Genetic cancer susceptibility is more frequent and variable in
`tumours of endocrine organs than in any other class of human
`neoplasms. Often, a variety of endocrine and non~endocrine
`tissues are involved, resulting in very complex clinical syn-
`dromes.
`
`Recent advancements in molecular genetics have made it pos-
`sible to establish inherited tumour syndromes (eg. the hyper-
`parathyroidism jaw tumour syndrome) which were previously
`included in major classical syndromes, such as multiple
`endocrine neoplasia (MEN1 and 2) and von Hippel—Lindau dis-
`ease (VHL).
`
`The increased understanding of clinical, pathological and
`genetic features has opened the door for sophisticated genetic
`counselling, preventive screening and prophylactic surgery.
`
`..
`
`'
`
`.. ..
`
`t
`
`•
`
`Roxane Labs., Inc.
`Exhibit 1012
`
`Page 040
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`Roxane Labs., Inc.
`Exhibit 1012
`Page 040
`
`

`
`Introduction
`
`c
`
`the clinical.
`This chapter discusses
`genetic and pathologic details of
`endocrine tumours which are compo(cid:173)
`nents of rnherited neoplasia
`In addition to the classical syndromes.
`such as the two multiple endocrine neo(cid:173)
`plasia syndromes (MEN) and von Hippei(cid:173)
`Lindau disease (VHL), this chapter also
`contains such entities. which in the past,
`had been part of sections dedicated to
`the more "traditional" syndromes. For
`example, there is a separate section on
`hyperparathyroidism Jaw tumour syn(cid:173)
`drome which would previously have
`been discussed with MEN 1. However,
`witll the recent isolation and partial char(cid:173)
`acterisation of the susceptiblity gene,
`HRPT2, this syndrome has come into its
`own. Similarly. non-MEN 2, non-VHL heri(cid:173)
`table phaeochromocytoma and paragan(cid:173)
`glioma would have been briefly dis(cid:173)
`cussed in the sections on MEN 2 and
`VHL. Given the discovery and character(cid:173)
`isa!Jon of the autosomal genes
`three of the four subunits of mitochondn(cid:173)
`al succinate dehydrogenase as suscep(cid:173)
`tibility genes for phaeochromocytomas
`and paragangliomas. this syndrome was
`felt to deserve a distinct sectron as well.
`Because this is a chapter in a book ded(cid:173)
`icated to endocrine tumours, a con(cid:173)
`scious decisron was made not to
`Cowden syndrome its own section, but
`instead to discuss it with all familral non(cid:173)
`medullary thyroid carcinoma predrsposi(cid:173)
`tion. Finally, there are two particularly
`Each
`notable features in this
`section discussing a
`type or
`group of inherited syndrome(s) ends with
`a subsection on
`counselling and
`preventative measures wr·itten by a can(cid:173)
`cer
`counselor or a practisrng
`physrcran-clrnrcal cancer genetrcrst
`Tnere is also a reference table (berow)
`differential
`whrch
`noses based on organ-specific endo(cid:173)
`crine
`
`Table 5.01
`Genetic differential diagnoses by endocrine organ system.
`
`Organ
`
`Histologic Type
`
`Syndromes
`
`Adrenal
`
`Adrenocortical neoplasia
`
`Phaeochromocytoma
`
`Li-Fraumeni syndrome
`Carney Complex
`Beckwith-Wiedemann syndrome
`MEN1
`Von Hippei-Lindau disease
`Pheochromocytoma-
`paraganglioma syndrome
`MEN2
`Neurofibromatosis type 1
`
`Gene
`
`TP53
`PRKARIA
`CDKNIC!NSDI
`MEN I
`VHL
`SDHD, SDHC,
`SDHB
`RET
`NFI
`
`Pancreas
`
`Islet cell neoplasias
`
`MEN1
`Von Hippei-Lindau disease
`
`MEN!
`VHL
`
`Paraganglia
`
`Paraganglioma
`
`Phaeochromocytoma-
`paraganglioma syndrome
`Von Hippei-Lindau syndrome
`MEN2
`Neurofibromatosis type 1
`
`SDHD, SDHC,
`SDHB
`VHL
`RET
`NFI
`
`Parathyroid
`
`Adenoma I Hyperplasia
`
`Carcinoma
`
`Pituitary
`
`Adenoma
`
`Thyroid
`
`Papillary carcinoma A
`
`Follicular carcinoma
`
`Medullary carcinoma
`
`MEN1
`MEN2
`Hyperparathyroidism-jaw
`tumour syndrome
`
`MEN1
`Carney complex
`
`Familial adenomatous
`polyposis syndrome
`Cowden syndrome*
`Carney complex ?
