A New Multi-Color FISH Assay for Brush Biopsy-Based Detection of Chromosomal Aneuploidy in Oral (Pre)Cancer in Patients with Fanconi Anemia
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Study Design and Samples
2.1.1. Cutoff Study
2.1.2. Validation Study
2.2. Re-Evaluation of Cytological Samples
2.3. FISH
2.3.1. Hybridization
2.3.2. Re-Hybridization
2.3.3. FISH Analysis
2.3.4. FISH Evaluation Protocol
- At least 3 FISH probes showed a gain (>2 signals) (definition 1 (D1));
- All 4 probes showed a gain (>2 signals) (definition 2 (D2));
- All 4 probes showed a gain (>2 signals) or 3 probes showed a gain (>2 signals) if one of these probes was MYC with a gain of ≥4 Signals (definition 3 (D3));
- All 4 probes showed a gain (>2 signals) or 3 probes showed a gain (>2 signals) if one of these probes was MYC with a gain of ≥5 Signals (definition 4 (D4)).
2.4. Reference Standard
2.5. Statistical Analysis
3. Results
3.1. Patients and Samples
3.2. Multi-Color FISH Assay
3.3. 9p21 FISH Assay
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Definition | Cutoff-Point | Sensitivity | Specificity | Youden Index | Definition | Cutoff-Point | Sensitivity | Specificity | Youden Index |
---|---|---|---|---|---|---|---|---|---|
D1 | 1 | 0.974 | 0.438 | 0.412 | D2 | 1 | 0.949 | 0.579 | 0.527 |
2 | 0.974 | 0.587 | 0.561 | 2 | 0.923 | 0.686 | 0.609 | ||
3 | 0.949 | 0.653 | 0.602 | 3 | 0.923 | 0.752 | 0.675 | ||
4 | 0.923 | 0.686 | 0.609 | 4 | 0.872 | 0.810 | 0.682 | ||
5 | 0.923 | 0.711 | 0.634 | 5 | 0.821 | 0.843 | 0.663 | ||
6 | 0.897 | 0.727 | 0.625 | 6 | 0.795 | 0.860 | 0.654 | ||
7 | 0.897 | 0.760 | 0.658 | 7 | 0.769 | 0.868 | 0.637 | ||
8 | 0.897 | 0.769 | 0.666 | 8 | 0.692 | 0.884 | 0.577 | ||
9 | 0.872 | 0.785 | 0.657 | 9 | 0.641 | 0.893 | 0.534 | ||
10 | 0.846 | 0.785 | 0.631 | 10 | 0.641 | 0.901 | 0.542 | ||
11 | 0.846 | 0.793 | 0.640 | 11 | 0.590 | 0.909 | 0.499 | ||
12 | 0.795 | 0.802 | 0.597 | 12 | 0.564 | 0.917 | 0.481 | ||
14 | 0.744 | 0.802 | 0.545 | 13 | 0.513 | 0.934 | 0.447 | ||
15 | 0.718 | 0.818 | 0.536 | 15 | 0.487 | 0.950 | 0.438 | ||
16 | 0.718 | 0.835 | 0.553 | 17 | 0.462 | 0.950 | 0.412 | ||
18 | 0.718 | 0.843 | 0.561 | 20 | 0.436 | 0.950 | 0.386 | ||
19 | 0.692 | 0.843 | 0.535 | 21 | 0.410 | 0.967 | 0.377 | ||
20 | 0.667 | 0.860 | 0.526 | 23 | 0.333 | 0.967 | 0.300 | ||
21 | 0.641 | 0.860 | 0.501 | 26 | 0.308 | 0.967 | 0.275 | ||
D3 | 1 | 0.974 | 0.529 | 0.503 | D4 | 1 | 0.949 | 0.570 | 0.519 |
2 | 0.949 | 0.636 | 0.585 | 2 | 0.949 | 0.686 | 0.635 | ||
3 | 0.949 | 0.711 | 0.659 | 3 | 0.949 | 0.736 | 0.684 | ||
4 | 0.923 | 0.752 | 0.675 | 4 | 0.897 | 0.785 | 0.683 | ||
5 | 0.872 | 0.802 | 0.673 | 5 | 0.872 | 0.826 | 0.698 | ||
6 | 0.872 | 0.818 | 0.690 | 6 | 0.872 | 0.835 | 0.707 | ||
7 | 0.872 | 0.826 | 0.698 | 7 | 0.846 | 0.843 | 0.689 | ||
