ACTH profiles in dogs after transsphenoidal
hypophysectomy as an indicator for recurrence.
Research study by Danielle Zierikzee
Supervisor: Drs. S van Rijn
Summary
One of the most encountered endocrinological disorders in dogs in the veterinary practice is
Cushing’s syndrome or hypercortisolism a disorder of the pituitary gland or adrenal glands
which causes the adrenal glands to secrete an excess of cortisol. Hypercortisolism causes a
wide variety of symptoms and not all are always seen in every patient.
Dogs with this disease can be treated with medication or in case of pituitary-dependent
hypercortisolism a surgical procedure called a transsphenoidal hypofysectomy can be
performed. In Europe this surgery is only performed at the University Clinic of companion
animals in Utrecht, the Netherlands. One of the most common complications of a
transsphenoidal hypophysectomy is the possibility of the recurrence of hypercortisolism due
to regrowth of the pituitary cells.
1
Table of contents
Introduction........................................................................................................................3
- Cushing’s syndrome.......................................................................................................3
- Clinical signs of hypercortisolism...................................................................................3
- Pituitary-dependent hypercortisolism...........................................................................4
The pituitary gland..............................................................................................................5
- Anatomy.........................................................................................................................5
- Function and physiology................................................................................................6
Diagnosis of hypercortisolism.............................................................................................7
- Urinary corticoid to creatinin ratios...............................................................................7
- High-dose dexamethasone suppression test.................................................................7
- Low-dose dexamethasone suppression test..................................................................8
Imaging of the pituitary gland.............................................................................................8
- Computed tomography..................................................................................................8
Treatment of pituitary-dependent hypercortisolism...........................................................9
- surgical treatment: Transsphenoidal Hypophysectomy................................................9
- recurrence....................................................................................................................10
- factors of prediction of recurrence..............................................................................10
Use of hormone profiles in the prognosis of recurrence....................................................11
Aims of the study..............................................................................................................12
Materials and Methods.....................................................................................................12
- Database......................................................................................................................12
- Patient follow-up information.....................................................................................12
- Animals.........................................................................................................................13
- Blood sampling and hormone values...........................................................................13
- Statistical analysis........................................................................................................14
Results..............................................................................................................................15
- Follow-up data.............................................................................................................15
- Box-plots ACTH values.................................................................................................15
- Cox’s regression univariate and multivariate analysis.................................................19
- Kaplan Meier survival analysis.....................................................................................20
Discussion.........................................................................................................................22
Conclusion........................................................................................................................24
References........................................................................................................................25
2
Introduction
Cushing’s syndrome
Cushing’s syndrome is a severe endocrinological disorder due to an excess of cortisol
secreted by the body. The chronic overproduction of cortisol is produced by the adrenal
glands. The pituitary gland produces ACTH (adrenocorticotrophic hormone) that stimulates
the adrenal glands to produce cortisol. Cushing’s syndrome occurs when this balance is
impaired by a overproduction of ACTH from the pituitary gland or by overproduction of
cortisol from the adrenal glands due to a neoplasia at one of these two sites. Therefore
hypercortisolism can be caused by the overproduction of adrenocorticotropic hormone due
to a neoplasia of the pituitary gland: pituitary-dependent hypercortisolism or by an adrenal
neoplasia that produces cortisol: adrenal-dependent hypercortisolism.(1)
Hypercortisolism can occur in all dogs of middle-age and older and all breeds can develop
the disease but the incidence is higher in some breeds. Poodles, dachshunds and small
terriers are more at risk to develop pituitary-dependent hypercortisolism. Adrenal tumors
are more frequently seen in larger breeds. There is no real difference in occurrence of
Cushing’s syndrome in sexes, male and female seem to be equally affected.(1)
The clinical signs of hypercortisolism
In a normal healthy body with a normal cortisol metabolism the effects of cortisol are
necessary and limited, in the event of hypercortisolism these effects are persistent and
cause severe problems in the body. The effects of hypercortisolism caused by a pituitary
neoplasia are the same as caused by a neoplasia in the adrenal glands, both cause the same
severity of hypercortisolism. Clinical signs accompanied by hypercortisolism are complex and
not all are presented in every patient. The Clinical symptoms can be divided in the effects it
has on various systems in the body. Metabolic changes are polyphagia, polyuria and
polydipsia, weight gain, abdominal enlargement and hepatomegaly. Skin and hair changes
like a thin coat, alopecia, hyperpigmentation and calcinosis cutis can be seen. Urinary
symptoms are polyuria and polydipsia and in some severe cases urinary tract infections, also
glucosuria can be present. Neuromusculair effects are lethargy, muscular weakness and
muscular atrophy, less common myotonia is seen. Panting is a symptom and in some
patients absence of estrus or testicular atrophy occurs.(1,2)
The hypercortisolism causes an increased gluconeogenesis and lipogenesis at the cost of the
body proteins, this gives the abdominal obesity and muscle atrophy. Polyuria is caused by
the interference of cortisol with the regulation and the work of action of vasopressin and the
effect it has on the kidneys. The skin and hair changes are caused by reduced proliferation of
keratinocytes, disturbed proteins metabolism and synthesis of lipids in the skin due to the
cortisol excess. These disturbances in the skin causes the coat to thin and the lack of
regrowth of fur also the skin can get thinner and wrinkled. The immune suppression caused
by the cortisol also makes the skin more sensitive to lesions and infections.(2)
3
Pituitary-dependent hypercortisolism
Pituitary-dependent hypercortisolism is caused by a tumor in the pituitary gland, a micro- or
macroadenoma. The tumor secretes excess ACTH which stimulates the adrenal glands to
overproduce cortisol, resulting in an excess plasma cortisol level. Due to the excess of ACTH
hyperplasia of the adrenal glands occurs and makes them produce even more cortisol.
