Prolactin-producing pituitary adenoma associated with prolactin cell hyperplasia

PRL Cell Adenoma and Hyperplasia 157Case Report

157

Department of LaboratoryMedicine (SV, EH, SC, KK),St. Michael’s Hospital, Toronto,Ontario, Canada; Departmentof Anatomy (SV), Laboratory ofHistology, University of Santiagode Compostela, Lugo, Spain;Departments of NeurosurgeryClinica Medellin (LVS),Hospital Pablo Tobon Uribe andUniversidad de Antioquia (HU),Medellin, Colombia.

Address correspondence toDr. Sergio Vidal, Department ofLaboratory Medicine, Divisionof Pathology, St. Michael’sHospital, 30 Bond Street,Toronto, Ontario, M5B 1W8,Canada. E-mail: [email protected]

Endocrine Pathology, vol. 13,no. 2, 157–165, Summer 2002© Copyright 2002 by HumanaPress Inc. All rights of anynature whatsoever reserved.1046–3976/02/13:157–165/$12.25

Prolactin-Producing Pituitary AdenomaAssociated with Prolactin Cell Hyperplasia

Sergio Vidal, DVM, Eva Horvath, PHD, Luis V. Syro, MD,Humberto Uribe, MD, Sandy Cohen, RT,and Kalman Kovacs, MD, PHD

AbstractA 24-yr-old woman with amenorrhea, galactorrhea, hyperprolactinemia, and sellar massunderwent transsphenoidal surgery. Histologic, immunohistochemical, and electronmicroscopic investigation revealed a well-differentiated, sparsely granulated prolactin(PRL) cell adenoma of the pituitary showing conclusive PRL immunoreactivity. In the nontu-morous adenohypophysis PRL cell hyperplasia was noted. Marked differences were evi-dent between the neoplastic and hyperplastic areas. The tumor consisted of sparselygranulated PRL cells immunoreactive only for PRL. As demonstrated by immunoelectronmicroscopy, the hyperplastic area comprised monohormonal sparsely granulated PRL cellsas well as bihormonal mammosomatotrophs immunoreactive for both PRL and growthhormone. The MIB-1 index was higher whereas microvessel density was lower in theadenoma as compared with the hyperplastic area. In addition, the nontumorous areashowed lymphocytic infiltration whereas inflammatory reaction was not seen in theadenoma. This case represents a rare association of a PRL cell adenoma and PRL cellhyperplasia. The fact that these two lesions were contiguous in the surgically removedmaterial raises the possibility that hyperplasia can precede and transform into adenoma.Key Words: Prolactin adenoma; pituitary hyperplasia; human; electron microscopy;immunohistochemistry.

changes implying feedback inhibition. Onetype is the Crooke’s hyaline change ofnontumorous corticotrophs in the pituitaryharboring a corticotroph adenoma inter-preted as the result of the negative feed-back effect of elevated blood glucocorticoidlevels [2]. The cell type, the morphologyof which is the most responsive to chang-ing functional demands, is the prolactin(PRL) cell. Accordingly, nontumorouslactotrophs display morphologic signs ofmarked functional suppression in the pres-ence of a PRL cell adenoma [3].

The case presented herein demonstratesthe rare coexistence of a PRL-producingpituitary adenoma and PRL cell hyperpla-sia. Immunohistochemical markers of cell

Introduction

Based on endocrine activity, pituitarytumors can be classified as functioning andnonfunctioning. Functioning tumors pro-duce and secrete hormones in excess,resulting in complex hypersecretory syn-dromes. Nonfunctioning tumors are hor-monally inactive, presenting as expandingsellar masses often accompanied by variousdegrees of hypopituitarism [1]. Function-ing pituitary adenomas are autonomoustumors and are relatively unresponsive tothe negative feedback effect of target glandhormones. By contrast, the appropriatenontumorous phenotypes outside theadenoma may show various morphologic

158 Endocrine Pathology Volume 13, Number 2 Summer 2002

proliferation and angiogenesis as well asimmunoelectron microscopy were used toclarify the behavior of this biphasic pitu-itary lesion. The possible histogenesis is alsodiscussed.

