Altered p16INK4 and RB1 Expressions Are Associated with Poor Prognosis in Patients with Nonsmall Cell Lung Cancer

p16INK4 and RB1 are two potent cell cycle regulators to control the G1/S transition by interacting with CDK4/6, E2F, and D-type cyclins, respectively. Depending on the tumour type, genetic alterations resulting in the functional inactivation have frequently been reported in both genes. By contrast, much less is known regarding the overexpression of these proteins in the tumor cells. In this study, expressions of p16INK4 RB1, and CDKN2A copy number variances (CNV) in the tumor cells were assessed by immunohistochemistry and fluorescence in situ hybridization (FISH), respectively, in 73 nonsmall cell lung cancer (NSCLC) with known 5-year survivals. The histologic type (P = 0.01), p16INK4 (P = 0.004), and RB1 (P < 0.001) were predictive of survivals. The CDKN2A CNV (P < 0.05) was also significant when compared to those cases without CNV. Therefore, among the molecular genetic prognostic factors, expressions of RB1 and p16INK4 in the tumor cells were the most strongly predictive of adverse outcomes in stage I and II nonsquamous NSCLC.


Introduction
Primary lung carcinoma is one of the leading causes of cancer death worldwide. Genetic and molecular alterations involving tumorigenesis have been extensively studied. Inactivation of tumor suppressor genes by deletion, mutations, altered splicing, promoter mutations, or epigenetic modifications are the common causes in lung cancers [1][2][3]. Amplification and activation mutations of oncogenes are often account for many malignant behaviors and worse clinical outcomes [4,5]. In fact, most of these genetic alterations might directly or indirectly affect the cell cycle and proliferation of the tumor cells. p16 INK4 and RB1 are two important tumor suppressor proteins and participate in negatively regulating the proliferation of normal cells [6][7][8]. Like other tumors, studies were focused on the genetic alterations resulting in either loss or decreased expressions and functions in the tumor cells because of their inhibitory roles in cell proliferation [9][10][11][12][13][14]. By contrast, studies were limited regarding the overexpression of these proteins and their effects on the tumorigenesis and prognosis in the tumor cells. Reports become more prominent in the head and neck squamous carcinomas in which p16 INK4 was overexpressed under the viral effect by the high-risk serotypes of the human papilloma virus (HPV), though sparse reports in tumors like basal-like breast carcinoma and NSCLC [15][16][17]. A single study showed that the combined RB-negative/p16positive/cyclin D1-negative tumors in NSCLC might relate to the adverse outcomes, but the independent role of each proteins (p16 INK4 and RB1) in the unfavorable prognosis was not confirmed [17]. In this paper, we studied p16 INK4 and RB1 protein expressions and CDKN2A gene copy variances in NSCLC with special reference to an association of the abnormal individual protein expression with clinical characters.

Case Selections and Tissue Microarray.
A tissue microarray (TMA) was prepared from formalin-fixed paraffin-embedded (FFPE) tissue specimens from 1985 to 1997 acquired through the pathology archive services of the Ohio State University Medical Center, Columbus, OH, USA. All the cases selected for this study meet following criteria: (1) nonsquamous NSCLC, surgically managed patients with stage I or stage II NSCLC at the time of diagnosis; (2) available clinical followup and outcome data; (3) adequate tissue (all surgical resection specimen) for immunohistochemical stains (IHC) or molecular studies. Patients selected for this study received no neoadjuvant chemotherapy or radiotherapy prior to surgery. Seventy-three NSCLC cases met the criteria and were included in this study. All the cases were reviewed, and the pathology diagnosis of each case was reclassified according to the current WHO classification. The study has been approved by the institutional human research committee. Additionally, tissues from human brain, lung, lymph node, kidney, placenta, thyroid, heart, liver, testes, and adrenal glands (1-2 samples each) were included in the TMA as normal controls.

Immunohistochemistry (IHC).
Immunohistochemistry was done using monoclonal p16 antibody clone INK4 (MTM laboratories) or pRB clone 13A10 (NovoCastra Laboratories) on a DAKO-automated staining instrument (Dako Scientific Systems, Tucson, AZ, USA) using an ABC-based detection kit (I View DAB, Ventana Medical Systems) or polymerbased detection kit (Mach3, Biocare Medical) as described previously [18,19]. Staining intensity was scored semiquantitatively separately for the cytoplasm and/or nucleus, using a scale from 0 to 3: 0, no staining; 1+, weak intensity in more than 25% of nuclei; 2+ moderate and 3+, strongly positive intensity in more than 75% of nuclei. Tumor cells with moderate (2+) or strong (3+) stainings were graded as overexpression or positive, while none (0) and weak (1+) stainings were negative. Specimens were scored in a blinded fashion by two pathologists (W. Zhao and M. E. Leon).

Interphase Fluorescence In Situ Hybridization (FISH).
To investigate the CDKN2A gene copy number variances (CNV), a dual color chromosome 9 centromere, CEP9 (spectrum green), and CDKN2A gene spectrum (orange) probe kit were used (Vysis, Abbott Laboratories, Abbott Park, IL) on the paraffin-embedded tissues (FFPE), either on the TMA or full sections at 2 to 4-μm-thickness as described previously with modifications [19]. Normally, each nucleus was expected to have 2 copies of each CEP9 (reference numbers of chromosome 9) and CDKN2A gene, that is, a cell without CNV should have ratio of 1 (2 CDKN2A/2 CEP9). The loss of CDKN2A might be homozygous (0/2, ratio = 0) or heterozygous (1/2, ratio = 0.5). The gains of CDKN2A might be amplification (>4/2, ratio >2.1) or polysomy 9 (both CDKN2A and CEP9 were amplified). All of the images and FISH slides were reviewed by a pathologist (W. Zhao) using a fluorescence microscope (Olympus BX51), and images were taken with a digital image camera (DP70, Olympus, USA).

