MARKERS OF MELANOGENESIS IN MALIGNANT MELANOMA

B. Matous, E, Bubnová, A. Budesínská, M. Kostirová

2nd Department of Medical Chemistry and Biochemistry,

1st Medical Faculty, CharIes University

Markery melanogeneze u maligního melanomu. Matous B., Bubnova E., Budesinská

A., Kositrova M. Sborn. Iék., Vol. 95 (1994) No. 4, p. 333-338.

Mailing address: Doc. MUDr. B. Matous, 2nd Department of Medical Chemistry and Biochemistry, 1st Medical Faculty, Charles University, U nemocnice 5, 128 53 Praha 2 (Czech Republic).

Univerzita Karlova, vydavatelstvi Karolinum, Praha 1994

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SUMMARY: Biochemical specificity of malignant melanoma is represented in part. by the formation of specific cytoplasmatlc particles of the pigment cell - melanosomes - in which the synthesis of pigment eumelanin and pheomelanin takes place and in part by the presence of a specific enzyme - tyrosinase - which catalyzes the formation of pigment eumelanin and pheomelanin and even the formation of specific metabolites (so called melanogens) which are excreted in increased amounts in the course of the disease. Tyrosinase and melanogens are specific for pigment cell and therefore can be used for monitoring of meIanogenesis in malignant meIanoma. When comparing our results and the resuIts of other authors we can conclude that following of specific markers of melanogenesis in malignant melanoma should serve for the evaluation of prognosis of the disease. The study of melanoma markers is by far not finished. We do hope that nearly future will be able to give: a sufficient answer to the question, whether melanogenuria is actually an expression of expected different biochemical or metabolic types of malignant melanoma on the one hand and/or biochemically or immunologically conditioned responses of the host organism on the other.

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Malignant melanoma of the skin is a tumor which has not only one of the worst prognosls of all skin tumours, but is aIso one of the most malignant tumour at all. Malignant melanoma is also explicitely different from other tumor diseases in its biochemical specificity. Biochemical specificity of malignant melanoma is represented in part by the formation of specific cytoplasmatic particles of the pigment cell - melanosomes - in which the synthesis of pigment eumelanin and pheomelanin takes place and in part by the presence of a specific enzyme - tyrosinase - which catalyzes the formation of pigrnent eumelanin and pheomeIanin and even the formation of specific metaboIites (so called melanogens) which are excreted in increased amounts in the course of the disease (6, 7). Tyrosinase and melanogens are, of course, specific for pigment cell and therefore can be used for monitoring of malignant melanoma(Fig. 1),

It is generally believed that melanogens i.e. the biochemical markers of melanogenesis are precursors and/or metabolite of these precursors an the tyrosine to melanin metabolic pathway and, according to the origin, can be divided into three principal groups:

1. DOPA (3,4-dihydroxyphenylalanine) and its metabolites (DOPA related compounds or metabolites);

2. Cysteinyldopa and its metabolites (Pheomelanin related compounds or metabolites);


Fig. 1 - 106 Ko- The formation of pheo- and eumelanin.


3. Indolic compounds and their metabolites (Eumelanin related compounds or metabolites). This group can be furthermore divided into two principal parts:

a) isomeric indolic acids (5 -hydroxy-6-methoxy- and 6-hydroxy-5-methoxyindole-2-carboxylic acids),

b) isomeric 5,6-hydroxymethoxyindoles (these melanogens are, according to their behaviour during Thormählen test, so called Thormählen positive melanogens - TPM) (6, 7, 8, 14, 15) (Fig. 2).

Two enzymes in the body produce DOPA, namely tyrosine hydroxylase in the nervous system and the adrenals and tyrosinase in the pigment cells. DOPA and the catechoIamines formed from DOPA and their metabolites are therefore of diagnostic importance as biochemical markers of certain tumors derived from the nervous system and the neural crest. In the pigment cell (melanocyte) DOPA is produced by the action of tyrosinase activity. Tissues containing tyroslne hydroxylase also contain dopa decarboxylase and in these tissues DOPA is decarboxylated, forming dopamine. In contrast, melanocytes do not contain DOPA decarboxylase, and dopamine and other catecholamines are therefore not produced in pigment cells. On the other hand, DOPA may accumulate in the pigment cell, because dopaquinone is reduced back to DOPA by indoles and cysteinyldopas, all of which are compounds with lower redox potential then DOPA-dopaquinone system. In the case of overproductíon of DOPA in malignant pigment cells, DOPA can be excreted from the tumor and then outside of melanocyte can be transformed to many metabolites by the action of monoaminooxidase, minotransferase and catechol- O-methyl transferase (COMT).