Report of a Case

Pituitary tissue was surgically removedfrom a 24-yr-old woman by using thetranssphenoidal approach, at the ClinicaMedellin, Medellin, Columbia. She had a2-yr history of amenorrhea and galactor-rhea. Magnetic resonance imaging dis-closed a sellar tumor of 15 mm withoutinvasion to the cavernous sinuses. Blood lev-els showed hyperprolactinemia at 286 ng/mL (normal: 3–18 ng/mL). She did nottolerate bromocriptine.

Materials and Methods

For histology, tissue obtained at surgerywas fixed in 10% buffered formalinimmediately after removal, dehydrated ingraded ethanol, paraffin embedded, andsectioned at 5 µm for histology andimmunohistochemistry. The sections werestained with the hematoxylin and eosin,periodic acid-Schiff (PAS), and Gordon-Sweetsilver methods. For immunohistochemicalstudies, the streptavidin-biotin-peroxidasecomplex technique was applied usingantibodies raised against growth hormone(GH), PRL, adrenocorticotropic hormone(ACTH), thyroid-stimulating hormone β(TSHβ), follicle-stimulating hormone β(FSHβ), luteinizing hormone β (LHβ),α-subunit of the glycoprotein hormones,S100 protein, and glial fibrillary acidic pro-tein (GFAP). The proliferative activity ofpituitary cells was evaluated using themonoclonal antibody (MAb) MIB-1,which reacts with the Ki-67 nuclear anti-gen present in proliferating cells. Vascular-

ization was determined using a specificMAb against CD-34, a sensitive marker ofendothelial cells. Details of immunohis-tochemical methods, sources of supplies,dilutions of antibodies, and control proce-dures have been described previously [4,5].The MIB-1 indices and microvessel densi-ties were quantitated as reported earlier [6].

For electron microscopy, small frag-ments of tissue were fixed in 2.5% glut-araldehyde, postfixed in 1% osmiumtetroxide, dehydrated in a series of gradedethanol, processed through propyleneoxide, and embedded in an Epon-Aralditemixture. The ultrathin sections werestained with uranyl acetate and lead cit-rate and examined with a Philips 410 LStransmission electron microscope.

Immunoelectron microscopy was per-formed using the double-immunogoldmethod of Bendayan [7] localizing GH andPRL in the same section. Details of theprocedure have been previously described[8]. After immunolabeling, sections werestained with uranyl acetate and examinedwith a Philips 410 LS electron microscope.

Results

Histology and Immunohistochemistry

By light microscopy, the submittedtissue consisted of a chromophobic,slightly acidophilic PAS-negative pituitaryadenoma and small fragments of nontu-morous adenohypophysis. The tumorshowed a diffuse pattern with no majorcellular and nuclear pleomorphism. Mitoticfigures were rare. The tumor was welldemarcated from the nontumorous adeno-hypophysis. In one nontumorous frag-ment, focal mononuclear cell infiltrationconsisting mainly of lymphocytes wasapparent. This alteration was similar to thefocal lymphocytic infiltrates of no appar-ent pathophysiologic significance seen in

PRL Cell Adenoma and Hyperplasia 159

various types of adenoma [9,10]. Thestreptavidin-biotin-peroxidase complexmethod demonstrated conclusive immuno-positivity for PRL in the cytoplasm ofalmost all adenoma cells. The immunopre-cipitate was located mainly in the conspicu-ous perinuclear Golgi zone, a characteristicfeature of PRL-secreting pituitary adenomas.Immunostaining within the tumor cellswas negative for GH, ACTH, TSHβ,FSHβ, LHβ, α-subunit, S-100 protein,and GFAP.

The cells in the nontumorous fragmentswere immunopositive for the variousadenohypophysial hormones, whereas S-100protein and GFAP immunostaining dem-

onstrated scattered positivity. Massivestaining for PRL was demonstrated in oneof the nontumorous fragments, whichshowed enlarged acini corresponding tohyperplasia (Fig. 1). In the same fragment,scattered GH immunopositive cells werealso apparent.