`Familial site specific
`non-medullary thyroid
`cancer syndromes
`Cowden syndrome*
`Werner syndrome
`MEN2
`
`MEN I
`RET
`HRPT2
`
`MEN I
`PRKARIA
`
`APC
`
`PTEN
`PRKARIA
`None yet
`
`PTEN
`WRN
`RET
`
`MENl/2, multiple endocrine neoplasia syndromes.
`*The great majority of epithelial thyroid carcinomas seen in Cowden syndrome are folllicular thyn.·
`carcinomas. Occasionally, papillary thyroid carcinomas (PTC) are seen in this syndrome. There is attn
`dotal evidence that what is commonly referred to as 'PTC' in familial adenomatous polyposis syndron•
`(FAP) is not identical to classic PTC. However, reclassification awaits systematic studies. For patlc.
`care, and hence purposes of differential diagnosis, FAP is still listed under the differential diagnosis '
`PTC.
`
`210
`
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`Exhibit 1012
`Page 041
`
`

`
`Multiple endocrine neoplasia type 2
`
`0 . Gimm
`CD . Morrison
`S. Suster
`
`P Komminoth
`L. Mulligan
`K.M . Sweet
`
`'Jefinition
`;·he multiple endocrine neoplasia type 2
`MEN 2) syndrome is an inherited tumour
`;yndrome with an autosomal dominant
`,>altern of
`inheritance, caused by
`Jermline mutations of the RET gene. It is
`:haracterised by the coexistence of vari(cid:173)
`'us endocrine tumours involving the thy-
`n;id, the adrenal , and the parathyroids
`21211
`In addi tion , abnormalities affect(cid:173)
`.ng various non-endocrine organs/tis(cid:173)
`;ues (e. g.
`intestine, mucosa, cornea,
`;keleton) may be present. MEN 2 has
`rJeen clinically subdivided into 3 groups:
`tamil ial medu llary
`thyroid ca rcinoma
`i FMTC), MEN 2A and MEN 2B.
`
`MIM Number
`fhe Mendelian Inheritance in Man (MIM)
`number for FMTC is 155240, the MIM
`number for MEN 2A is 171400, and the
`MIM number for MEN 2B is 162300.
`
`Synonyms
`MEN 2 has also been named multiple
`e ndocrine adenomatosis (MEA) type 2 or
`
`previously abbreviated MEN
`11 , which
`should no longer be used . MEN 2A is
`also known as Sipple syndrome 12064! .
`MEN 2B was also termed MEN 3 11092!;
`even
`less
`common
`is
`the
`term
`Wagenmann-Froboese syndrome 1665,
`2338!.
`
`Incidence I Prevalence
`The incidence of MEN 2 is unknown. The
`hereditary form of medullary thyroid car(cid:173)
`cinoma (MTC) has been assumed to
`account for about 25% of all MTCs. MTC
`is believed to account for 5-1 0% of all
`thyroid malignancies. The incidence of
`thyroid cancer has been assumed to be
`1-3/100,000 per year. Hence, MEN 2 may
`incidence of approximately
`have an
`1.25-7.5/10,000,000 per year. Its preva(cid:173)
`lence is believed to be about 1/35,000.
`The female to male ratio in MEN 2 is
`roughly 1: 1, in some studies a slight pre(cid:173)
`dominance of the female gender has
`been reported.
`
`{: 't ; .:i., ~:tl~d~
`
`S-100
`
`Fig. 5.01 Multiple endocrine neoplasia type 2b. A Ganglioneuromatosis of the tongue in a MEN2B patient
`(arrows). B Histology of ganglioneuroma of the tongue. C SlOO lmmunostaining of ganglioneuromatosis of
`the gallbladder. D Microscopic aspect of a ganglioneuromatosis in tha appendix of a MEN2B patient
`
`Fig. 5.02 Corneal nerves in a patient with MEN2B
`syndrome.
`
`Diagnostic criteria
`Besides MTC, patients with MEN 2A may
`develop phaeochromocytoma and/or pri(cid:173)
`mary hyperparathyroidism Patients with
`MEN 28 may develop phaeochromocy(cid:173)
`toma, neuromas of the tongue and/or
`ganglioneuromatosis of the intestine, a
`marfanoid habitus and/or medullated
`corneal nerve fibres . None of these latter
`phenotypes need be present in MEN 2B
`nor are they pathognomonic I 7 42,7 46,
`17781,
`i.e.
`they have been also been
`reported in patients without MTC or
`MEN 28-specific RET mutations although
`it is difficult to determine if such cl inical
`features are "ove r-called" once a c linical
`suspicion of MEN 2 is raised. Clinically
`evident primary hyperparathyroidism is
`not part of MEN 28. By definition.
