Background
Materials and methods
Patients and tissue samples
QuantiGenePlex DNA assay
DNA preparation and TaqMan copy number assays
Immunohistochemistry assay
Cell culture
Cell proliferation assays
Patient-derived xenograft (PDX) model and treatment
Code | Gender | Age | Ulceration | Anatomic site | Stage |
CDK4
|
CCND1
|
P16
INK4a
|
KIT
|
BRAF
|
NRAS
|
---|---|---|---|---|---|---|---|---|---|---|---|
PDX-001 | M | 49 | Yes | 1 | IV | Normal | Normal | Normal | WT | WT | WT |
PDX-002 | M | 57 | Yes | 4 | IV | Normal | Normal | Normal | W557R | WT | WT |
PDX-003 | F | 78 | Yes | 5 | II | Gain | Gain | Loss | WT | WT | WT |
PDX-004 | F | 43 | Yes | 5 | III | Gain | Gain | Loss | L576P | WT | WT |
PDX-005 | F | 57 | Yes | 5 | II | Gain | Gain | Loss | WT | WT | WT |
PDX-006 | M | 58 | Yes | 3 | I | Gain | Gain | Loss | WT | WT | WT |
PDX-007 | F | 71 | Yes | 2 | II | Gain | Gain | Normal | WT | WT | WT |
PDX-008 | F | 65 | No | 1 | II | Gain | Gain | Normal | WT | WT | G12C |
PDX-009 | F | 69 | Yes | 1 | II | Normal | Gain | Loss | WT | WT | Q61L |
PDX-010 | M | 55 | Yes | 2 | II | Normal | Gain | Normal | WT | WT | WT |
RNA-sequencing
Statistical analysis
Results
Aberrations of Cdk4, Ccnd1 and P16INK4a in MM
CNV status | N = 213 | Genetic mutation of therapeutic targets | ||
---|---|---|---|---|
N (%) | % (No. positive cases/no. examined cases) | |||
KIT
|
BRAF
|
NRAS
| ||
≥ 1 CNV | ||||
CDK4 gain | 100 (47.0) | 7.4 (7/95) | 5.2 (5/96) | 15.7 (11/70) |
2.5–5 copies | 76 (35.7) | 5.6 (4/71) | 5.6 (4/72) | 19.6 (11/56) |
5–10 copies | 14 (6.6) | 11.8 (2/14) | 0 (0/14) | 0 (0/10) |
>10 copies | 10 (4.7) | 10.0 (1/10) | 10.0 (1/10) | 0 (0/4) |
CCND1 gain | 59 (27.7) | 1.8 (1/56) | 8.9 (5/56) | 10.4 (5/48) |
2.5–5 copies | 49 (23.0) | 0 (0/46) | 10.9 (5/46) | 112.8 (5/39) |
5–10 copies | 9 (4.2) | 11.1 (1/9) | 0 (0/9) | 0 (0/8) |
> 10 copies | 1 (0.5) | 0 (0/1) | 0 (0/1) | 0 (0/1) |
P16INK4a loss | 123 (57.7) | 9.6 (11/114) | 4.3 (5/115) | 10.5 (9/86) |
Overall | 174 (81.7) | 7.0 (11/199) | 10.0 (10/200) | 13.7 (21/153) |
≥ 2 CNVs | ||||
CDK4 gain plusCCND1 gain | 13 (6.1) | 0 (0/12) | 0 (0/12) | 20.0 (2/10) |
CDK4 gain plus P16INK4aloss | 49 (23.0) | 12.8 (6/47) | 4.2 (2/48) | 12.1 (4/33) |
CCND1 gain plus P16INK4aloss | 16 (7.5) | 6.3 (1/16) | 0 (0/16) | 15.4 (2/13) |
Overall | 78 (36.6) | 9.3 (7/75) | 3.9 (3/76) | 14.3 (8/56) |
3 CNVs | ||||
Overall | 17 (8.0) | 0 (0/16) | 12.5 (2/16) | 0 (0/14) |
Correlation of CDK4 pathway aberrations to other driver genes
Correlation of Anatomic site to TNM stage of mucosal melanoma
Correlation of CDK4 pathway aberrations to clinicopathological features
Clinicopathologic factor | CDK4 aberration | CCND1 aberration | |||||
