From Molecular pathology

Detecting beta-catenin mutations in desmoid-type fibromatoses

Next generation gene sequencing is a very impressive technology that has a lot of potential but is only just beginning to find its uses in diagnostic histopathology. Adrienne Flanagan and her team at University College London have published an excellent study on the use of next-gen sequencing in the detection of beta-catenin mutations in desmoid-type fibromatoses. The clinical importance of detecting these mutations is that they define the entity of desmoid-type fibromatoses which are locally aggressive and tend to recur when compared to non-desmoid fibromatoses. Prof. Flanagan and her team found that next-gen sequencing detected beta-catenin mutations with a sensitivity of 92.36% and a specificity of 100%. All the mutations that were detected by the older technology of enzyme digestion and polymerase chain reaction were also detected by next-gen sequencing but additional, probably clinical-relevant, mutations were discovered by the sequencing methodology. This study begins to show how next-gen sequencing will affect diagnostic histopathology in the next few years.

Oncotype DX and Magee equations

In histopathology we are always trying to improve ways of predicting prognosis in tumours, especially tumours like breast cancer where overall stage is similar amongst many tumours (i.e. node negative breast cancer). Simple methods on H&E sections have been very effective, the Nottingham prognostic index in breast cancer being an excellent example, but the advent of molecular pathology has led to the development of other methodologies. A commercially-available test for prognostication in breast cancer is Oncotype DX which analyses the activity of 21 genes in tumour tissue. Oncotype DX is licensed for use on patients with node-negative, ER-positive HER2-unamplified breast cancer and there are well-validated studies of its prognostic power. The problem, in both private and NHS healthcare systems, is that the test is relatively costly (about £2500 in the UK) and although NICE originally sanctioned its use the NHS declined to fund it (it may now be available on a loss leader basis from Genomic Health, the parent company).

An interesting approach has been taken by the Department of Pathology at the University of Pittsburgh where they have ‘reverse engineered’ the Oncotype DX score using the readily available parameters of Nottingham prognostic index, ER, PR, HER2 and Ki67 staining. They sent over 800 samples for Oncotype DX and then used linear regression to derive equations that gave a surrogate ‘Oncotype DX score’ from the available parameters. They validated these equations on a further 200 cases and have made the equations available on a webpage. Interestingly they didn’t just use their equations to replace Oncotype DX but used them to screen out ‘obvious’ low and high recurrence risk cases and then sent the cases in the middle for Oncotype testing.

The problem with all of this type of prognostication is validation with large datasets. Although 800 cases to derive the equations and 200 test cases sounds a lot it really isn’t sufficiently large to prove entirely robust validation of the Pittsburgh equations. The correlation between the results from the Pittsburgh Magee equations and the real Oncotype score is statistically good but it isn’t perfect. Even the Oncotype DX validation studies do not have huge numbers with a total of 3707 patients in 5 separate studies (and one of those studies had a case control methodology) . I think the real value of these tests will only become apparent when clinical evidence has accumulated over a few more years.

Molecular testing on endoscopic gastrointestinal biopsies

As more molecular tests become available that stratify patients into different therapeutic groups it is likely that those tests will have to be performed on small endoscopic biopsies, especially where there is the possibility of neo-adjuvant therapy. As yet there have been few studies which have looked at the feasibility of this – do we need to ask endoscopists to take extra biopsies if they believe the lesion is a tumour? Should samples for potential molecular testing be sent in separate specimen bottles? In this month’s Histopathology Hieke Grabsch and colleagues review all the available published studies in this area. Their conclusions are that there is no hard evidence indicating an optimal number of biopsies to be taken if molecular testing is anticipated and whether these should be processed separately. In my experience of sending duodenal biopsies away for T cell receptor gene rearrangement studies (to detect refractory coeliac disease) the usual two or three standard endoscopic biopsies in formalin-fixed paraffin-embedded blocks are sufficient but obviously this might vary with the nature of the molecular test.