Correlation of positive RT-PCR for tyrosinase in peripheral blood of malignant melanoma patients with clinical stage, survival and other risk factors

The clinical value of the reverse transcription polymerase chain reaction (RT-PCR) assay for tyrosinase in peripheral blood of melanoma patients is still under debate. A total of 212 blood samples from 212 melanoma patients in all clinical stages (AJCC) were examined. Erythrocytes were lysed prior to RNA extraction by phenol precipitation from 2.7 ml of blood. cDNA for tyrosinase PCR was synthesized using random hexamers. Positive tyrosinase RT-PCR results were obtained in 11% of 106 stage I patients, 18% of 56 stage II patients, 31% of 26 stage III patients and 67% of 24 stage IV patients. After a median follow-up of 36 months (range 26–41), stage III patients with positive RT-PCR for tyrosinase had a shortened disease-free interval as compared to negative patients (P< 0.01). In stage IV patients, median overall survival was 8 months in case of a positive RT-PCR in contrast to 12 months in case of a negative test. While univariate analysis showed sex and primary tumour location associated with positive RT-PCR, multiple regression analysis revealed clinical stage and detection of tyrosinase transcripts in peripheral blood as best prognostic factors. Hazard ratios for disease-free survival were 19.7 (confidence interval (CI) 8.53–45.5, P = 0.0001) for metastatic vs primary disease and 2.96 (Cl 1.49–5.89, P = 0.002) for positive vs negative tyrosinase RT-PCR. The corresponding hazard ratios for overall survival were 97.0 (Cl 12.7–741, P = 0.0001) and 4.33 (Cl 1.69–11.1, P = 0.002). Our results emphasize the importance of tyrosinase RT-PCR testing in peripheral blood. © 2000 Cancer Research Campaig

Heat conduction from the exceedingly hot fiber tip contributes to the endovenous laser ablation of varicose veins

Lower-extremity venous insufficiency is a common condition, associated with considerable health care costs. Endovenous laser ablation is increasingly used as therapy, but its mechanism of action is insufficiently understood. Here, direct absorption of the laser light, collapsing steam bubbles and direct fiber-wall contact have all been mentioned as contributing mechanisms. Because fiber tips have reported temperatures of 800-1,300°C during endovenous laser ablation, we sought to assess whether heat conduction from the hot tip could cause irreversible thermal injury to the venous wall. We approximated the hot fiber tip as a sphere with diameter equal to the fiber diameter, having a steady state temperature of 800°C or 1,000°C. We computed venous wall temperatures due to heat conduction from this hot sphere, varying the pullback velocity of the fiber and the diameter of the vein. Venous wall temperatures corresponding to irreversible injury resulted for a 3 mm diameter vein and pullback velocities <3 mm/s but not for 5 mm and 1 mm/s. The highest wall temperature corresponded to the position on the wall closest to the fiber tip, hence it moves longitudinally in parallel with the moving fiber tip. We concluded that heat conduction from the hot fiber tip is a contributing mechanism in endovenous laser ablation

The heat-pipe resembling action of boiling bubbles in endovenous laser ablation

Endovenous laser ablation (EVLA) produces boiling bubbles emerging from pores within the hot fiber tip and traveling over a distal length of about 20 mm before condensing. This evaporation-condensation mechanism makes the vein act like a heat pipe, where very efficient heat transport maintains a constant temperature, the saturation temperature of 100°C, over the volume where these non-condensing bubbles exist. During EVLA the above-mentioned observations indicate that a venous cylindrical volume with a length of about 20 mm is kept at 100°C. Pullback velocities of a few mm/s then cause at least the upper part of the treated vein wall to remain close to 100°C for a time sufficient to cause irreversible injury. In conclusion, we propose that the mechanism of action of boiling bubbles during EVLA is an efficient heat-pipe resembling way of heating of the vein wall

Carbonized blood deposited on fibres during 810, 940 and 1,470 nm endovenous laser ablation: thickness and absorption by optical coherence tomography

Endovenous laser ablation (EVLA) is commonly used to treat saphenous varicosities. Very high temperatures at the laser fibre tip have been reported during EVLA. We hypothesized that the laser irradiation deposits a layer of strongly absorbing carbonized blood of very high temperature on the fibre tip. We sought to prove the existence of these layers and study their properties by optical transmission, optical coherence tomography (OCT) and microscopy. We analysed 23 EVLA fibres, 8 used at 810 nm, 7 at 940 nm and 8 at 1,470 nm. We measured the transmission of these fibres in two wavelength bands (450–950 nm; 950–1,650 nm). We used 1,310 nm OCT to assess the thickness of the layers and the attenuation as a function of depth to determine the absorption coefficient. Microscopy was used to view the tip surface. All fibres showed a slightly increasing transmission with wavelength in the 450–950 nm band, and a virtually wavelength-independent transmission in the 950–1,650 nm band. OCT scans showed a thin layer deposited on all 13 fibres investigated, 6 used at 810 nm, 4 at 940 nm and 3 at 1,470 nm, some with inhomogeneities over the tip area. The average absorption coefficient of the 13 layers was 72 ± 16 mm−1. The average layer thickness estimated from the transmission and absorption measurements was 8.0 ± 2.7 µm. From the OCT data, the average maximal thickness was 26 ± 6 µm. Microscopy of three fibre tips, one for each EVLA wavelength, showed rough, cracked and sometimes seriously damaged tip surfaces. There was no clear correlation between the properties of the layers and the EVLA parameters such as wavelength, except for a positive correlation between layer thickness and total delivered energy. In conclusion, we found strong evidence that all EVLA procedures in blood filled veins deposit a heavily absorbing hot layer of carbonized blood on the fibre tip, with concomitant tip damage. This major EVLA mechanism is unlikely to have much wavelength dependence at similar delivered energies per centimetre of vein. Optical–thermal interaction between the vein wall and the transmitted laser light depends on wavelength

