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Ultrastructural, Lectin Histochemical and Immunohistological Observations on Merkel Cell Tumors
Path. Res. Pract. 181,45-49 (1986)
Ultrastructural, Lectin Histochemical and Immunohistological Observations on Merkel Cell Tumors L. Pajor Department of Pathology, University Medical School of Pees,
M. Balazs Department of Pathology, Janos Hospital, Budapest,
J. Balogh Department of Pathology, Country Hospital, Szekszard,
F. Brittig Department of Pathology, Country Hospital, Szombathely,
L. Jo6s Department of Pathology, City Hospital, Nagykanizsa, Hungary
R. Linse Department of Dermatology, Medical Academy of Erfurt, GDR,
M. Scholz Department of Pathology, Weil Emil Hospital, Budapest,
Zs. Suba Department of Dermatology, Semmelweis University of Medicine, Budapest,
P. T6th Department of Pathology, Country Hospital and Szekesfehervar, Hungary
SUMMARY The clinicopathological and ultrastructural findings of 10 cases of Merkel cell tumor (MGT) are presented. Three patients died rapidly due to tumor dissemination. The tumor cells in eight out of the 10 cases were positive for neuron-specific enolase, however, all were negative for the ten polypeptide hormones examined, as well as for the argentaffin and argyrophil reactions. One of the patients had suffered from chronic lymphocytic leukaemia. Particularly in this case, but also in the others, the differentiation of MGT from a malignant lymphoma necessitated immuno-histological, lectin histochemical and ultrastructural studies. The focal peanut agglutinin positivity of the tumor cells in 6 out of the 10 MGTs seems to be a characteristic feature of these cells. © 1986 by Gustav Fischer Verlag, Stuttgart
0344-0338/8610181-0045$3.5010
46 . L. Pajor et al.
Introduction The Merkel cells (MCs) whose structure and function have been debated ever since they were described by Merkel in 1875 12 are nonkeratocytic cells in the basal layer of the epidermis. It is generally accepted that MCs together with the surrounding nerve endings serve as mechanoreceptors. However, definite electrophysiological evidence concerning their precise relationship to these nerve elements is still missing3, 19. Based on ultrastructural, embryological and immunohistochemical findings2\ it has been suggested that these cells are members of the APUD cell system 14 • The primary neuroendocrine tumor of the skin, called Merkel cell tumor (MCT)25, is likely to originate from this cell type, although, the histogenesis is not unambigously clarified23 . Because of the immature, blastic feature of this tumor a number of differential diagnostic difficulties arise in the course of histological examination. MCT might be misdiagnosed, among others, as a malignant lymphoma. In this paper 10 cases of MCT are reported, which were studied at the Malignant Lymphoma Reference Center9• Eight of 10 cases were positive for neuron specific enolase, but none of the cases was found to contain polypeptide hormones. All the 6 investigated cases were found to show focal positivity with peanut agglutinin.
examination was not carried out. The ultrathin sections were stained with lead citrate and uranyl acetate and examined in JEOL 100 C electron microscope.
Observation The age and sex of the patients, location of the tumors and other clinical findings are summarized in Table 1. In the cases studied the light and electron microscopic appearance of the tumors was very similar, they were located in the dermis without signs of epidermotropism. Under the atrophic epidermis the tumor cells were preferentially arranged into sheets (Fig. 1). No cribriform or alveolar arrangement was detected, pseudorosette-like structures were absent. In polarization microscope Congo red staining failed to show amyloid deposits in and around the tumor cells. Cytologically the cells were characterized by a very narrow cytoplasmic rim showing slight metachromasia at Giemsa staining, large, round to oval nucleus with finely
Table 1. The age and sex of the patients, location of the tumors, local recurrence, regional lymph node metastasis and survival No.
