Molecular Diagnostics - How Accurate Are you currently?

Precision medicine cancer
In the era of personalized medicine, the truth of molecular diagnostics is more important now than ever before.

Personalized medicine, using molecular diagnostics, is providing the exciting chance of cost-effective tailored therapies, based on an individual patient’s genetic code. Many of the true in the case of cancer the place where a single nucleotide polymorphism (SNP) out of a three billion-base genome can be the difference between having, and never having, an actionable drug therapy. However, identifying this one-in-a-billion can be tricky; with the multiple steps of the diagnostic workflow, any variability that creeps into each step is further compounded downstream potentially leading to incorrect diagnoses. The requirement of consistent accuracy to be able to provide a precise diagnosis and effective tailored therapy is therefore critical. What exactly progress is being made?
Companion diagnostic developments

Precision medicine statistics
Companion diagnostics are incredibly making good headway towards having this ultimate goal. As an example, the most recent collaboration between AstraZeneca and Qiagen provides first companion diagnostic method of guide the use of cell-free DNA (cfDNA) inside the treatment of patients with advanced non-small cell cancer of the lung (NSCLC). The therapy, Iressa (gefitinib), is the first epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor to possess a European label indicating the application of cfDNA obtained from a blood sample.

However, the clinical feasibility of employing cfDNA to detect EGFR mutations was assessed within a recent Phase III trial of an Japanese subset of patients (1). The trial discovered that the proportion of patients identified with mutant EGFR was lower when assessed in cfDNA (23.7 percent) compared with tumor tissue (61.5 %). A high rate of false negatives (56.9 %) was also observed. The big variance in concordance rates for mutation results between cfDNA and tumor tissue are shown in Figure 2

Although companion diagnostic technologies undergo thorough regulatory review before being released to the market, there is still a need to maintain clinical vigilance, particularly where limitations are identified inside a workflow approach, sampling method or limit of detection. As with every clinical protocol, sample handling requires clinical vigilance through audio quality assurance and control methodologies, including routine validation activities.

Outside cfDNA, the need for accuracy is shown in External Quality Assessment (EQA) schemes; by way of example, the worldwide EQA proficiency scheme (2014) reports that regarding laboratories tested, only 72 percent correctly identified EGFR mutations in patient samples (2).

While substantial advances remain made, it’s clear more is needed, and one technology containing seen an explosion in recent years is single-molecule sequencing (Figure 3). The brand new generation of these technologies (third-generation sequencing) has become emerging, with the possibility of even higher throughput, longer reads and shorter time for you to result, which will lead eventually into a lower overall cost. However, as with any new technology, new challenges arise along with new workflow steps and therefore new sources of variability. Similarly, because of the data now being furnished by next-generation sequencing (NGS) technologies in greater quantities, volume and speed, how's it actually being used?

How's Big Data used?

According to Boehringer Ingelheim’s recent ‘Let’s Test Campaign’ (4) - too few. The survey, conducted between December 2014 and January 2015, found out that, although 81 percent of newly diagnosed NSCLC patients received testing for EGFR mutations, only 50 % of oncologists reported their treatment decision was effected by a patient’s EGFR mutation subtype. It further discovered that they started a quarter of patients on first-line treatment before that they had even received results on mutation status.

Cited reasons state lack of tumor histology and insufficient tumor samples. The lack of tissue samples is a longstanding problem, particularly in hard-to-find lung cancers, which means the development of alternatives like cfDNA tests. But deficiency of material for both clinical testing and validation and hang up up of diagnostic tests is definitely an issue.

So what occurs when therapies go wrong? Consider colorectal cancer for instance: EGFRtargeting therapies have been intended for the treatment of patients with metastatic colorectal cancer to great effect. However, mutations from the KRAS gene are found in 30-40 percent of colorectal tumors (5) and those that have this particular mutation show an undesirable response to the popular therapies of cetuximab and panitumumab (6), with patients even experiencing worsening side-effects occasionally.

To put this into perspective; you'll find over 1.4 million people worldwide annually who are diagnosed with colorectal cancer (7). Combine this with the conservative number that 30 percent of these patients have a mutated KRAS gene, you can estimate that at a cost of $18,882 per treatment, it might potentially be costing payers over $8 billion worldwide per year because of incorrect tumor genotyping ends in molecular diagnostics.

As a result, since 2008, using EGFR-targeting antibodies in metastatic colorectal cancer has been restricted to patients with wild-type KRAS tumors with the European Medicines Agency, based on data showing too little efficacy and potential harm in patients with mutant KRAS tumors (Figure 5). To provide complexity, NRAS has also been given to be involved in the prognosis of inefficient treatment at ASCO (2013) (8), but that's another story. In any case, the variability between laboratories and methods means that some patients still receive medication when they do not need it, and more importantly, others do not receive potentially life-saving treatment when they do.
Figure 5. The wide range of EGFR testing methodologies utilised by labs in round two of the EQA scheme: Only four methods were the identical amongst 36 laboratories when identifying exactly the same mutation.

