Phospho-mTOR is not upregulated in metastatic SDHB paragangliomas

For Review Only

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European Journal of Clinical Investigation

For Review Only

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Phospho-mTOR is not upregulated in metastatic SDHB paragangliomas

Hans K. Ghayee, DO1, Alessio Giubellino, MD4, Arielle Click, MS2, Payal Kapur, MD3, Victoria

Martucci, BA4, Jerry W. Shay, PhD2, Rhonda F. Souza, MD1, Karel Pacak, MD, PhD, DSc4

1Department of Internal Medicine, University of Texas Southwestern Medical Center and VA

North Texas Health Care System, Dallas, TX; 2Department of Cell Biology, University of Texas

Southwestern Medical Center, Dallas, TX; 3Department of Pathology, University of Texas

Southwestern Medical Center, Dallas, TX; 4Program in Reproductive and Adult Endocrinology,

Eunice Kennedy Shriver National Institute of Child Health & Human Development, National

Institutes of Health, Bethesda, MD

Key Words: pmTOR, SDHB, paraganglioma

Word count: 1,674

Corresponding author:

Karel Pacak, MD, PhD, DSc

Chief, Section on Medical Neuroendocrinology

Professor of Medicine

Program in Reproductive and Adult Endocrinology

Eunice Kennedy Shriver NICHD, NIH

Building 10, CRC, Room 1E-3140

10 Center Drive, MSC-1109

Bethesda, Maryland 20892-1109

E-mail: [email protected]

Phone: 1-301-402-4594; Fax: 1-301-402-0884

Page 1 of 20 European Journal of Clinical Investigation

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Abstract

Background: Pheochromocytomas (PCCs)/paragangliomas (PGLs) are neuroendocrine tumors

that may cause arrhythmia and death if untreated. Treatment for patients with metastatic tumors

is lagging. As new PCC/PGL susceptibility genes are discovered that are associated with the

mTOR pathway, treatment targets focusing on this pathway are being intensively explored.

Design: 21 human PCCs/PGLs were analyzed from 2 tertiary care centers.

Immunohistochemistry (IHC) analysis was performed for phospho-mTOR (pmTOR), phospho-

S6K (pS6K), phosphoinositide 3-kinase (PI3K), phospho-4EBP1 (p4EBP1), HIF1�, and MIB-1

in 6 metastatic SDHB PGLs, 15 non-metastatic PCCs/PGLs, (including 1 TMEM127 PCC and 1

non-metastatic SDHB PGL), and 6 normal adrenal medullas. The product of the intensity of stain

and percentage of cells stained was calculated as an H score.

Results: Using a two-tailed t-test, pmTOR and pS6K had significantly higher H scores in non-

metastatic PCCs/PGLs than in metastatic SDHB PGLs. HIF1� had significantly higher H scores

in metastatic SDHB PGLs compared with non-metastatic PCCs/PGLs and normal adrenal

medulla. No difference in H scores was seen with p4EBP1, PI3K, and MIB-1 when comparing

metastatic SDHB PGLs and non-metastatic PCCs/PGLs. Significantly higher difference in

pmTOR was seen in non-metastatic PCCs/PGLs compared to normal adrenal medullas. Higher H

scores were seen in metastatic SDHB PGLs in HIF1� when compared to normal adrenal

medullas. pS6K was statistically different in normal adrenal medulla, non-metastatic

PCCs/PGLs, and metastatic SDHB PGLs.

Conclusion: The present results suggest that the use of mTOR inhibitors alone for metastatic

SDHB PGLs may not achieve good therapeutic efficacy in patients.

Page 2 of 20European Journal of Clinical Investigation

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Introduction:

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are neuroendocrine tumors that

arise from the adrenal medulla and produce catecholamines [1]. Patients with excessive

catecholamine secretion from these tumors may suffer from severe hypertension, myocardial

infarction, arrhythmia, and stroke, which are commonly associated with death [2, 3]. In addition,

patients with PCC/PGL can develop metastatic disease, for which there is currently no cure [4].

Furthermore, patients who are not surgical candidates or those with metastatic disease have very

limited treatment options.