Material and Methods For light microscopy the materials were fixed either in formalin or in Bouin-solution and they were embedded in paraffin. The following stainings were used: haematoxylin-eosin, PAS, Giemsa, reticulin, Congo red, argentaffin (Masson-Fontana) and argyrophil (Sevier-Munger) reactions. All cases were tested by the unlabelled antibody-enzyme (peroxidase-antiperoxidase, PAP) method for glucagon, pancreatic polypeptide, somatostatin, gastrin, substance-P, neurotensin, 5-hydroxytryptamin, ACTH, a-HCG, calcitonin and neuron specific enolase (NSE)4,5. In cases 7, 8 the immunohistological investigations for NSE were generously performed by ]. Polak, London, whereas all further tests were kindly carried out by H. Hofler, Graz. In case 7 the immunohistological investigations were supplemented by tests for cytoplasmic immunoglobulins (direct immunoperoxidase reaction, dilution 1: 30-50) and for J-chain, lysozyme, a-I-anti trypsin- and antichymotrypsin (indirect immunoperoxidase reaction, dilution 1: 50-60). The reaction products were visualized by peroxidase reaction using 3,3'diamino-benzidine (DAB). Expect anti J-chain (Nordic), antisera were purchased from Dakopatts. Horseradish-peroxidase labelled peanut agglutinin (PNA, Sigma) was used for lectin histochemistry. Sections were incubated with labelled lectin at a concentration of 10 IAg/ml, for 30 min at room temperature. Parallel sections were pretreated with neuraminidase (0.1 IU) before incubation. Lectin binding sites were visualized by peroxidase reaction using 3-amino-9ethyl-carbazol. For electron microscopy small blocks of the tumors were fixed in 2.5% glutaraldehyde, postfixed with 1% OS04 and dehydrated in acetone (cases 7 and 8). In cases 1, 2, 3, 4, 10 the tumor tissue was fixed in formalin or in Bouin solution, followed by the same dehydration processes. In cases 5, 6, 9 ultrastructural
1.
2.
3. 4. 5. 6. 7. 8. 9. 10.
Age/ Location Local Regional lymph node Survival sex recur- metastasis (month) renee 88/0" 74/0" 73/0" 53/2 49/2 63/2 56/2 75/2 65/2 83/2
eyelid knee eyelid neck wrist ear face chin face eyelid
+ + + + + + + +
+ (+ + + (+ + (+ +
20 15 dissemination) died (3) 34 31 19 dissemination) died (14) dissemination) died (10) 10 7
Fig. 1. Diffuse infiltration of tumor cells under the atrophic epidermis. H-E, x 150.
Ultrastructural, Lectin Histochemical Observations on Merkel Cell Tumors . 47
dispersed chromatin and 1-3 nucleoli associated with the nuclear membrane (Fig. 2). Mitoses were very frequent. The cells were completely negative in the PAS staining. Argentaffin and argyrophil reactions were also negative in all cases. Similarly, the cells failed to show any positivity for the 10 hormones and polypeptides (detailed in Methods). However, in 8 out of 10 cases the cells were positive for NSE, in one case the result of reaction was ambiguous, in the 6 cases examined for PNA reactivity the cells showed the same pattern with different intensity: there was a focal, paranuclear positivity corresponding to the Golgi region (Fig. 3).
Ultrastructurally the cytoplasm of the tumor cells was poorly differentiated. The most prominent feature of the cells was the presence of neurosecretory granules located at the periphery of cytoplasm with an average diameter of 120 nm. Few cytoplasmic intermediate filaments were also seen but "whirls" were not observed. At their periphery the cells showed a number of cytoplasmic projections.
Fig. 4. Blast type of cells with very narrow cytoplasmic rim and with neurosecretory granules ((-0) at the cytoplasmic membrane. X 8300. inset: neurosecretory granules, x 95000.
Fig. 2. Immature, blast type of cells. Semi thin section, toluidine blue, X 910.
Fig. 3. Focal paranuclear positivity (after neuraminidase treatment) with peanut agglutinin. x 300.
Fig. 5. Poorly differentiated cytoplasm contammg mainly mitochondria, Golgi profiles and neurosecretory granules ((-0) X 26000.
48 . L. Pajor et al. Table 2. The presence of neurosecretory granules, frequency of NSE and PNA positivity. No. 1.
2.
3. 4.
5.
NSE
Peanut Agglutinin
+ + + +
+ + +/+ + +
+ + + +
n.e. n.e.
6.
+ +
7. 8. 9.
n.e.
+
10.
n.e.
Neurosecretory granule
=
+ + +
n.e. n.e.
+
n.e. n.e.