MIB-1 immunostaining was detected inthe nuclei of some cells in both neoplasticand nontumorous fragments (Fig. 2). Suchimmunoreactive nuclei were easily identi-fied and quantified. Marked differences inMIB-1 labeling index were observed betweenPRL cell adenoma and nontumorous frag-ments (3.3 vs 0.2%).

Stains for CD34 showed the reaction tobe confined entirely to the endothelial lin-ing of the capillaries (Fig. 3). Morphomet-ric study demonstrated that microvesseldensity of the PRL-producing adenomawas lower (p < 0.05) than that of the non-tumorous fragments (1.5 vs 15.8%).

Electron Microscopy

The electron microscopy specimen con-tained two different areas: adenoma andnontumorous adenohypophysis. The tumorshowed the ultrastructural features of ahighly differentiated, sparsely granulatedPRL cell adenoma of the pituitary (Fig. 4).The adenoma cells were slightly larger thanmidsized, closely apposed, and rounded.The uniform, spherical, or slightly elon-gated nuclei contained a well-devel-oped nucleolus and small amounts ofstippled heterochromatin. The well-orga-nized lamellar rough endoplasmic reticu-lum (RER) was abundant. The extensivelydeveloped Golgi apparatus harborednumerous developing secretory granules,whereas storage granules were scant out-side the Golgi region. Granule extrusionswere readily noted, and the mitochondriawere unremarkable.

The fragments of nontumorous adeno-hypophysis contained several well-pre-

Fig. 1. Reticulin pattern in (A) PRL cell adenoma and (B) adjacent PRL cell hyper-plasia showing a few normal sized acini as well as enlarged, apparently confluentacini. Original magnification: ×100.


Fig. 2. MIB-1 immunoreactivity in (A) PRL cell adenoma and (B) PRL cell hyperplasia. Original magnifica-tion: ×400.Fig. 3. Immunostaining for CD34 in (A) PRL cell adenoma and (B) PRL cell hyperplasia. Original magnifica-tion: ×400.


served acini. Instead of the small, some-times barely recognizable, suppressednontumorous PRL cells usually seen inpituitaries harboring a PRL-producingadenoma, the acini were larger and con-tained numerous nonsuppressed PRL cells,

indicating PRL cell hyperplasia (Fig. 5).The PRL cells in the nontumorous adeno-hypophysis possessed a well-developedRER and Golgi complex. The secretorygranules were more numerous than in theadenoma cells. Granule extrusions were

Fig. 4. (top left) PRL cell adenoma. The tumor cells, having abundant RER, prominent, active Golgi complex, and “misplaced exocytosis”(arrows), display remarkable uniformity. Magnification: ×9700. Fig. 5. (top right) PRL cell hyperplasia in nontumorous adenohypophysis.Magnification: ×5300. Fig. 6. (bottom left) Nontumorous adenohypophysis. The two adjacent cells shown have secretory granules of thesame size. They label only for PRL in one cell but for both PRL and GH in the other. Magnification: ×27,550. Fig. 7. (bottom right) Nontumorousadenohypophysis. Adjacent to an immunonegative cell, the large secretory granules of a mammosomatotroph labeling for both GH andPRL are depicted. Magnification: ×30,150.


rare. In one area of the nontumorousadenohypophysis, a fairly intense accumu-lation of lymphocytes was noted. Macro-phages and mast cells were also recognized.The basal lamina was dissolved focally,and few lymphocytes were attached toPRL cells.

Immunoelectron microscopy usingdouble immunogold labeling for PRL andGH confirmed the ultrastructural findings.The monomorphous adenoma cells wereexclusively labeled for PRL, whereasmonohormonal PRL cells as well as fre-quent bihormonal mammosomatotrophswere identified in the nontumorous frag-ments (Figs. 6 and 7).

