Reduced FANCD2 influences spontaneous SCE and RAD51 foci formation in uveal melanoma and Fanconi anaemia.

Uveal melanoma (UM) is unique among cancers in displaying reduced endogenous levels of sister chromatid exchange (SCE). Here we demonstrate that FANCD2 expression is reduced in UM and that ectopic expression of FANCD2 increased SCE. Similarly, FANCD2-deficient fibroblasts (PD20) derived from Fanconi anaemia patients displayed reduced spontaneous SCE formation relative to their FANCD2-complemented counterparts, suggesting that this observation is not specific to UM. In addition, spontaneous RAD51 foci were reduced in UM and PD20 cells compared with FANCD2-proficient cells. This is consistent with a model where spontaneous SCEs are the end product of endogenous recombination events and implicates FANCD2 in the promotion of recombination-mediated repair of endogenous DNA damage and in SCE formation during normal DNA replication. In both UM and PD20 cells, low SCE was reversed by inhibiting DNA-PKcs (DNA-dependent protein kinase, catalytic subunit). Finally, we demonstrate that both PD20 and UM are sensitive to acetaldehyde, supporting a role for FANCD2 in repair of lesions induced by such endogenous metabolites. Together, these data suggest FANCD2 may promote spontaneous SCE by influencing which double-strand break repair pathway predominates during normal S-phase progression.

Resistance of uveal melanoma to the interstrand cross-linking agent mitomycin C is associated with reduced expression of CYP450R.

BACKGROUND: Uveal melanoma (UM) is the most common primary intraocular tumour of adults, frequently metastasising to the liver. Hepatic metastases are difficult to treat and are mainly unresponsive to chemotherapy. To investigate why UM are so chemo-resistant we explored the effect of interstrand cross-linking agents mitomycin C (MMC) and cisplatin in comparison with hydroxyurea (HU). METHODS: Sensitivity to MMC, cisplatin and HU was tested in established UM cell lines using clonogenic assays. The response of UM to MMC was confirmed in MTT assays using short-term cultures of primary UM. The expression of cytochrome P450 reductase (CYP450R) was analysed by western blotting, and DNA cross-linking was assessed using COMET analysis supported by γ-H2AX foci formation. RESULTS: Both established cell lines and primary cultures of UM were resistant to the cross-linking agent MMC (in each case P<0.001 in Student's t-test compared with controls). In two established UM cell lines, DNA cross-link damage was not induced by MMC (in both cases P<0.05 in Students's t-test compared with damage induced in controls). In all, 6 out of 6 UMs tested displayed reduced expression of the metabolising enzyme CYP450R and transient expression of CYP450R increased MMC sensitivity of UM. CONCLUSION: We suggest that reduced expression of CYP450R is responsible for MMC resistance of UM, through a lack of bioactivation, which can be reversed by complementing UM cell lines with CYP450R.

Angiopoietin-1, angiopoietin-2 and Tie-2 receptor expression in human dermal wound repair and scarring.

BACKGROUND: The angiopoietin (Ang)/Tie-2 ligand/receptor system is known to interact with the vascular endothelial growth factor (VEGF) pathway to determine the fate of blood vessels during angiogenesis. However, the precise contribution of this system to angiogenesis and the mechanisms of vascular maturation and remodelling in human tissue repair have yet to be elucidated. OBJECTIVES: To examine the spatial and temporal expression of Ang-1, Ang-2, Tie-2 and VEGF in relation to angiogenesis in human surgical wounds. METHODS: Punch biopsies were taken either from normal unwounded skin (controls) during surgery or from mastectomy scars between 3 days and 2 years postsurgery. Ang-1, Ang-2, Tie-2 and VEGF fibroblast/myofibroblast and endothelial expression were characterized by immunohistochemistry, analysed semiquantitatively and correlated with microvessel density (MVD) and scar age. RESULTS: The expression of VEGF, Ang-1, Ang-2 and Tie-2 in fibroblasts/myofibroblasts was increased significantly in early scars, decreased in older scars and was related to scar age (P < 0·001) and MVD (P < 0·0004), with strong correlations between all factors. In contrast, vascular expression of Ang-1 was decreased slightly in early scars, vascular Ang-2 remained constant and Tie-2 vascular expression increased, although there were no correlations with scar age or MVD. CONCLUSIONS: These data demonstrate that angiopoietins and their receptor, Tie-2, are expressed in both fibroblasts/myofibroblasts and endothelial cells in healing human wounds. Fibroblast/myofibroblast expression correlates with angiogenesis and VEGF expression, suggesting a role for the angiopoietin/Tie-2 system in normal wound repair and scarring.