Currently 30-35% of patients with PCC/PGL have an underlying germline mutation in

one of several well-described genes, including neurofibromatosis type 1 (NF1), von Hippel-

Lindau (VHL), transmembrane 127 (TMEM127), MYC associated factor X (MAX), RET

(responsible for multiple endocrine neoplasia type 2), hypoxia-inducible factor 2A (HIF2A) [5-

7], and genes that encode subunits of the mitochondrial enzyme succinate dehydrogenase

(SDHA, SDHB, SDHC, SDHD, and SDHAF2), which is responsible for the conversion of

succinate to fumarate in the Krebs cycle and oxidative phosphorylation [8, 9]. SDHB mutations

were found to be associated with aggressive and often metastatic behavior [10]. Mutations in

these mitochondrial genes cause pseudohypoxic conditions with an increase in hypoxia-inducible

factor alpha (HIF�) [10]. As a result, levels of angiogenic growth factors, like vascular

endothelial growth factor (VEGF), and glucose transporter 1 increase to allow sufficient blood

and nutrient supply for tumor growth [10]. In addition, tumor cell mitogenicity may increase

through the phosphatidylinositol 3-kinase (PI3K) pathway, which is also involved in the

activation of HIF [11] and the mammalian target of rapamycin (mTOR) pathway [12].

The mTOR pathway is involved in protein synthesis and cellular proliferation [13].


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Interestingly, the mTOR pathway components have signaling interactions with the succinate

dehydrogenase complex (SDHx) as well as with the TMEM127, VHL, MAX, and NF1 gene

products, reinforcing the rationale to use drugs targeting the mTOR pathway in PCCs/PGLs [5].

However, when the mTOR 1 inhibitor everolimus (Affinitor) was used for patients with

unresectable, metastatic PCCs/PGLs, the results were disappointing [14].

Thus, in the present study our aim was to explore protein expression of components of

the mTOR pathway, such as pmTOR and its downstream targets, including pS6K and p4EBP1,

in metastatic SDHB-related PGLs compared to non-metastatic PCCs/PGLs and normal adrenal

medulla. Other signaling components affecting mTOR, including PI3K and HIF1�, were also

evaluated in order to determine the relevance of future therapeutic options using mTOR

inhibitors in these tumors.

Materials and Methods:

Patients and tumors

PCCs/PGLs included 15 non-metastatic PCCs/PGLs (1 with a TMEM127 mutation, 1

with SDHB mutation), 6 metastatic SDHB PGLs, and 6 normal adrenal medullas collected at the

National Institutes of Health (NIH) and the University of Texas Southwestern Medical Center.

The present study was done in accordance with the Institutional Review Board (IRB) protocol

from both institutions.

Immunohistochemistry

Standard immunohistochemistry analysis was performed for the following mTOR and

related pathway members: pS6K (Ser 235/236), p4EBP1 (Thr37/46), pmTOR, PI3K, HIF1�, and


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MIB-1. Immunostaining was performed using the Benchmark XT automated stainer (Ventana)

for all antibodies. Briefly, formalin-fixed, paraffin-embedded tissue microarray sections were cut

at 3-4 micron and air-dried overnight. The sections were deparaffinized, rehydrated, and

subjected to heat-induced epitope retrieval. Sections were then incubated with the appropriate

primary antibody. For signal detection, the ultraView universal detection system (Ventana) was

used. The slides were developed using 3-3’-diaminobenzidine chromogen and counterstained

with hematoxylin-eosin. Appropriate positive and negative controls were utilized for each run of

immunostains and checked for validation of the assay.

Interpretation

Immunohistochemisty (IHC) stains were performed on sections of tumor and benign

tissue for each marker. The staining pattern (nuclear vs. cytoplasmic), extent (percentage of

positive cells: 10/high power field), and intensity (0 for negative, 1 for weakly positive, 2 for

moderately positive, and 3 for strongly positive) were evaluated by a clinical pathologist (P.K.).

p4EBP1 and HIF1� positivity were interpreted as nuclear and/or cytoplasmic expression; all

other antibodies were interpreted as exclusively cytoplasmic patterns of expression. An H score

was assigned to each section as the product of intensity of staining and the extent of

immunoexpression (percentage of cells staining). The final H scores for each were used during

statistical analyses for all markers.

Statistics

A Student t-test was used to compare non-metastatic PCCs/PGLs with metastatic SDHB

PGLs, as well as non-metastatic PCCs with normal adrenal medullas and metastatic SDHB PGLs


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� ��

with normal adrenal medullas. A P value of <0.05 was considered statistically significant.

Results:

Table 1 shows patient characteristics including age, tumor location, catecholamine

production, and treatments that the patients have received.

