THE PITUITARY
`
`THIRD EDITION
`Edited by
`
`SHLOMO MELMED
`Senior Vice President, Academic Affairs
`Dean of the Medical Faculty , Helene A. and Philip E. Hixon Chair in Investigative Medicine,
`Cedars~Sinai Medical Center, Los Angeles , CA, USA
`
`ELSEVIER
`
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`

`[
`
`J
`
`16
`
`Cushing's disease
`Xavier Bertagna 1
`2
`, Laurence Guignat 1, Marie ... Charles Raux ... Demay 3
`'
`, Fran~ois Girard 3
`Brigitte Guilhaume 1
`1 Service des Maladies Endocriniennes et Metaboliques, Centre de Reference des Maladies Rares de Ia Surrenale,
`hOpital Cochin, Paris, France 2Departernent Endocrinologie,Diabete, INSERM U,J016, Institut Cochin,
`Faculte de Medecine Paris Descartes, Universite Paris 5, Paris, France, 3 Explorations Fonctionelles Endocriniennes,
`hopital Trousseau, Paris, France.
`
`,
`
`PATHOPHYSIOLOGY
`
`Cushing's syndrome refers to the manifestations of
`chronic glucocorticoid excess and may result from
`various causes (Table 16.1). In Cushing's disease pitui(cid:173)
`tary adrenocorticotropic hormone (ACTH) oversecre(cid:173)
`tion induces bilateral adrenocortical hyperplasia and
`excess production of cortisol, adrenal androgens and
`11-deoxycorticosterone, which together provoke the
`clinical and biologic features of the disease.
`
`EPIDEMIOLOGY
`
`Cushing's disease is the most frequent cause of spon(cid:173)
`taneous Cushing's syndrome in adults. In most series its
`prevalence is approximately 70% with a definite female
`preponderance, the female/male ratio ranging between
`3:1 and 10:1 (1-4]. In our series of 809 adult patients
`with spontaneous Cushing's syndromes, Cushing's
`disease accounts for 68% of the cases, and the female/
`male ratio is 2.8 (Table 16.2). Distribution of the age at
`diagnosis shows a peak in adult females in the 25-
`45-year range (Figure 16.1).
`ln children, the causes of Cushing's syndrome have
`a different distribution. Primary adrenocortical tumors
`are more frequent and Cushing's disease accounts for
`about 50% of the cases. Children with this condition
`are usually older than 9 with an equal sex ratio (5-9).
`Cushing's disease accounts for 50% of Cushing's
`syndrome and is almost always caused by a pituitary
`microadenoma. The commonest age of presentation of
`pediatric Cushing's disease is during adolescence, and
`
`there is a strong predominance of males in prepubertal
`patients. Ectopic ACTH syndrome is extremely rare,
`occurring much less frequently than in adults. Unilateral
`adrenal tumors are an important cause of pediatric
`Cushing's syndrome (about 40%) and are almost always
`adrenal carcinoma in children, with rarely a pure hyper(cid:173)
`cortisolism, but usually associated virilization. Primary
`bilateral adrenocortical hyperplasia is a rare but impor(cid:173)
`tant cause of pediatric Cushing's syndrome, usually
`associated with the Carney complex, and typically
`occurs in adolescence or early adulthood. A total of
`398 cases of pediatric Cushing's syndrome are reported
`in the literature, with 182 Cushing's disease, 11 ectopic
`ACTH syndrome, 164 adrenocortical tumors, 16 Mc(cid:173)
`Cune Albright syndrome and 11 primary bilateral adre(cid:173)
`nocortical hyperplasia. The peak incidence was 14.1
`years in Cushing's d isease, 10.1 years in ectopic ACTH
`syndrome, 4.5 years in adrenocortical tumors, 1.2 in
`McCune-Albright syndrome and 13 in primary bilateral
`adrenocortical hyperplasia [8].
