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• W2084523207 abstract "The time course of microvascular changes in the environment of irradiated tumors was studied in a standardized human protocol. Eighty skin biopsies from 40 patients with previously treated primary breast cancer were taken from irradiated skin and corresponding contralateral unirradiated control areas 2 to 8 weeks, 11 to 14 months, or 17+ months after radiotherapy (skin equivalent dose 30 to 40 Gy). Twenty-two biopsies of 11 melanoma patients who had undergone lymph node dissection were used for unirradiated control. We found an increase of total podoplanin+ lymphatic microvessel density resulting mainly from a duplication of the density of smallest lymphatic vessels (diameter <10 μm) in the samples taken 1 year after radiation. Our findings implicate radiogenic lymphangiogenesis during the 1st year after therapy. The numbers of CD68+ and vascular endothelial growth factor-C+ cells were highly elevated in irradiated skin in the samples taken 2 to 8 weeks after radiotherapy. Thus, our results indicate that vascular endothelial growth factor-C expression by invading macrophages could be a pathogenetic route of induction of radiogenic lymphangiogenesis. The time course of microvascular changes in the environment of irradiated tumors was studied in a standardized human protocol. Eighty skin biopsies from 40 patients with previously treated primary breast cancer were taken from irradiated skin and corresponding contralateral unirradiated control areas 2 to 8 weeks, 11 to 14 months, or 17+ months after radiotherapy (skin equivalent dose 30 to 40 Gy). Twenty-two biopsies of 11 melanoma patients who had undergone lymph node dissection were used for unirradiated control. We found an increase of total podoplanin+ lymphatic microvessel density resulting mainly from a duplication of the density of smallest lymphatic vessels (diameter <10 μm) in the samples taken 1 year after radiation. Our findings implicate radiogenic lymphangiogenesis during the 1st year after therapy. The numbers of CD68+ and vascular endothelial growth factor-C+ cells were highly elevated in irradiated skin in the samples taken 2 to 8 weeks after radiotherapy. Thus, our results indicate that vascular endothelial growth factor-C expression by invading macrophages could be a pathogenetic route of induction of radiogenic lymphangiogenesis. Most knowledge on structural reorganization of microvessels after radiotherapy is derived from studies of acute radioreaction or on late changes that occur several years after radiation. In comparison with highly proliferative tissues, acute effects of radiation appear relatively late in the microvasculature of normal tissue. The nadir of microvessel density is to be found here within 50 to 70 days after a radiation dose equivalent to that usually used in solid neoplasms (50 to 60 Gy).1Archambeau JO Ines A Fajardo LF Correlation of the dermal microvasculature morphology with the epidermal and the endothelial population changes produced by single X ray fractions of 1649, 2231 and 2619 rad in swine.Int J Radiat Oncol Biol Phys. 1985; 11: 1639-1646Abstract Full Text PDF PubMed Scopus (22) Google Scholar The microvasculature is almost completely destroyed by very high doses (100 Gy) and must be rebuilt by the tissue following the principles of angiogenesis.2Takahashi S Sugimoto M Kotoura Y Sasai K Oka M Yamamuro T Long-term changes in the haversian systems following high-dose irradiation: an ultrastructural and quantitative histomorphological study.J Bone Joint Surg Am. 1994; 76: 722-738PubMed Google Scholar In contrast to posttraumatic wound healing, neovascularization is ineffective in irradiated tissue, despite high vascular endothelial growth factor (VEGF) levels and endothelial cell activation.3Tsai JH Makonnen S Feldman M Sehgal CM Maity A Lee WM Ionizing radiation inhibits tumor neovascularization by inducing ineffective angiogenesis.Cancer Biol Ther. 2005; 4: 1395-1400Crossref PubMed Scopus (47) Google Scholar Atrophy and fibrosis are typical late effects of radiotherapy. They frequently first become evident by ischemia and radiogenic ulcer 10 or more years after the radiogenic damage.4Fajardo LF The pathology of ionizing radiation as defined by morphologic patterns.Acta Oncol. 