Introduction
Molecular basis of ALK inhibition therapy
Effectiveness and safety of ALK inhibition therapy
Detection of ALK gene rearrangements
Tissue management
Standardized tissue and cell processing
Clinical diagnostic tests
IHC
Antibody | Source | HIER | Dilution | Incubation | Detection system | |
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Yi et al. [46] | ALK1 | Dako | EDTA, pH 8, 30 min | 1/100 | 30 min at RT | Advance (Dako) |
Yang et al. [43] | ALK1 | Dako | EDTA, pH 8, 30 min | 1/100 | 30 min at RT | Advance (Dako) |
Paik et al. [47] | 5A4 | Abcam | CC1 (Ventana), 1 h | 1/30 | 2 h at 42 °C | i-view (Ventana) |
McLeer-Florin et al. [48] | 5A4 | Abcam | CC1 (Ventana), 1 h | 1/50 | 2 h at 37 °C | i-view (Ventana) |
Hofman et al. [45] | 5A4 | Abcam | pH 9, 40 min, 97 °C | 1/50 | 30 min RT | EnVision Flex (Dako) |
Kim et al. [27] | 5A4 | Novocastra | CC1 100 °C, 20 min | 1/30 | 2 h at 42 °C | i-view (Ventana) |
Mino-Kenudson et al. [49] | D5F3 | CST | EDTA pH 8 pressure cooker | 1/500a
| Overnight | EnVision+ (Dako) |
D9E4 | CST | 1/100b
| ||||
ALK1 | Dako | 1/50a
| ||||
1/2b
|
Score | ||||
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0 | 1+ | 2 | 3 | |
Yi et al. [46] | No staining | Faint cytoplasmic staining | Moderate smooth cytoplasmic staining | Intense granular cytoplasmic staining in ≥10 % of tumour cells |
Kim et al. [27] | No staining | Faint or weak staining intensity with >5 % tumour cells or any staining intensity with ≤5 % tumour cells a
| Moderate staining intensity with >5 % tumour cells b
| Strong and granular staining intensity with >5 % tumour cells c
|
FISH
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Method—Use of diagnostic kits certified by the FDA (Abbott Vysis ALK Break Apart FISH Probe Kit, Abbott Molecular, Inc.) is recommended. The package insert contains a detailed protocol of the technical procedures and interpretation. In practice, the Vysis kit has some limitations: (1) appropriate optical filters are required for the probes used; (2) the green signal may fade earlier than the red signal, increasing the likelihood of false-positive single-red signals. Other, not yet FDA-approved, FISH assays are commercially available (e.g. ZytoLight® SPEC ALK/EML4 TriCheck™ Probe, ZytoVision). Every assay should undergo validation in the laboratory before clinical implementation. This validation can be conducted with five to ten positive and five to ten negative cases. The results are compared with a reference centre, and concordance of at least 95 % should be obtained. In addition, laboratories should participate in external quality assessment schemes as soon as they become available. When a laboratory authorized to conduct FISH adopts another bright-field ISH for diagnostic purposes, an internal validation can be conducted in the laboratory comparing the new technique with FISH. Again, a concordance of 95 % must be obtained. This 95 % threshold may change in time, after more experience is gained with the external quality assessment. The use of standardized kits requires strict adherence to the manufacturers' instructions, i.e. without any deviation.A minimum norm for ALK experience is not known yet, but for Her2 it was suggested that at least 150 ISH cases per annum (minimum of 100 breast and 50 gastric cases by ISH per annum) would be the optimal volume to ensure quality and cost effectiveness [53] (see “Proposal for an external quality assessment program” section).The number of annual tests considered optimal for guaranteeing the technical sufficiency of a laboratory is 150 ISH tests on any type of tumour [51]. Analysis of complete histological sections is recommended in order to minimize signal loss on the Z-axis. In FISH procedures, it is essential to use fluorescence filters with excitation and emission wavelengths appropriate to the fluorochromes of the probes contained in the kit.
