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Both c-Myc and Ki-67 expression are predictive markers in patients with Extranodal NK/T-cell lymphoma, nasal type: A retrospective study in China
Pathology - Research and Practice
Extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKTL) is an increasingly recognized disease entity of aggressive tumor progression. The objective of this study was to investigate the clinical and prognostic significance of c-Myc and Ki-67 expression in ENKTL. Immunohistochemistry for c-Myc and Ki-67 was performed on tissue sections from 53 patients diagnosed with ENKTL, and their correlation with clinicopathologic variables was assessed. We also made a comparison between c-Myc positive and negative ENKTL on proliferation index (PI); and analyzed relationship between expression of c-Myc and Ki-67 index. The survival was analyzed by Kaplan–Meier product-limit method. Positive expression of c-Myc was shown in 64.2% of the patients, and high expression of Ki-67 was found in 66%. The PI in c-Myc-positive tumor cell was significantly higher than that in c-Myc-negative ones (P = 0.005). The positive correlation between c-Myc expression and Ki-67 index was also found (r = 0.454, P = 0.001). Patients of c-Myc expression were shown to be associated with unfavorable overall survival (OS) and decreased progression free survival (PFS) (P = 0.000 and 0.013, respectively). High expression of Ki-67 was also shown to be correlated with worse OS and PFS (P = 0.014 and 0.016, respectively). And patients expression of c-Myc+/Ki-67 high had the worst OS and PFS (P = 0.002 and 0.027, respectively). c-Myc may play an important role in the carcinogenesis of NK/T cell lymphoma by promoting cell proliferation. Both c-Myc expression and Ki-67 high expression in ENKTL patients may be valuable indicators for predicting the survival of ENKTL patients.
Keywords: Extranodal NK/T-cell lymphoma, c-Myc, Ki-67, Prognosis.
Extranodal natural killer (NK)/T-cell lymphoma (ENKTL) is a distinct subtype of non-Hodgkin's lymphoma which is very rare in North America and Europe but rather common in Asia and South America. It has been reported that about for 8.7% of all non-Hodgkin's lymphomas and 74.1% of lymphomas which develop in the nasal cavity and paranasal sinuses are ENKTL in Korea. ENKTL has distinct morphology, immunophenotype, biologic features and clinical behaviors which are associated with an aggressive natural and poor survival rates. Pathologically, ENKTL has unique characteristics including vascular invasion, expression of cytoplasmic CD3, CD56 and cytotoxic molecules such as perforin, and is positive for Epstein–Barr virus (EBV) in situ hybridization. Clinically, it frequently occurs in middle-aged men, and usually presents as a localized disease involving the head and neck  . There is no clear guidance as its optimal therapy. It has been suggested that involved field radiotherapy ± anthracycline-based or asparaginase-based chemotherapy may add benefit for remission, but the overall prognosis of this disease is poor because of frequent relapse or resistance to treatment. In early years, Lee et al. developed a prognostic model which includes four risk factors: B symptoms, advanced stage, elevated LDH, and involvement of regional lymph nodes. Other unfavorable prognostic factors include elevated C reactive protein, anemia, thrombocytopenia and high serum EBV DNA levels  and . However, their prognosis role is still controversial and needed to be further investigated. In addition, other prognostic indicator reflecting biological characteristics, such as a rapid growth of tumor cells, may help to predict differences in survival among patients with ENKTL.
MYC is the most frequently amplified potent oncogene and the elevated expression of its gene product, the transcription factor c-Myc first identified as cancer-related factor in Burkitt's lymphoma, correlates with tumor aggression and poor clinical outcome. Elevated expression of c-Myc occurs through multiple mechanisms in tumor cells, including gene amplification, chromosomal translocation, mutation of upstream signaling pathways, and mutations that enhance the stability of the protein. In recent years, its prognostic value has also been verified in other non-Hodgkin's lymphoma, such as diffuse large B cell lymphoma (DLBCL) , , and . Though several gene expression studies of ENKTL have shown its enrichment, none of them emphasized on its prognostic value  and .
Ki-67 is a nuclear antigen expressed by dividing cells. Thus, the percentage of Ki-67-positive cells reflects the proportion of actively proliferating tumor cells. And it has been considered to be a useful prognostic index in various tumors including lymphoma. Although the prognostic impact of Ki-67 expression remains controversial in non-Hodgkin's lymphoma, several studies in ENKTL indicated that high Ki-67 expression seems to be correlated with poor prognosis  and .
Therefore, we performed this study to determine the value of c-Myc/Ki-67 expression in predicting prognosis in patients with ENKTL.
