Introduction
Materials and methods
Patients, controls and cell lines
Controls(187) | Sex* | ||
Male, N(%) | 157(83.9%) | ||
Female,(N%) | 30(16.1%) | ||
Mean Age# | 45.5 ± 5.2 | ||
HNSCC & Dysplastic lesions(147)
| Sex* | ||
Male, N(%) | 110(74.8%) | ||
Female,(N%) | 37(25.2%) | ||
Mean Age# | 52 ± 6.4 | ||
Lymp Node Positive | 26(17.7%) | ||
Lymp Node Negative | 121(82.3%) | ||
Tobacco User | 103(70%) | ||
Tobacco free | 44(30%) | ||
Alcohol User | 13(8.8%) | ||
Alcohol free | 134(91.2%) | ||
HNSCC (110)
|
Oral Cavity tumors(85)
| Buccal Mucosa, N(%) | 32(29.1%) |
Tongue, N(%) | 13(11.8%) | ||
Cheek, N(%) | 19(17.3%) | ||
Lip, N(%) | 3(2.7%) | ||
Tonsil, N(%) | 4(3.6%) | ||
Palate, N(%) | 2(1.8%) | ||
Alveolus, N(%) | 11(10%) | ||
Vocal cord, N(%) | 1(0.9%) | ||
Laryngeal Tumors(12) &
| Larynx, N(%) | 12(10.9%) | |
Nasopharyngeal Tumor(3)
| Nassopharynx, N(%) | 3(2.7%) | |
Orofacial Tumors(10)
| Maxilla, N(%) | 5(4.5%) | |
Mandible, N(%) | 5(4.5%) | ||
Histopathology(110)
| StageI | 15(14%) | |
StageII | 30(27%) | ||
StageIII | 34(31%) | ||
StageIV | 31(28%) | ||
Dysplastic lesions (37)
| Buccal Mucosa, N(%) | 37(100%) | |
Histopathology(37)
| Mild Dysplasia | 5(13.5%) | |
Moderate Dysplasia | 19(51.4%) | ||
Severe Dysplasia | 13(35.1%) |
Microdissection and DNA Extraction
Mutation analysis
Deletion analysis
Genotyping of hmlimD1 microsatellite marker
mRNA expression analysis
Immunohistochemical analysis
Detection of HPV-16 and HPV-18
Statistical analysis
Results
Alterations of LIMD1
Mutations | ||||||||
---|---|---|---|---|---|---|---|---|
Exon1 | Exon5 | |||||||
Nucleotide change | 470C → T | 660A del | 882C → T | 967T del | 1003C → A | 1068T → C | C del at intron4/exon5 | |
TNM stage | Amino acid change | Ser157 Phe | Frame shift | Thr294Thr | Frame shift | Pro335Thr | Gly356Gly | Splice-juction |
Samples showing mutations | ||||||||
Moderate | L66 | - | - | - | - | - | - | - |
Moderate | L139 | - | - | SNPa | - | - | + | - |
Severe | L144 | - | - | SNPa | - | - | - | - |
Severe | L126 | - | - | SNPa | - | - | + | - |
Severe | L153 | - | - | SNPa | - | - | - | - |
StageI | #292 | - | - | SNPa | - | - | - | - |
StageI | #2642 | - | - | - | - | - | + | - |
stageII | #2772 | - | - | + | - | - | - | - |
stageII | #7216 | - | - | - | + | - | - | - |
stageII | #2073 | + | - | SNPa | - | - | + | - |
stageII | #5303 | - | + | - | - | - | - | - |
stageII | #1234 | - | - | SNPa | - | - | SNPb | - |
stageII | #2323 | - | - | + | - | - | - | - |
stageII | #1108 | - | - | - | - | - | + | - |
stageII | #7077 | + | - | - | - | - | - | - |