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Controls—With ISH techniques, the case for study serves as a control when consistently presenting signals, both in tumour cells and in the accompanying normal cells. Nevertheless, the use of separate controls is strongly advised. A pre-hybridization assessment of digestion is useful in difficult samples (e.g. very small biopsies with low tumour content).
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Assessment of the results—A pathologist should interpret the results. When other persons score the specimens, they should be well trained and experienced in histo- and cytomorphology. In such a case, a pathologist should coordinate, validate, review and sign off the interpretation. A total of 50 nuclei should be scored using an epifluorescence microscope equipped with a DAPI, a Spectrum Orange, Spectrum Green and double-filter set, using a ×60–100 oil immersion objective lens. The spectral orange probe is mostly called the ‘red probe/signal’ in the literature, and this terminology has been used throughout the manuscript.Assessment must be conducted exclusively on nuclei with sufficient hybridization quality, in a consecutive manner, and with the microscope focus adjusted to each nucleus in order to correctly identify all the signals present in the nucleus of the cells.When conducting FISH as an ISH technique, given the difficulty of dark-field assessment, the following are recommended:a.Cells are considered ALK FISH positive when: (1) at least one set of red and green signals have a distance between the signal borders of ≥2 diameters of the largest of the two signals (break apart of the two differently coloured probes indicates gene fusion by inversion), or (2) when there is a single red signal without a corresponding green signal in addition to fused (normal) signals (indicating gene fusion by inversion and deletion) (Figs. 1 and 2). Note that a single green signal without a corresponding red signal is considered negative. Similarly, an increased copy number of non-rearranged ALK genes with fused signals corresponds to polysomy of chromosome 2 or ALK amplification, but is negative for rearrangement.b.A sample is considered negative if <5 cells (<10 %) are positive and positive if >25 cells (>50 %) are positive.c.A sample is considered equivocal if 5–25 cells (10–50 %) are positive. In this case, a second reader should evaluate the slide. If the average of the two readings contains at least 15 % positive cells, the sample is considered ALK FISH positive. It should be kept in mind that the digestion time needs to be adjusted for some samples. Examples of typical FISH findings are shown in Fig. 2.During interpretation, the spatial distribution of the distance between the two signals in the Z-direction also needs to be taken into account. In cases of only one signal, adjusting the plane of focus (by turning the microscrew) is frequently necessary when using the conventional FISH microscope. An advantage of a digitized automatic FISH analysis system is that fluorescent images may be compressed in the Z-direction (Z-stacks), often making this automated stage more convenient than live microscopy. The presence of all signals in one horizontal plane facilitates scoring and review, and allows permanent documentation. In our experience, a positive scoring result that cannot convincingly be illustrated by fluorescence photography is of questionable value.Cytological specimens offer the advantage of analysing entire cells/nuclei containing all chromosomal signals. Instead, a part of the nuclei in histological tissue sections are truncated, leading to a lower number of fluorescence signals, and this effect depends on the section thickness.Note that in most ALK-positive samples, not all alleles are abnormal (e.g. only one in a disomic cell or one or two in tetrasomic cells). Typically, there is one predominant type of rearrangement in one sample (either break apart or deletion). Great variability in