In Situ Hybridization of HPV High-and Low-Risk Probes.
The sample was processed and detected for HPV-H and -L risk probes as described previously [20].

Survival and Statistical Analysis.
Patient survival status at five years (60 months) after initial pathologic diagnosis was obtained from the institutional information warehouse at The Ohio State University and used as the outcome variable to calculate the five-year overall survival rate. Univariable analyses were performed using Chi-square (χ 2 ) test or Fisher's exact test to study the associations of categorical variables to the outcomes. P value of <0.05 was considered statistically significant. Statistical analyses were performed using MedCalc software (Frank Schoonjans, Mariakerke, Belgium).

Results
A total 73 cases of nonsquamous NSCLC at stage I and II were enrolled in this study. Overall, 32 out of 73 (43.8%) patients survived more than 5 years after the diagnosis. The factors with no correlation to the outcomes were age (median 65.0 years, 34-85) and gender (males: 57.5% versus females: 42.5%).
In the head and neck carcinoma, p16 INK4 positivity was positively associated with the infection of the high-risk human papilloma viruses (HPV) [20]. We examined this correlation by in situ hybridization (ISH) on the 73 tumor samples, and none (0/73, 0%) was positively detected with HPV-H and HPV-L probes (Data not shown).
RB1 expression was readily detected in the nuclei by IHC ranging from strong (3+ to 2+), weak (1+), to absent, as illustrated in Figure 3. In contrast to p16 INK4 , expression of RB1 (1+) was detected in the nuclei of the normal control tissues from heart, lung, thyroid, testes, adrenal gland, prostate, and kidney but variably in the stromal endothelial, fibroblast, and lymphoid cells. The expression of low level of RB1 in the normal tissues might be due to the fact that RB1 promoter are reminiscent which might be associated with housekeeping genes and result in the ubiquitous expression of the RB1 gene [21].

Discussion
In this study, we demonstrated that factors associated with poor outcomes in stage I and II nonsquamous NSCLC included nonadenocarcinoma, positive expression of p16 INK4 and RB1 by IHC, and with CNV of CDKN2A gene in the tumor cells. Consistent with previous reports, p16 INK4 was undetectable in the normal tissues or the stromal cells of the tumor tissues. The absent expression of p16 INK4 in the tumor cells, however, might be caused by homozygous loss of CDKN2A gene as demonstrated in this study or hypermethylation of the CDKN2 promoters as seen in other tumor types [22,23]. The causes of the overexpression in the tumor cells might resulted from genetic abnormalities, viral effect or a tumor-associated mutant of CDKN2A [24][25][26]. We demonstrated that in NSCLC, 32% of the p16 INK4 -positive cases was resulted from the increased copy numbers of CDKN2A. The HPV viral effect was ruled out by negative ISH results in this study. Therefore, studies for the mechanisms that result in an upregulated p16 INK4 expression should be sought in the future.
The overexpression of p16 INK4 protein in tumor cells is not uncommon findings. For example, p16 INK4 was increased in multiple ovarian cancer cell lines as well as in 7 of 10 clinical ovarian cancer specimens [27]. Previous reports also showed that p16 INK4 -positive prostate cancers were associated with early relapse and relapse, its association to an unfavorable prognosis in NSCLC is not known yet [28][29][30]. The paradoxical p16 INK4 positivity in NSCLC associated to the poor outcome is demonstrated in this study though interpretation needs to be cautious due to relatively small numbers included.
Like p16 INK4 , loss of RB1 function by genetic deletion is commonly seen to be an essential process of oncogenesis in wide ranges of human malignancy, such as retinoblastoma, breast cancer, and small cell carcinoma of the lungs. The increased RB1 in tumor cells was puzzling but was observed in colorectal carcinoma and bladder tumors [31,32]. In this study, we demonstrated that RB1 positivity in NSCLC was often seen (43.8%) in NSCLC. Furthermore, we demonstrated that its overexpression was significantly associated with the adverse outcomes. In addition, in those p16 INK4negative or adenocarcinoma tumors, the RB1 status stratifies them into favorable and unfavorable groups.
Abundant functionally defective mutant protein might be produced in the tumor cells, but their significance to the clinical outcomes is not clear yet [33,34]. The significance of increased expression of functionally intact tumor suppressor proteins such as p16 and RB1 in malignant cells remains poorly understood but might be explained by the concept of the cellular homeostasis in the cancer cells. For example, the apoptosis that normally resulted from Myc overproduction can be suppressed in tumor cells by oncogenic mutations that stimulate survival signals or directly inhibit the apoptotic machinery. Therefore, in order to couple with the hyperproliferation stress, the tumor cells might increase the production of cell-cycle inhibitory proteins such as p16INK4 to suppress the G1/S transition. The tumor cells with low proliferation might be more resistant to radiation and chemotherapy. Furthermore, the roles of overexpressed RB1 in suppressing the apoptosis might result in resistance to therapeutic radiation or chemotherapy, too [35,36].
In conclusion, we demonstrated that tumors with higher expression of p16 INK4 and RB1 were statistically significantly associated with unfavorable outcomes in patients with stage I and II nonsquamous NSCLC. The stratification of these patients by profiling p16 INK4 and RB1 protein expression in the tumors might provide predictive biomarkers for cancer prognosis. Further works to understand how these tumor suppressor genes were abnormally upregulated, and their roles in cancer homeostasis are needed to provide scientific bases for the prevention interference.