`patients with FMTC develop MTC only.
`Since the identification of RET as the
`MEN 2 susceptibility gene in 1993, the
`definitive d iagnosis of MEN 2 relies
`almost exclusively on germline RET
`mutation analysis. Of note, RET mutation
`analysi s has to be performed in any
`pati ent with MTC and is also recom(cid:173)
`mended in patients with phaeochromo(cid:173)
`cytoma 1 1585!, irrespective of age of the
`patient, the absence of accompanying
`disease features or family history in order
`to identify index patients (probands) and
`to enable at-risk relatives timely diagno(cid:173)
`sis and therapy.
`
`·. '
`
`211
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`Exhibit 1012
`Page 042
`
`

`
`A
`B
`Fig. 5.03 Multiple endocrine neoplasia type 2 (MEN 2). A Macroscopic aspect of a multifocal (arrows) familial medullary thyroid carcinoma. B Bilateral medullary
`thyroid carcinoma.
`
`Medullary thyroid carcinoma
`
`Age distribution I penetrance
`About 70% of patients with MEN 2A
`develop clinically apparent MTC by the
`age of 70 years (17621. By the age of 35
`years,
`the biochemical penetrance
`reaches 100%. Of note, MTC has been
`found in young children age 10 years
`and below (526, 1309,23731, in MEN 28
`even at the age of 6 months (22101 .
`Patients with FMTC diagnosed with thy(cid:173)
`roid tumours are on average older than
`patients with MEN 2A.
`
`Clinical features
`Thyroid nodules may be the first clinical
`sign of MTC. Routine preoperative calci(cid:173)
`tonin measurement in any patients hav(cid:173)
`ing a thyroid nodule may identify MTC
`preoperatively /1670,1828,23181. Medul(cid:173)
`lary carcinoma of the thyroid metasta(cid:173)
`sizes early to lymph nodes and distant
`organs (mainly liver, lung and bone) At
`primary operation, generally more than
`50% of index patients already have
`lymph node metastases. Hence, metas(cid:173)
`tases may be the initial symptom of
`patients with MTC. In the case of high
`serum calcitonin levels, symptoms may
`arise from diarrhoea not responding well
`to common anti-diarrhoeic drugs. It is rel(cid:173)
`atively rare to present with phaeochro(cid:173)
`mocytoma or hyperparathyroidism.
`
`Pathology
`The histopathologic features of MTC in
`MEN 2 are virtually Indistinguishable
`the sporadic
`those observed in
`from
`cases. Certain features, however. differ in
`the two ways As noted earlier. MTC in
`
`212
`
`MEN 2 tends to occur at a younger age
`and is typically bilateral and multicentric
`(190,2001. The tumours are typically well
`circumscribed but unencapsulated. with
`a tan-pink, soft to rubbery cut surface.
`The smaller lesions tend to arise at the
`junction of the upper and middle third of
`the thyroid lobes, corresponding to the
`areas containing the highest concentra(cid:173)
`tion of C cells. Larger lesions can occu(cid:173)
`py the entire lobe and infiltrate the
`perithyroidal tissue.
`Another feature that distinguishes the
`sporadic form of MTC from that in MEN 2
`is the frequent presence of C-ce/1 hyper(cid:173)
`plasia in the latter (200, 1728,24071
`In
`MEN 2 patients, foci of C-cell hyperplasia
`are typically present in the vicinity of the
`tumours as well as in areas away from
`the main tumour mass. The finding of C(cid:173)
`cell hyperplasia may thus serve as a
`morphologic marker for MEN 2-associat(cid:173)
`ed MTC . The definition of C-cell hyper(cid:173)
`plasia remains controversial. Morpho(cid:173)
`logically, the process can be nodular or
`diffuse (480,24061 . In diffuse hyperpla(cid:173)
`sia, C-cells are increased and diffusely
`scattered
`throughout
`the
`thyroid
`parenchyma. In nodular hyperplasia, C(cid:173)
`cells occur in clusters that may obliterate
`the
`follicular spaces . Most authors
`require the presence of clusters of more
`than 6 cells in several foci from both
`lobes to make the diagnosis of nodular
`C-cell hyperplasia (1470,24081. In the
`diffuse form , an increase of over 50 C(cid:173)
`cells per low-power field in both thyroid
`lobes is the most commonly accepted
`criterion 118621 Use of special stains.
`including calcitonin and chromogranin
`immunostains. may be of ai d for high-
`
`lighting these cells in cases of C-ce!!
