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IASLC ATLAS OF ALK TESTING IN LUNG CANCER
tumor cells (Thunnissen 2012b). The number of tumor cells required for IHC assessment of ALK protein remains undefined, but a minimum of 50 assessable tumor cells are required for FISH for the ALK gene rearrangement. Alternative approaches for cytology smear samples are available, but the most appropriate approach with cytology samples is usually the preparation of a cell block that allows sections to be prepared and treated in the same way as sections of tissue biopsy samples. In general, all of the tissue or cellular material received in the pathology laboratory should be processed. Surgical resection specimens are an exception, although, as a general rule, tumors with a diameter of 3 cm or less should be processed in toto. Large pleural effusions may also be processed in part; storage of fluid is recommended until all diagnostic procedures are complete.
Tissue Processing
Fixation by immersion, or where appropriate, by inflation, with 10% neutral buffered formalin is recommended. Pre-fixation in some alcohol-based fixatives may alter tissue antigenicity or DNA integrity. Acidic decalcifying solutions used on bone biopsy samples may interfere with IHC, frequently compromise FISH testing, and often degrade DNA, making mutation testing less reliable. Fixatives that are acidic (such as Bouin’s fluid) or based on hard-metal salts should also be avoided. In general, a period of fixation of more than 6 hours and less than 48 hours is recommended, especially when biomarker testing is to be done (for which DNA integrity is important) (Wolff 2007, Hunt 2007). Underfixation or overfixation may have deleterious effects on DNA and protein antigen epitopes (Werner 2000, Atkins 2004, Oyama 2007, Bussolati 2008, Eberhard 2008). One of the significant parts of this phase in tissue handling is the period of time beginning immediately after the sample is removed from the patient and placed in preservative. Most laboratories have neither control of nor data on how much time elapses between tissue removal and immersion in a fixative and its arrival in the laboratory. In addition, most tissue processing machines include a fixation step, which increases the fixation time. In practice, most laboratories will adjust their staining processes relevant to IHC and in situ hybridization (ISH) to allow for their own average fixation time. Determining the nature and duration of fixation is a greater challenge in laboratories that receive samples from many outside sources with widely differing fixation procedures.
Tissue Handling for Biomarker Testing
Most biomarker investigations (IHC, ISH, or RNA/DNA studies) are performed during the initial diagnostic workup. In these circumstances, freshly cut sections should be used for biomarker testing. Tissue stored on glass sections will deteriorate in a matter of days or weeks and certainly over months. Degradation depends on the storage conditions and most likely also on the specific biomarker (Atkins 2004). The stability of ALK protein on unstained cut sections has not yet been studied systematically. Therefore, similar to the case of HER2 testing in breast cancer, slides with tissue sections stored for longer than 6 weeks should not be used for IHC testing for ALK. If storage is necessary, the sections should be coated in wax or a similar medium to prevent air oxidation and the sections should be kept in cool, dry, dark conditions. Tissue in formalin-fixed paraffin-embedded (FFPE) blocks is less prone to deterioration, and recutting the tissue block as needed at a later time works well in most circumstances. Various strategies can help limit the number of times the block needs to be cut to provide material for initial morphologic assessment, IHC staining, and subsequent molecular analysis. For example, extra sections may be cut at the first cutting session, and although this strategy may