J Clin Pathol 1987;40:890-895

"Mucin secreting" and "mucinous" primary thyroidcarcinomas: pitfalls in mucin histochemistry applied tothyroid tumours

C RIGAUD, W V BOGOMOLETZFrom the Laboratoire d'Anatomie Pathologique, Institut Jean Godinot, Reims, France

suMMARY Forty primary carcinomas of the thyroid of different histological types were reviewedand studied histochemically, with the aim of identifying and assessing "mucin secretion". Thepatterns of extracellular "pure alcianophilia" and "mixed alcianophilia" were noted in 7-5% andabout 50% of these tumours, respectively. A critical review of the pitfalls in methods and inter-pretation of mucin histochemistry-as performed in previously reported cases of "mucin secreting"or "mucinous" primary thyroid tumours-is presented. The apparent "mucin secretion" describedin these unusual neoplasms could be due to histochemical staining of carbohydrate components orbreakdown products of thyroglobulin and colloid.

Cases of "mucin secreting" or "mucinous" primarycarcinomas and adenomas of the thyroid have beenincreasingly reported in the past decade.' -19 Thediagnosis of "mucin secretion" in these unusual thyr-oid tumours has essentially relied on the apparentpositivity of different histochemical methods formucins. These methods are more conventionally usedfor the study of epithelial mucins secreted by gobletcells of the gastrointestinal or bronchial tract. Themajor implication of these reports' -'9 has been thatthe follicular and parafollicular cells of the thyroid-an exclusive endocrine gland, by definition-couldalso produce "mucins" in primary tumours, in a man-ner similar to that of epithelial goblet cells in exocrineorgans. Does conventional mucin histochemistryapplied to thyroid tumours actually identify sub-stances, which could be regarded as being equivalentto gastrointestinal and bronchial mucins, or, alterna-tively, show up components or breakdown productsof thyroglobulin and colloid?2

In an effort to clarify further the nature of these socalled "mucin secreting" or "mucinous" primarythyroid tumours we carried out a histochemical studyon 40 primary carcinomas of the thyroid, using twocombined staining methods for mucin identification.

Accepted for publication 26 March 1987

Material and methods

The original haematoxylin and eosin stained slides of40 primary carcinomas of thyroid were retrieved fromour files and reviewed. These 40 cases were classifiedaccording to current histological nomenclatures20 21and included 22 papillary, nine follicular, five ana-plastic, and four medullary carcinomas. All the mate-rial had been fixed in formol saline (pH 7). Theparaffin blocks of these 40 cases were recut andnumerous additional sections stained by two com-bined histochemical methods for mucins:1 Alcian blue (pH 2.5) and periodic acid Schiff afterdiastase digestion (AB/PAS), which distinguishesbetween acidic mucins stained blue by AB and neutralmucins stained magenta by PAS (mixtures of acidicand neutral mucins produce a purple colour).2 High iron diamine and alcian blue pH 2-5(HID/AB), which differentiates sulphomucins stainedbrownish black by HID and sialomucins stained bluebyAB.

These two combined histochemical methods wereperformed in batches, with adequate positive andnegative controls, and strictly in accordance with themethods recommended by Filipe and Lake.22 Wehave been using this in our laboratory for many years,with consistent, reproducible, and reliable results.

In this study the term "alcianophilia" was definedas any positive blue staining reaction (extracellular orintracellular) caused by the AB component of

890

copyright. on O

ctober 25, 2020 by guest. Protected by

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.40.8.890 on 1 A

ugust 1987. Dow

nloaded from

Mucin secreting and mucinous primary thyroid carcinomas

AB/PAS or HID/AB, and regardless of the presumednature of the stained material.

Results

Three different staining and morphological patternscould be observed in the sections of the 40 primarythyroid carcinomas: "pure", "mixed", and "foreign".

PURE ALCIANOPHILIAThis was characterised by the presence of focalextracellular material, contained within scatteredneoplastic microfollicles, and showing a positive andhomogeneous blue staining with both AB/PAS andHID/AB. Apart from alcianophilia, no other stainingreaction was obtained with either the PAS or the HIDsequences of the two combined histochemical tech-niques. This pattern of pure alcianophilia wasidentified in only three (7.5%) of the 40 thyroid car-cinomas studied-one follicular and two medullarycarcinomas (fig 1).

