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.

Comprehensive evaluation of methods to assess overall and cell-specific immune infiltrates in breast cancer.

BACKGROUND: Breast cancer (BC) immune infiltrates play a critical role in tumor progression and response to treatment. Besides stromal tumor infiltrating lymphocytes (sTILs) which have recently reached level 1B evidence as a prognostic marker in triple negative BC, a plethora of methods to assess immune infiltration exists, and it is unclear how these compare to each other and if they can be used interchangeably. METHODS: Two experienced pathologists scored sTIL, intra-tumoral TIL (itTIL), and 6 immune cell types (CD3+, CD4+, CD8+, CD20+, CD68+, FOXP3+) in the International Cancer Genomics Consortium breast cancer cohort using hematoxylin and eosin-stained (n = 243) and immunohistochemistry-stained tissue microarrays (n = 254) and whole slides (n = 82). The same traits were evaluated using transcriptomic- and methylomic-based deconvolution methods or signatures. RESULTS: The concordance correlation coefficient (CCC) between pathologists for sTIL was very good (0.84) and for cell-specific immune infiltrates slightly lower (0.63-0.66). Comparison between tissue microarray and whole slide pathology scores revealed systematically higher values in whole slides (ratio 2.60-5.98). The Spearman correlations between microscopic sTIL and transcriptomic- or methylomic-based assessment of immune infiltrates were highly variable (r = 0.01-0.56). Similar observations were made for cell type-specific quantifications (r = 0.001-0.54). We observed a strong inter-method variability between the omics-derived estimations, which is further cell type dependent. Finally, we demonstrated that most methods more accurately identify highly infiltrated (sTIL ≥ 60%; area under the curve, AUC, 0.64-0.99) as compared to lowly infiltrated tumors (sTIL ≤ 10%; AUC 0.52-0.82). CONCLUSIONS: There is a lower inter-pathologist concordance for cell-specific quantification as compared to overall infiltration quantification. Microscopic assessments are underestimated when considering small cores (tissue microarray) instead of whole slides. Results further highlight considerable differences between the microscopic-, transcriptomic-, and methylomic-based methods in the assessment of overall and cell-specific immune infiltration in BC. We therefore call for extreme caution when assessing immune infiltrates using current methods and emphasize the need for standardized immune characterization beyond TIL.

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.

Assessing Tumor-infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method From the International Immunooncology Biomarkers Working Group: Part 1: Assessing the Host Immune Response, TILs in Invasive Breast Carcinoma and Ductal Carcinoma In Situ, Metastatic Tumor Deposits and Areas for Further Research.

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.

Assessing Tumor-Infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method from the International Immuno-Oncology Biomarkers Working Group: Part 2: TILs in Melanoma, Gastrointestinal Tract Carcinomas, Non-Small Cell Lung Carcinoma and Mesothelioma, Endometrial and Ovarian Carcinomas, Squamous Cell Carcinoma of the Head and Neck, Genitourinary Carcinomas, and Primary Brain Tumors.

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

Microvessel density and endothelial cell proliferation levels in colorectal liver metastases from patients given neo-adjuvant cytotoxic chemotherapy and bevacizumab.

The treatment of patients with colorectal liver metastasis has improved significantly and first line therapy is often combined chemotherapy and bevacizumab, although it is unknown who responds to this regimen. Colorectal liver metastases grow in different histological growth patterns showing differences in angiogenesis. To identify possible response markers, histological markers of angiogenesis were assessed. Patients who underwent resection of colorectal liver metastasis at Rigshospitalet, Copenhagen, Denmark from 2007 to 2011 were included (n = 254) including untreated and patients treated with chemotherapy or chemotherapy plus bevacizumab. The resected liver metastases were characterised with respect to growth pattern, endothelial and tumour cell proliferation as well as microvessel density and tumour regression. Tumour regression grade of liver metastases differed significantly between untreated/chemotherapy treated patients in comparison to chemotherapy plus bevacizumab treated patients (both p < 0.0001). Microvessel density was decreased in liver metastases from patients treated with bevacizumab in comparison to those from untreated/chemotherapy-treated patients (p = 0.006/p = 0.002). Tumour cell proliferation assessed by Ki67 expression correlated to a shorter recurrence free survival in the total patient cohort. In conclusion, liver metastases from patients treated with neo-adjuvant chemotherapy and bevacizumab had significantly lower microvessel densities and tumour regression grades when compared to liver metastases from untreated or chemotherapy treated patients. This may indicate that bevacizumab treatment results in altered vascular biology and tumour viability, with possible tumour reducing effect.