---|---|---|---|---|---|---|---|
Gain | Normal | P value | Gain | Loss | Normal | P value | |
Age (year) | 0.552 | 0.853 | |||||
Median (range) | 56.5 ± 11.6 | 54.9 ± 11.3 | 56.4 ± 11.3 | 55.3 ± 11.6 | 55.4 ± 11.6 | ||
Gender n (%) | 0.409 | 0.413 | |||||
Male | 41 (41.0) | 39 (35.5) | 20 (33.9) | 18 (47.4) | 45 (38.8) | ||
Female | 59 (59.0) | 71 (64.5) | 39 (66.1) | 20 (52.6) | 71 (61.2) | ||
Ulceration n (%) | 0.555 | 0.769 | |||||
Yes | 52 (52.0) | 49 (44.5) | 25 (42.4) | 20 (52.6) | 58 (50.0) | ||
No | 20 (20.0) | 26 (23.6) | 14 (23.7) | 9 (23.7) | 23 (19.8) | ||
NA | 28 (28.0) | 35 (31.8) | 20 (33.9) | 9 (23.7) | 35 (30.2) | ||
Depth of invasion | 0.981 | 0.853 | |||||
T1 ≤ 1 mm | 13 (13.0) | 16 (14.5) | 10 (16.9) | 4 (10.5) | 15 (12.9) | ||
T2 1–2 mm | 21 (21.0) | 24 (21.8) | 10 (16.9) | 11 (28.9) | 25 (21.6) | ||
T3 2–4 mm | 18 (18.0) | 20 (18.2) | 10 (16.9) | 6 (15.8) | 22 (19.0) | ||
T4 > 4 mm | 48 (48.0) | 50 (45.5) | 29 (49.2) | 17 (44.7) | 54 (46.6) | ||
Anatomic site n (%) | 0.153 | 0.885 | |||||
Head and neck | 51 (51.0) | 41 (37.3) | 27 (45.8) | 15 (39.5) | 52 (44.8) | ||
Oesophagus | 8 (8.0) | 11 (10.0) | 3 (5.1) | 5 (13.2) | 11 (9.5) | ||
Anorectum | 21 (21.0) | 23 (20.9) | 13 (22.0) | 9 (23.7) | 23 (19.8) | ||
Genitourinary | 20 (20.0) | 35 (31.8) | 16 (27.1) | 9 (23.7) | 30 (25.9) | ||
Stages n (%) |
0.004
| 0.239 | |||||
I | 5 (5.0) | 1 (0.9) | 0 (0.0) | 1 (2.6) | 5 (4.3) | ||
II | 46 (46.0) | 67 (60.9) | 30 (50.8) | 26 (68.4) | 58 (50.0) | ||
III | 38 (38.0) | 22 (20.0) | 18 (30.5) | 9 (23.7) | 34 (29.3) | ||
IV | 11 (11.0) | 20 (18.0) | 11 (18.6) | 2 (5.3) | 19 (16.4) | ||
Survival (months) | 0.054 | 0.702 | |||||
Median (95% CI) | 45.0 (39.3, 50.7) | 41.2 (35.4, 47.0) | 43 (40.2, 45.8) | 47.4 (38.9, 55.9) | 45.0 (40.0, 50.0) | ||
Median follow-up time (95% CI) | 38.1 (26.0, 50.2) | 37.0 (25.5, 48.5) | 0.322 | 44.5 (27.4, 61.6) | 39.5 (22.2, 56.8) | 31.0 (18.3, 43.7) | 0.453 |
Clinicopathologic factor | P16INK4a aberration | Overall aberration (≥ 1 CNV) | ||||
---|---|---|---|---|---|---|
Loss | Normal | P value | Yes | No | P value | |
Age (year) | 0.733 | 0.581 | ||||
Median (range) | 55.4 ± 11.5 | 56.2 ± 11.4 | 55.5 ± 11.4 | 56.3 ± 12.0 | ||
Gender n (%) | 0.903 | 0.771 | ||||
Male | 48 (39.0) | 34 (38.2) | 67 (38.5) | 16 (41.0) | ||
Female | 75 (61.0) | 55 (61.8) | 107 (61.5) | 23 (59.0) | ||
Ulceration n (%) |
0.014
| 0.189 | ||||
Yes | 52 (42.3) | 51 (57.3) | 81 (46.6) | 22 (56.4) | ||
No | 25 (20.3) | 21 (23.6) | 36 (20.7) | 10 (25.6) | ||
NA | 46 (37.4) | 17 (119.1) | 36 (20.7) | 7 (17.9) | ||
Depth of invasion | 0.053 | 0.348 | ||||
T1 ≤ 1 mm | 20 (16.3) | 9 (10.1) | 26 (14.9) | 3 (7.7) | ||