U~~~~~

Fig Medullary carcinoma ofthyroid. Focal pure

alcianophilia within one microfollicle (arrowed). (Combined

alcian blue, pH2 5, andperiodic acid Schff.)

MIXED ALCIANOPHILIAThis was characterised by the presence of focalextracellular material contained in the "colloid" ofneoplastic macrofollicles and showing a positive butheterogeneous staining with both AB/PAS (admixedblue and magenta positive staining) and HID/AB(admixed brownish black and blue positive staining)(figs 2 and 3). This pattern ofmixed alcianophilia wasseen in about 50% of the 40 tumours, more com-monly in follicular and papillary carcinomas.

FOREIGN ALCIANOPHILIAThis was characterised by a positive blue staining,obtained with both AB/PAS and HID/AB, ofdifferent tissue structures other than the intralumenalcontents of either thyroid macrofollicles or micro-follicles.The extracellular material of both pure and mixed

alcianophilia was scarce and scattered. Numeroussections stained by AB/PAS and HID/AB had to becarefully studied to identify positive stained material

"'74

.. . ....... .; i X

t H *(; ,-W -

Fig 2 Follicular carcinoma ofthyroid. Focal mixedalcianophilia with two macrofollicles (arrowed). (Combinedhigh iron diamine and alcian blue, pH2-5.)

891

copyright. on O

ctober 25, 2020 by guest. Protected by

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.40.8.890 on 1 A

ugust 1987. Dow

nloaded from

Rigaud, Bogomoletz

.0,4

;';e.!

Fig 3 Papillary carcinoma of thyroid. Focaialcianophilia within several macrofollicles. ((iron diamine and alcian blue, pH2-5.)in each case. No convincing intracelluAB positive, PAS positive (after diastor HID positive-could be shown.

Foreign alcianophilia was mostly clmicrocalcifications and the connectivein all of the 22 papillary carcinomas (alcianophilia, caused by the ABthe glycoconjugates of interstitial concould be abolished by prior enzymehyaluronidase,22 and did not interfere N

Discussion

An increasing number of articles oning" or "mucinous" primary tumourshave been published in the past decahave consisted mainly of single case rerseries, which have included primaryfollicular and parafollicular origin, asadenomas. These unusual thyroid tum4variously described as "mucinous aden

"mucinous carcinoma",5 "mucoepidermoid car-. cinoma",4 101214 "mucin producing medullary car-

cinoma"126 81318 "clear cell variant of thyroidV carcinoma with mucin secretion",9 "follicular, papil-

lary, medullary and anaplastic carcinomas withi1 mucinous substances",15 and "'mucin producing

microfollicular adenoma with signet ring cell pat-jFterns16 Other articles, however, have expressed

- contradictory opinions on the presence or absence ofmucin secretion in primary thyroid tumours.23 25 Inall the above reports the diagnosis of "mucin secre-tion" in these unusual neoplasms has relied on theapparent positivity of different histochemical meth-ods, the latter more often conventionally used for thestudy of epithelial mucins produced by goblet cells ofthe gastrointestinal or bronchial tract.A brief reminder of the histochemical classification

Si-tV m of epithelial mucins is pertinent. Epithelial mucinssecreted by goblet cells lining the gastrointestinaltract, bronchus, or endocervix are complex mucus

; b glycoproteins of high molecular weight.2 18 Mucinsare formed by a central protein core, to whichdifferent carbohydrates are attached, in the form of