`hyperplasia. However, at present, gene!·
`ic testing is the most accurate method fo1
`identifying familial and MEN 2-associal
`ed C-cell lesions , and is more reliablE
`than morphologic identification of C-cel'
`hyperplasia (1309,1734,23731 . C-ce!
`hyperplasia has also been described in
`association with non-medullary thyroid
`carcinoma, follicular adenoma , lympho·
`cytic thyroiditis and solid cell nest:-•
`(30.345,7971 . Such cases have bee1
`regarded as a reactive physiologic
`process unassociated with malignan:
`potential (17281
`The histologic appearance of MTC in
`MEN 2 mirrors that of the sporadic case ~.
`A wide range of cytologic features anc
`histologic growth patterns characteriz,
`the tumours . As with the sporadic cases
`a solid or compartmentalized ("orga
`noid") growth pattern predominates. Tht
`tumour cells may be round , oval , polygc
`nal or spindled , and generally contair
`abundant granular eosinophilic cyt o
`plasm. The nuclei are uniform, round tc
`oval, with occasional pleomorphism anc:
`multinucleation. Mitotic figures are usual·
`ly scarce, particularly in
`the small e:
`tumours. Stromal deposits of amyloi(
`and calcium deposits resembling psarn
`moma bodies are also trequent finding 5
`Unusual morphologic variants that ca· •
`mimic other tumours have also beer•
`described.
`The histologic diagnosis of MTC can bl
`confirmed with the use of immunohisto
`chemical stains . Calcitonin and cal c
`tonin gene-related peptide (CGRP) rep
`resent the most sensitive markers fu
`these tumours . although they are nc
`
`Roxane Labs., Inc.
`Exhibit 1012
`Page 043
`
`

`
`ntirely specific and can be expressed in
`;ther conditions !481 ,2051 ,2477) . Calci(cid:173)
`onin positivity can be focal and restric(cid:173)
`sd to only a few tumour cells . Also, a
`.~ mall subset of MTC (approximately
`1 .5%) may be negative tor calcitonin
`909)
`'wo additional markers of value for the
`_iiagnosis of MTC are chromogranin and
`.:::EA; strong expression of the latter has
`'Jeen identified in some studies as asso(cid:173)
`ciated with a worse prognosis ! 1981). A
`1ariety of other markers, including low(cid:173)
`;nolecular weight cytokeratin. vimentin,
`:1euron-specific enolase, synaptophysin ,
`somatostatin, and numerous other pep(cid:173)
`'ldes have been
`identified
`in
`these
`tumours but are not specific for MTC and
`are therefore of limited diagnostic value
`!1862)
`
`Prognosis and prognostic factors
`Patients with hereditary MTC overall
`appear to have a better prognosis than
`patients with sporadic MTC . However,
`this may be due to the younger age at
`diagnosis because of surveillance, i.e.
`lead time bias. Patients with MEN 28 are
`believed to develop the most aggressive
`form of MTC ; however, a recent study did
`not confirm this assumption ! 1259). The
`5-year survival rate in MTC is about 80-
`90%, the 10-year survival rate is about
`60-70%
`! 177,877) . Prognostic factors
`may be tumour stage ! 177.525), and
`postoperative calcitonin level !525,724) .
`The studies that
`tie
`the presence of
`somatic M91 8T mutation and prognosis
`! 817,134 7l are deeply flawed as somatic
`mutation analyses were performed on
`either primary MTC or metastases.
`
`Phaeochromocytoma
`
`Clinical features
`The most common symptoms are hyper(cid:173)
`tension, headache, tachycardia, and
`sweating. Patients with phaeochromocy(cid:173)
`toma may present with orthostatic dys(cid:173)
`In patients with MEN 2,
`regulation.
`phaeochromocytoma rarely (10%) pre(cid:173)
`cedes the development of MTC !3241
`and, hence, is either diagnosed synchro(cid:173)
`nously or metachronously during follow(cid:173)
`up . Due to the genetic origin of the dis(cid:173)
`ease, both adrenal glands may be affect(cid:173)
`ed .
`Biochemically, phaeochromocytoma can
`be diagnosed by measuring elevated
`levels of tree catecholamines (epineph(cid:173)
`rine , norepinephrine) in 24-hour urine.
`Alternatively, their metabolites (e .g. vanil(cid:173)
`lylmandelic acid) may be measured.