Predictive factors for skin reactions in patients treated with thermoradiotherapy.

In this study we performed univariate analyses to analyse the predictive factors for skin reactions, i.e. erythema, thermal blisters and ulceration, that occur during thermoradiotherapy. One hundred and twenty-six fields in 126 patients were treated with thermoradiotherapy using 915 MHz external microwave hyperthermia. Mean age of patients was 62 years. All but 11 lesions received previous therapy. Prior treatment included surgery (75%), chemotherapy (60%) and/or radiation therapy (51%). The mean previous radiation dose was 54 +/- 2 Gy. The concurrent tumour radiation dose was 45 +/- 1 Gy, in 16 fractions, over 35 elapsed days (dose per fraction of 1.6-4.8 Gy). The mean number of heat sessions administered was 5.5 +/- 0.2 (range 1-14). In 83% of cases hyperthermia was administered biweekly. Forty-two patients were treated without any skin reaction (33%), erythema occurred in 59 fields (47%), transient thermal blisters occurred in 25 fields (20%) and ulceration occurred in 23 fields (18%). In 25 cases, two or more skin reactions (20%) were observed concurrently. Concurrent radiation dose correlated with skin reactions (p = 0.02). The incidence of skin reactions was inversely correlated with previous radiation therapy (p = 0.04) and previous radiation therapy dose (p = 0.04) possibly due to fibrosis. None of the tumour or skin thermal parameters correlated with the reaction rate.

Use of manganin-constantan thermocouples in thermometry units designed for copper-constantan thermocouples.

Commercial ultrasound hyperthermia systems typically include thermometry units designed for copper-constantan thermocouples. Replacing these copper-constantan thermocouples with manganin-constantan thermocouples is advantageous in reducing the measurement error caused by the conduction of heat along the copper wire, but their performance in these thermometry units is uncertain. The accuracy of manganin-constantan thermocouples in the Labthermics LT-100, Clini-Therm TS1200/TM100, and Physitemp TM-12 thermometry units was investigated using a temperature controlled circulating water bath monitored by a mercury thermometer having a calibration traceable to NIST. The results demonstrate that an accuracy of +/- 0.2 degrees C can be achieved with manganin-constantan thermocouples over the range 35-55 degrees C without hardware modification provided specific calibration procedures are followed. With the Labthermics LT-100, a double point calibration should be carried out at 35 and 55 degrees C. With the Clini-Therm TS1200/TM100, a self-calibration of the unit using its internal calibration well plus a single point calibration using an external temperature standard provides sufficient accuracy. The Physitemp TM-12 requires an external computer for read out and the user must provide additional software to correct for the error by either a single or multiple point calibration.

A recommended revision in the RTOG thermometry guidelines for hyperthermia administered by ultrasound.

RTOG thermometry guidelines for clinical trials of hyperthermia using planar ultrasound recommended that temperatures be mapped in polyurethane catheters by use of single-junction copper-constantan thermocouples. These guidelines were based on an assumption that the error in temperature measurement due to thermal conduction would generally not exceed +/- 0.3 degrees C. The validity of this assumption was tested with a commercially available single-junction copper-constantan thermocouple. The width of the point spread function, an indicator of the relative magnitude of the conduction error, was five times greater than expected. As a result, the conduction error is projected to exceed 0.3 degrees C in a temperature gradient of only 1.5 degrees C/cm. This projection was confirmed by mapping a thermal peak which simulates a typical clinical temperature profile. This peak had an amplitude of 6 degrees C, a full-width at half-maximum of 3.5 cm, and a maximum gradient of approximately 3 degrees C/cm. Temperatures measured at 0.5-cm intervals over the span of this peak were in error by a mean of +/- 0.6 degrees C. It is strongly recommended that the RTOG guidelines be revised to replace copper-constantan thermocouples with manganin-constantan single- or multi-junction thermocouples which will assure that the conduction error will be < +/- 0.3 degrees C.

