. RESULTS: In both cohorts, results were similar; absolute CTC count was significantly associated to D-dimer level and inversely with platelet count. In the prospective cohort, quantification of tumor-derived extracellular vesicles (tdEVs) was associated with CTC count, and with coagulation abnormalities (low platelet count and increased D-dimers). tdEVs did not add to CTC count in predicting changes in platelets or D-dimers. In multivariate analysis only CTC ≥ 5 CTC/7.5 mL, ER status, HER2 status and lines of chemotherapy were associated with OS. In patients with terminally metastatic breast cancer, very high CTC counts are prevalent. CONCLUSION: A significant association exists between increasing CTC number and increased D-dimers and decreased platelet counts, suggesting a potential role for CTCs as a direct contributor of intravascular coagulation activation. In patients with advanced and progressive breast cancer, abnormalities in routine coagulation tests is the rule. In patients with terminally advanced breast cancer a "leukemic" phase with high CTC count is prevalent.

The CD151-midkine pathway regulates the immune microenvironment in inflammatory breast cancer.

The immune microenvironment in inflammatory breast cancer (IBC) is poorly characterised, and molecular and cellular pathways that control accumulation of various immune cells in IBC tissues remain largely unknown. Here, we discovered a novel pathway linking the expression of the tetraspanin protein CD151 in tumour cells with increased accumulation of macrophages in cancerous tissues. It is notable that elevated expression of CD151 and a higher number of tumour-infiltrating macrophages correlated with better patient responses to chemotherapy. Accordingly, CD151-expressing IBC xenografts were characterised by the increased infiltration of macrophages. In vitro migration experiments demonstrated that CD151 stimulates the chemoattractive potential of IBC cells for monocytes via mechanisms involving midkine (a heparin-binding growth factor), integrin α6ß1, and production of extracellular vesicles (EVs). Profiling of chemokines secreted by IBC cells demonstrated that CD151 increases production of midkine. Purified midkine specifically stimulated migration of monocytes, but not other immune cells. Further experiments demonstrated that the chemoattractive potential of IBC-derived EVs is blocked by anti-midkine antibodies. These results demonstrate for the first time that changes in the expression of a tetraspanin protein by tumour cells can affect the formation of the immune microenvironment by modulating recruitment of effector cells to cancerous tissues. Therefore, a CD151-midkine pathway can be considered as a novel target for controlled changes of the immune landscape in IBC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pathological features of vessel co-option versus sprouting angiogenesis.

Cancer cells can use existing blood vessels to acquire a vasculature. This process is termed 'vessel co-option'. Vessel co-option is an alternative to the growth of new blood vessels, or angiogenesis, and is adopted by a wide range of human tumour types growing within numerous tissues. A complementary aspect of this process is extravascular migratory tumour spread using the co-opted blood vessels as a trail. Vessel co-opting tumours can be discriminated from angiogenic tumours by specific morphological features. These features give rise to distinct histopathological growth patterns that reflect the interaction of cancer cells with the microenvironment of the organ in which they thrive. We will discuss the histopathological growth patterns of vessel co-option in the brain, the liver and the lungs. The review will also highlight evidence for the potential clinical value of the histopathological growth patterns of cancer. Vessel co-option can affect patient outcomes and resistance to cancer treatment. Insight into the biological drivers of this process of tumour vascularization will yield novel therapeutic strategies.

Inflammatory breast cancer cells are characterized by abrogated TGFß1-dependent cell motility and SMAD3 activity.