Discussion

The idiopathic form of PRL cell hyper-plasia is very rare in surgical material, andits association with PRL cell adenoma hasbeen reported only in a few cases ofautopsy-obtained pituitaries [11]. In thenontumorous adenohypophysis, an abnor-mal increase in the number of a specificcell type (i.e., hyperplasia) is a well-knownadaptation mechanism in situations requir-ing hypersecretion of a specific hormone.Hyperplasia can affect every pituitary celltype. Thyrotroph hyperplasia develops inpatients with protracted primary hypothy-roidism [12,13], corticotroph hyperplasiacan be demonstrated in the pituitaries ofpatients with untreated Addison diseaseand with corticotropin-releasing hormoneproducing extrapituitary tumors [14,15],somatotroph hyperplasia occurs in associa-tion with ectopic production of growthhormone–releasing hormone [16], andgonadotroph hyperplasia has been describedin relation to primary hypogonadism [17].Among pituitary cell types, hyperplasia ofPRL cells is the most common. It developsin response to the physiologic stimuli of

pregnancy and lactation, but also occursunder several other conditions such asestrogen administration, primary hypothy-roidism; or possibly compression ortranssection of the pituitary stalk, calledstalk section effect [13,18–21]. It has alsobeen demonstrated that hyperplastic PRLcells may derive from somatotrophs bytransdifferentiation [18]. The case pre-sented herein has the hallmarks of transdif-ferentiation: (1) there is the presence offrequent bihormonal cells labeling for bothGH and PRL, and (2) the immunoreac-tivities are not always true to phenotypeprobably because the morphologic changesare lagging behind the functional ones [18].

In the present case, the temporal rela-tionship between PRL cell hyperplasia andadenoma remains uncertain. The hyper-plastic area may represent a preneoplasticlesion, a potential precursor of neoplasia.Neoplastic transformation of PRL cellhyperplasia has been demonstrated in casesof protracted estrogen stimulation inrodents [22]. It has also been reported thatPRL cell adenomas develop after preexist-ing PRL hyperplasia in dopamine recep-tor (D

2) knockout mice [23]. The role of

PRL cell hyperplasia is suggestive in theinduction of pituitary tumors of trans-sexual men undergoing protracted estro-gen administration or in patients withsuprasellar lesions interfering with the syn-thesis, release, or transport of hypothalamicdopamine [19,21]. Although the molecu-lar events associated with the hyperplasia/neoplasia sequence are not well under-stood, it is generally assumed that hyper-plastic proliferating cells are at increasedrisk of genetic mutation, and alterationsin gene expression and chromosome struc-ture during mitosis. It was proposed thatgenetic alterations may be responsible forneoplastic transformation by activatingoncogenes, such as ras, and inactivatingtumor suppressor genes, such as the p-53


or the von Hippel–Lindau gene. Mutationmay also lead to disregulation in theexpression of cell-cycle-related proteins.It may well be that in the present case trans-formation of hyperplastic PRL cells toadenoma cells was owing to altered geneexpression [24–27].

Alternatively, the production of growthfactors may contribute to the neoplastictransformation within the hyperplasia.Missale et al. [28] have demonstrated thatoverexpression of transforming growth fac-tor-α and loss of nerve growth factor playimportant roles in the development andprogression of PRL-producing pituitaryadenomas.

Differences in cell proliferation andmicrovessel density between hyperplasticand neoplastic areas were striking in ourcase, indicating that hyperplasia and neo-plasia are well-defined distinct conditions.These findings contradict the recentlyemerging views attempting to merge thetwo pathologic processes. Based on theirthyroid studies, Derwahl and Studer [29]generalize and state that “most excessivegrowths in endocrine tissues are not sec-ondary to oversecretion of trophic hor-mones but are true, autonomous, benignneoplastic growths and there is no funda-mental difference between hyperplasia andadenoma.” In the human, pituitaryhyperplasias are histologically and, in sometypes, ultrastructurally well characterized.They occur in every cell type, but at mark-edly different rates. Pituitary adenomas arealso histologically and ultrastructurallywell-classified entities distinctly differentfrom hyperplastic proliferations. Further-more there is no correlation between fre-quencies of hyperplasias and adenomas ofcorresponding cell types. For example,hyperplasia of pars intermedia–derivedproopiomelanocortin cells is common,whereas their tumors (silent “corticotroph”adenomas) are rare [30]. Similarly, true

hyperplasia of gonadotrophs is rare [17],contrasted by the high frequency ofgonadotroph adenomas [3]. We agree withDerwahl and Studer [29] that hyperplasiais not a steady state, but an ongoing pro-cess with potential for neoplastic transfor-mation [17]. If an adenoma is formed,regardless of the circumstances, it is fun-damentally different from nonneoplasticproliferation not only in its causation andmorphology, but also in its prognosis andclinical management.