Materials and methods
From the archives of the Department of Pathology, Jinling Hospital, we selected 53 patients who had been diagnosed with ENKTL from January 2006 to December 2010. All biopsies of nasal ENKTL were taken from sites of the nasal cavity, without therapy before diagnosed. All cases were histological confirmed, with complete clinical and follow-up data. We randomly selected 20 normal lymph nodes from the same period as comparison The pathologic diagnosis of ENKTL was based on the following criteria according to the World Health Organization Classification: (i) the histological features of ENKTL, (ii) the expression of T cell markers (CD3 and/or CD43), CD56 and cytotoxic molecules (perforin and/or TIA1), (iii) the absence of B cell phenotype with CD20, iv) expression of Epstein–Barr virus encoded RNA (EBER) by in situ hybridization  .
The clinical data, gathered from hospital medical records, were available for all patients including: gender, age, serum lactate dehydrogenase (LDH), albumin, white blood cell (WBC), hemoglobin, platelet, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and clinical stage. The main clinicopathologic characteristics of patients with ENKTL are shown in Table 1 . There was a preponderance of male to female patients with a ratio of 4.3:1. The median age at diagnosis was 48 years (range 8–77 years) with a trend for more patients being younger than 60 years. On the basis of the Ann Arbor stage of lymphoma, 29 patients were in stage I, 9 in stage II and 15 in advanced stages (11 in stage III and 4 in stage IV) of the disease at the time of diagnosis. Therapy was known for all patients, 17 received radiotherapy alone, 19 received chemotherapy alone, and 17 received combination of radiotherapy and chemotherapy. Chemotherapy regimens include SMILE and VIPD regimens. All patients signed informed consent forms for this study, which was approved by the institutional Review Board.
|Variables||No.||c-Myc +(34) −(19)||χ 2||P-value||Ki-67 High(35)low(18)||χ 2||P-value|
|Male||43||25 18||26 17|
|Female||10||9 1||9 1|
|<60||44||31 13||29 15|
|≥60||9||3 6||6 3|
|Normal||32||21 11||18 14|
|High||21||13 8||17 4|
|Normal||32||19 13||20 12|
|Low||21||15 6||15 6|
|Normal||42||26 16||28 14|
|Low||11||8 3||7 4|
|Normal||41||25 16||25 16|
|High||12||9 3||10 2|
|Normal||51||32 19||33 18|
|Low||2||2 0||2 0|
|Normal||36||21 15||20 16|
|High||17||13 4||15 2|
|Normal||42||26 16||25 17|
|High||11||8 3||10 1|
|I–II||38||20 18||21 17|
|III–IV||15||14 1||14 1|
Lactate dehydrogenase (LDH), white blood cell (WBC), alanine aminotransferase (ALT), aspartate aminotransferase (AST). Statistical significance was estimated using the χ2 test. P-values (two-sided) < 0.05 were considered statistically significant.
Tissue sections (4 μm) from formalin-fixed and paraffin-embedded ENKTL samples were deparaffinized, rehydrated and subjected to pressure & heat-induced antigen retrieval in DakoCytomation Target Retrieval Solution, Citrate, pH8.0 (DAKO, Carpinteria, USA) at 100 °C for 20 min in water bath. The slices were then rinsed with PBS (0.01 mol/L, pH7.4) for three times, incubated with the primary antibody overnight at 4 °C and rinsed again with PBS three times as mentioned above. The antibodies we used were CD20 (clone L26; Novocastra, dilution 1:200), CD3 (clone PS1; Dako, dilution 1:100), CD43 (clone MT1; Novocastra, dilution 1:100), CD56 (clone 1B6; Novocastra, dilution 1:20), perforin (clone 5B10; Novocastra, dilution 1:50), c-Myc (clone Y69, Epitomics, dilution 1:80), Ki-67 (clone SP6; ZETA, dilution 1:200). They were then incubated with the anti-rabbit and mouse horseradish peroxidase polymer reagent for 20 min at ambient temperature and washed again with PBS three times as before. The reaction product was developed by diaminobenzidine tetrahydrochloride (DAB). Finally, the slices were counterstained in hematoxylin, dehydrated and coverslipped. The IHC results were reviewed by two independent certified pathologists without knowledge of clinical data. 20 normal lymph nodes were used as negative control in c-Myc immunostaining. Blank control was performed with PBS (0.01 mol/L, pH7.4) replacing the primary antibody and no staining was detected in negative control sections.
