[Show abstract][Hide abstract] ABSTRACT: Interstitial CD45+ cells and T lymphocytes have previously been demonstrated within infantile haemangioma (IH). This study investigated the expression of B and T lymphocyte markers by the CD45+ population, and the expression of Thy-1, a marker of thymocyte progenitors, which have the ability to give rise to both B and T cells.
Immunohistochemical (IHC) staining was performed on proliferating and involuted IHs for the expression of CD45, CD3, CD20, CD79a, Thy-1 and CD34. The presence of mRNA corresponding to CD45, CD3G, CD20 and Thy-1 was confirmed by reverse transcriptase-polymerase chain reaction in snap-frozen IH tissues. Cell counting of 3,3-diaminobenzidine IHC-stained slides was performed on CD45+ only cells and dually stained CD45+/CD3+ cells or CD45+/CD20+ cells and analysed statistically. In situ hybridisation and mass spectrometry were also performed to confirm the presence and abundance of Thy-1, respectively.
IHC staining showed a subpopulation of CD45+ interstitial cells that expressed the T lymphocyte marker, CD3, and another subpopulation that expressed the B lymphocyte marker, CD20, in proliferating and diminished in involuted IHs. The abundant expression of Thy-1 on the endothelium of proliferating, but not involuted IH, was demonstrated by IHC staining and confirmed by in situ hybridisation and mass spectrometry.
Both B and T lymphocytes are present within the interstitium of proliferating and involuted IH. The expression of Thy-1 by the endothelium suggests that B and T cells in IH may have originated from within the lesion, rather than migrating from the peripheral circulation.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
[Show abstract][Hide abstract] ABSTRACT: A 19 year-old male with a port wine stain on the base of his neck presented with a five-month history of gradual thickening of the involved skin which interfered with clothing and caused repeated bleeding. The lesion was excised and histopathologic examination revealed angiolymphoid hyperplasia with eosinophilia (ALHE) arising from the pre-existing port wine stain - a rare finding with only one previously reported case. Additionally the lesion was associated with elevated serum renin levels which virtually normalized following excision of the lesion. We further demonstrated the expression of angiotensin converting enzyme and angiotensin II receptors 1 and 2 by the lesion and discuss the possible role of the renin-angiotensin system in this condition.
This article is protected by copyright. All rights reserved.
Journal of Cutaneous Pathology 05/2015; DOI:10.1111/cup.12539 · 1.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cells expressing markers of mast cells, macrophages and dendritic cells have previously been demonstrated within the interstitium of infantile haemangioma (IH). This study characterised these myeloid cellular subpopulations within IH.
Immunohistochemical staining was performed on proliferating and involuted IHs for the expression of Nanog, tryptase, CD163, DC-SIGN and CD45. The presence of mRNA corresponding to Nanog, tryptase α/β-1, tryptase β-2, CD163 and DC-SIGN was confirmed by NanoString and RT-PCR in snap-frozen IH tissues.
Immunohistochemical staining showed expression of Nanog by interstitial phenotypical mast cells within proliferating IH, which were separate from the interstitial M2-polarised macrophages that also expressed DC-SIGN, a dendritic cell marker. These two myeloid cellular subpopulations in IH did not express the pan-haematopoietic marker, CD45.
There are two interstitial subpopulations of myeloid cells within IH: phenotypical mast cells which also express Nanog, indicating a primitive phenotype; and M2-polarised macrophages which also express DC-SIGN.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
[Show abstract][Hide abstract] ABSTRACT: Background
Infantile hemangioma (IH), is a common tumour of the vasculature in infants. Recent data has identified placental first trimester villous core cells as the origin of these tumours and established the capacity of cells derived from these tumours to undergo mesenchymal differentiation. We have previously shown a hemogenic endothelial signature of proliferating IH endothelium, and propose that these cells possess the plasticity for mesenchymal transition, processes similar to epithelial to mesenchymal transition (EMT).
To investigate the expression of EMT-associated proteins on IH endothelial cells and their ability to undergo mesenchymal differentiation.