Acknowledgments

We are indebted to Vidia Beharry, SandyBriggs, and Fabio Rotondo for technicalassistance, as well as the staff of St. Michael’sHospital Health Sciences Library for theircontribution to this study. We are alsograteful to the National Pituitary HormoneDistribution Agency for the kind donationof human pituitary antisera. This work wassupported in part by the generous dona-tions of the Jarislowsky Foundation, theLloyd Carr-Harris Foundation, and (forDrs. L. V. Syro and H. Uribe) CoomevaE.P.S., Medellin, Colombia. Dr SergioVidal was supported by a research grantfrom the University of Santiago deCompostela, Spain.

References

1. Kovacs K, Scheithauer BW, Horvath E, LloydRV. The World Health Organization classifi-cation of adenohypophysial neoplasms: a pro-posed five-tier scheme. Cancer 78:502–510,1996.

2. Kovacs K. The pathology of Cushing’s disease.J Steroid Biochem Mol Biol 45:179–182,1993.

3. Horvath E, Kovacs K. The adenohypophysis.In: Kovacs K, Asa SL, eds. Functional endo-crine pathology. 2nd ed. Malden, MA:Blackwell Science, 1998; 247–281.

4. Kovacs K, Stefaneanu L, Horvath E, LloydRV, Lancranjan I, Buchfelder M, Fahlbusch


R. Effect of dopamine agonist medication onprolactin producing pituitary adenomas:a morphological study including immuno-cytochemistry, electron microscopy and in situhybridization. Virchows Arch A Pathol AnatHistopathol 418:439–446, 1991.

5. Kovacs K, Lloyd R, Horvath E, Asa SL,Stefaneanu L, Killinger DW, Smyth HS. Silentsomatotroph adenomas of the human pitu-itary: a morphologic study of three casesincluding immunocytochemistry, electronmicroscopy, in vitro examination, and in situhybridization. Am J Pathol 134:345–353,1989.

6. Vidal S, Kovacs K, Horvath E, ScheithauerBW, Kuroki T, Lloyd RV. Microvessel densityin pituitary adenomas and carcinomas.Virchows Arch 438:595–602, 2001.

7. Bendayan M. Double immunocytochemicallabeling applying the protein A-gold tech-nique. J Histochem Cytochem 30:81–85,1982.

8. Vidal S, Horvath E, Kovacs K. Ultrastructuralimmunohistochemistry. In: Lloyd RV, ed.Morphology methods: cell and molecularbiology techniques. Totowa, NJ: Humana,2001; 375–402.

9. McConnon JK, Smyth HS, Horvath E. A caseof sparsely granulated growth hormone celladenoma associated with lymphocytic hypo-physitis. J Endocr Invest 14:691–696, 1991.

10. Heshmati HM, Kujas M, Casanova S, WollanPC, Racadot J, van Effenterre R, Derome PJ,Turpin G. Prevalence of lymphocytic infiltratein 1400 pituitary adenomas. Endocr J45:357–361, 1998.

11. Kovacs K, Corenblum B, Sirek AM, Penz G,Ezrin C. Localization of prolactin in chro-mophobe pituitary adenomas: study of humannecropsy material by immunoperoxidase tech-nique. J Clin Pathol 29:250–258, 1976.

12. Scheithauer BW, Kovacs KT, Young WF Jr,Randall RV. The pituitary gland in hyperthy-roidism. Mayo Clin Proc 67:22–26, 1992.

13. Scheithauer BW, Kovacs K, Randall RV, RyanN. Pituitary gland in hypothyroidism: histo-logic and immunocytologic study. Arch PatholLab Med 109:499–504, 1985.

14. Scheithauer BW, Kovacs K, Randall RV. Thepituitary gland in untreated Addison’s disease:a histologic and immunocytologic study of 18adenohypophyses. Arch Pathol Lab Med107:484–487, 1983.

