have  shown  that  the  mutations 
of  TP53  gene  is  the
frequent  event  in  many  human  cancers  and  is
associated  with  a  poor  clinical 
outcome  in  lung  cancer  patients.   Several studies have  revealed 
that  the  distinct 
TP53  mutational 
pattern  between  population 
groups  may  be  due
different  racial  or  exogenous  factors. 
In this report we studied the mutational spectrum of TP53 gene in 50 lung cancer patients of
a uniquely preserved ethnic population of Kashmir Valley.  Germline 
TP53  mutational 
analyses  were  also  performed  to  determine  the  inherited  cancer 
predisposition.  Exons  5-8  of   TP53  gene  were  analyzed 
by  sequencing  DNA  of  cancerous 
tissue  and  peripheral 
blood  leukocyte  samples 
from  50  nonsmall  cell  lung   cancer  patients. 
The  results  showed 
that   TP53  germline  mutation 
was  not  found  in  any  patient,  indicating 
that  TP53  germline  mutations 
were  not  responsible 
for  cancer  predisposition  in  this  group  of  patients. 
A  total  of  19  somatic 
mutations  were  found . 
No    smoking specific characteristic  hot  spot
were  observed except in two
patients.  The  data  thus  suggests  that  TP53 
mutations  in  this  study  group  are  induced 
by  exposure  to  substances
 besides tobacco  smoke. 
Pesticide  exposure  may  instead  be  related  to   tumorigenesis  of  lung  cancer 
via  TP53  mutations.












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High  incidence 
and  poor  prognosis 
of  lung  cancer 
make  it  a  major  health 
problem  worldwide  1,2. 
Lung  cancer,  which  was  initially 
considered  an  epidemic 
disease  among  men  in  industrialised  nations, 
has  now  become 
the  leading  killer 
cancer  in  both  sexes  in  the  United 
States  and  an  increasingly  common 
disease  of  both  sexes  in  developing  countries 
3.  In  Kashmir 
valley,  the  northernmost 
part  of  India, 
it  ranks  second 
among  all  cases  in  males. 
Non-small  cell  lung  cancer  accounts 
for  nearly  85%  and  small  cell  lung  cancer  accounts 
for  15%  to  20%  of  cases  4. 


The  multistage  process 
of  tumorigenesis  generally 
includes  the  gain  of  protooncogenes  activity 
and  the  loss  or  inactivity 
of  tumor  suppressor 
genes  5.  Among  tumor  suppressors,  TP53  gene  abnormalities  are  the  most  frequent  genetic 
events  illustrated  to  date  6-7.  TP53  mutation 
occurs  in  about  40-70%  of  lung  cancers 
8.  Mutations  at  codons  157,  158,  248,  249,  and  273  are  more  frequently 
detected  in  lung  tumors  than  mutations  at  other  positions 
and  are  considered 
”hotspots”  for  TP53  mutation 
9,10,6,11.  TP53  cancer-associated  mutation  in  the  highly 
conserved  DNA-binding  domain 
may  prevent  or  inhibit  p53-mediated 
cell  cycle  arrest, 
DNA  repair,  programmed 
cell  death,  and  other  protective 
responses  to  cell  stress  and  DNA  damage.12-14  The  causes  of  TP53  mutations 
include  both  endogenous 
factors  which  contribute 
to  the  infidelity 
of  DNA  synthesis 
and  exogenous  factors 
such  as  chemical 
mutagens  and  radiation 
exposure  9.  Various 
genotoxic  compounds  have  been  shown  to  selectively 
induce  alterations  of  specific  base  pairs  in  TP53,  that  are  related 
to  cancer  15.  These  alterations 
can  be  explained 
by  the  presence 
of  regionally  distinct 
carcinogens  in  both  smoke  and  air,  interacting 
with  local  environmental 
cofactors,  in  the  development  of  lung  cancer. 
Various  studies  have  been  conducted 
that  have  revealed 
a  distinct  TP53  mutation 
pattern  between  population 
groups  16-18,  suggesting 
the  involvement  of  a  potentially 
distinct  mutagenic  process 
in  each  population. 