In 80% of the cases with hypercortisolism the cause is pituitary-dependent, adenomas found
in the pars distalis of the pituitary gland are the most common. Adenomas can also exist in
the pars intermedia of the pituitary gland and in some rare cases a pituitary carcinoma is
found.(1,2) The tumors found in the pars intermedia are often larger than the tumors found
in the pars distalis.(3)
The pituitary gland
Anatomy
The pituitary gland or hypophysis consists of two different parts, the adenohypophysis and
the neurohypophysis. The adenohypophysis consists of the pars tuberalis, the pars distalis
and the pars intermedia. Another differentiation by anatomy can be made by dividing the
pituitary gland in the anterior lobe and the posterior lobe, respectively the adeno- and
neurohypophysis.(4) The pituitary gland has an average size of 1x0,75x0,75 cm in the
medium to large sized dog, but this differs greatly among different breeds. It is located
below the hypothalamus and attached by a narrow stalk suspended under the
hypothalamus. The pituitary gland lies in the hypophysial fossa ventrally to the large brain in
the basissphenoid bone and is covered by dura mater almost all around to the stalk where it
is suspended below the hypothalamus.(5)
Figure 1: Clinical endocrinology of dogs and cats., A. Rijnberk, H.S. Kooistra. 2nd edition,
2010.
4
Function and physiology
The main function of the pituitary gland is the production, storage and release of hormones
into the bloodstream. Figure 1 shows the pituitary gland and the hormones it secretes. The
adenohypophysis is the part of the pituitary gland that produces most of the hormones,
there are six different types of hormones. These are growth hormone (GH), gonadotrophic
hormones which are follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and
furthermore adrenocorticotrophic hormone (ACTH), thyroid-stimulating hormone (TSH) and
prolactin. In the intermediate part of the pituitary gland only one hormone is produced αmelanocyte-stimulating hormone (α-MSH). The neurohypophysis only stores and releases
two types of hormones directly derived from the hypothalamus, these are oxytocin and
vasopressin.(5)
The hypothalamus plays an important role in the production and release of the hormones of
the pituitary gland, by secretion of regulating hypophysiotrophic hormones. Some of these
regulating hormones are by example gonadotropin-releasing hormone (GnRH) the precursor
for FSH and LH and corticotropin-releasing hormone (CRH) the precursor for ACTH, all
hormones derived from the adenohypophysis are regulated by precursors from the
hypothalamus. When all these hormones are released into the bloodstream by the pituitary
gland they reach the target organs where they can exert their function. Table 1 shows an
overview of all the hormones, their origin, target organs and the secondary effect it has in
the body.(4,5)
The regulation of this so called hypothalamus-pituitary axis is done by the hormones
secreted by the target organs, they function as a negative feedback on the system, a
negative feedback loop. A loop is the regulation by the secondary gland, for example cortisol
is produced by the adrenal glands, when cortisol has reached a certain level the
concentration of cortisol in the blood gives a signal to the hypothalamus to stop releasing
CRH and the pituitary gland follows by declining ACTH release and therefore the release of
cortisol will stop.(4)
ACTH is produced in the pituitary gland from pro-opiomelanocortin a long strand of 265
amino acids. . After this process ACTH is ready to be secreted into the bloodstream when
necessary, ACTH has a short half-life only 10 minutes. ACTH is released in a pulsatile pattern,
therefore a short half-life period can be beneficial. When there is enough ACTH in the
bloodstream the release from the pituitary gland will stop and ACTH in the blood will also
disappear fairly quick.(2,4)
5
Table 1: An overview of the hormones of the hypothalamus-pituitary axis(4):
Hypothalamus
pituitary
target organ
secondary secretion
CRH
ACTH
adrenal glands
cortisol
TRH
TSH
thyroid
T4/T3
GnRH
FSH, LH
ovaries/testes
estrogens/progesterone
GHRH/GHIH
GH
lever
growth factor
PRH/PIH
prolactin
mammary gland
milk
Oxytocin and vasopressin are secreted by the hypothalamus and then stored and when
necessary released by the pituitary gland. Oxytocin stimulates the mammary glands to
release milk and vasopressin works on the kidneys ability of water resorption, antidiuresis.(4)
6
Diagnosis of hypercortisolism
Cushing’s syndrome is usually diagnosed by determining the urinary/corticoid to creatinin
ratios in the urine, it is also possible to diagnose Cushing by a bloodtest. Two test are
available the high-dose dexamethasone suppression test and the low-dose dexamethasone
suppression both can measure cortisol the high-dose suppression test in the urine and the
low-dose suppression test in blood plasma.(1,2,3) The dexamethasone suppression tests can
make a difference between pituitary-dependent or adrenal-dependent
hypercortisolism.(1,2,3)
Urinary corticoid to creatinin ratios
The urinary/corticoid to creatinin ratios determines cortisol in the urine and creatinin, which
is usually stable in the urine. Creatinin is used to determine the clearance of the kidneys and
is helpful in determining the cortisol concentration. The owner has to collect the urine at
home two to three consecutive days in the morning. These samples are sent to the
laboratory for determination of the cortisol:creatinin ratios. Collecting of the urine samples
can be done at home during the morning walks and therefore the amount of stress is much
lower than a consult and blood withdrawal at a veterinary clinic, this is beneficial for the