+
not examined
Desmosomes were not seen (Figs 4-5). The histochemical, lectin-histochemical, immunohistological and ultrastructural findings are summarized in Table 2. Discussion
In 1972 Toker described a peculiar skin tumor called trabecular carcinoma. The nature and histogenesis of this tumor were revealed by Tang and Toker (1978) in an ultrastructural study. In 1980 Wolf-Peeters et al. suggested the name of Merkel cell tumor (MCT) indicating an undifferentiated, blastic tumor occurring in the mid an lower dermis without involving the epidermis. Similarly to the cases reported elsewhere 13 ,22 in those presented here the tumors developed in elder patients and mainly on the head or on the extremities. Fifty percent of the cases had regional nodal metastases (developed within 2-6 months) which is consistend with that found by Pilotti et al (1982), however, Figerio et al (1983) published higher value (67%). Dissemination leading to death developed in 3 patients (30%) thus the mortality was practically the same as compared with that published by others 1• Disregarding the light microscopical characteristics, the diagnostical criteria of MCT are the neurosecretory granules, the cytoplasmic filaments and the NSE positivity. In the 7 cases examined electron microscopically, the ultrastructural findings were indicative of neuroendocrine tumor. Moreover, in 8 out of 10 cases the tumor cells were positive for NSE which enzyme has been assumed to be a useful marker of neuroendocrine (APUD) cells 18 • The negativity for NSE in two cases was considered to be due to poor fixation. In accordance with the observation of Rauch et al (1984), tumor cells in all cases were negative for the 10 hormones and polypeptides in our material. However, our data are in contrast with that of Johannessen and Gould (1980) who described a MCT associated with calcitonin production. The value of argyrophil reaction in MCTs is controversial. The majority of authors are of the opinion that argyrophilia is not a consistent feature of MCTs. Frigerio et al.
(1983) reported that "argyrophilia is common, but Bouin fixation is necessary to demonstrate it regularly". We did not find argyrophilia in one Bouin-fixed and in 9 formolfixed samples. The anaplastic appearance of the MCT in light microscope raises a number of differential diagnostic problems 2,6. Thus, MCTs have to be differentiated from anaplastic small cell bronchus carcinoma which, regarding its neuroendocrine characteristics, even at ultrastructural level, might be difficult. The melanocytic or histiocytic (Langerhans cell) origin can be ruled out by the absence of melanosomes and Birbeck granules. The light microscopical appearance (absence of Wright-Homes rosettes) and the age of the patients contradict the existence of neuroblastoma. On the basis of PAS reactivity the differentiation of MCT from an extraskeletal Ewing sarcoma is not difficultll. However, because of ultrastructural, immunohistological similarity, for the differentiation of MCT from metastases of different type of neuroendocrine tumors, a very careful clinical examination is necessary. The diffuse type of MCTs (which was observed in all of our cases) may resemble of malignant lymphomas 1,7. In case 7 the MCT developed in a patient who had suffered from chronic lymphocytic leukaemia (CLL). This raised the question wether it might have been a lymphoblastic or immunoblastic transformation of CLL. However, the absence of cytoplasmic immunoglobulin and of J-chain ruled out the existence of immature, immunoglobulin producting tumor lO • Rosati et al (1984) found that the surface binding sites of MCs for PNA were available only after neuraminidase treatment. In the 6 MCTs investigated by us the cytoplasmic positivity for PNA (after neuraminidase treatment) was consistent, however, it ranged from slight to strong. Although this rare tumor has a typical light microscopical appearance, because of its highly immature histological appearance, selective immunohistological and ultrastructural investigations have been suggested to confirm the diagnosis. Based on our lectin histochemical findings, the PNA reaction can be considered as a new, edditional tool in differential diagnosis of MCTs. Acknowledgements The authors thank J. M. Polak, Dsc, MD, MRC Path (Histochemistry Unit, Depart. of Histopathology, Royal Postgraduate Medical School, Hammersmith Hospital, London) and H. Hofler, M. D. (Karl-Franzens University, Institute of Pathological Anatomy, Graz) for the investigation of these cases for polypeptide hormones and for NSE. References 1
Frigerio B, Capella C, Eusebio V, Tenti P, Azzopardi JG
2
Gloor F, Heitz Ph U, Hofmann E, Hoefler H, Maurer R
(1983) Merkel cell carcinoma of the skin: the structure and origin of normal Merkel cells. Histopathology 7: 229-249 (1982) Das neuroendokrine Merkelzellkarzinom der Haut.
