Page 447 - Read Online

Dave et al. Nibrin expression in OSCC

aggressive metastases to loco-regional lymph embedded primary tumor tissue blocks (buccal mucosa:
nodes, strongly suggestive of an urge to improve the n = 39, tongue: n = 61) and histologically confirmed
treatment efficacy and diagnostic capabilities. Over the adjacent normal tissue blocks were collected from the
last decade, scientific research related to the specific histopathology department of our institute. The detail
pathways which are relevant to the development clinical history of the patients [age, gender, tobacco
and progression of this disease has been performed habit, site of disease, tumor-node-metastasis (TNM)
to investigate biological, diagnostic and prognostic stage, histopathological findings, treatment given,
parameters. [7-10] etc.] was obtained from the case files maintained at
our institute. In patients with OSCC the disease was
DNA damage is one of the underlying causes for staged according to the criteria of the American Joint
mutations which is very numerous and appears to be Committee on Cancer pTNM classification. Thus,
a fundamental problem for life leading to cancer. In the present study included 20 patients with stage I
human cells, the estimated average number of DNA disease, 22 patients with stage II disease, 18 patients
damages occurring per hour is about 800 which reach with stage III disease and 40 patients with stage IV
to 19,200 per day. If, such DNA damages are not disease. This study was approved by our institutional
[11]
repaired in dividing cells, cause errors during DNA review committee for dissertation/thesis/publications/
synthesis leading to mutations which can give rise to conference presentations and institutional ethics
cancer. Thus individuals are often at increased risk committee.
of cancer with an inherited damage in DNA repair
capability. [12] Immunohistochemistry
Immunohistochemistry of Nibrin was performed using
Nijmegen breakage syndrome (NBS) is a chromosomal- the avidine-biotin complex technique in which formalin
instability syndrome associated with cancer fixed paraffin embedded tissue sections (4 μm) were
predisposition, growth retardation, microcephaly, mounted on 3-aminopropyletriethoxy silane coated
radiosensitivity and immunodeficiency. [13-15] The NBS1/ glass slides. The sections were first deparafinized
Nibrin/p95 is a member of the DNA double-strand using xylene and then rehydrated using graded
break (DSB) repair complex (hMre11 complex) which alcohol. Endogenous peroxidase activity was blocked
is a product of the defective gene in NBS (the NBS using 3% hydrogen peroxide prepared in methanol for
gene) located on human chromosome 8q21. [14-16] The 15 min. Antigenicity of the processed tissue sections
Nibrin containing protein complex [Mre11-Rad50- was retrieved by cooking the sections in 10 mmol/L
Nbs1(MRN) complex] binds to the edges of the DNA tri-sodium citrate buffer (pH 6.0) solution with 0.05%
double stranded break and remains attached to this tween-20 for 20 min in a pressure cooker. Sections were
site until the break gets repaired. Nibrin is also then allowed to cool at room temperature. For Nibrin
[17]
involved in various signaling cascades other than immunostainings, a commercial mouse monoclonal
DSBs induced by irradiation such as mitotic V(D)J antibody (clone 1D7, Santa Cruz Biotechnology, Santa
rearrangements in T and B lymphocytes, maintenance Cruz, USA) at dilution of 1:100 prepared in tris buffered
of telomere function and meiotic recombination. [18,19] saline was applied to the sections and were then
Once ataxia-telangiectasia mutated protein incubated overnight at 4 ˚C. For immunohistochemical
phosphorylates NBS1, it then carries out its checkpoint detection of the antibody reaction, we used Novolink
functions following ionizing radiation. [20-22] However, in polymer detection kit from Novocastra. Sections were
certain types of human cancer rare or no mutations of then dehydrated, cleared in xylene and mounted in
NBS1 have been studied. [23-25] In addition, during the dibutylphthalate xylene. As positive controls, formalin-
process of carcinogenesis NBS1 is expressed in highly fixed paraffin-embedded tissue sections with intense
proliferating tissues. On the basis of this information, staining for a given marker were included with each
[26]
the aim of this study was to assess whether the Nibrin staining procedure.
expression would relate to clinicopathological variables
and if it could predict survival or recurrence in OSCC. Assessment of Nibrin expression
All sections were scored independently by two
METHODS independent researchers in a blinded fashion. The
staining intensities and the percentage of positive
Study population cells were separately assessed in primary tumor
A total of 100 untreated patients with histolpathologically tissues (n = 100) and their corresponding adjacent
confirmed OSCC of tongue and buccal mucosa normal squamous epithelium (n = 100). As the Nibrin
evaluated between 2011 and 2013 at our institute expression was not uniform in different parts of the
were included in this study. Formalin fixed and paraffin epithelium or cancerous tissue, we used modified
Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ November 25, 2016 437