`Even more sensitive (>95%) seems to be
`the determination of plasma meta(cid:173)
`nephrines or chromogranin A !550)
`the
`Once diagnosed biochemically,
`localization and
`the extent of
`the
`phaeochromocytoma needs to be deter(cid:173)
`mined. Enlarged adrenal glands are
`almost always present if the patient is
`symptomatic. In these cases , computed
`tomography and/or magnetic resonance
`imaging may be helpful in determining
`both
`localization and extent of
`the
`phaeochromocytoma. While both imag(cid:173)
`ing techniques have a high sensitivity
`(reaching up to 100%), their specificity is
`rather low (70%) Also. they may fail to
`identify extra-adrenal phaeochromocy(cid:173)
`toma. However, extraadrenal phaeochro(cid:173)
`mocytomas are a relatively unusual event
`tn MEN 2. In these instances, 131 1-meta-
`
`iodobenzylguanidine (MIBG) may be
`very helpful. While its sensitivity is only
`about 80%. its specificity reaches nearly
`100%.
`
`Pathology
`The majority of patients with MEN 2A and
`MEN 28 exhibit bilateral diffuse or nodu(cid:173)
`lar adrenal medullary hyperplasia as a
`precursor of phaeochromocytoma. Adre(cid:173)
`nomedullary involvement is not a feature
`of true FMTC. Diffuse medullary hyper(cid:173)
`plasia is defined as the expansion of the
`medulla into the alae or tail of the gland
`with or without nodule formation, enlarge(cid:173)
`ment of the adrenal medulla beyond the
`normal ratio ot cortical area to medullary
`area of 41 and a two-
`to three-fold
`increase in medullary volume and weight
`as compared to age- and sex-matched
`controls !483, 18811 Nodules larger than
`1 em in diameter are considered phaeo(cid:173)
`chromocytomas, while smaller nodules
`are defined as nodular medullary hyper(cid:173)
`plasia !3161 However, these criteria are
`arbitrary and remain to be confirmed by
`molecular analyses. Macroscopically,
`nodules of adrenomedullary hyperplasia
`are typically grey to tan and soft in tex(cid:173)
`ture. On microscopic examination there
`is often a mixed pattern of diffuse and
`nodular hyperplasia expanding into the
`tail of the gland and there may be inter(cid:173)
`mingling of medullary and adrenocortical
`cells. Nodules vary in size, usually lack
`true encapsulation, may show a "nodule
`in nodule" appearance and outlines of
`neighbouring nodules may be partially
`molded { 11921 Cellular, architectural
`and immunohistochemical features of
`hyperplastic lesions are similar to those
`
`•!. ,. ·. .
`
`213
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`Page 044
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`

`
`A
`Fig. 5.05 Multiple endocrine neoplasia type 2 (MEN 2). A Macroscopic view of multifocal phaeochromocytoma and adjacent nodular adrenomedullary hyperplasi u
`in a patient with MEN2. B Close up of nodular adrenomedullary hyperplasia (nodules < 1 em) adjacent to phaeochromocytoma in a patient with MEN2.
`
`seen in phaeochromocytomas.
`Phaeochromocytomas in MEN 2 patients
`are mostly multicentric and bilateral , vary
`in size and are confined to the adrenal
`medulla 12363) . However, some extraa(cid:173)
`drenal tumours and a phaeochromocy(cid:173)
`toma arising from an accessory adrenal
`gland have also been described
`11048,16101. Smaller tumours are often
`accompanied by diffuse and nodular
`adrenomedullary hyperplasia but larger
`tumours. as sporadic phaeochromocy(cid:173)
`tomas, show expansive growth with com(cid:173)
`pression of the adrenal cortex and over(cid:173)
`growth of
`the hyperplastic medulla .
`Histologically, MEN 2-associated phaeo(cid:173)
`chromocytomas are similar to sporadi(cid:173)
`cally occurring counterpart tumours.
`However, some studies reported a high(cid:173)
`er frequency of insular pattern, large or
`pleomorphic cells with vacuolated or
`granular cytoplasm, prominent nucleoli
`and hyaline globules in MEN 2A tumours
`than in sporadic phaeochromocytomas
`1751, 1121) . Occasionally. lipid degener(cid:173)
`ation may histologically mimic an adrenal
`cortical tumour (2282). Melanin pigmen(cid:173)
`tation,
`the presence of phaeochro(cid:173)
`moblasts as well as calcospherites have
`also been described I 1221).
`lmmunohistochemically, MEN 2-associ(cid:173)
`ated phaeochromocytomas exhibit a
`similar expression pattern of neuroen(cid:173)
`docrine and other markers as sporadic
`tumours 1 1132). However, some have
`reported differences including higher
`levels of corticotrophin hormone, lower
`levels of VIP and more S100 protein-pos(cid:173)
`itive cells in familial tumours when com(cid:173)
`pared to sporadic phaeochromocytomas
`I 1329 15341 RET immunostaining is not
`
`214
`
`helpful to distinguish MEN 2-associated
`from sporadic phaeochromoc ytomas
`since the latter may also harbour gain-of(cid:173)
`function RET mutations with consequent
`RET overexpression (1430) . In contrast to
`MEN 2-associated tumours, which har(cid:173)
`bour germline RET alterations , the RET
`mutations
`in sporadic
`tumours are
`somatic in nature (1131) .