4 oligosaccharide side chains. These carbohydrates (70to 90% by weight of mucins), vary in composition,length, branching, acidity and reactive groups. Ineffect the various histochemical methods used for epi-thelial mucin identification react directly with thesecarbohydrates. Depending on the type of carbo-hydrates present, epithelial mucins are classified his-tochemically into two broad groups namely, acidic

dand neutral mucins.22 28 29 Acidic mucins are divided7ombined high into sialomucins (with carbohydrates containing

sialic acid) and sulphomucins (with carbohydratesilar material- containing sulphate reactive groups). Enzymelar material- digestion by sialidase may further subdivide sialo-[ase digestion), mucins into sialidase labile (N-acetyl) and sialidase

resistant (0-acetyl) sialomucins. Sulphomucins areonfined to. the either strongly or weakly sulphated. Neutral mucinstissue papillae contain free hexoses but are devoid of acidic reactive55%). Foreign groups, such as sialic acid or sulphate radicals.staining of At least two types of difficulties are encounteredinective tissue, when reviewing previously published reports ondigestion with "mucin secreting"' or "mucinous"' primary thyroid'ith our study. tumours' 1823-25: problems of methods and of

interpretation.

"mucin secret-of the thyroid1de- 9 These)orts and a fewcarcinomas ofwell as benignours have beenocarcinoma",3

PROBLEMS OF METHODOLOGYVarious histochemical methods for showing the pres-ence of mucins have been used, although differentauthors have not necessarily performed the sametechniques. The PAS reaction used sin-gly,124-79 111215-171923-25 the mucicarminetechnique,3-6 8-12 14 15 1719 23 -25 and the copperphthalocyanin basic dye alcian blue (AB) usedsingly1 2 7 12 14 16 17 24 have been the most popular.

892

R

'Pooer

40",

Ok-

copyright. on O

ctober 25, 2020 by guest. Protected by

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.40.8.890 on 1 A

ugust 1987. Dow

nloaded from

Mucin secreting and mucinous primary thyroid carcinomas

Colloidal iron has been used in two reports."1 25 Twoother basic dyes, astra blue and alcian green (amixture of AB and alcian yellow), have each beenapplied on one occasion.1525 Some authors haveused the combined histochemical method ofAB/PAS.10 13 14 16 18 HID/AB, sialidase digestionfollowed by AB (Sial-AB), and the periodic acid-sodium borohydride saponification/periodic acid Schiffreaction (PB/KOH/PAS) have only been used once. 16

Precise information about certain crucial aspects orsteps of these histochemical methods has been lackingin some of the above papers, particularly the type offixative used'5 1"l 517-1923-25 and the pH of theAB solutions, the latter used singly or in combinationwith PAS.1 2 9 17 19 Prompt and optimal fixation isimportant for showing the presence of epithelialmucins; acqueous formalin fixatives aresatisfactory22 28 and certainly less deleterious forglycoproteins than picric acid fixatives. The specificaffinity of the carboxyl and sulphate groups acidicmucins for AB and the resulting staining intensityshown in tissue sections depend on full ionisation ofthese reactive groups, hence on the pH used. At pH2-5, sialidase labile sialomucins and weakly sulphatedsulphomucins will stain well with AB, but sialidaseresistant sialomucins and strongly sulphated sulpho-mucins will react better with AB at a lower pH (1-0and 1 5, respectively).28 When PAS was used singly,authors did not always make it clear whether priordigestion with diastase had been carried out to elimi-nate glycogen.' 2 12 15 Used singly, PAS is a usefulgeneral but non-specific indicator of epithelial mucinsin tissues. PAS will react positively withneutral mucins and sialidase labile sialomucins, butnegatively with sialidase resistant sialomucins;furthermore, PAS will give a variable stainingreaction with weakly sulphated sulphomucins.222829As mentioned earlier, mucicarmine has been exten-

sively used in most reports on "mucin secreting" and"mucinous" primary tumours of the thyroid. Muci-carmine, however, will stain some but not allmucins.28 29 Sialomucins and weakly sulphatedsulphomucins give positive results with mucicarmine,but neutral mucins and strongly sulphated sulpho-mucins tend to be negative with this stain. Recently,Cook questioned if mucicarmine had a real place inthe contemporary armamentrium of histochemicalmethods for mucins.28 He also suggested that thecombined AB/PAS technique, "will establish thepresence or absence of tissue mucins more certainlyand more informatively" (than mucicarmine). Fur-thermore, Drury and Wallington29 pointed out thatthe preparation of an optimally avid mucicarminestaining solution is difficult and may vary, dependingon batches; commercial mucicarmine tends to be lesssatisfactory than mucicarmine prepared in the labora-

tory. The pitfalls of mucicarmine staining areexemplified by the negative findings of two earlier andoften cited studies on "mucin secreting" or "mucin-ous" primary thyroid carcinomas.30 In contrast tothe apparent positivity of the mucicarmine techniquereported in recent papers,368'2 1415171923-25Foster and Levine30 and Klinick,3' searching for"mucins", failed to show carminophilic material withmucicarmine in their two extensive series of primarythyroid carcinomas.