8 | 0.846 | 0.826 | 0.673 | 8 | 0.744 | 0.843 | 0.587 | ||
9 | 0.821 | 0.835 | 0.655 | 9 | 0.718 | 0.860 | 0.577 | ||
10 | 0.769 | 0.835 | 0.604 | 10 | 0.692 | 0.876 | 0.568 | ||
11 | 0.718 | 0.843 | 0.561 | 11 | 0.615 | 0.884 | 0.500 | ||
12 | 0.667 | 0.843 | 0.510 | 12 | 0.590 | 0.901 | 0.491 | ||
13 | 0.641 | 0.860 | 0.501 | 13 | 0.590 | 0.926 | 0.515 | ||
14 | 0.641 | 0.868 | 0.509 | 14 | 0.538 | 0.926 | 0.464 | ||
15 | 0.641 | 0.901 | 0.542 | 15 | 0.538 | 0.934 | 0.472 | ||
16 | 0.590 | 0.909 | 0.499 | 16 | 0.538 | 0.950 | 0.489 | ||
17 | 0.590 | 0.934 | 0.524 | 18 | 0.513 | 0.950 | 0.463 | ||
19 | 0.564 | 0.942 | 0.506 | 21 | 0.487 | 0.967 | 0.454 |
Definition | Cutoff-Point | Sensitivity | Specificity | Youden Index | Definition | Cutoff-Point | Sensitivity | Specificity | Youden Index |
---|---|---|---|---|---|---|---|---|---|
D1 | 1 | 1.000 | 0.025 | 0.025 | D2 | 1 | 0.974 | 0.150 | 0.124 |
3 | 0.974 | 0.075 | 0.049 | 2 | 0.947 | 0.250 | 0.197 | ||
4 | 0.947 | 0.150 | 0.097 | 3 | 0.947 | 0.350 | 0.297 | ||
5 | 0.947 | 0.175 | 0.122 | 4 | 0.895 | 0.500 | 0.395 | ||
6 | 0.921 | 0.225 | 0.146 | 5 | 0.842 | 0.550 | 0.392 | ||
7 | 0.921 | 0.300 | 0.221 | 6 | 0.816 | 0.600 | 0.416 | ||
8 | 0.921 | 0.325 | 0.246 | 7 | 0.789 | 0.625 | 0.414 | ||
9 | 0.895 | 0.375 | 0.270 | 8 | 0.711 | 0.675 | 0.386 | ||
10 | 0.868 | 0.375 | 0.243 | 9 | 0.658 | 0.700 | 0.358 | ||
11 | 0.868 | 0.400 | 0.268 | 11 | 0.605 | 0.725 | 0.330 | ||
12 | 0.816 | 0.425 | 0.241 | 12 | 0.579 | 0.750 | 0.329 | ||
14 | 0.763 | 0.425 | 0.188 | 13 | 0.526 | 0.800 | 0.326 | ||
15 | 0.737 | 0.475 | 0.212 | 15 | 0.500 | 0.850 | 0.350 | ||
16 | 0.737 | 0.500 | 0.237 | 17 | 0.474 | 0.850 | 0.324 | ||
18 | 0.737 | 0.525 | 0.262 | 20 | 0.447 | 0.850 | 0.297 | ||
19 | 0.711 | 0.525 | 0.236 | 21 | 0.421 | 0.900 | 0.321 | ||
20 | 0.684 | 0.575 | 0.259 | 23 | 0.342 | 0.900 | 0.242 | ||
21 | 0.658 | 0.575 | 0.233 | 26 | 0.316 | 0.900 | 0.216 | ||
23 | 0.658 | 0.625 | 0.283 | 28 | 0.289 | 0.950 | 0.239 | ||
24 | 0.658 | 0.675 | 0.333 | 30 | 0.263 | 0.950 | 0.213 | ||
25 | 0.658 | 0.700 | 0.358 | 32 | 0.211 | 0.975 | 0.186 | ||
26 | 0.632 | 0.750 | 0.382 | 34 | 0.184 | 0.975 | 0.159 | ||
D3 | 1 | 1.000 | 0.075 | 0.075 | D4 | 1 | 0.974 | 0.125 | 0.099 |
2 | 0.974 | 0.125 | 0.099 | 2 | 0.974 | 0.250 | 0.224 | ||
3 | 0.974 | 0.250 | 0.224 | 3 | 0.974 | 0.325 | 0.299 | ||
4 | 0.947 | 0.350 | 0.297 | 4 | 0.921 | 0.450 | 0.371 | ||
5 | 0.895 | 0.425 | 0.320 | 5 | 0.895 | 0.500 | 0.395 | ||
6 | 0.895 | 0.475 | 0.370 | 6 | 0.895 | 0.525 | 0.420 | ||
7 | 0.895 | 0.500 | 0.395 | 7 | 0.868 | 0.550 | 0.418 | ||
8 | 0.868 | 0.500 | 0.368 | 8 | 0.763 | 0.550 | 0.313 | ||
9 | 0.842 | 0.525 | 0.367 | 9 | 0.737 | 0.600 | 0.337 | ||
10 | 0.789 | 0.525 | 0.314 | 10 | 0.711 | 0.650 | 0.361 | ||
11 | 0.737 | 0.550 | 0.287 | 11 | 0.632 | 0.650 | 0.282 | ||
