Electrochemical degradation of perfluoroalkyl and polyfluoroalkyl substances (PFASs) in groundwater.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) represent hazardous pollutants and are frequently detected in the environment, e.g. in contaminated groundwater. PFASs are persistent to biodegradation and conventional oxidation processes such as ozonation. In this study electrochemical degradation of PFASs on boron-doped diamond (BDD) electrodes is demonstrated. Experiments were performed with model solutions and contaminated groundwater with a dissolved organic carbon (DOC) content of 13 mg/L. The perfluorinated carboxylic acids (PFCAs) perfluorobutanoate, perfluoropentanoate, perfluorohexanoate, perfluoroheptanoate and perfluorooctanoate, and the perfluorinated sulfonic acids (PFSAs) perfluorobutane sulfonate, perfluorohexane sulfonate, perfluorooctane sulfonate and 6:2 fluorotelomer sulfonate were detected in the groundwater samples. At PFAS concentrations ranging from 0.26 to 34 mg/L (0.7 to 79 µM), the degradation of PFASs was achieved despite of the high DOC background. Pseudo first-order kinetic constants of PFSA degradation increased with the increase of carbon chain length. Fluoride formation as well as the generation of PFCAs with shortened chain lengths was observed. Inorganic byproducts such as perchlorate were also formed and have to be considered in further process optimization.

An experimental setup to evaluate innovative therapy options for the enhancement of bone healing using BMP as a benchmark--a pilot study.

Critical or delayed bone healing in rat osteotomy (OT) models is mostly achieved through large defects or instability. We aimed to design a rat OT model for impaired bone healing based on age, gender and parity. The outcome should be controllable through variations of the haematoma in the OT including a bone morphogenetic protein (BMP) 2 guided positive control. Using external fixation to stabilise femoral a 2 mm double OT in 12 month old, female Sprague Dawley rats after a minimum of 3 litters healing was characterised following in situ haematoma formation (ISH-group)), transplantation of a BMP charged autologous blood clot (BMP-group) and the artificial blood clot only (ABC-group) into the OT-gap. In vivo micro-computer tomography (µCT) scans were performed after 2, 4 and 6 weeks. After 6 weeks specimens underwent histological analyses. In µCT examinations and histological analyses no bony bridging was observed in all but one animal in the ISH-group. In the BMP group complete bridging was achieved in all animals. The ABC-group showed less mineralised tissue formation and smaller bridging scores during the course of healing than the ISH-group. In this pilot study we introduce a model for impaired bone healing taking the major biological risk factors into account. We could show that the in situ fracture haematoma is essential for bone regeneration. Using BMP as a positive control the presented experimental setup can serve to evaluate innovative therapeutical concepts in long bone application.

Time kinetics of bone defect healing in response to BMP-2 and GDF-5 characterised by in vivo biomechanics.

This study reports that treatment of osseous defects with different growth factors initiates distinct rates of repair. We developed a new method for monitoring the progression of repair, based upon measuring the in vivo mechanical properties of healing bone. Two different members of the bone morphogenetic protein (BMP) family were chosen to initiate defect healing: BMP-2 to induce osteogenesis, and growth-and-differentiation factor (GDF)-5 to induce chondrogenesis. To evaluate bone healing, BMPs were implanted into stabilised 5 mm bone defects in rat femurs and compared to controls. During the first two weeks, in vivo biomechanical measurements showed similar values regardless of the treatment used. However, 2 weeks after surgery, the rhBMP-2 group had a substantial increase in stiffness, which was supported by the imaging modalities. Although the rhGDF-5 group showed comparable mechanical properties at 6 weeks as the rhBMP-2 group, the temporal development of regenerating tissues appeared different with rhGDF-5, resulting in a smaller callus and delayed tissue mineralisation. Moreover, histology showed the presence of cartilage in the rhGDF-5 group whereas the rhBMP-2 group had no cartilaginous tissue. Therefore, this study shows that rhBMP-2 and rhGDF-5 treated defects, under the same conditions, use distinct rates of bone healing as shown by the tissue mechanical properties. Furthermore, results showed that in vivo biomechanical method is capable of detecting differences in healing rate by means of change in callus stiffness due to tissue mineralisation.

Seroprevalence of Salmonella and Mycoplasma infection in backyard chickens in the state of Entre Rios in Argentina.