Aiming for accuracy

It is possible to increase and ensure the accuracy of the laboratories’ tumor genotyping, including the using reference material, EQAs and ISO standards. Simon Patton, Director with the European Molecular Quality Network (EMQN), believes that EQA proficiency testing schemes would be the answer. His organization is in charge of coordinating many EQA schemes such as most recent EGFR EQA scheme (2), including three rounds. “EMQN continues to be organizing EQA schemes for rare single gene disorders for eighteen years. Because of this experience, we were approached by a few clinical oncologists working in Europe to offer EQA for lung cancer testing,” according to him.

“We had evidence coming from a pilot scheme that the quality of cancer of the lung testing and reporting with the results to clinicians was at need of improvement. This area of diagnostics has evolved quickly, and it’s been driven by pharma’s need to get their drugs to the clinical setting. This need has mainly been met by different diagnostic laboratories, predominantly genetics and pathology, that have been encouraged to set up testing for tumor markers, and the manufacturers have responded by developing new diagnostic kits and end-to-end diagnostic solutions. However working together with compromised FFPE samples is challenging and EQA schemes are required to ensure that the quality of testing delivers the right result, for the ideal patient at the correct time,” Patton adds.
The EQA scheme

A steering gang of five individuals was formed who planned, designed and assessed the outcomes of the pilot EQA scheme linked to NSCLC testing. It was coordinated and administered through the EMQN and three rounds were organized in just a period of 18 months. The 1st was restricted to at the most 30 laboratories to establish proof-of-principle and validate materials. A subsequent second round was organized with no restriction on participation. Laboratories that failed the 2nd round were supplied with another set of samples in a restricted third round. The steering group evaluated the outcomes according to a predefined scoring system, which assigned two points to correct genotype and zero suggests false-positive or -negative results (Figure 4).

After the data were analyzed, false-negative results were found to take into account 85 percent of all the genotype errors stated in the scheme, that may be a result of the low sensitivity of the method used for mutational analysis. For example, the expected minimum level of sensitivity is 15 percent for Sanger sequencing, and 5.43 percent for the p.(G719S) mutation as defined in version 1 of the Qiagen Therascreen kit packaging insert. Genotyping EGFR G719S especially showed a 35.Six percent error.

PCR/sequencing was the most frequent method used in the scheme for digitizing to detect point mutations. The main disadvantage of sequencing though would it be is not very sensitive (9), specially in samples with low tumor cell content. Real-time allele-specific exams are much more sensitive and particular, but only test for the subset of common mutations.

Following study, Patton commented, “There is still considerable room for improvement in the quality of genotyping of tumor genes along with the diagnostic error rate [an incorrect genotype that leads to a misdiagnosis] remains stubbornly high at 3.65 percent (as measured with the EQA). Errors are made by laboratories employing a broad range of methodologies (see Figure 5), but carry out have evidence that poor validation and/or verification of recent tests contributes significantly to this problem. This is especially true when implementing an NGS strategy, or using a ‘black box’ commercial diagnostic solution.”
Not all doom and gloom

Even though inaccuracies and wide range of methodologies are evident in diagnostics, Patton does highlight a few of the positives that have range from EQA scheme: “We are going to a significant improvement in clinical reporting with much less expensive ‘over interpretation’ of the genotyping results when it comes to treatment decision-making compared with previous EQA schemes. However, there still remains a tendency of participants to overstate value of the test result. EMQN continues to be pushing for standardization of reporting of sequence variants inside testing community by promoting best practice along with the use of the Human Genome Variation Society (HGVS) mutation nomenclature guidelines. These two activities play a crucial role in improving the company's test result.”

When inquired on his overall recommendations and future plans for that scheme, Patton felt that although the improvement of the quality of testing is happening, there’s still more to complete: “Annual participation in EQA needs to be seen as the norm for those laboratories offering a diagnostic test if they are serious about ensuring that they provide a high quality testing service.”

When applied correctly, personalized medicine may help identify not only patients that are most likely to benefit from a particular therapeutic product, but in addition those likely to be at increased chance of serious side-effects as a result of treatment. Furthermore, accurate diagnostics could also monitor a response to treatment with a particular therapeutic product, to realize improved safety. To ensure the accuracy and achieve confidence of diagnostic testing/tumor genotyping, a myriad of options are available that sustained evaluation and validation through reference materials, such as the EQA, are essential.