BRS REPORT DRAFT

Tissue and imaging biomarkers for hypoxia predict poor outcome in endometrial cancer

INTERNAL DEADLINE FOR DRAFT (Content draft, slides, script, meeting): August 21
REPORTING DATE: August 25

BSR Journal Discussion Rubric 23-24.pdf

PAPER GDOC: AccDxCA-oncotarget-07-69844
CANVA LINK

TRACKER
Part Assigned DRAFT SLIDE CONTENT SCRIPT Time Allotment
Overview (1) Celine Done Done Done 3 mins

Background/Object Hanby Done Done Done 5 mins

ives (1)
Approach (1) Zhiarlah Done Done Done 4 mins

Methods (2) Bernice, Nicole Done Done Done 16 mins

Results and Per Headers (Results) Done Done Done 10 mins

Discussion (6) Janella, Alyssa,
Myka, Johann

Tables and Figures

Ginny, Christine
Conclusions (1) Mar Done Done Done 4 mins

Limitations/Future Hannah Done Done Done 3 mins

Directions (1) (2 mins used)

Other Assignments
Slide Editors (2) Myka, Janella Not Started

Proofread/Script Johann, Christine, Not Started

Editors (3) Nicole
Questions for Not Started
Reactor Group (1
person - 1 question)
 DRAFT
Overview of the Problem
What aspect of the disease/condition is being addressed?

● In western countries, endometrial cancer (EC) is the most prevalent gynecologic

carcinoma, and its incidence is rising
● Hypoxia is linked to solid tumors, aggressive phenotypes and therapy resistance,
especially in Endometrial Cancer
● Patterns of HIF-1a in Endometrial Cancer were investigated to determine whether
preoperative functional imaging characteristics are related to tumor hypoxia in
endometrial cancer (EC).
● Reduced disease specific survival in EC was substantially correlated with low
epithelial HIF-1 expression and high stromal HIF-1 expression.
● High stromal HIF-1 expression is linked to higher tumor metabolism at FDG-PET/CT
and lower survival in EC.
Background
Elaborate on the specific state of the field prior to the study.
Objectives
● Studies from 2012-2016 reported reported HIF-1α protein's prognostic importance in EC
as unclear, possibly due to the lack in the assessment of tumor protein expression
through immunohistochemistry.
● Journals from 2002-2014 investigated that when looking at where the HIF-1α protein is
located within tumor cells in EC (either in the nucleus or the cytoplasm), the findings
are contradictory.
● In 2013-2015, the significant role of hypoxic microenvironments surrounding EC
tumors in aiding tumor growth and resistance to treatment has been recognized.
Within tumor stroma, HIF-1α plays a crucial part, leading to greater attention in
medical studies aimed at finding new treatments that can block this protein or its
effects.
● Additionally in 2014, patterns of protein expression in the stroma have been proven to
link with more severe clinical and pathological characteristics. Moreover, the
expression of genes in tumor cells is connected to the expression of downstream
proteins in the stroma, suggesting that the microenvironment plays a crucial role in
the development of EC.
obj:
- To investigate the link between HIF-1α expression in EC in relation to preoperative
functional imaging markers reflecting tumor microvasculature and metabolism
- To investigate to what extent markers of hypoxia in tumor tissue and from
preoperative imaging are reflected in a clinical phenotype
- To discuss the results would assist in the optimization and individualization of EC
treatment
 Approach
How was the experiment/s designed and why?

● To investigate the relationship between the expression of HIF-1α, tumor microvasculature and
metabolism, and how these markers are clinically presented in patients with endometrial
cancer (EC).
● Tumor samples obtained from hysterectomy specimens, collected in the Bergen
Gynecologic Cancer Biobank and linked to clinical and histopathological data
● To determine the expressions of genes related to HIF-1α expression and their gene
signatures, immunohistochemical staining and oligonucleotide microarray studies
were performed on the collected tissue samples.
● Preoperative radiologic imagings (MRI and FDG-PET/CT) were performed to obtain
information on the microvasculature, parameters (i.e. blood flow, tumor volume),
growth, and metabolism of tumors.
● Data collected from tissue samples and results from radiologic imagings were
correlated to the patient’s prognosis and disease presentation.
● Statistical analysis was used to find statistically significant associations.

Methods
Discuss methods that may not be widely understood and would benefit from an explanation. What is the
method in practical terms? What does it measure?
Who is included in the study?
● Patients surgically treated for endometrial cancer (EC) or complex atypical
hyperplasia (CAH) at Haukeland University Hospital between May 2001 and January
2015 were included in the study.
● Written informed consent was obtained from all patients.

Procedure Techniques What is measured Significance to the

(i.e. parameters)? study
Patient and tissue a. Tumor samples Samples will be used
sample collection obtained from for the imaging and
hysterectomy DNA analysis.
specimens and
collected in the
Bergen
Gynecologic
Cancer Biobank
b. Primary EC
Imaging
- DCE-MRI:
June 2009
- FDG-PET/CT:
June 2011
c. Subset of CAH
and EC patients
- Fresh frozen
tissue
d. Recording of
clinical
information

