Cancer Cachexia: Organ-specific Protein Synthesis 2

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 body mass index (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, they have never
been properly proven in humans. The availability of amino acids labelled with
stable isotopes creates the possibility to *trace* them at different points in
their metabolism using mass spectrometry. However, previous studies provide
conflicting data on protein metabolism in cachexia. Some show that there is
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 protein metabolism in muscle, protein metabolism in visceral organs
(e.g. liver, small intestine) might be an important factor in cachexia. A
recent bovine study showed that protein synthesis in several visceral organs is
in fact much higher than protein synthesis in muscle. Because of its visibility
and accessibility, muscle protein has been the main interest of most studies.
However, organ protein turnover is much higher than muscle protein turnover and
therefore possibly more affected by the negative protein balance in cachexia.
Common symptoms in cancer cachexia such as nausea, anorexia and insulin
resistance indicate multiple organ dysfunction. Also, tumour protein metabolism
has never been studied properly. An extremely high tumour protein turnover
would indicate that tumour protein consumption would strongly contribute to the
negative balance in cachexia. Some studies on tumour metabolism 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 our recent study (METC 13-3-068),
we assessed the protein synthesis rates in multiple organs during surgery in
cachectic patients with pancreatic cancer. Our first analyses indicate high
turnover rates as expected. However, because we only included cachectic
patients and due to the relatively short time of tracer infusion (around 6
hours), we will not be able to properly show a relation of the cachectic status
of a patient with tumour-specific and organ-specific protein synthesis rates.

Tracer techniques have been improved over the last few years and studies using
deuterium labelled water (2H2O) have been grown in popularity. Using 2H2O to
endogenously label the patient*s protein pool is an elegant method since it
allows for longer study duration (days rather than hours) and there is less
burden for the patient since and intensive tracer infusion day is no longer
necessary. Also, a recent study indicates that fractional synthetic rates
(FSRs) of plasma markers of muscle tissue (e.g. creatine kinase M) correlate
very well with muscle tissue FSR. Thought this has not been explored yet,
plasma protein coming from organs such as the liver or gut might be used to
assess organ FSR, which could be used to replace biopsies in many future
studies.

In the study proposed here, we will compare tissue protein synthesis of
cachectic with non cachectic patients with pancreatic cancer by assessing
fractional synthetic rates using deuterium labelled water in a two week period.
In addition, we will explore the correlation of plasma protein FSRs with tissue
protein FSRs.

Primary Objectives:
1. To compare protein synthesis rate of the tumour and normal pancreas between
cachectic and non-cachectic patients with pancreatic cancer.
Secondary Objective(s):
1. To compare protein synthesis rate of liver, intestinal, adipose tissue,
muscle tissue, and leukocytes between cachectic and non-cachectic patients with
pancreatic cancer and non-oncologic controls.
2. To assess the correlations between organ-specific plasma protein FSRs and
organ FSRs.
3. To assess regional tumour protein synthesis using mass spectrometry imaging

This study will be a cross-sectional study that will be conducted at Maastricht
University Medical Centre (MUMC, Maastricht, Netherlands).
In this study, protein turnover and protein incorporation into various organs
and tissues of patients with pancreatic cancer undergoing surgery will be
analysed by oral ingestion of 2H2O. In addition, non-oncologic patients
undergoing a cholecystectomy will be included into this study. After having
signed for informed consent, first data collection will take place. The
following patient characteristics will be collected from the patient*s medical
record:
• Age
• Sex
• American Association of Anesthesiologists (ASA) classification
• BMI
• Weight loss in the past six months
• Nutritional Status
• Abdominal computed tomography scan (CT-scan)
• 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 total study period will last two weeks. Fourteen days prior to surgery (day
0), the patient will come to the hospital or will be visited at home by the
investigator for sampling of 20ml of venous blood by venepuncture and a saliva
sample (using a swab). The patient will receive seven portions of 2H2O. The
patient will be instructed to 1) take a saliva swab daily and 2) ingest 60ml of
2H2O daily. After a week (day 7), the patient will meet the investigator. This
can either be at the hospital or at the patient*s home, which will be decided
by the patient*s preference. During this visit 1) an additional blood sample of
20ml will be drawn through standard venipuncture, 2) the empty water bottles
will be collected by the investigator, 3) the patient will receive 7 new 2H2O
bottles for the next week, 4) the saliva swabs will be collected, 5) the
patient will receive a movement meter and a nutritional diary. The patient
should keep the movement meter strapped on during the remaining study period
and to keep a nutritional diary to track every meal, snack, and/or drink that
is consumed during this week. All in all this visit will not take longer than
one hour. The patient will than continue with saliva swabs and ingestion of
60ml 2H2O daily, until the day of surgery. The day before surgery (day 13),
patients will be admitted to the hospital, as part of standard preoperative
care. The investigator will visit the patient at the ward in the evening and
will collect the patient*s saliva swabs, movement meter, nutritional diary, and
empty water bottles. Next morning, the investigator will take a blood sample
(20ml), through the intravenous catheter that has been placed as part of
preoperative care. After one hour of surgery there will be adequate exposure of
the abdominal organs. A muscle biopsy from the rectus abdominis and vastus
lateralis muscle will be collected as well as a 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. When the tumour and
adjacent pancreatic tissue has been removed, a biopsy of the pancreas and
pancreatic tumour will be taken. For non-oncologic patients all biopsies will
be taken directly after start of surgery. The gall bladder biopsy will be taken
after removal of the gallbladder. No pancreas, tumour, or intestinal biopsies
will be taken in non-oncologic patients. In total, patients will have to make
either two additional visit to the hospital or the investigator will visit the
patient two times at home (1 hour per visit). Patient*s discomfort is reduced
to the minimum since all biopsies are taken under general anaesthesia. Only two
additional venipunctures will be performed. After diagnoses by the pathologist
(usually one week after surgery), tissue that would be otherwise discarded will
be collected and stored for future analysis.

There are some small risks involved in participating in this study. A
venipuncture has a small risk of 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. Saliva swabs are collected by the patients themselves with a cotton
swab and do not form any kind of risk or discomfort. 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 labelled 2H2O tracers applied in this experiment are not radioactive and
are completely safe. The production of the tracers for oral administration will
occur in a sterile environment according to GMP guidelines.
Time investment of patients is low since only two extra one-hour visits are
required, which can also be scheduled at the patient*s home. Other sample
collection will take place during surgery when the patient is asleep. The
number of biopsies and blood samples has been reduced to the minimum for
minimal patient discomfort. All biopsies are taken while under general
anaesthesia.