`Cushing's disease is rare; its true incidence, which
`varies with age and sex, is difficult to evaluate. Incidence
`data are available on pituitary [10,11] and adrenocortical
`[12,13] tumors. The prevalence of corticotroph tumors in
`the former [14] and that of Cushing's syndrome in the
`latter [15- 17] provide an indirect means whereby the
`incidence of Cushing's disease may be roughly esti(cid:173)
`mated to be in the range of 1-10 new cases per million
`per year. European population-based studies reported
`an incidence of newly diagnosed Cushing's disease of
`0.7 to 2.4 cases per 1 million inhabitants per year
`[18,19]. In Vizcaya (Spain), the prevalence of known
`cases of Cushing's disease at the end of 1992 was
`
`n..l'ir.it.ry, TMd EJirion, 001: IO.IOI618978.Q.IZ•J80926- I.IOOI6-I
`
`533
`
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`534
`
`16. CUSHING'S DISEASE
`
`TABLE 16.1 Causes of Cushing's Syndrome
`
`SPONTANEOUS
`
`ACTH-dependent
`
`Pituitary ACTH oversecretion
`
`Cushing's disease
`
`Primary corticotroph
`
`Anterior pituitary adenoma
`
`Anterior pituitary mixed adenoma
`
`Anterior pituitary cancer
`
`Ectopic corticotroph adenoma
`
`Multiple endocrine neoplasia type 1
`
`Intermediate lobe pituitary adenoma?
`
`Primary hypothalamic dysfunction
`
`CRH-producing tumor
`
`Hypothalamic CRH-producing tumor
`
`Ectopic CRH-producing tumor
`
`Nonpituitary ACTH oversection
`
`Ectopic ACTH syndrome
`
`Endocrine tumors
`
`Mononuclear cells
`
`Cortisol hyperreactive syndrome?
`
`ACill-independent (Adrenal Cushing's Syndrome)
`
`Unilateral adrenocortical tumors
`
`Adrenocortical adenoma
`
`Adrenocortical carcinoma
`
`Bilateral adrenocortical disorders
`
`Primary pigmented nodular adrenal disease
`
`ACTH-independent macronodular adrenal hyperplasia (AI-MAH)
`
`Gonadal tumors
`
`IATROGENIC
`
`ExogenousGiucocorticoids
`
`Exogenous Cortrosyn
`
`CRH, corticotropin-releasing hormone.
`
`39.1 per million inhabitants [18). According to the
`Nationwide Inpatient Sample database, the largest all(cid:173)
`payer inpatient care database in the US which contains
`data from approximately 8 million discharges annually
`from 1004 hospitals located in 37 states, there were an
`estimated 3525 cases of transsphenoidal resection of
`Cushing's disease between 1993 and 2002 [20].
`Recent data suggest that Cushing's syndrome is more
`common than had previously been thought. In addition
`
`TABLE 16.2 Etiology of 809 Spontaneous Cushing's Syndromes
`in Adults
`
`Number of
`Patients (%)
`
`Female/Male
`Ratio
`
`Cushing's disease
`
`Primary adrenocortical tumor
`
`550 (68)
`
`199 (25)
`
`Benign adrenocortical adenoma
`
`111 (14)
`
`Adrenocortical carcinoma
`
`Ectopic ACTH syndrome
`
`Primary adrenocortical nodular
`dysplasia
`
`88 (11)
`
`58 (7)
`
`2 (0.2)
`
`2.8
`
`4.2
`
`0.5
`
`3.6
`
`1.4
`
`to the classical overt Cushing's syndrome, mild forms of
`Cushing's syndrome (named subclinical Cushing's
`syndrome or occult Cushing's syndrome) have been
`identified in patients with type 2 diabetes [21-24),
`hypertension (25), osteoporosis [26), and subjects with
`an adrenal incidentaloma. The reported prevalence of
`Cushing's syndrome is between 2% and 9.4% in over(cid:173)
`weight type 2 diabetic patients and reaches 10.8% in
`patients with T-scores of 2.5 or less and vertebral
`fractures, although a final diagnosis could not to be
`confirmed in all patients. For instance, definitive mild
`Cushing's syndrome was identified in four patients
`(2%) among 200 overweight, type-2 diabetic patients
`with poor metabolic control (HbA1C > 8%), with three
`Cushing's disease and one surgically proven adrenal
`adenoma. Definitive diagnosis remains to be established
`in seven additional patients (3.5%) [21).
