I think most people have had a form of medical testing at some point in their life, i.e. the sampling and testing of blood, urine, saliva, stool or body tissue. In a nutshell, the medical staff are just measuring the content of a ‘substance’ and then taking a view whether this is normal or not based on pre-determined ranges. These tests are normally done as a physician’s reaction to symptom presentation or maintenance/surveillance of an existing diagnosed condition. Sometimes, abnormal results will lead to more specialist tests.

In cancer, these tests are frequently called ‘markers’. Most tumour markers are made by normal cells as well as by cancer cells; however, they are produced at much higher levels in cancerous conditions. These substances can be found in the blood, urine, stool, tumour tissue, or other tissues or bodily fluids of some patients with cancer. Most tumour markers are proteins. However, more recently, patterns of gene expression and changes to DNA have also begun to be used as tumour markers.  Many different tumour markers have been characterized and are in clinical use. Some are associated with only one type of cancer, whereas others are associated with two or more cancer types. No “universal” tumour marker that can detect any type of cancer has been found.

There are some limitations to the use of tumor markers. Sometimes, noncancerous conditions can cause the levels of certain tumor markers to increase. In addition, not everyone with a particular type of cancer will have a higher level of a tumour marker associated with that cancer. Moreover, tumour markers have not been identified for every type of cancer. Tumour markers are not foolproof and other tests and checks are usually needed to learn more about a possible cancer or recurrence.

I’d also like to talk about a group of associated tests, in particular, hormone levels as these tests are really important to help determine the type of Neuroendocrine Tumour.  NETs will sometimes oversecrete hormones and this can give clues to the type.  The constraints mentioned above apply to hormone levels and other tests to a certain extent.

What this article will not cover

Routine Testing – the post will not cover routine blood tests (i.e. complete blood count etc).  Although they may point to a problem, these tests do not necessarily indicate a particular type of NET without other supporting evidence.

Biopsy Testing – Technically, the Immunohistochemical ‘stains’ used in biopsy testing are tumour markers but I’ll not be discussing that today. I did cover the output of biopsies in my blog on NETs – Stages and Grades.

Genetic Testing.  This is very specialised but you may find my Genetics and NETs article is of interest.

Sequencing of marker testing – diagnosis

The sequencing of marker testing may have been different for many patients.  In my own experience, I had a biopsy and then the biochemical checks were carried out. So regardless of the results of my marker tests, I was to be diagnosed with NETs. Those with lengthy and difficult diagnostic phases will perhaps have had a different sequence with the biochemical markers providing evidence for further tests to formally diagnose.  Markers alone will normally not be enough for a diagnosis but they do, however, feed into the treatment plan and provide a baseline at diagnosis and for tracking going forward.

Interpreting test results – International/National/Regional differences

The use of markers tends to be different on an international basis, e.g. specific marker tests can be developed in-country by independent labs. Testing can also vary in the same country as labs through the use of different commercially available ‘testing kits’.

Reference ranges are dependent on many factors, including patient age, gender, sample population, and test method, and numeric test results can have different meanings in different laboratories. The lab report containing your test results should include the relevant reference range for your test(s). Please consult your doctor or the laboratory that performed the test(s) to obtain the reference range if you do not have the lab report.

Moreover, the ‘normal’ test range can vary from hospital to hospital, even within the same tests. I can only imagine that clinical staff have their own versions of risk thresholds when dealing with test results. Even when results are just above or below, individual physicians can take their own view in a subjective manner. Testing is best done at the same lab each time if possible.

There’s a great website called LabTestsOnline which can describe each test. It’s peer-reviewed, non-commercial and patient-focused but just please note you should always refer to your own lab ‘normal ranges’ which will be printed on your test results.  For these reasons, you will not find reference ranges for the majority of tests described on this web site.  The link above will take you to the list of ‘country’ affiliated versions with specific information on a country basis.

Here’s some tips I always give people:

1 – Always try to get your own copy of results (preferably on paper) and track them yourself (I use a spreadsheet).

2 – When comparing results inside patient forums, always add the range and if possible, the unit of measurement (i.e. g/L, mmol/L, umol/L etc etc).  Failure to do this can at best confuse, and at worst frighten patients.  Compare apples with apples not with pears!