Schweiz med Wschr 112: 141-148
Ultrastructural, Lectin Histochemical Observations on Merkel Cell Tumors· 49 3 Gottschaldt KM, Vahle-Hinz C (1981) Merkel cell receptors: structure and transducer function. Science 214: 183-186 4 Gu J, Polak JM, Van Noorden S, Pearse AGE, Marangos PJ, Azzopardi JG (1983) Immunostaining of neuronspecific enolase as a diagnostic tool for Merkel cell tumors. Cancer 52: 1039-1043 5 Hofler H, Kasper M, Heitz Ph U (1983) The neuroendocrine system of normal human appendix, ileum and colon, and in neurogenic appoendicopathy. Virchows Arch (Pathol Anat) 399: 127-140 6 Hubner G, Remberger K, Pielsticker K (1983) Neuroendokrines Karzinom (Merkelzelltumor) der Haut. Pathologe 4: 212-218 7 Iwasaki H, Mitsui T, Kikuchi M, Imai T, Fukushima K (1981) Neuroendocrine carcinoma (trabecular carcinoma) of the skin with ectopic ACTH production. Cancer 48: 753-756 8 Johannessen]V, Gould VE (1980) Neuroendocrine skin carcinoma associated with calcitonin production: a Merkel cell carcinoma? Hum Pathol11 (Suppl): 586-589 9 Kelenyi G, Varbir6 M (1980) Malignant Lymphoma Reference Centre - Hungary, 1978. Virchows Arch (Pathol Anat) 388: 213-219 10 Kelenyi G (1985) Intracellular J chains in Iymphoproliferative disease. Virchows Arch (Pathol Anat) 405: 365-378 11 Meister P, Cokel M (1978) Extraskeletal Ewing's sarcoma. Virchows Arch (Pathol Anat) 378: 173-179 12 Merkel F (1875) Tastzellen und Tastkorperchen bei den Haustieren und beim Menschen. Arch Mikr Anat 11: 636-641 13 Nakashima N, Kishiko G, Takeuchi J, Maeda H (1983) Case report of neuroendocrine carcinoma of the skin, histochemical and electron microscopic study. Virchows Arch (Pathol Anat)
401: 261-273
14 Pearse AGE (1968) Common cytochemical and ultrastructural characteristics of the cells producing polypeptide hormones
(the APUD series) and their relevance to thyroid and ultimobronchial C cells and calcitonin. Proc. Roy Soc B 170: 71-80 15 Pilotti S, Rilke F, Lombardi L (1982) Neuroendocrine (Merkel cell) carcinoma of the skin. Am J Surg Pathol 6: 243-254 16 Rauch H-J, Hofler H, Ked H (1984) Das neuroendokrine (Merkel-Zell-) Karzinom der Haut. Hautarzt 35: 138-141 17 Rosati D, Nurse CA, Diamond J (1984) Lectin binding properties of the Merkel cell and other root sheats cells in perinatal rat. Cell Tissue Res 236: 373-381 18 Schmechel D, Merangos PJ, Brightman M (1978) Neuronspecific enolase is a molecular marker for peripheral and central neuroendocrine cells. Nature 276: 834-836 19 Tachibana T, Ishizeki K, Sakakura Y, Nawa T (1984) Ultrastructural evidence for a possible secretory function of Merkel cells in the barbels of a teleost fish, Cyprinus carpio. Cell Tissue Res 235: 695-697 20 Tang CK, Toker C (1978) Trabecular carcinoma of the skin (An ultrastructural study). Cancer 42: 2311-2321 21 Toker C (1972) Trabecular carcinoma of the skin. Arch Dermatol105: 107-110 22 Warner TFCS, Uno H, Hafez R, Burgess J, Bolles C, Lloyd RV, Oka M (1983) Merkel cells and Merkel cell tumors. Cancer 52:238-245 23 Wick MR, Goellner JR, Scheithauer BW, Thomas JR, Sanchez NP, Schroeter AL (1983) Primary neuroendocrine carcinomas of the skin (Merkel cell tumors). Am J Clin Pathol 79: 6-13 24 Winkelmann RK (1977) The Merkel cell system and a comparison between it and the neurosecretory or APUD cell system. J Invest Dermatol69: 41-46 25 De Wolf-Peeters C, Marien K, Mebis J, Desmet V (1980) A cutaneous APUDoma or Merkel cell tumor? Cancer 46: 1810-1816
Received January 18, 1985 . Accepted July 5, 1985
Key words: Merkel cell tumor - Neuron-specific enolase - Peanut agglutinin - Differential diagnosis L. Pajor, M.D., Department of Pathology, University Medical School of Pecs, H-7643 Pecs, POB 99, Hungary
Ultrastructural, Lectin Histochemical and Immunohistological Observations on Merkel Cell Tumors L. Pajor Department of Pathology, University Medical School of Pees,