`Most series report a very low malignancy
`rate for phaeochromocytomas in MEN 2
`patients when compared to sporadic
`tumours , but metastasizing tumours do
`occur 1370,697,888) . In two series of 100
`and 300 MEN 2 patients with phaeochro(cid:173)
`mocytomas, a frequency of 3% and 4% ,
`respectively, of malignant tumours was
`reported {324, 1521).
`As in sporadic tumours, histological cri(cid:173)
`teria to predict malignancy in tumours
`without metastases are not reliable in all
`cases and unequivocal immunohisto(cid:173)
`chemical or molecular markers of malig(cid:173)
`nancy are not yet available /197 4,2229) .
`Malignant tumours are usually heavier,
`show coarse nodularity, confluent necro(cid:173)
`sis, absence of hyaline globules, higher
`mitotic rates. small cell morphology,
`reduced numbers or absence of susten(cid:173)
`tacular cells and may exhibit a different
`immunohistochemical expression pattern
`than benign tumours 1392,469,790,1909,
`1910,1979,2281 ,2289)
`In addition to phaeochromocytomas, so
`called composite phaeochromocytomas
`(witr1 additional components of neurob(cid:173)
`lastoma, or ganglioneuroma. or gan(cid:173)
`glioneuroblastoma) arising in the adrenal
`medulla of MEN 2 patients have been
`described 1238.1431)
`
`Prognosis and prognostic factors
`Phaeochromocytoma as part of MEN 2 io
`almost always benign, with less than 5 ~.
`reported to be malignant 1324,1521 i
`Hence. the prognosis of patients witi
`MEN 2 is mainly determined by the clin •·
`cal course of
`their MTC. However
`patients with phaeochromocytoma har·
`bour a high risk of developing a hype•
`tensive crisis, which may be lethal. du•
`ing operations or childbirth. Hence, th r.
`presence of phaeochromocytoma need.·
`to be excluded prior to a surgical proce
`dure for MTC. If present. phaeochromc·
`cytoma has to be treated first.
`
`Hyperparathyroidism
`
`Clinical features
`The symptoms of primary hyperparathv
`roidism (pHPT) are very well known 8
`'moans, groans, stones and bones'. I·
`the 21st century, in countries where clin
`cal surveillance and genetic testing ar'
`routine, patients with MEN 2 rarely pre~ ·
`ent with these signs and symptoms. '''
`comparison to sporadic pHPT and pHP ;
`related to MEN 1, the disease appears\(.
`be rather mild and is generally dia\::
`nosed during follow-up .
`The diagnosis is made based on the cc·
`presence of elevated parathyroid ho•
`mone and elevated serum calcium lev
`els. The risk of untreated pHPT 1
`diverse. Severe osteoporosis and ostt:
`openia followed by bone fractures ma -.
`develop. Besides kidney stones. patient'
`may present with abdominal pain due tr
`peptic ulcers and/or panc reatitl ~:
`Further.
`fatigue and lethargy may b··
`
`Roxane Labs., Inc.
`Exhibit 1012
`Page 045
`
`

`
`resent in the case of severe
`)'~or to primary surgery. no
`techniques are necessary. In the
`or recurrent pHPT,
`.1se of
`hav1ng a
`Jwever, sestam1bi
`sensitivity (90%) and specificity
`imost 100% ), IS the nrt:>tt:>nrt:>ri
`to
`identify the
`uathyroid tissue (2364)
`
`;:sathology
`le gross findings of parathyroid hyper(cid:173)
`;lasia in MEN 2 do not differ from those
`hyperplasia in a non-familial associa(cid:173)
`'m All
`four glands are generally
`nlarged and hypercellular with a relative
`of intraparenchymal fat (classic
`usual type hyperplasia). There can be
`onsiderable variation in the size of indi(cid:173)
`rdual glands from a given case, as well
`variation
`in
`the degree of
`intra(cid:173)
`·arenchymal adipose
`tissue. Occa(cid:173)
`lonally, one may find two glands that are
`1arkedly enlarged and two glands of
`rear normal size (pseudoadenomatous
`ryperplasia), which does not preclude
`'le diagnosis of parathyroid hyperplasia
`Jarticularly in the setting of MEN 2. In a
`•;ubset of cases, there is little variation
`rrom normal parathyroid glands grossly
`1H histologically, with an abundance of
`ntraparenchymal fat in all glands exam(cid:173)
`ned (occult hyperplasia) (196,1892).