3 – Don’t get too excited about rises if the test is still inside the normal range – normal is normal!

4 – Don’t get too excited about rises taking you just outside of normal range – your doctors are looking for bigger spikes.

5. Don’t get too excited about a single test result, your doctors are looking for trends, a single test result is not much to go on.

NET Markers

Although some routine blood markers (complete blood count etc) are useful in NETs, it’s pretty much impossible to cover these in any general detail.  I’m going to focus on tumor and hormone associated markers

There are many markers involved with NETs. Some do different jobs and some are just variants measuring the same thing (more or less efficiently). You may also see something called ‘gold standard’ in reference to NET Tumour markers. Although thinking is changing (more on this below) and can vary from country to country, it is generally accepted that Chromogranin A and 5HIAA are the gold standard markers for tumour bulk and tumour functionality respectively.  These gold standard tests may not be applicable to every type of NET, particularly 5HIAA. I’m also aware that US doctors are reducing the dependency on CgA and using Pancreastatin instead (although many are measuring both).

NETs are known to be heterogeneous in nature (i.e. consisting of or composed of dissimilar elements; not having a uniform quality throughout).  Whilst some markers can be used widely, it follows that there are many very specialist marker tests for individual types of NET.  I think this applies to 3 broad categories of NETs: Tumours known to potentially oversecrete Serotonin and and perhaps others (mainly midgut), Pancreatic NETs (or pNETs) secreting various hormones by type; and other less common types and/or syndromes which might be considered by some to be even more complex than the former two and in some cases there are big overlaps.

Another interesting thing about NET markers is that an undiagnosed patient may undergo several specialist tests to eliminate the many possibilities that are being presented as vague and common symptoms.  Sometimes this is necessary to eliminate or ‘home in’ on a tumour type or syndrome/hormone involved (it’s that jigsaw thing again!).

Markers too can be divided into broad categories, those measuring how much tumour is in your body and its growth potential and those measuring how functional (or not) those tumours are.  The latter can probably be expanded to measure/assess excess hormone secretion and syndromes.

The Anatomy

Certain tests can be anatomy related so to add context and to prevent big repetitive lists when using the terms ‘foregut’, ‘midgut’ and ‘hindgut’, you may find this graphic useful.

Markers for measuring Tumour bulk or load/growth prediction

Chromogranin (plasma/blood test)

Chromogranin is an acidic protein released along with catecholamines from chromaffin cells and nerve terminals. This statement alone might explain why it is a good marker to use with NETs.  Depending on the test kit being used, you may see test results for Chromogranin A (CgA) and Chromogranin B (CgB) – the inclusion of CgB tends to be confined to Europe. There is also mention of Chromogranin C (CgC) in places but I’ve never heard of this being used in conjunction with NETs.

One of the disadvantages of CgA is that the results can be skewed by those taking Proton Pump Inhibitors (PPIs).  Many NET patients are taking PPIs to treat GERD (….and Zollinger-Ellison Syndrome). In the long-term, this has the result of increasing gastrin levels which can lead to an increase of CgA in the blood including for some months after discontinuing. CgB is said not be as influenced by the use of PPI as CgA. In addition to the issue with PPIs, CgA levels may also be elevated in other illnesses including severe hypertension and renal insufficiency. CgB is also said to be more sensitive to Pheochromocytoma.

Elevated CgA is a constant and somewhat excitable discussion point on patient forums and not just because of the lack of unit of measurement use I discussed above. Some people get quite excited about a single test result.  I refer to Dr Woltering et al (ISI Book) where it clearly states that changes in CgA levels of more than 25% over baseline are considered significant and a trend in serial CgA levels over time has been proven to be a useful predictor of tumour growth (i.e. a single test result with an insignificant rise may not be important on its own).  Dr Woltering also gives good advice on marker tests when he says “normal is normal” (i.e. an increased result which is still in range is normal).