T2 1–2 mm | 33 (26.8) | 13 (14.6) | 40 (23.0) | 6 (15.4) | ||
T3 2–4 mm | 19 (15.4) | 19 (21.3) | 29 (16.7) | 9 (23.1) | ||
T4 > 4 mm | 51 (41.5) | 48 (53.9) | 79 (45.4) | 21 (53.8) | ||
Anatomic site n (%) | 0.379 | 0.691 | ||||
Head and neck | 55 (44.7) | 39 (43.8) | 79 (45.4) | 15 (38.5) | ||
Oesophagus | 14 (11.4) | 5 (5.6) | 16 (9.2) | 3 (7.7) | ||
Anorectum | 22 (17.9) | 22 (24.7) | 34 (19.5) | 11 (28.2) | ||
Genitourinary | 32 (26.0) | 23 (25.8) | 45 (25.9) | 10 (25.6) | ||
Stages n (%) | 0.515 | |||||
I | 4 (3.3) | 2 (2.2) | 5 (2.9) | 1 (2.6) | 0.059 | |
II | 65 (52.8) | 49 (55.1) | 90 (51.7) | 24 (61.5) | ||
III | 39 (31.7) | 22 (24.7) | 56 (32.2) | 5 (12.8) | ||
IV | 15 (12.2) | 16 (18.0) | 23 (13.2) | 9 (23.1) | ||
Survival (months) | 0.528 | 0.169 | ||||
Median (95% CI) | 43.6 (40.0, 47.2) | 43.6 (32.3, 54.9) | 44.0 (40.4, 47.6) | 43.6 (18.6, 68.6) | ||
Median follow-up time (95% CI) | 39.7 (29.7, 49.7) | 33.7 (22.3, 45.1) | 0.137 | 38.1 (31.7, 44.5) | 21.9 (17.8, 26.0) | 0.206 |
Clinicopathologic factor | CDK4 gain + CCND1 gain | CDK4 gain + P16INK4a loss | ||||
---|---|---|---|---|---|---|
Positive | Negative | P value | Positive | Negative | P value | |
Age (year) | 0.866 | 0.722 | ||||
Median (range) | 57.6 ± 11.1 | 55.4 ± 11.5 | 56.2 ± 11.1 | 55.5 ± 11.6 | ||
Gender n (%) | 0.817 | 0.382 | ||||
Male | 12 (40.0) | 68 (37.8) | 28 (42.4) | 52 (36.1) | ||
Female | 18 (60.0) | 112 (62.2) | 38 (57.6) | 92 (63.9) | ||
Ulceration n (%) | 0.690 | 0.971 | ||||
Yes | 13 (43.3) | 88 (48.9) | 31 (47.0) | 70 (48.6) | ||
No | 6 (20.0) | 40 (22.2) | 15 (22.7) | 31 (21.5) | ||
NA | 11 (36.7) | 52 (28.9) | 20 (30.3) | 43 (29.9) | ||
Depth of invasion | 0.767 | 0.527 | ||||
T1 ≤ 1 mm | 3 (10.0) | 26 (14.4) | 8 (12.1) | 21 (14.6) | ||
T2 1–2 mm | 5 (16.7) | 40 (22.2) | 18 (27.3) | 27 (18.8) | ||
T3 2–4 mm | 6 (20.0) | 32 (17.8) | 10 (15.2) | 28 (19.4) | ||
T4 > 4 mm | 16 (53.3) | 82 (45.6) | 30 (45.5) | 68 (47.2) | ||
Anatomic site n (%) | 0.160 | 0.935 | ||||
Head and neck | 16 (53.3) | 76 (42.2) | 31 (47.0) | 61 (42.4) | ||
Oesophagus | 3 (10.0) | 16 (8.9) | 6 (9.1) | 13 (9.0) | ||
Anorectum | 8 (26.7) | 36 (20.0) | 13 (19.7) | 31 (21.5) | ||
Genitourinary | 3 (10.0) | 52 (28.9) | 16 (24.2) | 39 (27.1) | ||
Stages n (%) | 0.213 | 0.088 | ||||
I | 0 (0.0) | 6 (3.3) | 4 (6.1) | 2 (1.4) | ||
II | 12 (40.0) | 101 (56.1) | 32 (48.5) | 81 (56.2) | ||
III | 12 (40.0) | 48 (26.7) | 23 (34.8) | 37 (25.7) | ||
IV | 6 (20.0) | 25 (13.9) | 7 (10.6) | 24 (16.7) | ||
Survival (months) | 0.757 | 0.174 | ||||
Median (95% CI) | 44.0 (41.4, 46.6) | 43.6 (39.3, 47.9) | 44.0 (41.4, 46.6) | 43.0 (37.6, 48.4) | ||
Median follow-up time (95% CI) | 54.0 (29.2, 78.8) | 37.0 (30.0, 44.0) | 0.636 | 38.1 (25.9, 50.3) | 37.0 (29.1, 44.9) | 0.153 |