`
`CHRONIC ACTH AND
`PROOPIOMELANOCORTIN
`(POMC) PEPTIDE OVERSECRETION
`BY THE PITUITARY
`
`Normal Synthesis and Secretion of ACTH
`Mechanisms of ACTH Biosynthesis
`The mechanisms of ACTH biosynthesis have been
`fully elucidated in the last 30 years; a high-molecular(cid:173)
`weight ACTH-precursor molecule was identified and
`characterized in the ACTH-producing AtT-20/D16-v
`mouse tumor cell line [27). Recombinant DNA methods
`unravelled the primary structure of this precursor [28) -
`called POMC -
`in many species including humans
`[29-31). This is fully described in Chapter 2.
`The overall mechanism of POMC gene expression in
`man is shown schematically in Figure 16.2; a single
`POMC gene per haploid genome is present on the distal
`region (2p23- 25) of the short arm of chromosome 2
`[32,33]; it consists of three exons, the coding regions
`
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`CHRONIC ACfH AND PROOPIOMELANOCORTIN (POMC) PEPTIDE OVERSECRETION BY THE PITUITARY
`
`535
`
`EXON 1 (87 bp) EXON 2 (152 bp)
`lntron A
`lntron 8
`5' flanking (3.6 kbp)
`(3 kbp)
`-Q::----1~1 _. j
`"~:-..
`.................. sma 1
`/
`1
`"''\:.I (_.........-
`L
`•
`5' noncodingl
`(107 b)
`:
`l
`I '\ f
`-
`Signal 1/i-. •
`P -
`peptide
`(26 aa) ,_ .....
`s-_.. .....
`
`EXON 3 (833 bp)
`
`3' flanking
`I )--+-DNA
`Sal 1""
`Sma 1
`_...._...."'
`I AMA ___ mRNA (1.072 kb)
`IJ' noncoding
`1
`(164 b)
`I
`I
`jPre-POMC
`t
`LPOMC (241 aa)
`
`(801 b)
`
`~r---------------------------~
`60
`
`females
`
`malea
`
`FIGURE 16.1
`disease.
`
`Patient age at the time of diagnosis of Cushing's
`
`Years
`
`(in black) being present only on exons 2 and 3. After the
`splicing of the primary transcript a mature messenger
`RNA (mRNA) of 1072 nucleotides (nt) is generated
`and a poly (A)+ tail of about 200 nt is added. A pre(cid:173)
`POMC molecule
`is first
`translated starting with
`a 26-amino acid signal peptide necessary for the translo(cid:173)
`cation of the nascent protein through the membrane of
`the rough endoplasmic reticulum. Within the Golgi
`apparatus and the secretory granule the POMC mole(cid:173)
`cule undergoes a series of proteolytic cleavages and
`chemical transformations which together result in the
`maturation or processing of the precursor [34]: proteo(cid:173)
`lysis occurs at pairs of basic amino acids. Among the
`nine potential cleavage sites of the human POMC only
`four are utilized in the anterior pituitary, generating
`the N-terminal fragment [35,36], the joining peptide
`[37-39], ACTH [40-42], ~lipotropin (13-LPH), and
`a small amount of y-LPH and 13-endorphin (13-end)
`[42,43]. Other chemical transformations include glyco(cid:173)
`sylation of the N-terminal fragment [44], C-terminal
`amidation of the joining peptide [38,39,45,46], and
`partial phosphorylation of ACTH on Ser31 [47,48]. An
`alternate mode of nonprimate POMC processing takes
`place in the intermediate lobe of the pituitary, releasing
`smaller peptides such as 13-melanocyte-stimulating
`hormone (13-MSH), corticotrophin-like
`intermediate
`lobe peptide (CLIP) and a-MSH [49,50). It does not nor(cid:173)
`mally occur in the human pituitary where the interme(cid:173)
`diate lobe is only fully present in the fetus [51].