2005; 44: 13-22Crossref PubMed Scopus (235) Google Scholar In this, rarefaction of the blood microvascular densities of the terminal stream bed and formation of bypass vessels that are clinically visible as telangiectases play a central role.5Reinhold HS The influence of radiation on blood vessels and circulation. Chapter IV. Structural changes in blood vessels.Curr Top Radiat Res Q. 1974; 10: 58-74PubMed Google Scholar Interestingly, the time between acute and late effects, which can be regarded as the time window for development of local recurrences or metastases in most solid neoplasms, is not well studied. We partially close this gap in this study. The effect of an intermediate radio dose is another important aspect that has not yet been sufficiently investigated. Although the studies cited above mainly focused on tumoral target doses, investigation of the effect of a “tumor-environmental dose” (about two-thirds of the target volume dose) seems to be most promising, because the tumor environment is the major site of development of local recurrences or skin metastases.6Rofstad EK Mathiesen B Henriksen K Kindem K Galappathi K The tumor bed effect: increased metastatic dissemination from hypoxia-induced up-regulation of metastasis-promoting gene products.Cancer Res. 2005; 65: 2387-2396Crossref PubMed Scopus (76) Google Scholar The detection of molecules that are relatively specifically expressed by lymphatic endothelial cells, like podoplanin, lymphatic vessel endothelial hyaluronate receptor 1 (LYVE-1), vascular endothelial growth factor receptor (VEGFR)-3, prospero-related homeobox gene PROX-1, desmoplakin-1 (2.17), and β-chemokine receptor D6, has facilitated new insights into the molecular mechanisms that control lymphatic vessel development.7Jussila L Alitalo K Vascular growth factors and lymphangiogenesis.Physiol Rev. 2002; 82: 673-700Crossref PubMed Scopus (352) Google Scholar, 8Ordóñez NG Podoplanin: a novel diagnostic immunohistochemical marker.Adv Anat Pathol. 2006; 13: 83-88Crossref PubMed Scopus (150) Google Scholar, 9Breiteneder-Geleff S Matsui K Soleiman A Meraner P Poczewski H Kalt R Schaffner G Kerjaschki D Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis.Am J Pathol. 1997; 151: 1141-1152PubMed Google Scholar, 10Skobe M Hawighorst T Jackson DG Prevo R Janes L Velasco P Riccardi L Alitalo K Claffey K Detmar M Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.Nat Med. 2001; 7: 192-198Crossref PubMed Scopus (1498) Google Scholar, 11Joukov V Pajusola K Kaipainen A Chilov D Lahtinen I Kukk E Saksela O Kalkkinen N Alitalo K A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1153) Google Scholar, 12Hong YK Harvey N Noh YH Schacht V Hirakawa S Detmar M Oliver G Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate.Dev Dyn. 2002; 225: 351-357Crossref PubMed Scopus (430) Google Scholar Lymphangiogenesis is stimulated during embryogenesis, after trauma, in ischemic tissue, chronic wounds, acute lymphedema, or by malignant tumors.13Skobe M Hamberg LM Hawighorst T Schirner M Wolf GL Alitalo K Detmar M Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma.Am J Pathol. 2001; 159: 893-903Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 14Karpanen T Alitalo K Lymphatic vessels as targets of tumor therapy?.J Exp Med. 2001; 194: F37-F42Crossref PubMed Scopus (116) Google Scholar, 15Schoppmann SF Birner P Stockl J Kalt R Ullrich R Caucig C Kriehuber E Nagy K Alitalo K Kerjaschki D Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis.Am J Pathol. 2002; 161: 947-956Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 16Karpanen T Egeblad M Karkkainen MJ Kubo H Yla-Herttuala S Jaattela M Alitalo K Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth.Cancer Res. 2001; 61: 1786-1790PubMed Google Scholar Two members of the VEGF family, VEGF-C and VEGF-D, are important for the induction of lymphangiogenesis.7Jussila L Alitalo K Vascular growth factors and lymphangiogenesis.Physiol Rev. 2002; 82: 673-700Crossref PubMed Scopus (352) Google Scholar, 17Ferrara N VEGF: an update on biological and therapeutic aspects.Curr Opin Biotechnol. 