Clinicopathologic factor | P16INK4a loss + CCND1 gain | CDK4 gain + P16INK4a loss + CCND1 gain | ||||
---|---|---|---|---|---|---|
Positive | Negative | P value | Positive | Negative | P value | |
Age (year) | 0.531 | 0.807 | ||||
Median (range) | 58.1 ± 12.5 | 55.3 ± 11.2 | 59.2 ± 12.8 | 55.3 ± 11.4 | ||
Gender n (%) | 0.927 | 0.476 | ||||
Male | 13 (39.4) | 69 (38.5) | 8 (47.1) | 75 (38.3) | ||
Female | 20 (60.6) | 110 (61.5) | 9 (52.9) | 121 (61.7) | ||
Ulceration n (%) | 0.220 | 0.857 | ||||
Yes | 13 (39.4) | 90 (50.3) | 8 (47.1) | 95 (48.5) | ||
No | 6 (18.2) | 40 (22.3) | 3 (17.6) | 43 (21.9) | ||
NA | 14 (42.4) | 49 (27.4) | 6 (35.3) | 58 (29.6) | ||
Depth of invasion | 0.584 | 0.682 | ||||
T1 ≤ 1 mm | 7 (21.2) | 22 (12.3) | 1 (5.9) | 28 (14.3) | ||
T2 1–2 mm | 7 (21.1) | 39 (21.8) | 3 (17.6) | 43 (21.9) | ||
T3 2–4 mm | 5 (15.2) | 33 (18.4) | 3 (17.6) | 35 (17.9) | ||
T4 > 4 mm | 14 (42.4) | 85 (47.5) | 10 (58.8) | 90 (45.9) | ||
Anatomic site n (%) | 0.404 | 0.497 | ||||
Head and neck | 18 (54.5) | 76 (42.5) | 9 (52.9.6) | 85 (43.4) | ||
Oesophagus | 1 (3.0) | 18 (10.0) | 1 (5.9) | 18 (9.2) | ||
Anorectum | 6 (18.2) | 38 (21.1) | 5 (29.4) | 40 (20.4) | ||
Genitourinary | 8 (24.2) | 47 (26.1) | 2 (11.8) | 53 (27.0) | ||
Stages n (%) | 0.383 | 0.268 | ||||
I | 0 (0.0) | 6 (3.4) | 0 (0.0) | 6 (3.1) | ||
II | 16 (48.5) | 98 (54.7) | 6 (35.3) | 108 (55.1) | ||
III | 13 (39.4) | 48 (26.8) | 8 (47.1) | 53 (27.0) | ||
IV | 4 (12.1) | 27 (15.1) | 3 (17.6) | 29 (14.8) | ||
Survival (months) | 0.287 | 0.145 | ||||
Median (95% CI) | 42 (35.0, 49.0) | 45.5 (40.8, 50.2) | 42 (33.5, 50.5) | 45 (40.5, 49.4) | ||
Median follow-up time (95% CI) | 55.5 (20.3, 90.7) | 35.0 (27.6, 42.4) | 0.102 | 38.1 (25.9, 50.3) | 37.0 (29.1, 44.9) | 0.153 |
Sensitivity of MM cells to CDK4/6 inhibitors
Anatomic site | Patients (no. %) | TNM分期 (no.%) | P-value | |||
---|---|---|---|---|---|---|
I | II | III | IV | < 0.001 | ||
Head and neck | 94 (44.2) | 3 (3.2) | 57 (60.6) | 31 (33.0) | 3 (3.2) | |
Nasopharynx | 60 (28.2) | 1 (1.7) | 48 (80.0) | 10 (16.7) | 1 (1.7) | |
Oral cavity | 34 (16.0) | 2 (5.9) | 9 (26.5) | 21 (61.8) | 2 (5.9) | |
Oesophagus | 19 (8.9) | 1 (5.3) | 6 (31.6) | 8 (42.1) | 4 (21.1) | |
Anorectum | 45 (21.1) | 1 (2.2) | 15 (33.3) | 14 (31.1) | 15 (33.3) | |
Genitourinarya | 55 (25.8) | 1 (1.8) | 36 (65.5) | 8 (14.5) | 10 (18.2) | |
Total | 213 | 6 (2.8) | 114 (53.5) | 61 (28.6) | 32 (15.1) |
Sensitivity of PDX models to CDK4/6 inhibitors
Cells |
CDK4
|
CCND1
|
P16
INK4a
|
BRAF
a
|
NRAS
a
|
CKIT
a
|
---|---|---|---|---|---|---|
GAK | Gain | Gain | Normal | WT | Q61L | WT |
HMV II | Gain | Normal | Loss | G469L | Q61K | WT |