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chapter 3: Fluorescence in situ Hybridization
nucleus is not relevant for pattern classification.
SIGNAL CLASSIFICATION Patterns observed in native ALK Patterns observed in split 3'-5' ALK
Split (positive) pattern (Figure 7B). A cell is interpreted as having a split Red and green Red and green Red and green Red and green pattern (ALK positive) when separated by separated by separated by separated by ?2 signal <2 signal <2 signal ?2 signal the 5' and 3' signals are diameters diameters diameters diameters separated, regardless of the Classified Classified Classified Classified as negative as positive as positive as negative number of actual isolated signals (Figure 3). The sepaSPECIMEN CLASSIFICATION ration between the 5' and Nonrearranged tumors: Rearranged tumors: Rearrangement-positive cell rate Rearrangement-positive cell rate 3' signals must be two or <15% of cells ?15% of cells more times the diameter of the largest signal (Camidge Figure 6. Signal patterns in lung tumor nuclei hybridized with ALK break-apart FISH. 2010). The number of isolated 5' and 3' signals does not need to be equal; for example, a cell with two copies of isolated 5' signal and three copies of isolated 3' signals is classified as split. The number of accompanying fused 5'-3' signals in the cell is not relevant Patterns Examples for pattern classification. Isolated 3' (positive) pattern (Figure 7C). A cell is interpreted as having an isolated 3' pattern when isolated A. 3' signals are present with no isolated 5' sigFused nals. When a cell has both isolated 3' and 5' signals, with more 3' signals than 5' signals, the correct classification is the split pattern, not isolated 3'. The number of accompanyB. ing fused signals is not relevant for pattern Split classification. Isolated 5' (negative) pattern (Figure 7D). A cell is interpreted as having an isolated 5' pattern when isolated 5' signals are present with no isolated 3' signals. When a cell has both isolated 3' and 5' signals, with more 5' signals than 3' signals, the correct classification is the split pattern, not isolated 5'. The number of accompanying fused signals is not relevant for pattern classification. Note: The criteria for the split pattern are primarily based on testing of FFPE tumor sections
C. Isolated 3' ALK
D. Isolated 5' ALK
Figure 7. Tumor cell classification based on the ALK signal pattern on FISH.