The present work was conducted to study the seroprevalence of Salmonella, Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS) infection in backyard chickens located in Entre Ríos, Argentina, over 3 periods of time. A total of 2,441 sera samples were collected from backyard chickens belonging to 256 family farms in 16 counties in the state of Entre Ríos from January to May 2003 (first period), December 2004 to April 2005 (second period), and October 2006 to May 2007 (third period). The prevalence of family farms testing seropositive for Salmonella averaged 23.9, 15.9, and 28.6% during the first, second, and third period, respectively. The highest prevalence of Salmonella-seropositive farms recorded (66.7%) was on farms from Concordia county, and the lowest prevalence (0%) was on farms from La Paz county. In contrast, the prevalence of family farms seropositive for MG averaged 32.8, 55.1, and 76.2% during the first, second, and third periods, respectively. The highest prevalence of MG-seropositive farms (100%) was found in the counties of Victoria and Tala, and the lowest prevalence (8.7%) was found on farms on Colón county. The prevalence of family farms seropositive for MS averaged 68.6 and 100% during the first and second periods, respectively. The highest prevalence of MS-seropositive farms (100%) was on farms in 85% of the counties tested, and the lowest prevalence (21.7%) was on farms from Colón county. Salmonella, MG, and MS infection are present at high levels in backyard chicken farms, and this presents a high risk to commercial poultry production in Entre Ríos, the state with the highest chicken population and density in Argentina.

Osteochondral defect repair after implantation of biodegradable scaffolds: indirect magnetic resonance arthrography and histopathologic correlation.

BACKGROUND: Biodegradable scaffolds have become an important option in the treatment of osteochondral defects. Therefore, accurate and reproducible monitoring of scaffold repair tissue is crucial. PURPOSE: To assess the feasibility of indirect magnetic resonance (MR) arthrography in determining the quality of osteochondral repair after scaffold implantation using an MR imaging (MRI) scoring and grading system with histology as reference. MATERIAL AND METHODS: Osteochondral defects created at ovine condylar facets were treated with either a commercial poly (DL-lactide-co-glycolide) (PLG) scaffold or a modified softer one (n=6/group; 87% and 55% of the elastic modulus of ovine subchondral bone, respectively). Empty defects at the contralateral condyle served as control group. A 1.5T MRI scan was performed after 6 months with proton density (PD)-weighted (w) fat-saturated (fs) fast spin-echo (FSE), T1-w two-dimensional (2D), and 3D fs gradient echo (GE) sequences 30 min after intravenous Gd-DTPA administration and passive joint movement. Two independent radiologists evaluated the repair tissue. The MR findings were correlated with histological findings. RESULTS: MRI and histological grading correlated well (10/12 cases). The stiff-scaffold group showed significantly superior repair in comparison to the control group (P<0.05). The 3D fs GE sequence proved to be most valuable in evaluating morphologic status. Complete defect filling and integration, intact surface and isointense signal to the adjacent native cartilage, subchondral incorporation with bone marrow edema, and graft plug enhancement were associated with a good histological outcome. Histologically, we found a smooth fibrocartilaginous layer and osseous replacement of the scaffold. Incomplete cartilage repair and irregular subchondral structures on the MRI correlated histologically with fibrocartilage-like repair and subchondral sclerosis, due to substantial degradation of the scaffold. CONCLUSION: Indirect MR arthrography is an accurate, noninvasive monitoring tool in the follow-up of scaffold implants. The MRI scoring and grading system allows reliable assessment of normal and pathological repair, with high correlation to histological findings.

Digital image correlation: a technique for determining local mechanical conditions within early bone callus.

Local mechanical conditions are known to play a role during the regeneration of musculoskeletal tissues, and histomorphometrical investigations of the time course of healing have enabled specific conclusions regarding the mechanosensitivity of tissue differentiation. However, the mechanism for this influence is not clearly understood. In order to extend this analysis, it is essential to link local histological understanding with direct characterisation of the local mechanical environment. Digital image correlation (DIC) is a computer-based image analysis technique that enables the non-contact measurement of strains on material surfaces and is finding application in many areas of biomechanics. Here we report a DIC technique to investigate the local distribution of mechanical strain within regenerating soft tissue sections. We provide exemplary data from analysis of a section of sheep bone callus. An assessment of displacement measurement accuracy gave an RMS error of 4.2 microm, corresponding to an estimated strain error of 1.4%. The sections showed concentrations of up to four times the applied strain and comparison of the strain patterns with histological analysis confirmed that these concentrations reflected boundaries between hard and soft callus.