`POMC gene expression also occurs in many normal
`nonpituitary tissues [52,53]; it does so at a very low level
`and predominantly through an alternate mode of gene
`transcription (54- 56], generating negligible amounts of
`POMC peptide [57-59]. It is assumed that the highly
`predominant, if not the sole, source of circulating
`
`'\ Thr, 0-glycosylation
`
`'Asn, N-glycosylation
`
`r basic amino acid pair
`
`Schematic view of human proopiomelanocortin
`FIGURE 16.2
`(POMC) gene expression. Black bars denote the protein-coding
`regions of the DNA and messenger RNA (mRNA). Hatched bars
`denote the peptide fragments found in the human anterior pituitary.
`
`ACIH and POMC peptides in humans, under normal
`circumstances, is the anterior pituitary corticotroph cell.
`The coordinate proteolysis of POMC and the equi(cid:173)
`molar secretion of the various POMC peptides has two
`implications: any of the non-ACTH POMC peptides
`can be assayed in blood as an alternate marker of the
`overall pituitary corticotroph activity; a specific pattern
`of POMC peptides is associated with the pituitary corti(cid:173)
`cotroph, and any qualitative abnormality suggests
`a pathologic nonpituitary source [60-62]. Yet, in highly
`aggressive, poorly differentiated pituitary corticotroph
`adenomas, decreased expression of convertases indi(cid:173)
`cates that intact POMC may be secreted; these adenomas
`are therefore often "silent" [61,63].
`
`Regulation of ACTH Secretion
`The normal circadian rhythm of plasma cortisol is
`directly driven by pituitary corticotroph activity
`[64-66]. Its pattern is derived from variations in the
`number and amplitude of episodic ACTH bursts
`[67,68]. Pituitary corticotroph activity increases in the
`second half of the night, around 2-4 a.m., peaks on
`waking and gradually falls during the morning [69].
`Various physical and psychologic stresses can interrupt
`this normal rhythm, at any time, with an acute rise in
`
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`536
`
`16. CUSHING'S DISEASE
`
`ACTH. Both the normal circadian rhythm and the stress(cid:173)
`induced changes are central nervous system (CNS)
`mediated under the primary - although not exclusive
`-
`control of hypothalamic corticotropin-releasing
`hormone (CRH) (70).
`CRH [71] acts directly on the corticotroph cell through
`specific receptors that activate the adenylyl cyclase and
`increase intracellular cyclic adenosine monophosphate
`(cAMP) formation [72,73]. Arginine vasopressin (AVP),
`through its own specific V 1 type receptors, also acts on
`the corticotroph cell to activate phospholipase C, leading
`to increased phosphoinositide turnover, Ca2+ release and
`protein kinase C activation [74). The action of AVP poten(cid:173)
`tiates that of CRH [75] by further increasing cAMP
`formation [76-78]; cross-talk between the two trans(cid:173)
`ducing systems provides the synergistic action that
`promotes the maximal ACTH response by increasing
`both POMC gene transcription and secretory granule
`exocytosis [79-81]. This phenomenon, thoroughly
`studied in vitro on animal models, is also observed in
`humans; the simultaneous administration of CRH and
`AVP (or its analogue lysine vasopressin, LVP) induces
`a maximal ACTH rise, higher than that obtained by either
`secretagogue alone or their sum [82-84]. The specific
`A VP receptor of the corticotroph cell was recently cloned;
`this V3 (or V1b) receptor is closely analogous to the V1a
`receptor [85,86]. Interestingly,
`the AVP analogue,
`DDAVP or desmopressin, also has definite affinity for
`the V3 receptor, explaining that it is a powerful stimu(cid:173)
`lator of ACTH secretion in a vast majority (ca. 85%) of
`patients with Cushing's disease (87].
