Molecular diagnostics


The detection and (semi-) quantitative determination of specific molecules in body samples is generally summarized as molecular diagnostics. A good part of this is DNA-oriented looking for genetic alterations that cause or predispose for all kinds of pathological conditions. On top of that RNAs, proteins and metabolites are also monitored to get a better description of the overall or specific health condition of an individual. A special case of molecular diagnostics looks for a biomarker that is important especially for the applicability of a particular drug. This special case is called  “Companion diagnostics” and will be discussed in the next blog entry. 


Molecular diagnostics is not really new as clinical laboratories have been testing patient samples for molecules for decades. The new part his the increasing inclusion of genetic parameters and more specific biomarkers in addition to the general screenings already in place. There DNA-diagnostics comes in as well as advanced and disease-specific biomarkers all characterized at the molecular level. 


Basically everybody in the western world has undergone numerous blood screenings, where an MD (or a skilled assistant) draws blood samples (often several at the same time for different tests) to obtain a complete blood count. This usually encompasses counts of the most common blood cells types, usually three biomarkers for liver condition (enzymes, where excessive appearance in the blood indicates liver problems) as well as a number of metabolites (blood sugar, cholesterol, triglycerides) and in some cases a selection of vitamins (usually B and D vitamins). The latter substances all altogether molecular markers but usually not directly coupled to genomic of genetic events, which is a hallmark to which molecular diagnostics is gravitating these days. 


A good example for this gradual shift is the wide field of allergy diagnostics. Allergies are a wide-spread problem especially in the western societies as the natural desensitisation during early childhood is often suppressed by lack of exposure to natural amounts of common allergens, simply spoken lack of dirt due to extreme and often unhealthy overemphasis on cleanliness and hygiene. Both are very good things but as always overdoing turns the advantage into a disadvantage in this case fostering loads of allergic reactions. However, allergies are not all induced by lack of exposure, there are some strong genetic components that predispose for allergies. Hay fever, for example, is often inherited and a close cousin of hereditary asthma. Another related and very common allergy is directed agains animal hair especially cat hair, although the real problem with cats is not their hair. They produce a specific protein in their saliva which by relentless grooming and cleaning is spread all over their hair, which only serves as the vehicle to get the allergen to the humans. Cats lacking that protein usually do not provoke the allergic reaction. 


The most common test to detect allergies is the so-called “pricktest” where all kinds oaf allergies are placed on the skin (usually of a forearm) and brought to the attention of the immune system by a little prick in the skin. This is not a molecular diagnostics as none of the molecules involved in the ensuing inflammatory reaction is directly monitored. Doctors just look for the reddish puffs on the spots indicating allergic reactions. However, there are molecular markers for allergies that are tested for: histamine (a small molecule produced in the cause of an allergic reaction), leukotrienes (internal immune messenger molecules), or the eosinophil cationic protein (ECP) which correlates especially with the severity of an asthma attack. Asthma has a strong genetic basis and a review by Lang and Blake (2013) identified at least 15 different genetic loci (genes) correlated with asthma. An additional (partially overlapping) list of 10 genetic loci was reported that were specifically associated with childhood asthma. An additional 6 genes were associated with pharmacogenetics of asthma treatment all centered around leuotrienes. 


Allergies are all basically immune system problems, where the natural balance of an immune response gets out of control causing allergies in the mildest forms all the way to fatal autoimmune-diseases such as Multiple Sclerosis (MS). Therefore, immune biomarkers are of great interest for molecular diagnostics. Drugs (especially antibodies) directed against or towards particular immune reactions are very popular right now in treatment of a variety off cancers. However, cancer is a much simpler case than autoimmune-diseases in terms of immune-biomarkers. Autoimmune diseases usually affect a much broader network of immune cells than cancer, with a correspondingly more complex genomic variation (Willis and Lord, 2015).


Molecular diagnostics of immune-markers has increased dramatically in the last 10 years. Next to a variety of genetic markers (SNPs mostly, i.e. single nucleotide exchanges in genomic DNA), also epigenetic (changes at chromatin outside the DNA sequence), RNA-expression, a variety of individual proteins (e.g. cytokines), as well as a host of metabolic markers have been introduced into the clinical practice. The range of application is from stratification of patients into groups all the way down to individual diagnosis (especially for cancer patients prior to immune-therapy). 


There are many more molecular tests either focusing on detection of genetic background of predisposition to disease . Also markers for actual disease in progress are important such as the prostate-specific-antigen (PSA), which is routinely check in the ageing male population (one in seven males will contract prostate cancer during his lifetime). 


One especially important field of application for biomarkers are tumor markers, used to detect cancers or to monitor success or failure of cancer treatments. The National Cancer Institute (NCI) in Bethesda, USA defined tumor markers as follows: 


Tumor markers are substances that are produced by cancer or by other cells of the body in response to cancer or certain benign (noncancerous) conditions. Most tumor markers are made by normal cells as well as by cancer cells; however, they are produced at much higher levels in cancerous conditions.


This definition highlights a general problem of molecular diagnostics: It is often a quantitative rather than a qualitative measurement. Rarely, tumors produce substances entirely absent in normal cells. 


The NCI definition goes on: These substances can be found in the blood, urine, stool, tumor tissue, or other tissues or bodily fluids of some patients with cancer. Most tumor markers are proteins. However, more recently, patterns of gene expression and changes to DNA have also begun to be used as tumor markers. Many different tumor 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” tumor 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 tumor marker associated with that cancer. Moreover, tumor markers have not been identified for every type of cancer.



Figure 7: Tumor markers


The NCI lists 35 tumor markers currently in use, 3 of which are actually profiles of several genes (up to 70). There are many more and the list is a moving target as more tumor markers are detected and introduced into the clinical practice. This ever growing list exemplifies the biggest problem in cancer detection and treatment. Each and every cancer is a breed of its own, and while there are common features, no individual tumors are completely the same. 


Where is this heading? The trend clearly goes into the direction to use broader ranges of genes and proteins for characterization of tumors or diseases rather than relying on individual biomarkers. Biology is a complex and tightly interwoven system, that cannot be simplified so it can be described by just a few hallmarks. As already discussed earlier in this blog, only a sufficiently broad range of parameters allows getting a clearer picture of the health status. It does not matter if this is an allergy case, or an infection or cancer. Always the whole system (called human body) is involved to some extent.  




26    Lang, J. E. & Blake, K. V. Role of biomarkers in understanding and treating children with asthma: towards 

        personalized care. Pharmgenomics Pers Med 6, 73-84, doi:10.2147/PGPM.S30626 (2013).

27    Willis, J. C. & Lord, G. M. Immune biomarkers: the promises and pitfalls of personalized medicine. Nat Rev 

        Immunol 15, 323-329, doi:10.1038/nri3820 (2015).


What’s coming up next?


Next week I want to illustrate the principles of companion diagnostics on the example of herceptine, one of the first treatments that came with a companion test.