Magnetic resonance a. 20 mg ● ADC value: Structual MRI of

imaging protocol butylscopolamin measure of pelvis
and derived e bromide magnitude of ● Give the physician
imaging administered diffusion of information about
parameters through IV before water a woman’s uterus,
scanning to molecules ovaries, and
reduce motion within tissue fallopian tubes
b. Structural MRI,
Pelvic DWI, Pelvic DCE-MRI tumor Pelvic DWI:
DCE-MRI parameters: ● Reflects
c. Extraction of ● Blood flow (Fb): histological
tumor ADC ● Transfer structure of
values in regions constant from tissues and its
of interest (ROIs) extravaacular cellularity
d. Manual drawing extracellular ● Measures random
of ADC values on space (EES) to motion of free
ADC maps and blood (kep) water molecules
tumor ROI on ● Transfer from (Brownian
DCE images blood to EES motion)
e. Generation of (Ktrans) ● Differentiates
parametric maps ● EES volume (Vc) benig and
from DCE series ● Integrated area malignant pelvic
on the extended under the tumors
Tofts kinetic concentration
model time curve Pelvic DCE-MRI:
f. Calculation of (IAUGC) ● Potential tool for
DCE-MRI tumor (first 5 parameters are early diagnosis
 parameters: angionegensis and
- markers showing identification of
perfusion) EC
● Predict tumor
● Tumor volume: treatment
estimated by response
measuring the
largest tumor ADC Map
diameter in three ● Shows where
orthogonal measures of
planes (a, b, c) magnitude of
using the diffusion (of
following water molecules)
equation: Tumor are seen
volume = a × b ×
c/2 Parametric maps
● Help visualize
different aspects
of tissue
microstructure
(mean
diffusivity, tissue
anisotropy,
dominant fiber
orientation)

FDG-PET/CT and a. 6h fasting + ● SUVmax: index of FDG-PET/CT

derived imaging 18F-FDG glucose ● Identify precise
parameters (positron-emittin metabolism anatomical
g radiotracer) ● SUVmean location of a
given through IV ● MTV: lesion based on
60-120 mins preoperative accumulation of
before the CT prediction of FDG
scan lymph node
b. PET images metastases
fused with ● TLG (SUVmean x
diagnostic and MTV): can
low-dose CT predict
images recurrence of EC
c. Low-dose fusion (Both MTV and TLG can
images used for predict recurrence of
metabolic tumor EC)
measurements

Oligonucleotide a. RNA extraction ● Gene expression Microarray analysis

DNA microarray and hybridization levels: captured ● Identification of
analyses b. Scanning tissue through signal genes that are
microarrays intensity up-regulated or
 (TMAs) to detected by the down-regulated
measure signal microarray due to
intensities probes conditions, such
c. Correlation of as hypoxia (low
gene and protein oxygen levels)
expression data
for HIF-1α.
d. Analysis of gene
expression
patterns to
identify
differences
between groups.

Immunohistochemi a. Preparation and ● ERα, PR, and Immunohistological

cal staining scanning of HIF-1α levels and staining:
tissue distribution ● Visualization of
microarrays patterns the presence and
(TMAs) distribution of
b. Immunohistoche specific proteins
mistry (IHC) to (ERα, PR, and
detect ERα, PR, HIF-1α) within
and HIF-1α. endometrial
c. Double staining cancer tissue
procedure for samples
HIF-1α and CD45.
d. Evaluation of
staining
intensity and
area for each
protein.
e. Grade staining
results through
semi-quantitativ
e index

Statistical analysis a. Import the ● Differences in Correlation and

collected data continuous data association
into the SPSS (i.e. gene and analyses:
software. protein understand
b. Perform expression relationships
two-sided scores). between various
statistical tests ● Variances in factors.
(significance categorical data
level < 0.05) (i.e. patient Disease-specific
c. Assess characteristics, survival analysis:
differences in clinical assess how protein
continuous data variables). expression or other
through the ● Correlation variables influence
 Mann-Whitney-U between patient outcomes
test. epithelial and
d. Analyze stromal HIF-1α
variances in expression in
categorical data each patient.
through ● Associations
Pearson-Chi-squ between
ared test. continuous data
e. Explore the (e.g., correlations
correlation between different
between protein
epithelial and expressions).
stromal HIF-1α ● Disease-specific
expression using survival using
the McNemar Kaplan-Meier
test. analysis and Cox
f. Assess the proportional
association hazards
between regression.
continuous data.
g. Analyze
disease-specific
survival using
the Kaplan-Meier
method.
h. Multivariate
analyses.

Results
Highlight key results. How were these analyzed?

Table/Figure Set 2

Table 1 & 2: Immunohistochemical (IHC) staining in tissue micro array (TMA) slides was
performed to assess HIF-1a protein expression patterns of epithelial and stromal tumor cell
components and were shown to have an inverse prognostic impact. Both low epithelial HIF-1a
and high stromal HIF-1a expression were more frequent in patients with non-endometrioid
tumor subtype, high grade tumors, deep myometrial invasion, lymph node metastasis, high FIGO
stage and in tumors with loss of estrogen and progesterone receptors, thus showing its
association with aggressive/severe clinicopathological features in EC tumor cells.

Figure 1: Low epithelial HIF-1a expression and high expression of HIF-1a was significantly
associated with reduced survival in endometrial cancer (EC). High HIF-1a expression in stromal
cells was also seen to be rare in the premalignant lesions of complex atypical hyperplasia but
increased significantly to invasive cancer. Low epithelial HIF-1a expression and high
expression of HIF-1a therefore have a negative prognostic impact.

FIgure 2: High stromal HIF-1α expression was significantly associated with high total lesion
glycolysis (TLG) at FDG-PET/CT, indicating an increase in tumor metabolic activity.

Table 3: Cox proportional hazards model was used to glean the effect of variables to the survival
of EC patients. While all variables indicated increased risk of harm with each having an HR
greater than 1, high stromal HIF-1a expression notably indicated decreased survival when
assessed independently and even in other variables like lymph node metastasis were considered.