M. Balazs Department of Pathology, Janos Hospital, Budapest,
J. Balogh Department of Pathology, Country Hospital, Szekszard,
F. Brittig Department of Pathology, Country Hospital, Szombathely,
L. Jo6s Department of Pathology, City Hospital, Nagykanizsa, Hungary
R. Linse Department of Dermatology, Medical Academy of Erfurt, GDR,
M. Scholz Department of Pathology, Weil Emil Hospital, Budapest,
Zs. Suba Department of Dermatology, Semmelweis University of Medicine, Budapest,
P. T6th Department of Pathology, Country Hospital and Szekesfehervar, Hungary
SUMMARY The clinicopathological and ultrastructural findings of 10 cases of Merkel cell tumor (MGT) are presented. Three patients died rapidly due to tumor dissemination. The tumor cells in eight out of the 10 cases were positive for neuron-specific enolase, however, all were negative for the ten polypeptide hormones examined, as well as for the argentaffin and argyrophil reactions. One of the patients had suffered from chronic lymphocytic leukaemia. Particularly in this case, but also in the others, the differentiation of MGT from a malignant lymphoma necessitated immuno-histological, lectin histochemical and ultrastructural studies. The focal peanut agglutinin positivity of the tumor cells in 6 out of the 10 MGTs seems to be a characteristic feature of these cells. © 1986 by Gustav Fischer Verlag, Stuttgart
0344-0338/8610181-0045$3.5010
46 . L. Pajor et al.
Introduction The Merkel cells (MCs) whose structure and function have been debated ever since they were described by Merkel in 1875 12 are nonkeratocytic cells in the basal layer of the epidermis. It is generally accepted that MCs together with the surrounding nerve endings serve as mechanoreceptors. However, definite electrophysiological evidence concerning their precise relationship to these nerve elements is still missing3, 19. Based on ultrastructural, embryological and immunohistochemical findings2\ it has been suggested that these cells are members of the APUD cell system 14 • The primary neuroendocrine tumor of the skin, called Merkel cell tumor (MCT)25, is likely to originate from this cell type, although, the histogenesis is not unambigously clarified23 . Because of the immature, blastic feature of this tumor a number of differential diagnostic difficulties arise in the course of histological examination. MCT might be misdiagnosed, among others, as a malignant lymphoma. In this paper 10 cases of MCT are reported, which were studied at the Malignant Lymphoma Reference Center9• Eight of 10 cases were positive for neuron specific enolase, but none of the cases was found to contain polypeptide hormones. All the 6 investigated cases were found to show focal positivity with peanut agglutinin.
examination was not carried out. The ultrathin sections were stained with lead citrate and uranyl acetate and examined in JEOL 100 C electron microscope.
Observation The age and sex of the patients, location of the tumors and other clinical findings are summarized in Table 1. In the cases studied the light and electron microscopic appearance of the tumors was very similar, they were located in the dermis without signs of epidermotropism. Under the atrophic epidermis the tumor cells were preferentially arranged into sheets (Fig. 1). No cribriform or alveolar arrangement was detected, pseudorosette-like structures were absent. In polarization microscope Congo red staining failed to show amyloid deposits in and around the tumor cells. Cytologically the cells were characterized by a very narrow cytoplasmic rim showing slight metachromasia at Giemsa staining, large, round to oval nucleus with finely
Table 1. The age and sex of the patients, location of the tumors, local recurrence, regional lymph node metastasis and survival No.