`Glucocorticoids exert a negative feedback on pitui(cid:173)
`tary ACTH [88]. In patients with primary adrenal
`deficiency, basal and stimulated ACTH are increased.
`On the other hand, excess glucocorticoid administration
`or secretion by a primary adrenocortical tumor inhibits
`basal and stimulated ACTH. Prolonged glucocorticoid
`suppression of the hypothalamic-corticotroph axis
`characteristically induces long-lasting unresponsive(cid:173)
`ness, which may extend for months or years after the
`source of excess glucocorticoid has been withdrawn.
`Glucocorticoids inhibit hypothalamic CRH production
`[89] and also act directly at the corticotroph cell, as
`demonstrated in various animal models. They inhibit
`basal and stimulated ACTH release [90,91], as well as
`POMC gene transcription in a dose-dependent manner
`[92]. Interestingly this inhibition is not complete and
`a small proportion of POMC transcription is not sup(cid:173)
`pressed, even by very high amounts of glucocorticoids
`[80,93].
`is
`A proposed neuro-immuno-endocrine loop
`emerging which suggests that corticotroph function
`not .only acts on immunocompetent cells -
`through
`cortisol production - but is itself the target of various
`immunomodulators [94]. Data obtained in the rat
`
`show that interleukin-1 and -6 both exert a stimulatory
`action on ACTH release at the hypothalamic and pitui(cid:173)
`tary levels [95-97). It is suggested that they participate
`in the physiologic ACTH rise in acute infectious stress,
`as they experimentally mediate that which occurs after
`bacterial lipopolysaccharide injections. Both cytokines
`are normally present in the rat anterior pituitary, appar(cid:173)
`ently
`in a subpopulation of
`thyroid-stimulating
`hormone (TSH) cells and in folliculostellate cells for
`interleukin-1 and
`interleukin-6,
`respectively,
`thus
`raising the possibility that they act as local paracrine
`factors [98,99]. The role of the gp130-related cytokines,
`particularly LIF (leukemia inhibitory factor), has been
`convincingly established in the regulation of POMC
`expression and corticotroph cell development [100].
`Further studies are obviously needed to establish the
`exact significance of these data, the effects of other
`regulatory peptides found in the pituitary [101] and
`their possible implication on the physiology and patho(cid:173)
`physiology of ACTH release in humans [102,103].
`Similarly PPAR gamma [104] and retinoic acid [105]
`play a role in POMC gene regulation, at least in animal
`models.
`
`Oversecretion of ACTH in Cushing's Disease
`Cushing's Hypothesis
`The proposition that the pituitary was responsible for
`the clinical features of Cushing's disease was convinc(cid:173)
`ingly expressed for the first .time in Harvey Cushing's
`classic monograph of 1932; the basophil adenomas of
`the pituitary body and their clinical manifestations (pitu(cid:173)
`itary basophilism) [106]. Cushing was recognizing that. ..
`
`... striking clinical effects might be produced by minute,
`symptomatically predictable (pituitary) adenomas. So it is the
`degree of secretory activity of an adenoma which may be out of
`all proportion to its dimension, that evokes the recognizable
`symptom-complex in all hypersecretory states ...
`
`he was still wondering however:
`
`... if the polyglandular features of the disorder are partly
`due, as premised, to a secondary hyperplasia of adrenal cortex
`
`Much uncertainty remained at that time on the fine
`pathophysiologic mechanism of this disorder, yet the
`crucial clinical and pathologic observations had been
`made and the pertinent questions had been asked.
`Today it is recognized that chronic oversecretion of
`cortisol, androgens and 11-deoxycorticosterone by
`hyperplastic adrenocortical glands is directly respon(cid:173)
`sible for the clinical features of Cushing's disease,
`a phenomenon which is primarily driven by pituitary
`ACTH oversecretion.