Figure 3: Hypoxia gene signature was noted when blood flow in the tumor was low, as well as
when total lesion glycolysis, tumor maximum and mean standardized uptake values, HIF-1a,
VEGFA, and SLC2A1 mRNA expressions were high.

Table 4: Functional MRI and metabolic FDG-PET/CT were done to check the patterns of HIF-1a
expression. It showed that the expression of epithelial HIF-1a had no association with PET-CT and
MRI findings, and that MRI findings had no association with stromal HIF-1a expression either. On
to significant observations, it was observed that FDG-PET/CT parameters were higher in tumors
with high stromal HIF-1a expression including MTV. This means that HIF-1a expression is
higher in cases wherein tumor aggressiveness is high and survival is low.

Figure 4: To check if the HIF-1a expressed by cells is immune derived or due to CAF,
immunohistochemistry was done. Since most of HIF-1a expressing cells did not have CD45, the
surface marker of immune cells, it was determined that it was cancer associated fibroblasts
that expressed it. Further, the solid tumor-induced hypoxia leads to the expression of SLC2AI
mRNA as well to encourage energy production amid the hypoxic environment.

CK’s notes for Table/Figure Set 2

CAF - driver of tumor growth and angiogenesis, metabolic shift to glycolysis
HIF-1a is not immune derived since CD45 is not co-expressed
Tumor blood flow
SUV mean - average FDG uptake in an area
SUV max - pixel with highest FDG uptake
*marker of tumor glucose metabolism detected by [(18)F]-fluorodeoxyglucose ((18)F-FDG) PET/computed tomography (PET/CT) and reflects tumor aggressiveness

TLG - glycolysis is the preferred energy production pathway of tumors

HIF-1a mRNA - indicates early invasive type
VEGFA mRNA - angiogenesis - adaptation to hypoxia
SLC2A1 mRNA - glycolysis - adaptation to hypoxia

Population and Samples

● Large population based study of EC patients: 827 patients ​with comprehensive
clinical data available
○ Endometrial tissue from hysterectomy specimen were collected and
routinely investigated by pathologist at Haukeland University Hospital
○ Tissue specimens are classified as the ff:
■ Complex atypical hyperplasia (CAH) (n = 80)
■ Endometrioid EC (n = 612)
 ■ Non-endometrioid EC (n = 135)
○ Fresh frozen tissue of 282 patients (subset) for RNA extraction and gene
expression analysis
○ 164 had been subject to preoperative DCE-MRI
○ 108 had preoperative FDG-PET/CT

Stromal HIF-1α expression is an independent prognostic marker in endometrial

carcinomas
Recall: HIF = Hypoxia-Inducible Factor protein → Expression of genes important for cell
adaptation to hypoxia

Why stromal and epithelial tumor components in particular?

● Investigated expression patterns of HIF-1α in both the stromal and the epithelial
tumor component in EC
○ Not described before in EC, to the proponents’ knowledge
○ Assessing expression patterns separately in epithelial and stromal cells may address
discrepancies and gaps in literature

How was the stromal HIF-1α expression analyzed?

● Association between stromal/epithelial HIF- 1α protein expression and respective
clinicopathological phenotypes and receptor status in EC patients
○ See Table 1 and 2 for phenotype and receptor status used as variables for analysis
○ High stromal and low epithelial HIF- 1α protein expression (Inversely
correlated) ⇒ Reduced survival in EC
■ Associated with aggressive clinicopathological features
■ Negative prognostic impact of high stromal and low epithelial HIF-1α
expression
○ Protein expression scores in full sections correlate well with expression scores from
Tissue Microarrays (TMAs) for both epithelial and stroma HIF- 1α
● Cox proportional hazards model (Multivariate analysis)
○ High stromal HIF- 1α protein expression = Independent unfavorable
prognostic factor
■ Adjusted for epithelial HIF-1α expression, patient age, histological type
and grade, myometrial invasion and lymph node metastasis
■ Epithelial HIF- 1α failed to reach statistical significance
● High stromal HIF- 1α protein expression frequently observed in low grade
endometrioid EC lesions, almost non-existing in premalignant lesions (CAH) – Figure
1C
○ Suggests: HIF-1α positive cells in the tumor stroma are important in early steps
of endometrial carcinogenesis

Stromal HIF-1α expression correlates to gene sets representing inflammation and cell
cycle regulation and to high metabolic tumor activity measured by FDG-PET/CT
 ● The gene expression value of VEGFA and SLC2A1 in the tumor component to be
significantly positively correlated to stromal HIF-1α
○ VEGFA is strongly associated with angiogenesis
○ SLC2A1 is strongly associated with glycolysis
● NO difference in expression of HIF-1α mRNA in relation to histological type and grade
BUT
○ mRNA expression levels of VEGFA and SLC2A1 were significantly higher in the
more aggressive histologic phenotypes
● Inflammation and cell cycle regulation were identified as activated in lesions with
high stromal HIF-1α expression
● High stromal HIF-1α expression tended to be associated with higher tumor maximum
and mean standardized uptake value and metabolic tumor volume.

Hypoxia gene signature is associated with tumor mRNA expression of related genes and
functional imaging parameters and suggests treatment targets

Tumor Characteristics Relationship with Hypoxia Gene Signature

Tumor Blood Flow As the hypoxia gene signature score

increased, tumor blood flow decreased

Tumor Size When hypoxia gene signature score was

higher, tumor volume were larger

PET Imaging (SUVmean, SUVmax, and TLG)
SUVmean, SUVmax, and TLG are
measurements used in PET imaging to
measure and describe the activity and size
of a tumor. The research was able to see in
the result that there is a direct relationship
between these PET imaging measurements
and the hypoxia gene signature score. As the
PET imaging measurements increased so
did the hypoxia gene signature score.

mRNA Expression Gene expressions such as HIF-1ɑ, VGFA and

SLC2A1 also had a positive correlation with
the hypoxia gene signature score. Which
means that these genes are more active
when hypoxia gene signature score is
higher.