outcome of the test.(1,3) The UCCRs for healthy dogs are within 0.3 to 8.3 x 10-6, when the
ratios are above this range hypercortisolism is established.(3,6) The test has a high accuracy
rate, but the test should only be performed and interpreted when the patients clinical signs
are also fitting with Cushing’s disease, otherwise false positives are easily created. Dogs
suffering from a different condition than adrenal or pituitary can also have elevated UCCRs,
because of the stress of another disease that causes cortisol to rise.(1,3)
High-dose dexamethasone suppression test
It is possible to determine pituitary-dependent hypercortisolism by using the UCCRs in
combination with the administration of dexamethasone orally. Three urine samples of three
consecutive days need to be collected at the second day before taking the urine sample
dexamethasone is given orally. In the last urine sample on the third day a decrease in
cortisol/creatinin ratio of more than 50% from normal values confirms pituitary-dependent
hypercortisolism. In some cases the results of the UCCR’s are not conclusive for PDH, then
imaging is necessary to determine the location of the cause of the hypercortisolism, it still
can be in the pituitary gland but it can also be in the adrenal glands. Ultrasound imaging of
the abdomen for the adrenals and when no abnormalities are found, a CT-scan can be used
to visualise the pituitary gland and its surroundings. In the Netherlands the dexamethason
suppression test in the urine is recommended and mostly used to diagnose Cushing’s
syndrome, because it makes it possible to differentiate between adrenal-dependent and
pituitary-dependent.(2,3)
7
Low-dose dexamethasone suppression test
The low-dose dexamethasone suppression test measures plasma cortisol before and after
the administration of dexamethasone intravenously. The test measures the suppression of
the adrenal glands on the pituitary gland, the negative feedback loop, high cortisol (in the
test dexamethasone) will normally suppress the pituitary gland to produce ACTH and as a
result the adrenal glands produce less cortisol. After the first blood sample has been
collected 0.01 mg dexamethasone per kg bodyweight is injected intravenously, after 8 hours
another blood sample is collected. In both samples the plasma cortisol concentration is
measured. Plasma cortisol concentration after 8 hours should be at least 40 nmol/L to
establish hypercortisolism. In cases where the value of the second cortisol measurement is
50% lower than in the first blood sample, pituitary-dependent hypercortisolism is
confirmed.(2,3)
Imaging of the pituitary gland
The next step in diagnosing pituitary-dependent hypercortisolism is imaging of the pituitary
and its possible neoplasia.(6) Although the UCCRs can confirm a pituitary cause for the
hypercortisolism, it is necessary to establish this by visualising the tumor in the pituitary
gland. It is important to establish the differentiating between adrenal en pituitarydependent hypercortisolism by imaging for further treatment options. Size of the pituitary
gland and the size of its possible tumor are also very important to establish for diagnosis and
in the case of treatment by surgery. The scan also shows how big the tumor is and if it might
be invading the surrounding area of the pituitary gland this is important for the prognosis
and the possibility of an operation.(7) Imaging methods for the brain and pituitary gland are
computed tomography (CT) and multi-resonance imaging (MRI). The first choice for imaging
is a CT-scan because of the ability to look at different levels of the pituitary gland and to use
contrast enhancement.(7,8)
Computed tomography
Computed tomography (CT) is the first choice in imaging of the pituitary gland, it enables
direct and accurate imaging. It is possible to enhance the visualisation of the pituitary gland
and pituitary adenomas with radiographic contrast administration intravenously.(7)
The size of the pituitary gland varies between dogs of different breeds and individual dogs of
the same breed, this makes it impossible to evaluate the size just by measuring. The pituitary
height/brain area ratio is calculated to establish if the pituitary gland is enlarged. This ratio is
the height of the pituitary gland divided by the area of the brain, this differentiates for the
difference in brain and pituitary sizes among different dogs. A P/B ratio above 0.31 indicates
an enlarged pituitary gland.(9,10,11) An enlarged pituitary gland often predicts that there is
a tumor present in the pituitary gland, macroadenomas extend outside the normal
boundaries of the pituitary gland. Large pituitary tumors are therefore fairly easy to
8
diagnose on a CT-scan. However 40% of the tumors in the pituitary gland are
microadenomas, these do not change or enhance the shape of the pituitary gland.(9,10,11)
Dynamic computed tomography is used to diagnose small macroadenomas or
microadenomas. A series of transverse scans, thin slices through the pituitary gland in
combination with contrast enhancement may reveal these tumors. The difference in blood
supply between the neurohypophysis and adenohypophysis causes the contrast to be able
to differentiate between these two parts. A adenoma may cause displacement of the central
part of the neurohypophysis, when this is seen on a CT-scan the site of the adenoma can be
located in this way.(10,11)
Computed tomography is also used in the procedure where the pituitary gland and its tumor
are removed, a hypophysectomy. For the surgery it is necessary to have CT images to locate
the pituitary gland, also its size is important and therefore the pituitary/brain ratio is used.
