Interest of PS100 assay when 99mTc sestamibi scintigraphy failed to identify lymph node metastases of melanoma

ARTICLE

In the last ten years, the incidence of malignant melanoma has increased dramatically. In France, the incidence currently stands at about eight new cases per 100,000 inhabitants and per year. In Western countries, the incidence of melanoma is believed to double every ten years [1]. The main risk factor currently known is exposure to the sun. The most frequent secondary extension concerns lymph node area. During a previous study [2], we showed the value of combining a biological assay (protein S-100) with functional imaging (scintigraphy with sestamibi) in order to detect the lymph node metastases of malignant melanomas. The study was conducted in a population of 19 patients, with the hypothesis that protein S-100 could be a biological marker of metastatic malignant melanoma [3-8]. We extended that study (37 patients enrolled) and also conducted testing for gene MDR1 (by RT-PCR).

The aim of our study was to assess if a simple scintigraphic method in association with a PS100 assay could be used to differentiate between lymph node metastases (MLNM) of melanoma with and without PgP expression.

Material and methods

Patients

The study protocol was approved by the local ethics committee, and all patients gave informed consent. Thirty seven patients with a previously resected cutaneous melanoma were investigated. Patients were recruited by a dermatologist on the grounds of clinically questionable lymph nodes (CQLNs) in the areas of the malignant melanoma previously treated by surgery (6 months to 4 years previously). CQLNs were in the axilla n = 25, the groin n = 10, and the neck n = 2 cervical. For all patients, biological assay of PS100 and MIBI scintigraphy were performed. Furthermore, all CQLNs were histologically analysed to assess presence or absence of metastasis (M⁺ or M^-) on the one hand and to assess presence or absence of permeability glycoprotein (PgP) on the other hand.

Protein S-100

Protein S-100 has a molecular weight of 21 kDa. It binds calcium and is thermolabile. The protein consists of two subunits, alpha and beta, which may combine as alphabeta, betabeta or alphaalpha [9]. The protein has been isolated from the central nervous system in form beta. The protein is present in various types of cells in the central nervous system, in Langerhans' epidermal cells, melanocytes, adipocytes and chondrocytes [10]. The protein is rarely found in normal nevi. Most nodular melanomas contain subunits alpha and beta. In superficial melanomas, S-100beta have been found once vertical propagation has begun [11]. On the basis of the literature, the concentration in normal subjects is less than 0.1 µg.L^{- 1} [8]. Blood samples were collected in dry tubes. Serum was obtained by centrifuging the samples at 3,000 g for 5 min. The sera were stored at - 80° C. The assays were conducted in duplicate on a LIA-MAT (Nichols Institute Diagnostics). Protein S-100 assay was based on an immunoluminometric sandwich technique (LIA-mat* Sangtec* 100), in coated tubes, using chemoluminescence as detection system. The assay method employs three monoclonal antibodies (SMST 12, SMST 25 and SMST 28), specific to subunit beta of protein S-100. The reaction is two-phase: the protein S-100 contained in the samples reacts with the captured antibodies bound to the tube. The tube is washed. The tracer (isoluminol-labeled antibody) is added to the tube. Following a second washing, the presence of the sandwich is detected by chemoluminescence. The oxidation of isoluminol is induced by automatic injection of alkaline peroxide solution and a catalyst into the tube. The intensity of light emission at 425 nm is expressed in RLU and is directly proportional to the S-100 concentration contained in the standard and serum samples. A protein S-100 concentration greater than 0.10 µg.L^{- 1} is considered pathological.

^99mTc-Sestamibi scintigraphy

Technetium 99m Sestamibi (MIBI) is an isonitrile lipophilic cation used clinically as a myocardial perfusion imaging agent. MIBI has been found to be concentrated in different tumors including brain tumors, breast cancer, bone tumors, lung tumors and lymphoma [12-20]. Accumulation of MIBI in tumors cells may be related to increased mitochondrial activity of the tumor. Recent data show that 90% of the MIBI is found in mitochondrial tumor cells [21-23]. Few studies have shown the ability of the MIBI to localize in the lymph node metastases of malignant melanoma [24-27]. On the other hand, it is now well known that MIBI is a suitable transport substrate for PgP. More recently, a close relationship between the in vivo efflux rate of MIBI and PgP expression has been demonstrated [28-35]. It therefore does not reach the mitochondria and scintigraphic imaging is negative. In our study, planar imaging and single photon emission tomography (SPET) images were acquired 15 min after the injection of 1,110 MBq of ^99mTc-MIBI in order to explore the clinically-suspect lymph node areas. A normal image at 15-30 min showed symmetrical homogeneous radioactivity. Focal or multiple areas of increased uptake corresponding to palpable masses in the axillary, groin or neck areas were considered pathological. Quantitative analysis was done using planar images. Functional index ratios were taken in the areas of interest drawn from the MIBI uptake area and the contralateral site. When no MIBI uptake existed, the area of interest was the area corresponding with the clinically suspect site. Computer processing of the data enabled determination of the level of radioactivity in the suspect zone (C_max T) and the contralateral zone (C_max B). Dividing C_max T by C_max B yielded the uptake ratio (functional index).

Reverse transcriptase, polymerase chain reaction (RT-PCR)

RT-PCR was used to quantify gene MDR1 in surgically-removed lymph nodes. This radio-isotope method enabled a qualitative result (presence or absence of the gene) to be obtained. However, it also yielded a quantitative result in the form of the degree of overexpression of the gene. The various stages in gene MDR1 detection are shown graphically in Figure 1.