`Regardless of the disparity of size or
`jegree of cellularity between one or more
`parathyroid glands from a patient w1th
`known MEN 2 the diagnosis of parathy(cid:173)
`roid adenoma should not be used.
`the histological findings are
`lhe same as those described for hyper-
`occurring in a non-familial setting
`(478). The predominant cell type is the
`chief cell, which has faintly eosinophilic
`cytoplasm and a centrally placed round
`relatively monotonous nucleus without
`conspicuous nucleoli. There is generally
`some variation in the amount of cyto(cid:173)
`plasm present, which often occurs in dis(cid:173)
`crete foci creat1ng a slightly nodular
`appearance at low power magnification
`Some chief cells have more abundant
`cytoplasm with a
`lesser degree of
`in varying
`eosinophilia
`that
`results
`degrees of cytoplasmic clearing and are
`referred to as clear cells. This is not to be
`confused w1th
`diffuse water-clear
`(,wasserhelle") cell hyperplasia. which is
`not associated with Ma~ 2
`/478). Oxyphil cells are the result of an
`increased number of mitochondria in the
`
`cytoplasm that results 1n a granular
`eosinophilic appearance.
`of
`the varrat1on Ill cytoplasmic features the
`nuclear characteristics are remarkably
`similar among all cell types. although as
`in other endocrrne organs. it
`IS not
`uncommon to see focal nuclear atypia in
`benign parathyroid neoplasia. While
`nuclear atypia is more frequent in adeno(cid:173)
`mas than hyperplasia (12451. it is not
`uncommon to see nuclear enlargement
`w1th some degree of hyperchromasia or
`in
`occasional multinucleated cells
`parathyroid hyperplasia. Rarely does one
`see diffuse atypical nuclear changes
`from an MEN 2 family with particularly
`aggressive parathyroid disease.
`The architectural pattern in all cases of
`parathyroid hyperplasia consists of cords
`or nests or cells in a glandular pattern as
`well as foci of solid sheets of cells (nodu(cid:173)
`lar hyperplasia). although these findings
`are not specific for MEN 2 and can be
`seen in parathyroid adenoma as well as
`non-familial hyperplasia. Occasionally
`the overall architectural pattern is pre(cid:173)
`dominantly follicle-like with a single or few
`rows of chief cells surrounding a central(cid:173)
`ly located pseudolumen that generally
`contains a slight amount of eosinophilic
`debris. The intraparenchymal fat content
`is generally reduced
`in parathyroid
`hyperplasia, but there is a great deal of
`variation in this finding.
`The presence of mitotic figures in the
`absence of malignancy is a frequent
`finding in benign parathyroid
`(1924) and this applies equally well in
`MEN 2 associated parathyroid hyperpla(cid:173)
`sia (2073).
`
`Prognosis and prognostic factors
`MEN 2A-related primary hyperparathy(cid:173)
`roidism is generally mild, however, single
`cases w1th severe forms have been
`observed.
`
`of
`
`Hirschsprung disease
`Hirschsprung disease (HSCR) (MIM
`#142623) is a congenital abnormality
`characterised by absence or hypoplasia
`of neurons and ganglia of the submucos(cid:173)
`al and myenteric plexuses along variable
`of the hindgut /1698) Loss-of(cid:173)
`function mutations of the RET proto(cid:173)
`oncogene may be the single most com-
`
`Co-segrega(cid:173)
`mon cause of HSCR
`tion of MEN 2 and HSCR has been
`recognised in a small subset ot MEN 2
`(1533,21
`In
`these
`cases (<1%)
`cases. both disease phenotypes arrse
`from a s1ngle mutation of RET. generally
`the extracellular cysteine
`codons 609, 611, 618 or 620. Although
`the mutant protein is constitutively active,
`reduced levels are expressed on the cell
`surface, leading to both loss-of-function
`(HSCR) and gain-of-function (MEN 2)
`phenotypes associated with a single RET
`mutation ( 1553,2173).
`
`Cutaneous lichen amyloidosis
`In a few families with MEN 2, cutaneous
`lichen amyloidosis has been described
`In
`these instances, cuta(cid:173)
`/514, 1623).
`neous
`lichen amyloidosis has been
`assumed to be a paracrinopathy / 1151).
`
`MEN 2 is inherited as an autosomal dom(cid:173)
`inant disease, caused by germl1ne muta(cid:173)
`tions of the RET (REarranged during
`Transfection) proto-oncogene.