The CT-scan images determine the surgical landmarks needed to perform the
hypophysectomy.(8,9,10)
Treatment of pituitary-dependent hypercortisolism
There are different treatment options of pituitary-dependent hypercortisolism, a medical
treatment, a surgical treatment and radiotherapy. The treatment of PDH is focussed at
eliminating the cause for the hypercortisolism, the pituitary adenoma that causes an
excessive ACTH. The medical treatment can be focussed at the pituitary gland and the
adrenal glands. The success rates of the treatments vary, but for PDH the best treatment
option considered is a transsphenoidal hypophysectomy.(2)
Surgical treatment: transsphenoidal hypophysectomy
Dogs suffering from PDH with a pituitary adenoma can be cured by performing a procedure
called a transsphenoidal hypophysectomy. This procedure requires special skills and
experience from the surgeon and can only be performed at a hospital or clinic that has
access to CT or MRI imaging techniques and intensive postoperative care facilities. The goal
of the surgery is to remove the pituitary adenoma and the complete pituitary gland, this to
achieve total removal of all tumor cells and that no regrowth of the adenoma can occur.(2)
The procedure is performed with the patient in ventral recumbency and the head and neck
stretched and placed on a supportive cushion. The location of the surgery is in the mouth,
the pituitary gland is reached by making an incision through the soft palate in the region of
the hamulae of the patient, the exact location of the pituitary gland has been determined by
CT imaging, this is vital information for the surgeon. Carefully the surgeon reaches the
sphenoid bone and makes a hole in the bone to reach the dura mater that covers the
pituitary gland. The dura mater is opened and the pituitary gland is visualised, then it is
detached from its surroundings and gently removed.(12)
9
Dogs that underwent this procedure require excellent postoperative care, an intensive care
unit where they can recover under 24 hour surveillance. Complications or side-effects of a
total hypophysectomy are diabetes insipidus and keraconjuntivitis sicca these diseases need
to be monitored for weeks or months and treated, in most dogs these conditions resolve
over time. Lifelong supplementation with cortisone and thyroxine is required, these are
essential for the patient to function, because they can no longer produce these
themselves.(2)
Recurrence
The goal of the hypophysectomy is to remove the pituitary gland and its tumor whole, the
recurrence occurs when regrowth of tumor pituitary cells is possible. Regrowth is only
possible when during the surgery parts of the pituitary gland are left behind, in most cases
microscopically small cells are left behind and in the long-term they can cause
recurrence.(13,14) For the surgeon that is experienced in this procedure it is difficult to
determine of everything has been removed, because microscopically small tissue can be left
behind.