Immunohistochemistry was performed on six proliferating and six involuted IH specimens for the EMT markers, β-catenin, Twist, Slug, N-cadherin, E-cadherin and endoglin; and the mesenchymal markers, Vimentin and CD29. Six proliferating IH specimens were cultured in vitro and the derived cells were characterized using immuno-cytochemistry for the mesenchymal markers, and were grown in either adipocyte or osteoblast differentiation media. Terminal adipocyte and osteoblast differentiation was confirmed by oil o red and alizarin red, respectively.
The endothelium of proliferating IH expressed: the EMT markers, β-catenin, Twist, Slug, N-cadherin and endoglin, but not E-cadherin; as well as the mesenchymal markers Vimentin and CD29. Cells derived from proliferating IH lesions expressed Vimentin and CD29. These cells had the ability to terminally differentiate into either adipocytes or osteoblasts.
The co-expression of EMT-associated and mesenchymal markers on the endothelium of proliferating IH, as well as their ability for terminal mesenchymal differentiation confirms the plastic nature of IH hemogenic endothelium. These data support the ability of this plastic endothelial phenotype to undergo an EMT-parallel process.
[Show abstract][Hide abstract] ABSTRACT: Aims Recent reports on infantile haemangioma (IH) have demonstrated a primitive population of interstitial cells expressing the embryonic transcription factor, Nanog, with decreasing abundance during involution. In this report we investigated the expression of Nanog on mast cells in all three phases of IH progression.
Methods Paraffin-embedded sections of six proliferating, six involuting and six involuted IH lesions were used to investigate the expression of tryptase, Nanog, CD45, CD34 and GLUT-1 by immunostaining.
Results Mast cells, identified by their expression of tryptase, were located in the interstitium of IH lesions. 93%, 42% and 0% of these tryptase+ cells also expressed Nanog, in proliferating, involuting and involuted IH, respectively.
Conclusions The identification of an abundant population of tryptase+/Nanog+ cells in IH is novel. The relative loss of Nanog expression as IH involutes may be a result of maturation and/or proliferation of these cells. This report supports the primitive nature of IH.
[Show abstract][Hide abstract] ABSTRACT: Verrucous hemangioma (VH) presents clinically as a vascular malformation but has similar histopathologic features to infantile hemangioma. This study characterized the cell population within VH.
Paraffin-embedded sections from two male patients with VH were processed for immunohistochemistry. The expression of SMA, CD34, glucose transporter-1 (Glut-1), D2-40, brachyury, angiotensin converting enzyme (ACE), Oct-4, hemoglobin ζ chain (HBZ), Wilms tumor protein (WT-1) and CD45 was examined.
The lymphatic marker, D2-40, was not expressed in VH, whereas Glut-1 was widely expressed in infantile hemangioma, it was only focally expressed by the endothelium of VH. The endothelium of VH expressed the primitive markers, Oct-4, brachyury and ACE. The primitive marker, WT-1, was expressed predominantly on the pericyte layer of both VH and infantile hemangioma. However, HBZ was only expressed in infantile hemangioma. CD45, a mature hematopoetic marker, was expressed by cells within the interstitium, away from the endothelium of VH and infantile hemangioma.
The expression of the primitive markers, Oct-4, brachyury and ACE on the endothelium, and WT-1 predominantly on the pericyte layer of VH shows a primitive microvascular phenotype similar to infantile hemangioma. However, the absence of the embryonic marker, HBZ, expressed only in first trimester placenta and in proliferating infantile hemangioma, suggests a different cellular origin. HBZ could be used to distinguish between the two conditions.
[Show abstract][Hide abstract] ABSTRACT: The origin of infantile haemangioma (IH) remains enigmatic. A primitive mesodermal phenotype origin of IH with the ability to differentiate down erythropoietic and terminal mesenchymal lineages has recently been demonstrated.
To investigate the expression of human embryonic stem cell (hESC) markers in IH and to determine whether IH-derived cells have the functional capacity to form teratoma in vivo.