The  present 
study  aims  to  identify  potential 
genetic  risk  factors 
by  determining  the  TP53 
germline  and  somatic 
mutations  of  lung  cancer  patients 
residing  in  the  Kashmir  valley 
(INDIA).  The  mutation 
spectra  of  TP53  might  provide  important 
clues  for  cancer 
risk  assessment  in  molecular  epidemiology. 
Understanding  risk  factors 
and  genetic  predisposition  for  lung  cancer 
is  important  to  lung  cancer 


Subjects  and  methods

Study  subjects

Tumor  specimens 
(50  cases)  and  corresponding  normal 
tissues  were  obtained 
from  NSCLC  patients 
who  were  admitted 
to  the   Department 
of  Cardio-vascular  and  Thoracic  Surgery 
in  Sheri-Kashmir  Institute 
of  Medical  Sciences, 
Srinagar  (INDIA). 5ml  of  peripheral  blood  was  collected 
from  each  patient. 
This  research  project 
has  been  approved 
by  the  Research 
Ethics  Committee,  of  the  Institute. Subjects  enrolled 
in  the  study  were  asked  to  sign  informed  consent 
forms  before  having 
blood  samples  taken  and  being  interviewed.  A  detailed  questionnaire 
was  administered  by  interviewers  to  all  studied 
participants  about  personal 
life,  occupation,  lifestyle, 
health  and  disease, 
cigarette  smoking  and  environmental  exposures. 
The  diagnosis  of  lung  cancer 
was  performed  by  histological  evaluation 
of  tumour  biopsies 
and  chest  computed 
tomography  scans. 

Genomic  DNA  from  peripheral 
blood  leukocytes  and  cancerous  lung  tissues  of  each  patient 
was  extracted  by  inorganic  salting 
out  protocol19  or  phenol-chloroform  protocol 


Four  primer 
pairs  were  used  to  amplify 
exons  5,  6,  7  and  8  of  the  TP53 
gene  (Table I).    PCR  was  carried 
out  in  an  Biorad  Thermal 
cycler  at  each  primer  pair’s 
respective  annealing  temperature. PCR-SSCP  was  performed  as  follows:  initial 
denaturation  at  950C  for  5  min,  amplification  for  35  cycles 
with  denaturation  at  950C
40s,  primer  annealing 
at  550C  for  exon  5,  620C  for  exon  6  and  7  and  580C  for  exon  8  for  70s  and  elongation 
at  720C for  90s  in  each  cycle.  The  final  elongation 
was  performed  at  720C
for  7 min.  PCR  products  after  denaturation  were  analyzed  on  8%  polyacrylamide 
gel  (180V)  for  5h  and  stained  with  AgNO3.  Purified 
PCR  products  showing 
a  mobility  shift  (20  samples) 
in  SSCP  analysis ( Fig. I)  as  well  as  randomly  chosen 
samples were  used  for  direct  DNA  sequencing  (a  total  of  45  samples) using  the  ABI  prism  310  automated 
DNA  sequencer.  To confirm mutations reverse sequencing was
also done. 



Univariate  logistic 
regression  analysis  was  used  to  examine  the  association  between 
TP53  mutations 
and  potential    risk  factors. 
Odds  ratio  (OR)  and  their  95%  confidence 
intervals  (95%  CI)  were  estimated. 
Statistical analyses were performed using SPSS statistical software (version



The  characteristics  and  variable  factors 
of  50  NSCLC  patients  from  the  questionnaire 
are  summarized  in  Table
II.  A 
total  of  19  somatic  mutations (13 
different  types)  were  found  in  19  lung  cancer  patients 
(38%).  TP53  germline  mutations 
were  not  found  in  extracted 
DNA  from  peripheral 
blood  leucocytes  of  any  of  the  50  patients.  The  most  frequent 
type  of  mutation 
was  missense  (17  cases)  and  2  frameshift 
mutations.  The details of
mutations found are given in Table III.  No  characteristic  hot  spot  codon    were  observed  except 
175  and  282.  The  TP53 
mutation  pattern  consisted 
of  21.05%  of  A  >  G  transitions  
at  codon  193, 10.5% 
of  G >  A transitions at  codon  175,
A>  C transversions  at  codon  164  each,
15.8%  of 
G>  C  transversions 
at  codon  159  and  221  and  C>  T
transitions at codon  278, 282  and  294  each  and  5.26%  of  C>  G
transv ersions at codon  241, T>  C transitions at codon  163  and  G>  T
transversions at    codon  294
each.  Also  10.5%  of  insertion 
mutations  at  codon  260  (insertion 
of  A)  and  codon  296  (  insertion 
of  T)  were  identified  (Fig. II 
and  III).