Tumor tissue staining was performed for pmTOR, PI3K, p4EBP1, pS6K, HIF1�, and

MIB-1. Six metastatic SDHB PGLs, fifteen non-metastatic PCCs/PGLs (13 sporadic, 1 with a

TMEM127 mutation, 1 with SDHB mutation), and 6 normal adrenal medullas were stained.

pmTOR as well as pS6K were found to have higher H scores, whereas HIF1� had lower H

scores in non-metastatic PCCs/PGLs compared to metastatic SDHB PGLs. No differences in H

scores were observed between non-metastatic PCCs/PGLs and metastatic SDHB PGLs for MIB-

1, PI3K, and p4EBP1. When comparing metastatic SDHB PGLs with normal adrenal medulla,

HIF1� had higher H score in metastatic SDHB PGLs, while pS6K had statistically higher H

score in normal adrenal medullas compared with metastatic SDHB PGLs. Comparing non-

metastatic PCCs/PGLs with normal adrenal medulla, only pmTOR, had statistically different H

score. pmTOR was lower in normal adrenal medulla whereas, pS6K had higher H scores in

normal adrenal medullas compared with non-metastatic PCCs/PGLs. Table 2 shows summarized

results comparing the different tissue types. Figures 1 and 2 show a pictorial summary of the H

score results. Figure 3 shows representative IHC staining. Non-metastatic PCCs/PGLs had

positive staining for pmTOR and pS6K, and negative stains for HIF1�. On the other hand,

metastatic SDHB PGLs stained positively for HIF1� , but negatively for pmTOR and pS6K.


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Discussion:

Our results show that pmTOR expression in non-metastatic PCCs/PGLs is elevated

compared to metastatic SDHB PGLs, and that metastatic SDHB PGLs have elevated expression

of HIF1� compared to non-metastatic PCCs/PGLs. The statistically significant differences in

pmTOR expression in non-metastatic PCCs/PGLs compared to metastatic SDHB PGLs suggest

that mTOR inhibition alone may not reach good therapeutic efficacy for patients with metastatic

SDHB PGLs.

With the successful use of mTOR inhibitors in neuroendocrine tumors such as carcinoid

[15] and pancreatic neuroendocrine tumors [16], much hope has been placed in their application

towards treating other (neuro)endocrine tumors, including metastatic PCCs/PGLs. Further

enthusiasm for studying the mTOR pathway in these tumors occurred when the TMEM127 gene

was linked to their pathogenesis [5]. TMEM127 is a tumor suppressor gene whose product

modulates mTOR activity [5]. As a result, research interest in studying the mTOR pathway in

PCC patients is on the rise. In one study, which enrolled four patients with metastatic

PCCs/PGLs, a trial of everolimus (mTOR inhibitor) was given for 3 months in two patients, 5

months in the third patient, and 6 months in the fourth patient. Two of these patients received

chemotherapy in addition to everolimus. Unfortunately, none of the patients treated with

everolimus had any survival benefit, as tumor progression occurred in all cases [14]. Perhaps the

reason is that the TMEM127 gene mutation is found in non-metastatic tumors as opposed to

metastatic ones; however, the results of this treatment in a small sample population would need

confirmation in larger clinical trials.

We therefore hypothesized that mTOR pathway activity would be much more prevalent

in non-metastatic PCCs/PGLs than in metastatic ones, often linked to SDHB mutations. To test


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� �

this hypothesis, tissue staining for components of the mTOR pathway on SDHB mutated

metastatic tumors was compared to non-metastatic PCC/PGL tumors. The results showed that

HIF1��has intense staining in metastatic SDHB tumors, whereas the non-metastatic PCC/PGL

tumors have intense staining for pmTOR and pS6K. Both non-metastatic PCC/PGLs and

metastatic SDHB tumors have similar staining for p4EBP1. Our data suggests that the mTOR

pathway is prevalent in non-metastatic tumors as in tumors with TMEM127 gene mutations, but

not in metastatic SDHB PGLs, which may explain why using everolimus in the treatment of

metastatic tumors has been unsuccessful.

How can the mTOR pathway play a role in targeted therapy for PCCs/PGLs? It has been

found that the binding of growth factors to cell surface receptors activates PI3K, which increases

cellular glucose uptake, glycolysis, lactate production, and perhaps later the Warburg effect.

