Characterization of mTOR Activity and

Metabolic Profile in Pediatric

Rhabdomyosarcoma
Luca Felkai, Ildikó Krencz, Dorottya Judit Kiss, Noémi Nagy, Gábor Petovári, Titanilla
Dankó, Tamás Micsík, András Khoor, Tamás Tornóczky, Zoltán Sápi, Anna Sebestyén, and
Monika ókaCs

17 November 2021
Chairlady : Dr. dr. Hermin Aminah Usman, Sp.PA(K)
Opponent : dr. Anglita Yantisetiasti, Sp.PA(K), Mkes
Presenter : Okky Husain
 Rhabdomyosarcoma (RMS)
 Most common pediatric sarcoma, counts for 3-% of childhood malignancy
 Classified unto four main subtypes:
Alveolar RMS > PAX-FOXO fusion positive Worse prognosis in childhood
and adolescent RMS
Embryonal RMS
> PAX-FOXO fusion negative
Spindle cell RMS
Pleomorphic RMS RMS not found in childhood and
adolescent

 After initial therapy, RMS usually has good prognosis, however in relapse and
continuous tumor progression, prognosis are poor.
 Previous study suggest importance of mTOR activity in RMS proliferation
 PI3K/AKT/mTOR pathway
 mTOR RAPAMYCIN (sirolimus)
(mammalian target of rapamycin) originally antifungal that has immunosuppressant,
antiproliferative properties and use to prevent organ
rejection

Work by block mTOR

mTORC1
Detect nutrient and
energy availability
When active,
promote proliferative
activity
mTORC2
Similar function with
mTORC1, but has
RICTOR
RICTOR component Rapamycin-insensitive companion of mTOR
 PI3K/AKT/mTOR pathway

 PI3K
Insulin signaling pathway that activate
AKT protein

 AKT
Inhibiting apoptotic process, promote
growth factor mediated cell survival,
induce protein synthesis, and part of
insulin signaling pathway
 Samples
65 blocks out of 48 patients are collected (between 2007 to 2017)
Age range = 0 – 17.3 years old
Median = 5.7 years old

Histological Subtype Numbers Paired sample Numbers

Fusion Negative 43 Primary - pretreated 6
embryonal RMS 39
Primary-relapse 7
Botryoid RMS 1
Spindle cell RMS 3 Primary-pretreated-relapse 2
Fusion positive (alveolar RMS) 5
 Samples
65 blocks out of 48 patients are collected (between 2007 to 2017)
Age range = 0 – 17.3 years old
Median = 5.7 years old

IHC Numbers Paired sample Numbers

Fusion Negative 43 Primary - pretreated 6
embryonal RMS 39
Primary-relapse 7
Botryoid RMS 1
Spindle cell RMS 3 Primary-pretreated-relapse 2
Fusion positive (alveolar RMS) 5
 IHC assessment
Histo-Score (H-Score) = staining intensity (0, 1+, 2+, 3+) X positive cell percentages
positif if H-Score > 100

IHC used purpose

Phospho-mTOR (pmTOR) Evaluate active form of mTOR (cannot
differentiated mTORC1 and mTORC2)
Phosphorylated ribosomal S6 protein (pS6) Assessing mTORC1
Rapamycin-insensitive companion of mTOR
Assessing mTORC2
(RICTOR)
 results
pmTOR pS6 Rictor Fusion Fusion
mTOR activity
negative Positive
+ + - 1 0
mTORC1 activity
- + - 3 0
+ - + mTORC2 activity 17 4
- - + Potential mTORC2 activity 11 1
+ + + 2 0
mTORC1 and mTORC2 activity
- + + 2 0
+ - - Uncertain mTOR activity 1 0
- - - No mTOR activity 3 0

82% of primary sample shows high Rictor Expression

All studied Fusion positive (alveolar Rhabdomyosarcoma) shows high Rictor positivity
results
 Results (paired samples)
pmTOR staining intensity did not
alter in primary vs pretreated
pS6 significantly increase after
chemotherapy (p = 0.003)
Rictor expression decreased during
treatment (p = 0.019)

In combination of three IHCs,

chemotherapy doesn’t alter mTOR
activity
Results (paired samples)
 Results (paired samples)

Relapse sample pairs shows higher

mTOR activity with mTORC2
predominant compared to primary
sample.
Results (paired samples)
Could Rictor positivity not because of mTORC2 activity?
Rictor protein expression may caused by
RICTOR gene amplification. Therefore the Rictor number Mean Range copy
expression copy number
RICTOR gene copy number are examined by category number
FISH High (> 150) 17 2.33 -
Low (< 100) 6 2.39 -
general 23 2.35 1.33 - 4.70

One out of 23 shows equivocal RICTOR amplification (copy number 4.70). The case followed with
RICTOR/AP3B1 ratio