Immunohistochemical staining and quantitative reverse transcription PCR were used to investigate the expression of hESC markers in IH biopsies. The ability of cells derived from proliferating IH to form teratomas in a mouse xenograft model was investigated.
The hESC markers, Oct-4, STAT-3 and stage-specific embryonic antigen 4 were collectively expressed on the endothelium of proliferating IH lesions, whereas Nanog was not. Nanog was expressed by cells in the interstitium and these cells did not express Oct-4, stage-specific embryonic antigen 4 or STAT-3. Proliferating IH-derived cells were unable to form teratomas in severely compromised immunodeficient/non-obese diabetic mice.
The novel expression of hESC on two different populations of cells in proliferating IH and their inability to form teratomas in vivo infer the presence of a primitive cellular origin for IH downstream from hESC.
[Show abstract][Hide abstract] ABSTRACT: To investigate the expression of the placental cell-specific associated proteins in infantile haemangioma (IH).
Immunohistochemical staining was used to investigate the expression of human chorionic gonadotrophin (hCG), human placental lactogen (hPL), human leucocyte antigen-G (HLA-G), cytokeratin 7 (CK7) and smooth muscle actin in paraffin-embedded sections of proliferating and involuted IHs.
The proteins hCG and hPL were expressed by the endothelium but not the pericyte layer of proliferating IH, but these proteins were not detected in involuted lesions. There was no expression of CK7 and HLA-G in IH.
The expression of hCG and hPL, but not CK7 or HLA-G, by the endothelium of proliferating IH supports a placental chorionic villous mesenchymal core cellular origin for IH rather than a trophoblast origin.
[Show abstract][Hide abstract] ABSTRACT: Infantile haemangioma is a benign tumour of the microvasculature characterised by excessive proliferation of immature endothelial cells. It typically undergoes rapid proliferation during infancy followed by spontaneous slow involution during childhood often leaving a fibro-fatty residuum. In 2008, propranolol, a non-selective β-blocker, was serendipitously discovered to induce accelerated involution of a proliferating infantile haemangioma. However, the mechanism by which propranolol causes this dramatic effect is unclear. Using immunohistochemical staining, we show that the CD34+ endothelial progenitor cells of the microvessels in proliferating infantile haemangioma express angiotensin-converting enzyme and angiotensin II receptor-2, but not angiotensin II receptor-1. We have also shown using our in vitro explant model that the cells emanating from proliferating haemangioma biopsies form blast-like structures that proliferate in the presence of angiotensin II. We present here a plausible model involving the renin-angiotensin system that may account for the propranolol-induced accelerated involution of proliferating infantile haemangioma.
Journal of Plastic Reconstructive & Aesthetic Surgery 06/2011; 64(6):759-65. DOI:10.1016/j.bjps.2010.08.039 · 1.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND; Infantile haemangioma (IH) is a tumour of the microvasculature composed predominantly of proliferating endothelial cells. It expresses markers associated with endothelial, haematopoietic and mesenchymal lineages. We have previously shown that the cells forming the capillary endothelium of proliferating IH express cell surface markers and transcriptions factors consistent with it being a haemogenic endothelium.
We wished to determine whether the expression of transcription factors associated with the erythroid lineage was of physiological relevance and sufficient for IH tissue cultured in vitro to undergo erythropoiesis.
Immunohistochemical staining of paraffin-embedded sections of proliferating IHs was undertaken and expression of the embryonically associated haemoglobin ζ (HBZ) chain and the erythropoietin receptor (EPO-R) was determined. Relative expression of mRNA encoding these proteins was determined by quantitative reverse transcription-polymerase chain reaction using snap-frozen biopsy samples. Differentiation towards erythrocytes was investigated using freshly resected tissue cultured as explants in Matrigel.