PURPOSE: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with elevated metastatic potential, characterized by tumor emboli in dermal and parenchymal lymph vessels. This study has investigated the hypothesis that TGFß signaling is implicated in the molecular biology of IBC. METHODS: TGFß1-induced cell motility and gene expression patterns were investigated in three IBC and three non-IBC (nIBC) cell lines. Tissue samples from IBC and nIBC patients were investigated for the expression of nuclear SMAD2, SMAD3, and SMAD4. SMAD protein levels were related to gene expression data. RESULTS: TGFß1-induced cell motility was strongly abrogated in IBC cells (P = 0.003). Genes differentially expressed between IBC and nIBC cells post TGFß1 exposure revealed attenuated expression of SMAD3 transcriptional regulators, but overexpression of MYC target genes in IBC. IBC patient samples demonstrated a near absence of SMAD3 and -4 expression in the primary tumor compared to nIBC patient samples (P < 0.001) and a further reduction of staining intensity in tumor emboli. Integration of gene and protein expression data revealed that a substantial fraction of the IBC signature genes correlated with SMAD3 and these genes are indicative of attenuated SMAD3 signaling in IBC. CONCLUSION: We demonstrate attenuated SMAD3 transcriptional activity and SMAD protein expression in IBC, together with obliterated TGFß1-induced IBC cell motility. The further reduction of nuclear SMAD expression levels in tumor emboli suggests that the activity of these transcription factors is involved in the metastatic dissemination of IBC cells, possibly by enabling collective invasion after partial EMT.

Quantitative hormone receptor (HR) expression and gene expression analysis in HR+ inflammatory breast cancer (IBC) vs non-IBC.

BACKGROUND: The purpose of this study was to determine the prognostic role of hormone receptor (HR) on inflammatory breast cancer (IBC) to elucidate its aggressive biological behavior. METHODS: We evaluated the expression of estrogen receptor (ER) and progesterone receptor (PR) by immunohistochemical staining and determined the predictive and prognostic role of HR expression on 189 patients with HR+/HER2- IBC and 677 patients with HR+/HER2- stage III non-IBC. Furthermore, we performed gene expression (GE) analyses on 137 patients with HR+/HER2- IBC and 252 patients with HR+/HER2- non-IBC to detect genes that are specifically overexpressed in IBC. RESULTS: The expression of ER% was significantly associated with longer distant disease-free survival and overall survival. However, there was no significant relationship between ER% and neoadjuvant chemotherapy outcome. In the GE study, 84 genes were identified as significantly distinguishing HR+ IBC from non-IBC. Among the top 15 canonical pathways expressed in IBC, the ERK/MAPK, PDGF, insulin receptor, and IL-7 signaling pathways were associated with the ER signaling pathway. Upregulation of the MYC gene was observed in three of these four pathways. Furthermore, HR+/HER2- IBC had significantly higher MYC amplification, and the genetic alteration was associated with poor survival outcome. CONCLUSIONS: Higher ER expression was significantly associated with improved survival in both HR+/HER2- IBC and HR+/HER2- stage III non-IBC patients. HR+/HER2- IBC had several activated pathways with MYC upregulation, and the genetic alteration was associated with poor survival outcome. The results indicate that MYC may be a key gene for understanding the biology of HR+/HER2- IBC.

Oncological outcome, postoperative complications, and mammographic changes after intraoperative radiotherapy with electrons (IOERT) as a boost in a large single-institution cohort of breast cancer patients.

Advantages of using intraoperative radiotherapy with electrons (IOERT) as a boosting modality in breast-conserving therapy include the direct visualization of the tumor bed, a reduced skin dose, and patient convenience. We report oncological outcome, postoperative complication rate, and mammographic changes on follow-up imaging in women treated at our institution with IOERT as a boost modality in breast-conserving therapy for early-stage breast carcinoma. Between January 2007 and June 2018, 763 consecutive patients were enrolled. During breast-conserving surgery, an IOERT boost of 9 Gy was applied, followed by whole breast irradiation (WBI). At a median follow-up of 62.2 months (range: 0.5-135), 13 in-breast recurrences were observed, yielding a local tumor control rate of 98.4% at 5 years. In multivariable analysis, high tumor grading was predictive for local recurrence (HR = 5.6; 95%CI: 1.19-26.2). A total of 27 (3.5%) patients developed any kind of postoperative complication. None of the tumor characteristics nor any of the IOERT technical parameters were predictive for development of a postoperative complication. On follow-up imaging, 145 patients with mammographic changes BIRADS score ≥3 were found of which 50.3% required a biopsy. Only 17 patients had positive biopsies; none of the IOERT parameters were predictive for false-positive imaging. A 9 Gy IOERT boost combined with postoperative WBI provided outstanding local control rates, was well-tolerated, with limited postoperative complications. However, radiologists must be aware of a presumable higher prevalence of mammographic changes after IORT as a boost.