2000; 11: 617-624Crossref PubMed Scopus (355) Google Scholar, 18Achen MG Jeltsch M Kukk E Makinen T Vitali A Wilks AF Alitalo K Stacker SA Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4).Proc Natl Acad Sci USA. 1998; 95: 548-553Crossref PubMed Scopus (1017) Google Scholar, 19Veikkola T Jussila L Makinen T Karpanen T Jeltsch M Petrova TV Kubo H Thurston G McDonald DM Achen MG Stacker SA Alitalo K Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice.EMBO J. 2001; 20: 1223-1231Crossref PubMed Scopus (563) Google Scholar, 20Jeltsch M Kaipainen A Joukov V Meng X Lakso M Rauvala H Swartz M Fukumura D Jain RK Alitalo K Hyperplasia of lymphatic vessels in VEGF-C transgenic mice.Science. 1997; 276: 1423-1425Crossref PubMed Scopus (1111) Google Scholar, 21Karkkainen MJ Haiko P Sainio K Partanen J Taipale J Petrova TV Jeltsch M Jackson DG Talikka M Rauvala H Betsholtz C Alitalo K Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins.Nat Immunol. 2004; 5: 74-80Crossref PubMed Scopus (1054) Google Scholar These factors are ligands of the lymphatic endothelial VEGF receptor-3 that is expressed on the surface of lymphatic endothelial cells. VEGF-C is produced by macrophages, dendritic cells, endothelial cells, platelets, basophilic granulocytes, lymphocytes, and tumor cells.15Schoppmann SF Birner P Stockl J Kalt R Ullrich R Caucig C Kriehuber E Nagy K Alitalo K Kerjaschki D Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis.Am J Pathol. 2002; 161: 947-956Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 22Wartiovaara U Salven P Mikkola H Lassila R Kaukonen J Joukov V Orpana A Ristimaki A Heikinheimo M Joensuu H Alitalo K Palotie A Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation.Thromb Haemost. 1998; 80: 171-175PubMed Google Scholar Like other VEGFs, VEGF-C controls not only angiogenesis of blood and lymphatic vessels but also microvessel permeability and migration of endothelial cells.7Jussila L Alitalo K Vascular growth factors and lymphangiogenesis.Physiol Rev. 2002; 82: 673-700Crossref PubMed Scopus (352) Google Scholar, 11Joukov V Pajusola K Kaipainen A Chilov D Lahtinen I Kukk E Saksela O Kalkkinen N Alitalo K A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1153) Google Scholar, 17Ferrara N VEGF: an update on biological and therapeutic aspects.Curr Opin Biotechnol. 2000; 11: 617-624Crossref PubMed Scopus (355) Google Scholar, 23Kaipainen A Korhonen J Mustonen T van Hinsbergh VW Fang GH Dumont D Breitman M Alitalo K Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.Proc Natl Acad Sci USA. 1995; 92: 3566-3570Crossref PubMed Scopus (1185) Google Scholar In this study, we investigated the changes of the lymphatic and blood microvasculature during the first years after postoperative radiotherapy for breast cancer in a standardized group of patients by comparing intraindividual control samples of unirradiated skin taken from exactly symmetrically contralateral areas. In addition, we used for comparison a group of melanoma patients who received no radiotherapy but were treated by operation (lymph node dissection). The study received approval by the local Ethics Committee of the Medical Faculty at the University of Halle. Informed consent was obtained individually from the patients. We studied 80 samples of 40 consecutive female patients of the Breast Cancer Center of Martin Luther University Halle-Wittenberg after completed standard radiation therapy because of breast cancer (Table 1). Patients were treated by operation, chemotherapy, and/or hormone therapy according to the standards of care (St. Gallen protocol).24Goldhirsch A Glick JH Gelber RD Senn HJ International Consensus Panel on the treatment of primary breast cancer. V: update 1998.Recent Results Cancer Res. 1998; 152: 481-497Crossref PubMed Scopus (26) Google Scholar At the time of biopsy, patients were free of local recurrence or metastatic disease. Local exclusion criteria regarding the sampling regions were any clinical signs of radiation dermatitis or