`
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`537
`
`These observations are of utmost importance. Not
`only do they provide the basis for pathophysiologic
`understanding of ACTH oversecretion, but they also
`support the rationale of the diagnostic procedures [126].
`
`The Source and Mechanism of ACTH
`Oversecretion in Cushing's Disease
`The Classic Anterior Pituitary Corticotroph .Adenoma
`That Cushing's disease is a primary pituitary
`disorder caused by a corticotroph adenoma is based
`on the frequency with which such adenomas are found
`at surgery and the histologic, biochemical and clinical
`evidences for a suppressed hypothalamic CRH.
`
`PREVALENCE
`Since the late 1970s many groups have reported the
`high frequency of pituitary microadenomas found at
`surgery in patients who were systematically subjected
`to sellar exploration by the transsphenoidal route,
`whether or not a pituitary tumor had been suspected
`by prior X-ray, computed tomography (CT) scanning,
`or, more recently, magnetic resonance imaging (MRI).
`As a rule such tumors are found in more than 80% of
`the cases [127-135]. Although small, and "silent," corti(cid:173)
`cotroph tumors are sometimes found at autopsy of
`nonCushing's patients,
`the prevalence of such
`adenomas is definitively higher in patients with Cush(cid:173)
`ing's disease [136).
`
`HISTOLOGY
`The basophilic adenomas of Cushing's disease have
`variable sizes; a large majority of them are microadeno(cid:173)
`mas arbitrarily defined as being less than 10 mm with
`a mean of approximately 5 mm
`(Figure 16.4)
`[14,130,137]. Most are localized to a primary right- or
`
`LIGHTS our Fh :'._ I.
`Twenty-four-hour profile of
`cortisol (Cort.) and adrenocorticotrophic honnone
`(ACfH) in a nonnal woman (left panel) and a
`woman with Cushing's disease (right panel). From
`l..iu et a/. (54).
`
`CHRONIC ACTH AND PROOPIOMELANOCORTIN (POMC) PEPTIDE OVERSECRETION BY THE PllUITARY
`Demonstrating Acm Oversecretion
`~en plasma ACTH became measurable by bioassay
`[10!1 1t w~s found. to ,be normal or slightly elevated in
`patients w1th Cushing s disease [108-110]. ACTH radio(cid:173)
`~unoassay [64] came as an illuminating tool for the
`fine exploration of these patients.
`A majority of patients with Cushing's disease have
`normal plasma ~CTH values in the morning, although
`as a group theu mean ACTH value is significantly
`higher than that of normal subjects [109,111-113].
`~owever, even a normal ACTH value is inappropriately
`~gh. or not normally restrained in view of the hypercor(cid:173)
`tisohc state; repeated ACTH measurements over
`24 hours show that patients with Cushing's disease
`have high evening values with a lack of the normal circa(cid:173)
`dian rhythm [65,66,114]. Continuous sampling with
`a persistaltic pump has not been performed to study
`24-hour integrated plasma ACTH, as has been done
`for cortisol [115,116]. The fragility of the molecule in
`blood probably precluded this approach, which might
`be performed by measuring other, more stable, POMC
`peptides such as 13- and y-LPHs [117].
`
`ACm Secretion is Dysregulated, not Autonomous
`Besides being increased, corticotroph activity has
`acquired altered regulatory mechanisms that are the
`hallmark of Cushing's disease. Plasma ACTH - and
`cortisol - classically have lost their normal circadian
`periodicity; yet episodic fluctuations occur and in
`some cases a significant circadian variation may still
`be present (Figure 16.3) [66,118- 120]. They are unre(cid:173)
`sponsive to stress [121,122]; they have become partially
`resistant to the suppressive action of glucocorticoids
`[123]; they are - inappropriately - sensitive to the stim(cid:173)
`ulatory action of CRH and/ or A VP in spite of the hyper(cid:173)
`cortisolic state [111,124,125].