15. Carey RM, Varma SK, Drake CR Jr, ThornerMO, Kovacs K, Rivier J, Vale W. Ectopicsecretion of corticotropin-releasing factor asa cause of Cushing’s syndrome: a clinical, mor-phologic, and biochemical study. N Engl JMed 311:13–20, 1984.

16. Ezzat S, Asa SL, Stefaneanu L, Whittom R,Smyth HS, Horvath E, Kovacs K, FrohmanLA. Somatotroph hyperplasia without pitu-itary adenoma associated with a long standinggrowth hormone-releasing hormone-produc-ing bronchial carcinoid. J Clin EndocrinolMetab 78:555–560, 1994.

17. Horvath E, Kovacs K, Scheithauer BW. Pitu-itary hyperplasia. Pituitary 1:169–179, 1999.

18. Vidal S, Horvath E, Kovacs K, Lloyd RV,Smyth HS. Reversible transdifferentiation:interconversion of somatotrophs and lacto-trophs in pituitary hyperplasia. Mod Pathol14:20–28, 2001.

19. Cusimano MD, Kovacs K, Bilbao JM, TuckerWS, Singer W. Suprasellar craniopharyngiomaassociated with hyperprolactinemia, pituitarylactotroph hyperplasia, and microprolactinoma:case report. J Neurosurg 69:620–623, 1988.

20. Khalil A, Kovacs K, Sima AA, Burrow GN,Horvath E. Pituitary thyrotroph hyperplasiamimicking prolactin-secreting adenoma.J Endocrinol Invest 7:399–404, 1984.

21. Kovacs K, Stefaneanu L, Ezzat S, Smyth HS.Prolactin-producing pituitary adenoma in amale-to-female transsexual patient with pro-tracted estrogen administration: a morpho-logic study. Arch Pathol Lab Med 118:562–565, 1994.

22. van Nesselrooij JH, Szeverenyi NM, Ritter-Hrncirik C, Tillapaugh-Fay GM, Feron VJ.Rat pituitary changes observed with magneticresonance imaging following removal ofestrogen stimulus: correlation with histopa-thology and immunohistology. Carcinogen-esis 13:277–282, 1992.

23. Asa SL, Kelly MA, Grandy DK, Low MJ.Pituitary lactotroph adenomas develop afterprolonged lactotroph hyperplasia in dopam-ine D2 receptor-deficient mice. Endocrinol-ogy 140:5348–5355, 1999.

24. Schulte HM, Oldfield EH, Allolio B, KatzDA, Berkman RA, Ali IU. Clonal composi-tion of pituitary adenomas in patients withCushing’s disease: determination by X-chro-mosome inactivation analysis. J Clin Endo-crinol Metab 73:1302–1308, 1991.


25. Suhardja A, Kovacs K, Rutka J. Genetic basisof pituitary adenoma invasiveness: a review.J Neurooncol 52:195–204, 2001.

26. Suhardja AS, Kovacs KT, Rutka JT. Molecularpathogenesis of pituitary adenomas: a review.Acta Neurochir (Wien) 141:729–736, 1999.

27. Pal S, Claffey KP, Dvorak HF, MukhopadhyayD. The von Hippel–Lindau gene productinhibits vascular permeability factor/vascularendothelial growth factor expression in renal cellcarcinoma by blocking protein kinase C path-ways. J Biol Chem 272:27,509–27,512, 1997.

28. Missale C, Fiorentini C, Finardi A, Spano P.Growth factors in pituitary tumors. Pituitary1:153–158, 1999.

29. Derwahl M, Studer H. Hyperplasia versusadenoma in endocrine tissues: are they dif-ferent? Trends Endocr Metab 13:23–28,2002.

30. Horvath E, Kovacs K, Lloyd RV. Parsintermedia of the human pituitary revisited:morphologic aspects and frequency of hyper-plasia of POMC-peptide immunoreactivecells. Endocr Pathol 10:55–64, 1999.

Documents

Prolactin-producing pituitary adenoma associated with prolactin cell hyperplasia