During the years that more was learned about the procedure and the patients that
experienced a recurrence were studied, hormone profiles before and after surgery became
interesting values to look at in case of prognosis. ACTH, cortisol, α-MSH and urinary
corticoid/creatinin ratio’s are used and studied in the prognosis of recurrence in both
humans and dogs.(15,16)
Factors of prediction of recurrence of hypercortisolism
Different factors have been known to be of use in the prognosis of recurrence, like pituitary
size, high pre-operative α-msh plasma concentration, high urinary corticoid/creatinin ratios
and increased thickness of the sphenoid bone. Different studies also show that the rate of
recurrence is higher in macroadenomas then in microadenomas of the pituitary gland after
removal.(8,14)
ACTH is the most frequently used hormone in the perioperative evaluation of
hypercortisolism, where hypohysectomy is performed. The tumor, often an adenoma,
secretes this hormone, but it is also possible that the adenoma also produces more α-MSH,
because it can extend in the intermediate part of the pituitary gland where this hormone is
produced. Therefore α-msh and ACTH both can be used in the prognosis of recurrence. It
has been shown that dogs with a pulsatile excretion pattern of ACTH after performing the
surgery is a factor that increases the risk of recurrence of pituitary-dependent
hypercortisolism.(13) The value of ACTH after and before surgery can be a prognostic
indicator for the possibility of recurrence, high ACTH values may be at higher risk of
recurrence. The decline of ACTH after surgery in the next hours also is a prognostic factor, if
ACTH declines relatively fast this is more positive for the prognosis. This indicates that all
tumor and pituitary cells have been removed during the surgery, when all cells are gone
ACTH will decline more rapidly.(8,13,14)
10
Use of hormone profiles in the prognosis of recurrence
Cushing’s disease in humans is described as the same disease in dogs, mostly caused by a
pituitary adenoma that secretes an excess of ACTH that causes hypercortisolism.(17) In
humans the first choice of therapy in case of a pituitary adenoma is to remove the tumor
surgically, not the whole pituitary gland, in humane research there are different studies
done for the prognosis of recurrence, because recurrence is also a common risk after the
surgery in humans.(15,17) Post-operative cortisol and ACTH have been studied in the
prognosis after surgery and have been proven helpful in the prognosis of recurrence. Postoperative ACTH and cortisol values have been studied, usually approximately 24 hours after
surgery. The decline of both ACTH and cortisol after surgery in plasma following the removal
of the pituitary adenoma has been studied.(15,17,18) In patients with low cortisol within the
first 48 hours after surgery, the decline in ACTH was greater than in the patients with
persistent hypercortisolism. Plasma ACTH lower than 20 pg/ml in 3-36 hours after surgery
proved to be a indicator for hypocortisolism. The patients with these ACTH results developed
no recurrence later in life, this reduction in ACTH levels can differentiate for remission after
surgery and recurrence after surgery.(17)
In another study done by Acebes et al. the postoperative measurement of ACTH also seemed
helpful in the determination of a successful surgery, only here the value was considered to
be below 7,55 pg/l. Here it was also linked to the decline in cortisol and together with the
ACTH levels considered to be a factor of surgical success.(15)
In Czirjak et al. ACTH levels were measured 2 hours after surgery at multiple times, the
decline here was linked to the complete removal of the ACTH-producing pituitary adenoma.
A decline of 50% or more in the ACTH values indicated a complete removal and thus
remission after surgery.(18)
Although this ACTH levels prove to be significantly different in patients with recurrence after
surgery, it is very difficult to establish a threshold value of ACTH to go from and determine
at which point it is a higher risk of recurrence. Therefore the utility of post-operative ACTH
levels in humans also remains a important field of research.
In human studies other factors also have been studied that might be helpful in the prediction
of recurrence after removal of the pituitary gland. Postoperative plasma cortisol levels or
CRH levels from the hypothalamus have been studied and found useful in the prognosis after
surgery, however also in this studies the cutoff value is difficult to establish.(15,19,20) These
studies suggest that a prognosis after surgery is difficult to establish and an indication can be
made using the post-operative ACTH and cortisol values, but lifelong follow-up remains
necessary because the results are not inconclusive.(19,20)
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Aims of the study
The objective of this study is to assess immediate postoperative ACTH profiles of dogs that
underwent hypophysectomy and correlate peri-operative ACTH levels with the recurrence of
pituitary-dependent hypercortisolism in the long-term follow up. The following question will
be answered: Is it possible to use peri-operative ACTH levels in dogs with a pituitary
adenoma after hypophysectomy as a predictive value for the recurrence of hypercortisolism
in dogs.
Materials and methods
Database
This research study has been performed using a database available at the University clinic
for companion animals at Utrecht, the Netherlands. This database contains data from dogs
that underwent a transsphenoidal hypophysectomy as a treatment for pituitary-dependent
hypercortisolism at the University clinic for companion animals at Utrecht. All dogs were
operated by the same veterinary neurosurgeon. The database exists of a total of 321 dogs,
of all these dogs there is detailed data and follow-up information available, and when this
was not the case an attempt was made to achieve up to date information. The data available
at the database are the standard values as age, bodyweight, gender, breed and the date of
the surgery, also data of perioperative hormones measured in the blood are available. These
perioperative hormones include ACTH, α-MSH and cortisol, measured at different times
before and after the surgery. Other available information are values of the diagnosis such as
the UCCR’s and of the surgery such as the p/b ratio and detailed follow-up information of
the patients.
Patient follow-up information
The first period of this research study was spent on retrieving and collecting follow-up data
of the patients. In principal the goal was to complete all the data missing in the database,
using the patients information available at the University clinic of companion animals and by
calling the referring vets and owners of the patients. The main goal here was to achieve a
one-year follow up period of as many dogs as possible, these dogs can then be used in the
research for the recurrence of hypercortisolism. This also means that all hormone values
were checked and completed, in some cases these values are not complete, also these dogs
are further excluded from the research. Patients included in the research are dogs with a
minimal one-year follow up period and with complete data of the hormone values.