Here is a nice graphic explaining what else could be the cause of elevated CgA:

CgA appears to be a widely used tumour marker and is effective in most NETs (foregut, midgut and hindgut). It is also sensitive to Pheochromocytoma, particularly when correlated with a 131I-MIBG scan. Interestingly Chromogranin can also be used in the immunohistochemical staining of NET biopsy samples (along with other methods).

As for my own experience, my CgA was only elevated at diagnosis, remained elevated after intestinal surgery but returned to normal after liver surgery (indicating the effect of liver tumour bulk on results).  It also spiked out of range when some growth in a distant left axillary node was reported in Jan 2012.  Following a lymphadenectomy, it returned to normal again and has remained in range to this day.  It has been a good predictor of tumour bulk for me and I’m currently tested every 6 months.

Pancreastatin

In effect, this marker does the same job as CgA.  Interestingly, Pancreastatin is actually a fragment of the CgA molecule. There have been many studies (mainly in the US) indicating this is a more efficient marker than CgA, and not only because it is not influenced by the use of PPI.  It has also been suggested that it’s more sensitive than CgA and therefore capable of detecting early increases in tumour burden. It has also been suggested it can be an indication of tumour ‘activity’ (whatever that means). It is widely used in the US and some physicians will use it in preference to CgA (…..although from what I read, CgA also seems to be tested alongside).  I’m starting to see this mentioned in the UK.

Neurokinin A (NKA)

This is not a well publicised test. However, it is something used in USA but I’d like to hear from others to validate its use elsewhere.  In a nutshell, this test, which only applies to well differentiated midgut NETs, appears to have some prognostic indication.  I discovered this test in the ISI NET Guidance and it’s backed up by a study authored by names such as Woltering, O’Dorisio, Vinik, et al.  This is not a one-off test but one designed to be taken serially, i.e. a number of consecutive tests.  These authors believe that NKA can also aid in the early identification of patients with more aggressive tumors, allowing for better clinical management of these patients.  NKA is sometimes called Substance K.

Neuron-Specific Enolase (NSE)

In patients with suspected NET who have no clear elevations in the primary tumor markers used to diagnose these conditions, an elevated serum NSE level supports the clinical suspicion.

Markers for measuring Tumour functionality/hormone/peptide levels

So far, I’ve covered basic tumor markers which have a tumor bulk and/or prognostic indication.  This section is a slightly more complex area and many more tests are involved. There’s often a correlation between CgA/Pancreastatin and these type of markers in many patients i.e. a serial high level of CgA might indicate a high level of tumour bulk and therefore increased production of a hormone in patients with a syndrome or oversecreting tumor. However, it frequently does not work out like that, particularly when dealing with non-functioning tumours.

The type of marker for this element of NET diagnosis and surveillance will vary depending on the type of NET and its location (to a certain extent).  Like tumour bulk/growth, there might be different options or test variants on an international basis. There are too many to list here, so I’ll only cover the most common.

Serotonin Secreting Tumors

There are a few markers in use for measuring the functionality of this grouping of tumours. This tumour group has a tendency to secrete excess amounts of the hormone Serotonin although it differs depending on the area of the primary. For example, hindgut tumours tend to secret lower levels than foregut and midgut and therefore this test may present within range.  Please also note there may be other hormones of note involved. The antiquated and misleading term ‘Carcinoid’ is sometimes used as a descriptor for these tumours and more and more NET scientific organisations and specialists are now avoiding use of this term.

5HIAA.  5HIAA is a metabolite of Serotonin thus why it’s a useful thing to measure to assess functionality in this grouping of tumours. 5HIAA is actually the ‘gold standard’ test for functioning serotonin secreting tumours. It’s a key measure of the effects of carcinoid syndrome and the risk of succumbing to carcinoid heart disease.  However, there are two methods of testing:  Urine and Plasma. The latter is mainly used in USA but other countries are now looking at implementing the plasma version (in fact I’m now tested in both at my local hospital in UK).  The rather obvious key difference between the two is practicality. With the 24 hour urine, there are two key issues: 1.  The logistics (i.e. lug the jug).  2.  Fasting for up to 3 days prior to the test (4 if you count the day of the test). There are numerous variations on the fasting theme but most labs tend to say not to eat at least the following foods that contain high levels of serotonin producing amines: avocados, bananas, chocolate, kiwi fruit, pineapple, plums, tomatoes, and walnuts.  Some lists contain additional items. With the plasma version, the fasting period is reduced to 8 hours. There are also medicinal limitations including drugs that can also alter 5-HIAA urine values, such as acetanilide, phenacetin, glyceryl guaiacolate (found in many cough syrups), methocarbamol, and reserpine. Drugs that can decrease urinary 5-HIAA levels include heparin, isoniazid, levodopa, monoamine oxidase inhibitors, methenamine, methyldopa, phenothiazines, and tricyclic antidepressants. Patients should talk to their doctor before decreasing or discontinuing any medications.