Histopathological growth patterns as a candidate biomarker for immunomodulatory therapy.

The encroachment of a growing tumor upon the cells and structures of surrounding normal tissue results in a series of histopathological growth patterns (HGPs). These morphological changes can be assessed in hematoxylin-and-eosin (H&E) stained tissue sections from primary and metastatic tumors and have been characterized in a range of tissue types including liver, lung, lymph node and skin. HGPs in different tissues share certain general characteristics like the extent of angiogenesis, but also appropriate tissue-specific mechanisms which ultimately determine differences in the biology of HGP subtypes. For instance, in the well-characterized HGPs of liver metastases, the two main subtypes, replacement and desmoplastic, recapitulate two responses of the normal liver to injury: regeneration and fibrosis. HGP subtypes have distinct cytokine profiles and differing levels of lymphocytic infiltration which suggests that they are indicative of immune status in the tumor microenvironment. HGPs predict response to bevacizumab and are associated with overall survival (OS) after surgery for liver metastases in colorectal cancer (CRC). In addition, HGPs can change over time in response to therapy. With standard scoring methods being developed, HGPs represent an easily accessible, dynamic biomarker to consider when determining strategies for treatment using anti-VEGF and immunomodulatory drugs.

Methylglyoxal, a glycolysis metabolite, triggers metastasis through MEK/ERK/SMAD1 pathway activation in breast cancer.

BACKGROUND: Elevated aerobic glycolysis rate is a biochemical alteration associated with malignant transformation and cancer progression. This metabolic shift unavoidably generates methylglyoxal (MG), a potent inducer of dicarbonyl stress through the formation of advanced glycation end products (AGEs). We have previously shown that the silencing of glyoxalase 1 (GLO1), the main MG detoxifying enzyme, generates endogenous dicarbonyl stress resulting in enhanced growth and metastasis in vivo. However, the molecular mechanisms through which MG stress promotes metastasis development remain to be unveiled. METHODS: In this study, we used RNA sequencing analysis to investigate gene-expression profiling of GLO1-depleted breast cancer cells and we validated the regulated expression of selected genes of interest by RT-qPCR. Using in vitro and in vivo assays, we demonstrated the acquisition of a pro-metastatic phenotype related to dicarbonyl stress in MDA-MB-231, MDA-MB-468 and MCF7 breast cancer cellular models. Hyperactivation of MEK/ERK/SMAD1 pathway was evidenced using western blotting upon endogenous MG stress and exogenous MG treatment conditions. MEK and SMAD1 regulation of MG pro-metastatic signature genes in breast cancer cells was demonstrated by RT-qPCR. RESULTS: High-throughput transcriptome profiling of GLO1-depleted breast cancer cells highlighted a pro-metastatic signature that establishes novel connections between MG dicarbonyl stress, extracellular matrix (ECM) remodeling by neoplastic cells and enhanced cell migration. Mechanistically, we showed that these metastasis-related processes are functionally linked to MEK/ERK/SMAD1 cascade activation in breast cancer cells. We showed that sustained MEK/ERK activation in GLO1-depleted cells notably occurred through the down-regulation of the expression of dual specificity phosphatases in MG-stressed breast cancer cells. The use of carnosine and aminoguanidine, two potent MG scavengers, reversed MG stress effects in in vitro and in vivo experimental settings. CONCLUSIONS: These results uncover for the first time the key role of MG dicarbonyl stress in the induction of ECM remodeling and the activation of migratory signaling pathways, both in favor of enhanced metastatic dissemination of breast cancer cells. Importantly, the efficient inhibition of mitogen-activated protein kinase (MAPK) signaling using MG scavengers further emphasizes the need to investigate their therapeutic potential across different malignancies.