12
Animals
After completion of the follow-up information, 112 dogs were included in the research,
these dogs underwent transsphenoidal hypophysectomy over a period of 12 years, from
2001 till 2013. Dogs of 45 different breeds and cross-bred dogs were included. There were 1
Alaskan malamute, 1 American Cocker Spaniel, 2 American Staffordshire terriers, 3 Bassets,
8 Beagles, 2 Bearded Collies, 1 Belgian Shepherd dog, 1 Bernese Mountain Dog, 1
Bolognezer, 1 Border Collie, 1 Bouvier des Flandres, 5 Boxers, 1 Cavalier King Charles Spaniel,
1 Chesapeake Bay Retriever, 8 Dachshunds, 1 Dogo Argentino, 1 English Cocker Spaniel, 1
English Setter, 1 English Springer Spaniel, 1 French Bulldog, 3 German Pointers, 1 German
Shepherd Dog, 1 Golden Retriever, 2 Hovawarts, 4 Jack Russel Terriers, 9 Labrador
Retrievers, 9 Malteses, 1 Medium-sized Poodle, 1 Miniature Pincher, 1 Nova Scotia Duck
Tolling Retriever, 1 Rhodesian Ridgeback, 1 Riesenschnauzer, 1 Rottweiler, 1 Scottish Collie,
2 Scottish Terriers, 1 Shar Pei, 1 Shetland Sheepdog, 1 Shih Tzu, 1 Shipperke, 3 Stabyhounds,
1 Viszla, 1 Welsh Terrier, 3 Yorkshire Terriers and 20 cross-bred dogs. There were 54 male
dogs (22 castrated) and 58 female dogs (44 castrated). The age at the time of the surgery
ranged from 3 to 14 years (median 8,5 years). The body-weight ranged from 3,8 to 57,2 kg
(median 19,25 kg).
Blood sampling and hormone values
ACTH values were sampled before and after the patients underwent the surgery. ACTH was
determined in two samples 24 hours before the surgery, the mean ACTH value of these two
samples is used. Perioperative blood samples for ACTH determination were collected 1 hour
after removal of the pituitary gland, this sample was taken on the surgery table. The other
samples were collected at 2, 3, 4 and 5 hours after surgery. The last blood sample was
collected after 24 hours after the surgery. Blood samples were collected in EDTA tubes and
kept on ice and then sent to the laboratory for analysis. Analysis was performed at the
Veterinary Diagnostics Laboratory of the University of Utrecht, the Netherlands.
13
Statistical analysis
The statistical analysis made for this research study are performed by using SPSS statistical
program (SPSS Benelux BV, Gorinchem, The Netherlands). The analysis of all the ACTH values
is made by using box-plots and single t-tests to check for significance. Between two groups
the difference in ACTH values is analyzed, the recurrence group and the non-recurrence
group. ACTH was categorized in ACTH-24, ACTH-1, ACTH-2, ACTH-3, ACTH-4, ACTH-5 and
ACTH24. Respectively ACTH 24 hours before the surgery, 1,2,3,4 and 5 hours after surgery
and 24 hours after the surgery.
Survival analysis is made by using the cox regression univariate and multivariate analysis,
both with using the ACTH at different time points. Disease free intervals were used from all
the dogs in the research, when they developed recurrence the time was used from surgery
till the recurrence. Survival curves are made by using the Kaplan-Meier survival analysis.
14
Results
Follow-up data
112 dogs were included from the total database with complete hormone profiles and at
least a one year follow-up period. 31 dogs developed a recurrence of hypercortisolism 81
dogs stayed in remission. 77 dogs died and 35 are alive and might be at risk of developing a
recurrence in the future.
Median disease free period of all the dogs was 895,5 days (range: 80-3332 days). In the
recurrence dogs median disease free period was 588 days (range: 80-1688 days).
Of all the dogs 88 had enlarged pituitary glands before the hypophysectomy and 24 dogs had
normal sized pituitary glands. The median for the pituitary/brain ratio was 0,46 (range: 0,131,10) for all the dogs. In the recurrence dogs the median pituitary/brain ratio was 0,55
(range: 0,28-1,10).
Box-plots ACTH values
Box-plots made of ACTH-24, ACTH-1,2,3,4 and 5 and ACTH24 comparing the values for the
recurrence (1) and the non-recurrence (0) group. The values are normally distributed
therefore the significance is tested with T-tests between the recurrence and the nonrecurrence group for the different ACTH values.
Fig. 2: Box-plot of ACTH 24 hours before the surgery, the difference between the groups in a
independent sample T-test is not significant P=0,834. Mean ± SD for ACTH 24 is 111,3 ±
102,1.
15
Fig.3: Box-plot of ACTH values 1 hour after surgery, the difference between the groups in a
independent sample T-test is not significant P=0,294. Mean ± SD for ACTH 1 is 69,1 ± 87,4.
Fig.4: Box-plot of ACTH values 2 hours after surgery, the difference between the groups in a
independent sample T-test is not significant P=0,086. Mean ± SD for ACTH 2 is 36,5 ± 45,4.
16
Fig.5: Box-plot of ACTH values 3 hours after surgery, the difference between the groups is
significant P<0,05 tested with a independent sample T-test. Mean ± SD for ACTH 3 is 27,2 ±
32,5.
