For molecular pathology testing in the field of oncology, continuous and dynamic communication is fundamental between the laboratory and the clinician. When requesting the test in the pre-analytical phase, the laboratory expects the clinician to request the correct test and to provide sufficient information about the patient and the sample that was provided. In the post-analytical phase, it is important to formulate a correct answer to the clinician's question on the basis of the test results.
1. The total testing process in the visited laboratories
Because of its different process steps (macroscopic evaluation, microscopic evaluation, DNA extraction, DNA testing, data processing and result interpretation), molecular pathology typically requires a more complex set-up than, for example, classical clinical chemistry. The communication flows differ between the visited laboratories, with anatomical- and molecular pathology being part of one department or being a physically separate department. Potential problems were identified in both contexts. Each time a test is requested, errors can be made by both the clinician (e.g. requesting the wrong test, writing incorrect patient information on the request form) (21, 22), the person registering the request into the laboratory information system (LIS) and by the pathologist requesting molecular testing in case the molecular pathology laboratory is physically separate (either by location or by LIS system). Some copy-paste or transfer errors might be prevented by providing digital request forms via the LIS (23). It could be useful to attach the original pathology report to the request form for molecular testing, since this pathology rapport is likely to contain relevant patient information. Murphy et al. also identified several barriers for reporting the results to the clinicians, including the lack of a reliable process to contact clinicians (23) and to notify clinicians when all results are complete.
Testing strategies differed among visited laboratories. Although reflex testing was previously recommended (24), this is only done by two visited laboratories. Laboratories who used a combination of reflex and parallel testing often do their next-generation sequencing (NGS) testing in parallel with ALK and ROS1 IHC to reduce their turnaround time. In reflex to a positive ROS1 IHC result, a confirmatory FISH test is done, as this is still required according to the latest guidelines from the American Society of Clinical Oncology (25) and the European Society for Medical Oncology (26). Aside from one laboratory, all have tested for EGFR, ALK, ROS1 and PD-L1, which are recommended according to current guidelines for NSCLC (25, 26). These guidelines also concluded that multiplex platforms (NGS) are preferred over applying multiple targeted tests (25, 26).
2. What is currently included vs what should be according to guidelines?
Although (inter)national guidelines and standards for test requesting and reporting exist, these are often interpretable in different ways and not transferrable to molecular pathology. Although ISO 15189:2012 is an international standard, it was categorized as a standard ‘applicable in Belgium’ in Tables 2A and 2B since Belgian laboratories have to be accredited for most molecular tests according to a national reimbursement law (13). Overall, the analyzed forms and reports comply well with the guidelines applicable in Belgium (Tables 2A and 2B). Required elements that were often missing on the request forms include the address and the national reimbursement (RIZIV) number of the prescriber, patient gender and address and the histology of the tumor. The tumor histology is not literally required by ISO15189:2012, but can be an interpretation of ‘the anatomic site of origin’ (16). For reporting, sample arrival dates, page numbering, the DNA extraction method, pre-analytical conditions, overview of the variants tested and reference sequence were included by less than 70% of the laboratories.
At first sight, the analyzed template request forms for tissue (N = 4) do not closely resemble the template forms for liquid biopsies (N = 4). Major differences between the seven requests analyzed for tissue testing and the five for liquid biopsy testing included: whether the form is dedicated for molecular pathology or not, the name of the test prescriber, the contact person in the laboratory, the primary diagnosis of the patient, the original actionable variant and whether there is progression or not. The latter was included in 80% of the laboratories, which could be expected since plasma testing for EGFR variants is often done in the context of resistance to 1st and 2nd generation anti-EGFR TKIs. Nevertheless, when resistance mutations, such as the p.(Thr790Met) variant, could not be detected (26), the test should be repeated on a tissue sample. Since these resistance mutations can be present at a low variant allelic frequency (27), knowing that the patient progressed might impact the testing strategy and could thus be a clinically relevant item on the request form. Regardless of the differences between tissue and liquid biopsies, there are also items that are missing in both. A first example is the exact reason for testing. It would seem logical that the molecular laboratory can only give a conclusion when a question is asked, but in the majority of cases this is not explicitly formulated. Secondly, the progression type - and time of progression are also often lacking. As mentioned earlier, this is especially relevant for liquid biopsy testing. At the end of the molecular pathology report or during a molecular tumor board meeting, a conclusion has to be made in terms of therapy; which may differ between full-, local- and oligo progression (26).