PROBLEMS OF INTERPRETATIONPreviously published reports''9 have based theirclaim for "mucin secretion" in primary thyroidtumours, essentially on positive staining with theabove histochemical methods. One important fact,however, has largely been ignored. Humanthyroglobulin-stored as "colloid" in thyroidfollicles-contains different carbohydrates, suchas neutral sugar, acetyl hexosamine, sialic acidand fucose,32 representing 2 to 4% by weight ofthyroglobulin. The carbohydrates contained inthyroglobulin and colloid, or their breakdownproducts, could well be responsible for the positivestaining observed in the thyroid tumours reported as"mucin secreting" or "mucinous". Two findingsfavour this hypothesis. Firstly, "mucin production",whether intracellular or extracellular, has generallybeen described as scarce and scattered in most ofthese thyroid tumours.''1923 There has been onlyone instance in which pools of abundant extracellularmucin were noted.3 This observation would be inkeeping with the known low sugar contents of colloid;an excess of these carbohydrates, however, due toincreased tumour cell turnover, could still be envis-aged. Secondly, some authors have shown that the"mucinous" material stained by mucin techniquesalso shows positive immunostaining with thyro-globulin antiserum."16 23The above interpretation of "mucin secretion",

related to the carbohydrate contents of thyroglobulinand colloid, could understandably apply to thyroidtumours of follicular origin. But what about cases of"mucin producing" medullary carcinoma? It has beenincreasingly recognised that thyroglobulin immu-noreaction can be observed in medullary carcinoma,and several cases of so called "mixed medullary fol-licular thyroid carcinoma" have now beenreported.3336 The origin of this thyroglobulinimmunoreaction in medullary carcinoma, however, isyet unresolved: direct synthesis34 36 orphagocytosis3335 of thyroglobulin by neoplastic Ccells. When reviewing those previously publishedcases of "mucin producing" medullary carcinoma,which were also studied by immu-

893

copyright. on O

ctober 25, 2020 by guest. Protected by

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.40.8.890 on 1 A

ugust 1987. Dow

nloaded from

894 Rigaud, Bogomoletznohistochemistry,6 8 it is somewhat surprising torealise that only calcitonin immunostaining had beenperformed and that thyroglobulin had apparently notbeen tested.

Conventional electron microscopy has also beenperformed in a few instances of "mucin secreting" or"mucinous" primary thyroid tumours.' - 72 13 Theinterpretation of the ultrastructural nature of intra-cytoplasmic "mucinous" material, however, has oftenbeen based on the assumption that prior histo-chemical methods had identified mucins as such. Thepossibility that "clear vesicles",5 "mucus granules",6"mucin like vacuoles",7 "mucin like droplets"'12 and"vesicles of mucus"'13 could actually representthyroglobulin or colloid has not been fully enter-tained in these electron microscopic studies.

In our present study 40 cases of primary carcino-mas of the thyroid (papillary, follicular, anaplastic,and medullary) were stained with AB/PAS andHID/AB, according to a strict methodology. The pos-itive pure and mixed alcianophilia observed in 7.5%and about 50%, respectively, of our material could beinterpreted as representing genuine mixtures of acidicand neutral "mucin". The histogenesis of such gen-uine "mucin secretion" in thyroid tumours, however,remains difficult to explain. Of course, dualdifferentiation-that is, combination of mucin secre-tion and endocrine function-could be envisaged forthese unusual thyroid neoplasms."1 16 Certainly, it isconceivable that neoplastic follicular and para-follicular cells of the thyroid could undergo dualdifferentiation like other endodermally derived neo-plasms (lung, pancreas, or gastrointestinal tract)."