Fig. 2 - 122 Ko -The metabolic origin of melanogens


A metabolite of DOPA formed directly from DOPA by transamination, reduction and O-methylation - vanillactic acid is very important marker for melanoma, especially for a pigmented (amelanotic) forms (12, 19).

Among the cysteinyldopas, 5-S-cysteinyIdopa (5-S-Cysdopa) has attracted most attention since it is quantitatively most important (19). The formation of 5-S-Cysdopa in melanocyte depends on the presence of cysteine. It is known fact, that high quantíty of cysteine in melanocyte can prevent melanization. However, melanizations takes place in the melanosomes and the sulphur content of the melanin formed may be low, indicating a low ratio cysteinyldopa to DOPA. It seems therefore, that a large portion of free cysteinyldopas in the skin must be present outside the melanosomes. Cysteinyldopa may have been secreted from the melanosomes before melanization of the melanosomes occurs,or it may have been formed in tyrosine containing vesicules outside of melanosomes, in which compartments cysteine quantities may be higher and prevent melanization. Cysteinyldopa in the cytoplasm of the melanocytes is excreted to the surrounding tissue fluid, whence it passes to the circulation. 5-S-cysteinyldopa is partly O-methylated in the presence of O-methyltransferase and four different O-methylated cysteinyldopas have been found in the urine of melanorna patients. In some cases of melanoma the trichochromes have been detected in the urine (19).

The last group of melanogens is represented by the compounds of indolic structure. It has been identified several types of these indolic or eumelanin related melanogens in the urine of melanoma patients, as well as in the urine of healthy persons (8, 9, 11, 15, 16, 20). These indolic melanogens are formed from dopaquinone on the metabolic pathway leading to pigment eumelanin, it means that they have to be formed inside of melanosomes, where the process of melanization takes place. The O-methylation of indolic melanogens takes place most likely in the liver as well as conjugation and/or biotransformation with glucuronic or sulfuric acids (15, 20). The most of indolic melanogens are in the form of glucosiduronates or sulfates excreted in the urine (11, 15, 17, 20). It is also possible that indolic melanogens are in part methylated in pigment cell, because of the activity of enzyme, which is responsible for methylation has been detected in melanoma tissue. Two enzymes are capable to methylate 5, 6-dihydroxyindole . The first one catechol-O- methyltransferase catalyzes the methylation of 5,6-dihydroxyindole primarily at the position 6 of indole ring, whereas the second one hydroxyindole-O-methyltransferase catalyzes methylation mainly at 5 position of indole ring (4, 5). It is very difficult to study indolic melanogens, because of they are unstabIe and very easily polymerize in the urine forming secondary so called urinary melanin by oxidation (10, 15, 16, 17, 18, 19).

As it is follows from the metabolism of melanogens (pigment related-compounds), the elevated excretion of melanogens so called melanogenuria is one of the indications of the interaction of malignant pigment cells with host organism. The clinical significance of the determination of the urinary melanogens excretion level depends predominantly on their specificity for melanoma, the frequency of their occurrence and, finally, on the relation between the clinical stage and type of the disease and the corresponding analytical data.

The elevated excretion of indolic or eumelanin related melanogens and of cysteinyldopas or pheomelanin related melanogens is highly specific for melanoma, whereas elevated excretion of DOPA and DOPA related compounds is less specific (1, 6, 7, 10, 12). DOPA and DOPA related metabolites (homovanillic acid and vanillactic acid) may also occur in elevated levels in other tumour of neural crest origin, neuroablastomas, heochromocytomas and others and generally occur ìn all cases which are characterized by DOPA overproduction. This fact is understandable, since all this phenolic acids with the exception of cysteinyldopas are formed from DOPA itself, whereas all indolic compounds and cysteinyldopa arise only from dopaquinone which is formed by dopaoxidase activity of tyrosinase (6, 7, 9, 18, 19).