As for my own experience, my 5HIAA (urine) was elevated at diagnosis only returning to normal after removal of my primary and commencement of Lanreotide. It has been a good measure of tumour functionality for me and I’m currently tested every 6 months.

Other tests for the tumour subgroup include but not limited to:

Serum Serotonin (5-HydroxyTryptamine; 5-HT).  Firstly let’s deconflict between 5HIAA above and the serotonin (5-HT) blood test.  5HIAA is a metabolite of serotonin but the serotonin test is a measure of pure serotonin in the blood.  Morning specimens are preferred and this is a fasting test (10-12 hours).  There is always debate on forums about Serum Serotonin results.  I have Dr Liu on record as saying “a high serotonin level measured in the blood in isolation really isn’t that dangerous. It’s the 5HIAA (a breakdown product of serotonin, which is easily measured in the blood and urine) that is considered to be more indicative of persistent elevated hormone. It’s this test that is most closely related to the carcinoid heart disease”.

Substance P.   A substance associated with foregut and midgut tumours.  It is a vasoactive protein that can cause wheezing, diarrhea, tachycardia, flushing

Histamines – Usually associated with foregut tumors. Appears to be involved in patchy rashes and flushing.  The advice in the ISI NET book is no anti-histamine medication to be taken for 48 hours prior to blood draw.

Gastric NETs (Stomach)

Testing will be different depending on the Type:

• Type 1 – Typical Low Grade, tends to be caused by atrophic gastritis.
• Type 2 – Atypical Intermediate Grade and tends to be caused by gastrin secreting tumours.  Type 2 normally needs a check for MEN1/Zollinger-Ellison Syndrome.
• Type 3 – Tend to be larger and more aggressive tumours.

The key makers are CgA and Gastrin although Gastrin may not be elevated in Type 3. Gastrin ph is useful to differentiate between Type 1 and Type 2.  5HIAA can be considered but Carcinoid Syndrome is rare in Gastric NETs.

NETs of the Pancreas (pNETs)

pNETs can be very difficult to diagnose and not only because they share some presentational similarities to their exocrine counterparts.  Some pNETs actually comprise tumours arising in the upper part of the duodenum (small intestine) close to the Pancreas. Moreover, more than half of pNETs are non-functional which increases the difficulty in suspecting and then finding the tumours.  However, where there is clinical presentation or suspicion, these symptoms can lead to the appropriate testing to support the output of scans. The fasting gut profile mentioned above can be useful in identifying the offending hormones when the type of NET is not yet known.

Gut Hormones (Glucagon, Gastrin, VIP, Somatostatin, Pancreatic Polypeptide)

A gut hormone screen is used for the diagnosis of a variety of endocrine tumours of the pancreas area. Analysis includes gastrin, VIP, somatostatin, pancreatic polypeptide, and glucagon, but there may be others depending on processes used by your ordering specialist or hospital.

Notes:

1. You may see this referred to as a ‘Fasting Gut Profile’ or a ‘Fasting Gut Hormone Profile’.

2.  The individual hormones measured seem to differ between hospital labs.

3.  The fasting conditions also vary between hospitals and labs but all agree the conditions are critical to the most accurate results. Always ask for instructions if you’re offered this test.

The gastrin test is usually requested to help detect high levels of gastrin and stomach acid. It is used to help diagnose gastrin-producing tumours called gastrinomas, Zollinger-Ellison (ZE) syndrome, and hyperplasia of G-cells, specialised cells in the stomach that produce gastrin. It may be measured to screen for the presence of multiple endocrine neoplasia type I (MEN) It may be used if a person has abdominal pain, diarrhoea, and recurrent peptic ulcers. A gastrin test may also be requested to look for recurrence of disease following surgical removal of a gastrinoma.