The endothelium of the microvessels, but not the pericyte layer, was strongly immunoreactive for the EPO-R and the embryonically associated HBZ chain. Abundant expression of transcripts encoding these proteins was also detected, corroborating the immunohistochemical staining. When tissue was grown in culture the cells emanating from IH explants were able to generate enucleated erythrocytes in vitro. The erythrocytes were immunoreactive for the erythrocyte-specific marker glycophorin A.
The microvessels in IH are a functional haemogenic endothelium that expresses the embryonically associated HBZ chain and is able to form erythrocytes in vitro. IH thus represents a possible extramedullary site for tumour-associated primitive erythropoiesis.
British Journal of Dermatology 05/2011; 164(5):1097-100. DOI:10.1111/j.1365-2133.2010.10187.x · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proliferating infantile haemangioma (IH) is a tumour of the microvasculature composed predominantly of immature endothelial cells. The origin of IH is unclear, but it has been shown to express markers of both endothelial and haematopoietic lineages, and a role for endothelial progenitor cells in the aetiology of IH has been suggested. Haemangioblasts are precursors of both endothelial and haematopoietic cells, and their characterisation has identified the expression of cell surface and intracellular proteins that collectively can be used for assigning a haemangioblast phenotype.
The authors used immunohistochemical staining to characterise the expression of primitive haematopoietic-associated proteins in proliferating IHs.
The authors show that the cells forming the capillary endothelium express markers associated with primitive haematopoietic cells. Additionally, many of these cells express the transcription factors brachyury and GATA-2, indicating a primitive mesodermal origin. They hypothesise that the immature capillaries in IH are derived from primitive mesodermal cells with haemangioblastic differentiation capabilities. The expression of primitive mesodermal, endothelial and haematopoietic markers by the cells forming the endothelium suggests that the immature capillaries that predominate in proliferating IH are a haemogenic endothelium phenotype, derived from haemangioblasts.
[Show abstract][Hide abstract] ABSTRACT: Infantile haemangioma is a tumour of the microvasculature characterised by aggressive angiogenesis during infancy and spontaneously gradual involution, often leaving a fibro-fatty residuum. The segmental distribution of a subgroup of infantile haemangioma, especially those associated with midline structural anomalies that constitute posterior fossa malformations-hemangiomas-arterial anomalies-cardiac defects-eye abnormalities-sternal cleft and supraumbilical raphe syndrome (PHACES), led us to investigate whether neural crest cells might be involved in the aetiology of this tumour.
Immunohistochemical staining on paraffin embedded infantile haemangioma sections and immunocytochemical staining on cells derived from proliferating haemangioma cultures were performed.
The endothelium of proliferating infantile haemangioma contains abundant cells that express the neurotrophin receptor (p75), a cell surface marker that identifies neural crest cells, and also for brachyury, a transcription factor expressed in cells of the primitive mesoderm. The endothelium is also immunoreactive for the haematopoietic stem cell marker, CD133; the endothelial-haematopoietic stem/progenitor marker, CD34; the endothelial cell markers, CD31 and VEGFR-2; and the mesenchymal stem cell markers, CD29 and vimentin. Additionally, immunoreactivity for the transcription factors, Sox 9 and Sox 10, that are expressed by prospective neural crest cells was also observed. Cells from microvessel-like structures were isolated from in vitro cultured haemangioma tissue explants embedded in a fibrin matrix. Immunostaining of these cells showed that they retained expression of the same lineage-specific markers that are detected on the paraffin embedded tissue sections.
These data infer that infantile haemangioma is derived from primitive mesoderm and that the cells within the lesion have a neural crest stem cell phenotype, and they express proteins associated with haematopoietic, endothelial, neural crest and mesenchymal lineages. The authors propose a model to account for the natural progression of infantile haemangioma based upon the multipotent expression profile of the primitive mesoderm and their neural crest stem cell phenotype to form all the cell lineages detected during infantile haemangioma proliferation and involution.