signal patterns within one sample might suggest technical artefacts.There is evidence that ALK rearrangements are truly diffuse rather than heterogeneous within ALK-positive tumours [21, 54]. First, IHC typically shows diffuse ALK expression in ALK-positive tumours [47, 55]. Second, the distribution of cell positivity by FISH is diffuse; there are no discrete foci of ALK rearrangements within a tumour [21, 54]. This suggests that any observed variability is not biological, but rather caused by technical factors.Given the presumed homogeneous distribution of ALK rearrangements, the percentage of FISH-positive cells in an ALK-positive tumour lying between 20 and 80 %, and not reaching 100 % may appear contrary. The ‘single red’ signal is found in a higher fraction of tumour cells than the ‘break apart’ signal [21]. This discrepancy can readily be explained by the distribution of signals in three-dimensional space and their projection by the assessing pathologist to the horizontal plane. A separated signal can be located anywhere on a virtual sphere around the other signal. In a disomic tumour cell with one rearranged ALK gene copy, the probability of detecting break apart signals in the two-dimensional projection may therefore not exceed 25–30 %.In the authors' experience, the minimum number of tumor cells required in histological specimens is at least 100, as approximately half will not be stained with the fluorescence probes for reasons related to the stereological analysis. Less than 40 tumor cells/neoplastic nuclei with FISH signals are unreliable for determining positivity.Increased ALK gene copy number (3 to >6 gene copies), which is mostly due to polysomy of chromosome 2, has been reported in 80 % of NSCLC [56]. In cases of polysomy, one rearranged signal per nucleus is sufficient for a ‘positive’. High gene copy number of non-rearranged ALK does not appear to influence response to crizotinib. A possible predictive value of the copy number of the rearranged ALK gene in tumour cells and its influence on the level of ALK protein expression remains to be investigated.Overall, interpretation of the ALK FISH analysis is more complex than for other FISH assays, e.g. with amplifications or interchromosomal translocation. This is due to the fact that: (1) fusion inversion occurs on the same chromosome arm—in contrast to fusion processes in SYT-SSX and BCR-ABL FISH tests, for example, where genes from different chromosomes are involved; (2) in some ALK wild-type cells (especially with larger nuclei or after prolonged digestion) the red and green signals may be slightly separated and the fusion to yellow fluorescence is not apparent; (3) the process of cutting and subsequent signal separation may lead to a low number of false-positive break apart signals in cells without the ALK fusion. These characteristics mean that a low threshold of positive cells can still select patients who will react to ALK inhibitor treatment. However, this complexity highlights the need for specific training and experience with ALK FISH.
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Personnel—The number of laboratory technicians who conduct tests and the number of pathologists who interpret them must be as low as possible in order to guarantee the effectiveness of the undertaking. Both the technicians and the pathologists must have undergone training. For assessing the post-analytical (interpretation) phase, correlation with the reference result of at least 95 % in 10–20 cases is recommended. Periodically, the aforesaid training should be refreshed in dedicated working sessions.