other inflammatory disease, palpable or sonographically detectable axillary lymph node swelling, and lymphedema of the biopsy area and its direct neighborhood (axilla and pectoral region). In contrast, clinically detectable lymphedema restricted to the arm of the irradiated site was allowed and regarded in the analysis (10 of 40 patients). General exclusion criteria were diabetes mellitus, autoimmune disease, human immunodeficiency virus infection, and reduced general condition (Karnofsky index <80%). Co-morbidity included hypertension (n = 8), arrhythmia (n = 3), bronchial asthma (n = 2), previously treated inactive pulmonary tuberculosis (n = 2), rheumatic polyarthritis (n = 1), angina pectoris (n = 1), psoriasis (n = 1), or chronic hepatitis C (n = 1). Besides specific anticancer therapy (Table 1), patients were treated with l-thyroxin (n = 4), acetylsalicylic acid (n = 3), β-blockers (n = 2), angiotensin-converting enzyme inhibitors (n = 2), or calcium antagonists (n = 2).Table 1Characteristics of the Patients and TumorsAge (median, range)61 years (38 to 79)Sex (female/male)40/0Menopause29 of 40Histology T (DCIS/T1a/T1b/T1c/T2)47Singletary SE Allred C Ashley P Bassett LW Berry D Bland KI Borgen PI Clark GM Edge SB Hayes DF Hughes LL Hutter RV Morrow M Page DL Recht A Theriault RL Thor A Weaver DL Wieand HS Greene FL Staging system for breast cancer: revisions for the sixth edition of the AJCC Cancer Staging Manual.Surg Clin North Am. 2003; 83: 803-819Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar6/4/8/9/13 N*N state was histologically controlled in all patients except patients with DCIS (Nx); positive nodes were completely resected before radiotherapy. (Nx, N0, N1)6/22/12 M (M0/M1)40/0 Grading (G1/G2/G3)4/19/11 Estrogen receptor status (positive/negative/n.d.)23/10/7 Progesterone receptor status (positive/negative/n.d.)24/9/7Operative therapy Operative technique of the primary (breast-preserving/mastectomy)33/7 Axilla dissection (level I + II) (yes/no)34/6 Dissected nodes (median, range)17 (5 to 29) Positive nodes (median, range)0 (0 to 19) Complete carcinoma resection before radiotherapy (yes/no)40/0Radiotherapy Total dose (50.4 Gy/54.0 Gy/44.8 Gy)38/1/1 Single doses (1.8 Gy/1.6 Gy)39/1Adjuvant therapy Chemotherapy24Goldhirsch A Glick JH Gelber RD Senn HJ International Consensus Panel on the treatment of primary breast cancer. V: update 1998.Recent Results Cancer Res. 1998; 152: 481-497Crossref PubMed Scopus (26) Google Scholar17 Hormone therapy24Goldhirsch A Glick JH Gelber RD Senn HJ International Consensus Panel on the treatment of primary breast cancer. V: update 1998.Recent Results Cancer Res. 1998; 152: 481-497Crossref PubMed Scopus (26) Google Scholar26n.d., not done.* N state was histologically controlled in all patients except patients with DCIS (Nx); positive nodes were completely resected before radiotherapy. Open table in a new tab n.d., not done. Before sampling, patients had received standard radiotherapy with 6-MV photons via tangential fields (Table 1). Some patients with large breasts and larger dose inhomogeneities received part of the radiation dose with 10-MV photons, mainly about 40% of the total dose (20 of 50 Gy). Radiotherapy was delivered in conventional fractionation with five fractions per week in all patients. No bolus was used. Three of 40 patients with four or more positive nodes received additional radiotherapy to the supraclavicular fossa; treatment was delivered via a single anterior portal with 6-MV photons, and the radiation field did not cover the skin biopsy side (see below). These patients with additional supraclavicular radiotherapy belonged to the patient groups “2 to 8 weeks” (n = 1), “11 to 14 months” (n = 1), or “17 to 25 months after radiotherapy” (n = 1). In 38 of 40 patients, the total dose calculated at the International Commission on Radiation Units and Measurements reference point within the target volume was 50.4 Gy (in 28 single doses of 1.80 Gy). One patient of 40 received 54 Gy (30 × 1.8 Gy), and one patient of 40 received 44.8 Gy (28 × 1.6 Gy). Both of these dose-deviant patients belonged to the same patient