`
`Slrl
`
`! ::[
`
`LIGHTS OUT
`
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`ill. PITUITARY TIJMORS
`
`Teva Pharmaceuticals USA, Inc. v. Corcept Therapeutics, Inc.
`PGR2019-00048
`Corcept Ex. 2015, Page 7
`
`

`

`538
`
`16. CUSHING'S DISEASE
`
`,·
`
`FIGURE 16.4 Pituitary gland from a necropsy in a patient with
`Cushing's disease (horizontal section x 10). A prominent micro(cid:173)
`adenoma is located within the anterior lobe, in the vicinity of the
`posterior lobe (only a small portion of which is recognizable on this
`section: PL). Two invasive extensions (Ex) of the tumor are progress(cid:173)
`ing within the neighboring tissues of the sella turcica. Courtesy of
`L Olivier.
`
`left-sided position within the gland, but a significant
`proportion (ca. 15%) are situated centrally [130]. Some
`are found outside the pituitary fossa and develop from
`the uppermost part of the pituitary gland (pars tubera(cid:173)
`lis), above the diaphragma, out of reach of the neurosur(cid:173)
`geon via the transsphenoidal route, and several cases of
`Cushing's disease have been reported with an ectopic
`corticotroph adenoma [138- 140] in the mucosa of
`the sphenoidal sinus [141], and even in an ovarian
`teratoma [142].
`The classic basophilic adenoma is not encapsulated
`and is composed of compact cords of more or less homo(cid:173)
`geneous cells. The granule content is responsible for its
`basophilic and P~staining properties; the latter is
`now explained since non-ACTH POMC peptides (the
`N-terminal fragment) are glycosylated [44]. Electron
`microscopy shows secretory granules which are highly
`variable in size (from 100 to 700 nm) and in amount
`(Figure 16.5) [143]. Occasionally the paucity of the
`granule content explains why some adenomas appear
`chromophobe at the light microscope. Within the same
`tumor a variable pattern of granule load and size may
`
`be observed. In some adenomas [144] tumor cells show
`characteristic features of Crooke's cell [145], as depicted
`in the normal corticotroph of patients treated with corti(cid:173)
`costeroids: a ring-shaped homogeneous dense hyaline
`area constitutes an amorphous zone that repels the gran(cid:173)
`ules to the margin of the cell and dose to the nucleus;
`ultrastructural studies show that it is made of filaments
`[146].
`Immunocytochemistry has recently provided the
`ultimate means to recognize corticotroph cells by
`specific immunodetection of their content [143,148].
`For a given antibody the signal is generally correlated
`to the cell granule load (Figure 16.6A, B). The sensitivity
`of the method sometimes allows the detection of an
`immune signal in what appeared to be a chromophobe
`adenoma [143]. Many adenomas will, unsurprisingly,
`react with different antisera directed against different
`POMC fragments, though some will respond only to
`a given antiserum. Although this type of observation
`may point to some peculiar mode of POMC processing
`in a particular tumor which would not generate a gener(cid:173)
`ally accessible epitope to the antibody, as has been
`described for example in endorphin adenomas [148],
`it should be kept in mind that different antisera may
`show variable sensitivities. More recently the specific
`recognizition of POMC RNA by in situ hybridization
`has been achieved in human corticotroph adenomas
`(Figure 16.6C).
`The periadenomatous tissue shows a variable density
`of corticotroph cells, with frequent and typical Crooke's
`cells [146]. The coexistence of corticotroph hyperplasia
`and adenoma has been reported [149- 152].