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Report—While the report should be adapted to the different information systems used in different hospitals, it must include, at minimum, the data presented in Table 3. The recommended turnaround time is <7 working days.Table 3Recommended data in a report on ALK testing by in situ hybridizationClinical informationPatient identificationIdentification of doctor making requestDates of request and testIdentification of sample (case and block number)Type of sampleaAnatomical originGross specimen handling/molecular methodDate block received by laboratoryBlock used for analysisProbe used (supplier, approval by FDA or other agency)Method of assessment (semi-quantitative, image analysis)Threshold for positivityMicroscopy/molecular resultsNumber of nuclei assessedResultsPositive (%)Presence or absence of patterns indicating rearrangement (specify whether split or single)Negative (% of translocated cells, if any)Presence or absence of patterns indicating rearrangementInconclusive: Explanations for inconclusive answersbConclusionMolecular test outcome and interpretationRearrangement associated with sensitivityName and signature of the pathologist(s) responsible for the investigation
Concordance between IHC and FISH
Reference | Antibody | Source | Detection method | Samples, n
| ALK+ | ALK− | Comments | ||
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FISH | IHC | FISH | IHC | ||||||
Shaw et al. [25] | ALK1 | Dako | n.a. | NSCLC, 141 | 19 (13 %) | 19 (13 %) | n.a. | n.a. | Only FISH+ cases were confirmed with IHC |
Boland et al. [44] | ALK1 | Dako | n.a. | NSCLC, 335 | 6 (1.8 %) | 6 (1.8 %) | n.a. | n.a. | The 6 cases testing positive using IHC were positive with FISH. 8 of the IHC ALK− cases were also tested using FISH. All were negative |
Rodig et al. [24] | ALK1 | Dako | EnVision+ | Adeno, 358 | 20 (5.6 %) | n.a. | n.a. | n.a. | 8 of the 10 FISH ALK+ cases with sufficient tissue were also IHC ALK+ (sensitivity = 80 %); 1 case ALK– by FISH was ALK+ by IHC |
Mino-Kenudson et al. [49] | ALK1 | Dako | EnVision+ | Adeno, 153 | 22 (14.3 %) | n.a. | n.a. | n.a. | Sensitivity and specificity of IHC were 67 and 97 %, respectively |
D5F3 | Cell ST | EnVision+ | Adeno, 153 | 22 (14.3 %) | n.a. | n.a. | n.a. | Sensitivity and specificity of IHC were 100 and 99 %, respectively | |
Yi et al. [46] | ALK1 | Dako | Advance | Adeno, 101 | 10 (9.9 %) | 11a (10.9 %) | 91 (90.1 %) | 90a (89.1 %) | Of the 10 FISH+ cases, 8 were IHC 3+, 1 was IHC 2+ and 1 was IHC +; of the 91 FISH– cases, 2 were IHC 2+, 20 were IHC 1+, 69 were IHC 0; sensitivity and specificitya of IHC were 90 and 97.8 %, respectively |
Paik et al. [47] | 5A4 | Novocastra | i-view | NSCLC, 453 | 19 (4.2 %)a
| 26 (5.7 %) | 434 (95.8 %) | 427a (94.3 %) | Of the 19 FISH+ cases, 16 were IHC 3+, 3 were IHC 2+; of the 434 FISH− cases, 7 were IHC 2+, 14 were IHC 1+, 413 were IHC 0; sensitivity and specificitya of IHC were 100 and 92.5 %, respectively |
Mitsudomi et al. [64] | 5A4 | Santa Cruz | EnVision+ | NSCLC, 345 | n.a. | 12 (3.5 %) | n.a. | n.a. | All these 12 IHC ALK+ cases were also FISH ALK+; all IHC ALK– cases were also FISH ALK– |
Martinez et al. [65] | D5F3 | Cell ST | n.a. | NSCLC, 71 | 6 (8.5 %) | n.a. | n.a. | n.a. | All FISH ALK– negative cases were also IHC ALK–. 4/6 FISH ALK+ cases were also ALK IHC+; 1 case was IHC ALK–; 1 sample could not be analysed. |
Paik et al. [66] | 5A4 | Novocastra | n.a. | NSCLC, 735 | 28 (3.8 %) | 35 (4.8 %) | 707 (96.2 %) | 700 (95.2 %) | Of all cases, 15 were IHC 3+, 20 were IHC 2+, 20 IHC 1+, 700 were IHC0; all IHC0/1+ were ALK FISH–, all IHC 3+ cases were ALK FISH+, and 13 of the 20 IHC 2+ were FISH+; sensitivity and specificitya of IHC were 100 and 96.2 %, respectively |
Adeno, 395 | 27 (6.8 %) | n.a. | 368 (93.2 %) | n.a. | |||||
McLeer-Florin et al. [48] | 5A4 | Abcam | i-view | Adeno, 441 | n.a. | 29 (6.5 %) | n.a. | n.a. | Of 81 cases with interpretable IHC and FISH results, 21 were FISH ALK+; of these 21 cases, 19 were IHC ALK+ and 2 were IHC doubtfully positive; of the 60 FISH ALK– cases, 1 was IHC ALK+ and 59 were IHC ALK– |
Yang et al. [43] | ALK1 | Dako | Advance | Adeno, 300 | 22/216 (10.2 %) | 32 (10.7 %)a
| 194/216 (89.8 %) | 268 (89.3 %) | All IHC 0 cases were FISH ALK– and all IHC 3+ were FISH ALK+; 96.9 % of IHC 1+ cases were FISH ALK− and 85.7 % of IHC 2+ cases were FISH ALK+ |