12 | 0.684 | 0.550 | 0.234 | 12 | 0.605 | 0.700 | 0.305 | ||
13 | 0.658 | 0.575 | 0.233 | 13 | 0.605 | 0.775 | 0.380 | ||
14 | 0.658 | 0.600 | 0.258 | 14 | 0.553 | 0.775 | 0.328 | ||
15 | 0.658 | 0.700 | 0.358 | 15 | 0.553 | 0.800 | 0.353 | ||
16 | 0.605 | 0.725 | 0.330 | 16 | 0.553 | 0.850 | 0.403 | ||
17 | 0.605 | 0.800 | 0.405 | 18 | 0.526 | 0.850 | 0.376 | ||
19 | 0.579 | 0.825 | 0.404 | 21 | 0.500 | 0.900 | 0.400 |
Appendix B
Enhanced Follow-Up (Range 2–5 Years) without HG OED or SCC | ||
FP FISH | Histology | |
Oral Region | Result | Time Interval * |
Gingiva | --- | --- |
Gingiva | --- | --- |
Gingiva | --- | --- |
Gingiva | --- | --- |
Gingiva | --- | --- |
Cheek | --- | --- |
Tongue | --- | --- |
Tongue | --- | --- |
Tongue | --- | --- |
Gingiva | --- | --- |
HG OED or SCC Somewhere in the Oral Cavity but not in the Vicinity of the Brushed Lesion | ||
FP FISH | Histology | |
Oral Region | Result | Time Interval |
Gingiva | SCC | NR |
Gingiva | SCC, | <1 year |
HG OED | <1 year | |
SCC, | 2 Years | |
Tongue | HG OED | NR |
Gingiva | SCC | NR |
Tongue | SCC | NR |
HG OED or SCC in the vicinity of oral brushing or at the same place in the clinical course | ||
FP FISH | Histology | |
Oral Region | Result | Time Interval |
Gingiva | SCC, | <1 year |
Gingiva | HG OED | NR |
Gingiva | HG OED | NR |
Gingiva | SCC | <1 year |
Cheek | SCC | 2 Years |
Tongue | HG OED | 1 Year |
Lip | SCC | NR |
Cheek | SCC | 2 Years |
Gingiva | SCC | NR |
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Cutoff Study | ||||||
Characteristics | All | No HSCT | HSCT | |||
Negative FUP | Positive FUP | Negative FUP | Positive FUP | Negative FUP | Positive FUP | |
Total No. | 121 | 39 | 38 | 15 | 83 | 24 |
Sex, F/M | 56/65 | 13/26 | 20/18 | 11/4 | 36/47 | 2/22 |
Median age at brush cytology (Range), Y | 26 (10–46) | 30 (9–40) | 33 (10–46) | 33 (9–40) | 25 (10–41) | 29 (16–36) |
Median follow-up time (Range), Mo | 45 (9–86) | 31 (0–101) | 35 (9–66) | 9 (0–55) | 48 (14–86) | 58 (7–101) |
Validation Study | ||||||
Characteristics | All | no HSCT | HSCT | |||
Negative FUP | Positive FUP | Negative FUP | Positive FUP | Negative FUP | Positive FUP | |
Total No. | 129 | 23 | 39 | 9 | 90 | 14 |
Sex, F/M | 60/69 | 6/17 | 21/18 | 4/5 | 39/51 | 2/12 |
Median age at brush cytology (Range), Y | 27 (10–46) | 30 (16–36) | 32 (10–46) | 30 (16–35) | 26 (10–41) | 22 (16–36) |
Median follow-up time (Range), Mo | 33 (7–99) | 16 (7–58) | 32 (9–59) | 9 (7–31) | 36 (7–99) | 20 (7–58) |
Definition | Sensitivity | Specificity | Youden Index | AUC | AUC p-Value | AUC 5% Confidence Interval | ||
---|---|---|---|---|---|---|---|---|
Cutoff Study | All specimens | D1 | 89.7% (35/39) | 72.7% (88/121) | 0.625 | 0.812 | <0.001 | 0.738–0.886 |
D2 | 79.5% (31/39) | 86.0% (104/121) | 0.654 | 0.827 | <0.001 | 0.745–0.909 | ||
