4 The diagnostic tests

The interventions

RD‑100i OSNA system

4.1 The RD‑100i OSNA system analyses and amplifies mRNA from solubilised biopsy samples of sentinel lymph node tissue. It detects the level of expression of the cytokeratin‑19 (CK19) gene, an epithelial marker associated with breast cancer. CK19 is normally not present in healthy lymph node tissue. The OSNA technology involves the homogenisation of sentinel lymph node tissue followed by analysis of the CK19 mRNA using reverse transcription loop mediated isothermal amplification (RT-LAMP) on the automated analyser within the RD‑100i OSNA system. The system does not need the mRNA to be extracted from the tissue and purified before analysis. The expression level of CK19 mRNA correlates with the size of the metastatic foci. Since the metastatic foci may not be evenly distributed throughout the node, the system provides more accurate results if more of the node is analysed because there is less risk of tissue allocation bias (sample bias). Tissue allocation bias can occur when half of the lymph node is analysed using an intraoperative test and the other half using histopathology, if the metastasis is only contained in the tissue slices used for one of the methods. The RD‑100i OSNA system can be used with half of the lymph node (1 piece or alternate slices), allowing for the possibility of follow-up histopathology but potentially decreasing the accuracy of the results because of the increased risk of tissue allocation bias. The time to results depends on the number of lymph nodes analysed, but the test takes approximately 30 to 45 minutes. The RD‑100i OSNA system result is expressed both quantitatively and qualitatively: − for lymph node-negative test results; + for lymph nodes with a micro-metastatic tumour burden (that is, greater than 250 copies of CK19 mRNA/microlitre); and ++ for lymph nodes with a macro-metastatic tumour burden (that is, greater than 5000 copies of CK19 mRNA/microlitre). The analyser amplifies and detects the CK19 mRNA by using 6 different primers that have been designed to avoid the amplification of CK19 pseudogenes or their transcripts because amplification of these would lead to false positive results.

4.2 The manufacturer estimates that 1% of breast tumours do not express CK19 mRNA and so, if cancer spreads to the lymph nodes from these tumours, CK19 mRNA will not be detected even though the lymph nodes are metastatic. Pre-screening of tumour biopsies for CK19 expression could be carried out before using the RD‑100i OSNA system to reduce the small risk of false negative results for metastatic sentinel lymph nodes.

Metasin test

4.3 The Metasin test is an intraoperative molecular test developed within the NHS at the Princess Alexandra Hospital in Harlow, Essex. The test has similarities to a discontinued commercial test (Veridex GeneSearch breast lymph node [BLN] assay) and uses quantitative reverse transcription polymerase chain reaction (qRT‑PCR) to detect 2 predictive markers of metastases, CK19 and mammaglobin. Mammaglobin is expressed mainly by breast epithelial cells and high levels of mammaglobin are associated with breast cancer. A reference gene (porphobilinogen deaminase; PBGD) is used to confirm the validity of the mRNA used in the test and 2 other controls (positive and negative) are also included. The test uses reagents that can be purchased from commercial suppliers and can be used on the Cepheid Smartcycler platform. The Metasin test uses different primer-probe combinations to detect the CK19 and mammaglobin genes from the discontinued commercial test. It takes approximately 32 minutes to extract and purify mRNA from the tissue and then to produce results. The Metasin test is currently used in the NHS as an in-house test to analyse half of the lymph node. The results are confirmed by follow-up histopathology. The Metasin test could also be used as a replacement test for postoperative histopathology.

4.4 Pre-screening of tumour biopsies for CK19 mRNA and mammaglobin mRNA expression may be carried out before using the Metasin test because, like the CK19 biomarker, mammaglobin is not expressed in all breast tumours. The proportion of breast cancer tumours that do not express mammaglobin mRNA is not known.

The comparator: postoperative histopathology

4.5 Postoperative histopathology is the usual approach used in the NHS, in which the sentinel lymph nodes are fixed in paraffin blocks, sliced very thinly to produce sections that are mounted on slides, stained and then examined under a microscope by a consultant histopathologist. The time to receive results from histopathology is usually between 5 and 15 working days in the NHS. People who have macrometastases (defined as tumour deposits in which at least 1 dimension is above 2 mm) or micrometastases (tumour deposits that are only discernible microscopically and measure greater than 0.2 mm but have no dimension greater than 2 mm) detected in their sentinel lymph node are regarded as lymph node-positive, and will usually receive axillary lymph node dissection. People who have isolated tumour cells in their sentinel lymph node are regarded as lymph node-negative and will not receive axillary lymph node dissection.

4.6 The wait for histopathology test results can cause anxiety for patients and can also lead to the patient having a second operation to remove all of the relevant axillary lymph nodes if the test result is positive. This second operation can be more difficult and result in a higher risk of complications because it will involve operating on the same area of the breast and armpit as the first operation.

4.7 There are several levels of histopathology that can be performed. In one-level histopathology, 1 section of the lymph node is examined, in three-level, 3 sections and, in five-level, 5 sections. The level of histopathology used may affect the accuracy of histopathology because of the risk of tissue allocation bias. There is also uncertainty about the accuracy of histopathology associated with the different staining techniques used, the thickness of the section examined and the experience of the pathologist reading the section slides.

  • National Institute for Health and Care Excellence (NICE)