RICTOR/AP3B1 ratio less than 2, therefore RICTOR amplification negative

 Metabolic pathway-related proteins
Glycolysis marker (PFK, LDHA), oxidative
phosphorylation marker (ATPB), pentose-phosphate
pathway (G6PDH), and glutaminolysis marker (GLS) are
examined
Only one primary PAX-FOXO fusion positive shows PFK
increase.
LDHA expression high in most primary sample and even
higher in recurrent cases.
Less pronounced oxidative phosphorylation (low ATPB)
G6PDH high in 90% primary cases and only 45% in
recurrent cases (p = 0.004)
Glutaminolysis marker high in 71% primary cases and 55$
recurrent cases
 Metabolic pathway-related proteins
Glycolysis marker (PFK, LDHA), oxidative
phosphorylation marker (ATPB), pentose-phosphate
pathway (G6PDH), and glutaminolysis marker (GLS)
are examined
Only one primary PAX-FOXO fusion positive shows
PFK increase.
LDHA expression high in most primary sample and
even higher in recurrent cases.
Less pronounced oxidative phosphorylation (low
ATPB)
G6PDH high in 90% primary cases and only 45% in
recurrent cases (p = 0.004)
Glutaminolysis marker high in 71% primary cases and
55$ recurrent cases
 mTOR and metabolism marker correlation
Spearman correlation test indicated association
between mTOR activity, LDHA and GLS.

Positive correlation were found between pmTOR and

LDHA (r =0.403, p < 0.05)

Positive correlation were found between pmTOR and

GLS (r =0.302, p < 0.05)

mTOR promote utilization of alternate energy

source
 What we learn from the study?
Clinically and prognostically
High mTORC2 activity of RMS compare to mTORC1 responsible for rapalog resistance
Dual inhibitor needed to overcome this problem

Diagnostically
mTOR profiling by examine mTOR activity followed by Rictor examination should be done to evaluate
rapalog efficacy

Pathologically
Explain why alveolar RMS (PAX-FOXO fusion positive) has lower prognoses compared to embryonal RMS.
Shift of energy utilization unto Warburg phenotype explain RMS cell survival early on tumor progression which is
not alter by chemotherapy.
THANK YOU
 Rhabdomyoblast
Large round or oval cells with
abundant deeply eosinophilic
eccentric cytoplasm sometimes
with cross-striation

Better rhabdomyoblastic
differentiation is more
prominent in post
chemotherapeutic specimen
 Rhabdomyoblast
Large round or oval cells
with abundant deeply
eosinophilic eccentric
cytoplasm sometimes
with cross-striation

Better rhabdomyoblastic
differentiation is more
prominent in post
chemotherapeutic
specimen
 Rhabdomyoblast
Large round or oval cells
with abundant deeply
eosinophilic eccentric
cytoplasm sometimes
with cross-striation

Better rhabdomyoblastic
differentiation is more
prominent in post
chemotherapeutic
specimen
 Rhabdomyoblast
Large round or oval cells
with abundant deeply
eosinophilic eccentric
cytoplasm sometimes
with cross-striation

Better rhabdomyoblastic
differentiation is more
prominent in post
chemotherapeutic
specimen
 epidemiology
 Bimodal distribution
 First peak between 2-6 years mostly (60%)
 Mostly embryonal, botryoid, and spindle cell subtypes
 Second peak between 10 – 18 years old (up to 40%)
 Mostly alveolar RMS
 MOST COMMON SOFT TISSUE TUMOR IN CHILDREN UNDER 15
years old
 4.5% of all childhood cancers
 26% head and neck, 17% genitourinary, 15% extremity
 May arise in area lacking striated muscle such as bladder and
gastrointestinal!
 Embryonal rhabdomyosarcoma
60% of all Rhabdomyosarcoma. Most
below 10 years old (mean 7 years old)

most region is head and neck (orbit,

parameninges) followed by
genitourinary tract

Right, densely packed hypercellular

area of small undifferentiated cells
adjacent to less cellular myxoid area on
the left

Orbital embryonal rhabdomyosarcoma. Fleshy, pale-tan cut

surface that resulted in proptosis of the overlying globe
Embryonal rhabdomyosarcoma
60% of all Rhabdomyosarcoma. Most
below 10 years old (mean 7 years old)

most region is head and neck (orbit,

parameninges) followed by
genitourinary tract

Right, densely packed hypercellular

area of small undifferentiated cells
adjacent to less cellular myxoid area on
the left
Embryonal rhabdomyosarcoma
60% of all Rhabdomyosarcoma. Most
below 10 years old (mean 7 years old)

most region is head and neck (orbit,

parameninges) followed by
genitourinary tract

Alternating strongly stained

hypercellular and weakly stained
myxoid hypocellular area.
Embryonal rhabdomyosarcoma
60% of all Rhabdomyosarcoma. Most
below 10 years old (mean 7 years old)

most region is head and neck (orbit,

parameninges) followed by
genitourinary tract

Right, densely packed hypercellular

area of small undifferentiated cells
adjacent to less cellular myxoid area on
the left
 Embryonal rhabdomyosarcoma, botryoid