Activation of PI3K suppresses macromolecule breakdown in cells, whereas the mTOR protein

complex itself plays a key role in protein translation. All of these various components share the

common goal of cellular survival and/or proliferation. When the mTOR pathway is activated as a

result of growth factor stimulation, increased cellular transcription associated with cellular

proliferation occurs due to pS6K and 4EBP1. However, inhibiting the mTOR pathway to affect

tumorigenesis is not a simple task. The mTOR complex is composed of two components,

mTORC1 and mTORC2. mTORC1 is sensitive to the antibiotic rapamycin and is associated with

controlling protein synthesis. mTORC2, on the other hand, is not sensitive to rapamycin and is

associated with regulating the cell cytoskeleton as well as affecting the activation of mTORC1

[17]. Therefore, treating metastatic tumors with mTORC1 inhibitors alone will not be able to

control aggressive disease, such as those seen with metastatic SDHB PGLs. An effort is

underway to further study the successful role of dual mTORC1 and mTORC2 inhibitors in


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� ��

mouse models of PCC [18]. Nevertheless, in our study, we conclude that mTOR inhibition alone

may not be the favorable treatment target for metastatic SDHB-related PGLs.

We speculate that perhaps other targets in the mTOR pathway should be investigated in

metastatic PCCs/PGLs. Although HIF is known to regulate mTOR by activating a tumor

suppressor complex (TSC) which results in mTOR inhibition [19], [20], further studies are

warranted to examine these new possibilities, including HIF inhibitors.

Conflict-of-interest disclosure: The authors declare no conflict of interests.

Acknowledgements:

The authors are grateful to Amita Kathuria, MD at the University of Texas Southwestern

Medical Center for helpful discussions and for reviewing this manuscript. This study was funded

by the North American Neuroendocrine Tumor Society (NANETS) and the American Cancer

Society University of Texas Southwestern Institutional Grant (to HKG).

Contributions: HKG designed the experiment/analyzed data/wrote the manuscript; AG designed

the experiment/provided necessary reagents for experiments; AC performed the

experiment/analyzed data; PK performed the experiment/analyzed data; VM analyzed data/wrote

the manuscript; JWS designed the experiment /provided necessary reagents for experiments;

RFS designed the experiment/provided necessary reagents for experiments; KP designed the

experiment/provided necessary reagents for experiments/wrote the manuscript.


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REFERENCES

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�


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Table and figure legends:

Table 1: (A) Clinical characteristics of patients with non-metastatic PCC/PGL. 15 patients with

non-recurrent PCC/PGL are listed, including one with a TMEM127 gene mutation, and one with

SDHB gene mutation. (B) Clinical characteristics of patients with metastatic SDHB mutations.

Table 2: Comparison of non-metastatic and metastatic PCC/PGL and normal adrenal medulla.

Two-tailed t-test was performed comparing non-metastatic and metastatic tumors with normal

adrenal medulla controls.

Figure 1: mTOR pathway staining of non-metastatic PCC/PGL tumors. The H-score was

calculated by multiplying the staining intensity by the cell percentage. Darker colors correlate to

higher H scores.

Figure 2: mTOR pathway staining in metastatic SDHB tumors. The H-score was calculated by

multiplying the staining intensity by the cell percentage. Darker colors correlate to higher H

scores.

Figure 3: Immunohistochemistry (IHC) stains for normal adrenal medulla (NAM), SDHB

metastatic tumors, and non-metastatic PCC/PGL. Non-metastatic PCC/PGL have intense

staining for (A) pmTOR and (B) pS6K. SDHB metastatic tumors have intense IHC staining for

(A) HIF1� but no staining for pmTOR and (B) pS6K.


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For R

eview

Onl

y

Gen

der

Gen

etic

s Tu

mor

Lo

catio

n Ty

pe

Bio

chem

istr

y B

efor

e O

pera

tion

Late

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ease

O

ther

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atm

ents

Su

rviv

al

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adr

enal

pr

imar

y E

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ce

none

8

yrs

sinc

e su

rger

y

F pa

rasp

inal

pr

imar

y N

E, N

MN

no

kno

wn

recu

rren

ce

none

4

yrs

sinc

e su

rger

y

F R

adr

enal

pr

imar

y N

E, M

N, N

MN

no

kno

wn

recu

rren

ce

none

4.