`
`SUPPRESSED HYPOTHALAMIC CRH
`A crucial clue to the pathophysiologic mechanism of
`ACTH oversecretion in Cushing's disease is that pitui(cid:173)
`tary corticotroph adenomas are associated with a series
`of histologic, biochemical and clinical arguments that
`hypothalamic CRH is chronically suppressed: (1) histo(cid:173)
`logically, examination of the periadenomatous tissue
`in the vast majority of the cases -
`does not show -
`
`FIGURE 16.5
`Ultrastructural study of two
`surgically
`removed microadenomas, exhibiting
`completely different cytological features (same
`magnification, bars = 1 l!m). (A) This tumor is
`homogeneously constituted of poorly granulated
`cells (SG, secretory granules; L, lysosomal forma(cid:173)
`tions) with a large dear nucleus and a narrow ring of
`cytoplasm. (B) On the contrary, the second tumor is
`composed of granulated cells. The secretory granules
`(SG) vary in size, and are generally distributed along
`the plasma membrane. The tumor cells harbor
`a dense nucleus with a prominent nucleolus, and
`more or less developed bundles of filaments (F).
`Courtesy of E. Vila-Porc:ile.
`
`ill. PITUITARY TUMORS
`
`Teva Pharmaceuticals USA, Inc. v. Corcept Therapeutics, Inc.
`PGR2019-00048
`Corcept Ex. 2015, Page 8
`
`

`

`CHRONIC ACTH AND PROOPIOMELANOCORTIN (POMC) PEPTIDE OVERSECRFTION BY THE PITUITARY
`
`539
`
`Cytologic study of surgically
`FIGURE 16.6
`removed corticotroph microadenomas. (A) Immu(cid:173)
`nofluorescence with an anti-ACTH25-39 antibody; in
`immunoreactive cells are scattered
`this tumor,
`among unlabeled cei!s. Immunoreaction varies from
`cell to cell, and only concerns the cytoplasm, the
`thus appearing as dark dots
`nuclei
`(x350).
`(B) Immunofluorescence with an anti-~dorphin
`[!fD -t : ~- '
`antibody; in this second tumor, all the cells are
`·
`f-l.~ ;:,.,. :: ~ ;- . ·'·•~ ,\~ ."•
`heavily immunoreactive, and are densely clustered
`, ,.. ':.... ~: ~ ~.).~· · · }_.:: ·~~,...~ ;
`around a capillary (large dark area). The bright
`:•',.::~- :.:_ .-.r~ · .. ~ ~ · .. ~~ ·.:~t'lP' :;
`immunofluorescent labeling is homogeneous and is
`~· • i ='•' .....
`. ...
`... •
`· :· '
`· j
`-t. .,..\
`• l
`restricted to the cytoplasm (x350). Courtesy of L.
`,;:"•1-i-_~~"'\. . ~.' · . -~ .
`Olivier. (C) In situ hybridization of human cortic<r
`".;r t ·.·"f
`... ~:,:;_~ .. ~ '
`troph tumor cells with a 32P-labeled proopiomela-
`nocortin (POMC) DNA probe. Diffuse hybridization
`.
`•
`• • ' ·
`'
`signal indicated by the black silver grains localize high concentrations of POMC mRNA in the tumor cells. Courtesy of P. L. Texier.
`
`. -~ ~ !
`
`specific evidence of corticotroph cell hyperplasia
`[153-155]; (2) biochemically, measurement of POMC
`peptides by various radioi.mmunoassays (RIAs) reveals
`low concentrations in periadenomatous tissue in
`comparison with the adenoma and also with normal
`human pituitaries [156,157]; and (3) clinically, sup(cid:173)
`pressed hypothalamic CRH is supported by the lack of
`response to stress (insulin-induced hypoglycemia) in
`Cushing's disease in contrast to other situations of
`ACTH hypersecretion which are thought to be CRH(cid:173)
`dependent (e.g., depression) [121,122). It is supported
`also by clinical evaluation after selective pituitary
`surgery in case of both success and failure. In the former
`case, successful removal of the adenoma results in
`a state of selective pituitary corticotroph deficiency
`that spontaneously resumes its activity over months or
`years; all parameters of normality will be restored
`including perfect conservation of circadian rhythm
`[129,158-163]. Even the cases of failure are interesting;
`in such patients it was found that 24-hour urinary
`cortisol excretion and plasma ACTH were unchanged
`from preoperative values despite removal of a signifi(ci