Circulating tumor cells and survival in abiraterone- and enzalutamide-treated patients with castration-resistant prostate cancer.

BACKGROUND: The outcome to treatment administered to patients with metastatic castration-resistant prostate cancer (mCRPC) greatly differs between individuals, underlining the need for biomarkers guiding treatment decision making. OBJECTIVE: To investigate the prognostic value of circulating tumor cell (CTC) enumeration and dynamics, in the context of second-line endocrine therapies (ie, abiraterone acetate or enzalutamide), irrespective of prior systemic therapies. DESIGN, SETTINGS, AND PARTICIPANTS: In a prospective, multicentre study blood samples for CTC enumeration were collected from patients with mCRPC at baseline (n = 174). In patients who responded for minimally 10-12 weeks a follow-up sample was collected. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: For baseline analysis, patients were stratified in <5 or ≥5 CTCs/7.5 mL, whereas for the analysis of CTC dynamics at 10-12 weeks, in patients with stable, increasing or decreasing CTC counts. Progression-free survival (PFS), overall survival (OS), and PSA changes at 10-12 weeks were compared between groups. RESULTS: Patients demonstrating increasing CTCs on therapy had a shorter median PFS (4.03 vs 12.98 vs 13.67 months, HR 3.6, 95%CI 1.9-6.8; P < 0.0001) and OS (11.2 months vs not reached, HR 9.5, 95%CI 3.7-24; P < 0.0001), compared to patients with decreasing or stable CTCs. Multivariable Cox regression showed that prior chemotherapy (HR 4.1, 95%CI 1.9-8.9; P = 0.0003), a high baseline CTC count (HR 1.5, 95%CI 1.2-1.9; P = 0.002) and increasing CTCs at follow-up (HR 3.3, 95%CI 1.4-7.6; P = 0.005) were independent predictors of worse PFS. Previous chemotherapy (HR 7, 95%CI 1.9-25; P = 0.003), high baseline CTC counts (HR 2.2, 95%CI 1.4-3.7; P = 0.002) and increasing CTCs during therapy (HR 4.6, 95%CI 1.4-15; P = 0.01) were independently associated with shorter OS. ≥30% and ≥50% PSA responses less frequently occurred in patients with CTC inclines at 10-12 weeks on therapy (χ2 test: P < 0.01). CONCLUSIONS: CTC dynamics during therapy are associated with PSA response and provide independent clinical prognostication over PSA declines. Hence the study demonstrates the pharmacodynamic properties of CTCs.

Mammary stem cell and macrophage markers are enriched in normal tissue adjacent to inflammatory breast cancer.

INTRODUCTION: We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype. MATERIALS AND METHODS: Normal breast parenchyma ≥ 5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44+CD49f+CD133/2+ mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples. RESULTS: 8 of 8 IBC samples expressed isolated CD44+CD49f+CD133/2+ stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p = 0.001). Similarly, the median number of CD44+CD49f+CD133/2+ cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p = 0.007). 7 of 8 IBC samples expressed CD68 + histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p = 0.001). In the validation cohort, the median number of CD68 + cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p = 0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p = 0.02) and with a 79-gene IBC signature (p < 0.001). CONCLUSIONS: Normal tissue from IBC patients is enriched for both mammary stem cells and macrophages and has higher association with both a tumorigenic stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.

International consensus guidelines for scoring the histopathological growth patterns of liver metastasis.