subgroup “2 to 8 weeks after radiotherapy” (see below). The estimated skin dose was 60 to 70% of the reference dose (see Discussion). Punch biopsies (6 mm) were taken 2 to 8 weeks (median, 4 weeks; n = 13 patients), 11 to 14 months (median, 12 months; n = 11), 17 to 25 months (median, 24; n = 8), or 37 to 157 months (median, 43.5; n = 8) after radiotherapy from the skin of the superior anterior axillary line that was directly included in the target region of all fractions of the radiotherapy (2 to 3 cm proximal from lymph node dissection scar) and from the corresponding unirradiated skin area of the opposite anterior axillary line of each patient for control. Biopsies were divided into two parts. One was snap-frozen in liquid nitrogen and stored at −70°C, and the other was fixed overnight in 4% formalin and paraffin-embedded. Twenty-two samples from 11 melanoma patients who had not undergone radiation but underwent similar procedure of operation (lymph node dissection) were used for comparison. Eleven biopsies were taken 2 to 3 cm proximal to a lymph node dissection scar either from the axilla (9 of 11 cases) or from inguinal region (2 of 11), and 11 additional biopsies were taken from the exactly contralateral skin area of the same 11 patients: age, 61.8 ± 12.8 years; sex, 10 female, one male; time between operation and biopsy median, 14.4 ± 2.8 months (minimum, 9; maximum, 19 months); stage of disease (American Joint Committee on Cancer 200225Balch CM Buzaid AC Soong SJ Atkins MB Cascinelli N Coit DG Fleming ID Gershenwald JE Houghton Jr, A Kirkwood JM McMasters KM Mihm MF Morton DL Reintgen DS Ross MI Sober A Thompson JA Thompson JF Final version of the American Joint Committee on Cancer staging system for cutaneous melanoma.J Clin Oncol. 2001; 19: 3635-3648Crossref PubMed Scopus (2277) Google Scholar), 1b/2a/3b/3c, n = 1/1/8/1; type of operation, radical lymph node dissection (9 of 11) or sentinel node biopsy (2 of 11); and number of dissected lymph nodes, 13.9 ± 7.2. At the time of biopsy, patients were free of local recurrence or metastatic disease. Biopsies were fixed overnight in 4% formalin and paraffin-embedded. Polyclonal rabbit anti-human podoplanin IgG was raised against the recombinant human homolog of the rat 43-kd glycoprotein podoplanin. Rabbit sera were affinity-purified using nitrocellulose strips containing recombinant protein, as described previously.26Breiteneder-Geleff S Soleiman A Kowalski H Horvat R Amann G Kriehuber E Diem K Weninger W Tschachler E Alitalo K Kerjaschki D Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.Am J Pathol. 1999; 154: 385-394Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar Other primary antibodies were as follows: monoclonal mouse anti-human CD34, class II clone QBEnd10, isotype IgG1, κ (Dako, Glostrup, Denmark); monoclonal mouse anti-human CD68, clone KP1, isotype IgG1, κ (Dako); monoclonal mouse anti-human CD68, clone PG-M1, fluorescein isothiocyanate conjugate (Dako); rabbit anti-human LYVE-1 (AngioBio, Del Mar, CA); rabbit anti-human VEGF-C (Zymed Inc., South San Francisco, CA); and goat anti-human VEGF-D (R&D Systems, Minneapolis, MN). Fluorochrome-labeled secondary monoclonal antibodies were as follows: goat anti-rabbit IgG, F(ab′)2-tetramethylrhodamine isothiocyanate (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and donkey anti-goat IgG, F(ab′)2-tetramethylrhodamine B isothiocyanate (Santa Cruz). Paraffin sections (5 μm) of each sample were stained with hematoxylin and eosin (H&E). For immunohistology, consecutive sections were deparaffinized in a graded ethanol series. Endogenous peroxidase was blocked (0.3% H2O2 in methanol). Then, the sections were microwaved (10 mmol/L citrate buffer, pH 6, four times for 5 minutes, and 600 W) for antigen demasking. The sections were incubated with anti-podoplanin (1:250), anti-CD34 (1:40), anti-CD68/KP1 (1:50), or anti-VEGF-C (1:100) for 30 minutes at room temperature. In VEGF-C or CD68 staining, undiluted rabbit or mouse primary antibody isotype controls (08-6199 or 08-6599; Zymed) were used for negative control. Further steps were performed with the