All  characteristics  of  the  patients 
(Table  II),  were  tested  for  association  between 
potential  risk  factors 
and  TP53  mutations  in  a  univariate 
analysis  (Table  IV).  The  potential 
risk  factors  associated 
with  TP53  mutations  at  the  P  <  0.05  level  were  age  at  which  cancer  was  diagnosed  (P  value=0.03) and  pesticide  exposure  (P  value=0.01).  The  comparison  search  of  TP53  somatic  mutation  types  in  lung  cancer  reports  was  undertaken  using  the  most  updated  International  Agency  for  Research  on  Cancer  (IARC)  TP53  somatic  mutation  database  R12  21,  as  well  as  the  UMD  p53  database  2007_R1c  22.None  of  the  mutations  found  were  novel  although  the  pattern  and  frequency  of  different  mutations  were  different.    Discussion Mutations in TP53 widely occur in many types of human cancers  23. The majority of the mutations (more than 90%) occur in the core domain of the gene (exon 5 to exon 8), which contains the sequence-specific DNA binding activity of the p53 protein, and these mutations result in loss of DNA binding activity 24. Many studies have shown that mutation of the TP53 gene can contribute to lung cancer development, with mutations in this gene reported in over half of all lung cancers 25, 26. The protein product of the TP53 gene is involved in DNA damage response. Consequently, this gene may be a preferred target for environmental carcinogens, which act as DNA damaging agents. Moreover, carcinogens leave molecular fingerprints on the TP53 gene. Thus, the study of the TP53 mutational spectrum has been a useful approach for implicating suspected carcinogens to different human cancers. Besides somatic mutations, germline mutations have also been detected in TP53 gene. Although the carriers of the TP53 germline mutations have a high risk of developing the main Li-Fraumeni syndrome (LFS) component cancers 27, studies have reported that the TP53 mutation carriers also had increased risk of developing other types of cancers 28,29,30. The present work studied mutational spectrum of TP53 gene in 50 lung cancer (NSCLC) patients of Kashmir Valley. No germline mutations were detected in TP53 gene (exon 5-8) in extracted DNA from peripheral blood of the studied patients. The present study revealed somatic mutations of exons 5-8 of the TP53 gene in 38% of NSCLC cases examined. In NSCLC, the TP53 mutational frequency is variable among different populations and studies, ranging from 18 to 60% 31, possibly because of environmental factors and/or ethnic differences 31. The hot spot codons in NSCLC are codons 157, 158, 175, 245, 248, 249, 273 and 281/282  22, 6, which have been associated with smoking 9. In the present study, mutations at these hot spot codons were found in only two patients. Both were smokers, one had codon 175 mutation and other codon 282 mutation. Except for the two codons (175 and 282) no characteristic hot spot codon mutations were observed in all the cases studied. Most mutations in the studied cases were common in different patients indicating a unique pattern. Factors of ethnicity and specific environmental carcinogens might contribute to this unique pattern of mutations. Since environmental carcinogens have been linked with the distinct patterns of mutations found in TP53 gene 9 and no such information is available on the highly preserved ethnic population of Kashmir. Besides majority of the population here is associated with pesticide exposure through farming and gardening (major professions of the population), we tried to correlate the association between pesticide exposure and TP53 mutations and found a statistically significant association between the two. The reason for this could be that most patients were farmers and all of them had pesticide exposure. The IARC has classified insecticides and pesticides as probable human lung carcinogens 32. Previous reports have also suggested that pesticides currently in use in the World have been found to be significantly associated with lung cancer risk 33.  Our study also showed that TP53 mutations are associated with age of the patient, more mutations were detected in patients with age greater than fifty years than those with age less than fifty years, which is consistent with other studies.  The presence of TP53 somatic mutations did not correlate with the gender. The result is consistent with earlier lung cancer studies conducted on various patient populations 16,31,34. Both epidemiologic and experimental evidence have demonstrated that tobacco smoking is strongly associated with lung cancer 6,35. When examining the association between TP53 mutation and  smoking status as former, current, or never-smokers, the risk of mutation was found to be higher in current than former and never-smokers (without statistical significance), which is in conformity with other studies 36. This is supported by the observation that smokers with higher pack-years or longer duration of smoking had slightly higher risks of mutation 36. Polynuclear aromatic hydrocarbons and the tobacco-specific nitrosamines NNK and N- nitrosonicotine, known lung carcinogens present in mainstream tobacco smoke which have been linked with causing TP53 mutations via benzo(a)pyrene diol epoxide-DNA adduct formation 35,37. There was no association between mutation and histological type, but more squamous cell carcinoma patients had mutations than adenocarcinoma and other types (although statistically insignificant). Again these results are consistent with earlier studies 36,38,39. Also it could be because in our study group there were more squamous cell carcinoma patients compared to adenocarcinoma and other histological types. In conclusion, this study indicates that the TP53 germline mutation was not responsible for lung cancer predisposition in lung cancer patients of Kashmir Valley. Types and spectra of TP53 mutations in this patient group seems to be different from other  populations,  judging by comparison with the current TP53 database. This difference may be due to different mutagen origins or mechanisms of mutation and/or ethnicity. In this patient group, TP53 mutations appear to have been induced by chemical exposures or risk factors besides tobacco smoke. Pesticide exposure appears to be the most important factors related to the tumorogenesis of lung cancer via TP53 mutations in our study group. 


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