Vasoactive intestinal peptide (VIP) measurement is required for diagnosis of pancreatic tumour or a ganglioneuroma which secretes VIP. Administration of VIP to animals causes hyperglycaemia, inhibition of gastric acid, secretion of pancreatic bicarbonate and of small intestinal juice, and a lowering of systemic blood pressure with skin flush. These features are seen in patients with a tumour of this type which is secreting VIP.

Glucagon is measured for preoperative diagnosis of a glucagon-producing tumour of the pancreas in patients with diabetes and a characteristic skin rash (necrolytic migratory erythema).

Pancreatic polypeptide (PP) production is most commonly associated with tumours producing vasoactive intestinal polypeptide and with carcinoid syndrome and, less commonly, with insulinomas and gastrinomas.

When secreted by endocrine tumours, somatostatin appears to produce symptoms similar to those seen on pharmacological administration, i.e. steatorrhoea, diabetes mellitus and gall stones.

There are several types of pNETs, each with their own syndrome or hormone issue.  When they are suspected due to the presentational symptoms, the markers that could be used are listed below.  These types of tumours are complex and can be related to one or more syndromes.  A patient may be tested using multiple markers to include or exclude these.  Depending on other factors, some physicians may recommend additional marker testing in addition to the most common types below.

Insulinoma – Insulin, Proinsulin, C-peptide

Gastrinoma– Gastrin, Gastrin pH

Glucagonoma – Glucagon, Insulin, Pancreatic Polypeptide (PP), Adrenocorticotropic hormone (ACTH)

VIPoma – Vasoactive Intestinal Polypeptide (VIP), Electrolytes (due to profuse diarrhea)

Somatostatinoma – Somatostatin (plasma somatostatin like immunoreactivity)

PPoma – Pancreatic Polypeptide (PP)

Other NETs/Syndromes

Pheochromocytoma/Paraganglioma – Adrenaline-producing tumours. Plasma and urine catecholamines, plasma free total metanephrines, urine total metanephrines, vanillylmandelic acid (VMA)

Medullary Thyroid Cancer. Medullary thyroid cancer (MTC) starts as a growth of abnormal cancer cells within the thyroid – the parafollicular C cells. In the hereditary form of medullary thyroid cancer (~20% of cases, often called Familial MTC or FMTC), the growth of these cells is due to a mutation in the RET gene which was inherited. This mutated gene may first produce a premalignant condition called C cell hyperplasia. The parafollicular C cells of the thyroid begin to have unregulated growth. In the inherited forms of medullary thyroid cancer, the growing C cells may form a bump or nodule in any portion of the thyroid gland.  Unlike papillary and follicular thyroid cancers, which arise from thyroid hormone-producing cells, medullary thyroid cancer originates in the parafollicular cells (also called C cells) of the thyroid. These cancer cells make a different hormone called calcitonin, which has nothing to do with the control of metabolism in the way  thyroid hormone does.  The other test often seen in MTC is Carcinoembryonic Antigen (CEA). CEA is a protein that is usually found in the blood at a very low level but might rise in certain cancers, such as medullary thyroid cancer. There is no direct relationship between serum calcitonin levels and extent of medullary thyroid cancer.  However, trending serum calcitonin and CEA levels can be a useful tool for doctors to consider in determining the pace of change of a patient’s medullary cancer.

[please note there are extremely rare occurrences of elevated calcitonin from places outside the thyroid – read more here.

Parathyroid– Parathyroid hormone (PTH), Serum Calcium.  Parathyroid hormone (PTH) is secreted from four parathyroid glands, which are small glands in the neck, located behind the thyroid gland. Parathyroid hormone regulates calcium levels in the blood, largely by increasing the levels when they are too low.  A primary problem in the parathyroid glands, producing too much parathyroid hormone causes raised calcium levels in the blood (hypercalcaemia – primary hyperparathyroidism). You may also be offered an additional test called Parathyroid Hormone-Related Peptide (PTHrP). They would probably also measure Serum Calcium in combination with these type of tests. The parathyroid is one of the ‘3 p’ locations often connected to Multiple Endocrine Neoplasia – MEN 1 – see MEN below.