Material and Methods For light microscopy the materials were fixed either in formalin or in Bouin-solution and they were embedded in paraffin. The following stainings were used: haematoxylin-eosin, PAS, Giemsa, reticulin, Congo red, argentaffin (Masson-Fontana) and argyrophil (Sevier-Munger) reactions. All cases were tested by the unlabelled antibody-enzyme (peroxidase-antiperoxidase, PAP) method for glucagon, pancreatic polypeptide, somatostatin, gastrin, substance-P, neurotensin, 5-hydroxytryptamin, ACTH, a-HCG, calcitonin and neuron specific enolase (NSE)4,5. In cases 7, 8 the immunohistological investigations for NSE were generously performed by ]. Polak, London, whereas all further tests were kindly carried out by H. Hofler, Graz. In case 7 the immunohistological investigations were supplemented by tests for cytoplasmic immunoglobulins (direct immunoperoxidase reaction, dilution 1: 30-50) and for J-chain, lysozyme, a-I-anti trypsin- and antichymotrypsin (indirect immunoperoxidase reaction, dilution 1: 50-60). The reaction products were visualized by peroxidase reaction using 3,3'diamino-benzidine (DAB). Expect anti J-chain (Nordic), antisera were purchased from Dakopatts. Horseradish-peroxidase labelled peanut agglutinin (PNA, Sigma) was used for lectin histochemistry. Sections were incubated with labelled lectin at a concentration of 10 IAg/ml, for 30 min at room temperature. Parallel sections were pretreated with neuraminidase (0.1 IU) before incubation. Lectin binding sites were visualized by peroxidase reaction using 3-amino-9ethyl-carbazol. For electron microscopy small blocks of the tumors were fixed in 2.5% glutaraldehyde, postfixed with 1% OS04 and dehydrated in acetone (cases 7 and 8). In cases 1, 2, 3, 4, 10 the tumor tissue was fixed in formalin or in Bouin solution, followed by the same dehydration processes. In cases 5, 6, 9 ultrastructural
1.
2.
3. 4. 5. 6. 7. 8. 9. 10.
Age/ Location Local Regional lymph node Survival sex recur- metastasis (month) renee 88/0" 74/0" 73/0" 53/2 49/2 63/2 56/2 75/2 65/2 83/2
eyelid knee eyelid neck wrist ear face chin face eyelid
+ + + + + + + +
+ (+ + + (+ + (+ +
20 15 dissemination) died (3) 34 31 19 dissemination) died (14) dissemination) died (10) 10 7
Fig. 1. Diffuse infiltration of tumor cells under the atrophic epidermis. H-E, x 150.
Ultrastructural, Lectin Histochemical Observations on Merkel Cell Tumors . 47
dispersed chromatin and 1-3 nucleoli associated with the nuclear membrane (Fig. 2). Mitoses were very frequent. The cells were completely negative in the PAS staining. Argentaffin and argyrophil reactions were also negative in all cases. Similarly, the cells failed to show any positivity for the 10 hormones and polypeptides (detailed in Methods). However, in 8 out of 10 cases the cells were positive for NSE, in one case the result of reaction was ambiguous, in the 6 cases examined for PNA reactivity the cells showed the same pattern with different intensity: there was a focal, paranuclear positivity corresponding to the Golgi region (Fig. 3).
Ultrastructurally the cytoplasm of the tumor cells was poorly differentiated. The most prominent feature of the cells was the presence of neurosecretory granules located at the periphery of cytoplasm with an average diameter of 120 nm. Few cytoplasmic intermediate filaments were also seen but "whirls" were not observed. At their periphery the cells showed a number of cytoplasmic projections.
Fig. 4. Blast type of cells with very narrow cytoplasmic rim and with neurosecretory granules ((-0) at the cytoplasmic membrane. X 8300. inset: neurosecretory granules, x 95000.
Fig. 2. Immature, blast type of cells. Semi thin section, toluidine blue, X 910.
Fig. 3. Focal paranuclear positivity (after neuraminidase treatment) with peanut agglutinin. x 300.
Fig. 5. Poorly differentiated cytoplasm contammg mainly mitochondria, Golgi profiles and neurosecretory granules ((-0) X 26000.
48 . L. Pajor et al. Table 2. The presence of neurosecretory granules, frequency of NSE and PNA positivity. No. 1.
2.
3. 4.
5.
NSE
Peanut Agglutinin
+ + + +
+ + +/+ + +
+ + + +
n.e. n.e.
6.
+ +
7. 8. 9.
n.e.
+
10.
n.e.
Neurosecretory granule
=
+ + +
n.e. n.e.
+
n.e. n.e.