Fig.6: Box-plot of ACTH values 4 hours after surgery, the difference between the groups is
significant P<0,05 tested with a independent sample T-test. Mean ± SD for ACTH 4 is 28,9 ±
66,9.
17
Fig.7: Box-plot of ACTH values 5 hours after surgery, the difference between the groups is
significant P<0,05 tested with a independent sample T-test. Mean ± SD for ACTH 5 is 24,2 ±
32,3.
Fig.8: Box-plot of ACTH values 24 hours after surgery, the difference between the groups in a
independent sample T-test is not significant P=0,294. Mean ± SD for ACTH 24 is 11,7 ± 13,4.
The difference between the recurrence and the non-recurrence groups of the ACTH values
of 3,4 and 5 hours after surgery is significant, respectively P<0,05 for these values.
18
Cox’s regression univariate and multivariate analysis
The Cox regression method is used for investigating the effect of the different ACTH-values
in a period of time on survival, here the survival is the time until recurrence. A Cox
regression survival analysis was made for the absolute and relative ACTH-values. The
absolute values are the measured values of ACTH the relative values are calculated in
relation to all the values, to see them in perspective for their decline or if the measurement
was a very high value.
Table 2 shows the outcomes of the Cox-regression univariate analysis separate for all the
absolute ACTH-values. Table 3 shows the outcomes of the Cox-regression univariate analysis
separate for all the relative ACTH-values. The significant values of ACTH from the univariate
analysis are entered in a multivariate Cox-regression analysis. Table 4 shows the outcomes of
the Cox-regression multivariate analysis.
Table 2: Cox-regression univariate analysis absolute ACTH values.
Variable
P
95% CI
ACTH-24
0.491
0.998-1.005
ACTH 1
0.070
1.000-1.007
ACTH 2
0.002
1.003-1.015
ACTH 3
0.000
1.009-1.026
ACTH 4
0.000
1.004-1.013
ACTH 5
0.000
1.011-1.032
ACTH 24
1.000
Table 3: Cox-regression univariate analysis relative ACTH values.
Variable
P
95% CI
ACTH 1
0.767
0.938-1.049
ACTH 2
0.044
1.008-1.911
ACTH 3
0.047
1.004-1.778
ACTH 4
0.001
1.072-1.316
ACTH 5
0.001
1.154-1.793
ACTH 24
0.297
0.546-7.242
Table 4: Cox-regression multivariate analysis of all the significant absolute and relative
values.
Variable
P
95% CI
ACTH 3
0.002
1.006-1.030
ACTH 4
0.000
1.004-1.013
19
Analysis made with spss statistical program show that ACTH at -24,1,2,5 and 24 hours after
hypophysectomy are not significant for predicting a recurrence in the dogs. The ACTHvalues at 3 and 4 hours after surgery are significant for predicting a recurrence.
Kaplan Meier survival analysis
The results of the Cox-regression were entered in the Kaplan-Meier survival analysis. ACTH 3
and ACTH 4 are significant in the Cox-regression, of these variables the survival curves are
made. Both variables were divided in high and low ACTH values for all the dogs. The median
was used to establish a cut off point for a high value or a low value. Median for ACTH 3 is
18,5, median for ACTH 4 is 17,5, above these values was considered a high value. Dogs with
high ACTH values are group 1, dogs with low ACTH are group 0. The curves for both groups
are plotted in the same graph using the Kaplan-Meier survival analysis. The significance
between the two curves is tested with a log rank test for ACTH high/low. The log rank test
for ACTH 3 was not significant, P=0,197, the log rank test for ACTH 4 is significant P=0,004.
Fig 9: Kaplan-Meier survival curve for ACTH 3 high/low. The green line represent the dogs
with high ACTH 3, the blue line represents the dogs with low ACTH 3. The difference
between the two curves high and low ACTH 3 is not significant, Log rank test P=0,197.
20
Fig 10: Kaplan-Meier survival curve for ACTH 4 high/low. The green line represent the dogs
with high ACTH 4, the blue line represents the dogs with low ACTH 4. The difference
between the two curves high and low ACTH 4 is significant, Log rank test P=0,004.
21
Discussion
In this study ACTH 4 hours after surgery seems to be an indicator for the prognosis of
recurrence. The significance of ACTH 4 high/low indicates that dogs with high ACTH 4 are
more at risk for developing a recurrence then the dogs with low ACTH 4. Dogs with low ACTH
4 have a longer disease free period and stay longer in remission, dogs with high ACTH 4 have
a shorter disease free period, they develop a recurrence faster. Overall ACTH 4 seems to be
the best indicator for the prognosis for recurrence. It is hard to know why only ACTH at 4
hours after surgery comes out to be the only indicator for recurrence, the relative values
might be the cause for the significance. The box-plot of ACTH 4 with the absolute ACTH
values does not show that big of a difference between the recurrence and the nonrecurrence group, the use of the relative ACTH values in the Cox regression analysis have
been useful in the significance of ACTH 4.