Pituitary/Cushings – Adrenocorticotropic hormone (ACTH), Cortisol.

HPA AXIS – It’s important to note something called the HPA axis when discussing pituitary hormones as there is a natural and important connection and rhythm between the Hypothalamus, Pituitary and the Adrenal glands.

Adrenocorticotropic hormone (ACTH) is made in the corticotroph cells of the anterior pituitary gland. It’s production is stimulated by receiving corticotrophin releasing hormone (CRH) from the Hypothalamus. ACTH is secreted in several intermittent pulses during the day into the bloodstream and transported around the body. Like cortisol (see below), levels of ACTH are generally high in the morning when we wake up and fall throughout the day. This is called a diurnal rhythm. Once ACTH reaches the adrenal glands, it binds on to receptors causing the adrenal glands to secrete more cortisol, resulting in higher levels of cortisol in the blood. It also increases production of the chemical compounds that trigger an increase in other hormones such as adrenaline and noradrenaline. If too much is released, The effects of too much ACTH are mainly due to the increase in cortisol levels which result. Higher than normal levels of ACTH may be due to:

Cushing’s disease – this is the most common cause of increased ACTH. It is caused by a tumor in the pituitary gland (PitNET), which produces excess amounts of ACTH. (Please note, Cushing’s disease is just one of the numerous causes of Cushing’s syndrome). It is likely that a Cortisol test will also be ordered if Cushing’s is suspected.

Cortisol

This is a steroid hormone, one of the glucocorticoids, made in the cortex of the adrenal glands and then released into the blood, which transports it all round the body. Almost every cell contains receptors for cortisol and so cortisol can have lots of different actions depending on which sort of cells it is acting upon. These effects include controlling the body’s blood sugar levels and thus regulating metabolism acting as an anti-inflammatory, influencing memory formation, controlling salt and water balance, influencing blood pressure. Blood levels of cortisol vary dramatically, but generally are high in the morning when we wake up, and then fall throughout the day. This is called a diurnal rhythm. In people who work at night, this pattern is reversed, so the timing of cortisol release is clearly linked to daily activity patterns. In addition, in response to stress, extra cortisol is released to help the body to respond appropriately. Too much cortisol over a prolonged period of time can lead to Cushing’s syndrome.  Cortisol oversecretion can be associated with Adrenal Cortical Carcinoma (ACC) which can sometimes be grouped within the NET family.

Other hormones related to ACC include:

Androgens (e.g. Testosterone) – increased facial and body hair, particularly females. Deepened voice in females.

Estrogen – early signs of puberty in children, enlarged breast tissue in males.

Aldosterone – weight gain, high blood pressure.

Adrenal Insufficiency (Addison’s Disease) occurs when the adrenal glands do not produce enough of the hormone cortisol and in some cases, the hormone aldosterone. For this reason, the disease is sometimes called chronic adrenal insufficiency, or hypocortisolism.

A tumour outside the pituitary gland, producing ACTH (also called ectopic ACTH). With NETs, this is normally a pNET, Lung/Bronchial NET or Pheochromocytoma.

Multiple Endocrine Neoplasia (MEN).  Please note MEN is a group of distinct syndrome not a tumor.  Complex area and tends to be multiple instances of some of the tumours above.  For a breakdown of MEN types and locations, check out my MEN blog ‘Running in the Family’

Carcinoid Heart Disease(CHD) (Hedinger syndrome)  I’m not really talking directly about a tumour here but thought it would be useful to include a blood test called NT-proBNP.  I’ve left a link to my CHD article in the paragraph heading for those who wish to learn more about CHD in general.  For those not offered an annual Echocardiogram or are ‘non-syndromic’ there is a screening test that can give an indication of any heart issue which might then need further checks.

The Future – Molecular Markers?

This is testing using DNA and genes.  Exciting but complex – check out this article which involved some NETs.

Tumour Markers and Hormone levels – complex subject!