[Show abstract][Hide abstract] ABSTRACT: Purpose: We have recently shown mesenchymal stem cells (MSCs) isolated from haemangioma stained strongly for OPG. Vimentin immunoreactivity (IR) is an established marker for immature cells of mesenchymal origin. This study characterises haemangioma biopsies at different phases of progression to identify the presence of progenitor cells.Methods: Immunohistochemical staining was carried out on formalin-fixed paraffin sections of haemangioma at different phases of progression for vimentin, osteoprotegerin (OPG), von Willebrand factor and glucose transporter-1 (Glut-1).Results: Positive staining for OPG and vimentin was observed on progenitor cells in the proliferating and early involuting phases of haemangioma. Strong Glut-1 staining of mature capillaries of the involuted phase was inversely related to the degree of OPG and vimentin IR. Positive vimentin staining of cells in the interstitium of involuted heamangioma and weak vimentin IR of blood vessels was found suggesting that these vimentin-positive cells may be MSCs that eventually differentiate into adipocytes that characterise involuted lesions. OPG IR diminished as haemangioma involuted, with little staining seen in involuted lesions.Conclusions: OPG and vimentin IR are early progenitor cell markers that stain immature capillaries of proliferating haemangioma. In involuted haemangioma OPG IR is scant whereas vimentin-positive cells are present in the interstitium. This finding supports our hypothesis that OPG IR is a marker for progenitor cells present in immature capillaries of proliferating haemangioma that is lost as the lesions involute.
ANZ Journal of Surgery 05/2009; 79. DOI:10.1111/j.1445-2197.2009.04927_40.x · 1.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: HNcSCC is common in Australasia. Recent advances have led to new paradigms in our understanding of the complex interactions between tumour, stromal and immune cells, and the extracellular matrix. The deranged interplay between tumour and its microenvironment eventually spirals towards irreversibility. The repertoire of gene expression in the tumour becomes increasingly abnormal, and results in metastasis.Purpose: To examine the tumour microenvironment including, tumour immunosurveillance, angiogenesis and lymphangiogenesis, and investigate the expression of EGFR, which is fundamental to skin physiology, in HNcSCC.Methods: Archived HNcSCC specimens were stratified into groups of primary lesions with and without metastasis. (Immuno) histochemical analysis was performed for the differential parameters of peritumoural mast cell, microvessel and lymphatic vessel densities, VEGF and EGFR expression. The effects of mast cells on cell cycle and apoptotic gene expression were studied using cell co-culture and cDNA micro-array. The mechanism of EGFR over-expression in metastatic HNcSCC and its potential link with HER2 were assessed with fluorescence in-situ hybridisation.Results: Although mast cells demonstrate a tumour inhibitory effect by deregulating the cell cycle and apoptosis in vitro, this observation is not corroborated by study of tissue specimens. Lymphatic vessel density (but not microvessel density or VEGF expression) predicts metastatic potential. 75% of primary HNcSCC that subsequently metastasise over-express EGFR, with the majority harbouring HER2 polysomy.Conclusion: Improved understanding of the tumour microenvironment will hopefully lead to development of novel biologic therapy for HNcSCC.
ANZ Journal of Surgery 05/2009; 79. DOI:10.1111/j.1445-2197.2009.04927_11.x · 1.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is currently no prognostic tool that reliably predicts the risk of metastasis in cutaneous squamous cell carcinoma, most of which occur in the head and neck region. Epidermal growth factor receptor has received much interest in recent years with the advent of epidermal growth factor receptor-targeted molecular therapy in clinical oncology. We investigate the role of epidermal growth factor receptor as a biomarker for head and neck cutaneous squamous cell carcinoma. Using immunohistochemistry and fluorescence in situ hybridization, we assessed the epidermal growth factor receptor protein expression and gene copy in 3 groups of head and neck cutaneous squamous cell carcinoma: primary lesions not associated with metastasis (P), primary lesions associated with subsequent metastasis (PM), and metastatic nodal disease (M). Epidermal growth factor receptor overexpression was detected in 36% and 79% of P and PM cases, respectively. Epidermal growth factor receptor overexpression was significantly associated with PM (P = .03) and was found to be an independent prognostic factor for metastasis on multivariate analysis (P = .05). However, epidermal growth factor receptor overexpression was only maintained in 47% of cases in the M group. None of the 27 cases that overexpressed the epidermal growth factor receptor protein showed gene amplification: the results were uninterpretable in 2, and polysomy and balanced disomy were detected in 5 and 20 cases, respectively. These observations may have important prognostic and therapeutic implications for head and neck cutaneous squamous cell carcinoma.