Results

Protein S-100

Thirty-seven assays were conducted. For 15 assays, the result was less than 0.10 µg.L^{- 1}. The mean was 0.048 µg*L^{- 1} (0.01-0.09 µg.L^{- 1}). In 22 cases, the result was greater than 0.10 µg*L^{- 1}. The mean was 0.533 µg.L^{- 1} (0.10-6.3 µg.L^{- 1}). There were 13 true negative, 2 false negative, 20 true positive and 2 false positive results. The sensitivity and the specificity of the assay were 91% and 86.5%, respectively.

^99mTc-MIBI scintigraphy

Seventeen MIBI scintigraphy did not show any pathological increase in uptake of MIBI, with a mean ratio of the suspect zone to the contralateral background of 1.16 (0.80-1.2). Twenty MIBI scintigraphy were pathological, with a mean ratio of the suspect zone to the contralateral background of 1.65 (1.22-2.59). The difference between the two uptake ratios was statistically significant (p = 0.003). Results were summarized in Table I. Three false negatives and 1 false positive were observed. The sensitivity and specificity of the scintigraphy were thus 95% and 85%, respectively.

A concordance between the MIBI scintigraphy and biological assay true negative and true positive results was observed 30 times (13 concordances for true negatives, 17 concordances for true positives). In 7 cases, there was a discordance between the MIBI scintigraphy and biological assay findings (summarized in Table II).

RT-PCR

Tumor biopsy specimens were obtained from each patient and were assayed for MDR1 content. Hyperexpression of gene MDR1 was observed 6 times. Quantitatively, gene MDR1 expression ranged from 1.14 to 6.13 (one chemoresistant cell line, K562-DNR, used as internal control, showed an expression of 4.45). The results of the RT-PCR biological assay and scintigraphies for MDR⁺ patients are resumed in Table III.

Discussion

Compared to our previous study [2], the sensitivity value that we observed remained remarkably constant for PS100 assay (91%), although the population increased from 19 to 37 patients. The sensitivity of MIBI increased from 70% to 85%. This may be explained by the application of a quantitative method of analysis of the scintigraphic results (uptake ratio) in addition to the purely visual analysis, enabling detection of patients presenting with chemoresistance with moderate or weak expression of gene MDR1. For those patients, the C_max Tumor/C_max Background ratio was consistently greater than 1.2, and therefore pathological (normal if less than 1.1). The qualitative analysis alone was insufficient and could have led to a negative result. In addition, we did not have any new patient presenting overexpression of gene MDR1, compared to the population in the first study. This also certainly contributed to improving the sensitivity level of the scintigraphic examination. In fact, only those patients presented with a false negative scintigraphic result, and thus influenced the sensitivity of the investigation. The specificities of the scintigraphic investigation and biological assay were the same, at 93%. The value remained stable for the scintigraphic investigation, but fell slightly for the biological assay, from 100 to 93%. However, all the patients overexpressing gene MDR1 had a pathological PS-100 level, while the scintigraphic result was negative (no high uptake of MIBI in the clinically-suspect zone). Out of 37 assays, we only observed one false positive result (0.14 µg.L^{- 1}). The value was a veritable false positive, since the value found was markedly greater than the limit. Moreover, the control conducted confirmed the first assay result. For that patient, the scintigraphic findings were unambiguously negative, as were the histological findings. We also had two false negatives. The serum PS-100 levels were 0.09 and 0.08 µg.L^{- 1}, respectively. In both cases, the scintigraphic investigation enabled evidencing of pathological uptake sites in the clinically-suspect lymph node areas. It should be noted that the two PS-100 levels, while less than 0.10 µg.L^{- 1}, were nonetheless on the borderline of the upper normal limit. The results were therefore not clearly negative. The concomitant analysis of the biological and scintigraphic results for the MDR⁺ patients (all metastatic), on the one hand, and the MDR- patients (metastatic or non-metastatic), on the other hand, enabled the generation of Figures 2 and 3. These figures show that when the suspect site: uptake noise ratio is greater than 1.2, the PS-100 level is pathological (except for two patients) and that the patients present with metastatic disease. In contrast, if the uptake ratio is less than 1.2, Figures 2 and 3 show that the PS-100 level may be normal or pathological and the patients may or may not have metastases. Figure 2 shows a group of patients with an uptake ratio less than 1.2 and PS-100 level less than 0.10 µg.L^{- 1}. These patients were non-metastatic. Figure 3 shows that, for the MDR⁺ patients, all the PS-100 assay results were pathological, while the scintigraphic uptake ratios were, for four patients, less than 1.2, for three patients, greater than 1.2 but less than 1.8 (i.e. a slight increase in uptake on the scintigraphic images).

CONCLUSION

1) If an abnormal MIBI uptake exists, a lymph node metastasis is quite certain and surgical treatment should be proposed.
2) If no MIBI uptake is noted, additional PS 100 assay additional could be suggested to discriminate 2 groups of patients:

- group 1: if PS 100 > 0.10 µg.L^{- 1} then a MDR phenomenon mediated by overexpression of gene MDR1 at lymph node metastasis level must be suspected;
- group 2 : if PS 100 ¾ 0.10 µg.L^{- 1} then metastatic disease is highly unlikely. *

Article accepted on 12/4/01

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