Multiple field hyperthermia combined with radiotherapy in advanced carcinoma of the breast.

Extensive recurrences on the chest wall of advanced carcinoma of the breast in 20 patients were treated with multiple field patchwork hyperthermia combined with radiation therapy between 1987-1991. The objective of the study was to evaluate the feasibility, tumour response and complications of treating extensive lesions with multiple, overlapping fields of hyperthermia. All lesions were diffuse encompassing up to 2900 cm2 in area with or without multiple nodules < or = 3 cm deep. All lesions had failed previous therapy with all but three failing previous radiotherapy. Hyperthermia consisted of 282 hyperthermia applicator fields and 357 hyperthermia treatments with external 915 MHz microwaves using commercially available applicators. Hyperthermia applicator fields were defined by the surface 50% SAR distribution of a particular applicator, and hyperthermia fields were abutted to cover the entire tumour bearing area. Radiation therapy consisted of 81 fields to a mean dose of 40 +/- 1 Gy (SE), 88% of fields received between 30 and 50 Gy. The equivalent dose was 42 +/- 1 Gy, based on the linear-quadratic model and alpha/beta = 25 (Fowler 1989). Overlapping hyperthermia fields were separated by an interval of at least three days. Up to four heat sessions per week were required to cover the entire tumour in a rotating fashion. The hyperthermia treatment time was 60 min. Hyperthermia treatments were continued for the duration of radiation therapy. Each hyperthermia applicator field was heated at least once. Patients were exposed to a mean of 14 +/- 3 hyperthermia applicator fields (range of 3-46 fields) and a mean of 18 +/- 3 hyperthermia treatments (range of 6-61) delivered over a mean of 7.5 +/- 0.9 weeks (range of 3-17 weeks). Each field was heated an average of 1.3 times. The tumour complete response rate was 95% with a recurrence rate of 5%. Nevertheless, the mean survival of patients with a complete response was only 10.8 +/- 1.7 months (range of 2-28 months) because of the systemic tumour burden existing outside of the treated fields in these patients. Neither complete response, local control nor survival after thermoradiotherapy correlated with the disease free interval between initial mastectomy and recurrence. There was no evidence of increased thermal damage to skin nor evidence of tumour recurrence at junctions of hyperthermia field overlap. It is concluded that recurrent advanced carcinoma of the breast presenting as extensive, diffuse lesions on the chest wall can be treated as effectively with multiple field patchwork thermoradiotherapy as can nodular lesions treated with single hyperthermia fields.

Thermoradiotherapy for superficial tumour deposits in the head and neck.

Tumour deposits in the head and neck region were treated with hyperthermia using 915 MHz external microwave applicators and radiation therapy between 1986 and 1990. The mean (+/- SE) radiation dose was 47 +/- 2 Gy (range 21-77 Gy). All but four patients had failed previous therapy. Mean tumour volume was 40 +/- 10 cm3 (range 0.3-276 cm3). Hyperthermia was administered biweekly in 80% of the patients in 6.0 +/- 0.4 sessions (range 1-10); thermometry involved 3.6 +/- 0.4 catheters (range 1-9) and 5.7 +/- 0.4 sensors (range 1-12) per tumour. Of the 50 lesions evaluable for response, 29 had a complete response (58%), and 20 had a partial response (40%). Lesions were stratified by depth. In tumours considered potentially heatable (i.e. depth < or = 3 cm and lateral dimensions at least 2 cm less than boundary of applicator), the complete response rate was 81% (26/32, 47 +/- 2 Gy, 15 +/- 3 cm3); whereas for patients with tumours deeper than 3 cm, the complete response rate was 17% (3/18, 48 +/- 3 Gy, 110 +/- 21 cm3), p = 0.0001. Among lesions < or = 3 cm depth that exhibited a complete response, six recurred (24%, 5.8 +/- 1.8 months) while 20 lesions were recurrence free at last follow-up of 11.9 +/- 1.2 months). The overall survival of patients with lesions < or = 3 cm depth was 11.5 +/- 1.3 months (range 2.4-32.3 months) while for patients with lesions > 3 cm depth survival was 6.7 +/- 0.9 months (range 2.1-18.6 months), p = 0.01. In superficial lesions with depth < or = 3 cm, multivariate logistic regression analysis indicated that the model best correlating with complete response included radiation dose (p = 0.08) and tumour volume (p = 0.08, model p = 0.004). Multivariate proportional hazard analysis indicated that the model best correlating with duration of local control included tumour depth (p = 0.03) and previous radiation therapy (p = 0.08, model p = 0.006). Twenty-two fields were treated without any skin reactions (39%), 23 evidenced erythema (40%) and eight thermal blistering (14%). Ulceration occurred in 11 treatment fields but in all but one of these cases the ulceration may have been due to tumour breakdown as there was direct invasion of the skin by tumour prior to the initiation of treatment. The maximal skin temperature was the best predictor of morbidity although the correlation was not statistically significant (p = 0.19).