● The study also made use of connectivity map (Cmap) (version 2) to look for drugs that
can be used as treatments against tumors with high hypoxia gene signature scores.
The researchers were able to identify a couple of potential drugs which are: PI3K
inhibitor LY294002, HDAC inhibitor Trichostatin A, and HSP() inhibitor Tanespimycin.
● The study also noted that patients who had gone to radiotherapy and had high
hypoxia signature scores tended to have a reduction in their overall survival rates.
Cancer associated fibroblasts express HIF-1α in endometrial cancer lesions
● IHC staining was performed for HIF-1α and CD45 in a subset of tumor samples (n=95),
to exclude that HIF-1α expressing in stroma are either tumor associated
macrophages (TAMs) or tumor infiltrating lymphocytes (TILs).
○ “Despite some leukocytes and occasional granulocytes staining positive for
CD45, most of the HIF-1α positive cells did not express CD45 (Figure 4A).”

■ IHC staining reveals more HIF-1α positive Cancer associated fibroblasts

(CAF) than CD45 cells.
● RED: Cell is a cancer-associated fibroblast
● BROWN: Cell is positive for CD45 and indicates that it is
immune-derived and can either be TAM or TIL

● Results are supported by a study by Peng et al. where the gene signature was derived
from breast cancer exhibiting different gene expression patterns in CAFs and
fibroblasts from non-cancerous tissue
○ Significantly increase CAF gene signature score in endometrial lesions with
increased stromal HIF-1a expression (p=0.001)
 ○ CAF gene signature score was significantly higher in low grade EEC than in CAH
(p=0.001) (Figure C)
○ CAF gene signature score was directly correlated to HIF-1α and SLC2A1 mRNA
expression (p<0.001) (Figures D and E)

● HIF-1α positive stromal cells are significant in early endometrial carcinogenesis

○ As suggested by the high stromal HIF-1α expression in primary cancers vs
premalignant lesions (CAH)
○ High CAF gene signature score is observed in EC lesions with increased
stromal HIF-1α expression, more frequently in low grade EC, and directly
correlated with increased tumor mRNA expression

● CAFs may be a possible possible prognostic marker for EC

○ CAFs have a correlation with HIF-1α and SLC2A1 mRNA levels
○ CAFs are prominent in tumors and has a role in EC

Conclusions
Discuss major findings or breakthroughs.
● High stromal HIF-1α and low epithelial HIF-1α are associated with an aggressive
histological subtype and with reduced disease specific survival in endometrial cancer
(negative prognosis).
● Lesions with high stromal HIF-1α protein expression exhibited increased tumor
metabolism via angiogenesis and glycolysis due to overexpression of VEGFA and
SLC2AI genes, respectively, at FDG PET-CT.
● The expression of HIF-1α positive cells in tumor stroma is crucial during the early
stages of endometrial carcinogenesis.
● Imaging markers of increased tumor metabolism and low tumor blood flow are
associated with overexpression of stromal HIF-1α and a hypoxia gene signature,
confirming that tumor hypoxia may be important in endometrial cancer progression.
● Parameters from functional operative imaging by FDG PET-CT and DCE-MRI were
correlated to previously published hypoxia gene signature scores and tissue markers
of hypoxia are reflected in clinical phenotype and associated with functional operative
imaging markers in endometrial cancer.

Future Directions
What is the next important question? What technical hurdles need to be overcome, and how might it be
done?

As it was established in the study that high stromal HIF-1α expression is positively
 associated with tumor metabolism at FDG-PET/CT and PET/CT and DCE-MRI parameters,
the next big question could be: “How could preoperative imaging markers, in combination with
molecular tissue markers, further aid in the proper selection of patients for novel treatment
algorithms targeting hypoxia in endometrial cancer?”. Exploring this aspect of hypoxia-specific
EC treatment algorithms might be beneficial since the effect of hypoxia on the immune
tumor microenvironment is capable of promoting resistance to immune modulatory
approaches, which may interfere with treatment efforts (DeSouza et. al, 2022).

Although functional imaging methods such as dynamic contrast-enhanced magnetic

resonance imaging (DCEMRI) and positron emission tomography/computed tomography
(PET/CT) are able to depict and quantify changes in tumor hypoxia during therapy through
characterization of tumor microvasculature and metabolism, it could be challenging to
accurately estimate therapy response based alone on these changes. Aside from the tumor
microvasculature and metabolism, the tumor microenvironment, genetic heterogeneity,
and other biological processes may also have an impact on the tumor's response to
hypoxia-targeting therapy, and hence, must also be taken into consideration when
selecting patients for specific treatment algorithms. In addition, prognostic factors such as
histological type and grade, age, tumor size, and lymphovascular space involvement may
also be used to determine need for adjuvant therapies since it can aid in describing
patients by their risk of recurrence (Murali et. al, 2014). If done accordingly, hypoxia imaging
along with molecular profiling of tissue markers will also aid in redefining approaches to
maintenance therapies and surveillance of patients with endometrial cancer (DeSouza et.
al, 2022).