It is difficult to establish why the other values of ACTH at different time points in this study
don’t show a significance for the prognosis of recurrence. There are no veterinary studies
done before where they looked at ACTH at the different time points done in this study, this
makes it difficult to compare the results.
In this study dogs with an ACTH 4 value of 17,5 or higher were considered to be at higher risk
for developing a recurrence. In other veterinary studies there has not been a value of ACTH
that was established in the prognosis of recurrence. It is known that high ACTH values
increase the risk of a recurrence after surgery, or a faster decline after surgery of ACTH is
beneficial in the prognosis.(8,13,14) The pulsatile pattern of ACTH after surgery has also
been determined as a higher risk factor for recurrence.(13)
In humane research ACTH seems to be useful in the prognosis of recurrence, in the study by
Acebes et al. the postoperative measurement of ACTH seemed helpful in the determination
of a successful surgery, the value was considered to be below 7,55 pg/l.(15) in the study
done by Czirjak et al. a decline of 50% or more in the ACTH values indicated a complete
removal and thus remission after surgery.(18)
In this study a specific group of animals is used, the results of the ACTH levels of these dogs
have a certain outcome. Another group of animals would probably give a different
distribution and outcome of ACTH results. This is of course related to the nature of how
variable the different ACTH values are in different dogs. Different dogs with different ACTH
values might give another result of the ability to use them in the prediction of recurrence.
Another group would naturally give a different statistical median or mean and this might
change the cut-off point or threshold value for a certain ACTH value. In this study ACTH at 4
hours after surgery was significant for predicting a recurrence, more research is needed to
be able to determine if this is the only value that is useful. Humane research already showed
different outcomes of the hormone values in different studies with a different group of
patients. Therefore it is difficult to establish a threshold value from which a conclusion can
be made and a prediction for recurrence.(15,17)
22
The research might be expanded in the future with more patients that are presented for
transsphenoidal hypophysectomy, a larger group of dogs might increase the chance of
getting more significant results. A much more larger group then the one already discussed
might give significant results of other ACTH values.
The only value used in the prediction of recurrence in this research is ACTH, but as discussed
earlier there are a lot of different factors that influence the prognosis after transphenoidal
hypophysectomy. Different hormones like cortisol are also a factor in the prediction of
recurrence and especially the relation between ACTH and cortisol, this is not researched in
this study. When ACTH declines after surgery the most important is the relation with the
decline of cortisol. The decline in cortisol after surgery might be useful to include in the
prognosis for recurrence.(20)
The other factors that influence the possibility of a recurrence of hypercortisolism are also of
importance, these are all left out. Tumor type: micro- or macroadenoma, enlarged pituitary
gland, high pre-operative α-msh plasma concentration, high urinary corticoid/creatinin ratios
and increased thickness of the sphenoid bone are all factors that could be a predictive factor
for the recurrence. Especially a macro or microadenoma can be an important factor in the
prognosis, because large tumors are difficult to remove and they often secrete more ACTH.
(8,13,16)
As discussed there are a lot of factors that can be helpful in determining the prognosis for
recurrence after transsphenoidal hypophysectomy, only ACTH is a parameter that is possible
to measure in time at different points. This makes ACTH a factor that is worth studying in the
prognosis of recurrence in the future. This also true for cortisol and α-msh, these together
with ACTH might be the best indicators for the recurrence after transsphenoidal
hypophysectomy.(13)
23
Conclusion
In this study we researched the question if ACTH values of dogs that underwent
transsphenoidal hypophysectomy can be an indicator for the recurrence of clinical signs of
hypercortisolism. ACTH values at different times before and after the surgery have been
investigated from a database of 321 dogs. For 112 dogs of this database complete ACTH
profiles were obtained and used in this study. After statistical analysis the result for ACTH at
4 hours after surgery was the only value that was significant for predicting a recurrence. The
other time points of the ACTH values were not significant for predicting a recurrence, but
might be useful in the future with additional research.
ACTH 4 hours after surgery turns out to be the only possible time point that can be used in
the prediction of recurrence for clinical signs of hypercortisolism. In the future it might be
possible to use ACTH 4 as the most important indicator for recurrence.
The cut off point in this study for ACTH 4 hours after surgery was 17,5, when a dog had an
ACTH 4 value above this point it was considered high and increases the risk for recurrence.
This might serve as an indicator but other studies already have shown, mainly in human
research that it is difficult to establish a threshold value. Therefore more research is
probably needed in combination with other factors as already discussed.
Still recurrence after transsphenoidal hypophysectomy is a great risk and it happens often in
dogs, therefore lifelong monitoring is necessary. Dogs that had low ACTH 4 values are
probably at a lower risk for developing a recurrence but still owners should comply to the
fact that every 6 months their dogs need to be checked for hypercortisolism by determining
the UCCR’s. Owners of dogs that underwent transsphenoidal hypophysectomy for the
treatment of pituitary-dependent hypercortisolism should always be aware of the
recurrence of clinical signs of hypercortisolism.
24
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