Increased Angiogenesis and Lymphangiogenesis in Metastatic Sentinel Lymph Nodes Is Associated With Nonsentinel Lymph Node Involvement and Distant Metastasis in Patients With Melanoma.

Lymph node angio- and lymphangio-genesis have been shown to play an important role in the premetastatic niche of sentinel lymph nodes. In the current study we have investigated the association of angio- and lympangio-genesis related parameters in metastatic sentinel lymph nodes of patients with melanoma with the presence of nonsentinel and distant organ metastasis. Peritumoral and intratumoral relative blood and lymphatic vessel areas (evaluated by Chalkley method), blood and lymphatic microvessel densities, and the rates of blood and lymphatic vessel proliferation were assessed in primary tumors and sentinel lymph node metastasis of 44 patients with melanoma using CD34/Ki-67 and D240/Ki-67 immunohistochemical double staining. Primary melanoma exhibited significantly higher rate of lymphatic proliferation compared with its lymph node metastasis (P < 0.05), while lymph node metastasis showed significantly higher rate of blood vessel proliferation (P < 0.05). Using multivariate logistic regression model, the rate of peritumoral lymphatic proliferation was inversely associated with positive nonsentinel lymph node status (P < 0.05), whereas the rate of intratumoral blood vessel proliferation was associated with distant organ metastasis (P < 0.05). Using multivariate Cox regression analysis, the rate of intratumoral blood vessel proliferation was also inversely associated with overall survival of patients with melanoma (P < 0.05).

Peritumoral D2-40 Chalkley score independently predicts metastases and survival in patients with cutaneous malignant melanoma.

BACKGROUND: Many observational studies investigated the prognostic significance of angiogenesis and lymphangiogenesis in patients with melanoma. However, the obtained results are rather contradictory, probably due to the lack of the consensus methodology. METHODS: To investigate the prognostic significance of angiogenesis and lymphangiogenesis-related parameters in patients with melanoma, we performed a retrospective investigation following the consensus recommendations for angiogenesis and lymphangiogenesis quantification in solid tumors and reporting recommendations for tumor marker (REMARK) criteria for reporting the results. Blood and lymphatic vessel Chalkley scores, endothelial cell proliferation fractions and microvessel densities were quantified using a double immunostaining for endothelial marker CD34 or lymphendothelial marker D240 and the proliferation marker Ki-67 in 196 patients with melanoma. These parameters were evaluated separately for peritumoral (PT) and intratumoral areas and were correlated with outcome. RESULTS: In multivariate analysis PT D240 Chalkley score was identified as a strongest predictor for sentinel lymph node metastases, non-sentinel lymph node metastases, distant metastases, disease free survival and overall survival in patients with melanoma. CONCLUSIONS: If additional studies corroborate our findings, we believe that the inclusion of PT D240 Chalkley counts to the routine pathology examination of melanoma samples would provide additional information for identifying high-risk patients.

Melanomas arising on skin with chronic sun-induced damage exhibit low degree of angiogenesis and lymphangiogenesis.

Differences in gene expression between melanomas arising on skin intermittently and chronically sun-exposed areas were described. Additionally, several studies have shown differences in clinical characteristics and prognosis, suggesting distinct biological pathways in the development of these tumors. We performed a retrospective investigation aimed on evaluation of the differences in angiogenesis and lymphangiogenesis between melanomas arising on skin with and without signs of chronic sun-induced damage. For that purpose, we evaluated relative blood and lymphatic vessel areas, blood and lymphatic endothelial cell proliferation fractions, separately for peritumoral and intratumoral areas. We have shown that melanomas arising on sun-exposed skin exhibit lower angiogenic and lymphangiogenic potentials and better prognosis than those arising on skin without signs of chronic sun-induced damage.

Functional Th1-oriented T follicular helper cells that infiltrate human breast cancer promote effective adaptive immunity.