Reference:
DeSouza, N. M., Choudhury, A., Greaves, M., O’Connor, J. P., & Hoskin, P. J. (2022). Imaging
hypoxia in endometrial cancer: How and why should it be done? Frontiers in Oncology, 12.
https://doi.org/10.3389/fonc.2022.1020907

Murali, R., Soslow, R. A., & Weigelt, B. (2014). Classification of endometrial carcinoma: More
than two types. The Lancet Oncology, 15(7), e268-e278.
https://doi.org/10.1016/s1470-2045(13)70591-6

FOR PRESENTATION
SLIDE CONTENT SCRIPT
Overview
 Good morning, everyone! [Insert Introductions]

We’ll be sharing about the study entitled “Tissue and imaging

biomarkers for hypoxia predict poor outcome in endometrial
cancer”. Let’s dive in.

In western countries, Endometrial cancer is the most

common gynecologic carcinoma. Its frequency is continually
rising and hypoxia is considered an aggressive hallmark of
this condition.

Hypoxia is linked to solid tumors in Endometrial Cancer so

patterns of HIF-1α were investigated to determine the
functional imaging characteristics related to tumor hypoxia.
High stromal HIF-1α and low epithelial HIF-1α was
substantially correlated to Endometrial cancer.

Background
To elaborate on the specific state of the field prior to the study, we
have included a timeline of studies related to the paper

● Studies from 2012-2016 reported HIF-1α protein's

prognostic importance in EC as unclear, possibly due
to the lack in the assessment of tumor protein
expression through immunohistochemistry.
● Journals from 2002-2014 investigated that when
looking at where the HIF-1α protein is located within
tumor cells in EC (either in the nucleus or the
cytoplasm), the findings are contradictory.
● In 2013-2015, the significant role of hypoxic
microenvironments surrounding EC tumors in aiding
tumor growth and resistance to treatment was
recognized. Within tumor stroma, HIF-1α plays a crucial
part, leading to greater attention in medical studies
 aimed at finding new treatments that can block this
protein or its effects.
● Additionally in 2014, patterns of protein expression in
the stroma have been proven to link with more severe
clinical and pathological characteristics. Moreover, the
expression of genes in tumor cells is connected to the
expression of downstream proteins in the stroma,
suggesting that the microenvironment plays a crucial
role in the development of EC.

The objective of the researchers in conducting the study was:

- To investigate the link between HIF-1α expression in EC
in relation to preoperative functional imaging markers
reflecting tumor microvasculature and metabolism
- To investigate to what extent markers of hypoxia in
tumor tissue and from preoperative imaging are
reflected in a clinical phenotype
- To discuss how the results would assist in the
optimization and individualization of EC treatment

Approach
Just read it
 Tissue samples were obtained to investigate the relationships
between the expression of HIF-1α, radiologic markers for tumor
metabolism and microvasculature, and the clinical presentation of
these markers in patients with endometrial cancer.

These links were further studied via Immunohistochemical studies

and oligonucleotide microarrays, which were performed on collected
samples to determine the expression of genes related to HIF-1α
expression and their gene signatures.

Preoperative radiologic imagings were also performed to obtain

information on tumor size, markers for tumor growth, and
metabolism.

Data collected from tissue samples and results from radiologic

imagings were correlated to the patient’s prognosis and disease
presentation.

Lastly, Statistical analysis was used to find significant associations.

Methods
The participants of the study are patients surgically treated
for endometrial cancer (EC) or complex atypical hyperplasia
(CAH) at Haukeland University Hospital between May 2001 and
January 2015.

Tissue samples were collected, a written informed consent

was obtained from all patients for the imaging data and
specimens for biomarker studies.

Tumor samples were obtained from hysterectomy specimens

in the Bergen Gynecologic Cancer Biobank.

Imaging of patients with primary EC was done in the

preoperative protocol for patients from June 2009 for DCE-MRI
and from June 2011 for FDG-PET/CT.

Subsequently, fresh frozen tissue samples were obtained

from a subset of CAH and EC patients for RNA extraction and
mRNA analysis.

Clinical information such as age at primary treatment, BMI,

menopausal status, parity, primary surgical treatment and
additional therapy were also received.
For the magnetic resonance imaging protocol, 20mg of
butylscopolamine bromide was administered intravenously
prior to scanning in order to reduce motion artifacts. The
mean/median interval between MRI examination and surgery
was 1-98 days.

Pelvic DCE-MRI was acquired with 12 axial slices using 3D

spoiled gradient echo FLASH sequence. This helps in
assessing tumor perfusion, microvascular vessel wall
permeability, and extravascular-extracellular volume fraction

For the parameters being measured, we have the ADC values

that show the magnitude diffusion of water molecules within
the tissue.

Moreover, blood flow served as an angiogenesis marker

highlighting tumor perfusion.

Lastly, tumor volume is estimated by measuring the largest

tumor diameter in 3 orthogonal planes then multiplying
them.
The second major imaging procedure done is the FDG-PET/CT.
The scanning covered the caput to the proximal thigh.

The protocol included a 6hr fasting before the imaging.

18F-FDG, a positron-emitting radiotracer, was given
intravenously 60–120 min before the CT scan.

The PET images were fused with both the diagnostic

and the low-dose CT images while the metabolic tumor
measurements were performed using the low-dose fusion
images.

The mean/median interval between PET scanning and primary

treatment was 1–102 days.

Consequently, FDG-PET/CT helps in identifying the precise

anatomical location of a lesion from the accumulation of FDG.

The parameters being measured in this procedure include the

mean and maximum standardized uptake value (SUVmax)
which is used to measure the index of glucose. The metabolic
tumor volume (MTV) which is a prediction parameter for
lymph node metastases, and the total lesion glycolysis (TLG)
that can help predict the recurrence of EC.
RNA was extracted from fresh frozen tumor tissue with high
tumor purity. This was then hybridized to Agilent Whole
Human Genome Microarrays. The microarrays were
subsequently scanned using the Agilent Microarray Scanner
Bundle. Signal intensity was analyzed using the J-Express
software.