The haematoma and its role in bone healing.

Fracture treatment is an old endeavour intended to promote bone healing and to also enable early loading and regain of function in the injured limb. However, in today's clinical routine the healing potential of the initial fracture haematoma is still not fully recognized. The Arbeitsgemeinschaft für Osteosynthesefragen (AO) formed in Switzerland in 1956 formulated four AO principles of fracture treatment which are still valid today. Fracture treatment strategies have continued to evolve further, as for example the relatively new concept of minimally invasive plate osteosynthesis (MIPO). This MIPO treatment strategy harbours the benefit of an undisturbed original fracture haematoma that supports the healing process. The extent of the supportive effect of this haematoma for the bone healing process has not been considered in clinical practice so far. The rising importance of osteoimmunological aspects in bone healing supports the essential role of the initial haematoma as a source for inflammatory cells that release the cytokine pattern that directs cell recruitment towards the injured tissue. In reviewing the potential benefits of the fracture haematoma, the early development of angiogenic and osteogenic potentials within the haematoma are striking. Removing the haematoma during surgery could negatively influence the fracture healing process. In an ovine open tibial fracture model the haematoma was removed 4 or 7 days after injury and the bone that formed during the first two weeks of healing was significantly reduced in comparison with an undisturbed control. These findings indicate that whenever possible the original haematoma formed upon injury should be conserved during clinical fracture treatment to benefit from the inherent healing potential.

Do serological tissue turnover markers represent callus formation during fracture healing?

Serological parameters of bone and fibrous tissue turnover were demonstrated to monitor the course of fracture healing. The aim of this study was to evaluate the correlation between the serological parameter levels during fracture healing and callus development in a standardised ovine model of fracture healing. Two years old female sheep received a standardised 3 mm tibial bone defect stabilised by an external fixator. The serological levels of the C-terminal propeptide of procollagen type I (PICP), bone specific alkaline phosphatase (bALP), total alkaline phosphatase (tALP), osteocalcin, tartrate-resistant acid phosphatase (TRAP), calcium, phosphate and the N-terminal peptide of procollagen type III (PIIINP) were observed over a 9-week healing period. The course of fracture healing was monitored radiographically, and the callus composition was evaluated histologically at 2, 3, 6 and 9 weeks post-surgery. The serological results were compared with an untreated control group. Additionally, the maximum values during healing were compared with juvenile values to gauge the level of the serological response. The histological and radiographical results demonstrated callus formation without complications. All serological parameters showed broad inter-individual variations, and the response to the standardised fracture scenario was strongly individual. Maximum values during fracture healing did not reach the juvenile levels. The fractured as well as the control animals showed significant changes in the parameter levels. No correlations were observed between the histological course of healing and the course of bone formation markers whilst the TRAP level was reduced during bony callus formation. The PIIINP level increased when the amount of soft callus tissue decreased during healing. The observed bone formation markers were not suitable as general markers to detect the course of fracture healing, whilst PIIINP was able to reflect soft callus degradation.

The course of bone healing is influenced by the initial shear fixation stability.

Fracture healing is influenced by fixation stability and experimental evidence suggests that the initial mechanical conditions may determine the healing outcome. We hypothesised that mechanical conditions influence not only the healing outcome, but also the early phase of fracture healing. Additionally, it was hypothesised that decreased fixation stability characterised by an increased shear interfragmentary movement results in a delay in healing. Sixty-four sheep underwent a mid-shaft tibial osteotomy which was treated with either a rigid or a semi-rigid external fixator. Animals were sacrificed at 2, 3, 6 and 9 weeks postoperatively and the fracture callus was analysed using radiological, biomechanical and histological techniques. The tibia treated with semi-rigid fixation showed inferior callus stiffness and quality after 6 weeks. At 9 weeks, the calluses were no longer distinguishable in their mechanical competence. The calluses at 9 weeks produced under rigid fixation were smaller and consisted of a reduced fibrous tissue component. These results demonstrate that the callus formation over the course of healing differed both morphologically and in the rate of development. In this study, we provide evidence that the course of healing is influenced by the initial fixation stability. The semi-rigid fixator did not result in delayed healing, but a less optimal healing path was taken. An upper limit of stability required for successful healing remains unknown, however a limit by which healing is less optimal has been determined.