BACKGROUND: Liver metastases present with distinct histopathological growth patterns (HGPs), including the desmoplastic, pushing and replacement HGPs and two rarer HGPs. The HGPs are defined owing to the distinct interface between the cancer cells and the adjacent normal liver parenchyma that is present in each pattern and can be scored from standard haematoxylin-and-eosin-stained (H&E) tissue sections. The current study provides consensus guidelines for scoring these HGPs. METHODS: Guidelines for defining the HGPs were established by a large international team. To assess the validity of these guidelines, 12 independent observers scored a set of 159 liver metastases and interobserver variability was measured. In an independent cohort of 374 patients with colorectal liver metastases (CRCLM), the impact of HGPs on overall survival after hepatectomy was determined. RESULTS: Good-to-excellent correlations (intraclass correlation coefficient >0.5) with the gold standard were obtained for the assessment of the replacement HGP and desmoplastic HGP. Overall survival was significantly superior in the desmoplastic HGP subgroup compared with the replacement or pushing HGP subgroup (P=0.006). CONCLUSIONS: The current guidelines allow for reproducible determination of liver metastasis HGPs. As HGPs impact overall survival after surgery for CRCLM, they may serve as a novel biomarker for individualised therapies.

Proposal for magnetic resonance imaging-guided salvage radiotherapy for prostate cancer.

BACKGROUND: A subset of patients experience a biochemical recurrence following radical prostatectomy. Radiotherapy can salvage those patients, provided that all disease is encompassed within the target volume. We hypothesized that this can be achieved more adequately with magnetic resonance imaging (MRI)-guided treatment planning. MATERIAL AND METHODS: From January 2009 to April 2014, 183 patients were referred to our department for salvage radiotherapy (SRT). According to protocol, patients received a planning computed tomography (CT) as well as an MRI in treatment position. All MRI scans were retrospectively reviewed by an experienced uro-radiologist. RESULTS: Median prostate-specific antigen (PSA) value at time of referral was 0.3 ng/ml (range 0.02-4.7 ng/ml). MRI did not show any suspected macroscopic disease in 137 patients (75%). In 46 (25%) patients, MRI did indicate a pelvic recurrence. The mean PSA level was significantly higher in patients with a suspected recurrence on MRI (0.4 vs. 1.4 ng/ml, p < .001) on a Student's t-test. The mean follow-up was 33 months (range 5-69 months). Biochemical disease-free survival (bDFS) was significantly worse in patients with suspected disease on MRI [hazard ratio (HR) 2.9, p < .0001]. bDFS was significantly worse in the subgroup where the macroscopic recurrences on MRI received a lower radiation dose (HR 3.4, p = .01). CONCLUSION: MRI detects loco-regional disease in a substantial subset of patients with a biochemical recurrence after prostatectomy, especially in a PSA above 0.5 µg/l. Lack of MRI-based dose escalation on these macroscopic recurrences could explain some of the biochemical progression observed after SRT.

Comparison of the Adipose and Luminal Mammary Gland Compartment as Orthotopic Inoculation Sites in a 4T1-Based Immunocompetent Preclinical Model for Triple-Negative Breast Cancer.

Breast tumorigenesis is classically studied in mice by inoculating tumor cells in the fat pad, the adipose compartment of the mammary gland. Alternatively, the mammary ducts, which constitute the luminal mammary gland compartment, also provide a suitable inoculation site to induce breast cancer in murine models. The microenvironments in these compartments influence tumor cell progression, yet this effect has not been investigated in an immunocompetent context. Here, we compared both mammary gland compartments as distinct inoculation sites, taking into account the immunological aspect by inoculating 4T1 tumor cells in immunocompetent mice. Following tumor cell inoculation in the adipose compartment of non-pretreated/naive, hormonally pretreated/naive and non-pretreated/lactating mice, the primary tumors developed similarly. However, a slower onset of primary tumor growth was found after inoculations in the luminal compartment of non-pretreated/lactating mice. Despite this difference in tumor development rate, metastasis to the liver and lungs was equally observed and was accompanied by lymphatic spreading of tumor cells and progressive splenomegaly with both inoculation types. Chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) served as innovative biomarkers for disease progression showing increased levels in primary tumors and sera of the non-pretreated/lactating inoculation groups. A slower increase in circulating CHI3L1 but not LCN2 levels, was observed after inoculations in the luminal compartment which corroborated the slower tumor development at this inoculation site. Our results highlight the critical impact of different mammary gland compartments on tumor development in syngeneic murine models and support the use of novel tumor progression biomarkers in an immune-competent environment.