Thermoradiotherapy with combined interstitial and external hyperthermia in advanced tumours in the head and neck with depth > or = 3 cm.

Advanced tumours in the head and neck 3-6 cm depth are too deep to be completely heated by external 915 MHz microwaves. A preliminary study was performed using interstitial plus external hyperthermia combined with external beam radiation therapy to heat tumours to depths > or = 3 cm. Nine advanced metastatic lesions of squamous cell carcinoma located in the head and neck were treated between 1987 and 1990 with the combined hyperthermia technique and radiation doses of 38-60 Gy (mean of 49 +/- 3 Gy). The mean tumour volume was 58 +/- 9 (SE) cm3 (range 24-94 cm3) with a mean tumour depth of 3.9 +/- 0.3 cm (range 3-5.5 cm). The deeper aspects of the tumour were heated by interstitial 915 MHz microwave antennas and the superficial aspects heated by external 915 MHz applicators. A single plane of polyurethane closed-end catheters, 16 Ga, were inserted under local anaesthesia approximately 1.5-2 cm apart in parallel arrays at the base of a lesion behind the sternomastoid muscle, or an equivalent site in a dissected neck, extending forward and angled deeply no more than 15 degrees. Hyperthermia was administered twice weekly immediately after radiation therapy in a mean of 5.3 +/- 0.7 external heat sessions (range 3-7) and a mean of 3.5 +/- 0.6 interstitial heat sessions (range of 1-6). Interstitial hyperthermia was usually administered in alternating sessions with external hyperthermia, but in some patients all of the sessions of one modality were administered followed by all of the sessions of the other modality. In no case were both interstitial and external heatings performed on the same day. Surface thermometers were used to monitor skin temperature during external hyperthermia sessions. Results showed that by 8 weeks after completion of treatment, six lesions exhibited a complete response (67%) and three a partial response (33%). One of the partial responses continued to regress and became a complete response (78% complete response). The recurrence rate in complete responders was 14% (1/7) with time to recurrence of 7.7 months. Six lesions were recurrence-free at last follow-up of 21.3 +/- 8.8 months. Skin reactions were absent in four fields (44%), erythema was noted in five (56%) and thermal blistering in one (11%). Ulceration occurred only in association with tumour breakdown when the skin was infiltrated by tumour (three patients, 33%).(ABSTRACT TRUNCATED AT 400 WORDS)

[Chronic pancreatitis and the neighboring organs]. / Chronische Pankreatitis und Nachbarorgane.

The close topographic relationship between pancreas and the functional entity of the upper abdominal organs causes in about 20 percent of the patients with chronic pancreatitis alterations of the surroundings. In 531 patients compression of the common bile duct was found in 18.1%, duodenal stenosis in 17.7%, pleural thickening in 6.6%, splenic vein thrombosis in 5.1%, gastric outlet obstruction in 3.2% and colonic stenosis in 0.4%. In primary asymptomatic chronic pancreatitis these local complications may be the first hint to pancreatic disorders. Occasionally these changes demand operative interventions or alleviate the decision for resectional therapy.