Tyrosinase expression in the peripheral blood of stage III melanoma patients is associated with a poor prognosis: a clinical follow-up study of 110 patients

The aim of this study is to define the relationship between the tyrosinase expression in the peripheral blood and the clinical course of the disease in stage III disease-free melanoma patients after radical lymph node dissection. RT-PCR techniques were used to identify tyrosinase mRNA in 110 patients; a total of 542 blood samples were investigated. In all, 54 patients (49%) showed at least one positive result; 13 patients (11.8%) showed baseline positive results: six became negative thereafter, whereas seven showed follow-up positive results until disease progression occurred. One or more positive determinations were found during follow-up in 41 patients with negative baseline tyrosinase. No correlation was found between baseline results and the relapse rate or disease-free survival (DFS), whereas a significant correlation was found between positive tyrosinase results and disease recurrence during follow-up. In fact, 72.9% of positive patients relapsed, but only 19.3% of negative cases did so. The median interval between the positive results and the clinical demonstration of the relapse was 1.9 months (range 1-6.6). Disease-free survival multivariate analysis selected, as independent variables, Breslow thickness (P=0.05), lymph node involvement according to the AJCC classification (P=0.05) and tyrosinase expression (P=0.0001). In conclusion, RT-PCR tyrosinase mRNA expression is a reliable and reproducible marker associated with a high risk of melanoma progression and we encourage its clinical use in routine follow-up

Combination chemotherapy for choroidal melanoma: ex vivo sensitivity to treosulfan with gemcitabine or Cytosine arabinoside

Treatment of choroidal melanoma by chemotherapy is usually unsuccessful, with response rates of less than 1% reported for dacarbazine (DTIC)-containing regimens which show 20% or more response rates in skin melanoma. Recently, we reported the activity of several cytotoxic agents against primary choroidal melanoma in an ATP-based tumour chemosensitivity assay (ATP-TCA). In this study, we have used the same method to examine the sensitivity of choroidal melanoma to combinations suggested by our earlier study. Tumour material from 36 enucleated eyes was tested against a battery of single agents and combinations which showed some activity in the previous study. The combination of treosulfan with gemcitabine or cytosine arabinoside showed consistent activity in 70% and 86% of cases, respectively. Paclitaxel was also active, particularly in combination with treosulfan (47%) or mitoxantrone (33%). Addition of paclitaxel to the combination of treosulfan + cytosine analogue added little increased sensitivity. For treosulfan + cytosine arabinoside, further sequence and timing experiments showed that simultaneous administration gave the greatest suppression, with minor loss of inhibition if the cytosine analogue was given 24 h after the treosulfan. Administration of cytosine analogue 24 h before treosulfan produced considerably less inhibition at any concentration. While we have so far been unable to study metastatic tumour from choroidal melanoma patients, the combination of treosulfan with gemcitabine or cytosine arabinoside shows activity ex vivo against primary tumour tissue. Clinical trials are in progress. © 1999 Cancer Research Campaig

Quantitative methylation analyses of resection margins predict local recurrences and disease-specific deaths in patients with head and neck squamous cell carcinomas

This study sought to determine whether the presence of hypermethylated genes in the surgical margins can predict local recurrences in head and neck squamous cell carcinomas (HNSCCs). We prospectively collected tumour and surgical margin specimens from patients with HNSCCs who had undergone surgical resections. Quantitative methylation-specific PCR (QMSP) of CDKN2A, CCNA1 and DCC were performed in these specimens and correlated with clinical data. Of the 42 patients eligible for the study, 27 were hypermethylation informative for the above three genes. This latter group was associated with longer disease-free survivals (P=0.007) and longer time to disease-specific deaths (P=0.004). Multivariate analyses confirmed hypermethylation non-informative tumours as an independent prognosticating factor for disease-specific deaths (risk ratio 3.8, P=0.026). Quantitative MSP of the margins of 24 hypermethylation informative tumours revealed that 11 patients had molecularly positive margins, of which, five developed disease-specific events (DSEs, three local recurrences and two metastases), compared to none in patients with molecularly negative margins, after a median follow-up of 48 months. Log-rank analyses showed that molecularly positive margins were associated with shorter time to local recurrences and disease-specific deaths (P=0.03 and 0.01, respectively). This study demonstrated that QMSP of hypermethylated promoters in surgical margins predicted all the local recurrences in our series of HNSCC patients. We have also identified hypermethylation non-informative tumours as an independent predictor for the development of DSEs