Nevertheless, because of existing problems ofmethodology and interpretation, we feel that the evi-dence published so far to substantiate the concept ofgenuine "'mucin secreting" or "mucinous" primarythyroid tumours is not entirely convincing. This evi-dence is therefore also insufficient for comparingthese thyroid tumours with conventional mucinsecreting neoplasms derived from gastrointestinalor bronchial origin. Carbohydrates contained inthyroglobulin or colloid could well be responsible forthe apparent "positive" staining obtained with theconventional histochemical methods for mucins, andthis could apply to thyroid tumours of both follicularand C cell origin.Addendum

Since this paper was accepted for publication, twoadditional cases of signet ring cell "mucinous" ade-nomas of the thyroid have been described.3 Theauthor used 18 different histochemical methods (someproducing overlapping results), coupled with thyr-oglobulin immunostaining, and his overall conclusionlends support to our present study.

We thank Mrs MJ Bianchi for secretarial assistance,and Mrs MG Durand and Mrs B Didier for technicalassistance. We are also grateful to Dr MI Filipe (Lon-don) for reading the manuscript and for valuablecomments.

References

I Williams ED, Brown CL, Doniach I. Pathological and clinicalfindings in a series of 67 cases of medullary carcinoma of thethyroid. J Clin Pathol 1966;19:103-13.

2 Milhaud G, Calmettes C, Dreyfuss G, Mouktar MS. An unusualtrabecular thyroid cancer producing calcitonin. Experientia1970;26:1381-3.

3 Diaz-Perez R, Quiroz H, Nishiyama RH. Primary mucinousadenocarcinoma of the thyroid gland. Cancer 1976;38:1323-5.

4 Rhatigan RM, Roque JL, Bucher RL. Mucoepidermoidcarcinoma of the thyroid gland. Cancer 1977;39:210-14.

5 Deligdish L, Subhani Z, Gordon RE. Primary mucinouscarcinoma of the thyroid. Report of a case and ultrastructuralstudy. Cancer 1980;45:2564-7.

6 Fernandes BJ, Bedard YC, Rosen I. Mucus-producing medullarycell carcinoma of the thyroid gland. Am J Clin Pathol1982;78:536-40.

7 Martinelli G, Bazzochi F, Govoni E, Santini D. Anaplastic typeof medullary thyroid carcinoma. An ultrastructural and immu-nohistochemical study. Virchows Arch (Pathol Anat)1983;400:61-7.

8 Zaatari GS, Saigo PE, Huvos AG. Mucin production in medul-lary carcinoma of the thyroid. Arch Pathol Lab Med1983;107:70-4.

9 Civantos F, Albores-Saavedra J, Nadji M, Morales AR. Clearcell variant of thyroid carcinoma. Am J Surg Pathol1984;8:187-92.

10 Fransilla KO, Harach HR, Wasenius VM. Mucoepidermoidcarcinoma of the thyroid. Histopathology 1984;8:847-60.

11 Mendelsohn G. Signet-cell-simulating microfollicular adenomaof the thyroid. Am J Surg Pathol 1984;8:705-8.

12 Mizukami Y, Matsubara F, Hashimoto T, et al. Primarymucoepidermoid carcinoma in the thyroid gland. A case reportincluding an ultrastructural and biochemical study. Cancer1984;53:1741-5.

13 Golouh R, Us-Krasovec M, Auerspers M, Jancar J, Bondi A,Eusebi V. Amphircine-composite calcitonin and mucin-producing-carcinoma of the thyroid. Ultrastruct Pathol1985;8:197-206.

14 Harach RH. A study on the relationship between solid cell nestsand mucoepidermoid carcinoma of the thyroid. Histopathology1985;9:195-207.

15 Mlynek ML, Richter HJ, Leder LD. Mucin in carcinomas of thethyroid. Cancer 1985;56:2647-50.

16 Rigaud C, Peltier F, Bogomoletz WV. Mucin producing micro-follicular adenoma of the thyroid. J Clin Pathol1985;38:277-80.

