Cancer Cachexia: Organ-specific Protein Synthesis

Pancreatic cancer is a serious disease with high mortality. It is the eighth
leading cause of cancer deaths in men and the ninth in women with respectively
138.100 and 127.900 annual deaths worldwide. In the Netherlands, it is the
tenth most common form of cancer in men (1024 annual new patients) and eighth
in women (1052 annual new patients) with a general 1- and 5-year survival of
18% and 4% respectively. Cancer cachexia is a major problem in patients with
pancreatic cancer and greatly decreases survival and quality of life. It is
responsible for more than 80% of pancreatic cancer related deaths. Cancer
cachexia is a complex syndrome characterized by weight loss and muscle wasting
due to a negative energy and muscle protein balance caused by anorexia and
catabolic drivers such as systemic inflammation. It is defined as weight loss
of >= 5% or weight loss of >= 2% and a BMI of <= 19 or sarcopenia (muscle wasting)
in cancer patients.

Many theories have been proposed for the cause of weight loss and profound
muscle wasting in cachexia. Muscle protein metabolism is a dynamic process
characterized by the balance between the synthesis and breakdown of muscle
proteins. A disturbance of this equilibrium can lead to the loss of muscle mass
in cachexia. Some theories involve increased catabolic drivers whereas others
focus on anabolic resistance. Though both theories may be true, it has never
been properly proved in humans. The availability of amino acids labelled with
stable isotopes created the possibility to *trace* them at different points in
amino acid metabolism using mass spectrometry. However, previous studies
provide conflicting data. Some show that there is indeed increased (muscle)
protein breakdown whereas other studies in humans and animals show that this is
not the case and that there is in fact a slight decrease in muscle protein
synthesis.

Next to muscle protein metabolism, organ-specific protein metabolism (e.g.
liver, small intestine) might be an important factor in cachexia. A recent
bovine study showed that the organ-specific protein synthesis is in fact much
higher than muscle protein synthesis. Also, tumour protein metabolism has never
been studies properly. Some studies found a higher protein synthesis rate in
colonic and various gastrointestinal malignancies but used the *flooding dose*
tracer technique which has limitations compared with the *constant infusion*
tracer technique. In pancreatic cancer, the difference in metabolism of the
tumour and its normal stroma is important since the pancreatic stroma is
increasingly recognized to be a key factor with respect to patient prognosis
and cancer progression.

In the proposed study, we will analyse protein synthesis in muscle tissue as
well as various abdominal organs. In addition, we will analyse the difference
in protein synthesis of tumour tissue versus normal pancreatic stroma tissue.

Primary Objectives:
1. To compare the protein synthesis of pancreatic, liver, intestinal, fat and
muscle tissue and leukocytes in cachectic patients.
2. To compare pancreatic malignancy protein synthesis with the protein
synthesis in normal pancreatic tissue.

Secondary Objective(s):
1. To identify the effect of anaesthesia on muscle protein synthesis in
cachectic patients.

This study will be a prospective observational study that will be conducted at
Maastricht University Medical Center (MUMC, Maastricht, Netherlands).

Overview
In this study, protein turnover and protein incorporation into various organs
and tissues of pancreatic cancer patients undergoing surgery will be analysed
by intravenous infusion of amino acids labelled with stable isotopes
(L-[U-13C6]-phenylalanine an L-[3,5-2H2]-tyrosine). After having signed for
informed consent, first data collection will take place. A single venepuncture
of 10ml of blood will be performed for measurement of haemoglobin, leukocyte
count, HbA1C, fasting insulin and fasting glucose. The following patient
characteristics will be collected:
• Age
• Sex
• American Association of Anesthesiologists (ASA) classification
• BMI
• Weight loss in the past six months
• Intoxication (smoking, alcohol, drugs)
• Systemic steroid or non-steroidal anti-inflammatory drug (NSAID) use in the
last four weeks
• World Health Organisation (WHO) performance status
• Presence of diabetes mellitus
• Presence of cardiac comorbidity
• Presence of pulmonary comorbidity
• Neoadjuvant therapy
The screening will take about 30 minutes and will take place at the surgery
outpatient department.

