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Molecular Diagnostics - How Accurate Are You?



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

Personalized medicine, with the aid of molecular diagnostics, is providing the exciting possibility of cost-effective tailored therapies, depending 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 3 billion-base genome can be the difference between having, instead of having, an actionable drug therapy. However, identifying this one-in-a-billion may be tricky; with the multiple steps of your diagnostic workflow, any variability that creeps into each step is further compounded downstream potentially ultimately causing incorrect diagnoses. The requirement for consistent accuracy to be able to provide a precise diagnosis and effective tailored therapy is therefore critical. Just what exactly progress is being made?
Companion diagnostic developments

Precision medicine statistics
Companion diagnostics are very making good headway towards achieving the ultimate goal. By way of example, the most recent collaboration between AstraZeneca and Qiagen offers the first companion diagnostic method of guide the use of cell-free DNA (cfDNA) in 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 have 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 in the recent Phase III trial of the 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 %) compared with tumor tissue (61.Five percent). A high rate of false negatives (56.9 percent) was also observed. The larger 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 is certainly still a need to maintain clinical vigilance, particularly where limitations are identified within a workflow approach, sampling method or limit of detection. Just like any clinical protocol, sample handling will require clinical vigilance through sound quality assurance and control methodologies, including routine validation activities.

Beyond cfDNA, the need for accuracy is shown in External Quality Assessment (EQA) schemes; for 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 that more and more is needed, and one technology which has seen an explosion in recent years is single-molecule sequencing (Figure 3). The modern generation of these technologies (third-generation sequencing) is now emerging, with the possibility of even higher throughput, longer reads and shorter time to result, which will lead eventually into a lower overall cost. However, as with every new technology, new challenges arise as well as new workflow steps and thus new sources of variability. Similarly, with all the current data now being furnished by next-generation sequencing (NGS) technologies in greater quantities, volume and speed, how is it actually being used?

Bed mattress Big Data being utilized?

According to Boehringer Ingelheim’s recent ‘Let’s Test Campaign’ (4) - insufficient. 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 percent 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 they'd even received results on mutation status.

Cited reasons state insufficient tumor histology and insufficient tumor samples. Deficiency of tissue samples has become a longstanding problem, especially in hard-to-find lung cancers, to ensure the development of alternatives like cfDNA tests. But lack of material for both clinical testing and validation as well as set up of diagnostic tests is definitely an issue.

So what occurs when therapies go wrong? Consider colorectal cancer as an example: EGFRtargeting therapies have been developed for the treatment of patients with metastatic colorectal cancer to great effect. However, mutations inside the KRAS gene are found in 30-40 percent of colorectal tumors (5) and those that 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; you will find over 1.4 million people worldwide every year who are diagnosed with colorectal cancer (7). Combine this with all the conservative number that 30 percent of these patients have a mutated KRAS gene, you can estimate that for around $18,882 per treatment, it could possibly potentially be costing payers over $8 billion worldwide per year because of incorrect tumor genotyping results 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, depending on data showing a lack of efficacy and potential harm in patients with mutant KRAS tumors (Figure 5). To include complexity, NRAS has also been shown to be involved in the prognosis of inefficient treatment at ASCO (2013) (8), but that is another story. In any case, the variability between laboratories and techniques means that some patients still receive medication whenever they do not need it, and even more importantly, others do not receive potentially life-saving treatment after they do.
Figure 5. The wide range of EGFR testing methodologies used by labs in round a couple of the EQA scheme: Only four methods were precisely the same amongst 36 laboratories when identifying the identical mutation.

Aiming for accuracy

It is possible to increase and ensure the accuracy of an 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 could be the answer. His organization is liable for coordinating many EQA schemes like the most recent EGFR EQA scheme (2), including three rounds. “EMQN has become organizing EQA schemes for rare single gene disorders for eighteen years. For that reason experience, we were approached by a few clinical oncologists working in Europe to supply EQA for lung cancer testing,” he says.

“We had evidence from a pilot scheme the quality of united states testing and reporting of the results to clinicians was at need of improvement. El born area of diagnostics has evolved extremely fast, and it’s been driven by pharma’s should 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, and 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 should ensure that the quality of testing offers the right result, ideal patient at the perfect 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 involved with NSCLC testing. It was coordinated and administered through the EMQN and three rounds were organized in just a period of 18 months. The initial was restricted to no more than 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 other round were furnished with another set of samples inside a restricted third round. The steering group evaluated the final results 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 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 low sensitivity of the method employed for mutational analysis. For example, the expected minimum awareness is 15 percent for Sanger sequencing, and 5.43 percent for that p.(G719S) mutation as defined in version The Qiagen Therascreen kit packaging insert. Genotyping EGFR G719S in particular showed a 35.6 % error.

PCR/sequencing was the commonest method used in the scheme for scanning to detect point mutations. The main disadvantage of sequencing though is that it is not very sensitive (9), especially in samples with low tumor cell content. Real-time allele-specific exams are much more sensitive and certain, but only test for any subset of common mutations.

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

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

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

When applied correctly, personalized medicine can help identify not only patients who are most likely to benefit coming from a particular therapeutic product, but in addition those likely to be at increased probability of serious side-effects as a result of treatment. Furthermore, accurate diagnostics could also monitor a response to treatment using a particular therapeutic product, to accomplish improved safety. In order 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.

Post by personalizedgenes (2015-12-28 13:59)

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