Immunoreactivity for c-Myc and Ki-67
Both c-Myc and Ki-67 expression were observed in the nucleus of tumor cells. The percentage of cells with c-Myc or Ki-67 expression was calculated from the number of malignant cells in the highest labeling field under high magnification (400×). The expression of c-Myc was assessed according to the percentage of positive staining cells found in 1000 neoplastic cells (quantitative analysis). Immunoreaction was evaluated as follows: negative (−): <5% of stained neoplastic cells; positive (+): 5–25% of stained neoplastic cells; intense positivity (++): 25–50% of stained neoplastic cells; and extremely high positivity (+++): >50% of tumor cells stained. The percentage of Ki-67 expression was quantified in the same way, which also used as proliferation index (PI) in this study. The median percentage (50%) of Ki-67 expression was designated as a cutoff, thus cases higher than the median value (≥50%) were defined as high Ki-67 expression, and less than the median value (<50%) were defined as low Ki-67 expression for the survival analysis. All slides were evaluated independently by two pathologists who were blind to the clinical outcomes of the patients.
Statistical analyses were performed using Software Packages for Social Science 16.0 for Windows (SPSS, IBM, Chicago, IL, USA). Independent t test was used to make a comparison between c-Myc positive and negative ENKTL on PI. The correlation between c-Myc and Ki67 was assessed using the Spearman rank correlation analysis. The relationship between c-Myc/Ki-67 expression and clinical variables was evaluated using Pearson's χ2 test or Fisher's exact test when necessary. (Overall survival) OS rates and (Progression Free Survival) PFS rates were estimated using the Kaplan–Meier product-limit method and compared with the log-rank test. The Cox proportional hazards regression model was used for the multivariate analysis to compare the factors which were proven significant in the univariate analysis. OS was determined from the date of diagnosis until death. PFS was assessed from the first day of diagnosis to relapse, disease progression, death from any cause or last follow-up. The time points were reported in months. All statistical tests were 2-sided, and P-values of <0.05 were considered to be statistically significant.
Immunohistochemical stain is shown in Figure 1 , and the characteristics of patients in relation to c-Myc/Ki-67 expression are listed in Table 1 . Based on the aforementioned criteria, c-Myc expression was presented in 64.2% (34/53) of cases, and 66% (35/53) showed high expression of Ki-67 protein. The positive expression of c-Myc protein was shown to be correlated with patient's age (P = 0.034) and clinical stage (P = 0.009), but not with other clinicopathologic factors such as gender, LDH, albumin, WBC, hemoglobin, platelet, ALT, and AST. High expression of Ki-67 was correlated with ALT (P = 0.029) and clinical stage (P = 0.010), but not with other variables including gender, age, LDH, albumin, WBC, hemoglobin, platelet, and AST. The correlation of c-Myc and Ki-67 protein was also analyzed. The proliferation index in c-Myc-positive tumor cell was significantly higher than that in c-Myc-negative ones ((57.50 ± 20.97) % and (38.21 ± 26.07) %, respectively; P = 0.005). The positive correlation between c-Myc and Ki-67 was also noted ( Figure 1 D, r = 0.454, P = 0.001).
Among all cases, the 2-year OS was 47.2%, median OS was 18 months (95% CI, 10.079–25.921 months); 2-year PFS was 35.8%, median PFS was 13 months (95% CI, 9.529–16.471 months). The Kaplan–Meier analysis showed significantly lower survival rate and PFS rate in patients with c-Myc expression than in those without, indicating that expression of c-Myc protein was associated with decreased OS and PFS (P = 0.000 and 0.013, respectively). The 2-year survival rates in the high and low Ki-67 expression group patients were 34.3% and 72.2%, respectively; and 2-year PFS rates were 22.9% and 61.1%, respectively, suggesting that high Ki-67 protein expression was correlated with unfavorable prognosis (P = 0.014 and 0.016, respectively). When four different groups were divided (c-Myc+/Ki-67 high, c-Myc+/Ki-67 low, c-Myc-/Ki-67high, c-Myc-/Ki-67 low), we found that the c-Myc+/Ki-67 high group had the worst OS and PFS (P = 0.002 and 0.027, respectively).
Univariate survival analysis also showed that elevated LDH level, decreased albumin level, decreased platelets level, elevated AST level, and high clinical stage (III–IV) were significantly associated with poor OS (P = 0.012, 0.033, 0.002, 0.040 and 0.030, respectively). Elevated LDH level, decreased platelets level, and high clinical stage (III–IV) were significantly related to shorter PFS (P = 0.015, 0.002 and 0.013, respectively). Forward conditional multivariate Cox regression analysis was performed to assess the independent role of these variables ( Table 2 , P < 0.05). The expressions of c-Myc and elevated LDH levels were independent prognostic predictors of OS, because those positive expression of c-Myc had a 6.676-fold (95%CI: 2.238–19.916) high risk of death, and elevated LDH levels had a 3.272-fold (95%CI: 1.464–7.310) high risk of death. Decreased platelets level and high clinical stage were independent prognostic predictors of PFS, since those patients with decreased platelets level and high clinical stages (III–IV) had high risk of disease progress (P = 0.007 and 0.015, respectively). Kaplan–Meier curves of patients are shown in Fig 2, Fig 3, and Fig 4.