5 yr

s si

nce

surg

ery

M

R a

dren

al

prim

ary

EP

I, M

N, N

MN

no

kno

wn

recu

rren

ce

none

3

yrs

sinc

e su

rger

y

F L

rena

l vei

n pr

imar

y no

ne

no k

now

n re

curr

ence

no

ne

2.5

yrs

sinc

e su

rger

y

F R

car

otid

pr

imar

y no

ne

no k

now

n re

curr

ence

no

ne

1.5

yrs

sinc

e su

rger

y

F R

car

otid

pr

imar

y D

A no

kno

wn

recu

rren

ce

none

1.

5 yr

s si

nce

surg

ery

F R

adr

enal

pr

imar

y N

E, N

MN

, DA

no k

now

n re

curr

ence

no

ne

1 yr

sin

ce

surg

ery

F R

adr

enal

pr

imar

y M

N, N

MN

no

kno

wn

recu

rren

ce

none

11

mon

ths

sinc

esu

rger

y

M

L ad

rena

l pr

imar

y M

N

no k

now

n re

curr

ence

no

ne

11 m

onth

s si

nce

surg

ery

M

R a

dren

al

prim

ary

MN

, NM

N

no k

now

n re

curr

ence

no

ne

5 yr

s si

nce

surg

ery

F L

adre

nal

prim

ary

MN

, NM

N

no k

now

n re

curr

ence

no

ne

5 yr

s si

nce

surg

ery

F L

adre

nal

prim

ary

MN

, NM

N

no k

now

n re

curr

ence

no

ne

4 yr

s si

nce

surg

ery

F R

adr

enal

pr

imar

y E

PI,

MN

, NM

N

no k

now

n re

curr

ence

no

ne

4.5

yrs

sinc

e su

rger

y

F S

DH

B

iliac

bi

furc

atio

n m

ultip

le

NE

, NM

N, D

A no

kno

wn

recu

rren

ce

none

3

yrs

sinc

e su

rger

y

Tabl

e 1A

: Pat

ient

cha

ract

eris

tics

with

non

-met

asta

tic P

CC

/PG

L

Lege

nd: M

=mal

e; F

=fem

ale;

R=r

ight

; L=l

eft;

EP

I=ep

inep

hrin

e; N

E=n

orep

inep

hrin

e; M

N=m

etan

ephr

ine;

N

MN

=nor

met

anep

hrin

e; D

A=d

opam

ine;

TM

EM

12

7=t

rans

mem

bran

e 12

7; S

DH

B=s

ucci

nate

deh

ydro

gena

se B

Page

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

For R

eview

Onl

y

Gen

der

Gen

etic

sTu

mor

Lo

catio

n Ty

pe

Bio

chem

istr

y B

efor

e O

pera

tion

Late

r dis

ease

Oth

er T

reat

men

ts

Surv

ival

M

SD

HB

re

trope

riton

eal

met

asta

tic

none

m

etas

tatic

C

VD

; rad

iatio

n th

erap

y; R

FA o

f liv

er

lesi

ons

2.5

yrs

sinc

e su

rger

y

M

SD

HB

re

trope

riton

eal

met

asta

tic

NE

, NM

N, D

A m

etas

tatic

C

VD

de

ceas

ed (4

yrs

af

ter s

urge

ry)

M

SD

HB

na

soph

aryn

geal

m

etas

tatic

N

E, N

MN

, DA

met

asta

tic

none

4

yrs

sinc

e su

rger

y

M

SD

HB

in

traao

rtic,

righ

t re

nal h

ilar

met

asta

tic

NE

, NM

N

met

asta

tic

none

4.

5 yr

s si

nce

surg

ery

F S

DH

B

liver

m

etas

tatic

N

E, N

MN

m

etas

tatic

M

IBG

; CV

D; r

adia

tion

ther

apy

dece

ased

(3 y

rs

afte

r sur

gery

)

F S

DH

B

lung

m

etas

tatic

N

E

met

asta

tic

CV

D; R

FA to

m

etas

tatic

lesi

ons

3 yr

s si

nce

surg

ery

Tabl

e 1B

: Pat

ient

cha

ract

eris

tics

with

met

asta

tic S

DH

B m

utat

ion

Lege

nd: M

=mal

e; F

=fem

ale;

NE

=nor

epin

ephr

ine;

NM

N=n

orm

etan

ephr

ine;

DA

=dop

amin

e; S

DH

B=

succ

inat

e de

hydr

ogen

ase

B; R

FA=r

adio

frequ

ency

abl

atio

n; C

VD

=cyc

loph

osph

amid

e, v

incr

istin

e, d

acar

bazi

ne;