StageIII | #821 | - | - | - | - | - | + | - |
StageIII | #1367 | + | - | - | - | - | - | - |
StageIII | #1552 | - | - | SNPa | - | + | - | - |
StageIII | #615 | - | - | - | + | - | - | - |
StageIII | #5090B | - | - | + | - | - | - | - |
StageIII | #816 | - | - | + | - | - | - | - |
StageIII | #326 | - | + | SNPa | - | - | - | - |
StageIII | #1068 | - | - | SNPa | - | + | - | - |
StageIII | #3893 | - | - | - | + | - | - | - |
StageIII | #308 | - | + | SNPa | - | - | - | - |
StageIII | #6433 | - | + | SNPa | - | - | - | - |
StageIII | #1332 | + | - | SNPa | - | - | - | + |
StageIII | #2398 | - | - | - | + | - | - | - |
StageIII | #7059 | - | + | - | - | - | - | - |
StageIV | #1087 | - | - | + | - | - | - | - |
StageIV | #4075 | - | - | - | - | - | SNPb | - |
StageIV | #2508 | - | + | - | - | - | + | - |
StageIV | #5114 | - | - | + | - | - | - | - |
StageIV | #5219 | - | + | - | - | - | - | - |
StageIV | #1084 | + | - | - | - | - | SNPb | - |
StageIV | #5184 | - | + | - | - | - | - | - |
StageIV | #944 | - | - | - | - | - | - | + |
StageIV | #2785 | - | - | SNPa | - | - | - | + |
StageIV | #1774 | - | - | - | - | - | + | - |
Cell lines | Hep2 | - | - | SNPa | - | - | SNPb | - |
SCC084 | - | - | SNPa | - | - | SNPb | - | |
Mutation frequency in HNSCC | 6% (5/85) | 9% (8/85) | 7% (6/85) | 4% (4/85) | 2% (2/85) | 9% (8/85) | 4% (3/85) |
Dysplastic lesions | HNSCC | |||||
---|---|---|---|---|---|---|
Del+ | Del- |
P
| Del+ | Del- |
P
| |
Meth+ |
11
| 8 | 0.7022 |
21
| 15 | 0.6887 |
Meth- | 4 | 2 | 14 | 8 | ||
Mut+ |
1
| 1 | 0.7634 |
18
| 13 | 0.7036 |
Mut- | 14 | 9 | 17 | 10 | ||
Mut+ | Mut- | Mut+ | Mut- | |||
Meth+ |
1
| 18 | 0.3694 |
15
| 21 | 0.02139 |
Meth- | 1 | 5 | 16 | 6 |
Association of hmlimD1 polymorphism with risk of HNSCC
Allele | No of (CA)n repeats | Case | Control |
P
#
| ||
---|---|---|---|---|---|---|
No. of alleles | % | No. of alleles | % | |||
(CA)9 | 9 | 7 | 2.38 | 11 | 2.94 | 0.6571 |
(CA)13 | 13 | 4 | 1.36 | 35 | 9.36 | 0.00012 |
(CA)17 | 17 | 23 | 7.82 | 46 | 12.29 | 0.0592 |
(CA)19 | 19 | 97 | 32.99 | 169 | 45.19 | 0.00139 |
(CA)20 | 20 | 66 | 22.45 | 36 | 9.63 |
0.000005
|
(CA)21 | 21 | 21 | 7.14 | 15 | 4.01 | 0.075134 |
(CA)24 | 24 | 30 | 10.2 | 23 | 6.15 | 0.05427 |
(CA)26 | 26 | 2 | 0.68 | 15 | 4.01 | 0.0067 |
(CA)27 | 27 | 19 | 6.46 | 13 | 3.48 | 0.0728 |
(CA)30 | 30 | 10 | 3.4 | 6 | 1.6 | 0.1316 |
(CA)32 | 32 | 14 | 4.76 | 5 | 1.34 |
0.0082
|
(CA)38 | 38 | 1 | 0.34 | 0 | 0 | 0.259 |
Total no. of alleles | 294 | 100 | 374 | 100 | ||
χ2 = 29.175, df = 11, P= 0.0021 | ||||||