Angle stable locking reduces interfragmentary movements and promotes healing after unreamed nailing. Study of a displaced osteotomy model in sheep tibiae.

BACKGROUND: Large interfragmentary movements may delay bone-healing. The hypothesis of the present study was that a reduction of interfragmentary movements, especially of torsional rotation and bending angles, would support the healing process and lead to improved healing following unreamed tibial nailing. The objective of this study was to investigate healing of an unstable tibial osteotomy site following stabilization with unreamed nailing with a modified tibial device that had angle stable holes for the locking bolts. We compared those findings with healing after stabilization of such sites with standard unreamed tibial nailing. The duration of the study period was nine weeks. METHODS: The site of a standardized displaced osteotomy (3-mm gap) in twelve ovine tibiae was stabilized with unreamed tibial nailing: six animals were treated with a modified nail that had angle stable holes for the locking bolts, and six were treated with standard unreamed tibial nailing. In vivo gait analysis with optical measurements of interfragmentary movements and simultaneous measurements of ground reaction parameters were performed three days after the operation and once weekly afterward. After the animals were killed at nine weeks, the treated and contralateral tibiae were explanted, the implants were removed, and radiographs were made and evaluated for bridged cortices. Each pair of tibiae was also mechanically tested until torsional failure, after which the whole callus region was subjected to histological and histomorphometric analysis. RESULTS: Throughout the examination period, the interfragmentary movements in all directions were significantly smaller in the group treated with the angle stable tibial nail than they were in the group treated with standard unreamed tibial nailing. The limbs treated with the angle stable tibial nails returned to almost full weight-bearing during the period of the investigation, whereas those treated with standard nailing did not. Histomorphometric analysis, radiographic data, and mechanical testing showed superior bone-healing following treatment with the angle stable tibial nail. CONCLUSIONS: Use of an angle stable tibial nail may help to reduce interfragmentary movements in vivo and thus lead to superior bone-healing compared with that following standard unreamed tibial nailing.

Poly(D,L-lactide) coating is capable of enhancing osseous integration of Schanz screws in the absence of infection.

Pin loosening is a major complication in external fixation. Biological and mechanical conditions play an important role in the maintenance and enhancement of the implant-bone interface in fracture fixation. It is thought that biodegradable coatings may be capable of preventing pin track infection and pin loosening. The goal of this study was therefore to analyze the influence of a biodegradeable coating on the osseous integration of Schanz' screws during fracture treatment. Standardized osteotomies (3-mm fracture gap) of the right tibiae were performed on 16 sheep and stabilized by an AO mono-lateral external fixator. Additional, mechanically less loaded Schanz' screws were also mounted. All screws were randomly coated with biodegradable poly(D,L-lactide). The sheep were sacrificed after 9 weeks. All screws were removed and rolled on blood agar plates for microbiological analysis. Histological sections of the pin tracks were histochemically and morphometrically analyzed. Clinically, no signs of severe infection were visible. Microbiological analysis revealed 14.8% colonization by Staphylococcus aureus in the coated and 29% in the uncoated screws. Histomorphometry of the bone surrounding the Schanz' screws revealed that significantly more osseous integration had occurred on poly(D,L-lactide)-coated screws in the absence of bacterial colonization. Significantly more bone remodeling and a higher osteoclastic activity was seen near the screw-bone interface in the uncoated screw group. Up to a threefold increase in new bone formation and more severe remodeling was observed around the screw entry compared to the pin exit in all groups. Loaded screws showed significantly more callus formation around the exit sites than their less loaded counterparts. In the present study, poly(D,L-lactide) coating of Schanz' screws was found to enhance osseous integration in the absence of bacterial colonization in sheep by causing less cortical remodeling and less osteoclastic activity in the cortices compared to uncoated screws. Additionally, the coating appeared to reduce the instances of pin track infections. Mechanical loading showed an adverse effect on bone formation and remodeling. It has been shown that both biological and mechanical factors play an important role in the maintenance of osseous integrity of the pin-bone interface. Poly(D,L-lactide) coating of Schanz' screws does not prevent osseous destruction and severe bacterial colonization along the pin tracts, but can improve osseous integration of Schanz' screws in the absence of infection.