This was done to understand the transcriptional changes

occurring in the tumor tissue. By examining gene expression
patterns, researchers aimed to identify genes that are
upregulated or downregulated in relation to hypoxia.

Immunohistochemical (IHC) staining was performed on

tissue samples using tissue microarrays (TMAs) to assess the
protein expression of ERα, PR, and HIF-1α. A double staining
procedure was conducted for HIF-1α and CD45 in a subset of
patients. The stained slides were then evaluated using a
semiquantitative staining index that quantifies the protein
expression in terms of intensity and distribution.

Finally, statistical analyses were conducted. Using SPSS,

differences in continuous data, variances in categorical data,
correlation between epithelial and stromal HIF-1α expression,
and associations between continuous data and
disease-specific survival were analyzed.
Results
This is a large population based study of 827 EC patients.
Majority of endometrial tissue specimens from them were
classified as endometrioid EC.

Out of the 827 EC patients, 282 of them have fresh frozen

tissue available for RNA extraction and gene expression analysis,
164 had been subject to preoperative DCE-MRI, and 108 had
preoperative FDG-PET/CT.

These are the 4 key findings of the study that we will be

discussing today.
HIF stands for Hypoxia-Inducible Factor protein. This
promotes expression of genes important for cell adaptation to
hypoxia. As mentioned earlier, a hypoxic microenvironment of
an EC tumor aids in tumor growth and resistance to
treatment.

Analysis through chi-square showed that the

clinicopathological features and receptor status shown on the
slide were observed in patients with high stromal and low
epithelial HIF- 1α protein expression.

Also, Cox proportional hazards model was used to glean the

effect of variables to the survival of EC patients. While all
variables indicated increased risk of harm with each having
an HR greater than 1, high stromal HIF-1a expression notably
indicated decreased survival when assessed independently
and even when other variables like lymph node metastasis
were considered.

Here, epithelial HIF- 1α failed to reach statistical significance.

From those, it is gathered that the presence of LOW epithelial

and HIGH stromal HIF-1a expression indicate LOWER survival
in ECs.
Lastly, high stromal HIF- 1α protein expression was frequently
observed in low grade endometrioid EC lesions, but was
almost non-existent in premalignant lesions.

This suggests that HIF-1α positive cells in the tumor stroma

are important in the early steps of endometrial
carcinogenesis.

Next, it was also found that a high HIF-1α expression also

correlates with high gene expression of the VEGFA and SLC2A1
genes. The VEGFA gene is strongly associated with
angiogenesis, while the SLC2A1 gene is strongly associated
with glycolysis. It was also found that the HIF-1α mRNA was
expressed higher in aggressive histologic phenotypes rather
than the regular histological type.

Furthermore, the processes for inflammation and cell cycle

regulation were also activated with a high stromal HIF-1α
expression, as well as high tumor maximum, mean
standardized uptake value, and metabolic tumor volume.

Seen on this slide is the association between tumor

characteristics and the hypoxia gene signature score.

The gene score is anti-correlated with tumor blood flow,

meaning it is high when tumor blood flow is low.

On the other hand, the gene is positively correlated with all

the others–meaning it is high when tumor size, pet imaging
parameters, and HIF-1a, VGFA, and SLC2A1 mRNA expression
are high.
The study also used a connectivity map to look for drugs that
can be used as treatments against tumors with high hypoxia
gene signature scores. This identified PI3K inhibitor
LY294002, HDAC inhibitor Trichostatin A, and HSP90 inhibitor
Tanespimycin.

The study also noted that patients who had gone to

radiotherapy and had high hypoxia signature scores tended to
have a decrease in their overall survival rates.

Last but not the least for the results of the study, Cancer
associated fibroblasts express HIF-1α in endometrial
cancer lesions

IHC staining was performed for HIF-1α and CD45 in a subset

of tumor samples, to exclude that HIF-1α expressing in stroma
are either tumor associated macrophages (TAMs) or tumor
infiltrating lymphocytes (TILs).

It was revealed that there were more HIF-1α positive Cancer

associated fibroblasts (CAF) than CD45 cells.

These results supported by CAF gene signature set analysis

using a derived gene set* from the study by Peng et al. (2013).

Here on Figure B, There’s a significantly increased CAF gene

signature score (CGSS) in endometrial lesions with high
stromal HIF-1a expression

On Figure C, the CAF gene signature score was significantly

higher in low grade EEC than in CAH

and on Figure D and E, the CAF gene signature score was

directly correlated to HIF-1α and SLC2A1 mRNA expression
 So what are the implications of these results?

There was a high stromal HIF-1α expression in primary cancers

vs premalignant lesions (CAH) and a high CAF gene signature
score was observed in EC lesions with increased stromal
HIF-1α expression, more frequently in low grade EC, and
directly correlated with increased tumor mRNA expression.
This would imply that HIF-1α positive stromal cells are
significant in early endometrial carcinogenesis

Finally, CAFs, based on its link with HIF-1α and SLC2A1

mRNA level, may also be considered in the prognosis of EC

Seeing the prominence of CAFs in tumorigenesis and its role

in EC, it may now be considered as a possible prognostic
marker for EC
Conclusions
The following are the conclusions of the study:

First, high stromal HIF-1α is associated with an aggressive

histological subtype and with reduced disease specific
survival in endometrial cancer. This means that the prognosis
for patients with high expression of stromal HIF-1α is not
good. It must also be emphasized that there is an inverse
relationship between the expression of stromal HIF-1α and
epithelial HIF-1α. In the negative prognosis of endometrial
cancer, there is low expression of epithelial HIF-1α and high
expression of stromal HIF-1α.