It has been found that the elevated level of urinary melanogens occurs, on the average, in onIy 30 % of all patients. When we compared the results obtained by swedish group in the following of cysteinyldopa excretion and our results in the following of indolic (Thormählen positive) melanogens excretion in melanoma patients, we can conclude that the results are similar, and, in fact nearly the same. In 571 melanoma patients the level of cysteinyldopa in the urine was elevated in 182 cases, i. e. in 31,8 % (1, 2, 3). In our 690 melanoma patients, the level of eumelanin related (indolic) melanogens was elevated in 216 patients, i.e. in 31.3 % (10). From this is evident that the proof of increased melanogens urinary excretion has no great importance in an early diagnosis of malignant melanoma, since it can not substitute histological techniques and clinicaI observation. A dtfferent situation arises when the patients are divided according to the melanoma origin (cutaneous or ocular) and the stage of the disease. We have found increased excretion of indolic melanogens in the patients with ocular melanoma in Stage III (metastases and generalization of the disease) in all melanotic (pigmented) forms in contrast from Stage III with cutaneous melanoma, where increased urinary excretion of indolic melanogens was found in more than 80 % of patients (6, 7, 13). This fact, together with the differences in the absolute quantity of daily excretion of indolic melanogens indicates that metabolic differences could exist between the indlvidual types of malignant melanomas, just there exist differences in their biological behaviour. We assume on the basis of our results, that these metabolic variations could depend on the melanoma type and/or the degree of pigmentation (melanotic or amelanotic form). Eumelanin related melanogens can only arise from dopaquinone originating from tyrosine by means of catalytic activity of tyrosinase. Thus the differences in melanogens excretion could also be an indirect evidence of different tyrosinase activity in amelanotic and meIanotic melanomas. From this observation it follows, that the monitoring of indolic (eumelanin related) melanogens excretion in the course of malignant melanoma is very important for prognostication of the disease. A continuously increasing excretion of melanogens in the course of malignant melanoma is always an unfavourable sign of the patient (10). The variation of indolic melanogens excretion in the course of malignant melanoma is the dependence on its type and stage may aIso express an interaction of probably existing different biochemical types of malignant melanoma with different biochemically or immunologically conditioned responses of the host organism (10, 12, 13).

When we have studied the relation of eumelanin related melanogens (TPM) excretion to the excretion of DOPA related melanogens, represented by homovaniIlic acid (HVA) and vanillactic acid (VLA) in the advanced melanoma of different type and origin we found the differences in the ratio of eumelanin and DOPA related melanogens excretions. In amelanotic forms of advanced melanomas with Iiver metastases HIVA and VLA excretions predominated over eumeIanin related melanogens excretion, similarIy as in the advanced melanotic melanomas without Iiver metastases with the exception that in this form of malignant melanomas the average excretion of eumelanin related (indolic) melanogens was higher. In the advanced melanotic melanornas of both cutaneous and ocular origin, the total eumelanin related melanogens excretion predominated, although HVA and VLA excretions were elevated. The fact, that a low quantity of eumelanin related melanogens excretion has also been found in amelanotic forms of malignant melanoma is not at all surprising. All melanogens are excreted in the urine of healthy persons as well as in the urine of melanoma patients and differences are only quantitative. Since the eumelanin related rnelanogens are constantly excreted by healthy persons, it can be assumed that eumelanin related melanogens excreted in an amelanotic form of malignant melanoma are an expression of the metabolism of the normal pigment cells, whilst in amelanotic malignant pigment cells the dopaoxidase activity of tyrosinase is probably either strongly inhibited or missing (12).

When comparing eumelanin related melanogens (TPM) and HVA and VLA excretions we have found two significantly different relations for melanogens excretion, that is predominating eumelanin related melanogens urinary excretion in melanotic forms of malignant melanomas and predorminating HVA and VLA urinary excretion in amelanotic forms of malignant melanomas. We assume that it could be the indirect evidence of two biochemical and/or metaboIic types of malignant melanoma (12).

Study of melanogenesis and melanogenuria, as well as the study of biochemical monitoring of melanoma patients are by far not finished. But we do hope that we will be able in the nearly future to give a sufficient answer to the question, whether melanogenuria is actually an expression of expected different biochemical or metabolic types of malignant meIanoma on the one hand and/or biochemically or immunologically conditioned responses of the host organism on the other.

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