|Variable||B||Exp (B)||95% CI||P-value|
ENKTL is a distinct type of extranodal lymphoma with a unique immunophenotype of NK cell derivation, which mostly often occurs in male adults. Cell proliferation mechanisms may play a role in lymphoma-genesis and progression. In this study, we assessed 53 cases of ENKTL for the expression of proliferative indices, c-Myc and Ki-67, and examined their relationship to clinical characteristics and survival.
The proto-oncogene c-MYC encodes a transcription factor c-Myc which is implicated in various cellular processes—cell growth, proliferation, loss of differentiation and apoptosis  and . Early studies have already discovered that over-expression of c-Myc protein contributes to the development of series malignant tumors, such as Burkitt's lymphoma, metastatic breast cancer, esophageal squamous carcinoma, colon cancer, and DLBCL , , and . But the prognostic value of c-Myc has never been studied in ENKTL before. A recent study performed by Ng et al. has identified high expression of c-Myc as downstream target regulated by NF-κB in patients with ENKTL. But they have not investigated the prognostic value of c-Myc  . In the present study, we showed that c-Myc expression was positive in about 64.2% (34/53) of patients with ENKTL and significantly associated with patient's age and clinical stage. Moreover, positive expression of c-Myc was found to be correlated with significantly worse OS and PFS of patients. As c-Myc is a transcription factor and strong proto-oncogene, we suppose that it is activated through the following signal pathway: MAPK/ERK, JAK/STAT, or NF-κB, and then drives cell proliferation by regulating cell growth and apoptosis. It is of interest to investigate the possible mechanisms of c-Myc in the pathogenesis in ENKTL.
The Ki-67 protein is a cellular marker for proliferation. Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0). The proliferation index, which is the percentage of Ki-67-positive cells among the total tumor cells in a selected area, has been used to assess the proliferative activity of tumors. It is strictly associated with cell proliferation, and closely correlated with an unfavorable clinical course of many types of cancer including lymphoma. The prognostic value of Ki-67 in ENKTL was first studied by Kim et al. in 2007. However, in their study, its prognostic significance was only shown in patients with stage I/II ENKTL  . In our present study, high Ki-67 expression was defined by the median value (50%). High expression of Ki-67 was presented in 66% (35/53) of patients with ENKTL and significantly correlated with ALT and clinical stage. We also found that both OS and PFS were poor for patients with high Ki-67 expression.
In the current study, the positive correlation between c-Myc and Ki-67 was also found. When the two molecular markers were simultaneously analyzed, we found that c-Myc+/Ki-67 high group had the worst PFS. To the best of our knowledge, this is the first study to investigate the expression and prognostic value of combined c-Myc and Ki-67 in ENKTL. Although the possible mechanisms of their expression remain unclear, it seems that both c-Myc and Ki-67 have the potential of being molecular markers and might be used to predict the survival of ENKTL patients. Of course, further researches are needed to investigate the roles and possible mechanisms of c-Myc and Ki-67 in ENKTL.
Collectively, our data suggests that c-Myc protein expression is correlated with certain ENKTL clinical parameters, such as the high clinical stage and patient prognosis. And the high-level expression of Ki-67 links to poor OS and PFS. In addition, a significant positive association was found between the expression of c-Myc and Ki-67 protein, and the c-Myc+/Ki-67 high group had the worst PFS. The c-Myc expression, along with elevated LDH levels, can act as independent prognostic factors for the prediction of overall survival. In summary, our results indicate that the expression of c-Myc protein and high-level expression of Ki-67 may favor the progression of ENKTL. Since the sample size of this study is smaller, these results should be confirmed in a large prospective study.
Conflict of interest
The authors declare no conflicts of interest.
We thank Prof. Xiao-Jun Zhou and Mr. Bo Yu, Department of Pathology, Jinling Hospital, Nanjing, for their sincerely technical assistance with the immunohistochemistry procedures.
Appendix A. Supplementary data
The following are the supplementary data to this article.
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a Department of Medical Oncology, Jinling Hospital, Medical School of Nanjing University, 305 Zhong Shan Road East, Nanjing, Jiangsu Province, People's Republic of China
b Department of Pathology, Jinling Hospital, Medical School of Nanjing University, Nanjing, Jiangsu Province, People's Republic of China
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