Second, lesions with high stromal HIF-1α protein expression

exhibited increased tumor metabolism via angiogenesis and
glycolysis due to overexpression of VEGFA and SLC2AI genes,
respectively, as seen through the FDG PET-CT imaging. The
metabolic tumor marker TLG is significantly positively
correlated to stromal HIF-1α protein expression, which means
that glycolysis is the preferred energy source pathway of the
tumor in endometrial cancer.

Third, imaging markers of increased tumor metabolism and

low tumor blood flow are associated with overexpression of
stromal HIF-1α and a hypoxia gene signature. This confirms
that tumor hypoxia may play a crucial and important role in
the progression of endometrial cancer.

Fourth, tissue markers of hypoxia are reflected in clinical

phenotype and associated with imaging markers in
endometrial cancer, as exhibited by the positive correlation
between the hypoxia gene signature and HIF-1α, VEGFA, and
 SLC2AI. This means that the imaging parameters reveal real
functional aspects which reflect the oxygen status in the
tumor.

Fifth, the expression of HIF-1α positive cells in tumor stroma

is crucial during the early stages of endometrial
carcinogenesis. It must be noted that these cells are not
immune derived and are characteristically
carcinoma-associated fibroblasts (CAF), whose role in tumor
initiation and progression is well established.

Lastly, parameters from functional operative imaging by FDG

PET-CT and DCE-MRI were correlated to previously published
hypoxia gene signature scores and tissue markers of hypoxia
are reflected in clinical phenotype and associated with
functional operative imaging markers in endometrial cancer.
Future Directions
As it was established in the study that high stromal HIF-1α
expression is positively associated with tumor metabolism at
FDG-PET/CT and PET/CT and DCE-MRI parameters, the next big
question could be: “How could preoperative imaging markers, in
combination with molecular tissue markers, further aid in the
proper selection of patients for novel treatment algorithms targeting
hypoxia in endometrial cancer?”

However, if this idea was to be pursued, a possible technical

hurdle that might be encountered is the insufficiency of the
functional imaging methods such as dynamic
contrast-enhanced magnetic resonance imaging (DCE-MRI)
and positron emission tomography/computed tomography
(PET/CT) in accurately estimating therapy response for the
appropriate selection of patients for hypoxia-targeted
treatment algorithms. Aside from the tumor microvasculature
and metabolism determined by these preoperative imaging
markers, the tumor microenvironment, genetic heterogeneity,
and other biological processes also have an impact on the
tumor's response to hypoxia-targeting therapy, and hence,
must also be taken into consideration when selecting
patients for specific treatment algorithms. In addition,
prognostic factors such as histological type and grade, age,
tumor size, and lymphovascular space involvement may also
be used to determine need for adjuvant therapies since it can
aid in describing patients by their risk of recurrence. If done
accordingly, hypoxia imaging along with molecular profiling
of tissue markers will also aid in redefining approaches to
maintenance therapies and surveillance of patients with
endometrial cancer.
QUESTIONS FOR THE REACTOR GROUP
● Why were stromal and epithelial tumor components analyzed for the HIF-1α biomarker
expression, in particular?
○ The expression patterns of HIF-1α in specifically the stromal and the epithelial
tumor component in EC were not described before, to the proponents’
knowledge. It was always to a more generalized HIF-1α biomarker in tumors, no
distinction was made between stromal and epithelial tumor components. As
such, they found that there were a lot of discrepancies and gaps in literature
regarding the role of the biomarker, that’s why they opted to assess the
expression patterns separately for their study to possibly account for the
differences.
○ It was also found that the tumor microenvironment plays a significant role in
tumor progression. With this, it makes sense to us that they looked into the
stromal tumor component specifically since stromal cells affect the tumor
microenvironment. In this case, they were looking at hypoxic conditions
potentially driving the growth, resulting in poorer prognosis.

[On Introduction]
What is the significance of HIF proteins and how is it related to endometrial cancer??
- HiF proteins orchestrate the expression of numerous genes important for cell
adaptation to hypoxia.
- HIF-target genes have been demonstrated to encode proteins necessary for
angiogenesis, metabolism, epithelial-to-mesenchymal transition (EMT), invasion,
metastasis, stem cell maintenance, immune evasion and response to therapy
- HIF-1α is primarily regulated post-transcriptionally through proteasome degradation
when oxygen is available, and is recognized as a marker of hypoxia
- When the oxygen level drops, HIF-1α is stabilized and translocated to the nucleus and
it serves as a transcription-promoting factor. HIF-1α is recognized as a marker of poor
survival in many solid tumors.

● Why use DCE-MRI and FDG-PET/CT?

● Aside from HIF-1a, VEGFA and SLC2A1 were mentioned to be high. Can you clarify What
those tell us?
○ Those two reflect how the cells are attempting to adapt to the hypoxic
conditions. In particular, high VEGFA contributes to angiogenesis and SLC2A1 is
involved in glycolysisha

● [Results] In the discussion of the results a while ago, you mentioned some treatments
the researchers were able to identify a couple of drugs for treatment. I would like to
ask if you looked into any of these?
○ The drugs mentioned before targets different pathways in the body. So..:
■ LY294002 (PI3K Inhibitor)
 ● Targets the PI3K pathway which is involved in cell proliferation
and growth as well as inhibit cell apoptosis. If this enzyme is
inhibited then PI3K can potentially stop the growth and division
of cancer cells.
■ Trichostatin A (HDAC inhibitor)
● This drug inhibits the enzyme HDAC which regulates gene
expression and helps in modifying the structure of the
chromatin. If this enzyme is inhibited then certain activation of
genes that can suppress cancer growth may occur.
■ Tanespimycin (HSP90 inhibitor)
● This is an HSP90 inhibitor. HSP90 helps in maintaining the
shape and function of certain proteins required for cell survival.
If this enzyme is inhibited, it can lead to destabilizing the
cancer cells which can help eliminate them.