A subtype of embryonal rhabdomyosarcoma with better prognosis. Botryoid (Greek, grape bunches). Grow
beneath mucosal epithelial lined viscera such as bladder, vagina, or upper respiratory tract. Frequently shows
“cambium layer”, hypercellular zone immediately beneath epithelial surface meanwhile deeper layer is more
hypocellular
 Embryonal rhabdomyosarcoma, botryoid

A subtype of embryonal rhabdomyosarcoma with better prognosis. Botryoid (Greek, grape

bunches). Grow beneath mucosal epithelial lined viscera such as bladder, vagina, or upper
respiratory tract.
Embryonal rhabdomyosarcoma, botryoid
A subtype of embryonal
rhabdomyosarcoma with better
prognosis. Botryoid (Greek, grape
bunches). Grow beneath mucosal
epithelial lined viscera such as bladder,
vagina, or upper respiratory tract.
Frequently shows “cambium layer”,
hypercellular zone immediately
beneath epithelial surface meanwhile
deeper layer is more hypocellular
Embryonal rhabdomyosarcoma, botryoid
A subtype of embryonal
rhabdomyosarcoma with better
prognosis. Botryoid (Greek, grape
bunches). Grow beneath mucosal
epithelial lined viscera such as bladder,
vagina, or upper respiratory tract.
Frequently shows “cambium layer”,
hypercellular zone immediately
beneath epithelial surface meanwhile
deeper layer is more hypocellular
Embryonal rhabdomyosarcoma, anaplastic
Rare subtype of embryonal
rhabdomyosarcoma which difficult to
distinguish pleomorphic
rhabdomyosarcoma

Large bizzare cells at least three times

larger compared to neighboring cells

A negative prognostic factor, more

aggressive treatment
 Embryonal rhabdomyosarcoma, anaplastic
Left anaplastic variant of Embryonal rhabdomyosarcoma vs pleomorphic rhabdomyosarcoma

Pleomorphic rhabdomyosarcoma is commonly in people beyond 45 years with peak incidence sixth decades.
 Alveolar rhabdomyosarcoma
Second most common (40%) type of
rhabdomyosarcoma. Slightly older
patients compared to embryonal RMS

Microscopically monomorphic round

cells with skeletal muscle
differentiations. Fibrous septa may be
identified. Alveolar patterns and
multinucleated giant tumor cells are
common but not definite.

PAX3-FOXO1 or PAX7-FOXO1
fusion gene considered diagnostic
nowadays. No more PAX-FOXO1
fusion negative alveolar RMS
Septate with lacking alveolar pattern
 Alveolar rhabdomyosarcoma
Second most common (40%) type of
rhabdomyosarcoma. Slightly older
patients compared to embryonal RMS

Microscopically monomorphic round

cells with skeletal muscle
differentiations. Fibrous septa may be
identified. Alveolar patterns and
multinucleated giant tumor cells are
common but not definite.

PAX3-FOXO1 or PAX7-FOXO1
fusion gene considered diagnostic
nowadays. No more PAX-FOXO1
fusion negative alveolar RMS
Solid variant lacking alveolar appearance. If FOXO
fusion negative, diagnosed as embryonal RMS
Alveolar rhabdomyosarcoma
Second most common (40%) type of
rhabdomyosarcoma. Slightly older
patients compared to embryonal RMS

Microscopically monomorphic round

cells with skeletal muscle
differentiations. Fibrous septa may be
identified. Alveolar patterns and
multinucleated giant tumor cells are
common but not definite. Positivity in

PAX3-FOXO1 or PAX7-FOXO1
fusion gene considered diagnostic
nowadays. No more PAX-FOXO1
fusion negative alveolar RMS
Nest with
discohesive cells
 Alveolar rhabdomyosarcoma
Second most common (40%) type of
rhabdomyosarcoma. Slightly older
patients compared to embryonal RMS

Microscopically monomorphic round

cells with skeletal muscle
differentiations. Fibrous septa may be
identified. Alveolar patterns and
multinucleated giant tumor cells are
common but not definite. Positivity in

PAX3-FOXO1 or PAX7-FOXO1
fusion gene considered diagnostic
nowadays. No more PAX-FOXO1
fusion negative alveolar RMS

Nest with discohesive cells leaves behind

sieve-like of hyalinize fibrous trabeculae
 Alveolar rhabdomyosarcoma
Second most common (40%) type of
rhabdomyosarcoma. Slightly older
patients compared to embryonal RMS