Genotype | Case | % | Control | % |
P
#
| |
(CA)17/(CA)17 | 6 | 9.23 | 11 | 15.94 | 0.2434 | |
(CA)19/(CA)19 | 33 | 50.76 | 56 | 81.16 | 0.0002 | |
(CA)20/(CA)20 | 19 | 29.23 | 2 | 2.89 |
0.00003
| |
(CA)21/(CA)21 | 5 | 7.69 | 0 | 0 | 0.0247 | |
(CA)24/(CA)24 | 1 | 1.54 | 0 | 0 | 0.4851 | |
(CA)32/(CA)32 | 1 | 1.54 | 0 | 0 | 0.4851 |
Association of LIMD1 alterations with RB1 deletion in HNSCC
LIMD1
| |||||||
---|---|---|---|---|---|---|---|
Dysplasia | Stage I+II | Stage III+IV | |||||
ALT+ | ALT- | ALT+ | ALT- | ALT+ | ALT- | ||
RB1
|
Del+
| 2 | 0 | 7 | 0 | 13 | 0 |
Del-
| 21 | 2 | 11 | 1 | 24 | 2 | |
P
| 0.6637 | 0.4326 | 0.3046 |
Comparison in the mRNA expression of LIMD1 and RB1
Immuno-staining of pRB
RB1
|
LIMD1
| ||||
---|---|---|---|---|---|
Sample No.; HPV status | Genetic alterations | mRNA status | Protein expression | Genetic/epigenetic alterations | mRNA status |
Dysplastic lesions | |||||
L127; HPV- | D- | ND | Normal | D+Me+Mu- | ND |
L139; HPV- | D- | ND | Normal | D-Me+Mu- | ND |
L154; HPV- | D- | ND | Normal | D+Me+Mu- | ND |
L162; HPV- | D- | ND | Normal | D+Me+Mu- | ND |
L158; HPV16 | D- | ND | Medium | D+Me+Mu- | ND |
HNSCC samples | |||||
#1004; HPV- | D- | Normal | Normal | D+Me+Mu- | ↓ |
#5165; HPV- | D- | Normal | Normal | D+Me+Mu- | ↓ |
#6817;HPV16 | D+ | ↓ | Medium | D+Me+Mu- | ↓ |
#2884; HPV- | D- | Normal | Normal | D-Me-Mu- | Normal |
#1108; HPV - | D+ | ↓ | Medium | D-Me+Mu+ | ↓ |
#1774;HPV16 | D+ | ↓ | Low | D-Me+Mu+ | ↓ |
#872; HPV- | D- | Normal | Normal | D-Me-Mu- | Normal |
#5303; HPV- | D- | Normal | Normal | D-Me+Mu+ | ↓ |
#6907;HPV16 | D- | ↓ | Low | D-Me+Mu- | ↓ |
#1087; HPV- | D- | Normal | Normal | D-Me-Mu+ | Normal |
#944; HPV- | D+ | ↓ | Low | D+Me+Mu+ | ↓ |
#5733;HPV16 | D- | Normal | Normal | D+Me+Mu- | ↓ |
#6392; HPV- | D- | Normal | Normal | D-Me+Mu- | ↓ |
#7077; HPV- | D- | Normal | Normal | D-Me-Mu+ | ↓ |
#797; HPV- | D- | Normal | Normal | D+Me+Mu- | ↓ |
Hep2, HPV18 | D- | Normal | Normal | D-Me+Mu- | ↓ |
UPCI:SCC084, HPV- | D- | Normal | Normal | D-Me+Mu- | ↓ |
Clinical and prognostic implications of LIMD1 and RB1 alterations
Variable | Overall survival | ||
---|---|---|---|
P- value | Hazard ratio(HR) | 95% CI for HR | |
LIMD1 alteration |
0.0318
| 4.51 | 1.14-17.82 |
RB deletion | 0.1535 | 2.27 | 0.74-7.02 |
Stage | 0.5144 | 0.79 | 0.39-1.59 |
Grade | 0.5044 | 1.49 | 0.46-4.89 |
Age | 0.7786 | 0.84 | 0.25-2.85 |
Node | 0.4228 | 1.63 | 0.49-5.34 |
HPV |
0.0498
| 0.28 | 0.08-0.99 |
Tobacco |
0.0559
| 4.49 | 0.96-20.89 |
Alcohol | 0.2659 | 0.29 | 0.03-2.52 |