Clinicopathological significance of KAI1 expression and epithelial-mesenchymal transition in non-small cell lung cancer

Patients and clinical samples

Primary tumor tissues diagnosed NSCLC with pathologic stage I–IIIA from patients at
the First Hospital Affiliated of Bengbu Medical College from 2003 to 2007 were used
in this retrospective study. In total, 365 patients were registered from the hospital
database. Of these, 53 patients were excluded from the study due to the following:
(i) radiotherapy or chemotherapy before surgery (n?=?15), (ii) other malignancy within 5 years before NSCLC diagnosis (n?=?17), and (iii) inadequate paraffin-embedded fixed tissue blocks (n?=?20). Thus, 312 patients with complete medical records and adequate paraffin-embedded
tissue blocks were eligible. Due to financial or other reasons, 265 (84.9 %) patients
were to postoperative therapy (postoperative routine chemotherapy or radiotherapy).

This report includes follow-up data as of 31 October 2012. The median follow-up was
42 (range 8–105) months. All specimens were obtained from the archives of the Department
of Pathology at our hospital. The tumors were graded according to the World Health
Organization and staged according to the International Union Against Cancer’s tumor-node-metastasis
classification. This study was approved by the ethical committee of Bengbu Medical
College before it started.

Immunohistochemistry

All applied antibodies were subjected for immunohistochemistry (IHC) analysis on paraffin-embedded
tissue. The antibodies used in this study were as follows: KAI1 (mouse monoclonal;
Santa Cruz), E-cad (mouse monoclonal; LabVision), vimentin (mouse monoclonal; LabVision),
CD34 (mouse monoclonal; LabVision), and D2-40 (mouse monoclonal; LabVision). The IHC
procedures for these markers were previously described 21]. For each antibody, including negative controls, IHC was done in a single experiment.

Scoring of IHC

By light microscopy, representative tissue sections were scored semiquantitatively
for cytoplasmic and membrane staining. All samples were anonymized and independently
observed by two pathologists. If there is a disagreement, the observers would reexamine
and reach a consensus. In scoring expression of antibodies, both the intensity and
extent of immunopositivity were considered. The dominant staining intensity in tumors
was scored as follows: 0?=?negative, 1?=?weak, 2?=?moderate, and 3?=?strong. The extent
of positive staining tumor cells was scored as follows: 10 % is 1, 11–50 % is 2,
51–75 % is 3, and 75 % is 4. The final score was determined by multiplying the intensity
and the extent positivity scores, which yielded a range from 0 to 12. Mean score from
each individual was calculated in tumor cells. The positive expression for markers
was scored as follows: 2, KAI1; 3, E-cadherin; and 1, vimentin.

The positive expression of KAI1 and E-cad was found mainly on the membrane and cytoplasm
of NSCLC cells and normal lung tissues. The positive expression of vimentin was found
mainly on the cytoplasm of NSCLC cells and normal lung tissues. They were presented
as a brown granular material.

Microvessel and lymphatic vessel density

We assessed lymphatic vessel density (LVD) by D2-40 and microvessel density (MVD)
by CD34 immunohistochemical staining. Not only stained endothelial cell but also endothelial
cell cluster separated from other stromal elements was considered as a countable microvessel
or lymphatic vessel.

Statistical methods

All statistical analyses were done using the statistical software SPSS (SPSS inc.,
Chicago, IL), version 17. The Fisher’s exact test and Pearson chi-square test for
trends in proportions, Spearman’s correlate analysis, and Kaplan-Meier’s method with
log-rank test or Cox regression method for univariate or multivariate OS or disease-specific
survival (DSS) analysis were used to assess the associations among the positive staining
of KAI1, E-cad, vimentin, LVD, or MVD and clinicopathological indices. DSS was defined
from the date of surgery to the time of NSCLC death. A value of P 0.05 was considered statistically significant.