We previously demonstrated that tumor-infiltrating lymphocytes (TIL) in human breast cancer sometimes form organized tertiary lymphoid structures (TLS) characterized by CXCL13-producing T follicular helper (Tfh) cells. The present study found that CD4+ Tfh TIL, CD8+ TIL, and TIL-B, colocalizing in TLS, all express the CXCL13 receptor CXCR5. An ex vivo functional assay determined that only activated, functional Th1-oriented Tfh TIL (PD-1hiICOSint phenotype) provide help for immunoglobulin and IFN-γ production. A functional Tfh TIL presence signals an active TLS, characterized by humoral (immunoglobulins, Ki-67+ TIL-B in active germinal centers) and cytotoxic (GZMB+CD8+ and GZMB+CD68+ TIL plus Th1 gene expression) immune responses. Analysis of active versus inactive TLS in untreated patients revealed that the former are associated with positive clinical outcomes. TLS also contain functional T follicular regulatory (Tfr) TIL, which are characterized by a CD25+CXCR5+GARP+FOXP3+ phenotype and a demethylated FOXP3 gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-ß-dependent mechanism. The activity of tumor-associated TLS was dictated by the relative balance between functional Tfh TIL and functional Tfr TIL. These data provide mechanistic insight into TLS processes orchestrated by functional Th1-oriented Tfh TIL, including TIL-B and CD8+ TIL activation and immunological memory generation. Tfh TIL, regulated by functional Tfr TIL, are an expected key target of PD-1/PD-L1 blockade.

A low frequency of lymph node metastasis in clear-cell renal cell carcinoma is related to low lymphangiogenic activity.

OBJECTIVE: To assess ongoing lymphangiogenesis in renal cell carcinoma (RCC) by histomorphometry and by quantifying mRNA expression levels of lymphangiogenesis-related factors. MATERIALS AND METHODS: Using D2-40 antibody as a lymphatic marker, lymph vessels were counted in tissue sections of 150 clear-cell RCCs (ccRCC) and 61 non-neoplastic controls, using the Chalkley method, which measures the relative lymph vessel area (LVA). Double-staining with Ki67 and D2-40 was used to assess active lymphangiogenesis. In a subset of 25 ccRCCs and nine non-neoplastic controls mRNA expression levels of lymphangiogenic factors were determined by real-time quantitative reverse transcription-polymerase chain reaction. RESULTS: LVA was higher in normal renal tissue than in both intra- and peri-tumoral LVA (P < 0.001). LVA in the tumour periphery was higher than in the tumour parenchyma (P < 0.001). Lymphatic endothelial cell proliferation (LECP) was identified in 8.2% of the control sections and was higher than the intratumoral LECP fraction (LECP%, 2.6%; P = 0.02) and the peritumoral LECP% (6.5%; P > 0.05). Compared with controls, ccRCC specimens had higher mRNA expression levels of vascular endothelial growth factor (VEGF)-A and VEGF-C, but lower expression levels of VEGF-D and Prox-1 (all P < 0.001). CONCLUSION: Our results show that there is only limited ongoing lymphangiogenesis in ccRCC. Given that several growth factors stimulate both angiogenesis and lymphangiogenesis, our observation indirectly indicates that haemangiogenesis predominates in ccRCC. This finding might provide better understanding of why ccRCCs prefer haematogenous dissemination to lymphatic spread.

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-1alpha pathway. Furthermore, unsupervised hierarchical cluster analysis with hypoxia- and (lymph)angiogenesis-related genes showed that hypoxia-inducible factor-1alpha, 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.

TP53 Outperforms Other Androgen Receptor Biomarkers to Predict Abiraterone or Enzalutamide Outcome in Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: To infer the prognostic value of simultaneous androgen receptor (AR) and TP53 profiling in liquid biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) starting a new line of AR signaling inhibitors (ARSi).Experimental Design: Between March 2014 and April 2017, we recruited patients with mCRPC (n = 168) prior to ARSi in a cohort study encompassing 10 European centers. Blood samples were collected for comprehensive profiling of CellSearch-enriched circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Targeted CTC RNA sequencing (RNA-seq) allowed the detection of eight AR splice variants (ARV). Low-pass whole-genome and targeted gene-body sequencing of AR and TP53 was applied to identify amplifications, loss of heterozygosity, mutations, and structural rearrangements in ctDNA. Clinical or radiologic progression-free survival (PFS) was estimated by Kaplan-Meier analysis, and independent associations were determined using multivariable Cox regression models. RESULTS: Overall, no single AR perturbation remained associated with adverse prognosis after multivariable analysis. Instead, tumor burden estimates (CTC counts, ctDNA fraction, and visceral metastases) were significantly associated with PFS. TP53 inactivation harbored independent prognostic value [HR 1.88; 95% confidence interval (CI), 1.18-3.00; P = 0.008], and outperformed ARV expression and detection of genomic AR alterations. Using Cox coefficient analysis of clinical parameters and TP53 status, we identified three prognostic groups with differing PFS estimates (median, 14.7 vs. 7.51 vs. 2.62 months; P < 0.0001), which was validated in an independent mCRPC cohort (n = 202) starting first-line ARSi (median, 14.3 vs. 6.39 vs. 2.23 months; P < 0.0001). CONCLUSIONS: In an all-comer cohort, tumor burden estimates and TP53 outperform any AR perturbation to infer prognosis.See related commentary by Rebello et al., p. 1699.