+
not examined
Desmosomes were not seen (Figs 4-5). The histochemical, lectin-histochemical, immunohistological and ultrastructural findings are summarized in Table 2. Discussion
In 1972 Toker described a peculiar skin tumor called trabecular carcinoma. The nature and histogenesis of this tumor were revealed by Tang and Toker (1978) in an ultrastructural study. In 1980 Wolf-Peeters et al. suggested the name of Merkel cell tumor (MCT) indicating an undifferentiated, blastic tumor occurring in the mid an lower dermis without involving the epidermis. Similarly to the cases reported elsewhere 13 ,22 in those presented here the tumors developed in elder patients and mainly on the head or on the extremities. Fifty percent of the cases had regional nodal metastases (developed within 2-6 months) which is consistend with that found by Pilotti et al (1982), however, Figerio et al (1983) published higher value (67%). Dissemination leading to death developed in 3 patients (30%) thus the mortality was practically the same as compared with that published by others 1• Disregarding the light microscopical characteristics, the diagnostical criteria of MCT are the neurosecretory granules, the cytoplasmic filaments and the NSE positivity. In the 7 cases examined electron microscopically, the ultrastructural findings were indicative of neuroendocrine tumor. Moreover, in 8 out of 10 cases the tumor cells were positive for NSE which enzyme has been assumed to be a useful marker of neuroendocrine (APUD) cells 18 • The negativity for NSE in two cases was considered to be due to poor fixation. In accordance with the observation of Rauch et al (1984), tumor cells in all cases were negative for the 10 hormones and polypeptides in our material. However, our data are in contrast with that of Johannessen and Gould (1980) who described a MCT associated with calcitonin production. The value of argyrophil reaction in MCTs is controversial. The majority of authors are of the opinion that argyrophilia is not a consistent feature of MCTs. Frigerio et al.
(1983) reported that "argyrophilia is common, but Bouin fixation is necessary to demonstrate it regularly". We did not find argyrophilia in one Bouin-fixed and in 9 formolfixed samples. The anaplastic appearance of the MCT in light microscope raises a number of differential diagnostic problems 2,6. Thus, MCTs have to be differentiated from anaplastic small cell bronchus carcinoma which, regarding its neuroendocrine characteristics, even at ultrastructural level, might be difficult. The melanocytic or histiocytic (Langerhans cell) origin can be ruled out by the absence of melanosomes and Birbeck granules. The light microscopical appearance (absence of Wright-Homes rosettes) and the age of the patients contradict the existence of neuroblastoma. On the basis of PAS reactivity the differentiation of MCT from an extraskeletal Ewing sarcoma is not difficultll. However, because of ultrastructural, immunohistological similarity, for the differentiation of MCT from metastases of different type of neuroendocrine tumors, a very careful clinical examination is necessary. The diffuse type of MCTs (which was observed in all of our cases) may resemble of malignant lymphomas 1,7. In case 7 the MCT developed in a patient who had suffered from chronic lymphocytic leukaemia (CLL). This raised the question wether it might have been a lymphoblastic or immunoblastic transformation of CLL. However, the absence of cytoplasmic immunoglobulin and of J-chain ruled out the existence of immature, immunoglobulin producting tumor lO • Rosati et al (1984) found that the surface binding sites of MCs for PNA were available only after neuraminidase treatment. In the 6 MCTs investigated by us the cytoplasmic positivity for PNA (after neuraminidase treatment) was consistent, however, it ranged from slight to strong. Although this rare tumor has a typical light microscopical appearance, because of its highly immature histological appearance, selective immunohistological and ultrastructural investigations have been suggested to confirm the diagnosis. Based on our lectin histochemical findings, the PNA reaction can be considered as a new, edditional tool in differential diagnosis of MCTs. Acknowledgements The authors thank J. M. Polak, Dsc, MD, MRC Path (Histochemistry Unit, Depart. of Histopathology, Royal Postgraduate Medical School, Hammersmith Hospital, London) and H. Hofler, M. D. (Karl-Franzens University, Institute of Pathological Anatomy, Graz) for the investigation of these cases for polypeptide hormones and for NSE. References 1
Frigerio B, Capella C, Eusebio V, Tenti P, Azzopardi JG
2
Gloor F, Heitz Ph U, Hofmann E, Hoefler H, Maurer R
(1983) Merkel cell carcinoma of the skin: the structure and origin of normal Merkel cells. Histopathology 7: 229-249 (1982) Das neuroendokrine Merkelzellkarzinom der Haut.