17 Sobrinho-Simoes M, Nesland JM, Johannessen JV. A mucin-producing tumour in the thyroid gland. Ultrastruct Pathol1985;9:277-81.

18 Uribe M, Fenoglio-Preiser CM. Medullary carcinoma of thethyroid gland. Clinical, pathological and immunohisto-chemical features with review of the literature. Am J SurgPathol 1985;9:577-94.

19 Sobrinho-Simoes M, Stenwig AE, Nesland JM, Holm R,Johannessen JV. A mucinous carcinoma of the thyroid. PathRes Pract 1986;181:464-9.

20 Meissner WA, Warren S. Twnours ofthe thyroid gland. 2nd series.Washington, DC: Armed Forces Institute of Pathology, 1968.

21 Hedinger C. Histological typing of thyroid tumours. Geneva:World Health Organisation, 1974.

copyright. on O

ctober 25, 2020 by guest. Protected by

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.40.8.890 on 1 A

ugust 1987. Dow

nloaded from

Mucin secreting and mucinous primary thyroid carcinomas 89522 Filipe MI, Lake BD. Histochemistry in pathology. Edinburgh:

Churchill Livingstone, 1983:310-3.23 Carcangiu ML, Sibley RK, Rosai J. Clear cell change in primary

thyroid tumors. A study of 38 cases. Am J Surg Pathol1985;9:705-22.

24 Dayal Y, Uccik AA, Safaii H, Pohl D. Thyroglobulin and clear-cell tumors. Am J Surg Pathol 1986;10:70-2.

25 Schr6der S, Bocker W. Signet-ring-cell thyroid tumors. Folliclecell tumors with arrest of folliculogenesis. Am J Surg Pathol1985;9:619-29.

26 Carlstedt I, Sheehan JK, Corfield AP, Gallagher JT. Mucousglycoproteins: a gel of a problem. Essays in Biochemistry1985;20:40-76.

27 Laboisse CL. Structure of gastrointestinal mucins: searching forthe Rosetta stone. Biochimie 1986;68:611-7.

28 Cook HC. Carbohydrates. In: Bancroft JD, Stevens A, eds.Theory and practice of histological techniques, 2nd ed.Edinburgh: Churchill Livingstone, 1982:180-216.

29 Drury RAB, Wallington EA. Carbohydrates andmucosubstances. In: Carleton's histological technique. 5th ed.Oxford: Oxford University Press, 1980:232-59.

30 Foster EA, Levine AJ. Mucin production in metastatic car-cinoma. Cancer 1963;10:506-9.

31 Klinck GH. Structure of the thyroid gland. In: Hazard JB,Smith DE, eds. The thyroid. Baltimore: Williams & Wilkins,

1964:9.32 Fasman GD. Handbook of biochemistry and molecular biology.

3rd ed. Proteins. Vol II. Cleveland: CRC Press, 1976:271-3.33 DeLellis RA, Moore FM, Wolfe HJ. Thyroglobulin immuno-

reactivity in human medullary carcinoma. Lab Invest1983;48:20A.

34 Uribe M, Fenoglio-Preiser CM, Grimes M, Feind C. Medullarycarcinoma of the thyroid gland. Clinical, pathological, andimmunohistochemical features with review of the literature.Am J Surg Pathol 1985;9:577-94.

35 Perrone T. Mixed medullary-follicular thyroid carcinoma. Am JSurg Pathol 1986;10:362-3.

36 Holm R, Sobrinho-Simoes M, Nesland M, Johannessen JV.Concurrent production of calcitonin and thyroglobulin in thesame neoplastic cells. Ultrastruct Pathol 1986;10:241-8.

37 Gherardi G. Signet ring cell `mucinous" thyroid adenoma: a fol-licle cell tumour with abnormal accumulation of thyroglobulinand a peculiar histochemical profile. Histopathology1987;11:317-26.

Requests for reprints: Dr C Rigaud, Laboratoired'Anatomie Pathologique, Institut Jean Godinot, BP 171,51056 Reims Cedex, France.

copyright. on O

ctober 25, 2020 by guest. Protected by

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.40.8.890 on 1 A

ugust 1987. Dow

nloaded from