Elite-ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Finally, the sections were counterstained with 25% hemalum (3 minutes; Merck, Darmstadt, Germany), rinsed with tap water, and mounted (gelatin mounting medium; Dako). Cryostat sections (5 μm) were triple-stained with fluorescein isothiocyanate conjugate of anti-CD68/PG-M1 (1:50; 30 minutes; room temperature), anti-VEGF-C (1:100; 30 minutes; room temperature), or anti-VEGF-D (1:80; 30 minutes; room temperature) and DNA fluorochrome (Hoechst 33258; Sigma). Tetramethylrhodamine isothiocyanate-fluorochrome-labeled secondary monoclonal antibodies [goat anti-rabbit IgG, F(ab′)2; donkey anti-goat IgG, F(ab′)2] were both applied at 1:100 (30 minutes). For demonstration of LYVE-1+ lymphatic microvessels, 8-μm cryostat sections were double-stained with anti-LYVE-1 (1:100; 30 minutes; room temperature) and DNA fluorochrome. Tetramethylrhodamine isothiocyanate-fluorochrome-labeled goat anti-rabbit IgG, F(ab′)2 was applied 1:100 (30 minutes). In VEGF-C, VEGF-D, or LYVE-1 staining, 0.5 μg/ml primary antibody isotype controls of rabbit (Zymed) or goat (Abcam, Cambridge, UK) were used for negative control. In VEGF-C, negative controls were additionally preblocked with human recombinant VEGF-C (5 μg/ml; BioVision, Mountain View, CA). Sections were studied for general description of changes in comparison with the controls. Immunohistologically treated sections were evaluated in a “blinded” setting. At least three different sections of each sample were evaluated in the upper horizontal dermal plexus. Densities were calculated relative to skin surface (n/mm). The lymphatic vessels were grouped by diameter: <10 μm, 10 to 17 μm, 18 to 24 μm, and ≥25 μm (only in podoplanin investigations), as were the blood vessels: <12 μm, 12 to 15 μm, and ≥16 μm. For this, the shortest transversal axis of each vessel section was assessed under a measuring field ocular. The numbers of the visible microvessel sections were counted using the diameter groups. Only clearly identifiable microvessels were taken into account (Figure 1, j and k). The interobserver coefficient of variation of this method was calculated in sixfold counting of the podoplanin-stained sections of 10 different cases with a total of 57 sections at 0.076 ± 0.045. Statistics were performed by SPSS 12.0.1 (Chicago, IL). The following statistical tests were used: Pearson correlation coefficient (r); Spearman correlation coefficient (ρ); t-test for paired samples (t-test); Wilcoxon test, exact, two-sided significance (Wilcoxon test); Mann-Whitney U-test, exact, two-sided significance (U-test); and logistic regression. Categorical, ordinal covariates were median-dichotomized in logistic regression. In LYVE-1 immunofluorescence investigations, the number of positive cells or microvessel segments was calculated from five sections of each analyzed sample. In the H&E-stained sections of irradiated skin of the breast cancer patients, a sparse perivascular-oriented mononuclear infiltrate and qualitative degeneration of microvessels, hair follicles, or sebaceous glands were evident in the samples harvested 2 to 8 weeks after the end of radiation (Figure 1, a–d). Venules presented diameter enlargement. The basal epidermis showed single-cell necroses. Twelve months after radiation, a proportion of 10 to 30% disfigured microvessels was visible (Figure 1e). These changes were persistently found in other samples from later stages that included a time up to 157 months after radiotherapy (Figure 1f). In addition, basophilic degeneration of the subepidermal connective tissue could be found in some cases >3 years after radiation. The mean density of total podoplanin+ lymphatic vessels in the samples of the treated skin of the breast cancer patients proved 18% higher than that of the individual control skin. This difference was significant (Table 2). Detailed analysis showed that this was mainly a consequence of an increase of the smallest lymphatics (with a diameter <10 μm) in the irradiated skin. In all patient subgroups together, the mean density of small lymphatics <10 μm was 44% higher in the treated side versus control side (Table 2). The source of this effect could be localized by time-subgroup analysis: We found more than duplication (107%) of the mean density of small lymphatics <10 μm in the subgroup of patients who had undergone radiotherapy 11 to 14 months earlier (Table 2; Figure 1, g–k). This effect was not yet visible in samples gathered in the first weeks after therapy and tended to persist beyond 14 months after therapy (Table 2). Neither the larger lymphatic microvessel calibers (≥10 μm) nor the total CD34+ blood microvessels showed significant radiogenic effect regarding the vessel densities (Table 2).Table 2Densities of Microvessels, CD68+, VEGF-C+, or VEGF-D+ Cells in Skin of the Therapeutic and Contralateral Control Sides of 40 Cases of Breast CancerTherapy side (n/mm)*Mean ± SD.Control side (n/mm)*Mean ± SD.PnLymphatics total2.81 ± 0.142.38 ± 0.910.022†t-test.40 Lymphatics <10 μm total0.92 ± 0.840.64 ± 0.380.036†t-test.40 0.5 to 2 months after therapy0.64 ± 0.460.78 ± 0.300.279‡Wilcoxon test.13 11 to 14 months after therapy0.91 ± 0.650.44 ± 0.260.026‡Wilcoxon test.11 17+ months after therapy1.14 ± 1.090.67 ± 0.450.098‡Wilcoxon test.16 Lymphatics 10 to 17 μm total1.06 ± 0.630.90 ± 0.430.138†t-test.40 0.5 to 2 months after therapy0.96 ± 0.410.98 ± 0.470.917‡Wilcoxon test.13 11 to 14 months after therapy1.00 ± 0.560.80 ± 0.510.328‡Wilcoxon test.11 17+ months after therapy1.22 ± 0.790.89 ± 0.350.278‡Wilcoxon test.16 Lymphatics 18 to 24 μm total0.45 ± 0.390.50 ± 0.330.519†t-test.40 0.5 to 2 months after therapy0.50 ± 0.490.50 ± 0.300.861‡Wilcoxon test.13 11 to 14 months after therapy0.48 ± 0.180.54 ± 0.300.477‡Wilcoxon test.11 17+ months after therapy0.37 ± 0.430.47 ± 0.380.609‡Wilcoxon test.16 Lymphatics >24 μm total0.38 ± 0.360.34 ± 0.330.558†t-test.40 0.5 to 2 months after therapy0.40 ± 0.390.31 ± 0.350.328‡Wilcoxon test.13 11 to 14 months after therapy0.39 ± 0.320.35 ± 0.250.646‡Wilcoxon test.11 17+ months after therapy0.35 ± 0.370.36 ± 0.380.999‡Wilcoxon test.16Blood vessels total13.76 ± 3.2714.11 ± 3.650.598†t-test.40 Blood vessels <12 μm total8.15 ± 3.088.23 ± 3.220.860†t-test.40 0.5 to 2 months after therapy8.68 ± 2.128.13 ± 2.810.382‡Wilcoxon test.13 11 to 14 months after therapy8.67 ± 4.038.75 ± 3.690.859‡Wilcoxon test.11 17+ months after therapy7.26 ± 2.957.96 ± 3.350.605‡Wilcoxon test.16 Blood vessels 12 to 15 μm total2.24 ± 1.802.27 ± 1.740.883†t-test.40 0.5 to 2 months after therapy2.85 ± 1.773.05 ± 2.110.753‡Wilcoxon test.13 11 to 14 months after therapy2.53 ± 2.332.16 ± 1.520.374‡Wilcoxon test.11 17+ months after therapy1.55 ± 1.111.71 ± 1.370.301‡Wilcoxon test.16 Blood vessels >15 μm total3.38 ± 3.103.60 ± 3.540.558†t-test.40 0.5 to 2 months after therapy3.26 ± 3.372.37 ± 2.780.033‡Wilcoxon test.13 11 to 14 months after therapy2.85 ± 2.723.93 ± 4.390.049‡Wilcoxon test.11 17+ months after therapy4.03 ± 3.264.39 ± 3.400.363‡Wilcoxon test.16CD68+ cells§Time subgroups of CD68+, VEGF-C+, and VEGF-D+ cells are represented in Figure 3.85.1 ± 28.664.4 ± 25.2<0.001†t-test.40VEGF-C+ cells§Time subgroups of CD68+, VEGF-C+, and VEGF-D+ cells are represented in Figure 3.17.7 ± 8.810.2 ± 6.50.001‡Wilcoxon test.22VEGF-D+ cells§Time subgroups of CD68+, VEGF-C+, and VEGF-D+ cells are represented in Figure 3.4.4 ± 2.42.5 ± 1.7<0.014‡Wilcoxon test.20Subgroups of the microvessel density values reflect the influence of the time interval between end of radiotherapy and biopsy.* Mean ± SD.† t-test.‡ Wilcoxon test.§ Time subgroups of CD68+, VEGF-C+, and VEGF-D+ cells are represented in Figure 3. Open table in a new tab Subgroups of the microvessel density values reflect the influence of the time interval between end of radiotherapy and biopsy. There was neither a significant difference nor a recognizable trend in all investigated microvessel density parameters between the two “late” subgroups (17 to 25 months and 37 to" @default.
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• W2084523207 date "2007-07-01" @default.
• W2084523207 modified "2023-10-18" @default.
• W2084523207 title "Radiogenic Lymphangiogenesis in the Skin" @default.
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