Gait evaluation: a tool to monitor bone healing?

BACKGROUND: Current clinical methods for monitoring fracture healing are often invasive and inaccurate. This paper evaluates the use of a pressure sensitive platform to improve monitoring. METHODS: Standardised 3 mm diaphyseal bone defects were created in the right tibia of 64 female sheep and stabilised with either a rigid monolateral external fixator or a more flexible variant. Over a nine week healing period gait parameters were measured using a pressure sensitive platform and interfragmentary movements at the fracture site were monitored. Frequency spectra were calculated for the ground reaction forces. The tibiae were tested biomechanically after sacrifice and callus sections were analysed histomorphometrically. FINDINGS: All animals unloaded the operated and overloaded the contralateral hindlimb. Callus mineralisation and stiffness, as well as limb loading increased during healing whilst interfragmentary movements were reduced. Larger interfragmentary movements resulted in a slower fracture healing rate as documented histologically and biomechanically. Frequency analysis showed upto 14 dB loss of power at frequencies associated with bone mechanotransduction at four weeks postoperatively, reducing to a 3 dB loss at nine weeks. INTERPRETATION: Gait analysis is a valuable tool for monitoring the course of fracture healing. Different fixation stiffnesses caused different initial interfragmentary movements leading to different healing rates. Ground reaction forces were strongly related to the course of callus mineralisation and thus directly reflected the recovery of stiffness at the fracture site. Reduced levels of loading frequencies that may affect bone healing persist to nine weeks postoperatively.

BMP delivery complements the guiding effect of scaffold architecture without altering bone microstructure in critical-sized long bone defects: A multiscale analysis.

Scaffold architecture guides bone formation. However, in critical-sized long bone defects additional BMP-mediated osteogenic stimulation is needed to form clinically relevant volumes of new bone. The hierarchical structure of bone determines its mechanical properties. Yet, the micro- and nanostructure of BMP-mediated fast-forming bone has not been compared with slower regenerating bone without BMP. We investigated the combined effects of scaffold architecture (physical cue) and BMP stimulation (biological cue) on bone regeneration. It was hypothesized that a structured scaffold directs tissue organization through structural guidance and load transfer, while BMP stimulation accelerates bone formation without altering the microstructure at different length scales. BMP-loaded medical grade polycaprolactone-tricalcium phosphate scaffolds were implanted in 30mm tibial defects in sheep. BMP-mediated bone formation after 3 and 12 months was compared with slower bone formation with a scaffold alone after 12 months. A multiscale analysis based on microcomputed tomography, histology, polarized light microscopy, backscattered electron microscopy, small angle X-ray scattering and nanoindentation was used to characterize bone volume, collagen fiber orientation, mineral particle thickness and orientation, and local mechanical properties. Despite different observed kinetics in bone formation, similar structural properties on a microscopic and sub-micron level seem to emerge in both BMP-treated and scaffold only groups. The guiding effect of the scaffold architecture is illustrated through structural differences in bone across different regions. In the vicinity of the scaffold increased tissue organization is observed at 3 months. Loading along the long bone axis transferred through the scaffold defines bone micro- and nanostructure after 12 months.

Sex hormones and antiandrogens influence in vitro growth of dermal papilla cells and outer root sheath keratinocytes of human hair follicles.

