Molecular Diagnostics - How Accurate Are you currently?

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

Personalized medicine, by making use of molecular diagnostics, is providing the exciting potential for cost-effective tailored therapies, based on an individual patient’s genetic code. Almost all of the true in the case of cancer where a single nucleotide polymorphism (SNP) out of a three billion-base genome can be the difference between having, rather than having, an actionable drug therapy. However, identifying this one-in-a-billion could be tricky; with the multiple steps of the diagnostic workflow, any variability that creeps into each step is further compounded downstream potentially resulting in incorrect diagnoses. The requirement for consistent accuracy as a way to provide a precise diagnosis and effective tailored therapy is therefore critical. Precisely what progress is being made?
Companion diagnostic developments

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

However, the clinical feasibility of utilizing cfDNA to detect EGFR mutations was assessed inside a recent Phase III trial of your Japanese subset of patients (1). The trial found that the proportion of patients identified with mutant EGFR was lower when assessed in cfDNA (23.7 percent) compared with tumor tissue (61.5 percent). 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 in just a workflow approach, sampling method or limit of detection. Just like any clinical protocol, sample handling requires clinical vigilance through audio quality assurance and control methodologies, including routine validation activities.

Away from 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 relating to laboratories tested, only 72 percent correctly identified EGFR mutations in patient samples (2).

While substantial advances continue to be made, it’s clear more and more is needed, and one technology which has seen an explosion in recent years is single-molecule sequencing (Figure 3). The new generation of these technologies (third-generation sequencing) is now emerging, with the potential for even higher throughput, longer reads and shorter time and energy to result, which will lead eventually with a lower overall cost. However, just like any new technology, new challenges arise in addition to new workflow steps and for that reason new sources of variability. Similarly, with all the data now being supplied by next-generation sequencing (NGS) technologies in greater quantities, volume and speed, bed not the culprit it actually being used?

Bed mattress Big Data getting used?

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

Cited reasons state insufficient tumor histology and insufficient tumor samples. The lack of tissue samples is a huge longstanding problem, specially in hard-to-find lung cancers, therefore, the development of alternatives for example cfDNA tests. But not enough material for both clinical testing and validation and hang up up of diagnostic tests has always been 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 people who have this particular mutation show an unhealthy response to the popular therapies of cetuximab and panitumumab (6), with patients even experiencing worsening side-effects in some cases.

To put this into perspective; there are 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 possess a mutated KRAS gene, you can estimate that at a cost of $18,882 per treatment, it could possibly potentially be costing payers over $8 billion worldwide a year because of incorrect tumor genotyping ends in molecular diagnostics.

As a result, since 2008, the usage of EGFR-targeting antibodies in metastatic colorectal cancer may be restricted to patients with wild-type KRAS tumors through the European Medicines Agency, according to data showing deficiencies in efficacy and potential harm in patients with mutant KRAS tumors (Figure 5). To include complexity, NRAS has also been given to be involved in the prognosis of inefficient treatment at ASCO (2013) (8), that is another story. In any case, the variability between laboratories and techniques means that some patients still receive medication when they do not need it, and more importantly, others do not receive potentially life-saving treatment once they do.
Figure 5. The broad range of EGFR testing methodologies used by labs in round a couple of the EQA scheme: Only four methods were the same amongst 36 laboratories when identifying the same mutation.

Aiming for accuracy

You can increase and ensure the accuracy of an laboratories’ tumor genotyping, including the using reference material, EQAs and ISO standards. Simon Patton, Director of the European Molecular Quality Network (EMQN), believes that EQA proficiency testing schemes could be the answer. His organization is liable for coordinating many EQA schemes including the most recent EGFR EQA scheme (2), including three rounds. “EMQN may be organizing EQA schemes for rare single gene disorders for eighteen years. For this reason experience, we were approached by a few clinical oncologists working in Europe to deliver EQA for lung cancer testing,” he says.

“We had evidence from a pilot scheme that this quality of carcinoma of the lung testing and reporting with the results to clinicians what food was in need of improvement. El born area of diagnostics has evolved very fast, and it’s been driven by pharma’s should get their drugs in the clinical setting. This need has mainly been met by different diagnostic laboratories, predominantly genetics and pathology, which have been encouraged to set up testing for tumor markers, along with 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 necessary to ensure that the quality of testing offers the right result, for the ideal patient at the correct time,” Patton adds.
The EQA scheme

A steering number of five individuals was formed who planned, designed and assessed the outcome of the pilot EQA scheme associated with NSCLC testing. It was coordinated and administered through the EMQN and three rounds were organized within a period of 18 months. The first was restricted to at the most 30 laboratories to create proof-of-principle and validate materials. A subsequent second round was organized without any restriction on participation. Laboratories that failed the next round were supplied with another set of samples in the restricted third round. The steering group evaluated the outcome according to a predefined scoring system, which assigned two exactly what to correct genotype and zero exactly what to false-positive or -negative results (Figure 4).

Once the data were analyzed, false-negative outcome was found to be the cause of 85 percent of all the genotype errors made in the scheme, that may be a result of the lower sensitivity of the method used for mutational analysis. For example, the expected minimum awareness is 15 percent for Sanger sequencing, and 5.43 percent to the p.(G719S) mutation as defined in version 1 of the Qiagen Therascreen kit packaging insert. Genotyping EGFR G719S especially showed a 35.Six % error.

PCR/sequencing was the most common method used in the scheme for scanning to detect point mutations. The most important disadvantage of sequencing though is it is not very sensitive (9), particularly in samples with low tumor cell content. Real-time allele-specific tests are much more sensitive and specific, but only test to get a subset of common mutations.

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

Although the inaccuracies and number of methodologies are evident in diagnostics, Patton does highlight many of the positives that have range from EQA scheme: “We are traversing 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 propensity of participants to overstate the significance of the test result. EMQN continues to be pushing for standardization of reporting of sequence variants from the testing community by promoting best practice and also the use of the Human Genome Variation Society (HGVS) mutation nomenclature guidelines. Those two activities play an important role in improving the excellence of the test result.”

When asked about his overall recommendations and future plans to the scheme, Patton felt that even though improvement of the quality of exams are happening, there’s still more to perform: “Annual participation in EQA needs to be seen as the norm for all those laboratories offering a diagnostic test if they're serious about ensuring that they have a high quality testing service.”

When applied correctly, personalized medicine can help identify not only patients who are most likely to benefit from the particular therapeutic product, but in addition those likely to be at increased likelihood of serious side-effects as a result of treatment. Furthermore, accurate diagnostics can also monitor a response to treatment which has a particular therapeutic product, to realize improved safety. In order to ensure the accuracy and achieve confidence of diagnostic testing/tumor genotyping, all sorts of options are available ones sustained evaluation and validation through reference materials, for example the EQA, are essential.