On the day of pancreatic surgery, patients will already be admitted at the
hospital. Surgery will be planned to start around 9.00am (t=0). Two and a half
hours before start of surgery (t=-150), the principal investigator will insert
a polyethylene catheter in the antecubital vein for tracer pre-infusion. A
second polyethylene catheter will be inserted in the dorsal hand vein for
arterialized blood sampling. The hand will then be placed in a hotbox (60°C)
for stimulation of blood flow, preventing occlusion. A first arterialized blood
sample (10ml) will be drawn from the hand vein for measurement of basal blood
values. At t=-120 patients receive a single intravenous priming dose of
L-[U-13C6]-phenylalanine and L-[3,5-2H2]-tyrosine, and thereafter a continuous
infusion of both tracers. Subsequent arterialized blood samples (5ml) will be
drawn at regular time points, i.e. at t=-90, -60 and -30. Induction of
anaesthesia will be around 8.45am. As part of standard care, the anaesthetist
will place a polyethylene catheter in the radial artery for per-operative blood
testing. We will use this catheter for future arterial blood sampling (5ml),
i.e. at t= -15, 0, 15, 30, 45, 60, 90, 120, 150, 180. Arterial blood sampling
is more frequent one hour after induction of anaesthesia to measure
fluctuations of enrichments due to effects of medication given by the
anaesthesiologist. After one hour of surgery there will be adequate exposure of
the abdominal organs (t=60). A first muscle biopsy from the vastus lateralis
and rectus abdominis muscle will be collected as well as a first biopsy of the
liver, small intestine, subcutaneous fat and visceral fat. After the surgeon
has performed a cholecystectomy (which is part of the standard surgical
procedure), a single surgical biopsy will be taken from the gallbladder. After
one and a half hours of surgery (t=90), the surgeon will make the decision if
the pancreatic tumour will be resected or not (which depends on several
conditions that are checked per-operatively. If the surgeon decides to start
with the resection of the tumour, a biopsy of both the tumour and pancreatic
tissue (that will be resected) will be taken using a Tru-Cut® biopsy needle.
The resection will take about 60 to 90 minutes. When the tumour and adjacent
pancreatic and intestinal tissue can be removed, a final biopsy of the tumour,
pancreatic tissue, liver, small intestine, visceral fat, subcutaneous fat and
vastus lateralis muscle will be taken (t= ±180min). After this, tracer infusion
will be stopped. Since all measurements and data collection are done during
hospital stay and surgery, there will be no extra time-investment of the
patient for this study day. • After surgery, the tumour tissue and adjacent
structures will be examined by the pathologist to establish the diagnosis.
After diagnosis (usually one week), the majority of the tissue (frozen or
formalin-fixated) will be discarded. In close consultation with the pathologist
in charge, the tissue that would be otherwise discarded will be collected for
analysis. This tissue will be stored and used for additional analyses if
needed, since the amount of tissue taken during surgery is limited.

There are risks involved in participating in this study. Insertion of the
catheters in an arm vein and hand vein is comparable to a normal venipuncture
and the only risk is a small local hematoma. The same counts for the muscle
biopsy. The incision made for obtaining the muscle biopsy will be done by an
experienced physician and will heal completely. Within our research group we
have extensive experience with taking muscle biopsies. During the follow up
several days after taking the biopsy, no complications have been reported. The
biopsies of the small intestine, subcutaneous fat, visceral fat, pancreas and
tumour will be taken from parts that will be resected during the surgery, thus
preventing the risk for permanent complications. Potential peroperative
bleeding of the tissue will be electrocoagulated by the surgeon. The liver
biopsy is associated with a small chance of bleeding. This can be stopped by
electrocoagulation during surgery. There are no possible complications for the
gallbladder biopsy since this is taken after the gallbladder has been removed
from the patient*s body. All abdominal biopsies will be taken by skilled
hepatobiliarypancreatic surgeons.
The labeled amino acid tracers applied in this experiment are not radioactive
and are completely safe. The production of the tracers for intravenous
administration will occur in a sterile environment according to GMP guidelines.
Time investment of patients is minimal since testing and data collection will
be planned after a scheduled during a visit at the outpatient clinic (half an
hour) and during surgery. No additional hospital visits are required.