MIB

G=m

etai

odob

enzy

lgua

nidi

ne

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

For R

eview

Onl

y

Tabl

e 2:

Com

paris

on o

f non

-met

asta

tic &

met

asta

tic P

CC

/PG

L &

nor

mal

adr

enal

med

ulla

T-te

st re

sults

N

on-m

etas

tatic

vs

Met

asta

tic

Met

asta

tic v

s N

orm

al

Non

-met

asta

tic v

s N

orm

al

MIB

-1

0.19

53

0.19

71

0.92

99

HIF

1�

0.00

06*

0.00

08*

0.43

94

PI3

K

0.14

83

0.08

49

0.61

14

pS6K

0.

0144

* 0.

0000

08*

0.00

02*

pmTO

R

0.03

54*

0.38

88

0.00

50*

p4E

BP

1 0.

9094

0.

3319

0.

0806

Lege

nd: T

wo-

taile

d t-t

est p

erfo

rmed

com

parin

g no

n-m

etas

tatic

and

met

asta

tic tu

mor

s al

ong

with

no

rmal

adr

enal

med

ulla

con

trols

. * p

< 0

.05

is s

tatis

tical

ly s

igni

fican

t

Page

16

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n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

For R

eview

Onl

y

Hig

h In

tens

ity *

% C

ells

M

ediu

m In

tens

ity *

% C

ells

Lo

w In

tens

ity *

% C

ells

Lege

nd:

Figu

re 1

: Sta

inin

g of

mTO

R p

athw

ay o

f no

n-m

etas

tatic

tum

ors

PI3

K

HIF

1�

pmTO

R

p4E

BP

1 pS

6K

Cel

l Rec

epto

r

Pro

tein

Syn

thes

is

Cel

l Pro

lifer

atio

n

Ang

ioge

nesi

s

eIF4

E

Rel

ease

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17

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

For R

eview

Onl

y

Figu

re 2

: Sta

inin

g of

mTO

R p

athw

ay in

m

etas

tatic

SD

HB

tum

ors

Hig

h In

tens

ity *

% C

ells

M

ediu

m In

tens

ity *

% C

ells

Lo

w In

tens

ity *

% C

ells

Lege

nd:

HIF

1�

pmTO

R

p4E

BP

1 pS

6K

Cel

l Rec

epto

r

Pro

tein

Syn

thes

is

Cel

l Pro

lifer

atio

n

Ang

ioge

nesi

s

PI3

K

eIF4

E

Rel

ease

Page

18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

For R

eview

Onl

y

Figu

re 3

A: T

issu

e st

ains

of n

on-m

etas

tatic

PC

C/P

GL

& m

etas

tatic

SD

HB

tum

ors

Non

-met

asta

tic P

CC

/PG

L N

AM

M

etas

tatic

SD

HB

pmTO

R

pmTO

R

pmTO

R

PI3K

HIF

1�

PI3K

PI

3K

HIF

1�

HIF

1�

Lege

nd: I

mm

unoh

isto

chem

istry

(IH

C) s

tain

s fo

r nor

mal

adr

enal

med

ulla

(NA

M),

met

asta

tic

SD

HB

tum

ors,

and

non

-met

asta

tic p

heoc

hrom

ocyt

oma

(PC

C) a

nd p

arag

angl

iom

a (P

GL)

.

Page

19

of 2

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

For R

eview

Onl

y

Figu

re 3

B: T

issu

e st

ains

of n

on-m

etas

tatic

PC

C/P

GL

& m

etas

tatic

SD

HB

tum

ors

Non

-met

asta

tic P

CC

/PG

L N

AM

M

etas

tatic

SD

HB

pS6K

pS

6K

pS6K

MIB

-1

MIB

-1

MIB

-1

p4EB

P1

p4EB

P1

p4EB

P1

Lege

nd: I

mm

unoh

isto

chem

istry

(IH

C) s

tain

s fo

r nor

mal

adr

enal

med

ulla

(NA

M),

met

asta

tic

SD

HB

tum

ors,

and

non

-met

asta

tic p

heoc

hrom

ocyt

oma

(PC

C) a

nd p

arag

angl

iom

a (P

GL)

.

Page

20

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Phospho-mTOR is not upregulated in metastatic SDHB paragangliomas

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Transcript of Phospho-mTOR is not upregulated in metastatic SDHB paragangliomas