Anagen hair bulb papillae, interfollicular dermal fibroblasts, and interfollicular keratinocytes isolated from fronto-parietal scalp biopsies as well as outer root sheath keratinocytes from plucked anagen hairs were separately grown in subculture for 14 d. The effect of different concentrations (2.4 nM-17.3 microM) of testosterone, dihydrotestosterone, and the antiandrogens cyproterone acetate or 17 alpha-propylmesterolone on growth behavior of the mesenchymal and epithelial cell types of the hair follicle were comparatively studied by means of growth curves, cell doubling times, and 3H-thymidine incorporation. For control, all cell lines were subcultured in hormone-free medium. Testosterone and dihydrotestosterone (345 nM) significantly reduced proliferation of papilla cells compared with dermal fibroblasts (p < 0.01) and outer root sheath keratinocytes compared with interfollicular keratinocytes (p < 0.01), as well as compared with cells cultured in control medium. Low concentrations of 17 beta-estradiol were ineffective, whereas doses of 180 nM 17 beta-estradiol increased the growth velocities of all cell types, especially of papilla cells, compared with dermal fibroblasts. Low doses of either cyproterone acetate (24 nM) or 17 alpha-propylmesterolone (29 nM) induced a growth enhancement, especially of papilla cells and outer root sheath keratinocytes, whereas high doses of cyproterone (1.20 microM) and 17 alpha-propylmesterolone (1.45 microM) had opposite effects. These changes were significant between papilla cells and dermal fibroblasts as well as between outer root sheath keratinocytes and interfollicular keratinocytes. Applying increasing doses of androgens to cyproterone acetate (24 nM)- or 17 alpha-propylmesterolone (29 nM)-containing media neutralized the growth-stimulating effect of antiandrogens, particularly in papilla cells and outer root sheath keratinocytes. However, minor differences between testosterone and dihydrotestosterone effects on cell growth were found. The data clearly demonstrate that the changes of in vitro growth of hair follicle cells depend on the concentrations of androgens and antiandrogens, as higher doses of both antiandrogens tested retarded the cell proliferation similar to testosterone or dihydrotestosterone. The papilla cells and outer root sheath keratinocytes reacted more sensitively to the hormones tested, thereby confirming the concept of a distinct androgen sensitivity of these specialized hair follicle cells.

Segmental cell kinetics of the human anagen hair: a DNA-flow cytometric analysis.

The cell kinetics of anagen scalp hair taken by punch biopsies from 70 healthy male volunteers were determined at nine different defined bulbar and follicular hair segments using microdissection and DNA-flow cytometry. The highest mean proliferative activity (S-phase) was measured within the lowermost bulbar segment (14.0%), but decreased to 7.6% at Auber's segment and to 5.9% at the follicle isthmus. Notably, the S-phase data of the upper follicular segments (subdermal 2.4%, infundibular 2.4%) were found to be similar to those of the epidermis (2.5%). This study supporting and supplementing former autoradiographic investigations on human hair matrix epithelium clearly demonstrates the main proliferative activity of the anagen hair follicle being localized in the bulbar segments below Auber's level. Moreover, the method described is well suited for studying the effects of agents influencing cell growth (e.g., hormones or drugs) on the cell kinetics of different anagen hair compartments.

Cyclic nucleotides in human epidermis--diurnal variations.

In 12 healthy, adult males, the epidermal content of both cAMP and cGMP was determined radioimmunologically every 6 h over a period of 30 h, avoiding any ischemia, which can alter unphysiologically the in vivo levels of epidermal cyclic nucleotides. For cAMP a diurnal fluctuation with maximal level at midnight could be proved (p less than 0.05), whereas cGMP, in contrast to experiments in mice, revealed no significant variation during the test period. The presented results describe for the first time the diurnal course of human epidermal cGMP.

Growth and cell kinetics of human hair papilla cells in vitro. An autoradiographic and flow cytometric study.

Hair papilla, a distinct specialized dermal compartment, plays a fundamental role in the biology of hair growth. Recently some attention has been focused on hair papilla cells (HPC) as possible targets for drugs influencing the hair growth. Isolation and cultivation of the HPC facilitates screening for such drugs. In the present work, growth and cell kinetics of human occipital scalp follicle HPC have been studied in vitro. HPC grow according to a Gompertz function, i.e. with considerable growth delay long before becoming confluent cultures, due probably to elongation of the potential doubling time (Tpot) and to a parallel increasing cell loss rate. The [3H]dT labelling index of the HPC strongly depends on the age of the subculture; the cycle time being about 4 days. A potential doubling time of about 93 h, indicative of growth fraction (GF) = 1, and a duration of S phase and G2 + M phase of about 8 h each were found by the combined application of continuous labelling with [3H]dT and DNA flow cytometry.

Palliative radiotherapy for recurrent and metastatic malignant melanoma: prognostic factors for tumor response and long-term outcome: a 20-year experience.