● What do you think is the most important biomarker for carcinoma out of the tumor
derived parameters?
○ It depends on the FIGO score and the location of metastasis. In this study, the
primary metabolic tumor marker is TLG
● What are gene signature scores and why was it used in the study?
○ Gene signature scores are used to quantify the activity levels of genes. For each
tumor, It is calculated as the average expression of the overall signature genes
included in the study. Hypoxia gene signature scores in this case were
obtained so that it could be correlated with the functional imaging markers in
EC.

● [Mar]: What could be the possible reason why stromal HIF-1a cells were rare in
premalignant lesions and then suddenly increased in number in advanced stages of
endometrial cancer?
○ The researchers noticed this trend as well and from their investigation, it was
found out that the increase in the number of stromal HIF-1a cells were
positively correlated with the overexpression of genes regulated by HIF-1a,
which are VEGFA and SLMC2AI.
○ VEGFA is a gene for angiogenesis, which is the process of forming new blood
vessels, and is critical for tumor growth and progression. In early stages of
cancer, the blood supply might still be able to support the needs of the tumor.
However, as the tumor grows and its demand for nutrients and oxygen
increases, angiogenesis might not keep up with the pace, resulting in areas of
hypoxia and HIF-1α activation.
○ SLMC2AI gene, on the other hand, is a gene corresponding to the metabolic
uptake. There was an increase in the total lesion glycolysis, implying an
increase in the metabolic demand due to the growing tumor. The increased
consumption of oxygen and nutrients led to localized areas of hypoxia. HIF-1α
 is a key regulator of metabolic adaptations in hypoxic conditions, which could
contribute to the increased presence of HIF-1a in more advanced stages of
endometrial cancer.

● [On Future Directions]: Given that the paper was published 7 years ago, are there
already studies that might answer the question on how the imaging markers may aid
in the proper selection of patients for treatment algorithms?
○ In a study conducted by DeSouza et al in 2022, data showed that imaging
markers for hypoxia in EC aided in adjusting management strategies to
hypoxic status. Some of its findings include: (the ff are copy-pasted from the
paper)
■ Hypoxia imaging and its link to TP53 status offers potential to refine
management strategies by selecting patients through prognostic
stratification.
■ Pre-operative hypoxia imaging could identify the women who would
most benefit from adjuvant radiotherapy and select women who might
benefit from external beam radiotherapy (EBRT) rather than adjuvant
brachytherapy alone
■ In locally advanced endometrial cancer (stage III) treated primarily with
chemoradiotherapy, hypoxia imaging may also indicate those who
would benefit from hypoxia modification with a radiosensitiser.
○ Source: DeSouza, N. M., Choudhury, A., Greaves, M., O’Connor, J. P., & Hoskin, P. J. (2022).
Imaging hypoxia in endometrial cancer: How and why should it be done? Frontiers in
Oncology, 12. https://doi.org/10.3389/fonc.2022.1020907

MEETING NOTES 8/21/2023

● Background
● Objectives
○ No objectives given in the paper
○ Obtained from the last part of —---
● Approach
○ Outline of the study methods
○ Study is divided into two main procedures
● Patient and Tissue Samples
● MRI and Derived Imaging Parameters
○ Injected butylscopalamine bromine
○ Conducted series of MRI
○ Extracted ADC values
○ Manual drawing of ADC values
○ Generation of parametric maps
● FDG PET/CT
○ 6 hour fasting
○ 18 F-FDG
● Oligonucleotide DNA Microarray Analysis
○ RNA extraction and hybridization
○ Instruments used: *inaudible* - not in the slide but in Bernice’s notes
● Immunohistological staining
● Statistical analysis
 ○ SPSS
● Results
○ High stromal HIF1-a expression is Independent prognostic marker in endometrial carcinomas
■ High stromal HIF1-a expression is an unfavorable prognostic factor
■ Graphs A and B: epithelial
■ NEEC: Non endometrioid EC
■ Important in early steps of endometrial carcinoma
○ Stromal HIF 1a expression correlates to gene sets representing inflammation and cell cycle regulation and to
high metabolic tumor activity
○ Hypoxia gene signature is associated with tumor mRNA expression of related genes and functional imaging
parameters and suggests treatment targets
■ Ex. of interpretation:
■ Tumor blood flow (anti correlated) : decreased when there is high hypoxia activity (?)
■ Patients who had gone to radiotherapy and had high hypoxia signature scores tended to have a
decrease in their overall survival rates.
● Conclusions
● Future Directions
● Add meaning for abbreviations in the intro part: Celine Joy J. Juan
○ DEADLINE: 8/23/2023 (Wednesday) - 8 pm
● Add Johann Clive Go’s parts
● Bold some of Bernice’s parts
● For Zhiarlah B. Gatus: check how other groups did the approach part
○ "How was the experiment/s designed and why?"
● 1 question per person
○ DEADLINE: 8/23/2023 (Wednesday) - 8 pm
● DEADLINE: 8/23/2023 (Wednesday) - 5 pm: FINALIZE SLIDES
● Thursday: proofread