Schweiz med Wschr 112: 141-148
Ultrastructural, Lectin Histochemical Observations on Merkel Cell Tumors· 49 3 Gottschaldt KM, Vahle-Hinz C (1981) Merkel cell receptors: structure and transducer function. Science 214: 183-186 4 Gu J, Polak JM, Van Noorden S, Pearse AGE, Marangos PJ, Azzopardi JG (1983) Immunostaining of neuronspecific enolase as a diagnostic tool for Merkel cell tumors. Cancer 52: 1039-1043 5 Hofler H, Kasper M, Heitz Ph U (1983) The neuroendocrine system of normal human appendix, ileum and colon, and in neurogenic appoendicopathy. Virchows Arch (Pathol Anat) 399: 127-140 6 Hubner G, Remberger K, Pielsticker K (1983) Neuroendokrines Karzinom (Merkelzelltumor) der Haut. Pathologe 4: 212-218 7 Iwasaki H, Mitsui T, Kikuchi M, Imai T, Fukushima K (1981) Neuroendocrine carcinoma (trabecular carcinoma) of the skin with ectopic ACTH production. Cancer 48: 753-756 8 Johannessen]V, Gould VE (1980) Neuroendocrine skin carcinoma associated with calcitonin production: a Merkel cell carcinoma? Hum Pathol11 (Suppl): 586-589 9 Kelenyi G, Varbir6 M (1980) Malignant Lymphoma Reference Centre - Hungary, 1978. Virchows Arch (Pathol Anat) 388: 213-219 10 Kelenyi G (1985) Intracellular J chains in Iymphoproliferative disease. Virchows Arch (Pathol Anat) 405: 365-378 11 Meister P, Cokel M (1978) Extraskeletal Ewing's sarcoma. Virchows Arch (Pathol Anat) 378: 173-179 12 Merkel F (1875) Tastzellen und Tastkorperchen bei den Haustieren und beim Menschen. Arch Mikr Anat 11: 636-641 13 Nakashima N, Kishiko G, Takeuchi J, Maeda H (1983) Case report of neuroendocrine carcinoma of the skin, histochemical and electron microscopic study. Virchows Arch (Pathol Anat)
401: 261-273
14 Pearse AGE (1968) Common cytochemical and ultrastructural characteristics of the cells producing polypeptide hormones
(the APUD series) and their relevance to thyroid and ultimobronchial C cells and calcitonin. Proc. Roy Soc B 170: 71-80 15 Pilotti S, Rilke F, Lombardi L (1982) Neuroendocrine (Merkel cell) carcinoma of the skin. Am J Surg Pathol 6: 243-254 16 Rauch H-J, Hofler H, Ked H (1984) Das neuroendokrine (Merkel-Zell-) Karzinom der Haut. Hautarzt 35: 138-141 17 Rosati D, Nurse CA, Diamond J (1984) Lectin binding properties of the Merkel cell and other root sheats cells in perinatal rat. Cell Tissue Res 236: 373-381 18 Schmechel D, Merangos PJ, Brightman M (1978) Neuronspecific enolase is a molecular marker for peripheral and central neuroendocrine cells. Nature 276: 834-836 19 Tachibana T, Ishizeki K, Sakakura Y, Nawa T (1984) Ultrastructural evidence for a possible secretory function of Merkel cells in the barbels of a teleost fish, Cyprinus carpio. Cell Tissue Res 235: 695-697 20 Tang CK, Toker C (1978) Trabecular carcinoma of the skin (An ultrastructural study). Cancer 42: 2311-2321 21 Toker C (1972) Trabecular carcinoma of the skin. Arch Dermatol105: 107-110 22 Warner TFCS, Uno H, Hafez R, Burgess J, Bolles C, Lloyd RV, Oka M (1983) Merkel cells and Merkel cell tumors. Cancer 52:238-245 23 Wick MR, Goellner JR, Scheithauer BW, Thomas JR, Sanchez NP, Schroeter AL (1983) Primary neuroendocrine carcinomas of the skin (Merkel cell tumors). Am J Clin Pathol 79: 6-13 24 Winkelmann RK (1977) The Merkel cell system and a comparison between it and the neurosecretory or APUD cell system. J Invest Dermatol69: 41-46 25 De Wolf-Peeters C, Marien K, Mebis J, Desmet V (1980) A cutaneous APUDoma or Merkel cell tumor? Cancer 46: 1810-1816
Received January 18, 1985 . Accepted July 5, 1985
Key words: Merkel cell tumor - Neuron-specific enolase - Peanut agglutinin - Differential diagnosis L. Pajor, M.D., Department of Pathology, University Medical School of Pecs, H-7643 Pecs, POB 99, Hungary