PURPOSE: Radiotherapy is used as a "last resort" for patients with advanced cutaneous malignant melanoma. We have analyzed our 20-year clinical experience with respect to different endpoints and prognostic factors in patients with locally advanced, recurrent, or metastatic malignant melanoma. METHODS: From 1977 to 1995, 2,917 consecutive patients were entered in the melanoma registry of our hospital. Radiotherapy was indicated in 121 patients (56 females, 65 males) for palliative reasons in advanced malignant melanoma stages UICC IIB/III/IV. The histology of the primary lesion was nodular in 51 patients, superficial spreading in 35, acral-lentiginous in 8, and lentigo maligna melanoma in 4 patients. Eleven patients had primary or recurrent lesions which were either not eligible for surgery or had residual disease (R2) after resection of a primary or recurrent lesion (UICC IIB); 57 patients had lymph node (n = 33) or in-transit metastases (n = 24) (UICC III), and 53 had distant organ metastases (7 M1a; 46 M1b) (UICC IV). Time from first diagnosis to on-study radiotherapy averaged 19 (median: 18; range: 3-186) months. In most cases, conventional RT was applied with 2-6 Gy single fractions up to a median total radiation dose of 48 (mean: 45; range: 20-66) Gy. RESULTS: At 3 months follow-up, complete response (CR) was achieved in 7 (64%) and overall response [complete (CR) and partial response (PR)] in all (100%) UICC IIB patients, in 25 (44%) and 44 (77%) of 57 UICC III patients, and in 9 (17%) and 26 (49%) of 53 UICC IV patients. Tumor progression during radiotherapy occurred in 25 (21%) patients. Patients with CR survived longer (median: 40 months) than those without CR (median 10 months) (p < 0.01). At last follow-up (Dec 31, 1996), 26 patients were still alive: 6 (55%) UICC IIB, 17 (30%) UICC III, and 3 (6%) UICC IV patients (p < 0.01). Univariate analysis revealed the following prognostic factors for complete response and long-term survival: UICC stage (p < 0.001), primary location in the head and neck region, total radiation dose above 40 Gy (all p < 0.05), while age, gender, and histology had no impact. In multivariate analysis, UICC stage was the only independent prognostic factor (p < 0.001). CONCLUSION: External beam radiotherapy can provide long-term local control and effective palliation in malignant melanoma UICC stages IIB-IV. The current UICC staging system is an excellent prognostic factor for initial and long-term tumor response in metastatic melanoma. Therefore, prospective randomized trials using external radiotherapy with or without adjuvant therapy for advanced malignant melanoma are justified.

Chromosome gain and loss in paraffin sections from malignant melanomas of the skin.

Alphoid DNA probes specific for the chromosomes #6, #7, #9, and #17 were used to screen interphase nuclei for numerical chromosome aberrations in histologic thin sections obtained from archival paraffin material of 25 human melanomas of different type, thickness and stage of progression. An alphoid probe for chromosome #3 was applied in four of these tumors. Besides a general large variation of the number of subpopulations of cells characterized by gains and losses of the studied chromosomes, there was a trend to higher variability in metastatic melanomas as compared to small (<1.5 mm thickness) non-metastatic ones and, particularly, to normal skin tissue. Chromosomes #6 and #9 were those often affected by loss in thick melanomas (>2 mm), while subpopulations showing a gain of these chromosomes, and, in addition, of chromosome #7 seemed more frequently to be associated with thin and non-metastatic ones. The frequency of cases showing gain of chromosome #17 clearly exceeded those with loss of this chromosome in the studied melanomas, but was most pronounced in thicker metastatic tumors.

Interphase-FISH examinations in paraffin sections from benign, precancerous, and cancerous lesions of the skin and oral mucosa.

In a preliminary pilot study centromeric probes for chromosomes #7, #8, #11, and #17 and two-colour-FISH were applied on interphase nuclei of 10 coded histologic thin sections obtained from archival paraffin material from precancerous lesions and malignant tumors of the mouth epithelium. Brilliant signals could be obtained in this material without any computerized processing. Among the ten coded probes, localized malignant areas within grade 2 leukoplakias could be detected by their increased number of aneusomic cells, as could the samples from carcinomas. In extension of this study archival paraffin material from 30 epithelial tumors of the skin were examined. The studied squamous cell and Bowen carcinomas were characterized by a large number of chromosomally aberrant subclones and gains of chromosomes were the prevailing finding. In contrast, keratoacanthomas showed distinctly less clonal variation, their majority exhibiting small, but significant clones with chromosome loss, particularly of chromosome #7, less distinctly of chromosome #17.