`
`Chromosomal location
`All three disease phenotypes have been
`mapped to a single locus on chromo(cid:173)
`some 10q11.2 by linkage analyses, and
`RET mutations have been identified in
`each disease subtype /512,564,1556)
`
`Gene structure
`The RET gene spans 21 exons, and
`encodes a transmembrane receptor tyro(cid:173)
`sine kinase with three protein isoforms of
`1 072, 1106 and 1114 amino acids that
`differ in their C-terminal residues ( 1566.
`1697,2172). All RET receptors have a
`large extracellular domain, involved in
`recognition and binding of RET-ligands
`and co-receptors, a
`transmembrane
`domain, and an
`intracellular tyrosine
`kinase domain which is required
`for
`autophosphorylation of the receptor and
`initiation of downstream signalling.
`
`Gene expression
`The RET tyrosine kinase is essential for
`the development of the kidney. central
`and peripheral nervous systems, and
`some neuroendocrine tissues, as well as
`for maturation of spermatogonia /102,
`533,1485. 1669)1ts expression is highest
`in neural crest cells and neural crest
`
`215
`
`Roxane Labs., Inc.
`Exhibit 1012
`Page 046
`
`

`
`Table 5.02
`Genotype-phenotype correlation in MEN 2.
`
`Ex on
`10
`
`11
`
`13
`
`14
`
`15
`
`16
`
`Codon
`609
`611
`618
`620
`630
`634
`768
`790
`791
`804
`844
`883
`891
`918
`
`Mean age at diagnosis§ lyrs)
`Medullary thyroid carcinoma IMTC)
`Phaechromocytoma
`Primary hyperparathyroidism
`Ganglioneuromatosis
`Multiple mucosal neuromas
`Marfanoid habitus
`Thickened corneal fibers
`
`FMTC
`609
`611
`618
`620
`630
`634
`768
`790
`791
`804
`844
`
`891
`
`45-55
`90-100%*
`
`MEN2A
`609
`611
`618
`620
`
`634
`
`790
`791
`804#
`
`891~
`
`25-35
`90-100%*
`40-60%
`10-30%
`
`MEN2B
`
`804t
`
`883
`
`918
`
`10-20
`100%
`40-60%
`
`+
`+
`+
`+
`
`§ The age at diagnosis has become younger since the identification of RET
`#based on one report with MTC and adrenal and extra-adrenal phaeochromocytoma {1610}
`t based on several reports with additional germline RET mutation {1046, 1486, 1516}; however, it appears
`that the phenotype is rather MEN 28-fike than typical MEN 28
`~ based on one personal communication
`*since the identification of RET, many patients undergo surgery before MTC occurs
`-=disease/finding absent or frequency observed not higher that in the general population
`+=disease/finding present in most cases but neither required nor pathognomonic {742,746,1778}.
`
`derived structures during embryogene(cid:173)
`sis and in the earliest stages of kidney
`induction and morphogenesis, but
`decreases to low levels in adult tissues
`!102,5331669)
`
`Gene function
`RET plays a role in development and
`maturation of peripheral nerves and in
`kidney induction but is also an important
`mechanism of neuronal survival in the
`peripheral nervous system. and particu(cid:173)
`larly in the enteric nerve plexuses 1226).
`In normal cells. RET is activated by inter(cid:173)
`action with a multicomponent complex
`rncluding members of both a soluble lig(cid:173)
`and family, the glial cell line-derived neu(cid:173)
`rotrophic factors (GDNF) and also a
`family of cell surface bound co-recep(cid:173)
`family receptors a
`tors,
`the GDNF
`(GFRa) (1886)
`
`Mutation spectrum
`mutatrons of RET are
`
`identified in >95% of all MEN 2 cases
`(564). These are generally missense
`mutations clustered in only a few codons
`of RET, and there are strong correlations
`of specific disease phenotypes with sub(cid:173)
`sets of these changes. As RET acts as an
`oncogene, a second somatic mutation of
`that locus is generally not part of MEN 2
`tumourigenesis.
`
`Genotype vs phenotype
`Mutations of cysteine residues in the
`extracellular domain of RET(codons 609,
`611, 618. 620 and 634) are found in most
`patients with MEN 2A and are strongly
`linked to the presence of phaeochromo(cid:173)
`cytoma and hyperparathyroidism (HPT)
`(564, 1554) These mutations result in
`replacement of a cysteine normally in(cid:173)
`volved in intramolecular bonds, leaving
`an
`residue available to
`form
`intermolecular bonds with other
`RFT receotors.
`to constitutive
`dHllerizat1on and actrvat'on of RET
`
`nailing in the absence o