Human Pathlogy 04/2008; 39(3):344-9. DOI:10.1016/j.humpath.2007.07.004 · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Extra-skeletal Ewing's sarcoma (EES) is an uncommon malignancy, especially in the head and neck region that may arise in various extra-osseous tissues. We report a 22-year-old male with an EES of the submandibular gland, which to the best of our knowledge, has not been described previously. The patient who underwent combined treatment with surgical resection and chemo-irradiation was disease free for 22 months but succumbed to multi-organ metastases 14 months later. This case highlights the combined diagnostic role of immunohistochemical, cytogenetic and radiological evaluation of EES. EES is an aggressive cancer that requires multidisciplinary management with wide surgical excision and adjunctive chemo-irradiation for the best outcome.
Journal of Plastic Reconstructive & Aesthetic Surgery 02/2007; 60(12):1345-8. DOI:10.1016/j.bjps.2006.01.046 · 1.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metastatic parotid cutaneous squamous cell carcinoma (SCC) is the most common parotid gland malignancy in New Zealand and Australia. The current AJCC TNM staging system does not account for the extent of nodal metastasis. A staging system that separates parotid (P stage) from neck disease (N stage) has been proposed recently.
To review the outcome of patients with metastatic head and neck cutaneous SCC treated at our multidisciplinary Head and Neck Service using the proposed staging system.
Consecutive patients were culled from our Head and Neck/Skull Base Database, 1990-2004. These patients were restaged according to the proposed staging system: P stage: P0 = no disease in the parotid (i.e., neck disease only); P1 = metastatic node < or = 3 cm; P2=metastatic node > 3 cm and < or =6 cm, or multiple nodes; and P3 = metastatic node > 6 cm, or disease involving the facial nerve or skull base. N stage: N0=no disease in the neck (i.e., parotid disease only); N1 = single ipsilateral metastatic node < or = 3 cm; and N2 = multiple metastatic nodes, or any node > 3 cm, or contralateral neck involvement. Loco-regional recurrence and disease-specific survival were calculated using the Kaplan-Meier method and comparison of graphs made with the log-rank test. Multivariate analysis using the Cox regression model was carried out to assess the impact of various parameters.
Sixty-seven patients with metastatic head and neck cutaneous SCC were identified. Thirty-seven patients had parotid metastasis (of whom 13 also had neck disease) while 21 had neck metastasis alone. Nine patients had dermal or soft tissue metastasis. These nine patients were excluded from this series, and data analysis was carried out on the remaining 58 (46 men, 12 women, mean age 71 years) patients. Sixty-seven percent of the patients underwent post-operative adjuvant radiotherapy. The five-year disease-specific survival rate was 54%. Among 56 patients followed up to disease recurrence or for a minimum period of 18 months, the loco-regional recurrence rate was 52%. The presence of parotid disease was an independent prognostic factor on survival (p < 0.01), and P3 fared significantly worse than P1 and P2. Those patients who had both parotid and neck disease fared worse than those who had parotid or neck disease alone (p = 0.01). N2 had a significantly poorer outcome compared with N1 (p < 0.01). Immunosuppression (p = 0.01) and a positive surgical margin (p < 0.01) were significant adverse prognostic factors for survival. Adjuvant radiotherapy, extracapsular spread, and perineural and vascular invasion did not influence survival. Our study demonstrates that the extent of parotid disease is an independent prognostic factor for metastatic head and neck cutaneous SCC.
Journal of Plastic Reconstructive & Aesthetic Surgery 12/2006; 59(12):1288-93. DOI:10.1016/j.bjps.2006.03.043 · 1.42 Impact Factor