Microscopically monomorphic round

cells with skeletal muscle
differentiations. Fibrous septa may be
identified. Alveolar patterns and
multinucleated giant tumor cells are
common but not definite. Positivity in

PAX3-FOXO1 or PAX7-FOXO1
fusion gene considered diagnostic
nowadays. No more PAX-FOXO1
fusion negative alveolar RMS

Nest with discohesive cells leaves behind

sieve-like of hyalinize fibrous trabeculae
 Alveolar rhabdomyosarcoma
Second most common (40%) type of
rhabdomyosarcoma. Slightly older
patients compared to embryonal RMS

Microscopically monomorphic round

cells with skeletal muscle
differentiations. Fibrous septa may be
identified. Alveolar patterns and
multinucleated giant tumor cells are
common but not definite. Positivity in

PAX3-FOXO1 or PAX7-FOXO1
fusion gene considered diagnostic
nowadays. No more PAX-FOXO1
fusion negative alveolar RMS

Nest with discohesive cells some with giant

wreath like appearance nuclei
 Pleomorphic rhabdomyosarcoma
In people beyond 45 years old. Much aggressive, early metastasis. Mean survival below 2 years old. Confirmed
striated muscle differentiation, histologically or immunophenotype.
Genetic profile shows more similarity to Undifferentiated Pleomorphic Sarcoma compared to other embryonal,
alveolar, or spindle/sclerosing rhabdomyosarcoma
 Spindle/sclerosing rhabdomyosarcoma
 Previously separated, spindle and sclerosing
RMS now considered single entities.
 Uncommon, 5% of all RMS. Prognosis
favorable.
 Pediatric variant, most common
paratesticular than head and nect
 Adult type have predilection for head and
neck then extremitites
 Spindle cells neoplasm sometimes similar
appearance with fibrosarcoma as cellular
fascicles with herringbone pattern
 Spindle/sclerosing rhabdomyosarcoma

 Previously separated, spindle and

sclerosing RMS now considered
single entities.
 Uncommon, 5% of all RMS.
Prognosis favorable.
 Pediatric variant, most common
paratesticular than head and nect
 Adult type have predilection for
head and neck then extremitites
 Spindle cells neoplasm sometimes
similar appearance with
fibrosarcoma as cellular fascicles
with herringbone pattern
 Spindle/sclerosing rhabdomyosarcoma
 Sclerotic collagenous pattern with pseudovascular pattern of tumor (right)
 Striated muscle differentiations confirm by desmin, focal myogenin, and MYOD1 postivity
 Spindle/sclerosing rhabdomyosarcoma
molecular subtypes
Congenital / infantile form MYO-D1 mutation No reccurent identifiable
By VGLL2 or NCOA2 fusions. Adolescent and young adult genetic alteration

No histological difference between these three

subtypes. Only predilections between those.

Gross show well circumscribed tumor with tan-yellow

cut surface. Central necrosis found.
 Rhabdomyoma
Benign soft tissue neoplasm with skeletal muscle
differentiation

Median of 60 years old (range: 33 – 80 years old)

Distinctly rare! Lacking cambium layers

Positive for desmin, MSA, MYOD1 and Myogenin

Polypoid whole mount of vaginal rhabdomyoma

Rhabdomyoma
Benign soft tissue neoplasm with skeletal muscle
differentiation

Median of 60 years old (range: 33 – 80 years old)

Distinctly rare! Lacking cambium layers

Positive for desmin, MSA, MYOD1 and Myogenin

DESMIN MYOGENIN
 Prognosis
(based from International Classification of Rhabdomyosarcoma (ICR), 1995)

 1. superior prognosis (botryoid, spindle cell)

 2. intermediate prognosis (embryonal)
 3. poor prognosis (alveolar, undifferentiated sarcoma)
 4. prognosis not presently available (rhabdoid)

Important note! Since this classification in 1995, the knowledge of striated

muscle malignancy is keep expanding. Rhaboid classification is not listed in
5th WHO bone and soft tissue tumor. Molecular studies is also included in the
diagnosis of tumors
 PI3K/AKT/mTOR pathway
 Found in:
 Synovial Sarcoma (Nokitaka Setsu et al, 2013)
 Ewing Sarcoma (Passacantilli I et al, 2017)
 Leiomyosarcoma (Hernando E et al, 2007)
 Confirmed not found in:
 Angiosarcoma (Italiano A et al, 2012)
 General metabolism

Cells need for

Aerobic
energy CO2
Oxygen

Anaerobic Lactic Acid

No
Oxygen
 Warburg phenomenon

Cancer cells No Aerobic

No CO2
Oxygen

WARBURG
observation Lactic Acid

Oxygen

Oxygen