D3 | 87.2% (34/39) | 81.8% (99/121) | 0.690 | 0.845 | <0.001 | 0.772–0.918 | ||
D4 | 87.2% (34/39) | 83.5% (101/121) | 0.707 | 0.853 | <0.001 | 0.781–0.925 | ||
Atypical, suspicious and positive cytology only | D1 | 92.1% (35/38) | 22.5% (9/40) | 0.146 | 0.573 | 0.267 * | 0.446–0.700 | |
D2 | 81.6% (31/38) | 60.0% (24/40) | 0.416 | 0.708 | 0.002 | 0.591–0.825 | ||
D3 | 89.5% (34/38) | 47.5% (19/40) | 0.370 | 0.685 | 0.005 | 0.566–0.804 | ||
D4 | 89.5% (34/38) | 52.5% (21/40) | 0.420 | 0.710 | 0.001 | 0.593–0.826 | ||
Validation Study | All specimens | D2 | 82.6% (19/23) | 84.5% (109/129) | 0.671 | 0.836 | <0.001 | 0.739–0.932 |
D4 | 87.0% (20/23) | 82.9% (107/129) | 0.699 | 0.850 | <0.001 | 0.761–0.938 | ||
Atypical, suspicious and positive cytology only | D2 | 81.8% (18/22) | 55.2% (16/29) | 0.370 | 0.685 | 0.025 | 0.537–0.833 | |
D4 | 86.4% (19/22) | 51.7% (15/29) | 0.381 | 0.690 | 0.021 | 0.544–0.837 |
Cutoff Study | ||||||
FISH | Cytology | HSCT | ||||
Negative | Atypical | Suspicious | Positive | No | Yes | |
FN | 1 | 7 | 0 | 0 | 2 | 6 |
TN | 80 | 22 | 2 | 0 | 34 | 70 |
FP | 1 | 16 | 0 | 0 | 4 | 13 |
TP | 0 | 15 | 8 | 8 | 13 | 18 |
Validation Study | ||||||
FISH | Cytology | HSCT | ||||
Negative | Atypical | Suspicious | Positive | No | Yes | |
FN | 0 | 4 | 0 | 0 | 2 | 2 |
TN | 93 | 15 | 1 | 0 | 33 | 77 |
FP | 7 | 13 | 0 | 0 | 6 | 14 |
TP | 1 | 7 | 4 | 7 | 7 | 12 |
Cutoff | Sensitivity | Specificity | AUC | p-Value | AUC 5% Confidence Interval | ||
---|---|---|---|---|---|---|---|
Cutoff Study | All specimens | 16 | 77.3% | 63.9% | 0.752 | <0.001 | 0.662–0.842 |
Atypical, suspicious and positive cytology only | 16 | 55.3% | 65.7% | 0.587 | 0.200 * | 0.455–0.719 |
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Silva de Araujo, B.E.; Markgraf, M.; de Santana Almeida Araujo, I.K.; Velleuer, E.; Dietrich, R.; Pomjanski, N.; Schramm, M. A New Multi-Color FISH Assay for Brush Biopsy-Based Detection of Chromosomal Aneuploidy in Oral (Pre)Cancer in Patients with Fanconi Anemia. Cancers 2022, 14, 3468. https://doi.org/10.3390/cancers14143468
Silva de Araujo BE, Markgraf M, de Santana Almeida Araujo IK, Velleuer E, Dietrich R, Pomjanski N, Schramm M. A New Multi-Color FISH Assay for Brush Biopsy-Based Detection of Chromosomal Aneuploidy in Oral (Pre)Cancer in Patients with Fanconi Anemia. Cancers. 2022; 14(14):3468. https://doi.org/10.3390/cancers14143468
Chicago/Turabian StyleSilva de Araujo, Bruno Eduardo, Mona Markgraf, Isabela Karoline de Santana Almeida Araujo, Eunike Velleuer, Ralf Dietrich, Natalia Pomjanski, and Martin Schramm. 2022. "A New Multi-Color FISH Assay for Brush Biopsy-Based Detection of Chromosomal Aneuploidy in Oral (Pre)Cancer in Patients with Fanconi Anemia" Cancers 14, no. 14: 3468. https://doi.org/10.3390/cancers14143468