In addition to the analysis of the request forms, opinions from both the laboratory and requester side were gathered (Table 3). Although the number of participants was too low, a discrepancy seems to exist between what pulmonologists claimed they always enter on the request form versus what pathologists often miss, especially for the primary diagnosis, previous test performed, original actionable mutations and clinical question.
It was also analyzed whether sufficient information regarding the testing process was available online. In most laboratories, this was already the case for the sample recipient and transport medium, but not for the test specifications and indications for testing (Table 2A). In addition, the surveys show that the majority of pulmonologists would also like to see the indications, techniques, containers, transport media and transport delay on the application form (Table 3).
For the analyzed reports, the differences between tissue and liquid biopsy testing are less prominent. More abundant in the liquid biopsy reports are the request date, the planned line of therapy, the DNA extraction method, pre-analytical conditions, the reference sequence and a note regarding the result validity. This is to be expected somehow, since the previously mentioned testing context for progression and also because the pre-analytical conditions have more impact on the test result for liquid biopsies (28–30). Of the elements that are present in both report types (page numbering, concise title, planned line of therapy and whether the method is IVD/LDT), only the page numbering is crucial for the report, since loss of a page with critical information of the test can lead to wrong treatment decisions (4).
Overall, the elements described in the reports from the visited Belgian laboratories did not differ substantially from the reports submitted during EQA (Table 2B). However, these four laboratories had all participated in the ESP EQA scheme at least once, which might bias this observation. Nevertheless, several discrepancies existed. The patient’s address is less frequently included in EQA. There could be 2 possible explanations; 1) this information is in routine often retrieved from the online patient record, which is not available for EQA or 2) this data may have been anonymized for EQA purposes. Elements included at a higher frequency in the French reports are the presence of a title, the page numbering and the sample arrival date. This could be because French laboratories lose points if these elements are missed during the Gen&Tiss EQA scheme. A similar effect is seen for the overview of alterations tested and the reference sequence for the ESP EQA scheme.
In a similar way as for the request forms, we surveyed laboratory- and pulmonologists’ opinions regarding reporting. Both groups agree that the clinical interpretation is a critical element on the report. It might surprise pathologists, but pulmonologists also want to know the percentage of neoplastic cells, although this is a rather technical aspect. It must be noted that the genotyping result is not read by all, which emphasizes that the clinical interpretation must be certainly correct.
Based on these study results, proposed templates for requesting and reporting are shown in Additional file 3 and 4.
3. Relation between interpretations and request form
One of the aims of the study was to determine whether the question asked on the request form was fully answered in the report in 4 Belgium laboratories. Reports were studied for various clinical situations that are representative for NSCLC testing for tissue and liquid biopsies (Table 4) (26, 31). It was checked whether a clinical interpretation was given and whether this was written in a ‘general’ or ‘direct’ way. By general is meant, for example: “in general, patients with an activating mutation in the EGFR gene show response to 1st and 2nd generation anti-EGFR TKIs”, whereas ‘direct’ can be rather “this patient will respond to gefitinib”. The majority of laboratories gave a general interpretation. No guidelines regarding general interpretations exist, but this is recommended by several external quality assessment providers such as the European Society of Pathology. For ALK-, ROS1- and PD-L1 positive results, the reason for testing was not always reflected. For certain cases the request form only stated “EGFR?”. The laboratories claimed that they know the oncologist meant “test all relevant biomarkers for NSCLC”, among which ALK, ROS1 and PD-L1. For EGFR plasma testing at initial diagnosis, it was unclear in two laboratories whether progression occurred and therefore both interpretations for first- and second-line therapy were given. Especially in the interpretations for EGFR-positive cases (at initial diagnosis and after progression) different lines of therapy were considered. This makes sense in view of treatment with osimertinib which can be prescribed when the patient has a resistant mutation such as the p.(Thr790Met) variant (26). Note that at the time the study was conducted, osimertinib was not yet approved for first-line treatment. Whether the test result should be discussed in a molecular tumor board is not mentioned on any report. In Belgium, the implementation of molecular tumor boards is still under development. This may be because multidisciplinary oncological consults per cancer type are already mandatory for each patient before therapy can be reimbursed and the need for an additional tumor board, during which only rare gene variants are discussed, therefore seems less important (13, 32).