Molecular Diagnostics – How Accurate Do you think you’re?

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

Personalized medicine, with molecular diagnostics, is providing the exciting potential for cost-effective tailored therapies, according to an individual patient’s genetic code. Many of the true in the case of cancer in which a single nucleotide polymorphism (SNP) out of a 3 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 an diagnostic workflow, any variability that creeps into each step is further compounded downstream potentially resulting in incorrect diagnoses. The necessity for consistent accuracy as a way to provide a precise diagnosis and effective tailored therapy is therefore critical. Just what exactly progress is being made?
Companion diagnostic developments

Molecular diagnostics fundamentals
Companion diagnostics are extremely making good headway towards having this ultimate goal. For example, the most recent collaboration between AstraZeneca and Qiagen supplies the first companion diagnostic method of guide the use of cell-free DNA (cfDNA) from the treatment of patients with advanced non-small cell united states (NSCLC). The therapy, Iressa (gefitinib), is the first epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor to get a European label indicating using 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 learned that the proportion of patients identified with mutant EGFR was lower when assessed in cfDNA (23.7 %) compared with tumor tissue (61.5 percent). A high rate of false negatives (56.9 percent) was also observed. The big variance in concordance rates for mutation results between cfDNA and tumor tissue are provided in Figure 2

Although companion diagnostic technologies undergo thorough regulatory review prior to being released to the market, there’s still a need to keep up clinical vigilance, particularly where limitations are identified in just 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.

Away from cfDNA, the need for accuracy is shown in External Quality Assessment (EQA) schemes; as an example, the worldwide EQA proficiency scheme (2014) reports those of 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 times is single-molecule sequencing (Figure 3). The new generation of these technologies (third-generation sequencing) has become emerging, with the prospect of even higher throughput, longer reads and shorter time to result, which will lead eventually with a lower overall cost. However, as with every new technology, new challenges arise together with new workflow steps and therefore new sources of variability. Similarly, with all the data now being provided by next-generation sequencing (NGS) technologies in greater quantities, volume and speed, how is it actually being used?

Bed mattress Big Data getting used?

According to Boehringer Ingelheim’s recent ‘Let’s Test Campaign’ (4) – inadequate. The survey, conducted between December 2014 and January 2015, learned that, although 81 percent of newly diagnosed NSCLC patients received testing for EGFR mutations, only 1 / 2 of oncologists reported their treatment decision was effected by a patient’s EGFR mutation subtype. It further learned that they started one in four patients on first-line treatment before that they even received results on mutation status.

Cited reasons state lack of tumor histology and insufficient tumor samples. The lack of tissue samples has been a longstanding problem, specially in hard-to-find lung cancers, which means 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 within the KRAS gene are found in 30-40 percent of colorectal tumors (5) and those that have this particular mutation show an inadequate response to the popular therapies of cetuximab and panitumumab (6), with patients even experiencing worsening side-effects in some instances.

To put this into perspective; there are over 1.4 million people worldwide each and every year who are diagnosed with colorectal cancer (7). Combine this with all the conservative number that Thirty percent of these patients possess a mutated KRAS gene, you can estimate that for $18,882 per treatment, it could potentially be costing payers over $8 billion worldwide annually because of incorrect tumor genotyping leads to molecular diagnostics.

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

Aiming for accuracy

You are able to increase and ensure the accuracy of your laboratories’ tumor genotyping, including the utilization of reference material, EQAs and ISO standards. Simon Patton, Director in the European Molecular Quality Network (EMQN), believes that EQA proficiency testing schemes will be the answer. His organization is in charge of coordinating many EQA schemes such as the 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 number of clinical oncologists working in Europe to supply EQA for lung cancer testing,” he states.

“We had evidence coming from a pilot scheme the quality of lung cancer testing and reporting of the results to clinicians what food was in need of improvement. El born area of diagnostics has evolved extremely fast, and it’s been driven by pharma’s want to get their drugs into 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, along with the manufacturers have responded by developing new diagnostic kits and end-to-end diagnostic solutions. However dealing with compromised FFPE samples is challenging and EQA schemes are needed to ensure that the quality of testing provides the right result, for the right patient at the right 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 associated with NSCLC testing. It was coordinated and administered from the EMQN and three rounds were organized in a period of 18 months. The first was restricted to at the most 30 laboratories to establish proof-of-principle and validate the type of material. A subsequent second round was organized without having restriction on participation. Laboratories that failed the next round were furnished with another set of samples in a restricted third round. The steering group evaluated the outcome according to a predefined scoring system, which assigned two points to correct genotype and zero suggests false-positive or -negative results (Figure 4).

Once the data were analyzed, false-negative outcome was found to account for 85 percent of all the genotype errors stated in the scheme, that may be a result of period of time sensitivity of the method employed for mutational analysis. For example, the expected minimum level of responsiveness is 15 percent for Sanger sequencing, and 5.43 percent for that p.(G719S) mutation as defined in version One of the Qiagen Therascreen kit packaging insert. Genotyping EGFR G719S specifically showed a 35.6 % error.

PCR/sequencing was the most typical method used in the scheme for scanning to detect point mutations. The key disadvantage of sequencing though could it be is not very sensitive (9), particularly 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.

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

Even though the inaccuracies and number of methodologies are evident in diagnostics, Patton does highlight a few of the positives that have range from EQA scheme: “We are seeing a significant improvement in clinical reporting with much less ‘over interpretation’ of the genotyping results when it comes to treatment decision-making compared with previous EQA schemes. However, there still remains a bent of participants to overstate the significance of the test result. EMQN has been pushing for standardization of reporting of sequence variants inside the testing community your clients’ needs best practice as well as the use of the Human Genome Variation Society (HGVS) mutation nomenclature guidelines. Both these activities play a crucial role in improving the excellence of the test result.”

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

When applied correctly, personalized medicine might help identify not only patients that are most likely to benefit from your particular therapeutic product, but additionally those likely to be at increased risk 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 attain improved safety. In order to ensure the accuracy and achieve confidence of diagnostic testing/tumor genotyping, all sorts of options are available of which sustained evaluation and validation through reference materials, such as the EQA, are essential.

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