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 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). 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.

Differential expression of hypoxia and (lymph)angiogenesis-related genes at different metastatic sites in breast cancer.

INTRODUCTION: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

PTHrP, calcitonin and calcitriol in a case of severe, protracted and refractory hypercalcemia due to a pancreatic neuroendocrine tumor.

A patient with a primary neuroendocrine tumor of the pancreas, presented with severe hypercalcemia. This hypercalcemia of malignancy (HCM) failed to respond to intensive bisphosphonate treatment and needed continuous enhanced diuresis. Only after successful antitumor therapy did the hypercalcemia subside. Hypercalcemia was associated with increased concentrations of plasma PTHrP, calcitonin and 1,25-(OH)(2)D(3). Bone mineral density was markedly increased. We demonstrated the presence of both PTHrP and calcitonin in the tumor at the mRNA and protein level, using RT-PCR, immunohistochemistry and Western blotting. The high levels of plasma PTHrP and the demonstrated predominant renal mechanism in this case of HCM are suspected to be the cause for its refractoriness to bone resorption inhibitors. Our findings furthermore suggest that the tumoral production of calcitonin and PTHrP might have contributed to the increased bone mineral storage of calcium and thus probably attenuated the development of frank hypercalcemia.

Nuclear factor-kappaB signature of inflammatory breast cancer by cDNA microarray validated by quantitative real-time reverse transcription-PCR, immunohistochemistry, and nuclear factor-kappaB 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-kappaB (NF-kappaB) target genes with elevated expression in IBC versus non-IBC. This led to the hypothesis that NF-kappaB contributes to the phenotype of IBC. The aim of the present study was to further investigate the role of NF-kappaB in IBC. EXPERIMENTAL DESIGN: Immunohistochemistry and NF-kappaB DNA-binding experiments were done for all NF-kappaB subunits (RelA, RelB, cRel, NFkB1, and NFkB2) using IBC and non-IBC specimens. Transcriptionally active NF-kappaB dimers were identified by means of coexpression analysis. In addition, quantitative real-time reverse transcription-PCR for eight NF-kappaB 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-kappaB 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-kappaB DNA-binding experiments. Significant correlations between immunohistochemical data and NF-kappaB DNA binding for RelA, RelB, NFkB1, and NFkB2 were found. Transcriptionally active NF-kappaB dimers, composed of specific combinations of NF-kappaB 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-kappaB signaling pathway. NF-kappaB 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-kappaB 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.

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.

Breast cancer genome and transcriptome integration implicates specific mutational signatures with immune cell infiltration.

A recent comprehensive whole genome analysis of a large breast cancer cohort was used to link known and novel drivers and substitution signatures to the transcriptome of 266 cases. Here, we validate that subtype-specific aberrations show concordant expression changes for, for example, TP53, PIK3CA, PTEN, CCND1 and CDH1. We find that CCND3 expression levels do not correlate with amplification, while increased GATA3 expression in mutant GATA3 cancers suggests GATA3 is an oncogene. In luminal cases the total number of substitutions, irrespective of type, associates with cell cycle gene expression and adverse outcome, whereas the number of mutations of signatures 3 and 13 associates with immune-response specific gene expression, increased numbers of tumour-infiltrating lymphocytes and better outcome. Thus, while earlier reports imply that the sheer number of somatic aberrations could trigger an immune-response, our data suggests that substitutions of a particular type are more effective in doing so than others.